Polysaccharides Structural Diversity and Functional Versality

The rst edition was published as Polysaccharides: Structural Diversity and Functional Versatility, edited by Severian Dumitriu (Marcel
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1998).
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Foreword
Polysaccharides as natural polymers are by far the most abundant renewable resource on the earth with an annual formation
rate surpassing the world production rate of synthetic polymers by some orders of magnitude. In contrast to petroleum-based
synthetic polymers, plant polysaccharides are sustainable materials synthesized by the suns energy and fully biodegradable in
the original state. Thus, with decreasing supply of oil resources polysaccharides, including cellulose, starch, chitin, and
hemicelluloses, are expected to play an increasingly important role in industrial use. Polysaccharides are designed by nature to
carry out various specic functions. Examples comprise structural polymers such as cellulose and chitin, storage
polysaccharides such as starch and glycogen, and gel forming mono- and copolymers such as mucopolysaccharides
(glycosaminoglycans), agar, and pectins. Generally, polysaccharides are highly functional polymers with magnicent
structural diversity and functional versatility. Their structural and functional properties are often superior to synthetic
materials as demonstrated, for instance, by the cellulose based cell wall architecture of plants or the function of hyaluronic acid in
the human body.
It has been a true challenge to present state-of-the-art polysaccharide research from dierent aspects regarding the
macromolecular variety, function and structure in just one volume. In this book well-known and recognized authors describe
the current state of research in their specic elds of expertise in which many of them have been active for decades. With regard
to cellulose and starch as the most abundant polysaccharides, structure, chemical modication, physical chemistry, and
industrial aspects are being discussed. It is further demonstrated that cellulosic biomass conversion technology permits large
scale sustainable production of basic chemicals and derived products. The focus of other chapters are bacterial polysaccharides,
hemicelluloses, gums, chitin, chitosan, hyaluronan, alginates, proteoglycans, glycolipides, and heparan sulfate-like polysac-
charides. Some chapters deal with medical and pharmaceutical aspects including medical foods, anticoagulant properties and
the role of polysaccharides in tissue engineering. Furthermore, methodical aspects, including characterization by X-ray
scattering, spectroscopic methods, light scattering, and rheology are discussed.
In summary, the comprehensive, improved, and expanded second edition of Polysaccharides reects the current state
of knowledge of nearly the entire spectrum of polysaccharides with emphasis on structures, methods of structural analysis,
functions and properties, novel routes of modication, and novel application elds. With each chapter, the reader will nd
references for a deeper insight into a specic eld. Thus, this book is a very useful tool for scientists of both academia and
industry interested in the fundamental principles of polysaccharide functions and modications on one hand and novel
applications on the other. Having been involved in similar work mainly with industry-related issues of cellulose research for
many years, I would like to stress that the presented state of knowledge, as sophisticated as it might seem to be, should not be
understood as the nal stage, but as an invitation to add new knowledge to this eld and to explore additional applications of
polysaccharides. I would be delighted, if this monograph challenged and encouraged scientists to deal with polysaccharides as
fascinating polymers with a bright future.
Hans-Peter-Fink
Fraunhofer-Institute for Applied Polymer Research
Potsdam-Golm, Germany
iii
Preface
Polysaccharides are the macromolecules that belong to the means components of life. Together with nucleic acids and
proteins, the polysaccharides determine the functionality and specicity of the species. Polysaccharides have received little
such promotion even though they are widely distributed throughout nature and have highly organized structure. There are
important molecules involved throughout the body in signal transduction and cell adhesion. Polysaccharides can be broadly
classied into three groups based on their functions, which are closely related to their occurrence in nature: structural,
storage, and gel forming. The rst compounds used at the industrial level were the polysaccharides.
This work provides the most complete summary now available of the present knowledge of polysaccharide chemistry.
This book discusses eleven fundamental aspects of polysaccharides:
1. Progress in structural characterization. The structural analysis may oer the most fundamental knowledge to
understand the functions of polysaccharides, but the diversity and irregularity of polysaccharide chains make the structural
analysis a formidable task. The conformational analysis involves two aspects: (a) the characterization of a single chain
conformation and (b) the analysis of the chain assembly of polysaccharides. A remarkable progress has been achieved in recent
years with high-resolution, solution- and solid-state-1H- and 13C-NMR including cross-polarization-magic-angle-spinning
and two-dimensional techniques. Specic electron microscopy techniques can visualize single polysaccharide molecules and
can yield reliable information on their contour length distribution, persistence length and conformational aspects.
Some recent progress reports on computational methods for simulations and calculations associated with structure
elucidation of polysaccharides have demonstrated that these methods can contribute to a decision on the actual
conformational properties of oligosaccharides and linear polysaccharides.
2. Conformation and dynamic aspects of polysaccharide gels. The most important aspect of characterization of
polysaccharide gels seems to clarify their backbone dynamics together with conformations as viewed from their highly
heterogeneous nature. Backbone dynamics of polysaccharide gel network can be characterized by means of simple
comparative high-resolution 13C NMR measurements by cross-polarization-magic angle spinning (CP-MAS) and dipolar
decoupled-magic angle spinning (DD-MAS) techniques.
3. Rheological behavior of polysaccharides in aqueous systems. Rheology provides precious tools to explore and
understand the properties of polysaccharides in aqueous systems. The rheological behavior of polysaccharides systems
manifests the underlying structure of the systems. In the simplest case, that of polysaccharides solution, viscosity is directly
related to fundamental molecular properties (molecular conformations, molecular weight and molecular weight distribution,
intramolecular and intermolecular interactions). In the case of more structured polymer systems, gels, for example, their
viscoelastic properties are related to supramolecular organization. The main types of polysaccharide systems that are
encountered in the applications can be distributed schematically in three classes: solutions, gels, and polysaccharide/
polysaccharide (or polysaccharide/protein) mixtures in aqueous media.
4. Biosynthesis, structure, and physical properties of bacterial polysaccharides (exopolysaccharides). This part presents
the mechanisms of biosynthesis of bacterial polysaccharides and provides some information on the engineering of
polysaccharides that will allow in the near future the production of a polysaccharide with a choice chemical structure
having a set of predictable physical properties. This part covers also pertinent areas such as: bacterial and fungal
polysaccharides, cell-wall polysaccharides, production of microbial polysaccharides, industrial gums, and microbial
exopolysaccharides of practical importance.
v
vi Preface
The bacterial polysaccharides are described as: production and synthesis, composition and structure, physical
properties, degradation by polysaccharases and polysaccharide lyases, polysaccharides common to prokaryotes and
eukaryotes, biological properties and applications and commercial products.
One chapter is dedicated to the presentation of the order-disorder conformational transition of xanthan gum.
5. Hemicelluloses may function both as framework and matrix substances or reserve substances in seeds, where they
form independent wall layers which are mobilized when the seed germinates. In both hardwood and softwood, hemi-
celluloses fraction in lignied cell walls represents the matrix substance. This important part of the polysaccharides chemistry
is presented in three chapters: Hemicelluloses: Structure and properties; Chemical modication of hemicelluloses and gums;
Role of acetyl substitution in hardwood xylan.
6. In this edition a particular emphasis is placed on the presentation of the ionic polysaccharides (polyanion and
polycation) in the following chapters: AlginateA polysaccharide of industrial interest and diverse biological functions;
Characterization and properties of hyaluronic acid (hyaluronan); Structure property relationship in chitosans; Chitosan as
a delivery system for transmucosal administration of drugs; Pharmaceutical applications of chitosan; Macromolecular
complexes of chitosan.
7. Cellulose and starch are the two polysaccharides which constitute the majority of the polysaccharide production.
They are presented in four chapters: Chemical functionalization of cellulose; The physical chemistry of starch; Starch:
commercial sources and derived products; New development in cellulose technology.
8. The polysaccharides of a major importance in medicine and biology are extensively discussed in nine chapters:
Polysialic acid: structure and properties; Brain proteoglycans; Crystal structures of glycolipids; Synthetic and natural
polysaccharides with anticoagulant properties; Structural elucidation of heparan sulfate-like polysaccharides using
miniaturized LC/MS; Enzymatic synthesis of heparan sulfate; Synthetic and natural polysaccharides having biological
activities; Polysaccharide-based hydrogels in tissue engineering and Medical foods and fructooligosaccharides.
Polysialic acids form a structurally unique group of linear carbohydrate chains with a degree of polymerization up to 200
sialyl residue. Polysialic acids chains are covalently attached to membrane glycoconjugates on cells that range in
evolutionary diversity from bacteria to human brains.
Proteoglycans, a group of glycoproteins that are invested with covalently bound glycosaminoglycan chains, are one of
the important classes of molecules in brain development and maturation. The glycosaminoglycan chains that dene
proteoglycans are of four major classes: heparan sulfate; chondroitin sulfate, dermatan sulfate and keratan sulfate.
The glycolipids play roles as the structural holder of membrane proteins suspended in bilayer or bicontinuous cubic
phases and as the key code of the intercellular communication or immune system.
Anticoagulant polysaccharides as heparin, heparan sulfate and nonheparin glycosaminoglycans (dermatan sulfate,
chondroitin sulfates, acharan sulfate, carrageenas, sulfated fucans, sulfated galactan and nonheparin glycosaminoglycans
from microbial sources) have been of interest to the medical profession.
9. Renewable resources. Cellulosic biomass includes agricultural (e.g., corn stover and sugarcane bagase) and
forestry (e.g., sawdust, thin-nings, and mill wastes) residues, portions of municipal solid waste (e.g., waste paper) and
herbaceous (e.g., switch-grass) and woody (e.g., poplar trees) corps. They are appropriate materials used as renewable
resources for the production of building blocks for various industrial chemicals and engineering plastics polysaccharides.
The chapters Bioethanol production from lignocellulosic material, and Cellulosic biomass-derived products, describe
and evaluate the process for ethanol fuel production. The raw material, hydrolysis, and fermentation are described in detail
as well as the dierent possibilities to perform these process steps in various process designs. The chapter Hydrolysis of
cellulose and hemicellulose presents a comprehensive overview of the technology and economic status for cellulose and
hemicellulose hydrolysis describes the important structural features of cellulosic materials, applications, process steps, and
stoichiometry for hydrolysis reactions. The chapter then examines biomass structural characteristics that inuence cellulose
hydrolysis by enzymes, types of cellulose hydrolysis processes, experimental results for enzymatic conversion of cellulose,
and summarizes some of the factors inuencing hydrolysis kinetics.
10. New applications of polysaccharides. This section provides a selection of some new developmental products and
some recent applications, which might become of commercial interest in the near future. The polysaccharides are utilized as
gallants, thickeners, lm formers, llers, and delivery systems in pharmaceutical and cosmetic applications.
Immobilization. The use of ionic polysaccharides for the immobilization (enzymes, cells and other biocatalysts for
biotechnological production)
Ligand systems. Chitin, chitosan and other functional polysaccharides have also been widely used for the preparation
of metal chelators. Industrial application ranges from waste water treatment, ion exchange resins, and precious
metal recovery.
Separatory systems. Cellulose and chitosan derivatives are dominating the membrane market due to their favorable
stability and their selectivity in gas- and liquid-phase separations.
Biosurfactants. Numerous microorganisms (candida lipolytica, Acetinobacter calcoaceticus) produce extracellular
glycoconjugates with pronounced capabilities to modify interfacial and surface conditions.
Cellulose derivative composites for electro-optical applications. These studies present an optical cell formed by a trans-
parent solid matrix of mixed esters of cellulose with micrometer-sized pores lled with a nemantic liquid crystal.
Preface vii
11. Incorporation of the polysaccharides in the synthetic matrix oers on one hand the possibility to obtain a broader
application range of the usual polymers and, on the other hand, ways to optimize and control some properties and produce
new materials with unexpected performance at low cost.
The treatise is truly international with authors now residing in Austria, Brazil, Canada, Denmark, Egypt, Finland,
France, Germany, Greece, Japan, The Netherlands, Norway, Portugal, Romania, Sweden, United Kingdom, and the United
States. The editor is grateful to all the collaborators for their precious contributions.
Severian Dumitriu
Contents
Foreword Hans-Peter-Fink iii

Preface v
Contributors xiii
1. Progress in Structural Characterization of Functional Polysaccharides 1

Kanji Kajiwara and Takeaki Miyamoto
2. Conformations, Structures, and Morphologies of Celluloses 41

Serge Perez and Karim Mazeau
3. Hydrogen Bonds in Cellulose and Cellulose Derivatives 69

Tetsuo Kondo
4. X-ray Diraction Study of Polysaccharides 99

Toshifumi Yui and Kozo Ogawa
5. Recent Developments in Spectroscopic and Chemical Characterization of Cellulose 123

Rajai H. Atalla and Akira Isogai
6. Two-Dimensional Fourier Transform Infrared Spectroscopy Applied to Cellulose and Paper 159
Lennart Salmen, Margaretha Akerholm, and Barbara Hinterstoisser
7. Light Scattering from Polysaccharides 189

Walther Burchard
8. Advances in Characterization of Polysaccharides in Aqueous Solution and Gel State 237

M. Rinaudo
9. Conformational and Dynamics Aspects of Polysaccharide Gels by High-Resolution Solid-State NMR 253
Hazime Saito
10. Correlating Structural and Functional Properties of Lignocellulosics and Paper by Fluorescence 267
Spectroscopy and Chemometrics
Emmanouil S. Avgerinos, Evaggeli Billa, and Emmanuel G. Koukios
ix
x Contents
11. Computer Modeling of PolysaccharidePolysaccharide Interactions 281

Francois R. Taravel, Karim Mazeau, and Igor Tvaroska
12. Interactions Between Polysaccharides and Polypeptides 305

Delphine Magnin and Severian Dumitriu
13. Rheological Behavior of Polysaccharides Aqueous Systems 357

Jacques Lefebvre and Jean-Louis Doublier
14. Stability and Degradation of Polysaccharides 395

Valdir Soldi
15. Biosynthesis, Structure, and Physical Properties of Some Bacterial Polysaccharides 411
Roberto Geremia and Marguerite Rinaudo
16. Microbial Exopolysaccharides 431

I. W. Sutherland
17. OrderDisorder Conformational Transition of Xanthan Gum 459

Christer Viebke
18. Hemicelluloses: Structure and Properties 475

Iuliana Spiridon and Valentin I. Popa
19. Chemical Modication of Hemicelluloses and Gums 491

Margaretha Soderqvist Lindblad and Ann-Christine Albertsson
20. Role of Acetyl Substitution in Hardwood Xylan 509

Maria Grondahl and Paul Gatenholm
21. AlginateA Polysaccharide of Industrial Interest and Diverse Biological Functions 515
Wael Sabra and Wolf-Dieter Deckwer
22. Characterization and Properties of Hyaluronic Acid (Hyaluronan) 535

Michel Milas and Marguerite Rinaudo
23. Chemical Functionalization of Cellulose 551

Thomas Heinze
24. The Physical Chemistry of Starch 591

R. Parker and S. G. Ring
25. Starch: Commercial Sources and Derived Products 605

Charles J. Knill and John F. Kennedy
26. StructureProperty Relationship in Chitosans 625

Kjell M. Varum and Olav Smidsrd
27. Chitosan as a Delivery System for the Transmucosal Administration of Drugs 643
Lisbeth Illum and Stanley (Bob) S. Davis
28. Pharmaceutical Applications of Chitosan and Derivatives 661

M. Thanou and H. E. Junginger
29. Macromolecular Complexes of Chitosan 679

Naoji Kubota and Kei Shimoda
30. Polysialic Acid: Structure and Properties 707

Tadeusz Janas and Teresa Janas
Contents xi
31. Brain Proteoglycans 729

Russell T. Matthews and Susan Hockeld
32. Crystal Structures of Glycolipids 743

Yutaka Abe and Kazuaki Harata
33. Synthetic and Natural Polysaccharides with Anticoagulant Properties 773

Fuming Zhang, Patrick G. Yoder, and Robert J. Linhardt
34. Structural Elucidation of Heparan Sulfate-Like Polysaccharides Using Miniaturized LC/MS 795
Balagurunathan Kuberan, Miroslaw Lech, and Robert D. Rosenberg
35. Enzymatic Synthesis of Heparan Sulfate 811

Balagurunathan Kuberan, David L. Beeler, and Robert D. Rosenberg
36. Polysaccharide-Based Hydrogels in Tissue Engineering 817

Hyunjoon Kong and David J. Mooney
37. Synthetic and Natural Polysaccharides Having Specic Biological Activities 839
Takashi Yoshida
38. Medical Foods and Fructooligosaccharides 853

Bryan W. Wolf, JoMay Chow, and Keith A. Garleb
39. Immobilization of Cells in Polysaccharide Gels 867

Yunyu Yi, Ronald J. Neufeld, and Denis Poncelet
40. Hydrothermal Degradation and Fractionation of Saccharides and Polysaccharides 893

Ortwin Bobleter
41. Cellulosic Biomass-Derived Products 937

42. Bioethanol Production from Lignocellulosic Material 957

Lisbeth Olsson, Henning Jrgensen, Kristian B. R. Krogh, and Christophe Roca
43. Hydrolysis of Cellulose and Hemicellulose 995

Charles E. Wyman, Stephen R. Decker, Michael E. Himmel, John W. Brady, Catherine E. Skopec,
and Liisa Viikari
44. New Development in Cellulose Technology 1035

Bruno Lonnberg
45. Polysaccharide Surfactants: Structure, Synthesis, and Surface-Active Properties 1055

Roger E. Marchant, Eric H. Anderson, and Junmin Zhu
46. Structures and Functionalities of Membranes from Polysaccharide Derivatives 1087

Tadashi Uragami
47. Electro-optical Properties of Cellulose Derivative Composites 1123

J. L. Figueirinhas, P. L. Almeida, and M. H. Godinho
48. Blends and Composites Based on Cellulose Materials 1141

Georgeta Cazacu and Valentin I. Popa
49. Preparation and Properties of Cellulosic Bicomponent Fibers 1179

Richard D. Gilbert and John F. Kadla
Index 1189
Contributors
Yutaka Abe Process Development Research Center, Lion Corporation, Tokyo, Japan
Margaretha Akerholm STFI (Swedish Pulp and Paper Research Institute), Stockholm, Sweden
Ann-Christine Albertsson Royal Institute of Technology, Stockholm, Sweden
P. L. Almeida EST/IPS, Setubal, Portugal and FCT/UNL, Caparica, Portugal
Eric H. Anderson Case Western Reserve University, Cleveland, Ohio, U.S.A.
Rajai H. Atalla USDA Forest Service and University of Wisconsin, Madison, Wisconsin, U.S.A.
Emmanouil S. Avgerinos National Technical University of Athens, Athens, Greece
David L. Beeler Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and Beth Israel
Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A.
Evaggeli Billa National Technical University of Athens, Athens, Greece
Ortwin Bobleter University of Innsbruck, Innsbruck, Austria
John W. Brady Cornell University, Ithaca, New York, U.S.A.
Walther Burchard Institute of Macromolecular Chemistry, University of Freiburg, Germany
Georgeta Cazacu Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania
JoMay Chow Abbott Laboratories, Columbus, Ohio, U.S.A.
Stanley (Bob) S. Davis University of Nottingham, Nottingham, United Kingdom
xiii
xiv Contributors
Stephen R. Decker National Renewable Energy Laboratory, Golden, Colorado, U.S.A.
Wolf-Dieter Deckwer Biochemical Engineering, GBFNational Research Center for Biotechnology, Braunschweig,
Germany
Jean-Louis Doublier INRA-Laboratoire de Physico-Chimie des Macromolecules, Nantes, France
Severian Dumitriu Sherbrooke University, Sherbrooke, Quebec, Canada
J. L. Figueirinhas CFMC/UL, Lisbon, Portugal
Keith A. Garleb Abbott Laboratories, Columbus, Ohio, U.S.A.
Paul Gatenholm Biopolymer Technology, Department of Materials and Surface Chemistry, Chalmers University of
Technology, Goteborg, Sweden
Roberto Geremia Laboratoire dAdaptation et de Pathogenie des Microorganismes, Joseph Fourier University,
Grenoble, France
Richard D. Gilbert North Carolina State University, Raleigh, North Carolina, U.S.A.
M. H. Godinho FCT/UNL, Caparica, Portugal
Maria Grondahl Biopolymer Technology, Department of Materials and Surface Chemistry, Chalmers University of
Technology, Goteborg, Sweden
Kazuaki Harata Biological Information Research Center, National Institute of Advanced Industrial Science and
Technology, Ibaraki, Japan
Thomas Heinze Center of Excellence for Polysaccharide Research at the Friedrich Schiller University of Jena, Jena,
Germany
Michael E. Himmel National Renewable Energy Laboratory, Golden, Colorado, U.S.A.
Barbara Hinterstoisser BOKU-University of Natural Resources and Applied Life Sciences, Vienna, Austria
Susan Hockeld Yale University School of Medicine, New Haven, Connecticut, U.S.A.
Lisbeth Illum IDentity, Nottingham, United Kingdom
Akira Isogai Graduate School of Agricultural and Life Science, University of Tokyo, Tokyo, Japan
Tadeusz Janas University of Colorado, Boulder, Colorado, U.S.A.
Teresa Janas University of Colorado, Boulder, Colorado, U.S.A. and University of Zielona, Gora, Poland
Henning Jrgensen Center for Microbial Biotechnology BioCentrum-DTU, kgs. Lyngby, Denmark
H. E. Junginger Leiden University, Leiden, The Netherlands
John F. Kadla North Carolina State University, Raleigh, North Carolina, U.S.A.
Kanji Kajiwara Otsuma Womens University, Chiyoda-ku, Tokyo, Japan

Contributors xv
John F. Kennedy University of Birmingham Research Park and Chembiotech Laboratories, Birmingham, United
Kingdom
Charles J. Knill University of Birmingham Research Park and Chembiotech Laboratories, Birmingham, United
Kingdom
Tetsuo Kondo Kyushu University, Fukuoka, Japan
Hyunjoon Kong University of Michigan, Ann Arbor, Michigan, U.S.A.
Emmanuel G. Koukios National Technical University of Athens, Athens, Greece
Kristian B. R. Krogh Center for Microbial Biotechnology BioCentrum-DTU, kgs. Lyngby, Denmark
Balagurunathan Kuberan Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and Beth
Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A.
Naoji Kubota Oita University, Oita, Japan
Miroslaw Lech Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and Beth Israel
Jacques Lefebvre INRA-Laboratoire de Physico-Chimie des Macromolecules, Nantes, France
Margaretha Soderqvist Lindblad Royal Institute of Technology, Stockholm, Sweden
Robert J. Linhardt University of Iowa, Iowa City, Iowa, U.S.A.
Bruno Lonnberg Abo Akademi University, Turku/Abo, Finland
Delphine Magnin Sherbrooke University, Sherbrooke, Quebec, Canada
Roger E. Marchant Case Western Reserve University, Cleveland, Ohio, U.S.A.
Russell T. Matthews Yale University School of Medicine, New Haven, Connecticut, U.S.A.
Karim Mazeau Centre de Recherches sur les Macromolecules Vegetales, Grenoble, France
Michel Milas Centre de Recherches sur les Macromolecules Vegetales (CERMAV), CNRS, and Joseph Fourier
University, Grenoble, France
Takeaki Miyamoto National Matsue Polytechnic College, Matsue, Japan
David J. Mooney University of Michigan, Ann Arbor, Michigan, U.S.A.
Ronald J. Neufeld Queens University, Kingston, Ontario, Canada
Kozo Ogawa Osaka Prefecture University, Sakai, Osaka, Japan
Lisbeth Olsson Center for Microbial Biotechnology BioCentrum-DTU, kgs. Lyngby, Denmark
R. Parker Institute of Food Research, Norwich Research Park, Norwich, United Kingdom
Serge Perez Centre de Recherches sur les Macromolecules Vegetales, Grenoble, France
xvi Contributors
Denis Poncelet ENITIAA, Nantes, France
Valentin I. Popa Technical University of Jassy, Jassy, Romania
Marguerite Rinaudo Centre de Recherches sur les Macromolecules Vegetales (CERMAV), CNRS, and Joseph
Fourier University, Grenoble, France
S. G. Ring Institute of Food Research, Norwich Research Park, Norwich, United Kingdom
Christophe Roca Center for Microbial Biotechnology BioCentrum-DTU, kgs. Lyngby, Denmark
Robert D. Rosenberg Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and Beth Israel
Wael Sabra Microbiology Department, Faculty of Science, Alexandria University, Alexandria, Egypt
Hazime Saito Himeji Institute of Technology, Kamigori, Hyogo, Japan and Center for Quantum Life Sciences,
Hiroshima University, Higashi-Hiroshima, Japan
Lennart Salmen STFI (Swedish Pulp and Paper Research Institute), Stockholm, Sweden
Kei Shimoda Oita University, Oita, Japan
Catherine E. Skopec Cornell University, Ithaca, New York, U.S.A.
Olav Smidsrd Norwegian University of Science and Technology (NTNU), Trondheim, Norway
Valdir Soldi Federal University of Santa Catarina, Florianopolis, SC, Brazil
Iuliana Spiridon Petru Poni Institute of Macromolecular Chemistry, Jassy, Romania
I. W. Sutherland University of Edinburgh, Edinburgh, United Kingdom
Francois R. Taravel Centre de Recherches sur les Macromolecules Vegetales (CERMAV), CNRS, and Joseph
Fourier University, Grenoble, France
M. Thanou Cardi University, Cardi, United Kingdom
Igor Tvaros ka Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia
Tadashi Uragami Kansai University, Osaka, Japan
Kjell M. Varum Norwegian University of Science and Technology (NTNU), Trondheim, Norway
Christer Viebke The North East Wales Institute, Water Soluble Polymers Group Plas Coch, Wrexham, United
Kingdom
Liisa Viikari VTT Technical Research Centre of Finland, Finland
Bryan W. Wolf Abbott Laboratories, Columbus, Ohio, U.S.A.
Charles E. Wyman Dartmouth College, Hanover, New Hampshire, U.S.A.
Yunyu Yi Queens University, Kingston, Ontario, Canada

Contributors xvii
Patrick G. Yoder University of Iowa, Iowa City, Iowa, U.S.A.
Takashi Yoshida Kitami Institute of Technology, Kitami, Japan
Toshifumi Yui Miyazaki University, Miyazaki, Japan
Fuming Zhang University of Iowa, Iowa City, Iowa, U.S.A.
Junmin Zhu Case Western Reserve University, Cleveland, Ohio, U.S.A.

1
Progress in Structural Characterization of Functional
Polysaccharides
Kanji Kajiwara
Otsuma Womens University, Chiyoda-ku, Tokyo, Japan
Takeaki Miyamoto
National Matsue Polytechnic College, Matsue, Japan
I. INTRODUCTION (Fig. 1). In later sections, it will be shown that these basic
conformations of amylose and cellulose are supposed to
Oligosaccharides and polysaccharides are biopolymers be retained to some extent in aqueous solutions. The
commonly found in living organisms, and are known to dierence in the structure is reected by the respective
reveal the physiological functions by forming a specic physiological functions of edible amylose and nonedible
conformation. However, our understanding of polysaccha- cellulose.
ride chains is still in its premature state with respect to their There are some evidences that the higher-order struc-
structure in solid and in solution. Structural analysis may ture of polysaccharide chains is related to their physiolog-
oer the most fundamental knowledge to understand the ical function as exemplied by the triple-stranded helix of
functions of polysaccharides, but the diversity and irregu- scleroglucan, which is known to possess an antitumor
larity of polysaccharide chains make it a formidable task. activity. Many polysaccharide chains are able to assume
Polysaccharide chains are partly organized but are consid- an ordered or quasi-ordered structure such as a double-
ered to be mostly amorphous. No single crystal was made stranded helix, but the ordered structure is interrupted by
from polysaccharides up to now. Thus the crystallographic the irregularity of the primary structure in the polysaccha-
analysis of polysaccharide chains has been performed by ride chains. Many polysaccharide chains form gel in solu-
either using the small oligosaccharide single crystals or the tions by assuming an ordered or quasi- ordered chain
x-ray ber pattern diraction from drawn polysaccharide structure, which constitutes a cross-linking domain.
gels. The conformational analysis of polysaccharide chains
Although a monosaccharide unit is common to many involves two aspects: (1) the characterization of a single
polysaccharides, its linkage mode varies and characteristic chain conformation and (2) the analysis of the chain
functions/properties will appear accordingly. A good assembly (suprastructure) of polysaccharides. A single
example is demonstrated by simple poly-D-glucanswa- chain conformation of polysaccharides is primarily deter-
ter-soluble, digestible amylose and non-water-soluble, mined by the chemical structure specied by the types of
nondigestible cellulose. Both amylose and cellulose are sugar residues, sugar linkages, and side groups. A single
homopolymers composed of glucosidic residues, but they chain conformation accounts, to some extent, for the
dier in the mode of linkage. Amylose is a (1!4)-a-D- formation of suprastructures such as the complexing ca-
linked polyglucan, whereas cellulose is a (1!4)-a-D- pability of amylose and the fringed micelle formation of
linked polyglucan. The (1!4)-a linkage (amylose) and cellulose.
the (1!4)-a linkage (cellulose) of D-glucosidic residues Unlike cellulose and amylose, most polysaccharides
yield a wobbled helix and a stretched zigzag chain, have no regular homopolymeric structure, where the reg-
respectively, by joining the D-glucosidic residues in a ularity is interrupted by the random intrusion of dierent
simple manner so as to place the chain on a plane [1] types of linkage and/or sugar units. The introduction of
1
2 Kajiwara and Miyamoto
Figure 1 Wobbled helical conformation (a) and stretched zigzag conformation (b), representing the basic conformations of
amylose and cellulose, respectively.
such an irregularity hampers crystallization and promotes potential application of the crystallographic approach,
the formation of a suprastructure that is characteristic of which has been a main method of the structural analysis
the polysaccharide species. The interchain interaction of in protein science. Here we will describe two methods that
polysaccharides seems to be specic as exemplied by the are currently applied to the structural and conformational
suprastructure depending on the chemical structure and analysis of oligosaccharides and polysaccharides: small-
counterions (in the case of polysaccharides possessing angle x-ray scattering (SAXS) [2] and nuclear magnetic
carboxyl or sulfate groups). The formation of the supra- resonance (NMR) [3].
structure often results to gelation. Molecular modeling by computer is considered to
The complexity in characterizing polysaccharide chain supplement the analysis by small-angle x-ray scattering
conformation is due to the fact that the interchain interac- and NMR. Although an initial intention of molecular
tion of polysaccharides is so specic that polysaccharide modeling is to predict physical properties of carbohydrates
chains are seldom dispersed in solvent as a single chain. a priori [4], the ab initio calculation is limited to a small
Thus a rst task to understand the structurefunction monosaccharide and the semiempirical quantum method
relationship of polysaccharides is to evaluate the intrinsic can be applied for the structural characterization of mol-
chain (single chain) characteristics free from interchain ecules up to the size of disaccharides. Molecular mechanics
interaction. Once the intrinsic chain conformation is spec- or molecular dynamics is an alternative method applied to
ied, the interchain interaction can be analyzed in terms of the computer modeling of larger carbohydrate molecules,
the mode of suprastructure composed of several polysac- where the motion of constituent atoms is assumed to be
charide chains. described in terms of classical mechanics.
This review is intended to demonstrate the recent In the nal chapter, the structural and conformational
strategy in the structural and conformational characteriza- aspect is discussed from the chemical point of view. Here
tion of oligosaccharides and polysaccharides. Although the controlled chemical modication of cellulose is treated
various techniques are applied for the structural and and the physicochemical characteristics are discussed by
conformational analysis of oligosaccharides and polysac- taking into account the structural change due to chemical
charides, the general inability to crystallize excludes the modication of cellulose.
Progress in Structural Characterization of Functional Polysaccharides 3
II. STRATEGY AND METHODS OF ANALYSIS found in nature. The structural analysis of disaccharide
implies the identication, the linking order, the link posi-
Because many monosaccharides have a single, well-estab- tion, and the link mode of constituent monosaccharides.
lished conformation, the conformational analysis of oligo- Chemical and optical methods are available to determine
saccharides and polysaccharides starts from understanding the structure of disaccharide, but the recent development in
the energetic relationship when the monosaccharide resi- NMR has facilitated the assignment of specic protons or
dues are linked in a specic way. The entire geometry of carbons as well as the conformational determination of the
oligosaccharides and polysaccharide chains can be de- glucosidic linkage as shown in the next section. Fourier-
scribed in terms of a set of the pairs of dihedral angles of transform infrared (FTIR) and laser Raman spectroscopy
rotation about the monosaccharide links. If the rotation is are also useful tools for the characterization of glycosidic
independent at each monosaccharide link, the chain should bonds [5].
assume a random coil conformation. However, the con- X-ray and neutron diraction can be applied to deter-
formation of saccharide chains is found in most cases to mine the crystal structure and hydrogen bonding of mono-
assume nonrandom conformations due to intra- and inter- saccharides and disaccharides that form a single crystal. A
chain interactions that suppress the conformational space classic example will be found in the crystal structure
available for the chains linked by independent rotation. analysis of h-maltose monohydrate [6] (Fig. 4), where the
Even the crystal structure is partly retained in solution as in earlier structure determination using x-ray diraction [7]
the case of protein. Thus a single chain conformation may was rened to give a more accurate description of the
account, to some extent, for the mode of interactions and hydrogen bond structure. The x-ray diraction analysis
the formation of suprastructures. provides the most explicit information on structure in
This section gives a brief introduction on the structure terms of the precise atomic coordinates. The Cambridge
of monosaccharides and disaccharides as the basis of the Structural Database lists the crystal structure of about 40
structural and conformational analysis of oligosaccharides small oligosaccharides (cyclodextrins are omitted) includ-
and polysaccharides. The fundamentals of SAXS and ing about 10 trisaccharides, 2 tetrasaccharides, and 1
NMR together with the molecular modeling are also hexasaccharide. Here a number of crystal structures of
described. mono-, di-, and trisaccharides were determined from the
acetate derivatives because the acetylated derivatives are
A. Structure of Monosaccharide found to crystallize more easily than original (untreated)
and Disaccharide oligosaccharides. (1!3)-h-D-glucopyranosyl residues con-
sist of a main chain of a medically important class of
A monosaccharide is given by the chemical formula polysaccharides including curdlan, lentinan, schizophyl-
CnH2nOn, where n = 310. Pentose (n = 5) and hexose lan, scleroglucan, and grifolan, which possess branches at
(n = 6) are the most abundant in nature, and are C6 (except for curdlan). Glcph 1!3 Glc disaccharides are
composed of a pyranose or a furanose (Fig. 2) as a basic systematically synthesized and the crystal structures are
ring structure. A pyranose ring has two stable chair form determined. A rst attempt was made by Takeda et al. [8]
(C) conformers C1 and 1C where four atoms of O, C2, on 3-O-h-D-glucopyranosyl-h-D-glucopyranose (h-D-lam-
C3, and C5 are on the same plane. Fig. 3 lists some of inarabiose) ethyl hepta-O-acetyl-h-D-laminarabioside [9],
pyranose-type pentose and hexose, which appear in later followed by Perez et al. [10] on octa-O-acetyl-h-D-laminar-
sections, where the abbreviated description is given for abiose), and by Lamba et al. [11] on (methyl hepta-O-
each monosaccharide. acetyl-h-D-laminarabiose). Recently, 3-O-h-D-glucopyra-
A disaccharide is composed of two monosaccharides nosyl-h-D-glucopyranoside (methyl h-D-laminarabioside)
linked by any of the four modes of glycosidic linkages a,aV, [12] and methyl hepta-O-acetyl-h-D-laminarabioside [13]
a,hV, h,aV-, or h,hV-. Table 1 shows some disaccharides were prepared, and the crystal structures were determined
by x-ray diraction (Fig. 5). Table 2 summarizes two
dihedral angles, / and w, with respect to the glycosidic
bond for (1!3)-h-linked disaccharides, evaluated from the
crystallographic data of laminarabiose and laminarabio-
side derivatives. Here the dihedral angles are taken as / =
h[H(C1) ,C1 ,O1, C3V] and w = h[C1, O1, C3V, H(C3V)].
(See Sec. II.D for the denition of dihedral angles / and w.)
The angle / is almost invariant around 45j regardless of
the substituents, while the angle w is classied in two
groups of around 45j and 8j. When the intramolecular
hydrogen bond is formed between 04V and 05, the angle y
assumes a negative value. The introduction of acetyl
groups prevents the formation of intramolecular hydrogen
bonds as seen from the stereoview of the molecular struc-
tures of methyl h-D-laminarabioside and methyl hepta-O-
Figure 2 Pyranose and furanose. acetyl-h-D-laminarabioside in Fig. 5. The invariance of
Figure 3 Pyranose-type pentose and hexose.
the angle 4) is attributed to the exo-anomeric eect that Here the magnitude of the scattering vector is given by (4p /
restricts rotation around the bond between an anomeric k) sin(h / 2) with k and h being the wavelength and the
carbon atom and a glycosidic oxygen atom [14]. scattering angle, respectively. c(r) is a correlation function
representing the average of the product of two electron
B. Fundamentals of Small-Angle X-Ray density uctuations at a distance r. The distance distribu-
Scattering [2] tion function p(r) is dened as
Small-angle x-ray scattering is characterized by its small pr Vr2 cr 2

scattering angle. A scattering process obeys a reciprocal
law that relates the distance r in an ordinary (real) space which is characteristic of the shape of the scattering object.
with the scattering vector q in a Fourier (scattering) space The phase dierence between scattered rays becomes
by the phase factor dened by exp(q r); that is, the more prominent as the scattering angle increases. Thus the
scattered intensity I( q) is given by the Fourier transforma- scattered intensity is maximum at zero scattering angle and
tion of the electron density distribution in the object: proportional to the number of electrons in the object where
l the scattered rays are all in phase. The scattered intensity
Iq V 4pr2 cr expiq rdr 1 decreases with increasing scattering angle and diminishes
0 at a scattering angle of the order of k / D, where k and D
Table 1 Disaccharides in Nature

Mode of linkage Common name Structure Origin
(1!4) Linkage maltose Glcpa 1 ! 4 Glc starch
cellobiose Glcph 1 ! 4 Glc cellulose
lactose Galph 1 ! 4 Glc mammal milk
xylobiose Xylph 1 ! 4 Xyl xylan
chitobiose GlcNh 1 ! 4 GlcN chitin
cellobiouronic acid GlcUAph 1 ! 4 Glc D. pneumoniae
(1!6) Linkage isomaltose Glcpa 1 ! 6 Glc amylopectin, etc.
gentiobiose Glcph 1 ! 6 Glc gentianose
melibiose Galpa 1 ! 6 Glc ranose
planteobiose Galpa 1 ! 6 Fruf planteose
(1!3) Linkage nigerose Glcpa 1 ! 3 Glc mutan
laminaribiose Glcph 1 ! 3 Glc laminaran
turanose Glcpa 1 ! 3 Fruf meleziose (honey)
hyalobiuronic acid GlcUAph 1 ! 3 GlcN hyaluronic acid
chondrosine GlcUAph 1 ! 3 GalN chondroitin sulfate
(1!2) Linkage kojibiose Glcpa 1 ! 2 Glc Aspergillus orryzae
sophorose Glcph 1 ! 2 Glc Sophora japonica
sucrose Frufh 1 ! 2 aGlcp beet sugar
(1!1) Linkage a,a-trehalose Glcpa 1 ! 1 aGlcp yeast
p and f denote pyranose and furanose, respectively.
denote the wavelength of an incident beam and the average (Rt) of a at particle by describing approximately the
diameter of scattering objects. When x-ray is used as an scattering from the cross-section or the thickness in terms
incident beam (k = 0.154 nm), the limiting scattering angle of the exponential form. The scattering factor of a rod-like
to be observed is approximately equal to 0.450 when D = particle (a cylinder) consists of two components of the
10 nm, or to 0.0450 when D = 100 nm. height and the cross-section as
Because the phase factor exp(q r) can be replaced by p
its space average sin qr/qr for the statistically isotropic Pcylinder qc exp q2 R2c =2 6
2Hq
system according to Debye [15], Eq. (1) can be expanded in
the series of q2 at very small angles by expanding the sine where 2H denotes the height of the cylinder. The scattering
term to yield the particle scattering factor as factor of a at particle (a disk) is given by the product of
l two terms of the cross-sectional area and the thickness as
1
PquIq=I0 1 q2 4pr2 cr r2 dr=2 2p
3 0 Pdisk qc exp q2 R2t 7
3 Aq2
l
4pr2 crdr Oq4
0
where A denotes the cross-sectional area. Equations (6) and
(7) suggest that the cross-sectional radius of gyration Rc
where the second term on the right side represents the
radius of gyration RG, that is
l l
R2G pr r2 dr=2 prdr 4
0 0
in terms of the distance distribution function Eq. (2). The

sine expansion of Eq. (3) is approximately closed in the
exponential form, and the particle scattering factor is
reduced to the Guinier approximation [2,16]:
Pqcexpq2 R2G =3 5
suggesting that the radius of gyration can be evaluated

from the initial slope by plotting ln P( q) against q2 (the
Guinier plot). A similar argument can be applied to
evaluate the radius of gyration corresponding to the Figure 4 Stereoview of h-maltose monohydrate. (From
cross-section of a rod-like particle (Rc) or the thickness Ref. 6.)
Figure 5 Stereoview of methyl h-D-laminarabioside (top) and methyl hepta-O-acetyl-a-D-laminarabioside (bottom).
Table 2 Dihedral Angles of the Glycosidic Linkage for Glcph 1!3 Glc Disaccharides
Compound / = h[H(C1), C1, O1, C3V] w = h[C1, O1, C3V, H(C3V)]

Methyl h-D-laminarabioside 43 52
h-D-laminarabiose 28 38
Methyl hepta-O-acetyl-h-D-laminarabioside 43 5
Methyl hepta-O-acetyl-a-D-laminarabioside 43 2
Octa-O-acetyl-h-D-laminarabioside 42 14
Octa-O-acetyl-a-D-laminarabioside 54 11
and the thickness radius of gyration Rt are evaluated from equivalent to the particle scattering factor of such a mol-
the initial slope of the corresponding Guinier plots: ln ecule that freely moves in space. If a molecule (e.g., a
qPcylinder ( q) plotted against q2 or ln q2Pdisk( q) plotted exible polymer molecule) has a large internal freedom,
against q2. the distance dij uctuates with time due to the internal
Although the polymeric chain has an approximate motion of such a molecule. In this case, the particle
shape as represented by a sphere or an ellipsoid as a whole scattering factor should be calculated as an average over
in solution, the density distribution is not homogeneous a statistical ensemble generated by the Monte Carlo pro-
but decays exponentially from the center to the circumfer- cedure [18,19] according to the conditional bond confor-
ence. A simple OrnsteinZemike type is generally applied mation probability [20].
to the density correlation function for a Gaussian chain: When no molecular model is available, the scattering
prole can be analyzed in terms of a triaxial body model of
n
cric expr=n 8 homogeneous density representing the shape of the object
r [21] or by assuming a suitable pair correlation function for
where c is a concentration of polymer chains and n is a the electron density distribution in the object [22]. The
correlation length specifying the range of eective density scattering factor is explicitly calculated for some homoge-
uctuation. Introducing in Eq. (1) for a statistically isotro- neous triaxial bodies including a sphere, an ellipsoid, a
pic system, Eq. (8) yields the scattering prole as cylinder, and a prism. For example, the scattering factor
for a sphere is given by Eq. (14) as
cn3
Iqc 9 " #2
1 n2 q2 2 3sin Rq Rq cos Rq
Pq / qR 14
The volume term V in Eq. (1) is replaced by cn3, which Rq3
corresponds to the number of units in the correlated
density uctuation. Debye and Beuche [17] proposed a where R denotes the radius of a sphere. The observed
correlation function that species the density correlation scattering prole is compared with that calculated from
for a randomly associated system: an assumed triaxial model of a suitable dimension, which is
supposed to be composed of associated oligosaccharides or
criexpr=n 10 polysaccharide chains.
No interdomain (interparticular) interaction is con-
Equation (10) yields the scattering prole as sidered in the above argument, and the scattering is con-
sidered to be due solely to an isolated domain (or an
cn3 isolated particle). When the interdomain (interparticular)
Iqc 11
1 n2 q2 2 interaction becomes dominant, an interference peak will
appear at the q range corresponding to the interaction
which exhibits a faster decay of the scattered intensity with distance in the scattering prole. If the interdomain (inter-
q. particular) interaction is isotropic and spherically symmet-
The particle scattering from a single molecule is in ric, the scattering prole is decomposed into the product of
principle calculated from the coordinates of the constituent two terms of the particle scattering factor P( q) and the
atoms interference SI( q) [16]:
X
n n1 X
X n
IqcPq SI q 15
Iq g2i /2i q 2 gi gj /i q/j q
i1 t1 ji1
12 where the interference term is written as
sindij q
1
dij q SI qc 3=2
16
1 2p e=m1 bq
where q denotes the magnitude of the scattering vector with e and m1 being a constant close to unity and the average
given by (4p / k) sin(h / 2) with k and h being the wavelength volume allocated to each interacting domain, respectively.
of the incident beam and scattering angle, respectively, and b( q) represents the interaction potential in the Fourier
gi is an atomic scattering factor. dij is the distance between (scattering) space. When the interdomain interaction is
the ith and jth atoms, and the form factor for a single atom given in terms of a hard-sphere repulsion, b( q) is repre-
/i( q) is assumed to be given by the form factor for a sphere sented by the scattering amplitude of a sphere, Eqs. (13)
with a van deer Walls radius of the ith atom and (16) reduce to
3sinRi q Ri qcosRi q
1
/i 13 SI qc 17
1 8m0 =m1 e/2qR
Ri q3
where m0 is the volume of the sphere and the hard-sphere
where RI is the van deer Walls radius of the ith atom. If a interaction is represented by the sphere of a uniform radius
molecule is rigid, the distance dij is xed and Eq. (1) is 2R. The interaction potential b( q) is approximately given
by the Gaussian function when the interaction is softer 6 ppm for protons on the ring. The anomeric protons (Hi)
[22,23], and Eq. (16) is rewritten as have peaks in the region between 4.5 and 5.5 ppm, whereas
the chemical shifts for other protons (H2H6) ranges from
1
SI qc 18 2 to 4.5 ppm. The H1 chemical shift database will provide a
1 2A2 Mw c exp n2 q2 starting key to assign the chemical shifts of unknown
samples, although the chemical shift database for oligo-
where the Gaussian-type interaction potential is specied
saccharides and polysaccharides are still far from comple-
by the correlation length n of interaction.
tion with respect to the accumulation and systematization.
As the chemical shifts are also sensitive to the conforma-
C. Fundamentals of Nuclear Magnetic tional change, solvent, and temperature, it requires expe-
Resonance Spectroscopy Applied rience and skill to identify the 1H NMR peaks for unknown
to the Conformational Analysis oligosaccharides and polysaccharide samples. Various
two-dimensional NMR techniques have been developed
Nuclear magnetic resonance (NMR) spectroscopy has to facilitate the assignment and identication of the chem-
been widely employed in the structural analysis and the ical shifts as described in a later section.
conformational dynamics of polymers in solution, gel, or The 1H NMR chemical shift data are summarized for
solid states. However, its application is limited to the monosaccharides in Table 3 [24]. The data are shown for
polymers that are not entirely crystalline in general. It monosaccharides as the components of oligosaccharides in
provides information on microscopic chemical structures, which each is linked to an adjacent monosaccharide via a
including the primary structure, stereoregularity, confor- glycosidic bond oriented either below (a) or above (b) the
mation, and secondary structure of synthetic polymers, plane of the ring. The chemical shift values of monosac-
proteins, and polysaccharides. Various NMR techniques charides will assist the identication of oligosaccharides
have also been developed to investigate molecular motion and polysaccharides, but the values vary considerably with
through relaxation times, correlation times, and self-diu- the conguration and conformation of samples.
sion coecients. One of the advantages of NMR in the
structure analysis is its sensitivity to a microscopic struc- 2. Relaxation Time
ture within a short-range order in comparison with small-
The spin-lattice relaxation time (T1) and the spinspin
angle x-ray scattering. The other advantages of NMR are
relaxation time (T2) reect the conformational change
that (1) it is a noninvasive method where no probes are
and the local tumbling motion of oligosaccharides and
needed; (2) the sample for measurement can be liquid,
polysaccharide chains. The relaxation process has been
solid, or gel; (3) the NMR signals can be assigned individ-
observed to understand the structure-dependent molecular
ually to the main chain, the side chain, or the functional
motion, the helix-coil transition, the solgel transition, the
group of a sample and yield the structural information on a
crystalline structure, the amorphous structure, the aggre-
specic site; and (4) the molecular motion and dynamic
gation structure, and the hydration structure.
structure (time-dependent structure) can be observed.
The spin-lattice relaxation time T1 is measured with
However, NMR has some disadvantages: (1) the spatial
the repeated ps2/p radio frequency (RF) pulse sequence
position of atomic groups is not determined accurately; (2)
by the inversion recovery method [25]. T1 follows Eq. (19)
the information on the long-range and higher-order struc-
derived from Blochs equation:
ture will be lost; and (3) the duration time is long to observe
NMR peaks from polymer samples with a reasonable S/N
lnAl As ln 2Al s=T1 19
ratio and high resolution. Thus NMR spectroscopy com-
pliments other methods of the structural and conforma- where Al and As are the magnitude of the recovering
tional analysis of polymers, including x-ray diraction, vector of magnetization evolved by a p/2 RF pulse at time t
light scattering, and small-angle x-ray (neutron) scattering. = l and s, respectively. T1 is evaluated from the plot of
A variety of NMR techniques are available for the ln(Al As) against s. T1 is given in terms of the viscosity g
structure analysis of oligosaccharides and polysaccharides. and temperature T [26] as
The one-dimensional pulse NMR technique is mainly

applied for the analysis of the saccharide primary structure 1 128p3 l4 a3 g
in solution state and the determination of relaxation times. 20

T1 h2 r6 kT
The solid state, high-resolution NIVIR technique can be
applied for the structure analysis of oligosaccharides and where l denotes a nuclear moment, a is the eective radius
polysaccharides in viscose solution, gel, and solid state. The of a spherical molecule, and r is the distance from the
two- or three-dimensional techniques are used to determine observed nucleus to its magnetic neighbor. T1 decreases in
the primary and secondary structures and the conforma- proportion to g/T and a3 increases with r6. The eective
tion of oligosaccharides and polysaccharides. volume a3 is replaced with the molar volume in the case of
oligosaccharides and polysaccharides in solution. T1 as a
1. Chemical Shift function of the correlation time indicates the degree of
Oligosaccharides and polysaccharides show several 1H molecular motion, and T1 takes a minimum at the temper-
NMR signal peaks in the spectrum region between 2 and ature when the relaxation occurs according to the dipole
Table 3 Chemical Shifts (ppm) of Monosaccharides from Acetone at 2.225 ppm in D2O at 2227jC
Protons
Monosaccharidea H1 H2 H3 H4 H5 H6 H7 CH3 NAC

a-D-Glc-(1! 5.1 3.56 3.72 3.42 3.77 3.77 3.87
h-D-Glc-(1! 4.4 3.31 3.51 3.41 3.45 3.74 3.92
a-D-Man-(1! 1.9 3.98 3.83 3.70 3.70 3.78 3.89
h-D-Man-(1! 4.7 4.04 3.63 3.58 3.37 3.76 3.93
a-D-Gal-(1! 5.2 3.84 3.90 4.02 4.34 3.69 3.71
h-D-Gal-(1! 4.5 3.52 3.67 3.92 3.71 3.78 3.75
h-D-GlcNAc-(1! 4.7 3.75 3.56 3.48 3.45 3.90 3.67 2.04
a-D-GalNAc-(1! 5.2 4.24 3.92 4.00 4.07 3.79 3.68 2.04
h-D-GalNAc-(1! 4.7 3.96 3.87 3.92 3.65 3.80 3.75 1.23 2.01
a-L-Fuc-(1! 5.1 3.69 3.90 3.79 4.14.9b 1.28
a-L-Rha-(1! 4.9 4.06 3.80 3.46 3.74
c
h-D-Xyl-(1! 4.5 3.27 3.43 3.61 1.32
3-u-Me-a-L-Fuc-(1! 4.8 3.70 3.40 3.89 1.32
3-u-Me-a-L-Rha-(1! 5.0 4.24 3.59 3.52 3.77 1.32
2,3-di-u-Me-a-L-Rha-(1! 5.1 3.94 3.52 3.41 3.73
3,6-di-u-Me-h-D-Glc-(1! 4.7 3.34 3.31 3.51 3.51 3.66 3.78
a
These are average values for nonreducing terminal sugars linked by a glycosidic linkage to the adjacent monosaccharides. Signals for protons at
the ring carbons are shifted downeld when linked by another monosaccharide at the hydroxyl group of that carbon.
b
These signals considerably vary more than other signals due to conformational features.
c
H5ax 3.29; H5eq 3.93.
Source: From Ref. 24.
dipole interaction [27]. The correlation time, sc, is given power proton dipolar decoupling (DD) to improve the
approximately by resolution.
A magic angle spinning (MAS) method is employed to
sc 4p3 a3 g=3kT 21 diminish the chemical shift anisotropy [32]. A sample
1
placed in a cylindrical rotor is rotated about an axis making
H T1 varies with the spin diusion [28] and the value of T1 an angle a with the magnetic eld, H0, at 8005000 Hz by
is much inuenced by O2 gas. air. The chemical shift Hamiltonian is composed of a time-
The T2 experiments are performed to observe the independent term and a time-dependent term [33]. The
molecular motion in an extreme narrowing condition [27] time-dependent term yields side bands at the multiples of
where the viscosity of a sample solution is low and the the rotation rate in the spectrum, but the side bands
motion is fast. The T2 measurements are suitable especially disappear at a spinning rate faster than a half of the width
for 1H nuclei because the problem resulting from the spin of the chemical shift anisotropy powder pattern observed
diusion can be avoided in the T2 experiments. The T2 in the viscose solution or solid samples. When the sample is
value is determined by the CarrPurcell [29]/Meiboom rotated at the xed angle a being equal to 54.74j (magic
Gill [30] (CPMG) method. Here the pulse sequence (p/2) angle) with respect to the magnetic eld, the chemical shift
spy2spy2spyp. . .. (s is the pulse interval) is used to anisotropy vanishes and the time-independent term con-
avoid the cumulative error due to incorrect pulse lengths. tains only the isotropic chemical shift.
Due to long 13C T1, a long repetition time is needed to
3. High-Resolution Solid State Nuclear Magnetic observe an NMR spectrum with a sucient S/N ratio and a
Resonance high resolution in solid state experiments. The reduction of
A rapid isotropic tumbling molecular motion is restrained T1 can be achieved by transferring the energy of 13C spins in
in the viscose solution state or in the solid state of oligo- the excited state (at a high-spin temperature) to the NMR
saccharides and polysaccharides. The NMR spectrum lattice. The energy is transferred from 13C spins at a high-
shows a proton dipolar broadening of many kilohertz spin temperature to 1H spins in the cross-polarization (CP)
due to strong dipoledipole interaction and a chemical technique [34,35] where the HartmannHahn condition is
shift anisotropy as a result of the restraint of the molecular satised. The RF pulse sequence of the CP technique for
motion. A high-power, proton-decoupling eld [31] is measuring 13C nuclei is shown in Fig. 6a. SL denotes a
found to be eective to remove a proton dipolar broaden- pulse for spin locking and DD is a pulse for heteronuclear
ing. 13C1H scalar coupling can be removed by the high- dipolar decoupling. The HartmannHahn condition is
response of the nuclear spin system becomes a two-dimen-

sional function of two independent times t1 and t2. When
FID is two-dimensionally Fourier-transformed, a two-
dimensional spectroscopy is obtained as a function of
two independent frequencies.
The 2-D shift correlated spectroscopy (COSY) in-
forms the connection of nuclei. The pulse sequence of
COSY for 1H nuclei is shown in Fig. 6b. (p/2)/1 and
(p/2)/2 are the rst and second pulses, which dier in
phase. The time interval between two pulses, t1, is an
evolution time and t2 corresponds to an acquisition time.
A 1H1H COSY spectrum is represented by a square, where
both axes correspond to 1H chemical shifts. The signals in
the spectrum are classied in diagonal peaks and cross-
peaks. The diagonal peaks are equivalent to the 1-D NMR
spectroscopy. The cross-peaks appear symmetrically with-
respect to the diagonal peaks and correspond to the dif-
ference of the chemical shifts of two sites specied on the
diagonal line by the two coordinates of respective peak
position.
The 2-D homonuclear HartmanHahn spectroscopy
(HOHAHA) reveals a spinspin interaction network as a
totally correlated spectroscopy that is obtained by chang-
ing the duration of the spin-locking application [36]. When
the HartmanHahn condition is satised by spin locking,
the magnetization transfer takes place by the spinspin
coupling between I and S spins and its degree can be
adjusted by the duration of spin locking. Homonuclear
HartmanHahn spectroscopy is more sensitive than COSY
with respect to the line resolution, and facilitates the
assignment of 1H signals along covalent bonds. To satisfy
Figure 6 Timing diagrams for the NMR pulse sequence: (a)
the HartmanHahn condition over a wide range, a proton
CP, (b) COSY, (c) HOHAHA, and (d) NOESY. broadband decoupling is introduced by a specially
designed pulse sequence. Fig. 6c shows the pulse sequence
of HOHAHA, where SLy is a spin-locking pulse and sm a
mixing time. The MALEV-17 composite pulse [37] applied
satised by the pulse applied to 13C while applying the SL during the mixing time to lock spins over a wide frequency
pulse. The signal created by CP is four times the original range.
magnetization in an ideal condition. The DD and the MAS The nuclear Overhauser eect correlated spectroscopy
are usually combined with the CP technique to obtain high- (NOESY) observes the nuclear Overhauser eect due to the
resolution spectra (CP/MAS). magnetic dipoledipole interaction between nuclei in a
short distance, and reveals the conformation, congura-
4. Two-Dimensional Nuclear Magnetic Resonance tion, and chemical exchange of large molecules [38]. The
In interpreting the NMR spectra, the rst step is to identify pulse sequence of NOESY is basically the same as COSY
signal peaks. As mentioned above, the spectra for oligo- except for the additional p/2 pulse after a xed time sm as
saccharides and polysaccharides are complicated and two- shown in Fig. 6d, where sm denotes a mixing time. The
dimensional (2-D) NMR technique is commonly applied to distance between 1H nuclei is determined from the intensity
separate the NMR signals on the basis of J coupling. The 2- of cross-peaks, and oers a mean of investigating spatial
D NMR technique yields information on the spinspin relationships between nuclei through NOE. The cross-
coupling between heteronuclei, chemical exchange, and the relaxation rate for an I and S spin system, rIS, is a function
nuclear Overhauser eect (NOE). of the distance between the I and S spin:
The 2-D NMR technique involves several spectro-
scopic methods classied by the mode of pulse sequence c4 t2 6sc
(Fig. 6). The response of the nuclear spin system to the RF rIS sc 22
10r6 1 4x2 s2c
pulse is observed as FID (free induction decay) as a
function of a time t2, which is Fourier transformed to yield where x is the Larmor frequency and the sc is the correla-
an NMR spectrum in the conventional (1-D) spectroscopy. tion time of reorientation [39]. rIS is evaluated from the sm
By applying two RF pulses with a time interval t1, a second dependence of cross-peak intensities. The spatial informa-
time axis t1 (an evolution time) can be introduced where the tion obtained by NOESY is restricted within the distance of
about 0.5 nm. sc depends on the motility of molecules. The evaluation of a total conformational energy as a function
cross-peaks show negative and positive values for xsc < 1 of a pair / and w. / and w can take any value between 180j
and xsc > 1, respectively. When xsc c 1, the cross-peaks and +180j. The most likely conformation is expected to
of NOE are not observed. By applying spin locking, a have the lowest potential energy. For example, 38 pairs of /
positive NOB is observed over the wide time scale of and w evaluated from the crystallographic data of maltose
molecular motion. The rotating frame nuclear Overhauser Glcpa 1!4 Glc are found to lay within the low-energy
eect spectroscopy (ROESY) is developed [39,40] to ob- range of 2 kcal/mol above the absolute energy minimum on
serve NOB of the sample whose molecular weight ranges the energy map provided by molecular mechanical calcu-
from 1000 to 2000 and xsc c 1. lation, proving the validity of computer modeling. Here
molecular modeling permits to evaluate the range of attain-
D. Molecular Modeling able conformations in terms of the potential energy at each
point specied by a pair of / and w. The observed value of /
1. Monte Carlo Method and w will vary among the attainable conformations
Two dihedral angles / and w with respect to the glycosidic according to the crystal packing (in the solid state) or the
bond determine the conformation of a disaccharide, pro- type of solvent (in the solution). Fig. 7 shows the confor-
vided that a pyranose ring is rigid (Fig. 7). The conforma- mational energy map of maltose, cellobiose, xylobiose,
tional analysis of a disaccharide thus comprises the chitobiose, laminaribiose, and sphorobiose calculated by
Figure 7 Denition of two dihedral angles, / and w, to determine the conformation of a disaccharide, and 2-D contour energy
map (the potential energy as a function of two dihedral angles / and w) of (a) maltose, (b) cellobiose, (c) xylobiose, (d)
chitobiose, (e) laminaribiose (s = 112.5j), and (f ) sophorose.
Figure 7 Continued.
determined by a set of / and w when the bond angle s is

xed. The eect of excluded volume can be taken into
account by excluding the step that places a unit in a
specied vicinity of the space occupied already by the unit
in a previous step.
Because the polysaccharide chain undergoes thermal
uctuation in solution, the scattering from the solution is
observed as an average over space and time. Assuming the
ergodicity, several chains are independently generated to
constitute a microcanonical ensemble, and the particle
scattering function is then given by an ensemble average
over the scattering calculated from the atomic coordinates
of each generated chain according to Eq. (12). Fig. 9 shows
the scattering proles averaged over 500 chains of two 1,4-
glucans, (1!4)-a-D-glucan (amylose), and (1!4)-h-D-glu-
can (cellulose), generated by the scheme represented by Fig.
8 with varying the number of glucosidic residues in terms of
the Kratky plots by plotting q2I( q) against q. Here the
occurrence probability for a pair of / and w is provided by
Figure 7 Continued.
molecular mechanics (MM2 or MM3) with the force-eld

including bond vibration, bond stretching, angular torsion,
and van der Waals interaction (see Table 1 for the termi-
nology). Here the glucose residue is assumed to be rigid and
replaced with a virtual bond connecting the neighboring
oxygen atoms of the glycosidic linkage (Fig. 7). The bond
angle s is xed, for example, to 110j in the case of amylose so
as to yield a consistent value for the radius of gyration as
observed for high molecular weight amylose.
Longer chains are generated by the Monte Carlo
method according to the scheme summarized in Fig. 8,
where the assumption is made that the short-range inter-
action between two adjacent residues determines the range
of permissible values of a pair of the dihedral angles / and
w. That is, a pair of / and w is provided from the energy
map (Fig. 7) according to the occurrence probability P(/,
w) specied by the Boltzmann factor associated with the
potential energy E(/, w) of a disaccharide with a set of /
and w. Here P(/, w) is given as
P/; w c expE/; w=kB T

23
with kB being a Boltzmann constant, T an absolute tem-

perature, and c a normalization constant. A chain is con- Figure 8 Flow chart to generate polysaccharide chains and
structed step by step as the geometry of each unit is calculate the scattering factor.
Figure 9 Simulated scattering proles of (1!4)-h-D-glucan (a) and (1!4)-h-D-glucan (b) as a function of the number of
glucosidic residues with snapshots of a simulated structure of respective glucan chains composed of 40 glucosidic residues (a
stereo gure) on the right.
the energy map of Fig. 7. The snapshots of simulated chains chain conformation of (1!4)-a-D-glucan, which yields a
(DP = 40) are also shown on the right side of the Kratky thicker cross-sectional radius of gyration evaluated as
plots. The simulated amylose chain reveals the wobble approximately 5 A from the cross-sectional Guinier plot
helical conformation with localized highly ordered helical [Eq. (6)] of the simulated scattering proles for (1!4)-a-D-
regions, whereas the cellulose chain seems to have a rather glucan chains of over 20 glucosidic residues. The cross-
extended chain structure as expected from the primary sectional radius of gyration remains as small as 2.1 A in the
structure. The calculated scattering proles reveal a pro- case of (1!4)-h-D-glucan chains, whose extended chain
nounced peak in Kratky plots at q = 0.2 A1 for (1!4)-a- conformation promotes to assume the intermolecular hy-
D-glucan of higher degrees of polymerization, whereas drogen bonding to form non-water-soluble aggregates.
(1!4)-h-D-glucan exhibits a scattering prole typical to a Table 4 summarizes the radius of gyration of (1!4)-a-D-
rigid rod-like molecule. The intramolecular hydrogen glucan and (1!4)-h-D-glucan, each calculated from the
bonding is responsible for stabilizing the quasi-helical simulated proles and/or estimated from the observed
SAXS proles. Although some renement of the probabil-

ity map is necessary, the simulation accounts, at least
qualitatively, for the DP dependence of the radius of
gyration and the dierence in the radius of gyration due
to the glucosidic linkage mode.
When the saccharide chain is longer, the excluded
volume eect becomes more serious. The excluded volume
eect can be taken into account by considering the inter-
action between the nonbonded units. Conventionally, the
repulsive interaction is dealt with in the Monte Carlo
simulation by replacing the chain units (segments) with
hard spheres of a nite radius. Fig. 10 demonstrates the
snapshots of amylose chain generated by the Monte Carlo
method with and without excluded volume, which is rep-
resented by a sphere of a radius 4 A at the position of each
glycosidic oxygen. The excluded volume eect is seen to
expand the chain, but the helical nature of amylosic chains
is retained in both unperturbed and perturbed states.
2. Molecular Dynamics
The Monte Carlo method described in the preceding
section is based on the disaccharide conformation energy
map, and no water molecules are taken into account in the
model. Although the Monte Carlo method is capable of
simulating longer polysaccharide chains, it does not allow
including the solvation eect directly through water-medi-
ated hydrogen bonds. Molecular dynamics (MD) simula- Figure 10 Snapshots of amylose chain generated by the
tions [41] can be applied to the structural studies of various Monte Carlo method with (b) and without (a) excluded volume.
polysaccharides, where water molecules can be explicitly Here the excluded volume is taken into account by replacing the
included in the simulation. However, the MD simulation is glycosidic oxygen (denoted by a circle in the Figure) with the
restricted to relatively shorter chains owing to a present hard sphere of radius 4 A.
computational capacity. The results of the MD simulation
depend on the employed force-lled models such as Gro-
mos [42], Glycam93/99 [43], and C91/C [44], as well as on the glycosidic linkages. Here Gromos ignores the exo-
on the starting conformation. Among the force elds anomeric eect, while Glycam incorporates the eect
mentioned above, Gromos and Glycam is composed of a through the torsional terms determined from the ab initio
set of parameters specically developed for amylose; how- geometry optimization at the HF 6-31G* level. C91 is a
ever, they dier in the treatment of the exoanomeric eect general-purpose force eld for biomolecules, and C is
expanded from C91 to include the parameters with a
proper account of the anomeric eect on the glycosidic
linkages. The example of the MD simulations will be
Table 4 Radius of Gyration of (1!4)-a-D-Glucan and
(1!4)-h-D-Glucan Oligomers shown in the later section.
(1!4)-h-D-glucan (A) (1!4)-a-D-glucan (A)

DP (obs.) (cal.) (obs.) (cal.) III. STRUCTURAL AND CONFORMATIONAL
ANALYSIS OF OLIGOSACCHARIDES
1 3.47
AND POLYSACCHARIDES
2 3.53
3 4.85 4.55 4.43 The structural characterization of simple homoglucans is
4 6.22 5.28 5.32 mainly introduced in this section. The structural and
5 7.61 6.15 6.04 mechanical properties of the gels from various marine
6 9.00 6.69 6.56 polysaccharides, plant polysaccharides, microbial polysac-
7 10.40 7.26 7.06 charides, and animal polysaccharides are reviewed by
8 11.84 8.08 7.54 Clark and Ross-Murphy [45]. This chapter intends to
9 13.23 10.21 7.77
demonstrate the advanced methods applied for the struc-
10 14.70 10.11 8.83
tural and conformational characterization of oligosaccha-
20 28.53 13.89
rides and polysaccharides, particularly in solution, by
40 55.18 23.18
50 28.12 taking the examples of the homoglucans composed of
dierent modes of glucosidic linkage.
A. (1!4)-A-D-Glucan Represented by Amylose a single chain conformation of amylose. Here no adjustable

parameter is involved, except for the normalization with
The oligomers of (1!4)-a-D-glucans dissolve well in water. respect to the scattered intensity at q = 0.
The observed SAXS proles from maltohexaose and mal- Amylose is known to assume a double-stranded (B-
tooctaose are shown in Fig. 11, where no eect of associ- form) [46,47] or single-stranded helical (V-form) [48] con-
ation was observed. The simulated SAXS proles are also formation in a solid state from the analysis of the x-ray
shown in Fig. 11 to examine the consistency of simulation ber diraction, the x-ray powder, and the electron dif-
with the observed proles. The characteristics of wobbled fraction pattern of single crystals. Amylose aqueous solu-
helix represented by a pronounced peak in the Kratky plots tion forms gel by cooling. Gelation takes place through the
become more distinct in maltooctaose than in maltohexaose formation of nanocrystallites that serve as cross-linking
as expected from the molecular weight dependence of domains. Particle scattering from model nanocrystallites is
simulated SAXS proles (Fig. 9). A good agreement be- calculated [49] by assuming nanocrystallites composed of
tween simulated and observed SAXS proles assures that B-form double helices or single-stranded V helices. The
the simulation can be extended to a longer chain to elucidate model nanocrystallite is approximately represented by an
Figure 11 Simulated and observed scattering proles from maltohexaose (a) and maltooctaose (b). The Figures on the right
show a snapshot conformation of simulated maltohexaose and maltooctaose, respectively (a stereoview).
elliptical cylinder of 8.32-nm thickness (contains 42222

duplexes composed of 24 glucosidic residues per strand) or
a parallelepiped of 6.44-nm thickness (contains 120 helices
composed of 24 glucosidic residues per strand) for the B-
form or the V-form, respectively.
The SAXS prole from amylose aqueous solution
reveals a sharp upturn at q!0 in the Kratky plots (Fig.
12) [ln q2I( q) plotted against q] according to the solgel
transition. This pronounced upturn is ascribed on the
formation of an innite structure (gel) as expected by the
cascade theory of gelation [50]. At higher q regions, two
scattering proles from sol and gel coincide, indicating that
the local conformation is identical in the sol and gel states.
The local conformation is probably represented by a single-
stranded chain simulated by the Monte Carlo method
shown in Fig. 9a, considering that single-stranded chains
are present in amorphous region of gel or in solution [51].
The observed proles are t to the scattering prole from
Figure 13 Scattering prole of amylose gel decomposed into
two components. Iexcess denotes the excess scattering from
amylose gel with respect to amylose sol (= IGEL ISOL).
Imodel is the scattering calculated from the oblate ellipsoid of
revolution (12.9 13.1 4.3 nm), and Ical = ISOL + Imodel.
simulated (1!4)-a-D-glucan chains (DP = 40) in Fig. 12.

The Guinier plots for the cross section [Eq. (6)] yields the
cross-sectional radius of gyration as 0.45 nm in both gel
and sol. The value of 0.45 nm (close to 0.5 nm), which is
evaluated for the cross-sectional radius of gyration from
the model double-stranded helix [52], also corresponds to
an apparent cross-sectional radius of gyration of a single
(1!4)-a-D-glucan chain. Here the deviation at lower q
ranges is considered to be due to the presence of double-
stranded helices formed by the coupling of two neighboring
single-stranded helices without signicantly disturbing the
conformation. The SAXS prole from amylose gel was
analyzed in terms of two components representing nano-
crystallites and amorphous region [53], respectively, by
assuming that no interference would take place between
two components. The structure of the amorphous region in
amylose gel should be identical to that in the sol state. Thus
the excess scattering in the gel state with respect to the sol
state mainly resulted from the formation of nanocrystal-
lites that function as the cross-linking domain ( junction
zone). The oblate ellipsoid of revolution was found to yield
the scattering prole t to the excess scattering (Fig. 13),
and its dimension (three semiaxes 12.9 13.1 43 nm)
approximately corresponds to the nanocrystallite com-
posed of 42 B-form duplexes with 24 glucosidic residues
per strand.
The molecular dynamics simulation was performed on
maltopentaose with currently available force elds [54].
Figure 12 Small-angle x-ray scattering prole from amylose The results are compared with the small-angle x-ray scat-
gel and sol, where closed and open circles denote gel and sol, tering observed from maltopentaose in aqueous solution.
respectively. Solid lines represent the calculated scattering Fig. 14 compares the simulated proles with the observed
prole from simulated (1!4)-h-D-glucan chains of DP = SAXS proles, where Fig. 14a provides a series of results
40. simulated with available force elds, and Fig. 14b the
B. (1!4)-B-D-Glucan Represented by Cellulose

Cellopentaose cannot be completely dissolved in water
because of a strong intermolecular interaction by hydrogen
bonding through OH groups on C6. The SAXS from the
aqueous solution of cellopentaose (30 mg/mL) exhibits a
sharp upturn toward lower q due to the formation of large
aggregates (Fig. 16). If the aggregation is caused by inter-
molecular hydrogen bonding, the aggregates are con-
sidered to be formed by the side-by-side stacking of
cellopentaose chains. The simulated prole (a solid line in
Fig. 16) reects a chain stiness of a (1!4)-h-D-glucan
chain, but the observed prole signicantly deviates from
the simulated prole at a lower q region. The cross-sectional
radius of gyration is estimated as 3.5 A at the inter-
mediate q range and as over 70 A at the smaller q range. A
single (1!4)-h-D-glucan chain has the cross-sectional ra-
dius of gyration of 2.1 A, so that two cellopentaose chains
are considered to form a stable aggregate and some further
aggregate into a larger cluster. The intermolecular hydro-
gen bonds can be broken by adding urea in the aqueous
solution of cellopentaose. Fig. 16b observes a good agree-
ment between the observed and simulated SAXS proles,
and the cross-sectional radius of gyration is estimated as 2.1
A, which is expected for a single (1!4)-A-D-glucan chain.
Regarding the local conformation of (1!4)-h-D-glu-
can chain in the presence of water, the potential use of
multiple-RELAY-COSY is suggested from the analysis
of complex spin networks of 1H NMR spectra of cello-
oligosaccharides where the complete assignment of 1H
NMR resonance was achieved for cellotriose [56]. Solvent-
suppression COSY provides also a useful method to eluci-
Figure 14 Small-angle x-ray scattering proles observed
date the interaction of the hydroxyl groups with water [57].
from maltopentaose aqueous solution (open circles) of 20.13
The 1H NMR of methyl h-cellobioside in H2O-acetone-
mg/mL at 25jC with simulated proles (respective curves).
(a) MD results (the radius gyration and force eld are shown
d6 (85 :15) yields sharp signals due to the seven hydroxyl
in the Figure) and (b) Monte Carlo results and proles cal- groups at 20jC (Fig. 17), where all signals are identied
culated from crystalline regular helices (the radius of gyration [57].
and the source of other data are shown in the Figure).
Monte Carlo results with two probability maps (Monte

Carlo K denotes a rigid map employed in Fig. 9a) and the
proles calculated from the atomic coordinates of three
regular helices. Both Monte Carlo results show a satisfac-
tory agreement with observed SAXS proles, where a small
dierence due to the glucose geometry was observed at
higher q. The results of MD simulations vary with the force
elds, and the C91 seems to yield the best t to an
observed prole. Because the helix model of Goldsmith et Figure 15 Stereoviews of the snapshot conformations of
al. [55] ts satisfactorily well to the observed SAXS prole, maltopentaose as simulated by the Monte Carlo method and
maltopentaose seems to assume a quasi-helical conforma- MD, including regular amylose helices. (a) Regular helix (8.3
tion specied by a radius of 5.38 A, a rise of 2.44 A, a pitch A) [47], (b) regular helix (7.4 A) [55], (c) regular helix (5.9 A)
of 17.60 A, a repeat of 7.2 A, / = 105j, and w = 135j. A [48], (d) Monte Carlo/MM3 (7.57 A), (e) Glycam93 (8.85 A),
typical conformation of maltopentaose is shown in Fig. 15 (f ) Glycam99 (8.08 A), (g) modied Glycam93 (7.72 A), (h)
as simulated with various force elds. In fact, the confor- C91 (7.83 A), (i) C (8.30 A), ( j ) Gromos (8.32 A). The
mation observed by the MS simulation with the C91 is values in each bracket denote the radius of gyration, which
similar to the helix model of Goldsmith et al. was evaluated as 7.4 F 0.2 A from the SAXS prole.
Figure 16 Small-angle x-ray scattering prole from cellopentaose in water (a) and in 1 M urea aqueous solution (observed and
simulated as indicated in the Figures). A stereoview of a simulated cellopentaose chain is shown on the right.
The crystal structure of cellulose has been a subject of a found to vary its pattern, implying that the ratio of two
long-standing argument. Cellulose is known to have four allomorphs Ia/Ih diers by the origin of native cellulose
dierent polymorphic crystalline forms classied as cellu- [6264]. It is interesting to note that a single microbril of
lose I, II, III, and IV. Parallel chain packing is proposed for native cellulose is a composite of two crystalline phases, Ia
native cellulose I [58], and regenerated cellulose II is and Ih [65,66].
supposed to assume antiparallel chain packing [59,60] as The crystal structure of cellulose II is considered to
analyzed from the results of x-ray ber diraction pattern. consist of two antiparallel chains of almost identical con-
Because CP/MAS 13C NMR revealed cellulose I as being formation packed in a monoclinic unit cell, where the
composed of the allomorphic mixture of triclinic Ia and hydroxymethyl group at C6 assumes a tg or a gt confor-
monoclinic Ih, the renement of cellulose crystal structure mation in the up or down chain, respectively. How-
again became a main issue in the cellulose science [61]. Here ever, CP/MAS 13C NMR exhibits a singlet at 64 ppm for
the multiplicity at C4, C1, or C6 is due to magnetically the C6 resonance from cellulose II polymorph against the
nonequivalent sites present in crystalline domain, and is expected doublet to be observed at 64 and 66 ppm from the
tg and gt conformations [67]. The cellulose II crystal

structure is re-examined [68] from the crystal structure of
cellodextrin oligomers, including h-D-cellotetraose (Fig.
18) [69,70] and methyl h-cellotrio side [71]. In those cello-
dextrin oligomers, all the hydroxymethyl groups (C6O6
bonds) are in the gt position, but the two antiparallel chains
assume a dierent glucose ring conformation. This nding
accounts, at least qualitatively, a singlet for C6 and a
doublet for C1 and C4 observed for cellulose II by CP/
MAS 13C NMR.
C. (1!3)-B-D-Glucan
(1!3)-h-D-glucan consists of a backbone of a group
of extracellular plant/fungal glucans such as cinerean,
curdlan, krestin, laminaran, lentinan, schizophyllan, and
scleroglucan, which are known to aect the immune sys-
tem as an unspecic modulator [72]. Except for curdlan,
which is linear (1!3)-h-D-glucan, the (1!3)-h-D-glucan
family contains some amount of h(1!6) branched D-
glucose residues, and assumes a triple-helical conforma-
Figure 17 1H NMR spectra of methyl h-cellobioside in tion. Although the structural requirement is not expli-
H2Oacetoned6 (85:15) at 20jC. citly understood, the antitumor activity is said to be more
Figure 18 Molecular structure (stereoview) of two h-D-cellotetraose chains.

pronounced in lower h(1!6) branched (1!3)-h-D-glucans over the ensemble was calculated to compare with the
with a relatively high molecular mass [73]. Those (1!3)-h- observed SAXS proles. The simulated scattering proles
D-glucans form triple-stranded helices of high rigidity (in terms of the Kratky plots) exhibit characteristic maxima
in aqueous solution [74,75], and the TEM image revealed of helical conformation with increasing degree of polymer-
the macrocyclic species made of multiple triple-stranded ization (Fig. 19). Fig. 20 shows the observed and calculated
(1!3)-h-D-glucan chains in some cases after a cycle of de- SAXS proles of laminarahexaose together with a snap-
naturation-renaturation process [76]. shot of a simulated chain. Although laminarahexaose is not
Laminaran is produced by Laminaria seaweeds, and long enough to show the characteristics of helical confor-
contains a small amount of h(1!6)-branched D-glucose mation, the observed SAXS prole is in good agreement
residues and alkyl groups at reductive ends. The confor- with the simulated scattering prole. The observed prole
mation of laminara oligosaccharides was characterized in has a smooth shoulder at q = 0.20.25 A1, whereas a
aqueous solution by the combined method of small-angle simulated prole shows a slight peak at q = 0.2 A1 due to
x-ray scattering and Monte Carlo simulation [77]. The a quasi-helical structure. The deviation of the observed
conformational energy map of laminarabiose (Fig. 7e) prole from the simulated one is probably due to hydra-
shows four local minima including two global minima tion, which is not properly taken into account in the
around (/, w) = (0j, 50j) and (/, w) = (30j, 0j). The simulation. The radius of gyration RG and the cross-
crystallographic data of laminarabiose and laminarabio- sectional radius of gyration RG,c are consistent with the
side derivatives (except for methyl b-D-laminarabioside respective values evaluated from observed and simulated
and h-D-laminarabiose) conrm that two dihedral angles proles7.8 A (RG) and 3.0 A (RG,c) from the observed
/ and w with respect to the glycosidic bond fall in one of the prole for laminarahexaose, and 7.7 A (RG) and 3.4 A
global minima in the conformational energy map of lam- (RG,c) from the simulation.
inarabiose (see Table 2). w is twisted by the formation of A triple-helical structure has been proposed for (1!3)-
intramolecular hydrogen bond between O4V and O5, which h-D-glucan [78,79]. For example, the molecular and crystal
is prevented by introducing acetate substituents. The glob- structure of the anhydrous curdlan and its hydrated form
al minima indicate the helical conformation of laminaran, was determined by combined x-ray diraction from ori-
which will be interrupted by the other local minima at (20j, ented curdlan bers and stereochemical model renement
170j) and (160j, 10j). [80]. Here both hydrous and anhydrous forms assume a
Over 500 chains were generated to constitute a statis- right-handed triplex (sixfold triple-helical conformation)
tical ensemble of laminara-oligomers according to the crystallized in a hexagonal unit cell with the interstrand
scheme shown in Fig. 8, and the average scattering factor O2: : : O2 hydrogen bonds. Curdlan is believed to assume a
Figure 19 Scattering proles calculated for laminara-oligosaccharides as a function of the degree of polymerization.
Figure 20 Small-angle x-ray scattering prole from laminarahexaose in water (observed and simulated as indicated in the
Figure). A stereoview of a simulated laminarahexaose chain is shown on the right.
single- or triple-helical sevenfold conformation by swelling, Gel is formed in the aqueous solution of (1!3)-h-D-
where a chain is expanded along the chain direction to glucans, but its mechanism seems to dier from linear and
increase the helix repeat distance to 22.7 A from 17.6 A (in branched species. Curdlan low-set gel is prepared by
anhydrous form) or 18.8 A (in hydrous form). Regular or heating a slurry (>0.5% w/v) to above 60jC, and will be
irregular short-branch substitutions on the main chain O6 high-set with annealing at 95jC [84]. Gelation is suggested
hydroxyls seem not to aect the triplex structure as exem- to proceed with breaking hydrogen bonds to solubilize
plied by scleroglucan [81], schizophyllan [75], and len- curdlan and reforming intermolecular hydrogen bonds
tinan [82], which retain a triplex structure even in aqueous subsequently to consist the junction zones. Hydrophobic
solution. It is interesting to note that the dihedral angles / interaction promotes the intermolecular association of
and w of curdlan polymorphs are similar to those of the curdlan at elevated temperatures to form stronger high-
acetylated derivatives of laminarabiose or laminarabioside set gel. Thus curdlan gel is supposed to contain both
(Table 2). Similar / and w values are also evaluated from liquid-like (composed of exible chains) and solid-like
the molecular structure of the tetrasacharride (1!6) (composed of associated chains) domains. 13C NMR was
branched (1!3)-h-D-glucan [83]. applied to curdlan gel where various methods (including
CP/MAS, broadband coupling, and MAS) were employed of the anhydrous form appears at the early stage of
to obtain the signals from the domains of dierent molec- annealing, and eventually the transformation takes place
ular motions [85]. Fig. 21 shows the 13C NMR spectra of from the anhydrous to the hydrous form.
curdlan hydrate and gel recorded by various methods. The 13C NMR of branched (1!3)-h-D-glucan gel such
Here a conventional high-resolution NMR coupled with as lentinan and schizophyllan shows the characteristic
broadband decoupling conrms that the liquid-like do- peaks of the triple helix [87], but the peaks corresponding
main is composed of single-helical chains that are exible to the liquid-like domain disappear by gelation [82]. Thus
and undergo free molecular motion. The intermediate the gelation of h(1!6) branched (1!3)-h-D-glucans is
domain is also composed of single chains as indicated by mainly due to partial association of triple-helical chains.
high-power dipolar decoupling with magic angle spinning The gelation of schizophyllan is promoted by the
(MAS). The CP/MAS spectrum reveals a small amount of presence of sorbitol [88] where thermoreversible optically
triple helices visible in the solid-like domain as shown by transparent gel is formed by lowering the temperature.
an arrow (a C5 signal from the triple helix) in Fig. 21, but However, the small-angle x-ray scattering (SAXS) prole
otherwise gives the characteristics of the swollen sevenfold from the schizophyllan/sorbitol system shows less-marked
helical form of solid curdlan with C3 at 87 ppm and no change by the solgel transition. The 1.5% aqueous solu-
peak at 79 ppm. Annealing at elevated temperatures results tion of schizophyllan containing 4 M sorbitol is sol at 60jC
in the increase of the fraction of anhydrous (in a later stage but forms transparent gel at 5jC. The SAXS proles from
hydrous) sixfold helical domains and the decrease of the the solution at respective temperature were analyzed in
swollen form portion [86]. The NMR observation indicates terms of a modied broken rod model [89], which reads
that curdlan undergoes gelation by forming quasi-cross-
linking domain composed of single helical chains associ-
ated hydrophobically after swelling at lower temperatures, X 4J12 qRci
and subsequently, by increasing the triple-helical fraction q2 Iqc pqwi MLi const: 24
at elevated temperatures. The triple-helical conformation i qRci 2
Figure 21 13C NMR spectra of curdlan hydrate (A) and gel (BD), observed by CP/MAS (A, D), by broadband decoupling (B),
and by MAS (C).
where wi, MLi, and Rci denote the weight fraction, the broken rod model [Eq. (24)] where each component is
linear mass density, and the cross-sectional radius of the replaced with a triple helix and a single coil of the
rod-like component i, respectively. J1(x) is the rst-order schizophyllan molecular model. The atomic radius is
Bessel function, and the constant term accounts that the reduced to half of the van der Waals radius to account
rod-like components are connected by a free joint. The for the smaller cross-sectional radius. The inclusion of a
model takes into account the heterogeneity with respect to constant term is necessary, so that schizophyllan triple
the cross-section. The results are shown in Fig. 22 in two helices are speculated to disentangle into single chains that
types of Guinier plots. Schizophyllan assumes a triple- act as a free joint. Sorbitol breaks intramolecular hydro-
helical conformation in water, and undergoes no confor- gen bonds of schizophyllan triple helices, and solvates the
mational change by decreasing the temperature from 60jC broken parts to form a cross-linking junction by intermo-
to 5jC as shown in Fig. 22a,b, where the scattering prole lecular hydrogen bonding through sorbitol. An apparent
was calculated from the molecular model of schizophyllan smaller atomic radius observed at 5jC is probably due to
triple helix. The cross-sectional radius of gyration of solvated sorbitol reducing the contrast between solvent
schizophyllan is estimated as 6.4 A. When sorbitol is and solute.
added, the cross-sectional Guinier plots yield a smaller
apparent cross-sectional radius (5.1 A), which becomes
even smaller (4.6 A) by gel formation at a lower temper- D. Cyclic and Linear (1!2)-B-D-Glucan
ature (5jC). Here the SAXS scattering prole at 60jC was
tted with the molecular model of (1!3)-h-D-glucan triple Gram-negative bacteria such as Agrobacterium and Rhizo-
helices with no side chain (i.e., curdlan-type triple helix). bium [90,91] are known to produce a cyclic (1!2)-h-D-
The SAXS prole at 5jC can be tted by a modied glucan referred to as cyclosophoran. The DP value (the
Figure 22 Small-angle x-ray scattering proles from the 1.5% aqueous solution of schizophyllan and schizophyllan/4 M
sorbitol at 60jC (a) and 5jC (b). The solid lines represent the scattering proles calculated according to the molecular model (c)
and Eq. (23).
degree of polymerization) of cyclosophoran varies from 17

to 24 depending on the bacterial strain; the largest DP
reported is 40. Cyclosophoran is thought to act as a
regulator of the osmotic balance between the cytoplasm
and the periplasmic space for bacteria to adapt the change
in environmental osmolality [92] or to mediate the bacte-
riumplant host [93] interactions during the infection of the
host. Although the exact physiological role of cyclic (1!2)-
h-D-glucan is a matter of speculation, its physiological
function is assumed to be closely related to its conforma-
tion [94]. The conformation of (1!2)-h-D-glucan has been
extensively investigated by computer modeling and 13C/1H
NMR [95], but the homopolymeric nature and conforma-
tional identity of the glucose residues obscure the structure
determination by NMR.
The conformation of cyclic and linear (1!2)-h-D-
glucans was investigated by the combination of the Monte
Carlo simulation and SAXS [96]. Cyclosophoran mixtures
produced by Agrobacterium radiobactor and Rhizobium
fphaseoli were fractionated into nine fractions from DP
= 17 to 25 (each designated as CS17 to CS25) by high-
performance liquid crystallography (HPLC). Linear
(1!2)-h-D-glucans (designated as LS 19 and LS2 1 accord-
ing to DP) was prepared by acid hydrolysis of CS21 and
subsequent fractionation by HPLC.
Small-angle x-ray scattering (SAXS) was observed
from the aqueous solutions of cyclic glucans (CS17 to
CS24) and linear glucans (LS19 and LS21) at 25jC where
the concentration was varied from 10 to 40 mg/mL (for the
cyclic glucans) or from 12.5 to 25 mg/mL (for the linear
glucans). The observed range of the magnitude of the
scattering vector was from q = 2.50 102 A1 to q =
0.375 A1, which is equivalent to the Bragg spacing from
251 to 16.8 A. The observed SAXS proles reveal the
structural dierence of cyclic and linear (1!2)-h-D-glucan
chains in aqueous solution in the region of q > 0.15 A1
(Fig. 23). The radius of gyration, RG, was evaluated from
the initial slope of the Guinier plots as summarized in Table
5. The cross-sectional radius of gyration Rc was evaluated
from the Guinier plots for cross section [Eq. (6)] in the case
of linear (1!2)-h-D-glucan chains or the thickness T from
the Guinier plots for thickness [Eq. (7)] in the case of cyclic
(1!2)-h-D-glucan chains.
The results indicate that a cyclic (1!2)-h-D-glucan
chain assumes the shape of a at disk and a linear homolog
the shape of a cylinder. The compact conformation of a cy-
clic (1!2)-h-D-glucan chain is conrmed from the smaller
radius of gyration in comparison with a corresponding
linear (1!2)-h-D-glucan chain.
(1!2)-h-D-glucan chains were generated by the Mon-
te Carlo method, consistent with the disaccharide confor-
mational energy map (Fig. 7f ). The region of the energy
well is specied as the conformation +A for w > 20j, or
A for w < 20j according to York [95]. The glucose
residue is assumed to be rigid, and the conformational
energy map for h-sophorobiose is calculated by the mo-
lecular mechanics as a function of the dihedral angles /
Figure 22 Continued.
and w dened as / = h[H1, C1, O, C2V] and w = h[C1, O,
C2V, H2V]. Nonbonded van der Waals interactions and
Figure 23 Small-angle x-ray scattering proles of cyclic and linear (1!2)-h-D-glucan chains in (a) Guinier plots [ln P( q) plotted
against q2] and (b) Kratky plots [ q2P( q) plotted against q].
electrostatic interactions due to partial charges are taken The scattering factors are calculated according to Eq.
into account in the calculation. The occurrence probability (12) from the atomic coordinates of generated chains in an
is given by the Boltzmann factor for a pair of (/, w) ensemble, with the radii of carbon and oxygen atoms being
normalized with respect to the sum of the Boltzmann taken to be 1.67 and 1.50 A, respectively. Here all the O6
factors for all pairs of (/, w), whereas the bond angle s atoms of the glucose unit are axed to the pyranose ring at
at the glycosidic oxygen is xed at 113.6j. Among the a gauchetrans (gt) position with respect to the torsion
chains generated by the Monte Carlo method, those with angle h[O5, C5, C6, O6] and the torsion angle h[C4, C5, C6,
the end-to-end distance less than 1.5 A are collected to O6], respectively.
compose an ensemble of cyclic (1!2)-h-D-glucan chains. Fig. 24 shows a reasonable agreement of the simulated
An ensemble of linear (1!2)-h-D-glucan is composed of scattering prole for cyclic (1!2)-h-D-glucans with that
500 chains. observed by SAXS, where the scattering proles calculated
Table 5 The Radius of Gyration RG, the Cross-Sectional large molecule for the formation of an inclusion complex.
Radius of Gyration Rc, and the Thickness T of Cyclic and All the glucosidic linkage torsion angles are found within
Linear (1!2)-h-D-Glucan Chains Evaluated from the the region A of the conformational energy map (Fig. 7f )
Corresponding Guinier Plots of SAXS Data with 13 linkages in the region +A and 7 linkages in the
region A where no special mode is observed for arranging
Sample
code RG (A) Rc (A) T (A)
+A and A.
The Monte Carlo simulation for linear (1!2)-h-D-
CS17 7.8 10.0 glucans yields less satisfactory results with respect to the
CS18 8.1 10.0 scattering prole (Fig. 24). Although the Monte Carlo
CS19 8.5 10.0 simulation yields a consistent value of the radius of gyra-
CS20 8.3 10.0 tion with an observed one, the deviation in the scattering
CS21 8.6 10.5 prole becomes apparent at u (u qRG) > 1.3. A good
CS22 8.4 10.7 linearity observed in the cross-sectional Guinier plots [Eq.
CS23 8.9 10.8 (6)] indicates a cylindrical shape of a linear (1!2)-h-D-
CS24 10.6 9.8 glucan chain as shown in Fig. 26 with space lling models.
LS19 11.1 5.9 The cross-sectional diameter is evaluated as 11.8 A (LS 19)
LS21 12.0 6.6
from two elementary models (a rigid ring [97] and a exible

Gaussian ring [98]) are shown for comparison. The particle
scattering factors of two elementary models are analytical-
ly given, respectively, as
X
N
sin2usinpn=N
Prigid q N1 25
n1
2usinpn=N
and
2
u u
Pflexible q 2=uexp D 26
4 2
where u and D(x) denote the reduced scattering vector and
the Dawson integral dened as
uuqRG 27
x
Dx expt2 dt 28
0
The observed SAXS proles from cyclic (1!2)-h-D-

glucans exhibit a certain chain stiness in comparison with
the proles calculated from the elementary models, where
the conformational freedom is suppressed by linking end-
to-end. The Monte Carlo simulated scattering proles
reproduce the observed SAXS proles reasonably well
except for the deviation at the higher q region. The devia-
tion at the higher q region may indicate that the eect of
hydration should be taken into account, as no interference
term due to the solventsolute interaction is incorporated
in the scattering prole calculation. However, the intro-
duction of an apparent scattering unit smaller than 1.67 or
1.50 A (for a carbon atom or an oxygen atom, respectively)
reduces the deviation from the observed prole at u (u
qRG) > 3 without any physical signicance. Figure 24 Monte Carlo simulated and observed small-angle
A typical conformation is shown in Fig. 25 with space X-ray scattering proles (the Kratky plots) of CS17 (a) and
lling models, which reveal an irregular doughnut-like ring CS21 (b). The open circles denote the SAXS data, the solid
with the thickness of 10 A. The diameter of the CS21 ring lines the Monte Carlo simulated prole, the dotted lines the
annulus is about 45 A; that is, the cavity in a cyclic (1!2)- calculated prole for a rigid ring [Eq. (24)], and the broken
h-D-glucan chain seems too small to embrace a relatively lines the calculated prole for a Gaussian ring [Eq. (25)].
Figure 25 Space lling models of cyclic and linear (1!2)-h-D-glucan chains generated by the Monte Carlo method. CS21 (left)
and LS21 (right) are seen from the top and the side. Hydrogen atoms are not included.
and 13.2 A (LS21) from the SAXS data, or as 10.6 A (LS considered to account for the interference eect at the
19) and 11.3 A (LS21) from the Monte Carlo simulation. higher q region. We have observed an opposite tendency
The discrepancy between the two sets of corresponding of the interference eect at the higher q region in cyclic and
cross-sectional diameters evaluated independently ac- linear (1!2)-h-D-glucan aqueous solutions. Although no
counts to some extent for the deviation of the scattering physical signicance is known at this stage, the apparent
proles at the larger q region. When the observed thicken- dierence in the size of the scattering units may explain the
ing is compensated by introducing larger radii for C and O mechanism of the physiological function found only in
atoms than the equivalent van der Waals radii, the consis- cyclic (1!2)-h-D-glucans.
tency of the scattering proles at the larger q region also
improves. In Fig. 24, the scattering prole calculated from
larger apparent scattering units (C and O atoms) shows a IV. SUPRAMOLECULAR STRUCTURE OF
better tting to the observed SAXS prole. The cross- POLYSACCHARIDES IN SOLUTION
sectional diameter becomes approximately 12% larger by AND GEL
doubling the radius of the scattering units.
Although the Monte Carlo simulation yields reason- Polysaccharides assume not a completely random a but
ably consistent results as a whole, more detail inspection quasi-ordered conformation in solution as shown in the
reveals that the interaction with water (solvent) needs to be preceding sections. This particular characteristic results in
Figure 26 Monte Carlo simulated and observed small-angle X-ray scattering proles (the Kratky plots) of LS19 (a) and LS21
(b). The open circles denote the SAXS data, the solid lines the Monte Carlo simulated proles with the radii of scatterers 1.67 A
(C) and 1.50 A (O), the broken lines the Monte Carlo simulated proles with the radii of scatterers 3.34 A (C) and 3.00 A (O).
the formation of quasi-ordered domains in polysaccharide

solutions. As a consequence, many polysaccharides form
physical gel where the cross-linking domain is constituted
of the quasi-ordered assembly of polysaccharide chains.
The size of the quasi-ordered domain varies from a few
nanometers to a few hundred nanometers, and the overall
appearance of polysaccharide solutions and gels is deter-
mined by its size, structure, and the mode of its connection.
This section deals with the structural characterization of
quasi-ordered domains formed by oligo- and polysaccha-
rides in solution.
A. Thermotropic Liquid Crystal of Cellulose

Derivatives
Polysaccharide is hydrophilic and water-soluble in most
cases as one of the most fundamental molecular character-
istics. However, for example, cellulose is not water-soluble.
Figure 27 Isotropization temperature Ti and melting The lack of solubility of cellulose in water is caused by
temperature Tm as a function of side-chain length index for numerous intra- and intermolecular hydrogen bonds. Cellu-
fully substituted cellulose derivatives; tri-O-alkyl cellulose (.) lose is a linear polysaccharide consisting of anhydroglucose
and cellulose trialkanoate (E). units linked by (1!4)-h-glucosidic bonds. The equatorial
Figure 28 Representative structures of poly- and oligosaccharide-based liquid crystals: (a) chiral nematic (cholesteric), (b)
hexagonal columnar, (c) discotic hexagonal columnar, and (d) smectic A phase. Here (d) is supposed to be a structure for 1-O-
alkyl-h-D-cellobiosides.
other hand, MC samples prepared in a homogeneous phase

with a nonaqueous solvent system shows no solgel tran-
sition.
In general, the polymers possessing polar hydrophilic
and nonpolar hydrophobic groups can dissolve in water, if
water is a good solvent for the hydrophilic groups and any
neighboring hydrophobic group is hydrated. However,
when the temperature is increased, hydrogen bonds are
weakened and hydration is reduced in the aqueous solu-
tion; that is, solvated hydrophobic groups lose their weakly
bound water at higher temperatures. Consequently, they
coalesce into a water-insoluble phase and the polymer
precipitates at a certain temperature termed as a lower
critical solution temperature (LCST) [112,113]. The cloud
Figure 29 Transition temperature Ti (.) and Tm (o) for point is dened as the temperature at which turbidity is
narrow fractions of fully decanoated cellulose. observed while heating a dilute solution slowly.
Cellulose and chitosan are rich sources of lyotropic
and thermotropic liquid crystals (LCs) [114,115]. As al-
ready described, both are linear, stereoregular polymers of
conguration of the (1!4)-h-glucosidic bond predestines the D-glucose and D-glucosamine, respectively, linked by a
stretched chain conformation of cellulose, which in turn (1!4)-h bond. This bonding together with the bulky
promotes the intra- and intermolecular hydrogen bonds
among their chains. The conformation of (1!4)-h-glucans
is discussed in Sec. III.B in some detail. Another character-
istic of cellulose is that almost all cellulose derivatives can
form lyotropic liquid crystals because of the semirigidity of
cellulose main chains. Chitin and its deacetylated derivative,
chitosan, are also (1!4)-h-linked homopolysaccharides, and
they can form lyotropic liquid crystals. Both cellulose and
chitin can form bers having good mechanical properties.
Besides these, there occurs (1!4)-h-linked linear homopoly-
saccharide in nature.
The properties of cellulose derivatives depend not only
on the total degree of substitution (DS) and the molar
substitution (MS) but also on (1) the distribution of sub-
stituents in the anhydroglucose (AHG) units (i.e., the
relative DS and MS at three dierent types of hydroxyl
groups), (2) the distribution of substituents along the
cellulose chain, and (3) the distribution of DS and MS.
The distribution within glucose unit arises because the
three hydroxyl groups of the glucose residue generally
dier in reactivity. This distribution can be estimated by
1
H and 13C NMR methods. On the other hand, the
nonuniformity of the distribution along the chain is caused
by heterogeneous reaction. In the case of copolymers, the
control of compositional distribution is known to be very
important to control their physical properties. The prob-
lem is equivalent to the control of the distribution of DS
and MS values in the cellulose derivatives, although at
present it is not easy to estimate their distributions. At all
event, the substituent distribution control play a major role
for the higher functionalization of cellulose [96109].
An example is shown in the water solubility of the
derivatives. Commercial methyl cellulose (MC) is water-
soluble and shows a thermally reversible solgel transition
in aqueous solution [103,110112]. Commercial products
are usually prepared by the so-called alkali cellulose pro-
cess, which is based on a heterogeneous reaction. On the Figure 30 Four types of monomer units of xyloglucan.
Figure 31 Snapshots of xyloglucan monomer units with (1!4)-h-D-glucan spines on the right.
Figure 32 Small-angle X-ray scattering observed (black circles) and calculated (solid or dotted lines) from four types of
tamarind seed xyloglucan monomer units. Since octasaccharide has two isomers (i.e., XXLG and XLXG), two dotted lines
correspond to the respective scattering proles from XXLG (above) and XLXG (below) and the solid line represents the
calculated scattering prole as an average from the two isomers (30% XXLG and 70% XLXG in this instance).
monomeric units forces the molecules to assume an essen- the polymeric phase, it is parallel to the column axis. The
tially at and extended conformation, aording these transition from the perpendicular to the parallel orienta-
polymers and their oligomers mesogenic characters. Sev- tion of the molecular axis is expected to occur at a DP of
eral studies have been reported on lyotropic and thermo- around 10, as Fig. 29 suggests, but it cannot be actually
tropic cellulose derivatives. For a recent literature review observed, as the transition temperature will be well below
on the lyotropic and thermotropic systems of these deriv- the melting temperature Tm.
atives, the readers are referred to Ref. [116]. However, This behavior of the alkyl ester derivatives forms a
these studies are mostly concerned with HIPC-related remarkable contrast to that of the alkyl ether derivatives,
derivatives. Those are chemically disordered polymers, which, as already described, form a chiral nematic phase
the molecular structureproperty relationships of which when the chain lengths are suciently large. (Short alkyl
are dicult to establish. Here we describe the main ethers show no liquid crystallinity.)
features of thermotropic mesophases exhibited by fully Acylated derivatives of chito-oligosaccharides also
substituted derivatives, which are chemically ordered poly- form a discotic hexagonal phase [121]. Owing to the
mers [115]. hydrogen bonding interaction of the amide group, their
Fig. 27 demonstrates the phase behavior of two types phases have a higher stability than those of the cello-
of fully substituted cellulose derivatives, trialkyl celluloses counterparts. The stability of the discotic hexagonal phase
and cellulose trialkanoates [117]. The abscissa scale N of the chito-compounds decreases with increasing DP of
denotes the side chain length (i.e., the number of the C the main chain, and the derivative with a DP of 6 and a side
and O atoms forming the side chain skeleton). As N chain carbon number of 14 is likely to form a discotic
increases, the melting temperature Tm decreases drastically nematic phase [122].
at rst and seems to level o or slightly increases for N z 10,
in all cases. Thus it is evident that the introduction of alkyl B. Supramolecular Structure in Xyloglucan Gel
side chains eectively lowers the melting temperature of
cellulose, but as the side chains become longer and the side Xyloglucan is a general term applied to nonstarch plant
chain fraction becomes larger, the melting behavior of the polysaccharides composed of a (1!4)-h-D-glucan with
systems becomes more governed by that of the side chain (1!6)-a-branched xylose, which is partially substituted
components. by (1!2)-h-galacto-xylose [123]. Xyloglucan is normally
Although the melting behaviors of the two mentioned contained in plant seed, and its our has been traditionally
systems are similar to each other, their mesomorphic used as a food additive in everyday life. Four types of
properties are very dierent [118]. All cellulosic LCs, monomer units are allocated to xyloglucan as designated as
lyotropic or thermotropic, that were known in former XXXG, XXLG, XLXG, and XLLG. Each unit is com-
times were cholesteric (or nematic). A cholesteric (chiral posed of a sequence of four (1!4)-h-D-glucans but diers
nematic) phase is characterized by the director eld prop- in the number of galactose side chain (Fig. 30), so that the
agating in one direction, forming a helix of pitch P (Fig. total number of sugar residues is 7 (in XXXG), 8 (in XXLG
28a). The ether derivatives form a cholesteric phase in the and XLXG), or 9 (in XLLG). Tamarind indica (TSP) and
vertically hatched region in Fig. 27, while the ester deriv-
atives form quite a dierent phase in the horizontally
hatched region. This phase is of a columnar hexagonal
type [117].
The N dependence of the isotropization temperature
Ti is also dierent for the ether and ester derivatives. The Ti
of the ethers decreases with N rather monotonically,
whereas that of the esters goes through a small maximum
(Fig. 27). To be emphasized here is that though the
dierence in the chemical structures of these polymers is
rather small, the observed dierences in their mesophase
properties may be surprisingly large.
Fig. 29 shows the chain length dependence of transition
temperatures Ti and Tm obtained for narrow fractions of
fully decanoated cellulose and its oligomers [115,118,119].
This phase diagram consists of four regionsa crystalline
solid region K, an isotropic liquid region I, and two
mesomorphic regions D and C. The oligomeric phase D,
which is relevant to homologs with DP < 5, is a discotic
hexagonal columnar phase, as illustrated in Fig. 28c [120].
On the other hand, the polymeric phase C, relevant to
homologs with DPw > 20 corresponds to the structure
given in Fig. 28b [117]. Thus in the oligomeric phase, the Figure 33 Sol-gel transition temperature diagram of enzy-
molecular axis is perpendicular to the column axis, while in matically degraded tamarind seed xyloglucan solution (1%).
Detarium senegalense are two common xyloglucans com- are seen in Fig. 31, together with respective (1!4)-h-D-
mercially available as a food additive, which have the same glucan spines (without branches) shown on the right side.
fundamental (1!4)-h-D-glucan spine with the dierent Here (1!4)-h-D-glucan spines are seen to assume rather
content of galactose with respect xylose and glucose. at zigzag conformation, and xylose and galacto-xylose
Four types of xyloglucan monomers were simulated by branches to extend and fold upright on the (1!4)-h-D-
molecular dynamics, and the particle scattering function glucan at surface. In detail, nonasaccharide (xyloglucan
was calculated according to Eq. (12) from the atomic monomer composed of nine sugar residues) exhibits a at-
coordinates of the simulated xyloglucan monomer units arched spin conformation, while octasaccharide (xyloglu-
[124]. The snapshots of simulated xyloglucan monomers can composed of eight sugar residues) and heptasaccharide
Figure 34 Molecular model for xyloglucan aggregate.

Figure 35 Scattering prole from the model xyloglucan aggregates. The number of aggregated chains is indicated in the Figure.
Table 6 Evaluated Parameters for the Tree-Like Model as a Function of Reaction Time
Weight fraction of
Reaction time
(min) ( f1)a b (nm) Single chain 14-Chain aggregate
0 0.45 6.0 1.0 0.0

40 0.5 11.0 0.49 0.51
57 1.0 13.5 0.24 0.76
74 1.04 13.7 0.13 0.87
91 1.06 14.0 0.07 0.93
108 1.07 14.0 0.05 0.95
(xyloglucan composed of seven sugar residues) assume a course of the enzymatic degradation. As the reaction time
slightly twisted conformation. The loss of galactose side proceeds, the scattering prole at the medium q range
chains results in the increase of hydrophobicity, and seems becomes at in the Kratky plots, while the prole at a
to twist the backbone. A similar conformation of galacto- higher q region remains almost invariant, exhibiting the
xylose side chains is observed by Levy et al. [125] from the characteristics of the rod-like scattering. The scattering
simulation by assuming a xed at (cellulose-like) or curve at q ! 0 indicates to upturn after 57 min. This
twisted (cellobiose-like) backbone conformation. Simu- symptom is a typical behavior of the scattering from gelling
lated scattering proles are compared with the observed systems [50]. Here gelation is assumed to take place accord-
small-angle x-ray scattering proles from tamarind seed ing to the classic Flory-Stockmayer polyfunctional poly-
xyloglucan monomer units in Fig. 32. The consistency of condensation scheme [127], and the scattering intensity
the simulated and observed results conrms the reality of from such a system is given as
the chain conformation visualized in Fig. 31.
As gelation is prevented by the steric hindrance and Iq A2 a1 a/=1 f 1a/
hydrophilicity of (1!2)-h-D-galacto-xylose branches, the 29

enzymatic degradation by h-galactosidase promotes gela- / exp b2 q2 =6
tion of xyloglucan aqueous solution [126]. At room tem-
perature, tamarind seed xyloglucan aqueous solution Here f denotes the functionality of the cross-linking
forms gel at about 45% release of galactose residues, but domain (the number of branches from a domain), a the
this gel will melt at an elevated temperature. The resulted
gel is opaque and has a unique property to have two
melting points at lower and higher temperatures as shown
in Fig. 33. The loss of (1!2)-h-galactose proceeds with a
reaction time and more aggregation will take place to form
cross-linking domains during the course of enzymatic
degradation. Here the aggregation will take place laterally
at the portion of xyloglucan chains lacking in terminal
galactose. The aggregation is expected to form a quasi-
ordered domain composed of laterally arranged xyloglu-
can chains. Because the conventional analysis of the ob-
served small-angle x-ray scattering proles indicate the
formation of at objects with 1.1-nm thickness upon
gelation [50], the molecular model of a quasi-order domain
was constructed by stacking cellulose-like (1!4)-h-D-glu-
can chains in parallel as shown in Fig. 34, and the scattering
prole was calculated according to Eq. (12). Here the
model consists of 14 xyloglucan chains each composed of
40 (1!4)-h-D-glucans with 30 (1!6)-a-xylose branches in
the sequence of XXXG. (1!2)-h-galactose terminal
groups are eliminated because the quasi-ordered domains
are formed by the loss of these terminal groups. The Figure 36 Observed and calculated scattering proles at
scattering prole calculated from the model aggregate various reaction times (indicated in the Figure). Symbols
reveals distinguished peaks in the Kratky plots as the represent the observed small-angle x-ray scattering intensities
number of chains in the aggregate increases (Fig. 35). and solid lines the scattering proles calculated from Eq. (29)
The small-angle x-ray scattering was observed from with A2( q) corresponding to the domain composed of 14
1% tamarind seed xyloglucan aqueous solution during the aligned xyloglucan chains.
stacked in parallel to constitute a cross-linking domain),

( f 1)a should be regarded as a parameter to specify the
average branching degree, where ( f 1)a = 1 indicates a
gel point. The analysis involves three parametersb, ( f
1)a, and the weight fraction of the domain composed of 14
aligned xyloglucan chains. The evaluated parameters are
summarized in Table 6, and indicate that gelation takes
place after 57 min of the reaction time in the present system.
Table 1 also indicates that about 3/4 of chains are involved
in the quasi-ordered domain at a gel point, and more single
Figure 37 Chemical structure of lactose-carrying polystyrene. chains are incorporated into the quasi-ordered domains as
further reaction takes place mainly on single chains after
gelation. The thickness of the aggregated domain does not
grow from 1.1 nm, and thus the domain seems to be
composed of a single layer of stacked xyloglucan chains.
conversion (the probability that an arbitrary chosen unit is At the end of reaction, most of the chains are incorporated
reacted), b2 the mean square average of the distance
between the neighboring scattering units, and A2( q) the
scattering amplitude of each scattering unit [i.e., A2( q) = 1
in the case of a point]. The scattering amplitude A2( q) in the
gelling system could be represented by the scattering
factors of the domain composed of aggregated chains or
a single chain of a certain length (Fig. 35). For simplicity,
the gelling system of enzymatically degrading tamarind
seed xyloglucan is assumed to consist of two phases of
single chains (a dilute phase) and the domains of 14 parallel
stacked chains (a condensed phase), and the observed
scattering proles are analyzed according to Eq. (29).
The results are summarized in Table 6 and Fig. 36. The
calculated scattering proles are consistent with the ob-
served proles over the entire time course of enzymatically
degrading reaction, although the condensed phase is not
necessarily composed of 14 xyloglucan chains. Because no
explicit number is known for the functionality f of a
domain ( f should be equal to 24 if exactly 14 chains are
Figure 39 (a) Scattering proles for three degrees of po-

lymerization calculated from the molecular model of lactose-
Figure 38 Small-angle x-ray scattering prole of VLA29, carrying polystyrene and (b) the tting example for VLA92
VLA92, and PVLA. The concentrations of each solution are where the symbols denote the observed SAXS intensities and a
the same (2 wt.%). sold line represents the calculated prole for DP = 92.
in the thin domains, and the gel seems to be constituted of

the cell-like network.
C. Glycoconjugate Synthetic Polymer

Recent advances in the precise polymerization technique
has resulted in synthesizing novel functional polymers
mimicking biopolymers. Hybrids of synthetic polymers
and biopolymers are of a particular interest, as the hybrid
may enhance the characteristics of parent polymers. A
series of glycoconjugate polystyrene derivatives have been
synthesized with varying the types of pendant oligosac-
charides [128]. The synthesized glycoconjugate polystyrene
derivatives are amphiphilic with hydrophilic pendant oli-
gosaccharides densely grafted on hydrophobic polystyrene
main chain. Highly concentrated multiantennary glycol
signals along hydrophobic main chain were in fact found
to enhance the interaction with various types of carbohy-
drate-binding proteins, and the synthesized glycoconjugate
polymers to function as a highly sensitive ligand [129]. For
example, lactose-carrying polystyrene is suitable for the
incubation of liver cells and the drug delivery systems [130].
Amphiphilic glycoconjugate polystyrene derivatives
are water-soluble, as glycoconjugate polymers will form a
single-molecule micelle in water to prevent precipitation.
Three lactose-carrying polystyrene derivatives (the chemi-
cal structure is shown in Fig. 37) were prepared by radical Figure 40 Simulated molecular model of lactose-carrying
homopolymerization or living radical polymerization of polystyrene.
vinylbenzyl lactose amide [131]. The one prepared by
radical homopolymerization (PVLA) has a high degree of
polymerization with a broad molecular weight distribu-
use of Cerius2 ver 3.5, and the particle scattering factor was
tion, while the other two (VLA29 and VLA92) prepared by
calculated according to Eq. (12) for three lactose-carrying
living radical polymerization have the degrees of polymer-
polystyrenes from the atomic coordinates of simulated
ization of 29 and 92, respectively, with a narrow molecular
molecular models. The results are shown in Fig. 39 includ-
weight distribution around 1.2. Small-angle x-ray scatter-
ing the tting example for VLA92. The consistency of the
ing from the aqueous solutions of those samples (Fig. 38)
calculated prole to the observed SAXS is satisfactory in
reveals an identical prole at a high q region ( q > 0.1 A1)
all three cases, where VLA29 (a low degree of polymeriza-
[132], indicating that the conformation of lactose-carrying
tion) can be represented by a shape of an ellipsoid rather
polystyrene is almost the same regardless of the molecular
than a cylinder. The simulated molecular model consists of
weight and the SAXS prole dierence at a low q region is
a pseudohelical polystyrene backbone covered with lactose
due to the size of a whole molecule. The shape of both
side chains (Fig. 40). The simulated molecular model
VLA92 and PVLA is approximately represented by a
conrms that the pseudohelical conformation of polysty-
cylinder of the same cross-sectional radius as conformed
rene backbone is retained even at DP = 29. Because
from the cross-sectional Guinier plots of respective SAXS
polystyrene backbone is atactic, its conformation is ran-
intensities, whereas VLA29 is not long enough to reveal the
dom in principle but the backbone conformation is obliged
characteristics of a cylinder.
to assume pseudohelical by the amphiphilic character of
The cylindrical shape of VLA 92 and PLLA could be
the backbone (hydrophobic) and side chains (hydrophilic).
accounted for by a polystyrene spiral backbone with
In this context, lactose-carrying polystyrene forms a single-
protruding lactose side chains. Based on this conjecture,
molecule cylindrical micelle.
the molecular model of lactose-carrying polystyrene was
constructed rst by assuming an arbitrary sequence of
transgauche (TG observed in the crystalline phase of
isotactic polystyrene), transtrans (TT observed in the ACKNOWLEDGMENTS
crystalline phase of syndiotactic polystyrene), and trans
transgauchegauche (TTGG observed in syndiotactic The authors are indebted to Dr. Isao Wataoka, Dr.
polystyrene) for a polystyrene spiral backbone, and sec- Hidekazu Yasunaga, and Dr. Mitsuru Mimura for their
ondly by linking lactose side chains as shown in Fig. 37 valuable comments during the preparation of the manu-
[132,133]. Then, the molecular model was simulated by the script.
REFERENCES 33. Andrew, E.R. Philos. Trans. R. Soc. Lond. 1981, A299, 505.
34. Hartmann, S.R.; Hahn, B.L. Phys. Rev. 1962, 128, 2042.
1. Burchard, W.; Meuser, F., In Plant Polymeric Carbohy- 35. Pines, A.; Gibby, M.G.; Waugh, J.S. J. Chem. Phys. 1973,
drates; Manners, D.L., Seibel, W., Eds.; The Royal Soci- 59, 569.
ety of Chemistry: Cambridge, 1993. 36. Bax, A.; Davis, D.G.; Sarkar, S.K. J. Magn. Reson. 1985,
2. Glatter, O.; Kratky, O., Eds. Small Angle X-ray Scattering; 63, 230.
Academic Press: London, 1982. 37. Bax, A.; Davis, D.G. J. Magn. Reson. 1985, 65, 355.
3. Freeman, R. A Handbook of Nuclear Magnetic Resonance; 38. Macra, S.; Ernst, R.R. Mol. Phys. 1980, 41, 95.
Longman Scientic Technical: Harlow, U.K., 1988. 39. Bax, A.; Davis, D.G. J. Magn. Reson. 1985, 63, 207.
4. French, A.D.; Brady, J.W., Eds. Computer Modeling of 40. Bothner-By, A.A.; Stephens, R.L.; Lee, J.-M.; Warren,
Carbohydrate Molecules, ACS Symposium Series 430; C.D.; Jeanloz, R.W. J. Am. Chem. Soc. 1984, 106, 811.
American Chemical Society: Washington, DC, 1990. 41. Kollman, P. Chem. Rev. 1997, 93, 2395; van Gunsteren,
5. Kacurakova, M.; Mathlouthi, M. Carbohydr. Res. 1996, W.F. Weiner, P.K., Wilkinson, A.J., Computer Simu-
284, 145. lation of Biomolecular Systems: Theoretical and Exper-
6. Gress, M.E.; Jerey, G.A. Acta Crystallogr. 1977, B33, imental Applications; Kluwer Academic: Dordrecht, 1997.
2490. 42. Koehler, S.E.H.; Saenger, W.; van Gunsteren, W.F. Eur.
7. Quigley, G.J.; Sarko, A.; Marchessault, R.H. J. Am. Biophys. J. 1988, 15, 197; J. Mol. Biol. 1987, 203, 241.
Chem. Soc. 1970, 92, 5834. 43. Woods, R.J.; Dwek, R.A.; Edge, C.J.; Fraser-Reid, B. J.
8. Takeda, H.; Yasuoka, N.; Kasai, N. Carbohydr. Res. Phys. Chem. 1995, 99, 3832; Woods, R.J. Reviews in
1977, 53, 137. Computational Chemistry; Lipkowitz, K.B. Boyd, D.B.,
9. Takeda, H.; Kaiya, T.; Yasuoka, N.; Kasai, N. Carbo- Eds.; VCH: New York, 1995.
hydr. Res. 1978, 62, 27. 44. Maple, J.R.; Dinur, V.; Hagler, A.T. Proc. Natl. Acad.
10. Perez, S.; Vergelati, C.; Tran, V.H. Acta Crystallogr. Sci., U. S. A. 1998, 85, 5350; Hwang, M.-J.; Ni, X.;
1985, B41, 262. Waldman, M.; Ewig, C.S.; Hagler, A.T. Biopolymers
11. Lamba, D.; Burden, C.; Mackie, W.; Sheidrick, B. 1988, 45, 435.
Carbohydr. Res. 1986, 153, 205. 45. Clark, A.H.; Ross-Murphy, S.B. Adv. Polym. Sci. 1987,
12. Noguchi, K.; Okuyama, K.; Kitamura, S.; Takeo, K. 83, 57.
Carbohydr. Res. 1992, 237, 33. 46. Wu, H.-C.; Sarko, A. Carbohydr. Res. 1978, 61, 7.
13. Ikegami, M.; Noguchi, K.; Okuyama, K.; Kitamura, S.; 47. Imberty, A.; Perez, S. Biopolymers 1988, 27, 1205.
Takeo, K.; Ohno, S. Carbohydr. Res. 1994, 253, 29. 48. Rappenecker, G.; Zugenmaier, P. Carbohydr. Res. 1981,
14. Perez, S.; Marchessault, R.H. Carbohydr. Res. 1978, 65, 89, 11.
114. 49. Muller, J.J.; Gemet, C.; Schulz, W.; Muller, E.-C.;
15. Debye, P. Ann. Phys. (Leipz.) 1915, 28, 809. Vorweg, W.; Damaschum, G. Biopolymers 1995, 35, 271.
16. Guinier, A.; Foumet, G. Small-Angle Scattering of X-rays; 50. Kajiwara, K.; Kohjiya, S.; Shibayama, M.; Urakawa, H.
Wiley: New York, 1955. Polymer Gels; Fundamentals and Medical Applications;
17. Debye, P.; Bueche, A.M. J. Appl. Phys. 1949, 20, 518. DeRossi, D., Kajiwara, K., Osada, Y., Yamauchi, A.,
18. Zierenberg, B.; Carpenter, D.K.; Hsieh, J.H. J. Polymer Eds.; Plenum: New York, 1991.
Sci., Polym. Symp. 1976, 54, 145. 51. Leloup, V.M.; Colonna, P.; Ring, S.G.; Roberts, K.;
19. Kajiwara, K.; Burchard, W. Macromolecules 1984, 17, Wells, B. Carbohydr. Polym. 1992, 18, 189.
2669. 52. Miles, M.J.; Morris, V.J.; Ring, S.G. Carbohydr. Polym.
20. Flory, P.J. Statistical Mechanics of Chain Molecules; 1984, 4, 73.
Interscience: New York, 1969. 53. Reuther, F.; Pleitz, G.; Damaschun, G.; Purschel, H.-V.;
21. Kajiwara, K.; Hiragi, Y. In Applications of Synchrotron Krober, R.; Schijerbaum, F. Colloid Polym. Sci. 1983,
Radiation to Materials Analysis; Saisho, H., Goshi, Y., 261, 271.
Eds.; Elsevier: Amsterdam, 1996. 54. Shimada, J.; Kaneko, H.; Takaha, T.; Kitamura, S.;
22. Kajiwara, K. The Method of Small-Angle X-ray Scattering Kajiwara, K. J. Phys. Chem., B 2000, 104, 2136.
and Its Application to the Structure Analysis of Oligosac- 55. Goldsmith, E.; Sprang, S.; Fletterich, R. J. Mol. Biol.
charides and Polysaccharides in Solution; Rome Univer- 1982, 156, 411.
sity: Rome, 1997. 56. Ikura, M.; Hikichi, K. Carbohydr. Res. 1987, 163, 1.
23. Shimode, M.; Urakawa, H.; Yamanaka, S.; Hoshino, H.; 57. Hanessian, S.; Hod, H.; Tu, Y.; Boulanger, Y. Tetrahe-
Harada, N.; Kajiwara, K. Seni Gakkaishi 1996, 52, 293. dron 1994, 50, 77.
24. Hounsell, E.F. Progr. Nucl. Magn. Reson. Spectrosc. 58. Sarko, A.; Maggli, R. Macromolecules 1974, 7, 486.
1995, 27, 445. 59. Kolpak, F.J.; Blackwell, J. Macromolecules 1976, 9, 273.
25. Farrar, T.C.; Becker, E.D. Pulse and Fourier Transform 60. Stipanovic, A.J.; Sarko, A. Macromolecules 1976, 9, 851.
NMR: Introduction to Theory and Methods; Academic 61. Atalla, R.H.; VanderHart, D.L. Science 1984, 223, 283.
Press: New York, 1971. 62. Horii, F.; Hirai, A.; Kitamaru, R. ACS Symp. Ser. 1984,
26. Bovey, F.A. High Resolution NMR of Macromolecules; 260, 29.
Academic Press: New York, 1972. 63. Cael, J.J.; Kwoh, D.L.W.; Bhattacharjee, S.S.; Patt, S.L.
27. Bloembergen, N.; Purcell, E.M.; Pound, R.V. Phys. Rev. Macromolecules 1985, 18, 821.
1948, 73, 679. 64. Horii, F.; Hirai, A.; Kitamaru, R. Macromolecules 1987,
28. Bloembergen, N. Physica 1949, 15, 386. 20, 2117.
29. Carr, H.Y.; Purcell, B.M. Phys. Rev. 1954, 94, 630. 65. Sugiyama, J.; Vuong, R.; Chanzy, H. Macromolecules
30. Meiboom, S.; Gill, D. Rev. Sci. Instrum. 1958, 29, 688. 1991, 24, 4168.
31. Komoroski, R.A., Eds. High Resolution NMR Spectro- 66. Sugiyama, J. Cell. Commun. 1994, 1, 6.
scopy of Synthetic Polymers in Bulk; VCH, 1986. 67. Dudley, R.L.; Fyfe, C.A.; Stephenson, P.J.; Deslandes,
32. Andrew, B.R. In Progress in Nuclear Magnetic Resonance Y.; Hamer, G.K.; Marchessault, R.H. J. Am. Chem. Soc.
Spectroscopy; Emsley, J.W., Feeney, J., Sutclie, L.H., 1983, 105, 2469.
Eds.; Pergamon Press: Oxford, 1971; Vol. 8, Part 1, 1 p. 68. Raymond, S.; Chanzy, H. Cell. Commun. 1995, 2, 13.
69. Geissler, K.; Krauss, N.; Steiner, T.; Betzel, C.; Sand- 100. Miyamoto, T.; Sato, Y.; Shibata, T.; Tanahashi, M.;
mann, V.; Saenger, W. Science 1994, 266, 1027. Inagaki, H. J. Polym. Sci., Polym. Chem. Ed. 1985, 23, 1373.
70. Raymond, S.; Heyraud, A.; Tran Qui, D.; Kvick, A.; 101. Kamide, K.; Saito, M.; Akedo, T. Polymer Int. 1992, 27, 35.
Chanzy, H. Macromolecules 1995, 28, 2096. 102. Aiba, S. Makromol. Chem. 1993, 194, 65.
71. Raymond, S.; Henrissat, B.; Tran Qui, D.; Kvick, A.; 103. Takahashi, S.; Fujimoto, T.; Miyamoto, T.; Inagak, H. J.
Chanzy, H. Carbohydr. Res. 1995, 277, 209. Polym. Sci., Polym. Chem. Ed. 1987, 25, 987.
72. Tabata, K. Carbohydr. Res. 1981, 89, 121. 104. Daicel Chem. md., Ltd., Japanese Open Patent Sho 60
73. Gomaa, K.; Kraus, J.; Rokoph, F.; Roper, H.; Franz, 42241, 1985, Taguchi, A.; Omiya, T.; Shimizu, K. Cell.
G.; Cane, I. Res. Clin. Oncol. 1992, 118, 136. Commun. Jpn. 1995, 2, 29.
74. Yanaki, T.; Norisue, T. Polym. J. 1983, 15, 389. 105. Sikkema, D.J.; Jannsen, H. Macromolecules 1989, 22, 364.
75. Yanaki, T.; Norisue, T.; Fujita, H. Macromolecules 1980, 106. Fukuda, T.; Sato, T.; Miyamoto, T. J. Soc. Fiber Sci.
13, 1482. Technol. Jpn. 1992, 48, 320.
76. Stokke, B.T.; Elgster, A.; Brant, D.A.; Kitamura, S. 107. Klemm, D.; Heinze, T.; Stein, A.; Liebert, T. Macromol.
Macromolecules 1991, 24, 6349. Symp. 1995, 99, 129.
77. Kitamura, S.; Minami, T.; Nakamura, Y.; Isoda, H.; 108. Miyamoto, T.; Long, M.; Donkai, N. Macromol. Symp.
Takeo, K.; Kobayashi, H.; Mimura, M.; Urakawa, H.; 1995, 99, 141.
Kajiwara, K.; Ohno, S. J. Mol. Struct. 1997, 395396, 425. 109. Philipp, B.; Wagenknecht, W.; Nehls, I.; Klemm, D.;
78. Marchessault, R.H.; Deslandes, Y.; Ogawa, K.; Sundar- Stein, A.; Heinze, T. Polym. News 1996, 21, 155.
ajan, P.R. Can. J. Chem. 1977, 55, 300. 110. Kato, T.; Yokoyama, M.; Tukahashi, A. Colloid Polym.
79. Bluhm, T.L.; Sarko, A. Can. J. Chem. 1977, 55, 293. Sci. 1978, 256, 15.
80. Deslandes, Y.; Marchessault, R.H.; Sarko, A. Macro- 111. Donges, R. Br. Polym. J. 1990, 23, 315.
molecules 1980, 13, 1466. 112. Doelker, E. Adv. Polym. Sci. 1993, 107, 199.
81. Bluhm, T.M.; Deslandes, Y.; Marchessault, R.H. Carbo- 113. Mueller, K.F. Polymer 1992, 33, 3470.
hydr. Res. 1982, 100, 114. 114. Guo, J.-X.; Gray, D.G. In Cellulosic Polymers, Blends and
82. Sato, H.; Ohki, T.; Takasuka, N.; Sasaki, T. Carbohydr. Composites; Gilbert, R.D., Ed.; Hanser Verlag: Munich,
Res. 1977, 58, 293. Chap. 2, 1994.
83. Noguchi, K.; Kobayashi, E.; Okuyama, K.; Kitamura, S.; 115. Fukuda, T.; Takada, A.; Miyamoto, T. In Cellulosic
Takeo, K.; Ohno, S. Carbohydr. Res. 1994, 258, 35. Polymers, Blends and Composites; Gilbert, R.D., Ed.;
84. Harada, T. In Extracellular Microbial Polysaccharides; Hanser Verlag: Munich, Chap. 3, 1994.
ACS Symposium Series; Sanford, P.A., Laskin, A., Eds.; 116. Gilbert, R.D. Agricultural and Synthetic Polymers, ACS
Washington, DC: American Chemical Society, 1977; Vol. Symp. Ser.; American Chemical Society: Washington,
45, 265 pp. DC, 1990; Vol. 433.
85. Saito, H. Annu. Rep. NMR Spectrosc. 1995, 31, 157. 117. Yamagishi, T.; Fukuda, T.; Miyamoto, T.; Yakoh, Y.;
86. Stipanovic, A.J.; Giammatteo, P.J. In Industrial Poly- Takashina, Y.; Watanabe, J. J. Liq. Cryst. 1991, 10, 467.
saccharides: Genetic Engineering, Structure Property 118. Fukuda, T.; Tsujii, Y.; Miyamoto, T. Macromol. Symp.
Relations and Applications, Yalpani, M., Eds.; Elsevier: 1995, 99, 257.
Amsterdam, 1987; 281 pp. 119. Takada, A.; Fujii, K.; Watanabe, J.; Fukuda, T.;
87. Saito, H.; Yoshioka, Y.; Yokoi, M.; Yamada, J. Miyamoto, T. Macromolecules 1994, 27, 1651.
Biopolymers 1990, 29, 1689. 120. Itoh, T.; Takada, A.; Fukuda, T.; Miyamoto, T.; Yakoh,
88. Fuchs, T.; Richtering, W.; Burchard, W. Macromol. Y.; Watanabe, J. J. Liq. Cryst. 1991, 9, 211.
Symp. 1995, 99, 227. 121. Sugiura, M.; Minoda, M.; Fukuda, T.; Miyamoto, T.;
89. Guenet, J.-M. Thermoreversible Gelation of Polymers and Watanabe, J. J. Liq. Ciyst. 1992, 12, 603.
Bioploymers; Academic Press: London, 1992. 122. Sugiura, M.; Minoda, M.; Watanabe, J.; Miyamoto, T.
90. Amemura, A.; Hisamatsu, M.; Mitani, H.; Harada, H. Polym. J. 1994, 26, 1236.
Carbohydr. Res. 1983, 114, 277. 123. York, W.S.; van Halbeek, H.; Darvill, A.G.; Albersheim,
91. Dell, A.; York, W.S.; McNeil, M.; Darvill, A.G.; P. Carbohydr. Res. 1990, 200, 9.
Albersheim, P. Carbohydr. Res. 1983, 117, 185. 124. Yamanaka, S.; Mimura, M.; Urakawa, H.; Kajiwara, K.;
92. Miller, K.J.; Kennedy, E.P.; Rheinhold, V.N. Science Shirakawa, M. Seni Gakkaishi 1999, 55, 590.
1994, 231, Breedveld, M.W.; Miller, K.J. Microbiol. Rev. 125. Levy, S.; York, W.S.; Stuike-Prill, R.; Meyer, B.;
1986, 58, 145. Staehelin, A.L. Plant J. 1991, 1, 195.
93. Abe, M.; Amemura, A.; Higashi, S. Plant Soil 1988, 64, 126. Shirakawa, M.; Yamatoya, K.; Nishinari, K. Food
Staneld, S.W.; Jelpi, L.; OBrochla, D.; Helinski, D.R.; Hydrocoll. 1998, 12, 25.
Ditta, G.S. J. Bacteriol. 1982, 170, 3523. 127. Kajiwara, K.; Kohjiya, S.; Shibayama, M.; Urakawa, H.
94. Brant, D.A.; Mclntire, T.M. In Cyclic Polyssacharides in In Polymer Gels: Fundamentals and Biomedical Applica-
Large Ring Molecules; Semlyen, J.A., Ed.; Wiley: London, tions; DeRossi, D., Kajiwara, K., Osada, Y., Yamauchi,
1996. A., Eds.; Plenum: New York, 1991.
95. Palleschi, A.; Crescenzi, V. Gazz. Chim. Ital. 1985, 115, 128. See, for example Flory, P.J. Principles of Polymer
York, W.S.; Thomsen, J.U.; Meyer, B. Carbohydr. Res. Chemistry; Cornell UP: Ithaca, 1953.
1995, 248, 55; York, W.S. Carbohydr. Res. 1995, 278, 129. Kobayashi, K.; Sumitomo, H.; Ina, Y. Polym. J. 1985, 17,
205. 567.
96. Mimura, M.; Kitamura, S.; Gotoh, S.; Takeo, K.; 130. Kobayashi, K.; Tsuchida, A.; Usui, T.; Akike, T. Macro-
Urakawa, H.; Kajiwara, K. Carbohydr. Res. 1996, 289, 25. molecules 1997, 30, 2016.
97. Burchard, W. Theory of Cyclic Macromolecules. In Cyclic 131. Goto, M.; Yura, H.; Chang, C.-W.; Kobayashi, A.;
Polymers; Semylen, J.A., Ed.; London: Elsevier Applied Shinoda, T.; Maeda, A.; Kojima, S.; Kobayashi, K.;
Science Pub., 1986. Akaike, T. J. Control. Release 1993, 28, 223.
98. Casassa, E.F. J. Polym. Sci. 1980, A3, Burchard, W.; 132. Ohno, K.; Tusjii, Y.; Miyamoto, T.; Fukuda, T.; Goto, T.;
Schmidt, M. Polymer 1965, 21, 745. Kobayashi, K.; Akaike, T. Macromolecules 1998, 31, 1064.
99. Lukano, B.; Philipp, B.; Schleicher, H. Cellul. Chem. 133. Wataoka, I.; Urakawa, H.; Kobayashi, K.; Ohno, K.;
Technol. 1979, 13, 417. Fukuda, T.; Akaike, T.; Kajiwara, K. Polym. J. 1999, 31, 590.
2
Conformations, Structures, and Morphologies of Celluloses
Serge Perez and Karim Mazeau
Centre de Recherches sur les Macromolecules Vegetales, Grenoble, France
I. INTRODUCTION II. CELLULOSE AND ITS CELL WALL

ENVIRONMENT
Photosynthetic organisms such as plants, algae, and some
bacteria produce more than 100 million tons of organic Throughout their lifetime, the cells of living plants continue
matter each year from the xation of carbon dioxide. Half to divide with the production of certain cells, thus confer-
of this biomass is made up of the biopolymer cellulose, ring the unusual property of being able to grow indenitely
which, as a result, is perhaps the most abundant molecule while retaining the quality of young plants. These meriste-
on the planet. This carbohydrate macromolecule is the matic cells and those deriving from them grow and then
principal structural element of the cell wall of the majority dierentiate into specialized cells for various functions,
of plants. Cellulose is also a major component of wood as support, protection, ow of sap, etc. A collection of cells
well as cotton and other textile bers such as linen, hemp, specialized for one function constitutes a tissue.
and jute (ramie). For this reason, cellulose has always Plant cell walls are distinguished from animal cells by
played an important role in the life of man, and its appli- the presence, around the plasmalemma, of a wall within
cations could even represent a landmark in the under- which complex physicochemical and enzymatic phenome-
standing of human evolution. Both ne lingerie and na progress. In the course of cell growth, the dimensions of
rough cottons have been recovered from the tombs of the cell wall vary according to the type of macromolecule of
Egyptian pharaohs. Methods for the fabrication of cellu- which it is composed. The rst wall deposited after cell
lose substrates for writing and printing go back to the early division is called the middle lamella and is essentially
Chinese dynasties. Cellulose and its derivatives are one of composed of pectic material. The cell then lays down a wall
the principal materials of use for industrial exploitation composed of pecto-cellulosic material to supplant the
( paper, nitrocellulose, cellulose acetate, methyl cellulose, middle lamella of the primary cell wall (Fig. 1).
carboxymethyl cellulose, etc.) and they represent a consid- In fact, the primary cell wall is a glycoproteinaceous
erable economic investment. layer composed of pectin, cellulose, hemicellulose, and pro-
This article provides a synthesis of the developments teins. As the cell ages and dierentiates, it secretes new
and conclusions of many of the multitude of studies that materials, which form a mixture with the constituents of
have been conducted on cellulose. Several reviews have the primary cell wall, leading to the formation of a sec-
been published on cellulose research [18] Necessarily, we ondary cell wall. The nature of the constituents of the
have been selective and we have focussed on what we con- secondary cell wall depends on the cell type and tissue to
sider to be the most important events. Particular attention which the cell belongs. In general, totally dierentiated
has been paid to the impact that the accumulation of cells have stopped expanding and cannot divide further.
structural knowledge of cellulose at its various organiza- Young plant cell walls represent a structure that is
tional levels has had on the understanding of the biological simultaneously rigid and dynamic. Indeed, rigidity is re-
and commercial function and properties of this remarkable quired to counterbalance the eect of turgor pressure on
biological material. the plasmalemma. To allow cell extension to occur, the cell
41
42 Perez and Mazeau
content. In the course of dierentiation, cellulose micro-

brils, associated with smaller molecules, hemicelluloses,
and lignins, can provide a type of liquid crystalline matrix
in which microbrils can slide past one another, or else
cause a disordered arrangement that resists further cell wall
extension [9].
The hemicelluloses constituting a large number of
dierent polysaccharide molecules actually form a matrix
for the cellulose microbrils involving molecular interac-
tions such as hydrogen bonds and van der Waals forces. In
addition to structural properties, hemicelluloses may also
have other functions such as cell signalling, or as precursors
of signalling molecules, or as reserve substances. Xyloglu-
cans are major components of the hemicelluloses of higher
plant dicotyledons and represent 20% of the dry weight
primary cell wall material [5]. In monocotyledons, xyloglu-
can constitutes only 2% of dry material mass. In this case,
xylans and h(13)-h(14)-glucans represent the major
hemicellulose components with about 1520% of dry
weight cell wall mass. Xyloglucans, like the xylans, are
closely associated with cellulose microbrils through inter-
mediary hydrogen bonds.
Pectins constitute a major component of dicotyledon
higher plants, about 35% of dry weight cell wall. In mono-
cotyledons, their proportion is less and their type is dif-
ferent. Pectins represent a complex range of carbohydrate
molecules whose backbone is composed chiey of chains
of a-D-(14) galacturonan interrupted by units of a-L-(12)
Figure 1 Schematic representation of the plant cell walls rhamnose. The rhamnose-rich regions are frequently
along with the location of the main polysaccharides com- branched with side chains composed of neutral sugars of
ponents. (From Ref. 143.) the arabinan/arabinogalactan type. These segments con-
stitute the so-called hairy regions in contrast to un-
substituted galacturonan segments or smooth regions.
wall structure must be deformable. This dual functionality In addition to structural and developmental functions,
of cell walls is achieved through the mixture of polysac- pectins are responsible for the ion exchange capacity of
charides and proteins. Cellulose chains are formed into the cell wall and control of the ionic environment and pH of
microbrils, which constitute the basic framework of the the cell interior.
cell conveying a great resistance to tensile forces [9]. The The plant cell wall contains a range of proteins, which
cellulose microbrils represent about 2030% of dry are implicated in the organization and metabolism of the
weight cell wall material occupying about 15% of cell wall cell wall. The structural proteins can be gathered into ve
volume. In cell walls that have dierentiated and synthe- main families: extensins (rich in hydroxyproline), proteins
sized a secondary cell, the proportion of cellulose reaches rich in glycine (GRP), proteins rich in proline (PRP),
4090% of the wall biomass [10]. The stages of cellulose lectins, and proteins associated with arabinogalactans
biosynthesis involve transmembrane rosettes composed (AGP). Cell wall enzymes may also be grouped into
of hexamers of cellulose synthase [11]. The orientation and families according to function: (1) peroxidases that partic-
disposition of microbrils in the wall are important because ipate in the lignication processes of the cell wall; (2)
this more or less controls the capacity of the wall to deform transglycosidases that catalyze the breaking and making
and the direction in which the deformation can occur. In of glycosidic bonds in the cell wall; (3) a great number of
the nal stages of cell wall dierentiation, notably in the hydrolases (glycosidases, glucanases, cellulases, polygalac-
middle lamella and primary cell wall, other wall polymers turonases, etc.) and, just as important, esterasesa group
(lignins) are incorporated into the spaces around the of enzymes that constitute the machinery for the ecient
polysaccharide brillar elements to form lignin polysac- degradation of the cell wall; (4) expansinsproteins
charides. Lignins arise from free radical polymerization of capable of rupturing the hydrogen bonds between cellulose
alcohols of para-hydroxy cinnamic acid and constitute microbrils and xyloglucans [10]. A schematic representa-
between 10% and 30% of the dry weight of wood, placing tion of the primary cell wall of dicotyledons, showing the
them second to cellulose. They contribute to the mechan- possible relationships between the principal components,
ical strength of the plant cell wall and confer resistance to is shown.
pathogens. Due to their hydrophobicity, they also confer Higher plant tissues such as trees, cotton, ax, sugar
resistance to water and control solute transport and water beet residues, ramie, cereal straw, etc. represent the main
Conformations, Structures, and Morphologies of Celluloses 43
sources of cellulose. Cellulose is also synthesized by bacte- must consist of six glucan synthase molecules. The hydro-
ria such as Acetobacter. It is also found in a highly phobic regions coordinate the insertion of the hydrophilic
crystalline form in the cell walls of algae such as Valonia domains on the cytoplasmic side of the plasma mem-
and Microdicyon. The animal kingdom also provides brane, facilitating aggregation and association to form
examples of several types of cellulose, of which the best the rosette subunit particles. This particle is believed to
studied is the membrane of marine animals belonging synthesize glucan chain sheets [13], which have been
to the Ascite family commonly referred to as tunicates shown to be the rst products of the crystallization phase.
material [5]. Glucan chain sheets are then assemble to form the native
Available evidence suggests that cellulose is formed at, cellulose (Fig. 2).
or outside, the plasma membrane. Groups of rosette of
particles or terminal complexes are seen in the plasma
membrane. These groups of rosettes of particles can be
seen to be associated with the ends of microbrils (collec- III. CHEMICAL STRUCTURE OF THE
tion of cellulose chains) and are thought to be cellulose CELLULOSE MACROMOLECULE
synthase complexes involved in the elongation of whole
cellulose microbrils. The catalytic subunit is a transmem- Even though the early work of Braconnot [14] concerning
brane protein with a transmembrane region. At the initia- the acid hydrolysis of the substance constituting plant cell
tion of polymerization, two uridine 5c-diphosphate (UDP) walls goes back to the 19th century, it is with Payen [15]
glucose molecules are present in the substrate-binding that the honor lies of establishing that the brous compo-
pocket. As the chain elongates, glucose is added to the nent of all plant cells has a unique chemical structure. It is
nonreducing end. The globular region of the protein is also in the studies of Payen that the word cellulose was
thought to be located in the cytoplasm, the UDP glucose coined for the rst time. However, it required another 50
being in the cytosol. A general model has been set to years for the basic cellulose formula to be established by
explain the molecular organization of the cellulose syn- Willstatter and Zechmeister [16] and for the volume of the
thase molecules from the molecular level of organization to crystalline mesh to be evaluated in 1921. The concept of
the rosette terminal complex level [12]. This complex is cellulose as a macromolecule gave rise to a lively debate
responsible for the synthesis of a microfribril that has 36 because the generally accepted idea was that the crystalline
cellulose chains. Each of the six subunits of the rosette mesh corresponded exactly to the volume occupied by one
molecule or a restricted number of molecules. It was due to
the contribution of Staudinger [17] that the macromolecu-
lar nature of cellulose was nally recognized and accepted.
Following this, Irvine and Hirst [18] and then Freu-
denberg and Braun [19] showed that 2,3,6-trimethyl glu-
cose was the sole quantitative product resulting from
methylation and hydrolysis of cellulose. This work showed
that in cellulose, carbon atoms 2, 3, and 6 carried free
hydroxyls available for reaction. Complementing these
investigations were those in which, on one hand, the
structure of glucose and cellobiose [2022] was established
and, on the other hand, those in which it was determined
that cellulose was a homopolymer of h-(14)-linked D-
glucopyranose. Crystallographical investigations of D-glu-
cose and cellobiose [23] established unambiguously that the
4
D-glucose residues had the C1 chair conformation.
All these investigations led to the establishment of the
primary structure of cellulose as a linear homopolymer of
glucose residues having the D conguration and connected
by h-(14) glycosidic linkages (Fig. 3).
The two chain ends are chemically dierent. One end
has a D-glucopyranose unit in which the anomeric carbon
atom is involved in a glycosidic linkage, whereas the other
end has a D-glucopyranose unit in which the anomeric
carbon atom is free. This cyclic hemiacetal function is in
an equilibrium in which a small proportion is an aldehyde,
which gives rise to reducing properties at this end of the
Figure 2 General model of the molecular organization of chain so that the cellulose chain has a chemical polarity.
the cellulose synthase complex: the so-called rosette ter- Determination of the relative orientation of cellulose
minal complex, from which the crystalline cellulose I chains in the three-dimensional structure has been and
emerges. (From Ref. 144.) remains one of the major problems in the study of cellulose.
44 Perez and Mazeau
Figure 3 Schematic representation of the cellulose chain.
X-ray diraction patterns obtained from brillar sam- external environments. The exocyclic primary hydroxyl
ples do not provide sucient experimental information to groups (O6) can adopt three low-energy conformations
resolve the crystallographical structure unambiguously. (gauchegauche, gauchetrans, and transgauche) depend-
Indeed, a ber is composed of an assembly of crystallites ing on a gauche stereoelectronic eect (Fig. 7).
having a common axis but random orientation. To this Although the transgauche conformation is rarely
source of disorder, that arising from the disorientation of observed in the crystalline structures of oligosaccharides
the chains in the interior of the crystallite domains, along
with their small dimensions, needs to be added. These
various sources of disorder are the origin of the low number
of reections found in the ber diagrams. On these dif-
fractograms, the reections are distributed in horizontal
rows, the spacing of which corresponds to the ber repeat
when the polymer axis is parallel to the ber axis. Thus, this
periodicity is a geometrical parameter that can be deter-
mined unambiguously from a ber diagram, and this
usually corresponds to the c dimensions of the unit cell.
Systematic absences of (0,0,1) reections also provide
information about the helical symmetry of the polymer
chain. The possibility of unambiguous determination of
the other unit cell parameters, as well as systematic ab-
sences in all the reciprocal space, depends on the ability to
index the observed reections, which, in turn, depends on
the quality of the samples (Fig. 4).
The conformation of the cellulose chain can be deter-
mined by means of molecular modelling, taking into
account experimental data such as the helical parameters
derived from the x-ray ber diraction diagrams. In the
case of the cellulose chain, the conformational variations
depend principally on the rotations around the glycosidic
linkage. The rst step involves construction of a map of the
energies corresponding to the variations of the angles (U,
W) that make up the glycosidic linkage. In the same way, it
is possible to superimpose values of helical parameters on
the iso-energy maps to permit the construction of a stable
model (Fig. 5). Figure 4 Idealized ber diraction diagram from x-ray or
The representation of the three-dimensional structure Neutron scattering. An assembly of partially oriented blocks
of microcrystallites (A) diracts to produce large diraction
of the cellulose chain shows some key structural character-
arcs (B). A perfectly oriented specimen (C) diracts to give
istics. As a consequence of the 4C1 chair conformation and
Bragg reections on layer lines (D). (From Ref. 145.) The
the (14) glycosidic linkage of the h-D-glucopyranose
meridian reection of the second layer lines indicates two-
residues, the structure is very much extended and corre- fold helix symmetry. The periodicity along the linear macro-
sponds to a twofold helix having a periodicity of 10.36 A molecule shows up a series of diracting layer lines having a
(Fig. 6). regular spacing, corresponding to the dierent orders of dif-
This conformation is situated in the low-energy zone in fraction. The equator corresponds to the layer line 0, inter-
which van der Waals interaction and anomeric eect are secting the non-diracted central beam. The meridian is
optimized. An intramolecular hydrogen bond between O3 perpendicular to the equator and lies parallel to the ber axis.
and the ring O5 of another residue provides additional The spacing along the meridian provides information about
stabilization (O5. . .O3: 2.75 A). This linkage is standard in the periodicity of the macromolecule and its helical sym-
cellulose chains with twofold symmetry, but is absent when metry. The so-called helical parameters, n and h, are directly
other less stable conformations are derived under dierent related to the symmetry of the macromolecular chain.
Figure 5 Potential energy surface computed for cellobiose as a function of U and W glycosidic torsion angles. The iso-energy
contours are drawn by interpolation of 1 kcal mol1 with respect to the energy minimum. (From Ref. 146.)
46 Perez and Mazeau
Glycosidic Torsion Angles and O3. . .O5V Distances for Cellobiose Fragments in Crystal Structures
O3. . .O5V O6V O6

Compound U (j) W (j) distance (A) H-bond conformation conformation Reference
h-cellobiose 76.3 132.3 2.77 Yes gt gt 23

Me-h-cellotrioside 94.4 146.6 2.864 Yes gt gt 147
(average of eight)
Cellotetraose (average of six) 94.4 146.8 2.875 Yes gt gt 80
Me-4O-Me cellobioside 88.1 151.3 2.81 Yes gt gg 148
(monoclinic form)
Me-4O-Me cellobioside 90.0 159.2 2.76 Yes gt gg 148
(triclinic form)
Me-h-cellobioside MeOH 91.1 160.7 2.76 Yes gt gg 149
Cellulose II mercerized 96.8 143.5 2.79 Yes gt gt 83
93.3 150.8 2.75 Yes gt gt
Cellulose II regenerated 95.4 147.7 2.78 Yes gt gt 82
95.1 150.6 2.76 Yes gt gt
Cellulose Ih 98.8 141.9 2.77 Yes tg tg 68
88.7 147.1 2.70 Yes tg tg
Cellulose Ia 98.0 138.0 2.47 Yes tg tg 150
99.0 140.0 2.92 Yes tg tg
Figure 5 Continued.
[24], this conformation would yield a second hydrogen molecular and intermolecular hydrogen bonds, which may
bond between chains (O2H. . .O6 = 2.87 A) that brings give rise to various ordered crystalline arrangements. In the
an extra stabilizing factor to the cellulose chain conforma- case of cellulose, these crystalline arrangements are usually
tion. Nevertheless, it should be mentioned that cellulose imperfect to the extent that, in terms of crystal dimensions,
can adopt other low-energy conformations, in particular at even chain orientation and the purity of the crystalline
the interface of crystalline and amorphous zones where form must be taken into consideration. The crystal density
stacking constraint is less strong. Obviously, the possibil- can be gauged from the crystallographical data, as can the
ities for the formation of intrachain and interchain hydro- importance of the amorphous components generally pres-
gen bonds can give rise to various possibilities for the ent. The density of the crystalline phase is 1.59 g cm3, but
formation of stable three-dimensional structures. The pos- when determined for natural samples is on the order of 1.55
sibilities are also reected in dierences of reactivity of the g cm3 [28], which corresponds to a value of about 70% for
dierent functional groups, in particular in etherication the crystalline component. The degree of crystallinity can
reactions, because it has been shown that the hydroxyl also be estimated by infrared spectroscopy as a function of
groups O3 and O6 are much less reactive than O2. the relative intensity of certain bands [29]. Four principal
The degree of polymerization (DP) of native celluloses allomorphs have been identied for cellulose: I, II, III, and
depends on the source and it is not well established. In fact, IV [30]. Each of these forms can be identied by its
the combination of procedures required to isolate, purify, characteristic x-ray diraction pattern. Progress achieved
and solubilize cellulose generally causes scission of the in the characterization of cellulose ultrastructure has
chains. The values of DP obtained are therefore minimal shown that within these four allomorphic families, sub-
and depend on the methods used [25,26]. Values of DP groups exist. The relationships among the various allo-
ranging from hundreds and several tens of thousands have morphs are shown schematically in Fig. 8. From Ref. [31].
been reported [26]. For the same reasons, the distribution The natural form of cellulose, called cellulose I or
of chain lengths of cellulose is not well established. None- native cellulose, apparently is the most abundant form. Its
theless, some authors suggest that the molecular mass three-dimensional structure is highly complex and not yet
distribution must be homogeneous for a cellulose of a given completely resolved as a result of the coexistence of two
source [27]. distinct crystalline forms, cellulose Ia and Ih. This was a
major discovery and led to a revival of interest in the study
of cellulose structure [32]. Cellulose I can be made to
IV. CRYSTALLINITY AND POLYMORPHISM undergo an irreversible transition to a stable crystalline
OF CELLULOSE form, cellulose II, by two distinct processes: regeneration
and mercerization. Cellulose II allomorph is known by the
The free hydroxyl groups present in the cellulose macro- term regenerated cellulose. Regeneration involves either
molecules are likely to be involved in a number of intra- preparing a solution of cellulose in an appropriate solvent,
form cellulose IIII) or from cellulose II (which leads to the

form IIIII). Cellulose III treated at high temperature in
glycerol is transformed into cellulose IV. Here again, two
types exist: cellulose IVI and IVII, respectively, obtained
from cellulose IIII and IIIII. It is generally accepted that
cellulose IVI is a disordered form of cellulose I. This could
explain the reported occurrence of this form in the native
state in some plants as determined by x-ray diraction
[33,34].
V. CRYSTALLINE STRUCTURES OF NATIVE

CELLULOSES
X-ray investigations of native cellulose samples were made
20 years ago following the early observations by optical
microscopy, which suggested the existence of submicro-
scopical birefringent and oriented domains [35,36]. The
analysis of x-ray diraction patterns has played and con-
tinues to play a major role in structural studies of cellulose
[28]. Prior to the discovery of the crystalline dimorphism of
cellulose, most crystallographical studies concentrated on
the determination of a basic unit cell. The controversy
concerning the cellulose I unit cell dimensions and space
group (believed to be unique) has lasted for many years in
spite of the observations of various workers [37,38] who
reported experimental data showing that diraction inten-
sities and spacings varied greatly depending on sample
Figure 6 Selected helical parameters n (number of residues origin. For this reason, the literature is especially confusing
per turn) and h (A) (projection of the residue on the helical on these points and is overloaded with conicting experi-
axis) computed for a regular cellulose chain as a function of mental data and structural models.
glycosidic torsion angles U and W. The iso-n and iso-h
contours are superimposed on the potential energy surface
for cellobiose. Arbitrarily, positive values of n and h
designate a right-handed helix, and opposite signs will
correspond to left-handed chirality. The screw sense of the
helix changes to the opposite sign whenever the values n = 2
are interchanged. In practice, the regular parameters are
readily derived from the observed ber diraction pattern
(n = 2, h = 5.18 A). Values of the torsional angles consistent
with the observed parameters are found at the intersection of
the corresponding iso-h and iso-n contours. Discrimination
between possible solutions is based on the magnitude of the
potential energy.
or of an intermediate derivative followed by coagulation

and recrystallization. This process is used to produce rayon
bers. Mercerization involves intracrystalline swelling of
cellulose in concentrated aqueous NaOH followed by
washing and recrystallization. This process is used to Figure 7 4C1 chair conformation of a hexopyranose and
improve the properties of natural yarns and fabrics. The Newman projections of the three staggered conformations
transition from cellulose I to cellulose II is not reversible, about the C 5C6 bond. In this gure, g and t are
and this implies that cellulose II is a stable form compared abbreviations of gauche (160j) and trans (180j), respectively,
with the metastable cellulose I (Fig. 9). indicating qualitatively the value of a dihedral angle. The
Treatment with liquid ammonia or certain amines angle of the O6C6C5O5 moiety is indicated by the rst
such as ethylene diamine (EDA) allows the preparation character and the angle of the O6C6C5C4 moiety by the
of cellulose III either from cellulose I (which leads to the second character.
48 Perez and Mazeau
Figure 8 Relationships among the dierent cellulose allomorphs.
A. Cellulose I arrangements with dierent dimensions [43,44]. It was 10

more years before the existence of two families of native
From the x-ray diraction pattern of cellulose from ramie, cellulose was conrmed by the application of solid-state
Meyer and Mark [39] proposed a monoclinic unit cell nuclear magnetic resonance (NMR) (13C CP-MAS) to a
[a = 8.35 A, b = 7.0 A, and c = 10.3 A (ber axis), c = range of cellulose samples of dierent origins. From a
84j] that served as a point of reference for a long time. The detailed analysis of the carbon atom couplings observed
symmetry elements in the space group P21 are compatible
with a twofold helicoidal symmetry for the cellulose chain
and the authors proposed a structural model in which the
chains were oriented in antiparallel fashion. Later, more
elaborate studies, which took advantage of methods for
resolving crystal structures and taking the packing energies
into account, showed that the original proposal of Meyer
and Misch represented an approximation. However, the
principal modication to the original proposal concerned
the chain orientation, which was concluded to be parallel in
the crystalline lattice [40,41].
Studies on highly crystalline algal cellulose led to a
reopening of the question of unit cell and space group
proposed by Meyer and Misch. In particular, electron
diraction studies, made at low temperature on Valonia
cellulose, produced results that were incompatible both
with the unit cell dimensions and the space group symmetry
proposed previously. The results, conrmed by indepen-
dent studies, contradicted the twofold symmetry of the
chain and suggested that Valonia cellulose had space group
P1 and a triclinic unit cell [41,42].
B. Celluloses IA and IB
Away from the main controversy, other works suggested
that celluloses from Valonia and bacterial sources had the
same crystalline unit cell. Native celluloses of dierent Figure 9 Diraction patterns of cellulose I and II after
origins might, in the same way, crystallize in dierent intracrystalline deuteration. (Courtesy of Dr. Y. Nishiyama.)
in the solid-state NMR spectrum, Atalla and VanderHart chain, whereas cellulose Ih has a monoclinic unit cell
[32] and VanderHart and Atalla [45] established that native containing two parallel chains, similar to the approximate
cellulose was a composite of two distinct crystalline phases unit cell proposed previously for cellulose I [40,41]. The
named Ia and Ih (Fig. 10). parallel-up chain packing organization favored by
The crystalline phases Ia and Ih can occur in variable Koyama et al. [59] has been conrmed by an electron
proportions according to the source of the cellulose. The microscopy study. These results have allowed a number
celluloses produced by primitive organisms (bacteria, al- of molecular descriptions for Ia and Ih to be produced by
gae, etc.) are enriched in the Ia phase, whereas the cellulose molecular modelling investigations [6064]. There are also
of higher plants (woody tissues, cotton, ramie, etc.) consists been a reexamination of the cellulose Ih structure deter-
mainly of the Ih phase. Study of the cellulose of the outer mined from x-ray patterns of Valonia cellulose [65].
membrane of marine animals showed that this is uniquely The experimental revision of the structure of cellulose
composed of the Ih phase. Hence, this cellulose may be I, in light of this dimorphism, awaited the development of
considered to be the standard for the Ih phase [46]. new structural tools such as those provided by synchro-
Cellulose from Glaucocystis has been shown to consist of tron, and neutron techniques. To achieve this, methods
essentially cellulose Ia [47]. The discovery of the crystalline have been developed for deuteriation of the intracrystalline
dimorphism of cellulose was the starting point for a regions of native cellulose without aecting the overall
number of research projects of which the aim was to structural integrity [66,67]. The neutron diraction dia-
evaluate the properties of each allomorph and procedures grams obtained in these studies are presented in Fig. 11 for
for their interconversion [4852]. The observed reections cellulose I and II. These ber diraction diagrams are
could be indexed to a monoclinical unit cell having space recorded at a resolution of 0.9 A, and several hundred
group P21 and dimensions a = 8.01 A, b = 8.17 A, c = independent diraction spots can be measured by oering
10.36 A, c = 97.3j. This unit cell is close to that proposed the promise of the establishment of unambiguous three-
originally by Meyer and Misch from their work on cellu- dimensional structures. The deuterated bers give high-
lose from ramie, now known to be enriched in phase Ih. resolution neutron diraction patterns with intensities that
Phase Ia corresponds to a triclinic symmetry with space are substantially dierent from the intensities observed on
group P1 and dimensions a = 6.74 A, b = 5.93 A, c = neutron diraction patterns obtained from hydrogenated
10.36 A, a = 117j, b = 113j, and c = 97.3j. bers.
The discovery of the crystalline dimorphism of cellu- The crystal structure and hydrogen-bonding system in
lose and the existence of two families of native cellulose cellulose Ih was elucidated by the combined use of syn-
explained the number of inconsistencies that has charac- chrotron x-ray and neutron ber diraction [68]. Oriented
terized 50 years of the crystallographical study of cellulose. brous samples were prepared by aligning cellulose micro-
Thus, the eight-chain unit cell [53] can be explained as an crystals from tunicin, reconstituted into oriented lms.
artifact arising from the superimposition of the diraction These samples diracted both synchrotron x-rays and
diagrams of phases Ia and Ih, which are both present in the neutron to better than 1 A resolution, yielding more than
Valonia cellulose. 300 unique reections and an unambiguous assignment of
The occurrence of the dimorphism in native cellulose the monoclinic unit cell dimensions, (a = 7.784 A, b =
has been conrmed by systematic investigations of a wide 8.201 A, c = 10.380 A, c = 96.5j) in the space group P21.
range of samples by x-ray and neutron diraction. The The x-ray data were used to determine the C and O atom
dimorphic concept has also allowed elucidation of several positions. The positions of hydrogen atoms involved in
features of the spectra reported in infrared [54,55] and hydrogen bonding were determined from Fourier dier-
Raman spectroscopic studies [56]. In recent x-ray and ence analysis using neutron diraction data collected from
electron diraction studies, the space group and the chain hydrogenated and deuterated samples.
packing of the Ia and Ih phases have been characterized The chains are located on the 21 axes of the mono-
[57,58]. Cellulose Ia has a triclinic unit cell containing one clinic cell. Therefore, they are not linked by any sym-
metry operation. The resulting structure consists of two
parallel chains having slightly dierent conformations,
both in terms of backbone and glucose conformations.
All the hydroxymethyl groups adopt the transgauche
conformation, which allows the formation of intrachain
hydrogen bonding involving O2 and O6 groups interacting
throughout multiple possibilities. In contrast, the O3. . .O5
intramolecular hydrogen bond is unambiguously well
organized. Such a multiple hydrogen bonding scheme
explains the complex OUC stretching bands observed in
the infrared spectra of cellulose Ih [69]. The cellulose
chains are organized in sheets packed in a parallel-up
fashion. There are no intersheet OUH. . .O hydrogen bonds
in cellulose Ih and, therefore, the cellulose sheets are held
Figure 10 Solid-state NMR spectrum of cellulose Ia and Ih. together by only hydrophobic interactions and weak
(From Ref. 32.) CUH. . .O bonds.
50 Perez and Mazeau
hydrogen atoms involved in hydrogen bonding was deter-

mined from a Fourier dierence analysis using neutron
diraction data collected from hydrogenated and deuter-
ated samples. The resulting structure is a one-chain triclinic
unit cell of dimensions: a = 6.717 A, b = 5.962 A, c =
10.400 A, a = 118.08j, b = 114.80j, c = 80.37j, space
group P1. The resulting structure consists of a parallel
chain arrangement of the parallel-up type packed in
very ecient way, the density being 1.61. Contiguous
residues along the chain axis adopt a conformation re-
markably close to a twofold screw, which is not required by
the space group symmetry, all the hydroxymethyl groups
being in a transgauche conformation. The occurrence of
the intrachain hydrogen bond O3. . .O5 is found all over the
structure with an alternation of two slightly dierent
geometries. The hydrogen bonds associated with O2 and
O6 are distributed between a number of partially occupied,
Figure 11 Description of the three-dimensional structure of

cellulose Ih. (From Ref. 68.) (A) Neutron ber diraction
pattern recorded on a hydrogenated sample (left) and on a
deuterated sample (right). (B) Corey, Pauling, Koltun (CPK)
representation and ball-and-stick representation of the layers
of cellulose chains packed in a parallel up fashion in the
monoclinic unit cell. (C) Details of the conformation of
the two crystallographically independent chains, along with
the hydrogen-bonding schemes. All the primary hydroxyl
groups are in a trans-gauche orientation.
The occurrence of nonequivalent chains may provide

an explanation for the ne details displayed by the 13C
Cross Polarization-Magic Angle Spinning (CP-MAS)
spectra of cellulose Ih [70]. The resonances assigned to
the C1, C4, and C6 atoms exhibit distinct splitting. The
dierent conformations of the glycosidic linkages and at Figure 12 Description of the three-dimensional structure of
cellulose Ia. (From Ref. 150.) (A) Details of the conforma-
the primary hydroxyl groups for the nonequivalent chains
tion of two cellulose chains, made up of the alternation
provide a structural explanation for these splittings.
of slightly dierent conformations of the glycosidic linkages
The crystal and molecular structures of the cellulose Ia (U = 98j, W = 140j) and (U =98j, W = 138j), All
allomorph have been established using synchrotron and the primary hydroxyl groups are in a transgauche orienta-
neutron diraction data recorded from oriented brous tion. (B) Projection of the relative orientation of the parallel
samples prepared by aligning cellulose microcrystals from chains of cellulose arranged in a parallel-up fashion in the
the cell wall of fresh water alga Glaucocystis nostochinea- triclinic unit cell. (C) CPK representation and ball-and-stick
rum. The x-ray data recorded at 1 A resolution were used to representation of the parallel layers of cellulose chains along
determine the C and O atom positions. The position of the ber axis.
but still well-dened, positions. As with cellulose Ih, these structures that could arise from parallel associations of
partially occupied positions can be described by two mu- cellulose chains. Within the framework of these studies,
tually exclusive hydrogen-bonding networks, and there is three-dimensional models have also been proposed,
no hint of intersheet OUH. . .O hydrogen bonds (Fig. 12). which allows comparison of the similarities and the dier-
Given the relationship between monoclinic and tri- ences that characterize the two allomorphs of native
clinic unit cells, as well as the Ia!Ih transformation by cellulose [63].
annealing in the solid state, it is likely that cellulose Ia also Several hypotheses have been proposed to account
packs in a parallel-up fashion. The projections of the for the occurrence of two phases in native cellulose. In
crystal structures of cellulose Ia and Ih down the chain axes general, samples that are rich in Ia are biosynthesized by
are remarkably similar. As the projection perpendicular linear terminal complexes containing a number of cellulose
to the chain axis in the plane of the hydrogen-bonded sheets synthases assembled in biological spinneret at the cell
shows, the main dierence is the relative displacement of membranes. Those rich in Ih are organized in a rosette
the sheets in the chain direction. In both Ia and Ih, there fashion [72]. However, a notable exception is tunicin,
is a relative shift of about c/4 in the up direction between where linear terminal complexes produce almost pure Ih
neighboring sheets. The most likely route for solid-state [91]. Obviously, the comparison between the morphology
conversion of cellulose Ia!Ib is the relative slippage of of Ih tunicin microbrils with those of Ia-rich seaweed
the cellulose chains past one another. Such a movement would be instructive. The former have a parallelogram
does not require the disruption of the hydrogen-bonded shape, whereas the latter have a square shape. Therefore,
sheets (along the 010 planes for cellulose Ih, and 110 despite their common linear geometry, the terminal com-
planes for cellulose Ia, but slippage by c/2 at the interface plexes of tunicates and those of seaweeds produce micro-
of the sheets). brils of dierent shapes and crystalline polymorphism.
The exact location of the Ia and Ih phases along the Obviously, other factors may play a key role in inducing
crystalline cellulose microbrils is another subject of inter- cellulose crystalline structures.
est. The respective components could be identied as
alternating along the microbril of the highly crystalline
algal cellulose in the Microdiction cell wall [71] (Fig. 13).
VI. CELLULOSE II
Modelling studies have established that the two crys-
talline arrangements correspond to the two low-energy Early work on the solid-state structure of cellulose dates
from 1929 [73] from which it was proposed that the unit cell
had dimensions: a = 8.14 A, b = 14 A, c = 10.3 A, c = 62j
and contained two cellulose chains. This proposal has
caused little controversy in spite of the diculty in indexing
the x-ray diraction reections precisely. However, a larger
unit cell (a = 15.92 A, b = 18.22 A, c = 18.22 A, c = 117j)
was proposed on the basis of a neutron diraction study [74],
which called into question the previous assignment of the
monoclinic space group P21. These variations could arise
from the use of neutrons, which are sensitive to structural
defects and disorder arising from the occurrence of various
factors aecting the conformations of the positions of the
hydrogen atoms in the hydroxyl groups. Besides, it could
also be argued that the methods of preparation of this
allomorph might account for some of the dierences. There
are indeed two main routes to cellulose II: mercerization,
which involves treatment with alkali, and solubilization
followed by regeneration (recrystallization). In spite of the
similarities in unit cell dimensions, there are some dierences
that seem to be signicant. For example, the values of the a
dimension in a cellulose regenerated from ramie are 8.662
and 8.588 A in the case of a cellulose obtained by mercer-
ization. Similarly, the value of the angle g is always more
signicant for mercerized celluloses than for regenerated
celluloses. It also seems likely that the degree of sample
purity has a bearing on the quality of the crystalline domains
and unit cell parameters, leading, as in the case of regener-
ation, to elevated rates of conversion [75]. There are few
reports of the occurrence of the type II allomorph in native
celluloses [76]. However, a structure corresponding to cel-
Figure 13 The relationship between the unit cell of cellulose lulose II has been proposed for a mutant strain of Aceto-
Ia and Ih. bacter xylinum [77].
52 Perez and Mazeau
Several studies have been dedicated to structural the h-D-cellotetraose structure. The intermolecular hydro-
determinations using a combined approach of x-ray dif- gen bonding diers substantially from that observed in h-
fraction data and modelling methods for minimizing the D-cellotetraose. One consequence of the dierence is that
packing energies of cellulose chains in the unit cell O6 of the origin chain can donate a hydrogen bond to three
[41,42,75,78]. In spite of some minor dierences, the results possible acceptors, the major component being O6 of the
agree suciently well to propose a model in which the center chain. These three acceptors already interact with
cellulose chains have almost perfect twofold symmetry and one another through a three-center hydrogen bond. It is
are compatible with occurrence of two intermolecular not clear as to what extent of disorder of the O6 group of
hydrogen bonds between consecutive residues [OH- the center chain is responsible for this intricate hydrogen-
3V. . .OU5 (2.70 A) and OH-2. . .O6V (2.70 A)]. Within bonding arrangement (Fig. 14).
the crystalline mesh, a network of hydrogen bonds ensures The use of synchrotron x-ray data collected from
the formation of layers composed of cellulose chains. A ramie bers after ad hoc treatment in NaOH provided a
notable feature of this three-dimensional arrangement is revised crystal structure determination of mercerized cel-
the antiparallel orientation of the cellulose chains. lulose II at 1 A resolution [83]. The unit cell dimensions of
The similarities that exist between x-ray powder dif- the P21 monoclinic space group are a = 8.10 A, b = 9.04
fraction diagrams of cellulosic oligomers and that of A, c = 10.36 A, c = 117.1j. As with the regenerated
cellulose II have excited the curiosity of crystallographers
because it seemed likely that high-quality structural data
from single crystals could be used to construct a model for
the polymer. However, in spite of early success in crystal-
lizing cellotetraose [79] and attempts at simulation, it was
not until 1995 that the structure was resolved by two
independent research groups [80,81]. Cellotetraose crystal-
lizes in a triclinical unit cell (a = 8.023 A, b = 8.951 A, c =
22.445 A, a = 89.26j, b = 85.07j, c = 63.93j) having
space group P1 containing two independent molecules. A
major conclusion of these studies concerned the signicant
dierences in the geometry of the two cellotetraose mole-
cules, which were oriented in antiparallel fashion in the unit
cell. The application of these new data to the resolution of
cellulose II [80,81] has conrmed the conclusions of these
studies with regard to the relative chain orientation, net-
work of hydrogen bonds, chain conformation, and unam-
biguous assignment of the gauchetrans conformation of
the primary hydroxyl groups, all in accord with spectro-
scopic data.
Recent progress with methods of intracrystalline deu-
teration [66,67] has also made an important contribution to
the elucidation of the cellulose II structure. Indeed, the
combination of x-ray and neutron diraction data has
allowed the precise analysis of the complex network of
intermolecular and intramolecular hydrogen bonding in
cellulose II obtained by regeneration. This is the best model
available for cellulose II [82]. In this model, the structure of
cellulose II is based on a two-chain unit cell of dimensions
a = 8.10 A, b = 9.04 A, c = 10.36 A, c = 117.1j. The
Figure 14 Description of the three-dimensional structure of
chains are located on the 21 axes of the monoclinic cell
cellulose II. (From Ref. 82.) Neutron diraction patterns
and are antiparallel. The two chains have dierent back-
collected from two ax samples: once mercerized in NaOH/
bone and glucose conformations. The glucoses of the
H2O (left) and the other mercerized in NaOD/D2O (right). The
central chain are strained and the chains are displaced ber axis is vertical. Details of the conformation of the two
relative to each other by about one fourth of the ber crystallographically independent chains (origin: (U =
repeat. The hydroxymethyl groups of the central chains are 96.8j, W = 143.5j); center chain (U = 93.3j, W =
disordered and occupy both transgauche and gauche 150.8j). These chains are arranged in an antiparallel fashion
trans positions. The precise location of hydrogen atoms in the unit cell. The hydroxymethyl group displays a gauche
provides the detailed description of the hydrogen-bonding trans orientation. A schematic representation of the hydrogen
system. A systematic three-center intrachain hydrogen bonds in sheets containing only origin chains (C), only
bond is observed in both chains. This bond has a major center chains (D), and center and origin chains.
component between O3 and O5, with O3 as a donor. A Intramolecular hydrogen bonds are O3UH. . .O5 in each
similar three-center hydrogen bond interaction occurs in molecule, with a minor component involving O6 as acceptor.
was accompanied by an important decrystallization and

fragmentation of the cellulose crystal. The reverse transi-
tion resulted in partial recrystallization, but this did not
allow complete restoration of the damage done to the
morphological surface. Characterization by electron dif-
fraction revealed that the uniplanaruniaxial orientation of
the crystalline cellulose microbrils was lost completely
during the stage of swelling and washing necessary for the
conversion into cellulose IIII. Washing with methanol
resulted in the formation of irregularities into which were
inserted crystalline domains of small dimensions. The nal
material that crystallized in the cellulose I form was
obtained by treatment with hot water and characteristically
displayed an increase in the accessible surface and conse-
quently reactivity.
Solid-state NMR studies have shown a signicant
decrease in the lateral crystallite dimensions during the
transition of cellulose IIIII. At the same time, the cellulose
chains show conformational changes arising from the
primary hydroxyl groups that change from a transgauche
Figure 15 Projections of the structure of cellulose II along arrangement in cellulose I (65.7 ppm) to gauchetrans in
the ber axis, along with the location of the chains in the the cellulose IEDA (62.2 ppm) in the allomorph IIII.
monoclinic unit cell. CPK representation and ball-and-stick Thus, the regenerated cellulose I provides a spectrum that
representation of the antiparallel layers of cellulose chains diers from that of the native form. Electron microscopy
along the ber axis. shows that cellulose I complexed with EDA is composed of
nonuniform crystalline domains, whereas the III I
allomorph is characterized by well-dened crystalline
cellulose, the chains are located on the 21 axes of the cell. zones. The conformational changes observed for the pri-
This indicates that the dierent ways of preparing cellulose mary hydroxyl groups are of interest because they provide
II result in similar crystal and molecular structures. The possible markers for study of the various conformational
crystal structure consists of antiparallel chains with dier- transitions associated with cellulosic systems.
ent conformations but with the hydroxymethyl groups of
both chains near the gauchetrans orientation. There are,
nevertheless, some signicant dierences between the con- VIII. CELLULOSE IV
formations of the hydroxymethyl group of the center chain
compared to that found in regenerated cellulose. This may Cellulose IVI and IVII allomorphs originate from cellulose
be related to the dierence observed in the amount of I and II, respectively. The conversions are never totally
hydroxymethyl group disorder: 30% for regenerated cel- complete, which explains the diculties in the production
lulose and 10% for mercerized cellulose. Whether this of good-quality x-ray diraction patterns [88]. However,
disorder is conned to the surface of the crystallites or is unit cell dimensions have been obtained for the two
pervasive is not known for the time being (Fig. 15). allomorphs of which IVI has a = 8.03 A, b = 8.13 A,
c = 10.34 A, which is close to those found for form IVII
(a = 7.99 A, b = 8.10 A, c = 10.34 A) [89]. In both these
VII. CELLULOSE III cases, the poor quality of the diraction patterns does not
allow determination of the space group. The authors
The crystalline forms of cellulose III (IIII and IIIII) are
suggest space group P1 but this is not compatible with the
reversible and this suggests that, as with allomorphs I and
proposed unit cell dimensions.
II, the chain orientation is the same as in the starting
material [84,85]. From unit cell dimensions a = 10.25 A,
b = 7.78 A, c = 10.34 A, c = 122.4j, a structural model in IX. ALKALI CELLULOSE
which the chains did not have strict twofold symmetry was
proposed. Several research investigations have focussed on Up to the present, research reports have tended to focus on
the reversible transformations between cellulose I and III the relative arrangement of cellulose chains in the cellulose
using techniques such as electron microscopy [86], solid- I and II allomorphs. Whereas in native architectures the
state NMR [50,51], x-ray diraction [85], and molecular chains are parallel, regenerated or mercerized cellulose has
modelling [87]. antiparallel arrangements. Elucidation of the detailed
From one study of the cellulose III transformation events that take place during the transformation of cellu-
involving an intermediate cellulose IEDA complex, it was lose I and II is of great interest, especially as the process of
concluded that a liquid crystalline phase was involved [87]. mercerization does not appear to require solubilization of
In the case of Valonia, the conversion from form I to II the cellulose chains. It would seem therefore that the
54 Perez and Mazeau
cellulose structure should be preserved. To understand the molecular and crystallographical structure of native cellu-
mechanisms that come into play, a great number of studies lose was lacking.
have been dedicated to the study of intermediate structures It was during the 19th century that Hermans and
[90,91]. By means of characterization by x-ray diraction, Weidinger [28] developed a theory to deal with the
Okano and Sarko [92] put forward evidence for the occur- birefringent materials in plant cells and starch grains.
rence of ve dierent types of structure that could be This theory led to the introduction of the concept of
classied as a function of the cellulose chain conformation. crystalline micelles having submicroscopical dimensions.
Soda celluloses of types I, III, and IV were characterized by This in turn led to the proposal that crystalline micelles
a repeat of 10.3 A, whereas types IIA and IIB had a repeat were separate, well-dened entities that were stacked like
of 15.0 A, corresponding to a helicoidal repeat of 3, a value bricks, whose length coincided with the axis of the
not seen in previous studies. In addition, all the soda constituent cellulose molecules. In order to take account
celluloses showed a reasonable degree of crystallinity and of the amorphous content of cellulose, the idea of indi-
orientation. Hence, it seems dicult to reconcile a change vidual micelles evolved into the hypothesis of fringed
of orientation with the mercerization process. These work- micelles [97]. In this model, the micelles are considered
ers proposed that, because Na-cellulose I could not be to be ordered regions statistically distributed in a mass of
converted back into cellulose I, the chains must be ar- chains that are more or less parallel. The interface
ranged in antiparallel fashion (i.e., as in cellulose II). between crystalline zones and amorphous zones is blurred
Despite the failure to identify the mechanisms that come and the micelle length need not necessarily correspond to
into play during the transformation from a parallel ar- the constituent chain length and a single chain may even
rangement (cellulose I) to an antiparallel arrangement pass through several micelles. The microbrillar structure
(cellulose II), these reports nevertheless have the merit of of cellulose has been established beyond doubt through
identifying the intermediate stage (Na-cellulose) from the application of electron microscopy [98,99] and great
which the structural rearrangement could arise without variations in dimensions, depending on origin, have been
chain solubilization. reported [1,33,34]. The question of whether or not inter-
An extension of this research can be found in the mediate structural elements called elementary brils exist
studies of Hayashi et al. [91] in which nine types of Na- has been a topic of great controversy. However, the appli-
cellulose, which could be formed from allomorphs I, II, III, cation of transmission electron microscopy [100,101] has
and IV, were identied [93,94]. From these studies, it was established with certainty that the microbril is the basic
concluded that the irreversibility probably depended more crystalline element of native cellulose [5,100102]. It
on conformational changes of the cellulose chain than appears that the dierent levels of structural organization
chain rearrangements. This argument seems unconvincing of cellulose are now well characterized.
because the energy dierences between twofold and three-
fold helices are too small to account for the irreversibility A. Polarity of Cellulose Crystals
observed for type I and II structures. Another tentative
explanation was made from the occurrence of two types of The cellulose chain possesses a polarity that arises from the
microbril in samples of Valonia: one of which had the chemical dierence of the two ends of the molecule and this
chains oriented along the Oz crystal axis, the other having confers particularly interesting properties to the crystalline
chains oriented in the opposite sense [95]. Most of the architecture. In eect, two types of arrangement can be
investigations dealing with mercerization of cellulose have envisaged, depending on whether the reducing groups are
focused on global measurements recorded on whole bers all located at the same end of the chain assembly (parallel
(i.e., assembly of a large number of organized microbrils). arrangement), or whether the reducing and nonreducing
The structural and morphological changes accompanying ends are arranged in alternating fashion within the assem-
mercerization of isolated cellulose microbrils have been bly (antiparallel arrangement). The answer to this question
followed by transmission electron microscopy, x-ray, has been the aim of numerous investigations but has
Fourier transform infrared spectroscopy (FTIR) and 13C equally given rise to a number of controversies. In their
CP-MAS NMR. The changes in morphology when going original model, Meyer and Misch [103] had proposed an
from cellulose I to cellulose II were spectacular as all the antiparallel arrangement, which was supported by the
microbrillar cellulose morphologies disappeared during observations of Colvin [104] on the production of bacterial
the treatment. The outcome of this investigation is that is cellulose in which the reducing ends of cellulose were
impossible for isolated cellulose microbrils to become stained with silver nitrate [105]. However, other attempts
mercerized while keeping their initial morphology [96]. to identify reducing chain ends using conditions similar to
those of Colvin were interpreted as supporting a parallel
chain arrangement. It was from the use of exocellulases
X. CRYSTALLINE MICROFIBRILS OF NATIVE that nal experimental proof of parallel arrangement [106
CELLULOSE 108] in the family of native celluloses was nally obtained
[109]. Recent investigations using complementary enzy-
Much research has been devoted to experimental and matic and chemical staining of reducing ends have sup-
theoretical studies of the crystalline component of native ported this model and, at the same time, produced precise
cellulose often in a context in which knowledge of the descriptions of the orientation of the chains relative to the
crystal axes [59]. Hence, the crystalline microbrils possess

the same polarity as the chains of which they are composed.
These conclusions are in agreement with the body of
crystallographical and molecular modelling studies and
reect the constraints imposed by the biosynthetic require-
ments of native cellulose.
B. The Crystalline Morphology of Native

Celluloses
The availability of an accurate description of the crystalline
structure of cellulose Ih, along with the predicted features
of cellulose Ia, provides new insights into the crystalline
morphology of native celluloses. These models can be used
to generate dierent ordered atomic surfaces, and evaluate
their occurrence along with their respective features. The
schematic representation of the crystalline arrangements of
cellulose Ia and Ih in relation with their respective unit cells
is shown on Fig. 16. Irrespective of the ne structural
dierences, the same gross features are exhibited by the
two polymorphs. This indicates that the same morphologi-
cal features are expected to occur in the native celluloses.
From such structural arrangements, well distinct types of
crystalline surfaces can be identied.
The type 1 surface represents the faces that run Figure 17 CPK representation of the main crystalline faces
through the diagonal of the of the ab plane of the Ih mono- for cellulose I.
clinical unit cell or through the a and b axis of the Ia tri-
clinical unit cell. These surfaces are tortuous, displaying
grooves extending parallel to the c axis. They are created of these surfaces. The type 2 surface represents the faces
by free spaces between the chains. Hydroxyl groups that run through either the b axis of the Ih crystal or the
point outward, emphasizing the hydrophilic character rst diagonal of the ab plane of the Ia crystals. The
cellulosic chains exhibit CH groups at the surface. This
surface is at and hydrophobic. The type 3 surface repre-
sents the faces that are parallel to the a axis of the Ih unit
cell or to the second diagonal of the ab plane of the Ia
crystals (Fig. 17).
C. Whiskers and Cellulose Microfibrils

Depending on their origin cellulose microbrils have diam-
eters from 20 to 200 A, whereas their length can achieve
several tens of microns [5] (Fig. 18).
These characteristics confer very interesting mechan-
ical properties on microbrils. Transmission electron dif-
fraction methods have made a contribution to the
quantication of the degree of crystallinity. Thus, using
the technique of image reconstruction it was shown that,
in the microbril of Valonia, which has a diameter of about
200 A, there could be more than 1000 cellulose chains all
aligned in parallel in an almost perfect crystalline array.
Some imperfections arose from dislocations at the inter-
face of microcrystalline domains along the microbril
length [100,110]. These imperfections were used to advan-
tage by treatment with acid to produce monocrystals called
whiskers having the same diameter as the starting micro-
Figure 16 Projection of the structure of cellulose Ia and Ih brils but much shorter length.
along the ber axis. The triclinical and monoclinical unit cells These cellulose whiskers possess a mechanical modu-
are shown along with the main crystallographic directions, lus of about 130 GPa, which is close to calculated value for
relevant for the crystalline morphologies. cellulose [111]. These characteristics (microscopical dimen-
56 Perez and Mazeau
allows disruption of the microbrils without aecting the

original length. As a result, microbrils several microns
long and 2030 A wide are obtained (Fig. 20).
Analysis of these dierent specimens by x-ray dirac-
tion allows appreciation of the crystalline variation and the
extent to which the amorphous components occur.
The diraction patterns of tunicin microcrystals are
clearly of the allomorph I; they are detailed with well-
dened rings. Those of cotton are less well dened and the
rings are signicantly more diuse. In the case of paren-
Figure 18 Range of microbril size from dierent sources. chyma microbrils, the resolution of the rings is less good
and they begin to mergea reection of decreased lateral
order and small size of the microbril diameter. In contrast,
sions, form, and exceptional mechanical properties) made longitudinal order is maintained along microbrils of large
whiskers a choice ingredient in the manufacture of dimensions. The amorphous phase increases as a result of
nanocomposite materials [112] (Fig. 19). decrease in microbril diameter and the increase in the
Celluloses of dierent origin yield whiskers of diverse number of surface chains. The noncrystalline component
structural quality suitable for a range of applications. essentially corresponds to the surface chains of which there
Hydrolysis of bleached tunicin cellulose (Halocynthia ro- will be more when the microbrils are small. Fig. 21 is an
retzi) with sulphuric acid yields monolithic microcrystals idealized representation of the organization of cellulose
with a smooth appearance and lengths varying from chains, displaying morphology and dimensions typical of
hundreds of nanometers to several micrometers. The lat- those of microbrils of parenchyma. In such a case, the
eral dimensions of these monocrystals range from 50 A to total number of cellulose chains would be 26; among them,
more than 200 A, which makes them 100 times superior 16 could be considered as surface chains. This nding has
with regard to form. The microcrystals obtained by hydrol- been corroborated by CP-MAS NMR applied to ultrathin
ysis of cotton linters are shorter than those from tunicin cellulose microbril extracted from sugar beet pulp [113].
and reach lengths of 0.1 Am and widths from 10 to 50 A, It can be estimated that the surface chains in tunicin
and have a shape value of 20. At the other end of the microbrils constitute no more than 5% of the total
spectrum, there are cellulose microbrils from parenchyma number of tunicin chains, whereas surface chains can
that are quite dierent in appearance from those of cotton represent 70% of the total number in parenchyma micro-
and tunimycin. These microbrils are produced by a brils [5]. The result of increasing number of surface chains
mechanical treatment, which, contrary to hydrolysis, and decrease in whisker diameter can also be seen by
Figure 19 Electron micrograph of cellulose whiskers. (Courtesy of H. Chanty.)

Figure 20 Transmission electron micrographs of tunicin (A) microcrystals negatively stained with uranyl acetate. (B) Ultrathin
section of microbrils in bright eld. Transmission electron micrographs of (C) cellulose microcrystals negatively stained with
uranyl acetate. (D) Ultrathin section of a cotton ber in bright eld. Transmission electron micrographs of (E) parenchyma
cellulose microbrils negatively stained with uranyl acetate (F). (G) X-ray diraction diagrams of cellulose microcrystals: (a)
tunicin cellulose; (b) cotton; (c) parenchyma. (From Ref. 151.)
58 Perez and Mazeau
interactions; furthermore, they are often delicate to imple-

ment and the obtained data are generally not easily ex-
ploitable.
The organization of the surface chains has been ini-
tially studied by microstructural chemical analysis in which
the reactivity of the exposed hydroxyl groups toward
various chemical agents is tested [116122]. Such experi-
ments are analyzed under the assumption that only the
exposed hydroxyls can react with an external chemical
agent. Furthermore, hydroxyl group reactivity may be
correlated with the degree of organization at the surface.
An equivalent reactivity is expected for the dierent
hydroxyls in the case of amorphous structure, in which
the surface chains are not organized; this is a direct
consequence of similar accessibilities. The specic reactiv-
ity of the O2H, O3H, and O6H hydroxyl groups toward
various agents was measured [117] on cellulose samples
diering in crystalline content. Such studies showed that
hydroxyl group O2H is almost always available, in contrary
to O3H hydroxyl, whose reactivity is strongly dependent on
the crystalline index of the studied cellulose sample. The
O3H is almost not susceptible toward chemical agents in
highly crystalline Valonia or bacterial celluloses, in contrast
Figure 21 Molecular model of a microbril of cellulose to its measured reactivity in cotton cellulose for which the
projected along the bril axis compared to the typical degree of organization is far less perfect. Hydroxyl group
morphologies observed for Tunicin and Valonia samples.
O6H shows an intermediate reactivity that also depends on
the crystal index of cellulose. Those results are in good
agreement with hydrogen-bonding network revealed from
analysis of the neutron diraction data of a deuterated
infrared spectroscopy by a broadening of the absorption tunicin sample [68]. Absence of reactivity of the O3H
bands with loss of resolution. It is believed that a peak at hydroxyl suggests that the strong O3H. . .O5 hydrogen
1635 cm1 can be attributed to vibrations arising from bond observed in the crystalline structure persists at the
water molecules absorbed in the noncrystalline regions of surface of the cellulose materials, whereas larger reactivity
cellulose [114,115]. of the O2H and O6H suggests that the hydrogen bonds
involving those hydroxyls are at last partially disrupted at
XI. SURFACE FEATURES OF CELLULOSES the surface. A larger conformational dynamics is therefore
expected at the surface as compared to the one of the bulk.
Many properties of native cellulose depend on the inter- Organization of the surface chains of cellulose has
actions that occur at the surface of the brils. As compared been observed by atomic force microscopy (AFM) [123
to bulk chains, surface chains are accessible and reactive. 127]. The recorded AFM images obtained on Valonia
This is due to the dense packing of the chains within the samples highlighted that the surface chains are organized
crystal in which all the hydroxyl groups participate in alike those of the bulk. A triclinic organization has been
crystalline cohesion through intramolecular and intermo- observed on Valonia ventricosa [123125], whereas Valonia
lecular hydrogen bonds. This structural understanding is macrophysa displays a monoclinic organization of the
supported by the many reported selective modications of observed surface chains [127]. High-resolution images
the cellulose brils, which occur only at the surface of the obtained by Baker et al. showed the (100) face of the Ia
cellulose material. triclinical allomorph. Periodicities of 10.4 and 5.2 A were
Surface chains play a fundamental role in the interac- recorded, corresponding to cellobiose repeat along the ber
tion processes (adsorption and adhesion) of the cellulose axis and interchain spacing (the distance between interre-
brils with other molecules. Such surface interactions play ticular planes has been measured at 5.3 A), respectively.
a key role in many scientic areas: biology (interaction with Triclinic organization was conrmed by the typical super-
the plant cell wall polymers, adsorption of cellulolytic molecular arrangement of the chains, a diagonal shift close
enzymes), industrial (paper and textile industries), and to 65j, corresponding to this allomorph, whereas discrim-
technological (compatibilization and adhesion a thermo- ination between the (100) and (010) surfaces is based on the
plastic amorphous matrix on cellulose). Unfortunately, to interreticular distance (Fig. 22).
date, very little information has been gathered on the Finally, comparison between high-resolution images
organization, conformation, and dynamics of the surface and reconstituted AFM image from crystallographic
chains. As a matter of fact, few experimental techniques coordinates showed that the surface hydroxymethyl groups
can access the morphology of the material and surface adopt a conformation that is dierent from the trans
if all the dierent studies attempt to highlight the intrinsic

characteristics of the surfaces (stability, hydrophilic and
hydrophobic character, morphology, etc.), only few of
them explicitly unravel the interactions between the sur-
faces of native cellulose and a guest molecule.
A. Congo Red
Work performed by Woodcock et al. [130] aimed at
energetical and geometrical characterization of the adsorp-
tion process of the aromatic organic dye, Congo red, on
crystalline surfaces of cellulose with the help of molecular
Figure 22 Atomic force microscopy of native cellulose. mechanics program, Assisted Model Building with Energy
(From Ref. 124.) Renement (AMBER). The considered surfaces are the
(100) and (010) of the Ia phase, and the (110) and (110) of
the Ih allomorph. Results suggest a preferential adsorption
gauche conformation of the buried groups. Molecular on the (010) of Ia and the (110) of Ih surfaces.
models of the surface of cellulose having either gauche Adsorption is mainly governed by the electrostatic
trans or gauchegauche orientation of the hydroxymethyl contribution of the total energy between polar groups of
group have a better agreement with the observed images. the dye molecule and available hydroxyls and acetal oxy-
Such conformational dierences disrupt the intramolecu- gens of cellulose. Correlation between the calculated data
lar (O2H. . .O6) hydrogen bond of the chains that are and electronic microscopical experimental evidence
located at the surface of the cellulose material. It should [136,137] on adsorption of the cellulose-binding modules
be noted that all these AFM studies require an acid of cellulolytic enzymes on cellulose suggested that specic
treatment of the samples to remove the disorganized chains adsorption of cellulase might occur on the same surfaces
initially present at the surface of the materials. Such of the modelled adsorption of Congo red. Such hypothesis
chemical treatment allows an experimental observation of has been recently revisited [138]; the cellulose-binding
the surface in which the backbone conformation and the modules specically adsorb on the (110) of the Ia phase
super molecular organization of the chains are close to that of cellulose from Valonia. Nevertheless, preferential ge-
of the crystal. ometry of the complex has been reported from the ar-
The majority of the AFM observations are supported rangement that gives the lowest potential energy. Congo
by solid-state NMR [128,129]. In particular, NMR suggests red is oriented parallel to the cellulose chains. In this study,
that the orientation of the exposed primary (O6) group is in the surface of the cellulose crystals is considered rigid;
the transgauche and gauchegauche orientations, in con- therefore, it does not consider possible structural disorga-
trast with the transgauche orientation of the buried nization and the enhanced mobility of the surface chains.
hydroxymethyl groups. NMR is a powerful tool to evi- Note also that no water molecules are included in the
dence the conformational disorder of the surface chains; it modelling protocol.
allows an estimation of the relative proportion of the
dierent organization state of the brils of cellulose: crys-
talline bulk, organized surfaces, less-ordered surfaces, and B. Benzophenone
amorphous domains [128]. It has been shown that the Surface chain mobility and structural disorganization have
purication process, together with acid treatment, aects been considered in the work of Mazeau and Vergelati [135],
the ultrastructural organization of the chains [113]. which revealed the dependence of the cellulose surface
The dierent experimental results show that the sur- characteristics on the adsorption behavior of benzophe-
face chains are partially disorganized; their conformational none molecules. With the help of the Consistent Force
freedom is larger than the one of the bulk chains. The lesser Field (CVFF) force eld, the geometrical and energetical
amount of hydrogen bonds of the surface chains, as com- characteristics of adsorption of benzophenone on crystal-
pared to the bulk ones, is consistent with a larger reactivity line (110), (110), and (200) of the Ih allomorph and an
of those chains toward reactants and also for adsorbed amorphous surface have been studied. Adsorption of
species: water molecules, hemicelluloses, lignins, etc. benzophenone molecules has been modelled by using
Monte Carlo and molecular dynamics protocols. Both
XII. INTERMOLECULAR INTERACTIONS (110) and (110) crystalline surfaces give similar results,
whereas notable dierences could be observed between
In spite of the many structural investigations of cellulose, these two surfaces and the (200). Among all the crystalline
studies by molecular modelling remain surprisingly far surfaces, adsorption occurs preferentially on the (200)
from numerous. Furthermore, most of the eorts are surface. Interaction energies are of the same order of
devoted to the bulk: crystal packing prediction and esti- magnitude for all those crystalline surfaces. The principal
mation of mechanical properties. Studies concerning the energetical component that stabilizes the adsorption on the
surfaces of cellulose are extremely rare [130135]. Finally, (200) surface is the van der Waals term, whereas the
60 Perez and Mazeau
electrostatic contribution is important for the (110) and (1 the experiment. On the contrary, for the amorphous sur-
10) surfaces. Many adsorption sites have been identied for face, benzophenone molecules signicantly penetrate with-
each surface. Adsorption of benzophenone has no prefer- in the cellulose material.
ential geometry on the (200) surface; on the contrary, the
(110) and (110) surfaces for the geometry of adsorption C. Water
appear strict. Finally, independently of the considered
surface, a hydrogen bond is systematically observed be- Heiner and Teleman [132] and Heiner et al. [133] performed
tween the carbonyl oxygen of benzophenone and a hy- molecular dynamics simulations on an interface between
droxyl group of cellulose (Fig. 23). crystalline cellulose and water, by using the Gronengen
Benzophenone adsorbs at on the (200) surface; the Molecular Simulation System (GROMOS) force eld. The
aromatic rings lie parallel to the surface of cellulose. In this considered surfaces are the (010) and (100) of the triclinical
preferential geometry, the aromatic rings are located above phase and the (100) and (110) of the monoclinical one.
the apolar CH groups of the surface chains, favoring Each modelled system is composed of six layers of cellulose
hydrophobic stacking interactions. In spite of light struc- chains; each layer is constituted by six chains of six glucose
tural dierences between the (110) and (110) surfaces, the residues. This cellulose material is solvated by explicit
adsorption process of benzophenone is the same for these water molecules. Analysis of the geometrical and confor-
two surfaces. The authors also studied the formation of a mational parameters shows that only the topmost cellulose
benzophenone monolayer on (110), (200), and amor- layer is aected by the presence of water molecules. This
phous surfaces. On the crystalline surfaces, the adsorption result is in good agreement with solid-state NMR data,
sites are qualitatively identical and are periodically re- which show that the surface eective component of the
peated on the surface. On the contrary, the amorphous spectra corresponds to a single layer [128]. Dynamics and
surface of cellulose shows sites that are topologically conformation of the surface chains dier from the bulk
extremely favorable to the benzophenone adsorption for chains. In particular, hydroxymethyl group conformation
which the interaction energy is large. Once those remark- possesses a marked rotational freedom; gauchetrans is the
able sites are fullled, benzophenone molecules adsorb on preferred orientation, in opposition with the transgauche
sites that are energetically comparable to the crystalline conformation of the hydroxymethyl group of the bulk
ones. Finally, stability and time evolution of the adsorbed chains. The decrease of hydrogen bonds at the surface of
monolayer of benzophenone at the interface with liquid the cellulose is compensated by hydrogen bonds between
water were tested by recording molecular dynamics trajec- cellulose and water. The O3HO5 intramolecular hydrogen
tories. Dierences in the density proles between the initial bond persists at the surface, in agreement with the con-
structures and the structures obtained after 1 nsec of clusions derived from microstructural chemical analysis
molecular dynamics experiment show a light reorganiza- (Fig. 24).
tion of the crystalline surfaces and benzophenone mole- Hydration of the surfaces has been estimated from
cules remain located at the surface within the time scale of density proles of the water molecules, which clearly
Figure 23 Molecular representation of the adsorption of benzoophenone on crystalline cellulose. (From Ref. 135.)
same approach, but for an argon atom, which is a model of

lipophilic molecules. Data showed that the two surfaces are
equivalent, nonhydrophilic, and mainly lipophilic. Water
molecules are not attracted by the surface.
D. Lignin
The hypothesis of association between lignin building blocs
on the surface of the cellulosic matrix was tested by
modelling. Houtman and Atalla [131] studied the dynam-
ical behavior of model compounds that are lignin precur-
sors (monolignols and trilignols) in the presence of a
hydrated surface of crystalline Ih cellulose. Although
initially located in the aqueous phase, at about 13 A of
the cellulose surface, modelling evidenced a rapid adsorp-
tion of the monolignol on the cellulose surface. Results
showed that the driving force responsible for adsorption is
mainly of electrostatic nature. Interactions between the
monolignol and the cellulose chains are strong enough to
inuence the dynamics of the adsorbate close to the
surface. Mobility of the monolignol is considerably de-
creased when the molecule reaches the surface. As a
consequence of this limited mobility, the preferred adsorp-
tion geometry of the lignols is parallel to the cellulose ber
axis and the aromatic moieties of lignols are parallel to the
cellulose surface; thus, hydrophobic interactions are max-
imized through stacking-type interactions. Also, a fast
adsorption of the trilignol model was observed. It adsorbs
at on the surface and two of the three aromatic rings are
oriented parallel to the surface. On the basis of those
results, authors conrm that the cellulose bers and, more
generally, the polysaccharide matrix, can inuence mono-
lignol polymerization and ultrastructural organization of
lignin in the plant cell walls. Such adsorption leads to a
reorganization of the lignin structural units before poly-
merization, which could inuence the primary structure of
Figure 24 Hydration of cellulose as seen from molecular the lignin polymer through selected distribution of the
dynamics simulations. (From Ref. 132.) monomer units and also could inuence the conformation
of the lignin polymer. Work performed by Jurasek [139]
also suggests that cellulose might inuence the structure of
indicate water structuring close to the surfaces. Because of the lignin polymer. The growing of a bidimensional model
the interactions between cellulose and water, the dynamics of the secondary cell wall is faster in the direction parallel to
of the water molecules decreases by a factor of 23 close to the ber axis than in the perpendicular direction. Spatial
the surface. It is also found that monoclinic (110) and
triclinic (010) surfaces behave similarly just as the mono-
clinic (110) and triclinic (100) ones. These two last
surfaces have a larger hydrophilic character than the two
others. Hydrophilic character of the surface has been
shown to be dependent on hydroxyl group distribution
on the surface together with their ability to maximize
hydrogen bonds. Hydrophilic and lipophilic character of
the cellulose surfaces has been independently estimated by
Biermann et al. [134], using molecular dynamics calcula-
tions. The modelled systems consist of an interface of
cellulose, exposing their monoclinic (110) and (110)
surfaces, and water. Hydrophilic character was estimated
from local values of the chemical potential of water close to Figure 25 Schematic representation of wood ber structure.
the surface with respect to its value far from the surface, in P, primary cell wall; S2, middle secondary cell wall; S1, outer
the bulk. Lipophilic character was estimated by using the secondary wall; S3, inner (tertiary) cell wall.
62 Perez and Mazeau
Figure 27 Structural levels of organization of cellulose in the plant cell wall.
Figure 26 Microbril organization in wood. (From Ref. 142.) (A) Wood cells in cross section were scanned by a microbeam
smaller than the thickness of a single cell wall. The tilt angle of the cellulose brils with respect to the beam corresponds to the
microbril angle l. (B) A perfect alignment of all crystallographic axes of cellulose would yield sharp diraction spots
corresponding to the (0 2 0), (1 2 0), (1 1 0) reections in the plane perpendicular to the bril axis in the reciprocal space. A
random orientation of the brils around their longitudinal axis would result in a smearing of the reections to rings. (C) Principle
for the measurement of local bril orientations. (i) Cellulose bril tilted by an angle l with respect to the incoming X-ray beam: a
/ denotes the orientation of the bril in the plane perpendicular to the beam. (ii) In reciprocal space, the smearing of the
reections [(0 2 0), large ring; (1 1 0) + (1 1 0), small ring] is caused by random orientation of the parallel cellulose brils
around their longitudinal axis. A and B are the points of intersection between the Ewald sphere and the DebyeScherrer rings.
(iii) Scattering pattern as it would appear on the area detector in the plane perpendicular to the beam. The scattering pattern is
asymmetrical. The orientation af of the cellulose brils (indicated by an arrow) can be extracted directly form the peak position.
(D) Mesh scan over a complete wood cell in cross section with part of neighboring cells; pixel size: 2 2 mm. Dark region
correspond to lumina; bright region showing a scattering signal corresponding to cell walls. (b) Two typical diraction patterns
(with greater magnication) showing the local orientation of the cellulose brils af, denoted by arrows. (E) Map of local cellulose
bril orientations. Following the arrows readily yields the trace of the brils around the cell. Longer arrows denote a more
pronounced asymmetry of the diraction patterns corresponding to smaller local brils angles; shorter arrows denote larger bril
angles. (F) Translation of the arrow map into a three-dimensional model; the cellulose brils trace a helix around the cell.
64 Perez and Mazeau
constraints due to the presence of the cellulosic matrix where knowledge is incomplete. The structure and dynam-
impose a certain degree of regularity in the nal structure of ics of chains at the crystallite surface are also not well
lignin. Molecular mechanics calculations performed by established and the study of outer chains is likely to become
Faulon and Hatcher [140] suggest that the helical confor- an important topic for future research using techniques
mations of lignin oligomers are energetically preferred than such as solid-state NMR, near-lled microscopy, molecu-
random coils. This is in agreement with experimental lar modelling, mechanical spectroscopy, etc. The nature of
observation of lignin by scanning tunneling microscopy; the topological changes that accompany the transitions
images show an ordered structure [141]. between the dierent cellulose polymorphs also remains to
be more rmly established.
Without doubt, better knowledge of the dierent
XIII. MICROFIBRIL ORGANIZATION structural levels in which cellulose participates will permit
In nature, cellulose is most commonly found as part of an better use of this unique and metastable molecular assem-
architectural complex whose ultrastructural organization bly, which is produced by biosynthesis. The numerous
depends on the organism under consideration. In a mate- classical applications of cellulose, depending on factors
rial such as wood, which is rich in cellulose, the cell walls such as the macromolecular nature of the chain or as a
are composed of cohesive, interlaced crystalline micro- component of wood, will soon be complemented by appli-
brils that are themselves composed of cellulose (Fig. 25). cations involving whiskers. Thus, the industrial applica-
The cellulosic bers are 12 nm long and about 35 A tions for cellulose will continue to grow. For this to be put
wide and the microbrils are composed of 3040 cellulose into operation, understanding of the modication of the
chains. Application of new approaches using synchrotron heterogeneous phase by chemical and enzymatic means will
radiation has made a momentous contribution to the be needed to confer new properties on the macromolecular
characterization of their structural organization [142] chain assemblies of cellulose.
(Fig. 26).
X-ray diraction diagrams have been recorded, using
ACKNOWLEDGMENTS
wavelengths of 0.78 A, on wood sections about 10 Am thick
oriented perpendicularly to the incident beam. The speci- The authors express their gratitudes to Drs. H. Chanzy,
men under investigation (52 42 Am) was scanned in Y. Nishiyama and J. F. Sassi.
increments of 2 Am, resulting in a collection of 26 21
diraction patterns, which provided a distribution map of
the orientation of the axes of the cellulose microbrils. In REFERENCES
eect, each diraction diagram is characterized by strong
1. Preston, R.D. X-ray analysis and the structure of the com-
intensities, which were attributed to (0, 2, 0), (1, 1, 0) and
ponents of plant cell walls. Phys. Rep. 1975, 21, 183226.
(1, 1, 0), so that the orientation of the microbril along 2. Preston, R.D. Natural cellulose. In Cellulose: Structure,
the direction of propagation could be deduced. The out- Modication and Hydrolysis; Young, R.A., Rowell, R.M.,
come of such an exploration is shown schematically, in Eds.; John Wiley and Sons: New York, 1986, 327.
which the dark areas where no diraction is recorded are 3. Marchessault, R.H.; Sundararajan, P.R. In Cellulose. In
considered to arise from the lumen. Analysis of each The Polysaccharides; New York: Academic Press, 1983,
diagram for its part allows determination of the local 1195.
4. Sarko, A. Cellulosehow much do we know about its
orientation of the microbril axis. Integration of the structure? In Wood and Cellulosic Industrial Utilization,
individual observations gives an image of the degree of Biotechnology, Structures and Properties; Kennedy, J.F.,
disorientation. The big arrows indicate a marked local Ed.; Ellis Horwood: Chichester, UK, 1987, 5570.
asymmetry in the microbrils and thus that amplitude is 5. Chanzy, H. In Cellulose Sources and Exploitation, Aspects
of less importance than the local orientation of the micro- of Cellulose Structure. Kennedy, J.F., Phillips, G.O.,
bril. Translated into three dimensions, these results lead Williams, P.A. Eds.; Ellis Horwood Ltd.: New York,
1990, 312.
to an ultrastructural model in which it is established that 6. Okamura, K. Structure of cellulose. In Wood and Cellulosic
the orientation of the cellulose brils is aligned with the cell Chemistry; Hon, D.N.S.-S., Shiraishi, N., Eds.; Marcel
axis in a superhelicoidal fashion. Dekker: New York, 1991, 80111.
7. OSullivan, A. Cellulose: the structure slowly unravels.
Cellulose 1997, 4, 173207.
XIV. CONCLUSIONS 8. French, A.D. Structure and biosynthesis of cellulose: I.
Structure and biosynthesis of cellulose. Discov. Plant Biol.
The data gathered together in this article have followed the 2000, 3, 163197.
development of knowledge of the dierent structural levels 9. Jarvis, M.C. Structure and properties of pectin gels in plant
of cellulose. These concern: chain conformation, chain cell walls. Plant Cell Environ. 1984, 7, 153164.
polarity, chain association, crystal polarity, microbril 10. MacQueen-Mason, S.J.; Durachko, D.M.; Cosgrove, D.J.
Two endogenous proteins that induce cell wall extension in
structure, and organization. All these structural levels are plants. Plant Cell 1992, 4, 14251433.
presented schematically, including their relationship to the 11. Taiz, L.; Zeiger, E. Plant Physiology. The Benjammin/
plant cell wall (Fig. 27). Many structural features have been Cumming Publishing Company, 1991.
rmly established, and there remains relatively few areas 12. Malcom Brown, R.; Saxena, I.M., Jr. Cellulose biosyn-
thesis: a model for understanding the assembly of 35. Ambronn, H. Uber das Zusammenwirken von Stabchen-
biopolymers. Plant Physiol. Biochem. 2000, 38, 5767. doppelbre-chung und Eigendoppelbrechung I. Kolloid-Z.
13. Cousins, S.K.; Brown, R.M., Jr. Cellulose I microbril 1916, 18, 9097.
assembly: computational molecular mechanics energy 36. Ambronn, H. Uber das Zusammenwirken von Stabchen-
analysis favour bonding by van der Waals forces as the doppelbre-chung und Eigendoppelbrechung II. Kolloid-Z.
initial step in crystallization. Polymer 1995, 36, 38853888. 1916, 18, 273281.
14. Braconnot, H. Sur la conversion du corps ligneux en 37. Kubo, T. Untersuchungen Uber die Umwandlung von
gomme, en sucre, et en un acide dune nature particulie`re, Hydratcellulose in naturliche Cellulose: VII. Die Kristall-
par le moyen de lacide sulfurique; conversion de la meme struktur des Umwandlungs-produktes sowie eines hochst
substance ligneuse en ulmine par la potasse. Ann. Chim. orientierten naturlichen Cellulosepraparates. Z. Phys.
1819, 12, 172195. Chem. 1940, 187, 297312.
15. Payen, A. Memoire sur la composition du tissu propre des 38. Wellard, H.J. Variation in the lattice spacing of cellulose. J.
plantes et du ligneux. Compt. Rend. 1838, 7, 10521056. Polym. Sci. 1954, 13, 471476.
16. Willstatter, R.; Zechmeister, L. Zur Kenntnis der Hydro- 39. Meyer, K.H.; Mark, H. Uber den Bau des krystallisierten
lyse von cellulose, I. Be 1913, 46, 24012412. Anteils der Cellulose. Ber 1928, 61, 593614.
17. Staudinger, H. Die Chemie der hochmolekularen organ- 40. Gardner, K.H.; Blackwell, J. The structure of native
ischen Stoe im Sinne der Kekule`schen Strukturlehre. BER cellulose. Biopolymers 1974, 13, 19752001.
1926, 59, 30193043. 41. Sarko, A.; Muggli, R. Packing analysis of carbohydrates
18. Irvine, J.C.; Hirst, E.L. The constitution of polysaccha- and polysaccharides: III. Valonia cellulose and cellulose II.
rides: Part VI. The molecular structure of cotton cellulose. Macromolecules 1974, 7, 486494.
J. Chem. Soc. 1923, 123, 518532. 42. Claey, W.; Blackwell, J. Electron diraction of Valonia
19. Freudenberg, K.; Braun, E. Methylcellulose. Mitt. Lignin cellulose. A quantitative interpretation. Biopolymers 1976,
Cellulose Ann. 1928, 5 (460), 288304. 15, 19031915.
20. Charlton, W.; Haworth, W.N.; Peat, S. A revision of the 43. Fischer, D.G.; Mann, J. Crystalline modication of
structural formula of glucose. J. Chem. Soc., 1926; 89101. cellulose: Part VI. Unit cell and molecular symmetry of
21. Haworth, W.N. Revision of the structural formula of cellulose I. J. Polym. Sci. 1960, 62, 189194.
dextrose. Nature 1925, 116, 430. 44. Nieduszynski, I.A.; Preston, R.D. Crystallite size in natural
22. Haworth, W.N. The structure of carbohydrates. Helv. cellulose. Nature 1970, 225, 273274.
Chim. Acta 1928, 11, 534548. 45. VanderHart, D.L.; Atalla, R.H. In Cellulose: Structure,
23. Chu, S.S.C.; Jerey, G.A. The renement of the crystal Modication and Hydrolysis; Young, R.A., Rowell, R.M.,
structures of b-D-glucose and cellobiose. Acta Crystallogr. Eds.; New York: Wiley Interscience, 1986; 88118.
1968, 24, 830838. 46. Belton, P.S.; Tanner, S.F.; Cartier, N.; Chanzy, H. High
24. Perez, S.; Gautier, C.; Imberty, A. Oligosaccharide con- resolution solid state 13C NMR spectroscopy of Tunicin,
formations by diraction method. In Oligosaccharides in an animal cellulose. Macromolecules 1989, 22, 16151617.
Chemistry and Biology; Ernst, B., Hart, G., Sinay, P., Eds.; 47. Sugiyama, J.; Persson, J.; Chanzy, H. Macromolecules.
Wiley/VCH, 2000; 9691001. Combined infrared and electron diraction study of the
25. Philipp, B.; Linow, K.J. Untersuchungen zur Kettenlan- polymorphism of native celluloses. Macromolecules 1991,
gendierenz zwischen Nitrat- und Cuoxam-DP der Cellu- 24, 24612466.
lose and ihrer Anderung im Viskoseprozelb. Papier 1960, 48. VanderHart, D.L.; Atalla, R.H. Further carbon-13 NMR
20, 649657. evidence for the coexistence of two crystalline forms in
26. Mark, R.E. Adhesion in cellulosic and wood-based com- native celluloses. ACS Symp. Ser. 1987, 340, 88118.
posites. In Molecular and Cell Wall Structure of Wood; 49. Atalla, R.H.; Whitmore, R.E.; VanderHart, D.L. A highly
Oliver, J.F., Ed.; Plenum Press: New York, 1981; 751. crystalline a cellulose from Rhizoclonium hieroglyphicum.
27. Marx-Figini, M. Uber die Kinetik der Biosynthese der Biopolymers 1985, 24, 421423.
Cellulose in der Baumwolle. Papier 1964, 18, 546549. 50. Chanzy, H.; Henrissat, B.; Vincendon, M.; Tanner, S.;
28. Hermans, P.H.; Weidinger, A. X-ray studies on the Belton, P.S. Solid-state 13C-NMR and electron microscopy
crystallinity of cellulose. J. Polym. Sci. 1949, 4, 135144. study on the reversible cellulose I to cellulose IIII trans-
29. Fengel, D. Characteristics of cellulose by deconvoluting the formation in Valonia. Carbohydr. Res. 1987, 160, 111.
OH valency range in FTIR spectra. Holzforschung 1992, 51. Hirai, A.; Horii, F.; Kitamaru, R. Transformation of
46, 283288. native cellulose crystals from cellulose Ih to cellulose Ia
30. Howsmon, J.A.; Sisson, W.A. High polymers, structure through solid-state chemical reactions. Macromolecules
and properties of cellulose bers: B. Submicroscopic struc- 1987, 20, 14401442.
ture. In Cellulose and Cellulose Derivatives, Part I; Ott, E., 52. Tanahashi, M.; Goto, T.; Horii, F.; Hirai, A.; Higuchi, T.
Spurlin, H.M., Eds.; Interscience: New York, 1963; Vol. V, Characterization of steam-exploded wood: III. Trans-
231346. formation of cellulose crystals and changes of crystallinity.
31. Isogai, A. Allomorphs of cellulose and other polysaccha- Mokuzai Gakkaishi 1989, 35, 654662.
rides. In Cellulosic Polymers, Blends and Composites; 53. Honjo, G.; Watanabe, M. Examination of cellulose bre
Gilbert, R.D., Ed.; Hanser Publishing: Munich, 1994; 124. by the low-temperature specimen method of electron
32. Atalla, R.H.; VanderHart, D.L. Native cellulose: a com- diraction and electron microscopy. Nature 1958, 181,
posite of two distinct crystalline forms. Science 1984, 223, 326328.
283285. 54. Marrinan, H.J.; Mann, J. Infrared spectra of the crystalline
33. Chanzy, H.; Imada, K.; Vuong, R. Electron diraction modications of cellulose. J. Polym. Sci. 1956, 21, 301311.
from the primary wall of cotton bers. Protoplasma 1978, 55. Mann, J.; Marrinan, H.J. Crystalline modications of
94, 299306. cellulose: Part II. A study with plane-polarized infrared
34. Chanzy, H.; Imada, K.; Mollard, A.; Vuong, R.; Barnoud, radiation. J. Polym. Sci. 1958, 32, 357370.
F. Crystallographic aspects of sub-elementary cellulose 56. Wiley, J.H.; Atalla, R.H. Bands assignments in the Raman
brils occurring in the wall of rose cells cultured in vitro. spectra of celluloses. Carbohydr. Res. 1987, 160, 113129.
Protoplasma 1979, 100, 303316. 57. Imai, T.T.; Sugiyama, J.; Itoh, T.; Horii, F. Almost pure Ia
66 Perez and Mazeau
cellulose in the cell wall of Glaucocystis. J. Struct. Biol. drates and polysaccharides: 6. Molecular and crystal
1999, 127, 248257. structure of regenerated cellulose II. Macromolecules
58. Sugiyama, J.; Vuong, R.; Chanzy, H. Electron diraction 1976, 9, 851857.
study on the two crystalline phases occurring in native 79. Poppleton, B.J.; Mathieson, A. McL, crystal structure of h-
cellulose from an algal cell wall. Macromolecules 1991, 24, D-cellotetraose and its relationship to cellulose. Nature
41684175. 1968, 219, 10461049.
59. Koyama, M.; Helbert, W.; Imai, T.; Sugiyama, J.; Hen- 80. Gessler, K.; Krauss, N.; Steiner, T.; Betzel, C.; Sarko, A.;
rissat, B. Parallel-up structure evidences the molecular Saenger, W. h-D-celloteraose hemihydrate as a structural
directionality during biosynthesis of bacterial cellulose. model for cellulose: II. An X-ray diraction study. J. Am.
Proc. Natl. Acad. Sci. USA 1997, 94, 90919095. Chem. Soc. 1995, 17, 1139711406.
60. Heiner, A.P.; Sugiyama, J.; Teleman, O. Crystalline cel- 81. Raymond, S.; Heyraud, A.; Qui, A.; Kvick, H. Crystal
lulose Ia and Ih studied by molecular dynamics simu- and molecular structure of h-D-cellotetraose hemihydrate
lations. Carbohydr. Res. 1995, 273, 207223. as a model of cellulose II. Macromolecules 1995, 28,
61. Aabloo, A.; French, A.D.; Mikelsaar, R.H.; Perstin, A. 20962100.
Studies of the crystalline native celluloses using potential- 82. Langan, P.; Nishiyama, Y.; Chanzy, H. A revised structure
energy calculations Cellulose 1995, 1, 161168. and hydrogen bonding system in Cellulose II from a
62. Kroon-Batenburg, L.M.J.; Bouma, B.; Kroon, J. Stability neutron ber diraction analysis. J. Am. Chem. Soc. 1999,
of cellulose structures studied by MD simulations. Could 121, 99409946.
mercerized cellulose II be parallel? Macromolecules 1996, 83. Langan, P.; Nishiyama, Y.; Chanzy, H. The X-ray struc-
29, 56955699. ture of mercerized cellulose II at 1 A resolution. Bio-
63. Vietor, R.J.; Mazeau, K.; Lakin, M.; Perez, S. A priori macromolecules 2001, 2, 410416.
crystal structure prediction of native celluloses. Biopoly- 84. Sarko, A. What is the crystalline structure of cellulose?
mers 2000, 54, 342354. Tappi 1978, 61, 5961.
64. Simon, I.; Scheraga, H.A.; Manley, R.St.J. Structure of 85. Sugiyama, J.; Okano, T. Electron microscopic and X-ray
cellulose: 2. Low energy crystalline arrangements. Macro- diraction study of cellulose IIII and cellulose I. Cellulose
molecules 1988, 21, 990998. and Wood: Chemistry and Technology, Proceedings of the
65. Finkendstadt, V.L.; Millane, R.P. Crystal structure of Tenth Cellulose Conference; 1989; 119127.
Valonia cellulose Ih. Macromolecules 1998, 31, 7776 86. Roche, E.; Chanzy, H. Electron microscopy study of the
7783. transformation of cellulose I into cellulose IIII in Valonia.
66. Nishiyama, Y.; Isogai, A.; Isogai, O.; Muller, T.M.; Macromolecules 1981, 3, 201206.
Chanzy, H. Intracrystalline deuteration of native cellulose. 87. Sarko, A.; Southwick, J.; Hayashi, J. Packing analysis of
Macromolecules 1999, 32, 20782081. carbohydrates and polysaccharides: 7. Crystal structure of
67. Nishiyama, Y.; Okano, T.; Langan, P.; Chanzy, H. High cellulose III, and its relationship to other cellulose
resolution neutron bre diraction data on hydrogenated polymorphs. Macromolecules 1976, 9, 857863.
and deuterated cellulose. Int. J. Biol. Macromol. 1999, 26, 88. Buleon, A.; Chanzy, H. Single crystals of cellulose IVII.
279283. Preparation and properties. J. Polym. Sci. 1980, 18, 1209
68. Nishiyama, Y.; Langan, P.; Chanzy, H. Crystal structure 1217.
and hydrogen-bonding system in cellulose Ih from 89. Gardiner, E.S.; Sarko, A. Packing analysis of carbohy-
synchrotron X-ray and neutron ber diraction. J. Am. drates and polysaccharides: 16. The crystal structures of
Chem. Soc. 2002, 124, 90749082. cellulose IVI and IVII. Can. J. Chem. 1985, 63, 173180.
69. Marechal, Y.; Chanzy, H. The hydrogen bond network in 90. Okano, T.; Sarko, A. Mercerization of cellulose: II. Alkali-
Ibeta cellulose as observed by infrared spectrometry. J. cellulose intermediates and a possible mercerization
Mol. Struct. 2000, 523, 183196. mechanism. J. Appl. Polym. Sci. 1985, 30, 325332.
70. Atalla, R.H. Celluloses. In Comprehensive Natural Chem- 91. Hayashi, J.; Yamada, T.; Shimizu, Y.-L. Memory phe-
istry, Carbohydrates and Their Natural Derivatives Includ- nomenon of the original crystal structure in allomorphs of
ing Tanins, Cellulose and Related Lignins; Pinto, B.M., Ed.; Na-cellulose, 77-102. In Cellulose and Wood: Chemistry and
Elsevier: Cambridge, 1999; Vol. 3, 529598. Technology; Schuerch, C., Ed.; Wiley: New York, 1989.
71. Sugiyama, J.; Vuong, R.; Chanzy, H. Electron diraction 92. Okano, T.; Sarko, A. Mercerization of cellulose I. X-ray
study of the two crystalline phases occurring in native diraction evidence for intermediate structures. J. Appl.
cellulose from an algal cell wall. Macromolecules 1991, 24, Polym. Sci. 1984, 29, 41754182.
41684175. 93. Nishimura, H.; Okano, T.; Sarko, A. Mercerization of
72. Wada, M.; Sugiyama, J.; Okano, T. Native cellulose on the cellulose: 5. Crystal and molecular structure of Na-
basis of the two crystalline phase (Ia/Ih) system. J. Appl. cellulose I. Macromolecules 1991a, 24, 759770.
Polym. Sci. 1993, 49, 14911496. 94. Nishimura, H.; Okano, T.; Sarko, A. Mercerization of
73. Andress, K.R. The X-ray diagram of mercerized cellulose. cellulose: 6. Crystal and molecular structure of Na-
Zietschrift Plysikali. Sohe Chem. Abstr., B 1929, 4, 190 cellulose I. Macromolecules 1991b, 24, 771778.
201. 95. Revol, J.F.; Goring, D.A.I. Directionality of the ber c-axis
74. Ahmed, A.U.; Ahmed, U.; Aslam, J.; Butt, N.M.; Khan, of cellulose crystallites in microbrils of Valonia ventricosa.
Q.H.; Atta, M.A Neutron diraction and studies of the unit Polymer 1983, 24, 15471550.
cell of cellulose: II. Polymer letters (edition 14). J. Polym. 96. Dinand, E.; Vignon, M.; Chanzy, H.; Heux, L. Mercer-
Sci. 1976; 561564. ization of primary wall cellulose and its implication for the
75. Kolpak, F.J.; Blackwell, J. Determination of the structure conversion of cellulose I!cellulose II. Cellulose 2002, 9,
of cellulose II. Macromolecules 1976, 9, 273278. 33113314.
76. Nyburg, S.C. X-ray analysis of organic structures. Fibrous 97. Kratky, O.; Mark, H. Zur Frage der individuellen
Macromol. Subst. 1961; 302314. Cellulosemicellen. Z. Phys. Chem., B 1937, 36, 129139.
77. Kuga, S.; Takagi, S.; Brown, R.M.J. Native folded-chain 98. Preston, R.D.; Nicolai, E.; Reed, R.; Mallard, A. Electron-
cellulose II. Polymer. 1993, 34, 32933297. microscopic study of cellulose in the wall of Valonia
78. Stipanovich, A.J.; Sarko, A. Packing analysis of carbohy- ventricosa. Nature 1948, 162, 665667.
99. Frey-Wyssling, A.; Miihlethaler, K.; Wycko, R.W.G. 120. Rowland, S.P.; Howley, P.S. Structure in amorphous
Mikrobrillenbau der panzlichen Zellwande. Experientia regions, accessible segments of brils, of the cotton ber.
1948, 4, 475476. Text. Res. J. 1988, 58, 96101.
100. Revol, J.F. Change of the d spacing in cellulose crystals 121. Verlhac, C.; Dedier, J.; Chanzy, H. Availability of surface
during lattice imaging. J. Mater. Sci. Lett. 1985, 4, 1347 hydroxyl groups in Valonia and bacterial cellulose. J.
1349. Polym. Sci., Part A Polym. Chem. 1990, 28, 11711177.
101. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N. High 122. Tasker, S.; Badyal, J.P.S.; Backson, S.C.E.; Richards,
resolution observations of cellulose microbrils. Mokuzai R.W. Hydroxyl accessibility in celluloses. Polymer. 1994,
Gakkaishi 1985, 30, Sugiyama, J.; Harada, H.; Fujiyoshi, 35, 47174721.
Y.; Uyeda, N. Observations of cellulose microbrils in 123. Baker, A.A.; Helbert, W.; Sugiyama, J.; Miles, M.J. High-
Valonia macrophysa by high resolution electron micro- resolution atomic force microscopy of native Valonia
scopy. Mokuzai Gakkaishi 1985, 31, 6167. cellulose I microcrystals. J. Struct. Biol. 1997, 119, 129138.
102. Marchessault, R.H. Cellulosics as advanced materials. In 124. Baker, A.A.; Helbert, W.; Sugiyama, J.; Miles, M.J. Sur-
Cellulose and Wood: Chemistry and Technology; Schuerch, face structure of native cellulose microcrystals by AFM:
C., Ed.; Wiley: New York, 1989; 12. Part 1. Scanning tunneling microscopy/spectroscopy and
103. Meyer, K.H.; Misch, L. Position des atomes dans le related techniques. Appl. Phys., A Mater. Sci. Process., A
nouveau module spatial de la cellulose. Helv. Chim. Acta 1998, 66 (Supplement), S559S563.
1937, 20, 232244. 125. Baker, A.A.; Helbert, W.; Sungiyama, J.; Miles, M.J. New
104. Colvin, J.R. Oxidation of cellulose microbril segments insight into cellulose structure by atomic force microscopy
by alkaline silver nitrate and its relation to the ne structure shows the Ia crystal phase at near-atomic resolution. Bio-
of cellulose. J. Appl. Polym. Sci. 1964, 8, 27632774. phys. J. 2000, 79, 11391145.
105. Colvin, J.R. Tip-growth of bacterial cellulose microbrils 126. Hanley, S.J.; Giasson, J.; Revol, J.F.; Gray, D.G. Atomic
and its relation to the crystallographic ne structure of force microscopy of cellulose microbrils: comparison with
cellulose. J. Polym. Sci. 1966, 4, 747754. transmission electron microscopy. Polymer. 1992, 33,
106. Kuga, S.; Brown, R.M.J. Silver labelling of the reducing 46394642.
ends of bacterial cellulose. Carbohydr. Res. 1988, 180, 345 127. Kuutti, L.; Peltonen, J.; Pere, J.; Teleman, O. Identication
350. and surface structure of crystalline cellulose studied by
107. Hieta, K.; Kuga, S.; Usuda, M. Electron staining of re- atomic force microscopy. J. Microsc. 1995, 178, 16.
ducing ends evidences a parallel-chain structure in Valonia 128. Newman, R.H.; Hemmingson, J.A. Carbon-13 NMR dis-
cellulose. Biopolymers 1984; 18071810. tinction between categories of molecular order and disorder
108. Maurer, A.; Fengel, D. Parallel orientation of the mo- in cellulose. Cellulose (London) 1995, 2, 95110.
lecular chains in cellulose I and cellulose II deriving from 129. Heux, L.; Dinand, E.; Vignon, M.R. Structural aspects in
higher plants. Holz Roh-Werkst. 1992, 50, 493. ultrathin cellulose microbrils followed by 13C CP-MAS
109. Chanzy, H.; Henrissat, B. Unidirectional degradation of NMR. Carbohydr. Polym. 1999, 40 (2), 115124.
Valonia cellulose microcrystals subjected to cellulose 130. Woodcok, S.; Henrissat, B.; Sugiyama, J. Docking of
action. FEBS Lett. 1985, 184, 285288. Congo red to the surface of crystalline cellulose using
110. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N. Lattice molecular mechanics. Biopolymers 1995, 36, 201210.
imaging from ultrathin sections of cellulose microbrils in 131. Houtman, C.; Atalla, R.J. Celluloselignin interactions: a
the cell wall of Valonia macrophysa. Planta 1985, 166, 161 computational study. Plant Physiol. 1995, 107, 977984.
168. 132. Heiner, A.P.; Teleman, O. Interface between monoclinic
111. Sakurada, L.; Nukushina, Y.; Ito, T. Experimental de- crystalline cellulose and water: breakdown of the odd/even
termination of the elastic modulus of crystalline regions duplicity. Langmuir 1997, 13, 511518.
in oriented polymers. J. Polym. Sci. 1962, 57, 651660. 133. Heiner, A.P.; Kuutti, L.; Teleman, O. Comparison of the
112. Dufresne, A.; Kellerhals, M.B.; Witholt, B. Transcrystal- interface between water and four surfaces of native
lization in Mc1-PHAs/cellulose whiskers composites. crystalline cellulose by molecular dynamics simulations.
Macromolecules 1999, 32, 73967400. Carbohydr. Res. 1998, 306, 205220.
113. Heux, L.; Dinand, E.; Vignon, M.R. Structural aspects in 134. Biermann, O.; Hadicke, E.; Koltzenburg, S.; Muller-
ultrathin cellulose microbrils followed by 13C CP-MAS Plathe, F. Hydrophilicity and lipophilicity of cellulose
NMR. Carbohydr. Polym. 1999, 40, 115124. crystal surfaces. Angew. Chem., Int. Ed. 2001, 40, 3822
114. Liang, C.Y.; Marchessault, R.H. Infrared spectra of 3825.
crystalline polysaccharides: I. Hydrogen bonds in native 135. Mazeau, K.; Vergelati, C. Atomistic modeling of the
celluloses. J. Polym. Sci. 1959, 37, 385395. absorption of benziphenone onto cellulosic surfaces.
115. Tsuboi, M. Infrared spectrum and crystal structure of Langmuir 2001, 18, 19191927.
cellulose. J. Polym. Sci. 1957, 25, 159171. 136. Chanzy, H.; Henrissat, B.; Vuong, R. Gold labelling of 1,4-
116. Rowland, S.P.; Roberts, E.J.; Wade, C.P. Selective h-D-glucan cellobiohydrolase absorbed on cellulose sub-
accessibilities of hydroxyl groups in the microstructure of strates. FEBS Lett. 1984, 172, 193197.
cotton cellulose. Text. Res. J. 1969, 39, 530542. 137. Gilkes, N.R.; Kilburn, D.G.; Miller, R.C.; Warren, R.A.J.;
117. Rowland, S.O.; Roberts, E.J. The nature of accessible Sugiyama, J.; Chanzy, H.; Henrissat, B. Visualization of
surfaces in the microstructure of native cellulose. J. Polym. the adsorption of a bacterial endo-h-1,4-glucanase and its
Sci., Part A 1972, 10, 24472461. isolated cellulose-binding domain to crystalline cellulose.
118. Rowland, S.P.; Roberts, E.J. Disposition of D-glucopyr- Int. J. Biol. Macromol. 1993, 15, 347351.
anosyl units on the surfaces of crystalline elementary brils 138. Lehtio, J.; Sugiyama, J.; Gustavsson, M.; Fransson, L.;
of cotton cellulose. J. Polym. Sci., Polym. Chem. Ed. 1972, Linder, M.; Teeri, T.T. The binding specicity of family 1
10, 867879. and family 3 cellulose binding modules. PNAS 2003, 100,
119. Rowland, S.P.; Howley, P.S. Hydrogen bonding on ac- 484489.
cessible surfaces of cellulose from various sources and 139. Jurasek, L. Experimenting with virtual lignins. ACS Symp.
relationship to order within crystalline regions. J. Polym. Ser. 1998, 697 (Lignin and Lignan Biosynthesis), 276
Sci., Part A Polym. Chem. 1988, 26, 17691778. 293.
68 Perez and Mazeau
140. Faulon, J.L.; Hatcher, P.G. Is there any order in the surfaces of crystalline disaccharides. J. Mol. Graph.
structure of lignin? Energy Fuels 1994, 8, 402407. Modell. 2000, 18, 95107.
141. Radotic, K.; Radotic; Simic-Krstic, J.; Jeremic, M.; 147. Raymond, S.; Henrissat, B.; Tran-Qui, D.; Kvick, A.;
Trifunovic, M. A study of lignin formation at the molecular Chanzy, H. The crystal structure of methyl h-cellotrioside
level by scanning tunneling microscopy. Biophys. J. 1994, monohydrate 0.25 ethanolated and its relationship to
66, 17631767. cellulose II. Carbohydr. Res. 1995, 277, 209229.
142. Lichtenegger, H.; Muller, M.; Paris, O.; Riekel, C.; Fratzl, 148. Rencurosi, A.; Rohrling, J.; Pauli, J.; Potthast, A.; Jager,
P. Imaging of the helical arrangement of cellulose brils C.; Perez, S.; Kosma, P.; Imberty, A. Polymorphism in the
in wood by synchrotron X-ray microdiraction. J. Appl. crystal structure of the cellulose fragment analogue methyl
Crystallogr. 1999, 32, 11271133. 4-O-methyl-h-D-glucopyranosyl-(14)-h-D-glucopyrano-
143. McCann, M.; Weels, B.; Roberts, K. Direct visualization of side. Angew. Chem., Int. Ed. 2002, 22, 42774281.
cross-links in the primary cell walls. J. Cell. Sci. 1990, 96, 149. Ham, J.T.; Williams, D.G. The crystal and molecular
323334. structure of methyl h-cellobioside methanol. Acta Crys-
144. Brown, M.R.; Saxena, I.M. Jr. Cellulose biosynthesis: a tallogr., B 1970, 26, 13731383.
model for understanding the assembly of biopolymers. 150. Nishiyama, Y.; Sugiyama, J.; Chanzy, H.; Langan, P. Crys-
Plant Physiol. Biochem. 2000, 38, 5767. tal structure and hydrogen bonding system in cellulox Ia
145. Chandrasekaran, R. Molecular architecture of polysac- from synchrotron x-ray and neutron ber diraction. J.
charide helices in oriented bers. Adv. Carbohydr. Chem. Am. Chem. Soc. 2003, 125, 1630016306.
Biochem. 1997, 52, 311439. 151. Sassi, J.F. Etude ultrastructurale de lacetylation de la cel-
146. French, A.D.; Kelterer, A.-M.; Johnson, G.P.; Dowd, lulose. Application a` la preparation de nanocomposites,
M.K.; Cramer, C.J. Constructing and evaluating energy The`se de doctorat de lUniversite Joseph Fourier, 1995.
3
Hydrogen Bonds in Cellulose and Cellulose Derivatives
Tetsuo Kondo
Kyushu University, Fukuoka, Japan
I. HYDROGEN BONDS IN CELLULOSE formation of hydrogen bonds in parallel direction to a

glucopyranose ring, and to van der Waals interaction per-
The native biopolymer assembly has been shown to be a pendicular to the ring.
complex process involving two separate but integrated Another important point for the hydroxyl groups is
steps of polymerization and crystallization [1,2]. In the type of hydroxymethyl conformation at the C-6
particular, cellulose has shown to be assembled by a position, because the conformation of C(5)C(6) and the
macromolecular complex of enzymes located on the cell resulting interactions including inter- and intramolecular
surface. Nature has designed an ecient system for regu- hydrogen bonds in the present cellulose structure may
lating the molecular weight, crystallinity, size, and shape of dier from that in crystallites as described in the following
the nanostructure of cellulose (called cellulose micro- section, and it is also assumed to make up the extent of
brils). Then the microbrils are self-assembled to form cell crystallization, as well as the nal morphology of cellulose
walls maintaining tree-frame structure. In this manner, [35]. In the noncrystalline regions, the rotational position
cellulose molecules biosynthesized at angstrom scale as- of hydroxymethyl groups at the C-6 position may be
semble to be microbrils at nanoscale, and the microbrils considered as indeterminate or totally nonoriented,
assemble to be cell walls at micronscale, then they scale up whereas all of those are identical in the crystallites.
with growing (Fig. 1). Hydrogen bonds are no doubt a Therefore it was important to conrm the type of O(6)
major interaction to stabilize this hierarchical architecture rotational position with respect to the O(5) and C(4) in a
of higher plants. Therefore considering hydrogen bonds h-glucan chain, employing CP/MAS 13C NMR [6]. The
of cellulose requires in your mind a picture of the size type of hydroxymethyl conformations, gauchetrans (gt),
(angstrom, nano, or micron) of the subject that you are transgauche (tg), or gauchegauche (gg) at the C-6 posi-
looking at. tions in carbohydrates is shown in Fig. 4. In the gt
First, cellulose is considered as a single molecule conformation, hydroxyl groups (OH) at the C-6 position
(primary or chemical structure): cellulose owns an ex- locate at the opposite side to OH at the neighboring C-2
tended structure with a 21 screw axis composed of the position, and thus they are supposed to form intermolec-
h-1,4 glucosidic linkages between anhydroglucose units. ular hydrogen bonds with the neighbors, whereas tg
Thus it would be natural to accept the dimer called cello- conformation may provide intramolecular hydrogen
biose as a repeating unit. The present three kinds of bonds engaged between OH groups at the C-2 and C-6
hydroxyl groups within an anhydroglucose unit exhibit positions. As for the noncrystalline states, they are con-
dierent polarities, which contribute to the formation of sidered as the gg conformation.
various kinds of inter- and intramolecular hydrogen bonds As described above, the dierence in polarity among
among secondary OH at the C-2, secondary OH at the hydroxyl groups and hydroxymethyl conformation at the
C-3, and primary OH at the C-6 position (Fig. 2). In addi- C-6 position in relation to equatorial bonding of them to an
tion, all the hydroxyl groups are bonded to a glucopyra- anhydroglucose ring is strongly attributed to inter- and
nose ring equatorially. This causes the appearance of intramolecular hydrogen bonds of cellulose. Further, the
hydrophilic site parallel to the ring plane. On the con- hydrogen bonding patterns in cellulose are considered as
trary, the CH groups are bonded to a glocopyranose ring one of the most inuential factors on the physical proper-
axially, resulting in a hydrophobic site perpendicular to ties of cellulose and its derivatives shown in Fig. 2. This
the ring as shown in Fig. 3. These eects lead to the issue will be treated later in this chapter.
69
70 Kondo
Figure 1 Hierarchical system of cellulose self-assembly.
Of course, cellulosic bers and materials are made of

inter- and intramolecular hydrogen bonds, and turning to
the molecular structure of cellulose, intramolecular hydro-
gen bonds are primarily to be employed as a most inuen-
tial interaction to the molecule. Cellulose is supposed to
have at most two dierent intramolecular hydrogen bonds,
which are between the OH-3 and adjacent ring O-5V and
between the OH-6 and OH-2V when the hydroxymethyl
conformation at the C-6 position is tg (Fig. 5) [711]. The
intramolecular hydrogen bonds inuence rst on the mo-
lecular main chain stiness. Is the main chain for each
cellulose single molecule that seems to be sti really sti ?
For this question, extensive studies of solution properties
and liquid crystalline properties for cellulose and cellulose
derivatives have been carried out. At the moment it is
explained that the main chain for each cellulose molecule is
not so sti, and rather the molecule itself is a semiexible
one in which the worm-like chain model by Heine et al. [12]
can be well applied to [1315]. Therefore one single mole-
cule is considered relatively mobile. Only a single molecule,
however, cannot exist in normal states. The molecules are
mutually inuenced and interacted with each other. There
may be two stabilizing ways for each molecule, depending
on the initial step. One is that after a single molecule
interacts with other molecules, then each molecule com-
pensates the potential energy to be stabilized. Another is
that rst the potential energy for each molecule is mini-
mized and after the minimization, it starts interacting with
each other by hydrogen bonding, van der Waals force, and
dipole moment interaction. In any case, every single cellu-
lose molecule interacts with each other. Therefore it Figure 2 A general idea on the correlation of basic
becomes important to characterize the interaction that is characters for each hydroxyl group with physicochemical
engaged between molecules and further the relationship properties.
Hydrogen Bonds in Cellulose and Cellulose Derivatives 71
Figure 3 Hydrophilic and hydrophobic nature in cellulose chemical structure.
between the interaction and physical properties of cellulose the state such as crystals, noncrystalline and amorphous
and the derivatives in order to anticipate both the physical solids, gels, liquid crystals, and solutions.
and chemical characteristics of new cellulosic materials
[1628]. A. Hydrogen Bonds in Cellulose Crystals
Among the interactions in cellulose, the hydrogen
bonding interaction should be most frequently observed. In this section, the object is up-scaled from a single
Fig. 2 shows a general idea on the correlation of basic molecule of cellulose up to a microbril as a molecular
characters for each hydroxyl group with the physicochem- assembled state. As shown in Fig. 6, after the biosynthesis
ical properties. Cellulose exhibits really versatile properties of cellulose molecular chain, each single glucan chain
ranging from transformation of crystalline form to the starts associating with each other to self-assemble into a
regiochemical dierence of the reactivity for the chemical microbril at a nanoscale (3.54.0 nm for wood micro-
derivatization and enzymatic hydrolysis. We have to dis- brils). Biosynthesis is an integrated step to form crystal-
tinguish the situation for the hydrogen bonds depending on line form of cellulose as a microbril. The process mostly
Figure 4 Schematic diagram of the hydroxymethyl conformations at the C-6 position, namely, the orientation of the C6O6
bond, gauchetrans (gt), transgauche (tg), or gauchegauche (gg) with a cellobiose unit.
72 Kondo
backbone conformation, with the two glucose residues

repeating in approximately 1.03 nm. Recently, native cel-
lulose (cellulose I) was found to be a composite of cellulose
Ia and cellulose Ih crystalline forms by Atalla and Vender-
Hart [30]. However, some of the crystalline structures such
as cellulose IV are still in question so far. Therefore we only
focus on conventional crystalline structure, cellulose I and
cellulose II. In particular, for cellulose I, we will employ
cellulose Ih, which is two-chain monoclinic [31] and is close
to the proposed models on the basis of X-ray and electron
Figure 5 Possible intramolecular hydrogen bonding forma- diraction data as well as chain packing energetics [32,33].
tion in tg conformation of hydroxymethyl groups in cellulose. Thus we can easily understand the formation of hydrogen
bonds using the models. On the other hand, because, to
date, we cannot have pure cellulose Ia, it is still dicult to
produces highly crystalline domains, but not necessarily a explain the hydrogen bonding formation for the cellulose
perfect process to secrete a cellulose crystalline ber. Ia crystalline form. Just recently, a revised structure and a
Therefore a cellulose bril contains a certain amount of hydrogen bonding system in cellulose Ih [34] and cellulose
noncrystalline domains as illustrated in Fig. 6. About II [35] have been proposed. In this section, the new insight
cellulose biosynthesis, there are many books from dierent as well as the previous one will be compared historically.
viewpoints (see, for example, Ref. [29]). In this way, Before getting into the details, we will conrm how to take
crystalline cellulose domain is an idealistic assembly of the crystallographic axes (a, b, and c) and a certain angle, g,
cellulose molecules in the biological system. Then hydrox- between the ab axes. Now many people use the recent
yl groups equatorially bonded to the glucose ring become crystallographic rule, namely, c axis is a molecular axis, but
closer enough among neighboring h-glucan chains to form still some researchers take b axis for the molecular chain
intermolecular hydrogen bonding. Therefore it would be axis. In fact, the number of researchers who use the recent
of more importance to understand intermolecular inter- crystallographic rule is increasing gradually. In this chap-
actions in crystalline cellulose. ter, we will follow the recent rule.
Crystalline cellulose has dierent crystalline forms
from cellulose I to cellulose IV, and even in alkaline 1. Hydrogen Bonds in Native Cellulose (Cellulose I)
conditions it shows so-called alkalicellulose crystals, The crystalline nature of cellulose was revealed almost a
which still gives a question as to the manner of existence. century ago when Nishikawa and Ono recorded the rst
In each form, the chains have approximately the same X-ray diraction patterns from ber bundles originated
Figure 6 Crystalline and noncrystalline regions of cellulose microbrils.

from various plants [36]. Since then, in addition to X-ray, 2: : : O6V, to give an even more rigid four-ring layer cong-
many powerful tools have appeared for investigating cel- uration. In these structures there is an intermolecular
lulose crystalline structures such as electron diraction and hydrogen bond, OH-6: : :O3 (Fig. 7C), linking the layers
microscope, FT-IR, Raman spectroscopy, and 13C CP/ laterally, but no bonding between layers. In other words,
MAS NMR. The development of high-resolution 13C there is no intermolecular hydrogen bonding along the
solid-state NMR techniques in the 1980s has brought a (110) and (110) planes, but there is only one along the (200)
new dimension to determining the crystal structure of plane as shown in Fig. 7A. Investigating hydrogen bonds in
cellulose. In fact, 13C CP/MAS NMR of highly crystalline cellulose using IR was rst performed by Marrinan and
cellulose samples such as Valonia showed the presence of Mann [44,49], and then Liang and Marchessault [46,47]
two crystalline allomorphs (cellulose Ia and Ih) in cellulose proceeded to assign the whole area of OH stretching
I. However, FT-IR is still one of the best tools to study frequencies in IR spectra for celluloses I and II. They used
hydrogen bonding formation with consideration of the polarized IR measurements for oriented lms having cel-
two-chain unit cell models (21 axis) [37]. In particular, lulose I or cellulose II crystalline structures and assigned
not only FT-IR but also advanced FT-IR technique in some typical maxima for the OH regions of the IR spectra
combination with suitable attachments could provide us on the basis of the dierence between the parallel and the
with further information on cellulose supermolecular perpendicular bands. Now let us take a look at Table 1
structure. Many reports using IR analyses have appeared [46,48,5052] and you would nd that two intramolecular
to date. Fengel analyzed the hydroxyl absorption bands by hydrogen bonds and an intermolecular hydrogen bond
deconvoluted FT-IR spectra of celluloses [3840]. Michell have already been assigned. Some experimental assign-
used the second-derivative mode in order to improve the ments correspond to the calculated wavenumbers [48].
FT-IR resolution [4143]. The assignments for the hydrox- In the 1980s, the crystalline dimorphism of native
yl frequencies had been established from the late 1950s to celluloses was found [30,53], and the two phases, cellulose
the early 1960s on the basis of the modied MeyerMisch Ia and Ih, have been considered to dier in their hydrogen
model [4447]. In addition, the OH stretching frequencies bonding rather than in the conformation on the basis of
due to intra- and intermolecular hydrogen bonds in cellu- Raman [51] and FT-IR [31,43] spectral data. According to
lose I were calculated [48]. Table 1 lists these reported Sugiyama et al. [52], a characteristic hydroxyl absorption
band-assignments for native cellulose. To make an easy band due to Ia crystalline phase is 3240 cm 1, whereas a
understanding of the hydrogen-bonding network, we will band at 3270 cm 1 is due to Ih crystalline phase. There are
use the ab and bc projection of the unit cell for cellulose I a number of literatures reporting on the IR data of native
(21 screw) originally proposed by Gardner and Blackwell cellulose [4046,49,52,54]. Most of these studies give more
[32] as shown in Fig. 7. This permits the formation of two or less complete lists of IR band assignments on the
intramolecular hydrogen bonds, OH-3: : :O5V and OH- hydrogen bonding system. Most of these earlier IR studies,
Table 1 IR Assignments for OH Regions Reported in Native Cellulose
Interpretation
(Liang et al. 1959:L) Calculated
Frequency (Ivanova et al. 1989:I) Interpretation by Raman wavenumbers
(cm 1) (Sugiyama et al. 1991:S) (Wiley et al. 1987) (Tashiro et al. 1991)
32303310 I: O(6)HO(3)
Inter H-bond
3231 Cellulose Ia (?)
appeared in Valonia
3240 S: Cellulose Ia
3270 S: Cellulose Ih
3305 L: OH Inter H-bond
3309 OH Inter H-bond
33403375 L&I: O(3)HO(5)
intra H-bond
3372 OH Intra H-bond
interO(3)HO(5)
3405 L: OH Inter H-bond
34103460 I: O(2)O(6)
Intra H-bond
O(2)O(6)inter
3429 Cellulose Ih (?)
appeared in Ramie
74 Kondo
Figure 7 Proposed structure of cellulose I before the discovery of native cellulose allomorphs, Ia and Ih, in 1983 by Atalla and
VanderHart [29]. (A) ab projection (looking along the chain axis); (B) bc projection; (C) hydrogen-bonding network in the sheet
parallel to the bc plane. This gure is modied on the basis of the Gardner and Blackwell model [31].
however, were made before the discovery of the two crys- requires data of pure Ia and Ih bers. Recently, the revised
talline phase system of cellulose. Thus a number of the data have been proposed using synchrotron and neutron
earlier band assignments should be reevaluated in light of diraction for oriented brous samples from tunicate
the two-phase system. Recently, Marechal and Chanzy [55] cellulose microcrystals [34]. The study proposed not only
have reported the revised assignments of the IR bands in the revised Ih crystalline structure, but also the tg confor-
cellulose Ih from hydrothermally treated Valonia micro- mation of hydroxymethyl groups and the hydrogen bond-
brils. Their assignments are illustrated in Fig. 8. Accord- ing fashion. The hydrogen bonding scheme is represented
ing to them, hydroxymethyl moieties were found adopting schematically in Fig. 9: There is no hint of intersheet OH
three conformations (a dominant one and two minor ones) O hydrogen bonds in cellulose Ih, indicating that the
allowing the formation of dierent hydrogen bonding on cellulose sheets are held only by hydrophobic interactions
adjacent chains. Most probably, the primary hydroxyl and weak CHO bonds. Within the sheet, the intramo-
groups (OH) that accept a hydrogen bond from the adja- lecular O3O5 hydrogen bonds were well dened, whereas
cent OH at the C-2 position were not the ones adopting the those corresponding O2O6 hydrogen bonds were indi-
dominant conformation. However, there are still some cated in a variety of the fashion.
questions remaining as OH bands due to dierent modes
overlap with each other, causing diculties in interpreta-
tion even in crystalline structures. 2. Hydrogen Bonds in Cellulose II
The crystal and molecular structure of cellulose I need Native cellulose can be easily transformed into cellulose II
to be revised also in light of this dimorphism. This revision after an alkaline treatment at more than 18 wt.% and
Figure 8 Assignment of IR bands of cellulose Ih, with stretching bands drawn as two-headed arrows and bending bands of
alcoholic groups as single-headed arrows. Assignments of bending bands to a particular alcohol group are tentative and may be
the object of permutations between the three types of alcohols. Three conformations of hydroxymethyl groups at the C-6 position
are displayed labeled I, II, and III. Conformations of II and III are deduced from conformation I by rotation of them around the
C5C6 bond. Conformation I represents at least 2/3 of the total conformations, conformation II (tg) less than 10%. Hydrogen
bonds are supposed to be established by these primary alcohols on O atoms of other chains. For commodity hydroxyl groups at
the C-2 position establishing weak hydrogen bonds is drawn with free OH groups [55].
subsequent washing thoroughly with distilled water. For hydrogen bond OH-2: : :OH-2V to the next chain along the
cellulose II crystalline structure, some models have been long ab diagonal. This bond is shown in Fig. 10E and is
proposed and they have dened the crystals as consisting of indicated by the dashed lines in Fig. 10A. This extra
two antiparallel and crystallographically independent intermolecular bonding is a major dierence between
chains. The proposed structure has a monoclinic cell where cellulose II and native cellulose and probably goes a long
the chains are aligned on the 21 screw axes. Both chains way to explain the higher stability of the regenerated form
have equivalent backbone conformation but dier in the [58]. Table 2 lists the experimental IR assignments [47] and
conformation of hydroxymethyl groups proposed by two
groups, Kolpak and Blackwell [56] and Stipanovic and
Sarko [57]. Their antiparallel model for cellulose II is
shown in Fig. 10. The ab projection shows that the chains
have approximately the same orientation about their axes
and are stacked along the short ab diagonal (Fig. 10B).
These stacks will be stabilized by hydrophobic (van der
Waals) forces, more so than between the sheet in native
cellulose. The relative stagger of the chains is 0.216c, again
close to the quarter-stagger position. The renement has
led to dierent conformations of the CH2OH groups on
the center and corner chains. Adjacent center chains along
the a axis are shown in Fig. 10C. The CH2OH groups are
oriented like those in native cellulose, so as to allow the
formation of a second intramolecular hydrogen bond OH-
2V: : :OH-6 and an intermolecular hydrogen bond OH-
6: : : OH-3 along the a axis. The sheet of corner chains is Figure 9 Schematic representation of the hydrogen bonds
shown in Fig. 10D. Here the CH2OH group is swung in the origin (top) and center (bottom) sheets of cellulose Ih.
round so that it forms an OH-6: : :OH-2 intermolecular Hydrogen bonds are represented by dotted lines. Only the
hydrogen bond. Intramolecular bonding for OH-2 is now oxygen atoms involved in hydrogen bonding have been
not possible, and this group forms another intermolecular labeled for clarity. (From Ref. 34.)
76 Kondo
Figure 10 Structure of cellulose II proposed by Kolpak and Blackwell [56]. (A) ac projection; (B) ab projection (- - - hydrogen
bonds between center and corner chains); (C) hydrogen-bonding of the center chains; (D) hydrogen-bonding of the corner chains;
(E) hydrogen-bonding between corner and center chains. This gure is modied on the basis of their model.
Table 2 IR Assignments for OH Regions Reported in B. Hydrogen Bonds in Regioselectively

Cellulose II Substituted Cellulose Derivatives in Solid-State
Noncrystalline Films
Frequency Calculated
(cm 1) wavenumbers 1. Characterization Using FT-IR and Solid-State
(Tashiro (Marchessault CP/MAS 13C NMR Spectra
et al. 1991) Interpretation et al. 1960)
The formation of hydrogen bonds in cellulosics is consid-
3175 OH stretching ered as one of the most inuential factors on the physical
(Corner chain)
3350 ?
3435 (center chain)
3447 OH stretching
(corner chain)
calculated wavenumbers [48] for cellulose II. The model for

the crystalline structure indicated the formation of intra-
and intermolecular hydrogen bonds for cellulose II. In this
model, hydroxymethyl moieties are near gt conformation
for the glycosyl residues located at the origin of the cell as
opposed to tg conformation for those at the center chain.
The model of cellulose II has been further investigated
using h-cellotetraose hemihydrate and methyl h-cellotrio-
side monohydrate 0.25 ethanolate [5962]. Their molecular
conguration was also similar to that of the cellulose II
model except in two main respects: all hydroxymethyl
groups are in gt conformation and the sugar pucker was
dierent for the two chains. In addition, the proposed
hydrogen bonding schemes using oligosaccharides were
signicantly dierent from the previous ones.
Recently, Langan et al. have reexamined the structure
of cellulose II using a neutron ber diraction analysis [35].
In crystalline bers of cellulose II, a 3-D network of
hydrogen bonds exists. This new model as shown in
Fig. 11 indicates a new substantially dierent hydrogen
bonding network from previous proposals. In Fig. 11,
intermolecular hydrogen bonds are O2-D-O6 in sheets
containing only origin molecules and O6-D-O2 in sheets
containing only center molecules. In the sheets containing
both center and origin molecules there are O6-D-O6 and
O2-D-O2 intermolecular hydrogen bonds. The former has
minor components involving O5 and O3 as acceptors.
Intramolecular hydrogen bonds are O3-D-O5 in each
molecule with a minor component involving O6 as accep-
tor. However, you would notice that there are still dicul-
ties in the interpretation of reactivity, properties of
cellulose in various forms, and transformation process of
cellulose from one to the other as well as even crystalline Figure 11 A schematic representation of hydrogen bonds
structures, as OH groups with dierent modes overlap with on cellulose II proposed by Langan et al. [35]. Only atoms
each other. involved in hydrogen bonds are represented by dotted lines.
78 Kondo
cellulose can be useful in controlling the formation of

hydrogen bonds without causing a resultant change in
the glucose ring conformation. Thus the regioselectively
methylated celluloses have been proven to be satisfactory
model compounds for cellulose.
In continuation, the formation of hydrogen bonds in
the regioselectively substituted cellulose derivatives was
characterized by FT-IR and solid-state CP/MAS 13C
NMR spectra of the lm samples [67]. Fig. 13 shows OH
frequencies for lm samples of typical regioselectively
substituted cellulose ethers, namely, 2,3-di-O- and 6-O-
methylcellulose (23MC and 6MC), compared with that of
pure cellulose. They were all predominantly noncrystalline
lms with ca. 5-Am thickness prepared by casting from their
dimethyl acetoamide (DMAc) solutions for methylated
derivatives and the DMAc-LiCl cellulose solution for pure
cellulose, respectively. Therefore we do not have to take
into account specically oriented intermolecular hydrogen
bonds, dierently from the crystalline samples. Thus we
Figure 12 The chemical structures of regioselectively sub- have only to consider the following hydrogen bonds for the
stituted cellulose derivatives prepared in Ref. [67]. above amorphous lm samples: isotropic intermolecular
hydrogen bonds and two dierent intramolecular hydro-
gen bonds, which are between the OH-3 and adjacent ring
O-5V and between the OH-6 and OH-2V (Fig. 14A). Look-
properties of not only cellulose itself but also its derivatives. ing at the spectra in Fig. 13 under these assumptions, we
The previous section explained the possible formation of will nd that they exhibited characteristic band shapes in
hydrogen bonds in native cellulose and cellulose II crystal- the OH stretching vibration regions. The shape of the 6-O-
line structures. As for cellulose derivatives, they show either methylcellulose (6MC) having two OH groups in a unit is
crystalline structure or noncrystalline structure, depending rather symmetric and sharp, compared with that of 2,3-di-
on the degree of substitution (DS) and the structural O-methylcellulose (23MC), which has one OH group per
regularity or the distribution pattern of substituted units anhydroglucose unit. Generally, increasing the number of
along the molecular chain. For cellulose derivatives we can
use NMR spectroscopy to observe hydrogen-bonding
engagements [63]. In this case, we can mostly use solution
NMR as solid-state NMR still has a problem on the
resolution for signal splitting. However, most of the inves-
tigations on hydrogen bonding formation in cellulose as a
polymer have diculties in interpreting the chemical shift
for the same reason as those studies using IR spectroscopy.
Therefore we had better simplify the formation of hydro-
gen bonds to be more easily analyzed if possible. How can
we do this? Kondo and Gray developed the methods for
synthesizing regioselectively substituted cellulose ethers,
2,3-di-O-, 6-O-, and tri-O-substituted cellulose derivatives
[6467] as shown in Fig. 12. As the hydroxyl groups should
form controlled intra- and intermolecular hydrogen bonds
particularly in regioselectively methylated celluloses, the
cellulose derivatives are thought to be cellulose model
compounds to investigate the relationships between for-
mation of hydrogen bonds and physical properties of
cellulose, as well as the cellulose derivatives. When the
OH groups within the anhydroglucose units are blocked by
methyl groups, it remains a question whether the pyrano-
glucose ring conformation still keeps the original shape
(4C1 chair conformation) or not. To answer this question, it
has already been reported [25] that the OH groups in the
anhydroglucose units, despite being blocked by methyl Figure 13 IR spectra of noncrystalline lm samples of (A)
groups, do not aect the structure of the glucose ring. In cellulose, (B) 2,3-di-O-, and (C) 6-O-methylcellulose deriva-
other words, blocking hydroxyls by methyl groups in tives in the region of OH stretching vibration.
biological structures. When the donor group is cation-like

or the acceptor group is anion-like, as in OH+O or O
HO , strong and almost symmetrical hydrogen bonds
are also observed [1]. Because in 2,3-di-O-substituted cellu-
lose free OH groups at the C-6 position are comparatively
exible, intermolecular hydrogen bonds may be formed
favorably. The OH groups may also form intramolecular
hydrogen bonds with the ether oxygen at the adjacent C-2
position (Fig. 14B). Thus, mixture of the inter- and the
intramolecular hydrogen bonds is considered to cause
the broadening of the OH band in the IR spectra. On the
other hand, two intramolecular hydrogen bonds may
form in the 6-O-substituted cellulose derivatives as shown
in Fig. 14C. The two intramolecular hydrogen bonds (O6-
HO-2V and 3-OH-O5V) have similar type of formation,
which is between the OH group and the ether or acetal
oxygen. Therefore the sharp and symmetric IR spectra of 6-
O-substituted cellulose derivatives in Fig. 15 indicate that
the two intramolecular hydrogen bonding structures may
give a similar OH absorption band, and intermolecular
hydrogen bonding formation which broadens the OH band
may be not signicant. Furthermore, the strengths of the
two hydrogen bonds may be almost equivalent, and hence
both of the intramolecular hydrogen bonds between the
OH and the ether oxygen appear at almost the same wave
number around 3465 cm 1 in OH stretching frequencies of
the IR bands.
In another experiment [68], the curve tting procedure
Figure 14 Schematic representation of possible hydrogen was performed for the regions due to OH stretching
bonds in cellobiose units of (A) cellulose, (B) 2,3-di-O-, and vibration in the IR spectra of the noncrystalline lms from
(C) 6-O-substituted cellulose derivatives. 2,3-di-O-methylcellulose (2,3-di-O-MC) and 6-O-methyl-
cellulose (6-O-MC). The results are shown in Fig. 16. The
OH bands for 2,3-di-O-MC are resolved into two Lorent-
OH groups per unit should show the diversity of OH fre- zian bands, sharper (3472 cm 1) and broad (3382 cm 1)
quencies in IR spectra, and then the absorption band ones; 6MC has one major Lorentzian OH absorption band
should become broader. In the present case, however, (3460 cm 1) and a small subband (3270 cm 1). These bands
the appearance of OH band of 6MC, which has two OH may correspond to the presence of specic inter- and
groups per unit, is contradictory. This phenomenon intramolecular hydrogen bonds involved in 2,3-di-O-MC
appeared in other relationships between 6-O- and 2,3-di- and 6-O-MC as mentioned above. Thus the bands with
O-substituted cellulose derivatives with the same substitu- peak positions at higher wavenumbers of 34603470 cm 1
ent [67]. It is assumed that the phenomenon is attributed for both 2,3-di-O-MC and 6-O-MC result in the assign-
mainly to the manner of formation of hydrogen bonds. ments of the following intramolecular hydrogen bonds,
Schematic representations of possible inter- and intra- because in general for cellulosic materials the intramolec-
molecular hydrogen bonds in the cellobiose unit are shown ular hydrogen bonds tend to appear at relatively higher
in Fig. 14. Cellulose may possibly have two types of wave numbers (34103460 cm 1 in cellulose I [50] and
intramolecular hydrogen bonds and some intermolecular 34603480 cm 1 in cellulose II [47,68,69]) in the IR spectra.
hydrogen bonds, depending on the phase. While the inter- There may be between OCH3 at the C-2 position and OH at
molecular hydrogen bonds are not specied, the two the C-6 position for 2,3-di-O-MC. There may be between
intramolecular hydrogen bonds are assumed to be either OH at the C-3 position and the adjacent ring oxygen and
OH-3-OV5 or OH-6-OH-2V (Fig. 14A). In the 2,3-di-O- between OH at the C-2 position and OCH3 at the C-6
substituted cellulose derivatives, the main reason for the position for 6-O-MC. It has been conrmed that such
broader OH bands due to the diversity of OH frequencies intramolecular hydrogen bonds can be formed in either
appears to be the formation of intermolecular hydrogen solid lm or homogeneous solution states [6972]. The
bonds associated with OH-6. In the case of 6-O-substituted formation of such intramolecular hydrogen bonds has also
cellulose, specic intramolecular hydrogen bonding for- been observed in a DMSO solution state of 23MC using
mation, which makes the IR band of the OH region NMR analyses with deuteration [73].
symmetric and sharper, may exist. Hydroxyl and ring ether To investigate the intramolecular hydrogen bonds,
(C-O-C) groups correspond to the hydrogen bonding the lm samples were also analyzed by CP/MAS 13C
donor and acceptor, respectively, which are common in NMR [67]. Fig. 17 shows the CP/MAS spectra of (1)
80 Kondo
2,3-di-O-MC, (2) 6-O-MC, and (3) Tri-O-MC. The intro-

duction of an O-alkyl group promotes strong deshielding
of the 13C nucleus of the substituted carbinol group,
usually by ca. 9 ppm in solution NMR [74,75]. In the
spectra of cellulose ethers, this characteristic should be
reected in the chemical shifts of carbons at C-2, C-3, and
C-6 positions bearing alkoxy substituents. Thus peak
assignment was carried out in the CP/MAS 13C NMR
spectra of the lm samples using the solution-state 13C
NMR results of Parfondry and Perlin [76]. Unsubstituted
C-2, C-3, and C-5, and substituted C-6 carbon signals in
6-O-MC overlapped to some extent with each other in the
range of 7770 ppm. Signals of the C-2, C-3, and C-6
carbons shifted to downeld (ca. 10 ppm) by methyl
substitution. In the 2,3-di-O-MC and Tri-O-MC, the C-4
carbon signals, which are assignable in cellulose, shifted
upeld by substitution of the adjacent OH groups at the
C-3 position and overlapped with the C-5 carbon signal.
Signals of C-2 and C-3 carbons in the two MCs overlapped
with each other and cannot be identied because of their
similar and strong deshielding of 13C nuclei of completely
substituted methoxy groups at both positions.
Considering a weak deshielding eect of the intramo-
lecular hydrogen bonds between the methoxy oxygen at the
C-2 position and the OH groups at the adjacent C-6
position as described later, the C-2 carbon may resonate
Figure 15 IR spectra of lm samples of cellulose, 2,3-di-O-

and 6-O-substituted cellulose derivatives in the region of OH
stretching vibration. MC: O-methylcellulose; EC: O-ethyl- Figure 16 Curve tting for OH stretching regions in 2,3-di-
cellulose; AC: O-allylcellulose; and BC: O-benzylcellulose. O-MC and 6-O-MC.
13
Figure 17 CP/MAS C NMR spectra of regioselectively substituted O-methylcelluloses (MCs).
upeld to the C-3 carbon. Only the C-1 signal is easily bonding formation eects cannot be simply applied to the
assignable as it is completely separated from other signals. C-1 signals of the tri-O-substituted derivatives. Because the
The presence of an ether substituent at the C-2 position substitution of both C-2 and the adjacent C-6 hydroxyls
causes an upeld shift of the C-1 resonance relative to that can cause a mutual repulsion between the two substituents,
of the glucose residue [76]. Therefore it is assumed that the the interaction occurring among them can be of dierent
C-1 resonances may have an upeld shift similarly by the types such as hydrophobic linkage and hence the confor-
hydrogen bonding formation at the adjacent C-2 position. mation of the main chain should change.
Naturally, the change in the hydrogen bonding arrange- As discussed in the previous section using FT-IR, CP/
ment should be more weakly attributed to the chemical MAS 13C NMR may suggest the type of the hydroxymethyl
shift of C-1 carbon than the eect due to the substitution of conformations, gt or tg at the C-6 positions in carbohy-
the OH at the C-2 position. Kamide et al. [70] reported that drates. Horii et al. indicated [6] that the C-6 carbon reso-
the chemical shift of the C-1 carbon may appear to have an nance occurs only as a singlet near 64 ppm in the case of the
upeld value of more than 105 ppm in the case of the gt conformation, whereas a resonance band near 66 ppm
intramolecular hydrogen bond between the C-2 and C-6 will appear when the tg conformation is present within
positions (Fig. 14A and C). They also mentioned that the the crystalline structures, as shown in Fig. 18 [6]. According
C-1 chemical shift may have a eld value lower than 105 to them, the chemical shifts fall into three groups of 60
ppm in the case of the free OH at the C-2 position that did 62.6, 62.564.5, and 65.566.5 ppm, which are related to
not include the hydrogen bond between the C-2 and C-6 gg, gt, and tg conformations, respectively. In fact, the
positions. The latter case was based on the assumption that chemical shift of the C-6 for cellulose II [7780] indicated
a seven-membered kjelectron conjugate system is formed the gt conformation, which agrees with the recent result
in a cellobiose unit [C-4-O-C-1V-O5V-HO-3-C-3-C-4: con- from the neutron ber diraction analysis as described
sider the case without the intramolecular hydrogen bond above [35]. In the regioselectively methylated cellulose
between C-6 and C-2V in Fig. 14(A)]. In Table 3, the C-1 ethers shown in Fig. 17 and other experiments, the chem-
chemical shifts of 6-O-substituted cellulose derivatives ical shifts of the C-6 were 61.58 and 61.66 ppm for 2,3-di-O-
except 6-O-tritylcellulose show smaller values than 105 MC and 3-O-MC [81], respectively. These results show that
ppm, suggesting the existence of intramolecular hydrogen the conformation of the OH groups at the C-6 position for
bonds between the C-2 and C-6 position. From this result the two regioselectively methylated cellulose derivatives
and FT-IR analyses, it is indicated that in the 6-O-substi-
tuted cellulose derivatives except 6-O-tritylcellulose, two
intramolecular hydrogen bonds (O6-HO-2V and 3-OH- Table 3 Comparison of Chemical Shifts at the C-1 Positions
O5V) form predominantly and the strengths of the two of Anhydroglucose Unit in CP/MAS 13C NMR Spectra of
bonds may be almost equivalent (Fig. 14C). Various Cellulose Derivatives
The C-1 chemical shifts of 2,3-di-O-substituted cellu-
lose derivatives in Table 3 also show smaller values than Sample 6-O- 2,3,di-O- Tri-O-
105 ppm. This indicates the formation of the intramolec-
ular hydrogen bonds between O2-HO-6V (Fig. 14B) in Methyl cellulose 104.1 103.9 106.5
addition to intermolecular hydrogen bonds at the C-6 Ethyl cellulose 103.8 103.5 102.2
Propyl cellulose 102.8 104.5 102.6
position. As for tri-O-substituted cellulose ethers, the C-1
Decyl cellulose 104.2 104.4 LCa
signals appeared more upeld than those of 2,3-di-O- and
Allyl cellulose 103.1 103.9 102.5
6-O-substituted cellulose derivatives. Only tri-O-methyl- Benzyl cellulose 103.2 102.4 102.0
cellulose shifted downeld compared with other tri-O-sub- Trityl cellulose 105.5
stituted cellulose derivatives. To explain these phenomena,
a
the above explanation of chemical shifts with the hydrogen LC: Liquid crystal at room temperature.
82 Kondo
number, two intramolecular hydrogen bonds expected to

be formed in 6-O-MC were maintained in 6-O-alkylcellu-
loses. Only in 6-O-decylcellulose there appeared a small
shoulder around 3600 cm 1. In the case of electron-with-
drawing substituents such as allyl and benzyl groups that
are easy to form cations (Fig. 15C and D), the hydroxyl
frequencies also appeared sharp and symmetric, although
there was a slight shoulder at around 3580 cm 1. However,
a remarkable shoulder of hydroxyl frequencies appeared at
around 3580 cm 1 in both an electron-withdrawing and
bulky trityl substituent as shown in Fig. 20. In general,
hydroxyl frequencies at 35843650 cm 1 are considered as
absorption band of free OH groups [54,71]. Hydroxyl
frequencies due to the intermolecular hydrogen bonds were
reported as 3305, 3350, and 3405 cm 1 by Marchessault
and Liang [46,47]. The shoulder at 3580 cm 1 in the
hydroxyl absorption band of the tritylcellulose was found
to be due to rather free hydroxyl groups. The OH bands
should be broad because of the diversity. Thus an intra-
molecular hydrogen bond and free OH appear to be
formed at the C-2 position of the tritylcellulose.
The 6-O-tritylcellulose was then multiple-methylated
to investigate the behavior of OH groups at the C-2 and C-
3 positions. The change of distribution of the methyl group
as a block of OH groups in a step is monitored in Table 4.
Figure 18 13C chemical shifts of the CH2OH carbon vs. tor- As OH groups at the C-2 position were rapidly methylated
sion angles v around the exo-cyclic CC bonds. a: a-D-glu- in the rst methylation, the DS of methyl groups at the C-3
cose; b: a-D-glucose H2O; c: h-D-glucose; d: h-D-cellobiose; position increased slowly in the step. This dierence
e: a-D-lactose H2O; f: h-lactose; g: sucrose; h: a-melibiose indicates a quick break of hydrogen bond and methylation
H2O; i: h-methyl cellobioside CH3OH.
may be gg. However, the indication due to the chemical

shifts [6] was derived from mono- and oligosaccharides,
which have dierent hydrogen bonding engagements from
the present samples. As mentioned already, even hydrogen
bonds for celluloses I and II may be totally dierent from
those of 2,3-di-O-MC and 3-O-MC, judging from the OH
stretching frequencies in the IR spectra. As the relatively
large scattering of data within 2 ppm depending on the
conformation at the C-6 may be due to other additional
eects such as packing [82] and hydrogen bonding [83,84],
the chemical shifts of the C-6 for the regioselectively
methylated cellulose derivatives, which are expected to
have controlled and specic hydrogen bonds, may not
agree with the indication. In addition, the conformation
of the glucopyranose ring may be somehow changed by
the regioselective substitution by methyl groups. Further
studies will be required.
2. Inuence of Substituent on the Hydrogen Bonding

Formation
Hydroxyl frequencies in the IR spectra of 6-O-alkylcellu-
loses with dierent lengths of alkyl chains are shown in
Fig. 19. The sharp and symmetric shape of the IR spectrum Figure 19 IR spectra of 6-O-alkylcellulose lms in the
for 6-O-MC did not show a signicant change with in- region of OH stretching frequencies: (a) 6-O-methylcellulose;
creasing of alkyl chain length. It suggests that irrespective (b) 6-O-ethylcellulose; (c) 6-O-propylcellulose; and (d) 6-O-
of the alkyl chain length with the range of 1 to 10 in carbon decylcellulose.
Table 4 Degree of Substitution at Individual 2,3-di-O-methyl-6-O-tritylcellulose prepared in another

Positions in 6-O-trityl- and Methylated Tritylcellu- way [64]. This indicates that free OH groups coming
lose Derivatives from the hydroxyl group at the C-2 position and scission
of intramolecular hydrogen bonds at the C-3 position were
Sample X2 X3 X6 methylated.
6-O-TC 0 0 Trityl CP/MAS 13C NMR spectra of the above samples also
MTC1 0.74 0.55 Trityl showed the same behavior of the hydroxyl groups through
MTC2 0.82 0.66 Trityl methylation (Fig. 21). Broad C-1 signal at 105.5 ppm in the
MTC3 0.84 0.73 Trityl tritylcellulose shifted to upeld peak that has three peak
tops at 105.5, 102.5, and 101.4 ppm in MTC1 (Fig. 21 (2)).
Xn: DS on the OH of Cn (n=2, 3, and 6). Each peak top at the three values is due to free OH, the
intramolecular hydrogen bond, and methylation of hy-
droxyl groups at the C-2 position, respectively. The broad
of the free hydroxyl groups at the C-2 position, and a slow C-1 signal of the tritylcellulose also has a shoulder at
scission of the intramolecular hydrogen bond between C-3 around 102.5 ppm due to the intramolecular hydrogen
and O5 through three methylation steps. Namely, the rst bond. Judging from the C-1 peak shape, the region around
apparent change in the IR spectrum of methylated trityl- 105.5 ppm is main and the vicinity at 102.5 ppm is relatively
cellulose is attributed to the behavior of the hydroxyl small. This indicates that the free OH is the one, rather than
group at the C-2 position. In the traces of Fig. 20, as the the OH engaged in the intramolecular hydrogen bonds at
methylation step preceded, the intensity of the absorption the C-2 position.
band at 3484 cm 1 decreased gradually and the shoulder When the methylation proceeded, the peak top at
at 3580 cm 1 got smaller and sharper. In Fig. 20, the 105.5 ppm decreased as the other two peak tops appeared
absorbance of the OH bands for the MTCs was normal- distinguishably. The peak top at 102.5 ppm decreased and
ized on the basis of the internal standard band at 1596 eventually the C-1 signal became one peak at 101.4 ppm
cm 1 due to the trityl group which was not aected by the in 2,3-di-O-methyl-6-O-tritylcellulose whose OH groups
methylation, and the peak heights between 6-O-TC (tri-
tylcellulose) and MTC1-3 were not comparable with each
other. Eventually, OH frequencies mostly disappeared and
all hydroxyl groups were almost completely methylated in
Figure 21 Change of the C-1 chemical shift of tritylcellulose

Figure 20 Change of OH stretching frequencies in IR and methylated tritylcellulose derivatives: (1) 6-O-tritylcellu-
spectra of tritylcellulose through the multiple methylation lose; (2) MTC1*; (3) MTC2*; (4) MTC3*; and (5) 2,3-di-O-
step. 6-O-TC: tritylcellulose; MTC1-3: see Table 4. methyl-6-O-tritylcellulose. *See Table 4.
84 Kondo
were almost completely blocked by methyl. This phenom-

enon also indicates that free OH at the C-2 position
is rapidly methylated and then scission of the intramolec-
ular hydrogen bonds at the C-2 position occurred and
nally all hydroxyl groups at the C-2 position were com-
pletely methylated.
3. Assignment of Free Hydroxyl Groups

in Cellulose
As described in the above section, Kondo et al. have ex-
tensively characterized intra- and intermolecular hydrogen
bonds involved in cellulose derivatives using regioselec-
tively methylated cellulose derivatives as model com-
pounds by FT-IR and CP/MAS 13C NMR analyses. It
mentioned that not only the assignments due to inter- and
intramolecular hydrogen bonds but also the IR interpre-
tation of free or non-hydrogen-bonded hydroxyl groups
should be of importance to characterize the formation
of hydrogen bonds. So far not many papers reported on
the IR interpretation of free or non-hydrogen-bonded
hydroxyl groups. Kondo attempted to assign the free
hydroxyl groups in the IR absorption bands [68].
In the multiple-methylated derivatives (MTC1V-3V)
from 6-O-tritylcellulose (6TC), which have a dierent
distribution pattern from the above MTC1-3, the methyl
substitution behavior of hydroxyl groups at the C-2 and
C-3 positions in 6TC was monitored by FT-IR (Fig. 22)
together with the change of the DS of methyl and the Figure 22 Change of IR spectrum for the methylated 6-O-
tritylcellulose (MTC1V3V) and 2,3-di-O-methyl-6-O-trityl-
remaining hydroxyl groups at each position for the above
cellulose (23M6TC) prepared from 6-O-tritylcellulose (6TC).
four samples determined by gas-chromatographic analyses
for the hydrozates of the polymer (Table 5). All spectra in
Fig. 22 were normalized on the basis of the internal
standard band at 1596 cm 1 due to trityl groups that are Considering that the product was very similar to these
not aected by the methylation. In the CH stretching tri-O-substituted derivatives and that the remaining OH
regions from 2800 to 3100 cm 1, the methylation had an groups were quite small, the IR absorption bands cannot
inuence obviously on each spectrum. Even in the be considered simply due to hydrogen-bonded OH groups;
23M6TC spectrum shown at the bottom of Fig. 22, the they may be due to free or non-hydrogen-bonded hy-
hydroxyl absorption band was slightly observed. This band droxyl groups. In other words, a trace amount of hydroxyl
cannot be noise because S/N ratio in this FT-IR machine groups in the original tritylcellulose remained intact during
was better than 1/15,000. The same band shape was also this methylation process, possibly because of heterogeneity
observed carefully in the other tri-O-substituted derivatives in the reaction mixtures. Thus when the lm sample for
that were already prepared in previous papers [65,67]. FT-IR measurements was cast from the clear solution of
Table 5 Distribution of Methyl and Hydroxyl Groups in Regioselectively

Methylated 6-O-tritylcellulose Derivatives
DS at positions DS of OH at each position

Overall
Sample DS X2 X3 X6 OH-2 OH-3 OH-6
MTC1V 1.39 0.82 0.57 0.18 0.43 Xtri

MTC2V 1.67 0.91 0.76 0.09 0.24 Xtri
MTC3V 1.68 0.91 0.77 0.09 0.23 Xtri
23M6TC 1.80 0.92 0.88 0.08 0.12 Xtri
Overall DS equals X2+X3+X6. Each Xn was determined by a gas chromatographic
analysis. Xn is the mole fraction of glucitol derivatives substituted on the OH of Cn (n=2, 3,
and 6). DS of OH-n = 1-Xn; Xtri: degree of trityl substitution.
Figure 23 Curve tting and peak assignments for OH stretching regions in 23M6TC.
the product powder, the molecules in the powder may have able. After the best curve tting, the peak positions of the
rearranged to give free hydroxyl groups in the lm. The three deconvoluted bands were 3579, 3558, and 3489 cm 1,
probability of the formation of intermolecular hydrogen respectively. The two bands (3579 and 3558 cm 1) were
bonds in this lm is fairly low because neighboring hy- assigned to the free hydroxyl groups because they appeared
droxyl groups are rare; the intramolecular hydrogen bonds at higher wavenumbers than those for inter- or intramo-
can occur: between OH at the C-3 position and the adjacent lecular hydrogen-bonded hydroxyl groups in cellulose
ring oxygen, between OCH3 at the C-2 and OH at the C-6 (Tables 1 and 2). As a reference for the IR band due to
positions, and between OH at the C-2 position and OCH3 free hydroxyl groups, it is reported that the free OH in
at the C-6 position. As reported by Kondo [18], the secondary alcohol appears at 36203635 cm 1, whereas the
intramolecular bonds may still be maintained even in the free OH for primary alcohol shows bands at 36303645
homogeneous solution state. The formation of such intra- cm 1 [85]. The dierence in the band positions between
molecular hydrogen bonds has also been observed in a cellulose and alcohol can be due to the regiochemical eects
DMSO solution state of 2,3-di-O-methylcellulose (23MC) in cellulose.
using NMR analyses with deuteration [81]. To assign the
IR band due to free OH groups, it is necessary to evaluate
the contribution of the intramolecularly hydrogen-bonded
OH groups. In addition, the three hydroxyl groups at each Table 6 The Results from a Curve Fitting Method Ap-
position are quite dierent in the sense that secondary plied to the Methylated Tritylcelluloses, MTC1MTC3 and
hydroxyl groups at the C-2 position are inuenced by the 23M6TC. Each Correlation Coeciency (R2) is Better than
0.99
anomeric C-1 carbon, secondary hydroxyl groups at the C-
3 position are favorable to form the intramolecular hydro- Three peaktops of the deconvulated IR bands
gen bonds, and the hydroxyl groups at the C-6 position are
primary OH. Therefore the hydroxyl groups at each posi- a (cm 1) b (cm 1) c (cm 1)
tion can exhibit three dierent IR absorption bands.
The IR absorption bands for OH stretching regions in MTC1V 3579 3513 3480
23M6TC of Fig. 22 were deconvoluted into three bands for MTC2V 3577 3552 3486
MTC3V 3578 3559 3487
the curve tting as shown in Fig. 23. To improve the
23M6TC 3579 3558 3489
calculation, the peaks needed to be well resolved, and the
number of peaks, the positions, and the areas were accu-
rately determined. Three was employed as the number of
peaks and the position was determined by the point at
which the second derivative of the spectrum contained OH2 OH-6 Intramolecular
peaks. All other parameters in the calculations were vari- H-Bonds
86 Kondo
To assign the three OH bands, the corresponding molecular association can be categorized in cellulose. A
deconvoluted IR bands in partially methylated tritylcellu- major consideration is that the predominant crystalline
loses as cast lms, MTC1VMTC3V, were compared with state of cellulose is included as a component in the ordered
those for 23M6TC. The results are shown in Table 6. IR state. This means that the ordered state also contains non-
absorption bands in the OH stretching region for each crystalline-ordered states. The category ordered is in
sample were also deconvoluted into three IR bands (a, b, contrast to the nonordered state which, to date, has been
and c in Fig. 23) with peak tops at around 3580, 3555 considered as amorphous cellulose for lack of a useful
(free hydroxyl groups), and 3485 cm 1, respectively. The way to characterize the product. It should be noted that the
wave number of band c with a lower peak top at 3485 cm 1 amorphous state being categorized as the nonordered
coincides with that calculated for intramolecular hydro- state should be distinguished from the noncrystalline state
gen-bonded hydroxyl groups in regenerated cellulose (Ta- of cellulose. Thus in this classication it becomes crucial
ble 2) and is very close to the assignment (34703480 cm 1) whether the state is ordered or nonordered. The
proposed for intramolecular hydrogen-bonded hydroxyls relationship is schematically illustrated in Fig. 24 [5].
using regioselectively methylated cellulose derivatives in Historically, it has been dicult to nd an appropriate
Fig. 15 [17] and Fig. 14B and C, between OH at the C-3 method to characterize noncrystalline or amorphous
position and the adjacent ring oxygen, between OCH3 at domains of cellulose. Often, wide-angle X-ray diraction
the C-2 position and OH at the C-6 position, and between (WAXD) has been used for determining the crystalline
OH at the C-2 position and OCH3 at the C-6 position. forms and the crystallinity; however, in most cases when
Considering these results, band c in Fig. 23 may involve not WAXD provides a diuse diraction pattern, it is consid-
only the intramolecular hydrogen bonds at the C-3 posi- ered simply as amorphous cellulose. However, as men-
tion, but also possible intramolecular hydrogen bonds tioned above, noncrystalline state does not necessarily
between the functional groups at the C-2 and C-6 positions. indicate amorphous state. Noncrystalline state includes
Therefore some of the free hydroxyl groups at the C-2 and both liquid crystalline and nematic-ordered cellulose [5]
C-6 positions may contribute to band c, which may be the that exhibits a certain order state, whereas amorphous state
reason for the inconsistency between FT-IR results and does not own any preferred orientation. Thus we have
OH values determined by the gas chromatographic method attempted to determine the noncrystalline regions of
as shown in Table 5. noncrystalline cellulose lms using FT-IR monitoring of
Bands a and b, because of the remaining free OH the deuterated hydroxyl groups [86]. We have found that
groups at either C-2 or C-6 position which were not such noncrystalline regions may comprise at least three
tritylated in 6TC (although some of them may contribute dierent domains. The study [86] also indicated the pres-
to intramolecular hydrogen bonds), can be assigned in the ence of ordered domains in the noncrystalline regions.
following way: The original IR bands in Fig. 23 show two As the regioselectively methylated celluloses, 23MC
peak tops at 3575 and 3485 cm 1, where bands a and c are (64), 6MC(66), and tri-O-methylcellulose (236MC)(65)
mainly indicated, and their relative intensities change had a controlled distribution of substituents and thus
depending on the samples (MTC1V-3V in Fig. 22). In hydrogen bonds, they were considered to be model com-
contrast, the relative intensity of band b does not change pounds for amorphous cellulose and hence ideal in exam-
signicantly. During this multiple methylation process, the ining the relationship between polymer structure and the
OH groups at the C-2 and C-3 positions are replaced and
their FT-IR spectra are aected. On the other hand, for the
OH at the C-6 position, which was a trace amount from the
beginning, the inuences are small. Thus bands a and b
with peak tops of 3580 and 3555 cm 1 are assigned to free
OH at the C-2 and C-6 positions, respectively.
Furthermore, the two peak positions of bands a and c
did not shift signicantly from 23M6TC to MTC1V as
shown in Table 6. However, the peak position of band b at
3558 cm 1 changed to a lower wave number between
MTC2V (3552 cm 1) and MTC1V (3513 cm 1). This indi-
cates that intramolecular hydrogen bonding at the C-6
position starts to perturb the OH bands.
C. Hydrogen Bonds in Noncrystalline

or Amorphous Cellulose
1. Noncrystalline Cellulose and Amorphous Cellulose
Cellulose, which is a h-1,4-linked glucan homopolymer, is
normally classied according to how the h-glucan chains
associate. We expand the concept of how various states of Figure 24 Concept of glucan chain association for cellulose.
physical properties of cellulose and its derivatives in terms

of hydrogen bond formation. It is noted that pure cellulose
tends to order somehow in a certain way, so that it is
dicult to keep amorphous state in cellulose if it can be
obtained.
In the previous sections, FT-IR spectroscopy was used
to identify intra- and intermolecular hydrogen bonds in the
regioselectively synthesized 23MC and 6MC. Films of
23MC and 6MC exhibited narrow OH stretching bands
in their IR spectra because of the controlled hydrogen
bonding (Fig. 13). Morphologically, lms cast from these
methylcellulose derivatives were also found to be predom-
inantly noncrystalline or rather amorphous than crystal-
line. Therefore the narrow OH absorbance bands and the
amorphous homogeneity of the sample microstructure
enabled us to clarify and classify the interchain hydrogen
bond interactions found in the samples. It is believed that a
characterization of the hydrogen bonds found in amor-
phous cellulose would be of fundamental value and, fur-
Figure 25 Wide-angle X-ray diraction patterns for cast
thermore, that a structural study of amorphous cellulose in lms: (A) cellulose, (B) 23MC, (C) 6MC, and (D) a blend of
light of hydrogen bonding might be a rst step in uncover- 23MC and 6MC (1.08/1 w/w).
ing details of how molecules rearrange in going from the
liquid to the crystalline state.
So-called amorphous cellulose samples are usually
prepared by ball milling of cellulose [87,88] by deacetyla-
tion of cellulose acetate with sodium methoxide in anhy- studied, and Table 7 shows the most probable assignment
drous methanol [89], or by precipitation from nonaqueous for these bands, as well as those found in a real amorphous
solvent systems into nonaqueous regeneration media with cellulose sample. Figs. 26 and 27, respectively, show OH
the avoidance of stress [9094]. To date, most of these and CO stretching vibration regions resulting from the
samples have been studied by WAXD [8795], FT-IR glucose ring skeletal vibration. In comparing the real and
spectroscopy [95], and solid-state NMR [94]. Hatakeyama the articial spectra for the amorphous cellulose, there is
and Hatakeyama [96] have previously reported on the no signicant dierence in the ring stretching vibration
formation of interchain hydrogen bonds with increasing region as clearly illustrated in Fig. 27. This indicates that
temperature for amorphous regions in cellulose bers. any absorption contributions by methyl groups may be
More recently, Kondo and Sawatari [24] have tried to precluded in the methylated samples to the ring stretching
analyze and comment on the types of hydrogen bonds vibrations. Thus the articial spectrum mirrors the glucose
formed in amorphous cellulose. The methodology was ring structure found in real amorphous cellulose. The data
threefold: (1) to quantitatively produce an articial IR contained in Table 7, which lists typical absorption fre-
spectrum for amorphous cellulose by using a combination quencies for the two spectra, also seem to support this
of amorphous methylcellulose model compound IR spec- hypothesis. In regard to the stretching and bending vibra-
tra; (2) to characterize the dierence between the real and tions for methine and methylene groups in cellulose, the
the articial spectra in terms of the formation of hydrogen quantitative mathematical model could not completely
bonds; and, nally, (3) to compare the result of (2) with the remove the contribution by methyl groups in the methyl-
IR spectra of propyl alcohol solutions, which can serve as ated samples to totally match the articial spectrum for
model systems for intermolecular hydrogen bonding. This amorphous cellulose.
approach lets us draw a number of conclusions about the
hydrogen bonds formed at the C-2 and C-3 positions in the Dierence Between the Real and the Articial Spectra
anhydroglucose repeating units of amorphous cellulose. There is a marked dierence in the OH stretching
For this investigation, all lm materials (cellulose, 23MC, region of the real and the articial IR spectra. The dier-
6MC) used should have a nonordered amorphous micro- ence spectrum (real articial; Fig. 26ab) is shown in Fig.
structure at the level of WAXD patterns as shown in 28 with the region between 3750 and 3000 cm 1 expanded
Fig. 25. to clearly show this marked dierence. Considering that
Fig. 13 shows the OH frequency region of the IR the articial spectrum was constructed assuming a linear
spectra for each amorphous homopolymer lm sample contribution by the intra- and intermolecular hydrogen
investigated. By using these four spectra and by quantita- bonds at the C-6 position, the dierence spectrum should
tive manipulation, the articial spectrum could be con- thus contain peaks arising from intermolecular hydrogen
structed as illustrated in Figs. 26b and 27b. The IR bonds at the C-2 and C-3 positions and any free hy-
spectrometer software produced a list of the most promi- droxyl groups. Of course, the bands resulting from com-
nent bands in this articial IR spectrum in the region being mon hydrogen bonds in both the real and the articial
88 Kondo
Figure 26 IR spectra in the OH stretching vibration (37503000 cm 1) region: (a) real spectrum of amorphous cellulose lm, (b)
articial spectrum, and (c) spectrum for the blend sample (23MC/6MC=1.08/1 w/w).
spectra are not completely canceled out by subtraction light at 3558 and 3580 cm 1 described in a previous section
because in the two spectra the magnitude for each OH (see Assignment of Free Hyroxyl Groups in Cellulose
absorbance band is not necessarily of equal value. The under Section B) [68], which is a higher wave number than
dierence spectrum can thus include, to a greater or lesser the two peaks. A shoulder around 3580 cm 1 with an ab-
extent, all the hydrogen bonds present in pure amorphous sorption of 0.06 may be due to the free OH. As all the
cellulose. However, the marked dierences in the two OH groups in 23MC and 6MC are engaged in some form of
spectra cannot adequately be explained simply in terms
of unequal contributions from common hydrogen bonds:
the two positive peaks and negative valley in the dierence Table 7 Comparison of Typical Absorption Frequencies
spectrum are attributed to intermolecular hydrogen bonds Between the Real and the Synthesized IR Spectra of
at the C-2 and C-3 positions as well as free OH groups. Amorphous Cellulose
The main negative and positive peaks in Fig. 28
appeared at two particular wavenumbers, 3472 and 3352 Frequency (cm 1)
Relative
cm 1. Unbonded or free OH groups absorb infrared
Realb Synthesizedc intensitya Interpretation
669 671 W OH out-of-phase

bending
899 892 M Antisymmetric
out-of-phase
ring stretching
1040 1040 S CO stretching
1070 1075 S Skeletal vibrations
involving CO
stretching
1108 1108 S Antisymmetric
in-phase ring
stretching
1159 1154 S Antisymmetric
bridge COC
stretching
1374 1375 M CH bending
1420 1425 W CH2 symmetric
bending
2892 2903 M CH stretching
3420 3457 S OH stretching
Figure 27 IR spectra in the CO stretching vibration (1500 a
Key: S, strong; M, medium; W, weak.
700 cm 1) region: (a) real cellulose spectrum and (b) articial b
Real spectrum of amorphous cellulose lm prepared by casting.
c
cellulose spectrum. Synthesized spectrum of amorphous cellulose.
Figure 28 Dierence IR spectrum obtained by subtracting the articial (Fig. 26b) from the real (Fig. 26a) cellulose spectrum.
hydrogen bonding, the articial spectrum formed by com- and WAXD patterns for the amorphous cellulose gave no
bining the two contributing spectra is thought to have few support to the idea that microcrystallites were present. It is
free OH groups. Therefore the almost negligible signal at therefore concluded that the amorphous cellulose must
3580 cm 1 in the dierence spectrum indicates that there include, to some degree, domains formed by the intermo-
are very few free OH groups in the amorphous cellulose lecular hydrogen bonds at the C-2, C-3, and C-6 positions.
itself. A negative absorbance valley at 3472 cm 1 (assigned Kondo and Sawatari therefore proposed a model for
to intramolecular hydrogen bonds) indicates that the signal amorphous cellulose in which some amorphous domains
was overcanceled in the articial spectrum, whereas the pos- are partly interacted by intermolecular hydrogen bonds as
itive band around 3352 cm 1 was attributed to the intermo- illustrated in Fig. 29. This is similar to a fringed micellar
lecular hydrogen bonding at the C-2, C-3, or C-6 position. structure. In a dilute and semidilute cellulosic solutions
The negative valley (3472 cm 1) in the dierence spec- Buchard et al. proposed the formation of the fringed
trum appeared to reect an excess of the intramolecular micelle to explain the rheological behavior of the cellulo-
hydrogen bonds either at the C-2 position and the OCH3 at sics as shown in Fig. 30 [97100]. Considering the above
the C-6 position, or at the C-6 position and the OCH3 at the results, there may be correlation on the aggregation states
C-2 position, or at the C-3 position and the ring oxygen in between the solution states and amorphous states of cel-
the articial spectrum. lulose. In other words, from a dilute solution state and a
Interestingly, as the solvent is changed for the same concentrated lyotropic liquid crystalline state to an amor-
23MC sample, the crystallinity of the lm varies from sol- phous and a crystalline solid state there might be a con-
vent to solvent and the OH groups at the C-6 position may nection in terms of rearrangements through inter and
be favorably involved in an intermolecular hydrogen bond- intramolecular hydrogen bonding formation.
ing. The extent of crystallization may be dependent upon
the behavior of the primary OH group located at the C-6
position. Stated dierently, the OH group at the C-6 2. Hydrogen-Bonded Domain in Amorphous Cellulose
position may be signicant in determining the nal mor- Currently, there is a shortage of structural information
phological make-up of cellulose. This indicates that when about the noncrystalline regions including amorphous
the C-6 hydroxyls in cellulose are, to a great extent, en- state, perhaps partly because terminology suggests and
gaged in the intermolecular hydrogen bonding, the result- impression persists that molecular chains in these regions
ing cellulose should exhibit high crystallinity. Invoking are completely without structure and partly because meth-
this hypothesis, either a random distribution of micro- odology has been limited to WAXD and CP-MAS 13C
crystallites or a series of domains arising from intermolec- NMR for measuring order in the presence of substantial
ular hydrogen bonds will result in a highly amorphous state amount of disorder. It is not uncommon for substrates that
for cellulose. To conrm the existence of these postulated are not identiable as crystalline by a method such as X-ray
microcrystallites, small angle X-ray scanning (SAXS) in- diraction to be labeled amorphous, but the denition
tensity distributions were measured using a PSPC system. of amorphous goes beyond noncrystalline to unorganized
The SAXS pattern for the amorphous cellulose showed no and having no pattern of structure as described above
signicant scattering maxima, indicating that there was [5,101]. Therefore in this section details of the noncrystal-
no lamella structure present in the cellulose. Thus SAXS line regions are particularly examined in the noncrystalline
90 Kondo
only the noncrystalline regions in the cellulose model com-

pounds are focused using the FT-IR monitoring of the
deuteration process. The main advantage of this method
is that the use of a reaction cell has enabled us to obtain
not only a change in reproducible and correct IR spectra
for noncrystalline regions in cellulose, but also the rate
constant for the deuteration reaction which is assumed to
correspond to the nature of amorphous regions [86]. Thus
it is possible to characterize the morphology of noncrys-
talline regions by analyzing the diusion behavior of D2O
as a probe. As already described in a previous section, each
polymer, 23MC and 6MC, is considered to have dierent
types of inter- and intramolecular hydrogen bonds. Before
analyses, it was conrmed that the morphology (smooth or
rough) on the surface of the lm does not aect the rate
constant by comparison of the surface observation by
atomic force microscopy (AFM) with the rate constants
for dierently prepared two lm samples with dierent
morphologies on the surfaces.
Fig. 31 shows the change in IR spectra for 6MC
accompanied by the deuteration process. When the deu-
teration proceeded, OH stretching vibration around 3470
Figure 29 Schematic model for amorphous cellulose. cm 1 decreased, and instead the bands at 2559 cm 1 due to
OD at the C-2 and C-3 positions appeared and then
increased. Similar behaviors were observed for the OH
and OD bands in the amorphous 23MC and cellulose.
However, the deuteration was not completed regardless of
cellulose lm samples, regioselectively methylated 23MC the amorphous samples, giving intact OH groups. The
and 6MC amorphous lm samples used above, as model amount of the unexchangeable OH was 14.3%, 10%, and
components of amorphous cellulose using FT-IR methods 13.3% for 6MC, 23MC, and noncrystalline (amorphous)
[86]. cellulose, respectively. These unexchangeable hydroxyl
Applications of deuteration methods to IR have so far bands may also indicate the presence of domains involving
focused on the separation of IR spectra for cellulose the intermolecular hydrogen bonds in a proposed amor-
structure into two parts of crystalline and noncrystalline phous model of Fig. 29 [24].
regions, respectively, and then only the discriminated Here the OHOD exchange reaction can be postulated
crystalline regions have been studied [44,102,103]. Here, to obey pseudo-rst-order kinetics because of the large
amount of D2O to the amount of OH in each lm sam-
Figure 30 Schematic drawing to demonstrate various size

of particles visualized by changing the angular dependence of
light scattering: qRg<<1 (whole particle seen); qRg > 1
(substructures approved). (From Ref. [99]; Courtesy of
Prof. Burchard; Hermann Schtaudinger Institute, Freiburg, Figure 31 Changes of OH and OD in IR absorption bands
Germany.) for monitoring the deuteration of amorphous 6MC.
ple. The general equation, d[OH]/dt=k[OH], can be em- Table 8 Rate Constants (k) from OH to OD for Every Single
ployed. Thus the derived relationship, ln{[OH]t /[OH]0 }= Reaction in the Deuteration Processes of the Amorphous
kt, was drawn in Fig. 32 for the three amorphous Three Film Samples
samples, 23MC, 6MC, and cellulose, respectively. Each
of the three samples showed similar kinetic behaviors that Time course (hr) k (hr 1) R2
were divided by three single reactions. In other words, Reaction 1
three kinds of exchange reaction (1, 2, and 3 in Fig. 32) can 23MC 0.085 0.136 0.98
competitively coexist. Table 8 shows the rate constants for 6 MC 0.084 0.247 0.99
each reaction. The rate constant for reaction 1 was rela- Cellulose 0.082 0.264 1.00
Reaction 2
23 MC 612 0.085 0.98
6 MC 513 0.057 0.99
Cellulose 312 0.075 0.98
R: Correlation coecient.
tively dierent among the three samples. On the contrary,

the rate constant for two reactions seems to be similar
among them. Of course, this is still speculative. In general,
adsorption and diusion in crystalline polymers are con-
sidered to occur in noncrystalline or amorphous regions.
However, it is very complex because the structure for the
crystalline regions may somehow have inuences on it
although the morphological eects on the surface have no
inuence.
Considering that the samples exhibited noncrystalline
for the X-ray measurements, noncrystalline regions in these
cellulosics may be discriminated into three phases: (1)
deuteration is fast, corresponding to reaction 1; (2) deuter-
ation is slower, corresponding to reaction 2; and (3) almost
undeuterated, corresponding to reaction 3. Interestingly,
the second rate constants among the three samples were
very similar although the rst rate constants for them
diered. This indicates that the second one may correlate
with the deuteration for the intramolecular hydrogen
bonds (Fig. 6) in noncrystalline or amorphous phases
which are common interactions among the above three
samples. Further investigation will be needed because the
type of intermolecular hydrogen bonds found at the C-2, C-
3, and C-6 hydroxyl positions participates, to some extent,
in determining the structure of amorphous cellulose [25].
D. The Relationship Between Intramolecular

Hydrogen Bonds and Certain Physical
Properties of Regioselectively Substituted
Cellulose Derivatives
To date, the author has focused on clarifying the hydrogen
bonding formation involved in cellulose molecules in var-
ious states, such as blends, gels, amorphous, and liquid
crystals [1728,67,68,104], by using regioselectively substi-
tuted methylcelluloses such as 23MC(64) and 6MC(66) as
cellulose model compounds. These investigations were
Figure 32 The relationships between decreasing ratio of OH performed mainly by using tools such as FT-IR, DSC,
groups, ln{[OH]t/[OH]0} and time, t, during the deuteration uorescence spectroscopy, and solid-state NMR. Interest-
process for the three amorphous samples, 23MC, 6MC, and ingly, the OH absorption band in the IR spectrum of 6MC
pure cellulose. lms was sharper and more symmetric than those for
92 Kondo
23MC, which has only one OH group per anhydroglucose Table 9 Solubility of Various Methylcellulose (MC)
unit. This phenomenon was also observed for other sub-
stituents with longer alkyl, benzyl, and allyl groups. To Commercial MC
clarify this behavior, a model was proposed to explain the DS DS DS 23MC 6MC
formation of intramolecular hydrogen bonds in 6-O-sub- Solvent 0.11.1 1.42.0 2.42.8 DS 2.0 DS 1.0
stituted cellulose derivatives. Specically, these cellulose
derivatives have two intramolecular hydrogen bonds, one Water D o
between the OH at the C-3 position and an adjacent ether Aq. alkali o D D D
oxygen of the glucose ring, and the second between the (pH 9.5)
ether oxygen at the substituted C-6 position and an adja- Aq. acid o o D o oD
cent OH at the C-2 position (Fig. 14). Accordingly, the (pH 5.5)
aggregation state of 6-O-substituted compounds should be Acetone o
interesting to study particularly in terms of any relation- Methanol D o
ship between the formation of the hydrogen bonds and THF o o
their direct eect on certain physical properties. Chloroform o D o
In general, the formation of inter- and intramolecular DMSO oD o oD o o
hydrogen bonds in cellulose and its derivatives is consid- DMAc oD o oD o o
ered to have a strong inuence on their physical properties. o: Soluble; D: swelling; : insoluble; oD: partially soluble.
However, only a few published reports [48,105,106] have
dealt directly with the above point of view, although many
papers [107, 108] have referred indirectly to this relation-
ship between structure and physical properties. Therefore
a DS of 1.0 with other methylcelluloses having a
one 6-O-substituted cellulose ether, namely, 6-O-methyl-
corresponding DS, respectively, the solubility of both
cellulose, which has been revealed to have mainly intramo-
23MC and 6MC exhibited remarkable dierences. In
lecular hydrogen bonds, was chosen and its physical
particular, 6MC, which has mainly intramolecular rather
properties including solubility, hydroxyl reactivity, and
than intermolecular hydrogen bonds, exhibited more fa-
crystallinity, which should be inuenced directly by any
vorable solubility in solvents having dierent polarities.
intramolecular hydrogen bonds, were investigated as fol-
These results indicated that the solubility of 6MC is mostly
lows [19].
determined by the absence of interchain hydrogen bonds
Three typical properties chosen to study were the
that can obstruct swelling and solvation of the polymer.
solubility, the relative reactivity of the remaining OH
Primary OH at the C-6 position can favorably form
groups at the C-2 and C-3 positions in a repeating unit,
intermolecular hydrogen bonds [1820,23,24], causing
and the crystallinity in order to answer the following four
poor solubility of the material to solvents. In other words,
questions: (1) Can the polymer be easily dissolved in various
the amount of free OH groups at the C-6 position may
solvents due to a lack of intermolecular hydrogen bonds?
contribute more critically to determining the solubility of
(2) Is it possible to maintain intramolecular hydrogen
the material than that of free OH groups at the C-2 or C-3
bonds after dissolution to allow the solution to have an
position. In fact, comparing 6MC with MC of DS 2.42.8
inuence on the OH reactivity mentioned above? and (3) Is
in Table 9, primary OH-6 was fully substituted for 6MC
the solution less likely to crystallize than other ordinary
whereas for the MC with DS 2.42.8 unsubstituted OH-6
cellulose and cellulose derivatives because of a restriction in
was more or less remaining. Therefore it may cause the
the intermolecular hydrogen bonding which is available to
superior solubility for 6MC to the MC with 2.42.8
the crystallizing polymer? (4) Is there any inuence of
although total DS for the latter MC is higher.
intramolecular hydrogen bonds on the determination of
the handedness of cholesteric twist sense of ethylcellulose Relative Reactivities of the Remaining OH
lyotropic mesophases? Groups at the C-2 and C-3 Positions
Concerning the dierence in relative reactivity of the
Solubility hydroxyl groups (OH-2, OH-3, and OH-6, respectively) at
Table 9 lists the solubility of methylcelluloses, 23MC, the two (C-2), three (C-3), and six (C-6) positions of the
6MC, and three kinds of methylcelluloses normally pre- anhydroglucose unit, it is well known [109,110] that the
pared commercially in an aqueous system so as to have order of the relative reactivity is OH-2>OH-6>OH-3 for
dierent DS. In general, for cellulose derivatives, the MC prepared from alkali-cellulose in an aqueous solvent
higher the DS the higher the solubility of the derivative system. The higher reactivity of OH-2 has been postulated
to organic solvents. However, interestingly enough, 6MC to be due to its high acidity that is enhanced by its
having a lower DS showed a higher solubility than did proximity to the anomeric center, C-1 [111]. Therefore, if
23MC with a DS of 2.0. As shown in a previously an intramolecular hydrogen bond was still present at the
published paper [67,68] the FT-IR spectra for 23MC and C-2 position even in the reaction mixture, then the reac-
6MC showed this was due to the presence of interchain tivity of OH-2 should be altered, and it would probably be
hydrogen bonds at the C-6 position of 23MC. Further, reduced. Within this denition, the relative reactivity of
in comparing both 23MC with a DS of 2.0 and 6MC with the remaining hydroxyl groups (OH-2 and OH-3) in 6MC,
6TC, and 6BC was examined. As reported previously [67], exhibited noncrystalline (amorphous) patterns similar to
the two kinds of 6-substituted derivatives, 6TC and 6BC, those obtained from noncrystalline celluloses obtained
are assumed not to have any intramolecular hydrogen from the DMAc-LiCl and SO2-diethyl amine-DMSO
bonds; only 6MC has them. solutions, and further the 6MC did not show a crystal-
From the change in the DS values at the C-2 and C-3 line pattern even after heat treatment at 160jC. Thus
positions before and after the reaction, the relative reac- 6MC shows poor crystallinity irrespective of the homo-
tivity was found to be in the following order: 6TC> geneity of the structural unit along a molecular chain. In
6BC>6MC. This is apparently due to the dierence in general, crystallization depends not only on the regular-
reactivity at the C-2 position. Moreover, this dierence is ity of chemical structure but also on sucient chain ex-
directly attributed to the presence of intramolecular hy- ibility for coordinated molecular motion to form nuclei.
drogen bonds at the C-2 position. As described in a The 6MC chain may be suciently sti to form a high
previous section, the introduction of electron-withdrawing viscosity medium during evaporation of the solvent that
and bulky functionalities such as trityl and benzyl groups at would prevent nucleation. Therefore precipitation/crys-
the C-6 position changes the structure of the intramolecu- tallization in a dilute solution was tried. However, crys-
lar hydrogen bonds to give free OH groups at the C-2 tallized 6MC could not be obtained after this process. On
position and, in addition, the substituent eect of the more the other hand, 23MC showed dierent patterns depend-
bulky trityl group shows itself more in the appearance of ing on the solvents. As indicated in previous papers on
free OH-2 than in the smaller benzyl group [67]. Further, crystallization [25] and gel formation [20,26] of cellulo-
the bulkiness of the trityl groups at the C-6 position for sics, the primary OH groups at the C-6 position may be
6TC may change the conformation of the glucose back- favorably involved in interchain hydrogen bonding. The
bone to cause, to some extent, a break of the intramolecular extent of crystallization may depend on the behavior of
hydrogen bonds between the OH at the C-3 position and its the primary OH groups. Thus cellulose derivatives whose
neighboring ring oxygen (O-5). Thus, this produced free OH groups at the C-6 position are blocked like in 6MC
OH-3 for 6TC which can be more methylated than 6BC. In may prevent crystallization in the same manner as crys-
the case of 6MC, this deformation of the intramolecular tallization resulting from interchain hydrogen bonding in,
hydrogen bonds is not expected by the substitution at the say, 23MC. Stated dierently, 6MC, which shows strong
C-6 position. Therefore the order of 6TC>6BC>6MC intramolecular hydrogen bonds, can perhaps possibly
above can be considered as the reverse order of preference form a crystalline state simply from van der Waals force
for the formation of intramolecular hydrogen bonds. In- by a minimization of the system potential energy. How-
deed, in 6MC that has strong intramolecular hydrogen ever, the poor crystallinity exhibited by 6MC described
bonds, the relative reactivity at both the C-2 and C-3 above suggests that interchain hydrogen bonds at the OH
positions exhibited almost the same values, which was groups of the C-6 position may be more advantageous
distinctly dierent for both 6TC and 6BC. This does not in aiding crystallization in cellulosics than van der Waals
mean an enhanced reactivity of the OH-3 reactivity, but force. The fact that the uniform structure of 6MC in
rather a reduction in the OH-2 reactivity probably due to which every structural unit is completely and regioselec-
the formation of intramolecular hydrogen bonds at this tively substituted can engage in intramolecular hydrogen
position. Simultaneously, the hydrogen bonds at the C-2 bonds is not an advantage for crystallization, which diers
position which form between OH-2 and the ether oxygen of from other synthetic polymers such as polyolens and
a methoxy group at the C-6 position seem to be very similar polyesters. This might be due to an induced stiness in
to the intramolecular hydrogen bonds at the C-3 position, the main chain of 6MC, which results from the presence
between OH-3 and the glucose ring oxygen. Therefore of intramolecular hydrogen bonds.
reactivity at both the C-2 and C-3 positions shows similar Concerning precedence for the primary OH at the C-
values. In contrast, 6TC and 6BC exhibited relative reac- 6 position to the secondary OH at the C-2 and C-3
tivity in the order of OH-2>OH-3. Taking hydrogen positions in crystallization, one reason is that, as de-
bonding into account, this order is reasonable and scribed above, 23MC, which has only free primary OH
coincides with that usually exhibited in aqueous systems at the C-6 position, was easier to be crystallized than 6MC
as mentioned previously. In this study, it is noted that the and yet the crystallized 23MC did not show a melting
methylation was performed in homogeneous DMSO solu- point. This appears probably due to the strong hydrogen
tion, and therefore the hydroxyl groups in 6TC and 6BC bonding engagements in the crystallized 23MC. As for the
may be solvated to prevent further involvement of the favorableness of the interaction, Kondo et al. have al-
hydrogen bonds. In 6MC, as stated above, the relative ready reported [18,23] that in the comparison of 23MC
reactivity at the C-2 and C-3 positions indicates that the with 6MC for the blend with poly(ethylene oxide) (PEO)
intramolecular bonds were still maintained even in the which has oxygen in the polymer backbone, only primary
homogeneous solution state [19,26]. OH at the C-6 position for 23MC was engaged in
intermolecular hydrogen bonds, whereas secondary OH
at the C-2 or C-3 position for 6MC did not form the
Crystallinity interaction with PEO. Therefore it is considered that the
The X-ray diraction patterns for 6MC lms cast primary 6-OH may contribute to the crystallization more
from both DMAc and CHCl3/CH3OH(4/1 v/v) solvents than the secondary hydroxyls.
94 Kondo
The Inuence of Intramolecular Hydrogen Bonds

on Handedness in Ethylcellulose/CH2CL2
Liquid Crystalline Mesophases
In 6MC samples there was a direct correlation between
the physical properties observed and the formation of
intramolecular hydrogen bonds that can even be main-
tained in solution [19,27]. The intramolecular hydrogen
bonds were also found to have an inuence on the enzy-
matic hydrolysis of 6MC [21,22].
Kondo and Gray [64] succeeded in preparing a series
of methyl- and ethyl celluloses having a systematically
controlled distribution of substituents and DS. These
polymers would appear to form an ideal set of complete
samples for determining what type of correlation exists
between the physical properties and the distribution of
substituents for alkylcellulose derivatives in terms of hy-
drogen bond formation. Thus the eect of substituent
distribution on the liquid crystalline properties of EC is
the focus in this section. The majority of cellulosic meso-
phases that have been studied to date are right-handed Figure 34 Change in DS pattern at the individual C-2, C-3,
cholesteric, but a few left-handed systems have also been and C-6 positions for series B samples prepared from a
reported [112116]. Specically, lyotropic ethylcellulose commercially available EC.
(EC) mesophases were observed to show both types of
handedness depending on the polymer volume fraction and
solvent [112]. Guo and Gray [115,116] reported that cho-
lesteric liquid crystalline solutions of acetylated EC in DS, the inuence of intramolecular hydrogen bonds, with-
chloroform exhibited a change in handedness from a left- in the same chiral backbone, in determining the handedness
handed to a right-handed helicoidal supermolecular ar- of these lyotropic mesophases was investigated [27].
rangement with an increasing acetyl content in the EC. The Two series (A and B) of ethylcellulose samples were
twist sense of EC mesophases in chloroform and CH2Cl2 used in this experiment to cover the entire range of hy-
was also observed to change from left-handed to right- droxyl substitutions possible in EC. For this set A of sam-
handed with an increasing degree of ethyl-substitution in ples (Fig. 33), all hydroxyl groups at the C-2 and C-3
EC [117]. However, the driving force for this structural positions were almost completely ethylated and only the
reversal is still not clear. Using ethylcelluloses having a ethyl DS at the C-6 position hydroxyl was systematically
systematically controlled distribution of substituents and increased with increasing sample code number. The
hydroxyls at the C-6 position in cellulosics easily form
intermolecular hydrogen bonds, resulting in poor sample
solubility in many solvents. In this series as seen in Fig. 33,
samples 1 to 4, which had a DS at the C-6 position of less
than 0.78, did not give clear CH2Cl2 solutions even at
concentrations of 1 wt.%. A high degree of substitution
at the C-6 position was required for the polymer to dis-
solve in CH2Cl2. For series B (Fig. 34), the DS at the C-3
position was somewhat lower than that for the other posi-
tions. The C-2 and C-6 hydroxyl groups were easily and
completely ethylated to give a saturated point with a DS
of 1.0. Thus only the ethyl DS for the C-3 hydroxyls was
individually increased up to a limit of 1.0. In contrast to
series A, the C-6 position hydroxyl groups in series B were
almost completely ethylated and all samples dissolved in
CH2Cl2 at 1 wt.% to yield clear solutions.
Circular dichroism (CD) was used to determine the
handedness of the cholesteric structure by the sign of the
induced CD band that results from the selective reection
of circularly polarized light. A positive CD band corre-
sponds to a left-handed cholesteric twist, whereas a neg-
Figure 33 Change in the degree of substitution (DS) pattern ative CD band corresponds to a right-handed twist.
at the individual C-2, C-3, and C-6 positions for series A Samples from series A, as shown in Fig. 33, showed a
samples prepared from synthesized 23EC. totally dierent behavior in cholesteric handedness from
that of series B samples. Series A samples that were II. CONCLUSION

prepared from 23EC have free hydroxyls only at the C-6
position. As the sample code number increases, the free In this chapter, the author has attempted to explain the
hydroxyl groups at the C-6 position are gradually replaced characterization of hydrogen bonds in various states from
by ethyl groups. As already noted, hydroxyl groups at the crystal to solution. It is clear that using regioselectively
C-6 position in cellulosics contribute favorably to the substituted cellulose derivatives some specic intramolec-
formation of intermolecular hydrogen bonds and this ular hydrogen bonds can be characterized. However, hy-
results in poor solubility of the polymer. This can be seen drogen bonds themselves are still a very dicult subject to
for samples 1 to 4 in Fig. 33, which do not dissolve clarify. Further extensive study will be desired to realize the
completely even in very dilute solution. Instead, they were correlation between the formation of hydrogen bonds and
found to swell and form gels. Samples with a DS of more their inuence on the properties found in cellulosics. In
than 0.8 at the C-6 position, namely, samples 5 to 9, show addition, interchain hydrogen bonds in cellulosics/synthet-
induced CD spectra for concentrated anisotropic solutions ic polymer blends [18,23,24,104] and aggregation in gels
from 40 to 50 wt.% polymer (Fig. 33) and all are right- are dropped in this chapter. Of course, these subjects are of
handed chiral nematic liquid crystals. importance in terms of polymerpolymer interactions. The
The above results strongly suggest that the distribu- author could not nd enough space to mention about it.
tion of ethyl substituents among C-2, C-3, and C-6 posi- We will wait for another review.
tions of the anhydroglucose unit in EC samples can aect
the cholesteric handedness of their anisotropic solutions.
In particular, the substitution of hydroxyl groups at the C- REFERENCES
3 position may play an important role in determining the
handedness, whereas hydroxyl groups at the C-6 position 1. Jerey, G.A.; Saenger, W. Hydrogen Bonding in Bio-
are important and contribute to the solubility of the logical Structures; Springer-Verlag, 1991, Chapters 1
and 2.
sample in various solvents. The intramolecular hydrogen
2. Cousins, S.K.; Brown, R.M., Jr. Photoisomerization of a
bonds formed between hydroxyls at the C-3 position and dye-altered h-1,4 glucan sheet induces the crystallization
adjacent ether oxygen of the glucose ring may even be of a cellulose-composite. Polymer 1997, 38, 903912.
maintained in the solution state. Further, the intramolec- 3. Togawa, E.; Kondo, T. Change of morphological
ular hydrogen bonds at the C-3 position may play a role in properties in drawing water-swollen cellulose lms
determining the conformation of the extended glucose prepared from organic solutions. A view of molecular
chain structure, causing greater chain stiness. Therefore orientation in the drawing process. J. Polym. Sci. B:
Polym. Phys. 1999, 37, 451.
once the hydroxyl groups at the C-3 position are highly 4. Kondo, T.; Sawatari, C. A Fourier transform infra-red
substituted, then the intramolecular bonds must be cut, spectroscopic analysis of the character of hydrogen
and the glucose unit is now freer and may rotate easily to bonds in amorphous cellulose. Polymer 1996, 37, 393.
alter the torsion angle between two consecutive units and 5. Kondo, T.; Togawa, E.; Brown, R.M., Jr. Nematic
as a result the molecular chain will be more exible. In fact, ordered cellulose: A concept of glucan chain association.
T1 relaxation time of the C-1 carbon in a glucose unit in Biomacromolecules 2001, 2, 1324.
6. Horii, F.; Hirai, A.; Kitamaru, R. Solid-state 13C-NMR
cellulose derivatives whose hydroxyl groups at the C-3 study of conformations of oligosaccharides and cellu-
position remain unsubstituted is longer than that for lose-conformation of CH2OH groups about the exo-
2,3-O-substituted cellulosics even in the solution state. cyclic CC bond. Polym. Bull. 1983, 10, 357.
13
C NMR chemical shifts and relaxation time at the C-1 7. Liang, C.Y.; Marchessault, R.H. Infrared spectra of
carbon also change after deuteration of the hydroxyl crystalline polysaccharides. I. Hydrogen bonds in native
groups in the same system [73]. These results indicate celluloses. J. Polym. Sci. 1959, 37, 385.
8. Liang, C.Y.; Marchessault, R.H. Infrared spectra of
not only the engagements of the intramolecular hydrogen
crystalline polysaccharides. II. Native celluloses in the
bonds in the solution state, but also the positive contribu- region from 640 to 1700 cm 1. J. Polym. Sci. 1959, 39, 269.
tion of the interaction to the molecular chain stiness. For 9. Hermans, P.H. Physics and Chemistry of Cellulose
all samples in series A, the hydroxyl groups at the C-3 Fibres; Am. Elsevier: New York, 1949.
position are assumed to be almost fully substituted and 10. Marchessault, R.H.; Liang, C.Y. Infrared spectra of
thus as long as the sample is dissolved, the anisotropic crystalline polysaccharides. III. Mercerized cellulose. J.
solution shows right-handedness. As shown in Fig. 34 Polym. Sci. 1960, 43, 71.
11. Blackwell, J.; Marchessault, R.H. In Cellulose and
(series B), the samples with an ethyl DS of more than 0.9 Cellulose Derivatives; Bikales, N., Segal, L., Eds.; Wiley
at the C-3 position, EC 5 to 9, form a lyotropic right- Interscience: New York, 1971 Part IV, Chapter XII, 1
handed chiral nematic mesophase. In relating the intra- 37.
molecular hydrogen bonds at the C-3 position with chain 12. Marchessault, R.H.; Sundararajan, P.R. In The Poly-
stiness, the above results indicate that the less sti the saccharide; Aspinall, G.O., Ed.; Academic Press, Inc.:
chain is the more favorably the right-handed chiral ne- New York, 1983; Vol. 2, 1295.
13. Heine, S.; Kratky, O.; Porod, G.; Schmit, P.J. Eine
matic structure is formed. These results indicate that the verfeinerte Theorie der Rontgenkleinwinkelstreuung des
contribution of intramolecular hydrogen bonds should be verknauelten Fadenmolekuls und uhre Anwendung auf
considered when trying to explain or account for the Cellulosenitrat in Losung. Makromol. Chem. 1961, 44,
chiroptical properties of cellulosic mesophases. 682.
96 Kondo
14. Gray, D.G. Liquid crystalline cellulose derivatives. J. and hydrogen-bonding system in cellulose Ih from
Appl. Polym. Symp. 1983, 37, 179. synchrotron X-ray and neutron ber diraction. J.
15. Flory, P.J. Liquid crystal polymers. Adv. Polym. Sci. Am. Chem. Soc. 2002, 124, 90749082.
1984, 59, 1. 35. Langan, P.; Nishiyama, Y.; Chanzy, H. A revised
16. Fukuda, T. Liquid crystals of cellulosics (Japanese). Sen-i structure and hydrogen-bonding system in cellulose II
Gakkaishi 1990, 46, P-504. from a neutron ber diraction analysis. J. Am. Chem.
17. Kondo, T. Hydrogen bonds in regioselectively substi- Soc. 1999, 121, 99409946.
tuted cellulose derivatives. J. Polym. Sci.: B, Polym. 36. Nishikawa, S.; Ono, S. X-ray diraction of cotton
Phys. 1994, 32, 1229. cellulose bers. Proc. Tokyo Math, Phys. Soc. 1913, 7, 131.
18. Kondo, T.; Sawatari, C.; St-J. Manley, R.; Gray, D.G. 37. Meyer, K.H.; Misch, L. Uber den Bau des krystallisiert-
Characterization of hydrogen bonding in cellulose- en Anteils der cellulose, V. Mitteil. Ber. 1937, 70B, 266.
synthetic polymer blend systems with regioselectively 38. Fengel, D. Characterization of cellulose by deconvolu-
substituted methylcellulose. Macromolecules 1994, 27, tion the OH valency range in FTIR spectra. Holzfor-
210. schung 1992, 46, 283.
19. Kondo, T. The relationship between intramolecular 39. Fengel, D. Inuence of water on the OH valency range in
hydrogen bonds and certain physical properties of deconvoluted FTIR spectra of cellulose. Holzforschung
regioselectively substituted cellulose derivatives. J. Polym. 1993, 47, 103.
Sci.: B, Polym. Phys. 1997, 35, 717. 40. Fengel, D. Inuence of the alkali concentration on the
20. Itagaki, H.; Takahashi, I.; Natsume, M.; Kondo, T. formation of cellulose II. Holzforschung 1995, 49, 505.
Gelation of cellulose whose hydroxyl groups are 41. Michell, A.J. Second-derivative F.T.-I.R. spectra of
specically substituted by the uorescent groups. Polym. celluloses I and II and related mono- and oligo-
Bull. 1994, 32, 7781. saccharides. Carbohydr. Res. 1988, 173, 185.
21. Kondo, T.; Nojiri, M. Characterization of the cleavage 42. Michell, A.J. Second-derivative F.T.-I.R. spectra of
of h-glucosidic linkage by Trichoderma viride cellulase native celluloses. Carbohydr. Res. 1990, 197, 53.
using regioselectively substituted methylcelluloses. 43. Michell, A.J. Second-derivative FTIR spectra of native
Chem. Lett. 1994; 10031006. celluloses from Valonia and tunicin. Carbohydr. Res.
22. Nojiri, M.; Kondo, T. Application of regioselectively 1993, 241, 47.
substituted methylcelluloses to characterize the reaction 44. Marrinan, H.J.; Mann, J. Infrared spectra of the
mechanism of cellulase. Macromolecules 1996, 29, 2392 crystalline modication of cellulose. J. Polym. Sci.
2395. 1956, 21, 301.
23. Kondo, T.; Sawatari, C. Intermolecular hydrogen 45. Tsuboi, M. Infrared spectrum and crystal structure of
bonding in cellulose/poly(ethylene oxide) blends: Ther- cellulose. J. Polym. Sci. 1957, 25, 159.
modynamic examination using 2,3-di-O- and 6-O-meth- 46. Liang, C.Y.; Marchessault, R.H. Infrared spectra of
ylcelluloses as cellulose model compounds. Polymer. crystalline polysaccharides: I. Hydrogen bonds in native
1994, 35, 4423. cellulose. J. Polym. Sci. 1959, 37, 385.
24. Kondo, T.; Sawatari, C. Interchain hydrogen bonds in 47. Marchessault, R.H.; Liang, C.Y. Infrared spectra of
cellulose/poly(vinyl alcohol) characterized by DSC and crystalline polysaccharides: III. Mercerized cellulose. J.
solid-state NMR analyses using cellulose model com- Polym. Sci. 1960, 43, 71.
pounds. ACS Symp. Ser. 1998, 680. 48. Tashiro, K.; Kobayashi, M. Theoretical evaluation
25. Kondo, T.; Sawatari, C. A Fourier transform infra-red of three-dimensional elastic constants of native and
spectroscopic analysis of the character of hydrogen regenerated celluloses: Role of hydrogen bonds. Polymer.
bonds in amorphous cellulose. Polymer. 1996, 37, 393. 1991, 32, 1516.
26. Itagaki, H.; Tokai, M.; Kondo, T. Physical gelation 49. Mann, J.; Marrinan, H.J. Crystalline modication of
process of cellulose whose hydroxyl groups are regiose- cellulose: Part II. A study with plane-polarized infrared
lectively substituted by the uorescent groups. Polymer. radiation. J. Polym. Sci. 1958, 32, 357.
1997, 38, 42014205. 50. Ivanova, N.V.; Korolenko, E.A.; Korolik, E.V.; Zban-
27. Kondo, T.; Miyamoto, T. The inuence of intramolec- kov, R.G. Mathematical processing of the IR spectrum
ular hydrogen bonds on handedness in ethylcellulose/ of cellulose. Zh. Prikl. Spektrosk. 1989, 51, 301. Russian.
CH2Cl2 liquid crystalline mesophases. Polymer 1998, 39, 51. Wiley, J.M.; Atalla, R.H. Band assignments in the Ra-
11231127. man spectra of cellulose. Carbohydr. Res. 1987, 160, 113.
28. Sekiguchi, Y.; Sawatari, C.; Kondo, T. A gelation mech- 52. Sugiyama, J.; Persson, J.; Chanzy, H. Combined infrared
anism depending on hydrogen bonding formation in and electron diraction study of the polymorphism of
regioselectively substituted O-methylcelluloses, Carbo- native cellulose. Macromolecules 1991, 24, 2461.
hydr. Polym. 2003, 53, 145153. 53. VanderHart, D.L.; Atalla, R.H. Studies of microstruc-
29. Cellulose and Other Natural Polymer Systems; Brown, ture in native celluloses using solid-state 13C NMR.
R.M. Jr., Ed.; Plenum: New York, 1982; 412 pp. Macromolecules 1984, 17, 1465.
30. Atalla, R.H.; VanderHart, D.L. Native cellulose: A 54. Kondo, T. The assignment of IR absorption bands due
composite of two distinct crystalline forms. Science 1984, to free hydroxyl groups in cellulose. Cellulose 1997, 4,
223, 283. 281.
31. Sugiyama, J.; Vuong, R.; Chanzy, H. Electron dirac- 55. Marechal, Y.; Chanzy, H. The hydrogen bond network
tion study on the two crystalline phases occurring in in Ih cellulose as observed by infrared spectrometry. J.
native cellulose from an algal cell wall. Macromolecules Mol. Struct. 2000, 523, 183196.
1991, 24, 4168. 56. Kolpak, K.; Blackwell, J. Determination of the structure
32. Gardner, K.H.; Blackwell, J. The structure of native of cellulose II. Macromolecules 1976, 9, 273.
cellulose. Biopolymers 1974, 13, 1975. 57. Stipanovic, A.; Sarko, A. Packing analysis of carbohy-
33. Sarko, A.; Muggli, R. Packing analysis of carbohydrates drates and polysaccharides 6. Molecular and crystal
and polysaccharides: III. Valonia cellulose and cellulose structure of regenerated cellulose II. Macromolecules
II. Macromolecules 1974, 7, 486. 1976, 9, 851.
34. Nishiyama, Y.; Langan, P.; Chanzy, H. Crystal structure 58. Blackwell, J. In Cellulose and Other Natural Polymer
Systems; Brown, R.M., Jr., Ed.; Plenum: New York, 79. Isogai, A.; Usuda, M.; Kato, T.; Uryu, T.; Atalla, R.H.
1982; 412 pp. Solid-state CP/MAS 13C NMR study of cellulose
59. Gessler, K.; Krauss, N.; Steiner, T.; Betzel, C.; Sand- polymorphs. Macromolecules 1989, 22, 3168.
mann, C.; Saenger, W. Crystal structure of h-D- 80. Horii, F.; Hirai, A.; Kitamaru, R.; Sakurada, I. Cellulose
cellotetraose hemihydrate with implications for the Chem. Technol. 1985, 19, 513.
structure of cellulose II. Science 1994, 266, 1027. 81. Unpublished data.
60. Gessler, K.; Krauss, N.; Steiner, T.; Betzel, C.; Sand- 82. VanderHart, D.L. J. Magn. Reson. 1981, 44, 117.
mann, C.; Saenger, W. h-D-cellotetraose hemihydrate as 83. Imashino, F.; Maeda, S.; Takegoshi, K.; Terao, T.;
a structural model for cellulose II. An X-ray diraction Saika, A. Intermolecular hydrogen-bonding eect on 13C
study. J. Am. Chem. Soc. 1995, 117, 11397. NMR shielding for enol forms of diketones in the solid
61. Raymond, S.; Heyraud, A.; Tran Qui, D.; Kvick, A.; state. Chem. Phys. Lett. 1982, 92, 642.
Chanzy, H. Crystal and molecular structure of h-D- 84. Horii, F.; Hirai, A.; Kitamaru, R. Solid-state high-
cellotetraose hemihydrate as a model of cellulose II. resolution 13C-NMR studies of regenerated cellulose
Macromolecules 1995, 28, 2096. samples with dierent crystallinities. Polym. Bull. 1982,
62. Raymond, S.; Henrissat, B.; Tran Qui, D.; Kvick, A.; 8, 163.
Chanzy, H. The crystal structure of methyl h-cellotrio- 85. Bellamy, L.J. The Infrared Spectra of Complex Mole-
side monohydrate 0.25 ethanolate and its relationship to cules; Chapman and Hall: London, 1975.
cellulose II. Carbohydr. Res. 1995, 277, 209. 86. Hishikawa, Y.; Togawa, E.; Kataoka, Y.; Kondo, T.
63. Gagnaire, D.; St-Germain, J.; Vincendon, M. NMR Characterization of amorphous domains in cellulosic
evidence of hydrogen bonds in cellulose solutions. J. materials using a FTIR deuteration monitoring analysis.
Appl. Polym. Sci. Appl. Polym. Symp. 1983, 37, 261. Polymer. 1999, 40, 7117.
64. Kondo, T.; Gray, D.G. The preparation of O-methyl- 87. Hess, K.; Kiessig, H.; Gundermann, J. Z. Phys. Chem.
and O-ethylcelluloses having controlled distribution of 1941, B49, 64.
substituents. Carbohydr. Res. 1991, 220, 173. 88. Hermans, P.H.; Weidinger, A. On the recrystallization
65. Kondo, T.; Gray, D.G. Facile method for the prepara- of amorphous cellulose. J. Am. Chem. Soc. 1946, 68,
tion of tri-O-(alkyl)cellulose. J. Appl. Polym. Sci. 1992, 2547.
45, 417. 89. Wadehra, I.L.; St. J. Manley, R. Recrystallization of
66. Kondo, T. Preparation of 6-O-alkylcellulose. Carbo- amorphous cellulose. J. Polym. Sci. 1965, 9, 2627.
hydr. Res. 1993, 238, 231. 90. Jezirny, A.; Kepka, S. Preparation of standard amor-
67. Kondo, T. Hydrogen bonds in regioselectively substi- phous specimens for X-ray analysis of ber crystallinity.
tuted cellulose derivatives. J. Polym. Sci.: B, Polym. J. Polym. Sci. Polym. Lett. Ed. 1972, 10, 257.
Phys. 1994, 32, 1229. 91. Jeries, R. Preparation and properties of lms and bers
68. Kondo, T. The assignment of IR absorption bands due of disordered cellulose. J. Appl. Polym. Sci. 1968, 12, 425.
to free hydroxyl groups in cellulose. Cellulose 1997, 4, 92. Atalla, R.H.; Ellis, J.D.; Schroeder, L.R. Some eects of
281. elevated temperatures on the structure of cellulose and
69. Blackwell, J.; Marchessault, R.H. Cellulose and Cellulose its transformation. J. Wood Chem. Technol. 1984, 4,
Derivatives; John Wiley and Sons: New York, 1971. Part 465.
IV. 93. Schroeder, L.R.; Gentile, V.M.; Atalla, R.H. Non-
70. Kamide, K.; Okajima, K.; Kowsaka, K.; Matsui, T. CP/ degradative preparation of amorphous cellulose. J.
MASS 13C NMR spectra of cellulose solids: An expla- Wood Chem. Technol. 1986, 6, 1.
nation by the intra-molecular hydrogen bond concept. 94. Isogai, A.; Atalla, R.H. Amorphous celluloses stable in
Polym. J. 1985, 17, 701. aqueous media: Regeneration from SO2amine solvent
71. Silverstein, R.M.; Bassler, G.C.; Morrill, T.C. Spectro- systems. J. Polym. Sci. Polym. Chem. Ed. 1991, 29, 113.
metric Identication of Organic Compounds, Fourth Edi- 95. Nelson, M.L.; OConnor, R.T. Relation of certain
tion; John Wiley & Sons: New York, 1981; 112 pp. infrared bands to cellulose crystallinity and crystal lattice
72. Dudley, R.L.; Fyfe, C.A.; Stephenson, P.J.; Deslandes, type: Part II. A new infrared ratio for estimation of
Y.; Hamer, G.K.; Marchessault, R.H. High-resolution crystallinity in celluloses I and II. J. Appl. Polym. Sci.
13
C CP/MAS NMR spectra of solid cellulose oligomers 1964, 8, 1311.
and the structure of cellulose II. J. Am. Chem. Soc. 1983, 96. Hatakeyama, H.; Hatakeyama, T. Structural change of
105, 2469. amorphous cellulose by water- and heat-treatment.
73. Nojiri, M.; Kondo T. unpublished work. Macromol. Chem. 1981, 182, 1655.
74. Dorman, D.E.; Roberts, J.D. Nuclear magnetic reso- 97. Burchard, W. Lichtstreuuntersuchungen an Polysaccha-
nance spectroscopy. Carbon-13 spectra of some pentose ridlosungen. Das Papier 1994, 48, 755.
and hexose aldopyranoses. J. Am. Chem. Soc. 1970, 92, 98. Burchard, W.; Schulz, L. Functionality of the h(1,4)
1355. glycosidic linkage in polysaccharides. Macromol. Symp.
75. Perlin, A.S.; Casu, B.; Koch, H.J. Congurational and 1995, 99, 57.
conformational inuences on the carbon-13 chemical 99. Burchard, W. Polymer structure and dynamics, and
shifts of some carbohydrates. Can. J. Chem. 1970, 48, polymerpolymer interactions. Adv. Coll. Int. Sci. 1996,
2596. 64, 45.
76. Parfondry, A.; Perlin, A.S. 13C-N.M.R. spectroscopy of 100. Seger, B. Ph.D. Thesis, University of Freiburg, 1996.
cellulose ethers. Carbohydr. Res. 1977, 57, 39. 101. Rowland, S.P.; Howley, P.S. Structure in amorphous
77. Dudley, R.L.; Fyfe, C.A.; Stephenson, P.J.; Deslandes, regions, accessible segments of brils, of the cotton
Y.; Hamer, G.K.; Marchessault, R.H. High-resolution ber. Text. Res. J. 1988, 58, 96.
13
C CP/MAS NMR spectra of solid cellulose oligomers 102. Jeries, R. An infra-red study of the deuteration
and the structure of cellulose II. J. Am. Chem. Soc. 1983, of cellulose and cellulose derivatives. Polymer 1963, 4, 375.
105, 2469. 103. Taniguchi, T.; Harada, H.; Nakato, K. Accessibility of
78. Fyfe, C.A.; Stephenson, P.J.; Veregin, R.P.; Hamer, hydroxyl groups in wood. Mokuzai Gakkaishi 1966, 12,
G.K.; Marchessault, R.H. Carbohydr. Chem. 1984, 3, 215.
663. 104. Shin, J.-H.; Kondo, T. Cellulosic blends with
98 Kondo
poly(acrylonitrile): Characterization of hydrogen 111. Haines, A.H. Relative reactivities of hydroxyl groups in
bonds using regioselectively methylated cellulose carbohydrates. Adv. Carbohydr. Chem. 1976, 33, 11.
derivatives. Polymer. 1998, 29, 6899. 112. Zugenmaier, P.; Haurand, P. Structural and rheological
105. Yano, S.; Hatakeyama, H.; Hatakeyama, T. Eect of investigations on the lyotropic, liquid-crystalline system:
hydrogen bond formation on dynamic mechanical O-ethylcellulose-acetic acid-dichloroacetic acid. Carbo-
properties of amorphous cellulose. J. Appl. Polym. Sci. hydr. Res. 1987, 160, 369.
1976, 20, 3221. 113. Ritcey, A.M.; Holme, K.R.; Gray, D.G. Cholesteric
106. Hatakeyama, T.; Ikeda, Y.; Hatakeyama, H. Eect of properties of cellulose acetate and triacetate in triuoro-
bound water on structural change of regenerated acetic acid. Macromolecules 1988, 21, 2914.
cellulose. Macromol. Chem. 1987, 188, 1875. 114. Pawlowski, W.A.; Gilbert, R.D.; Fornes, R.E.; Pur-
107. Bikales, N.M., Segal, L., Eds.; In Cellulose and Cellulose rington, S.T. The thermotropic and lyotropic liquid-
Derivatives. Part IV, Chapter XIII; New York: John crystalline properties of acetoacetoxypropyl cellulose. J.
Wiley & Sons, 1971. Polym. Sci.: B Polym. Phys. 1987, 25, 2293.
108. French, A.D. In Cellulose Chemistry and its Applications; 115. Guo, J.-X.; Gray, D.G. Preparation and liquid crystal-
Nevell, T.P., Zeronian, S.H., Eds.; Ellis Horwood: line properties of (acetyl)(ethyl)cellulose. Macromole-
Chichester, 1985. Chapter 3. cules 1989, 22, 2082.
109. Croon, I.; Lindberg, B. The distribution of substituents 116. Guo, J.-X.; Gray, D.G. Chiropitical behavior of (acetyl)
in partially methylated celluloses part 1. Svensk Papper- (ethyl)-cellulose liquid crystalline solutions in chloro-
stidn. 1957, 60, 843. form. Macromolecules 1989, 22, 2086.
110. Croon, I.; Lindberg, B. The distribution of substituents 117. Budgell, D.R.; Gray, D.G. In Polymer and Fiber Science:
in partially methylated celluloses part 3. Svensk Papper- Recent Advances; Fornes, R.E., Gilbert, R.D., Eds.;
stidn. 1958, 61, 919. VCH: New York, 1992; 145 pp. Chapter 12.
4
X-ray Diraction Study of Polysaccharides
Toshifumi Yui
Miyazaki University, Miyazaki, Japan
Kozo Ogawa
Osaka Prefecture University, Sakai, Osaka, Japan
I. INTRODUCTION In comparison with other biopolymers, polysaccha-

rides are characterized by their diversity, the presence of a
The requirement for information regarding the three-di- large number of functional groups, and their conforma-
mensional structure of polysaccharides at the molecular tional rigidity. Even unsubstituted pyranoglycans contain
level is growing for a number of reasons: these molecules three hydroxyl groups per sugar residue. In addition, most
have been regarded as biodegradable polymer materials, polysaccharides can be found in the form of linear or
compared to the usual synthetic polymers; polysaccharides branched homopolymers or copolymers based on two or
are the most abundant organic materials in nature; and a more dierent sugars as constituents. Some copolymers
great variety of polysaccharides composed of various also result from variations of the linkage structure in the
monosaccharide residues and linkages have been found. sequence of the same sugars. Several extracellular poly-
Polysaccharides can be broadly classied into three groups saccharides have more complicated chemical structures
based on their functions, which are closely related to their composed of one to as many as seven kinds of sugars in
occurrence in nature: structural, storage, and gel forming. their chemical repeating unit.
Structural polysaccharides, typical examples being cellu- The above characteristics are easily interpreted, con-
lose in plant cell walls and chitin in exoskeletons of many sidering the basic stereochemical features of sugars. For
insects, form long brils or sheets which play a supporting example, in the case of homoglucans, the hydroxyl group at
role in various organisms. Generally, their molecular C(1) of glucose can chemically bond with one of the four
chains form extended twofold helical conformations. Stor- hydroxyls of another glucose (i.e., 1!2, 1!3, 1!4, and
age polysaccharides characterized by highly branched 1!6 linkages), and the position of the glycosidic oxygen is
chains are thought to be folded back on themselves to yield either axial or equatorial to the pyranose ring (a- or h-
compact structures. Amylose, amylopectin, and glycogen anomer), leading to two stereoisomers for each bond type.
are examples of this type of polysaccharides. The gel- Thus polymerization of D-glucose residues provides eight
forming, network polysaccharides, such as alginic acids types of homoglucans, each with a dierent glycosidic
and mucopolysaccharides, which are found in the cell walls linkage structure, all of them having been found in nature.
and intercellular regions of certain algae and seaweeds or Given the fact that many kinds of sugar in pentose or
the amorphous matrix material of animal connective tis- hexose could form six dierent types of homoglycans from
sues, serve as water-holding substances in these organisms. the former or eight from the latter, various homoglycans
In addition, some polysaccharides have recently been have been found.
found useful as biomedical materials, such as chitin/chito- A theoretical treatment of the homopolysaccharide
san showing antibacterial action and a branched (1!3)-h- conformations was carried out by Rees and Scott [1], who
D-glucan having antitumor activity. These physiologically proposed that the typical molecular shapes of homoglu-
active polysaccharides are considered to enhance the im- cans and other homoglycans, such as galactan, mannan,
mune system systematically, resulting in antitumor and xylan, and arabinan, could be characterized in terms of
antibacterial activities. four conformational types: type Aextended and ribbon-
99
100 Yui and Ogawa
like; type Bexible and helical; type Crigid and crum- II. X-RAY STRUCTURE ANALYSIS
pled; type Dvery exible but, on the average, rather
extended. X-ray and electron diraction studies of various A. Sample Preparation
homoglycans have demonstrated that the chain conforma-
tions of these polysaccharides in crystals are reasonably Some brous materials are naturally present as a highly
consistent with the predictions of Rees and Scott [1]. oriented assembly of microcrystals, such as ramie and
The conformational rigidity of polysaccharides is cotton bers and tendon chitosan. Otherwise, the diract-
revealed by inspecting the conformational probability ing specimen for a given polysaccharide must be prepared
map of the polymer where a potential energy contour by organizing the molecular chains. Only the ber diagram
map is drawn as a function of the two rotational angles of high quality allows one the crystal structure analysis of
of the glycosidic linkage. The allowed area for a polysac- high resolution, and therefore a well-oriented and highly
charide where no steric overlaps occur between residues is crystallized ber sample is essential. Unfortunately, no
generally much smaller than that for a polypeptide. The common methodology has been established to prepare
polyfunctional nature of polysaccharides as polyalcohols such a good ber sample valid for all polysaccharides.
explains their ability to form a multitude of intermolecular The following two methods have mostly been adopted to
hydrogen bonds in the solid state. Coupled with the obtain a uniaxially oriented ber sample: spinning a ber
conformational rigidity of the chains, which results in a and stretching a lm. The former is obtained by extruding a
self-ordering tendency, almost all polysaccharides readily concentrated polymer solution into a precipitant solvent or
form microcrystalline phases in the solid state, where the solution. The latter is prepared by stretching the lm cast
crystal structures are marked by extensive hydrogen-bond- from a polymer solution. Usually, the ber-forming poly-
ing network. saccharides of the former class are able to form continuous
In the x-ray structure analysis of a biopolymer, it lms from their solutions as well. However, the opposite is
should be noted that a polysaccharide crystal is simpler not always true. In order to facilitate a ber or lm
but far less complete than for a globular protein. One can stretching, a polysaccharide of higher molecular weight is
obtain a single crystal of a globular protein large enough, desirable and the lms must be continuous, soft, and of low
say 0.5-mm diameter, for x-ray analysis. Even smaller crystallinity in advance of stretching. Several methods for
crystals can be analyzed using synchrotron radiation. stretching polysaccharide lm have been reported. A poly-
The number of x-ray reections observed from the single mer lm prepared by casting is cut into strips approximate-
crystal may be more than a few thousands. In contrast, a ly 2 mm wide, and the strip is stretched under constant load
polysaccharide, as well as most of other polymers, never using a weight of a few grams [2]. Another common method
provides a single crystal large enough for x-ray diraction is to use a stretching tool by which one can manually
analysis. It sometimes forms a microscopic single crystal stretch the strips. The former method requires more skills
which only can serve an electron diraction study. The x- but is more likely to provide a sample of high orientation.
ray diraction pattern from a polysaccharide crystal is In both cases, stretching must be performed under a
referred to as a ber diagram since it is diracted from a desirable atmosphere, such as in air, under controlled
ber sample. A ber sample is a polycrystalline material relative humidity, in various solvents (or a mixed solvent),
consisting of uniaxially oriented microcrystallites along at a certain temperature, or under the combination of some
the stretched direction of it. They are randomly oriented of them, which depends on polysaccharide. Water, water
about the lateral directions and comprise noncrystalline alcohol (often isopropanol) mixture, or glycerin are often
region to some extent. Thus a resolution of crystal struc- used as solvent for stretching, although this aspect depends
ture analysis is signicantly aected by quality of a ber on polymer properties such as solubility. Even having
diagram, depending on the degrees of crystallization and obtained a well-oriented lm or ber, the sample to be
of orientation of microcrystallites. The helix axis of a x-rayed must be of high crystallinity. To improve crystal-
molecular chain coincides with the oriented direction of linity, the uniaxially oriented sample is annealed at a high
microcrystallites and, consequently, of a ber sample. In temperature or rinsed with an acid solution, such as
addition, there is the simplifying fact that the molecular aqueous hydrochloride. The former procedure is done in
chain axis in microcrystallites is parallel to one of the three any solvent (e.g., water), a mixed solvent (e.g., water
axes of the unit cell, usually assigned to the c-axis. This axis isopropanol), or water vapor, usually not in air, except
is called the ber axis, and the unit cell length along it is for some polysaccharide derivatives. A sealed bomb is
called the ber repeat. Experimentally, the ber repeat is recommended for annealing at temperatures higher than
readily derived from the layer line spacing of the ber the boiling point of the solvent. At any rate, it should be
diagram. noted that there is practically no recipe to achieve success-
The present article describes briey the general tech- ful sample preparation except for trial and error.
niques of the x-ray ber diraction analysis, which includes It seems that ambivalent properties are required for
the computer-aided model building and structure rene- the polymer material to form the oriented polycrystalline
ment methodology specialized in polysaccharide crystals. phase. For example, the lm of excessive crystallinity is not
The rest of the article introduces the molecular and crystal appropriate for succeeding stretching. The crystallinity
structures of several topical polysaccharides followed by depends on many factors. Chemical composition in terms
the recent advances in the cellulose crystal structures. of polysaccharide residue and linkage type is principal.
X-ray Diffraction Study of Polysaccharides 101
The molecular weight (Mw) of the polymer is also essen- atives, such as (1!3)-a-D-glucan tribenzoate, are swollen
tial. A low Mw is favorable for crystallization but disad- or dissolved by these solvents, in which case an inorganic
vantageous for orientation, whereas high Mw is just the salt solution, such as aqueous NaI solution, can be used. A
opposite. Other factors are the solvent, temperature, and graduated glass column in which the mixture ratio (con-
so on. A typical example of how to solve the ambivalent centration gradient) of the two solvents changes continu-
problem is the case of (1!3)-a-D-glucan [3]. This glucan is ously from the top of the column to the bottom can also
not soluble in water but soluble in aqueous alkali and serve for the density measurement. The ber sample is put
cellulose solvents, such as hydrazine hydride and N-meth- into the column top and is allowed to go down until
ylmorpholine N-oxidedimethyl sulfoxide. Attempts to reaching the zone with the same density; at this point, the
prepare a continuous lm or a well-oriented ber from graduation is read.
such solutions were not successful. However, the glucan
was acetylated, dissolved in chloroform, and cast into lm, B. X-ray Fiber Diffraction Measurements
from which a well-oriented ber sample was obtained by
stretching in hot glycerin. The ber was deacetylated in The ber diraction diagram is usually taken using a at-
sodium methoxidemethyl alcohol, while keeping the lm camera with which the diraction beams arising
length of the ber constant. The crystallinity of the regen- through the ber sample are collected on an x-ray photo
erated glucan ber was remarkably improved by annealing lm. When the irradiation is done in the air, the x-ray beam
in water in a sealed bomb. The resultant sample of (1!3)- scattered by the air causes the occurrence of a (sometimes
a-D-glucan gave a ber diraction pattern of high quality serious) background scattering on the x-ray lm. In addi-
[3]. In conclusion, preparation of the x-ray ber sample tion, not only polysaccharides, but also other biopolymers
requires a challenging spirit. Finally, it depends on luck, often have water molecules in their crystals. They often
the challenger sometimes encounters polysaccharides change the crystalline polymorphs with relative humidity.
which have never been crystallized. A good example is the case of (1!3)-h-D-glucan. As
Knowledge of the density of the polysaccharide lm or described later, the glucan exhibits both the hydrated and
ber to be x-rayed is necessary in order to know what kind anhydrous polymorphs and they transform readily and
and how many molecules are packed in the crystalline unit reversibly to each other by changing the relative humidity
cell. The density measurement is performed using a mixture in the x-ray camera [4]. Not only to avoid x-ray scattering
of two solvents which do not dissolve nor swell the poly- by air, but also to control relative humidity, a box camera is
saccharide, such as xylene and carbon tetrachloride. The recommended to use for obtaining good ber diagrams of
powder-like samples obtained by grinding the polymer lm polysaccharides and other brous biopolymers. By passing
or ber are put into a mixture of xylene and carbon humidity-controlled helium gas through the camera, the
tetrachloride in a stoppered measuring cylinder in a ther- ber diagram can be obtained under any relative humidity.
mostat bath at, for example, 25jC. Xylene or CCl4, as A helium-gas atmosphere causes practically no back-
necessary, is added to the suspension until the sample ground scattering because the helium atom has only two
settles in the cylinder. Then, the solution and the sample electrons.
are of the same density, and the density of the solution is Fig. 1 shows typical x-ray ber diagrams of the
measured with a picnometer. If the density of the sample is oriented crystalline samples of a polysaccharide. Although
higher than that of CCl4, a mixture of CCl4 and ethylene a ber diagram corresponds to the rotation diagram of
dibromide may be employed. Some polysaccharide deriv- single crystals where the rotation axis coincides with the
Figure 1 Fiber diagrams of three polymorphs of chitosan. Left: tendon (hydrated) polymorph; middle: annealed (anhydrous)
polymorph; right: type II form which was obtained with chitosan HCl salt.
102 Yui and Ogawa
ber axis, it has much poorer quality than a single-crystal obtaining ber patterns by x-ray irradiation. Then, the
data. Three-dimensional reection data are reduced to a intensities of all the reections appearing on all the lms are
two-dimensional pattern, generally accompanied by an measured. This procedure requires tough work and is time-
overlapping of reection peaks. The diraction spots are consuming, and the multiple lm pack technique often
broad and diminish rapidly in intensity with increasing provides erroneous evaluation for some of the intensities.
diraction angles due to the small size of each microcrystal. An advance in x-ray diraction measurement is rep-
Therefore most reections may disappear into the back- resented by development of an imaging plate (IP) to replace
ground scattering arising from noncrystalline regions. the x-ray photo lm [811]. The IP has been widely used
Disorders of arrays of microcrystals along the ber axis in the x-ray analysis of a globular protein single crystal and
cause severe arcing in a prole of diraction spotsin in the synchrotron x-ray diraction studies. The plate is a
particular, at larger diraction angles. The diraction plastic disk coated with photostimulable phosphor crystals
angle, 2h, of each reection is obtained by measuring the and is a new type of two-dimensional detector for x-ray
distance from the center of the pattern to the diraction beams. The diraction pattern recorded on the IP is read by
maximum using a comparator. Crystalline powder of measuring the uorescence intensity stimulated by a He
sodium uoride is lightly dusted on the sample in order Ne laser beam, and all the data can be saved in various
to provide a calibration diraction ring (0.2319 nm) on the storage media. In addition, the plate can be used repeatedly
ber diagram. The spacing between adjacent reection by using an erasing machine. The IP is characterized by a
planes, d, and h are related by the Bragg law: wide dynamic range (106) and high sensitivity, which
readily accomplishes about 10 times shorter exposure time
nk 2dsinu; n 1; 2; 3; . . . and 1060 times higher accuracy in measuring diraction
intensity than the use of conventional x-ray lms. Particu-
where k is the wavelength of the x-ray radiation. It is larly, the wide dynamic range of the IP provides a great
sometimes dicult to dene the diraction maximum of advantage in measuring the ber diraction data where a
some spotsfor instance, those giving the peak proles relative intensity of each spot often varies by a factor of 103
that unsymmetrically broaden or diuse out in the back- so that only a single IP can serve for measuring the
ground scattering. The former case may arise from the intensities of all reections occurring from a ber crystal.
combined spot consisting of several diraction peaks with Obata and Okuyama [911] reported the ber diraction
the 2h values being close each other. A set of d-spacing data collection and processing system using IP.
values is then used to determine unit cell parameters The relative intensities, I0, are converted to the ob-
(lengths of a-, b-, and c-axes and angles of a, b, and c) served structure amplitudes, jF0j, by the equation:
and the space group of a given crystal by a trial and error
method. AF0 A KI0 =Lp1=2
The next step is measuring the relative intensity of each
reection on a ber diagram in order to get the structure In the equation, Lp is the Lorentz polarization correction
amplitude of each reection plane, which will be described factor for the ber diraction data, and the scale factor K
in the next section. The intensities of diraction peaks are incorporates the geometric corrections.
generally obtained by radial scans of a microdensitometer
on x-ray lms to trace the diraction maximum of each C. Refinement of Molecular and Crystal Structure
spot. The combined diraction peaks may have to be
resolved into individual peaks by the least-squares curve- Unlike an ordinary single-crystal structure analysis in
tting procedure. However, when dealing with such one- which a large amount of reection intensity data on order
dimensional peak proles, one sometimes nds that a of 103104 is available, the exact positions of individual
measurement for severely deformed or arced peak is likely atomic coordinates in the unit cell cannot be determined
to result in unreasonable value. The two-dimensional scans solely from the ber diraction data. Usually, in the case of
over the whole diraction pattern should provide more polysaccharide crystal structures, the geometry of the sugar
appropriate proles of diraction peaks; this requires a residue is rst dened based on the structural data of
computer-controlled microdensitometer to process a large relevant small molecules that have been determined by
amount of digitized data. Compared with one-dimensional the single-crystal data. As shown in Fig. 2, with the xed
scanning data, further sophisticated mathematical tech- residue geometry, the helical conformation of a polysac-
niques are necessary for the two-dimensional data in the charide is described by either a pair of glycosidic torsional
background removal and the peak prole resolutions. The angles (U, W) and glycosidic bridge angle, s, or the rotation
details of the two-dimensional collection and processing of of an entire residue, h, around a virtual bond (an
a ber diraction diagram were discussed by Millane and imaginary bond connecting the glycosidic oxygens). In
Arnott [57]. As shown in Fig. 1, a large variation of the the case of a regular helical chain model, the former
intensity of reection from strong to weak is observed, parameters must be adjusted under the constraints of a
depending on the number of electrons present on the set of the helix parameters: h, an axial rise per residue, and
reection plane. However, the dynamic range of the usual n, a number of residues per helix repeat. These values,
x-ray photo lm is around 102. Consequently, a set of lms related to the ber repeat distance, c, such that c=hn, can
where four to ve lms are piled up must be used for be obtained directly from a measurement of meridional
In this equation, the rst term describes the dierences

between observed, Fom, and calculated, Fcm, structure
amplitudes, each being given the weight, Wm. The second
term represents the sum of the nonbonded interaction
energies, eij, that estimate stereochemical acceptability of
the crystal model. The third term, Gq, is a set of constraint
relationships among the parameters that are to be zero, and
kq is initially undetermined Lagrange multipliers. The
minimizing function installed in PS79 is
( )
X X
2
P fR 1 f Dk w eij
k ij
where the fractional weight, f, balances the x-ray crystal-

lographic and stereochemical terms. In addition to the
nonbonded interactions (third term), the second term also
involves the bonded interactions, Dk, to evaluate the
amount of deviations of any bond length, bond angle, or
Figure 2 Chain conformational and helix parameters for conformation angle from their standard values. The rst
the twofold helical chain (n=2) represented by the regular term can be either the x-ray crystallographic residuals
helical structure of chitin. All hydrogen atoms are omitted. X X
R AAFom A AFcm AA= AFom A
m m
or the weighted one

reections of a ber diagram. Exocyclic rotational groups,
v, such as a hydroxyl methyl group on C(5) and N-acetyl " #1=2
X X
and O-acetyl groups for sugar derivatives, can be rotated RW 2
Wm AAFom A AFcm AA = Wm AFom A2
when necessary. Although these parameters are called m m
chain conformational parameters, those that dene the
positions of an entire molecular chain in a unit cell are These residuals are also calculated for crystal models in
the chain-packing parameters. They are chain rotation LALS. Minimizing these functions are carried out by the
about the helix axis, the z-translation of the chain along constrained least-squares process in LALS and by Com-
the ber axis, and the xy positions of the helix axis on an plex method in PS79. The latter method performs a direct
ab plane of a unit cell. search for the minimum of a multidimensional function by
The two representative programs that are principally evaluating the function at several trial points, while varia-
aimed at studying the molecular and crystal structures of bles as well as the functions are conned within given
biopolymers have been developed: LALS [12] and PS79 constraints or limits. Advantages to this method, therefore,
[13]. Both programs, in addition to the ordinary procedure over those of PS79 in structure determination, are that the
for x-ray diraction analysis, are equipped with the stereo- number and type of variable and their limits and con-
chemical renement approach to complement the ber straints are readily introduced and changed in the course of
diraction data of poor quality. The algorithms and back- the minimization. A disadvantage is that the optimization
ground theories adopted by the programs have been de- proceeds very slowly as it approaches the minima, in
scribed in detail by their respective authors, along with the particular, in the nal stage of the structure renement
strategies in solving polymer crystal structures [12,13]. The where more variables should be involved. In spite of above
discussion herein will focus on the comparisons between dierences in the model building and minimizing proce-
the two methods. With regard to helix model building and dures, it was suggested that the two methods were able to
its renement, LALS uses the glycosidic linkage parame- produce essentially similar structures when the renement
ters U, W, and s to describe chain conformation, whereas was carried appropriately [14].
PS79 adopts the virtual bond method. Either method The general strategy of crystal structure analysis
allows a monomer geometry to vary in the course of using the above programs is that it proceeds on a trial-
structure analysis. The virtual bond approach of PS79 and-error basis. Possible crystal models are established
has been originally developed for solving a polysaccharide and each is evaluated against the stereochemical restraints
conformation. LALS is designed to be more exible and and the x-ray diraction data at individual steps of the
readily applicable to the other biopolymer systems such as structure analysis. As the structure determination pro-
polypeptides and polynucleotides. The quantity of the ceeds, inferior models are eliminated, and the most prob-
following function is minimized in LALS: able one is nally selected. The rst stage of structure
X X X determination is to construct the helical model of a
V Wm jFom Fcm j2 S eij kq Gq polysaccharide chain. As mentioned earlier, the starting
m ij q geometry of a monomer residue can be taken from the
104 Yui and Ogawa
atomic coordinates obtained by the x-ray single-crystal factors by placing a single water molecule on each grid
studies. When appropriate experimental data are not point over the ab plane in turn. These xy positions are
available, the optimized structure by molecular mechanics subsequently extended to the three-dimensional case by
(MM) calculations may be an alternative source of mono- introducing the hk1 diraction data. Similar methodology
mer geometry. As for a- and h-D-glucoses, their standard can be applied to the case of polysaccharide salts in
residues whose atomic coordinates were averaged from determining the locations of ions. An alternative method
the crystal structures of several relevant sugars were for evaluating the eect of water molecules is the use of the
proposed [15]. The chain conformation is rened by water-weighted atomic scattering factors [17]. This ap-
minimizing the stereochemical interactions with respect proach assumes that water molecules do not reside at
to the chain conformational parameters mentioned above, dened crystallographic positions and that their electrons
by imposing the helical symmetry dened by n and h. In are smeared out throughout the unit cell. The crystal
the second stage, molecular chains are placed based on the structure of curdlan hydrate introduced later was deter-
information on the density of crystals and the space group mined by combining the above methods; for 36 water
suggested forms the diraction pattern. The poor quality molecules present in the unit cell, one-half was found at
of diraction pattern often prevents one from discrimi- the dened positions near monomer residues, and the other
nating a unique space group among the possible ones. In half was distributed in a statistical manner [18].
such a case, all packing models having possible symme-
tries are constructed and tested. The authors of both PS79
and LALS suggested that an initial search for chain- III. MOLECULAR CONFORMATIONS
packing parameters should proceed solely with the stereo-
OF TOPICAL POLYSACCHARIDES
chemical constraints, not based on the x-ray diraction
data [13,16]. The primarily reason for this was that the A. Polyaminosugars
calculations of structure amplitudes were considered to be
more time-consuming than those of stereochemical func- 1. Chitosan
tions. At present, however, such a problem becomes So far, one of the most promising polysaccharides is
virtually negligible owing to the drastic development of chitosan, a linear polymer of h-(1!4)-linked 2-amino-2-
computer hardware in the last decades. In fact, LALS, deoxy-D-glucose residues, which is readily prepared from
which was originally designed for a main-ame computer, chitin by chemical N-deacetylation. Chitin, chitosan, and a
can be implemented by a commonly used PC! Further- partially N-acetylated chitosan have been widely developed
more, because the potential surface of the steric energy is to be used as antimicrobials, biomedical materials, cos-
generally more complicated and consists of more local metics, food additives, separators, sewage disposal, agri-
minima than that of the x-ray crystallographic residuals, cultural materials, and so on. The chemical and
R and RW, the minimum position may be more easily as biochemical reactivity of chitosan and the partially acety-
well as quickly identied on the latter surface. The stereo- lated chitosan are higher than those of chitin because
chemical search is virtually useless in determining hydrat- chitosan has free primary amino groups distributed regu-
ed crystal structures where the nonbonding, interchain larly in its chain. Crystal structures of chitin had been
interactions are mostly negligible due to the involvement analyzed by Gardner and Blackwell [19] for the h-chitin
of water molecules. It therefore seems to be more appro- polymorph and by Minke and Blackwell [20] for a-chitin
priate that crude structure with regard to chain-packing and have been presented in many books (e.g., Ref. [21]).
positions is determined by using the x-ray diraction data. The rst ber diagrams of chitin and chitosan were
Chain rotational positions are rst investigated against reported by Clark and Smith [22] in 1937. However, the
the hk0 diraction data, which is followed immediately by rst complete crystal structure of a chitosan polymorph
a search of the z-translational position using the hk1 data. was published in 1994 [23], although the base (ab) plane
The stereochemical constraints should be introduced, by structure of chitosan crystal had been reported in 1985 [24].
minimizing the total values of the above minimizing So far, three crystalline polymorphs (x-ray ber diagrams)
functions, in the nal stage of structure renement where of chitosan have been found. One, the most abundant, is
the packing and conformational parameters are simulta- called the tendon chitosan polymorph (Fig. 1 left) [25],
neously rened. Care must be taken in introducing attrac- which is prepared from chitin of a crab tendon by N-
tive interaction, such as the hydrogen-bonding deacetylation. It is a hydrated form found by Clark and
interaction, into the crystal structure model. Introducing Smith, where the chitosan molecule forms an extended
it on an improper renement step may lead to prejudiced twofold helix (Fig. 3a) in the crystal [26]. The second is the
models. annealed polymorph (Fig. 1 middle) which is anhydrous
The crystal structure analysis of hydrated polysac- [27]. Cairns et al. [28] found a dierent ber pattern of
charides requires extra parameters for the locations of chitosan but similar to that for a hydrochloride salt of
water molecules. After placement of molecular chains in chitosan [29] (Fig. 1 right), and they proposed an extended
the unit cell, initial xy locations of waters are generally 8/5 (a left-handed eightfold) helix for the chitosan confor-
obtained by calculating either a dierence Fourier or R mation. When the chitosan sample was stored at 98%
factor map of the ab projection plane with the hk0 dirac- relative humidity, it exhibited another pattern of the more
tion data. The latter map represents variations in R (or RW) conventional twofold helix [28].
hydrogen bonds contribute to stabilizing the three-dimen-

sional structure in the crystal. The anhydrous crystal of
chitosan neither dissolves in any aqueous acid solution nor
forms a complex with any transition metal ion [23]. There-
fore the anhydrous chitosan may be considered an inert
material. The reason this polymorph is called annealed is
that it has been obtained by heating a stretched chitosan
lm in the presence of water [27]. Later, no annealing was
found to be necessary to obtain the anhydrous crystal for a
chitosan sample having lower molecular weight [25].
Having a regular distribution of the aliphatic primary
amino groups, chitosan exhibits a remarkable ability to
form salts with acids and to form complexes with transition
metal ions [28]. As shown in Table 1 [31], the crystalline
polymorphs of chitosan salts with both inorganic [32] and
organic [3336] acids are divided into two types, depending
on the acid used. In the case of some salts, they depend on
Figure 3 Packing structure of hydrated chitosan projected

along the a-axis (a) and along the c-axis (b). Filled circles
denote nitrogen atoms. For the sake of clarity, only three
polymer chains of the lower layer in (b) are shown in (a).
(From Ref. 26.)
As shown in Fig. 3, the chitosan molecules in the

hydrated form have the twofold helical symmetry rein-
forced by an O(3)UO(5) hydrogen bond with the repeating
period of 1.034 nm [26]. This is a typical structure for the
h-(1!4)-linked polysaccharides such as cellulose, mannan,
and chitin. The orientation of O(6) has a gt conformation
[30]. Adjacent chitosan chains along the b-axis are arranged
in an antiparallel fashion and are linked to each other by
two N(2)UO(6) hydrogen bonds along the b-axis (Fig. 3a).
These sheets are piled up along the a-direction (Fig. 3b). No
direct hydrogen bond is present between the sheets, but the
hydrogen bonds via water molecules hold the chain sheet to
stabilize the whole packing structure.
In the annealed polymorph, the extended twofold
helical conformation of chitosan is also stabilized by
intramolecular O(3)UO(5) hydrogen bonds, and an O(6)
atom is rotated at near gt position as shown in Fig. 4 [23], Figure 4 Projections of the crystal structure of chitosan in
which are the same features to those observed in the the anhydrous polymorph on the ab (top) and bc (bottom)
hydrated polymorph. Two chains pass through the unit base planes. All hydrogen atoms are omitted, and hydrogen
cell in an antiparallel fashion. Intermolecular N(2)UO(6) bonds are shown as dashed lines. (From Ref. 23.)
106 Yui and Ogawa
Table 1 Classication of the Inorganic and Organic Acid A Spontaneous Water-Removing Action of the
Salts of Chitosan Type II Salts
Type Characteristics Acid An interesting phenomenon has been observed in the
type II salts, in particular, with the monocarboxylic acid
Type I Extended twofold HNO3 (high salts [31,34,36], which was rst found for a chitosanacetic
helix (anhydrous) concentrationa), acid salt [34]. The chitosan salt samples freshly prepared
HBr, HI show the x-ray diagram similar to the typical type II salt
L-ascorbic acid (Fig. 1 right) where the chitosan chain forms the less-
L- or D-lactic acid extended twofold helix. A completely dierent ber pattern
(high temperatureb) is then observed when the salt specimen is stored at room
Maleic acid temperature of around 80% RH for 3 months. The dia-
Type II Less extended HNO3 (low gram appears very similar to that of the annealed
twofold helix concentrationc),
polymorph of chitosan (Fig. 1 middle). This indicates that
(hydrated) HF, HCl, H2SO4
acetic acid is spontaneously removed from the chitosan salt
Succinic acid
accompanied by water molecules present in the salt crystal,
Fumaric acid
L-tartaric acid resulting in the transformation to the anhydrous crystal of
L- or D-lactic acid chitosan. Measurements of the density and the FT-IR
(low temperatured) spectrum of the specimen also supported the crystalline
Monocarboxylic transformation [34]. This change is accelerated when the
(formic, acetic, or salt was stored at higher humidity, e.g., at near 100% RH
propionic) acids for approximately 1 month. When a formic acid salt of
chitosan is kept at 100% RH, the crystalline transforma-
a
The acid concentration was 7 M at the salt preparation (Chanzy, tion requires approximately 3 months. In contrast, the
private communication). crystalline transformation for a chitosan propionic acid
b
The salt was prepared at 50jC.
c
The acid concentration was 2.8 M at the salt preparation (Chanzy,
private communication).
d
The salt was prepared at 15jC.
the preparation temperature or on the acid concentration.

The type I salts provide dierent ber patterns to one
another, but all of them correspond to an anhydrous form
and involve the unreacted chitosan chains of the extended
twofold helical conformation [29]. All the type II salts
indicate similar ber patterns to that of chitosan HCl salt
(Fig. 1 right). This indicates that these acid ions are not
located in a regular position in each crystal [29]. Accord-
ingly, it is not likely that the acid ions contribute to the ber
diraction pattern. The molecular and crystal structure of
chitosanformic acid salt (a type II salt) is shown in Fig. 5
[37]. The less-extended twofold helix conformation with
the tetrasaccharide repeat (ber axis length=4.08 nm) as a
helical asymmetric unit was proposed for the chitosan
chain in the type II salt. There are two antiparallel chitosan
chains, one at the corner and the other at the center of the
unit cell (Fig. 5b). Neither acid nor water molecule has been
dened because they are not arranged in a regular position
in the crystal structure.
This shrinking of the molecular conformation ob-
served in the type II salts seems to be unique behavior of a
chitosan chain, which has not been detected for any other
h-(1!4)-linked polysaccharide, such as cellulose or chitin.
The latter two exhibit the fully extended twofold helical Figure 5 Projections of the crystal structure of chitosan in
conformation in all of their crystal forms. The regular the type II on the ac (a) and ab (b) base planes. All hydrogen
distribution of cation, NH+ 3 , along the chitosan chain atoms are omitted. Chitosan chains located at the corner and
would be a primary cause for the chain shrinking with the center of the unit cell are of opposite polarity. (From Ref.
presence of particular anion species. 37.)
salt proceeds faster, requiring 3 weeks. In the case of sample, more moderate method based on spontaneous
butyric acid, only the annealed pattern is observed water-removing action to obtain an inert chitosan sample
because, probably, the transformation is too fast to detect. has been proposed [31]. Instead of adopting a direct
These results indicate that the behavior of the water- transformation from the hydrated to anhydrous poly-
removing action by acid depends on the hydrophobicity morphs, the procedure detours via the type II salt forma-
of it [36]. Recently, the action was found to occur in all the tion, which proceeds under room temperature throughout
type II salts when they are immersed in an isopropanol the process. Whereas the transformations to the anhydrous
water mixture, and, in the case of the monocarboxylic acid polymorph are irreversible from both the hydrated poly-
salts, the procedure considerably accelerates the transfor- morph and the type II salts, the type II salt is readily
mation compared with simply storing them in air [31]. converted to the original hydrated tendon chitosan by
The Crystalline Transformation of Chitosan neutralization with an aqueous alkali such as sodium
hydroxide solution.
As described earlier, the neighboring chitosan chains
In the case of the type I salts, no such transformation
are arranged with antiparallel polarity along the chain
occurs although they are stored for a prolonged period. On
sheet in the hydrated (Tendon) crystal (Fig. 6a), while the
the contrary, all the type I salts change to the original
chain sheet in the anhydrous (Annealed) polymorph con-
hydrated (tendon) chitosan when they are immersed in an
sists of parallel chains (Fig. 6b). The sheetwatersheet
isopropanolwater mixture. The behavior of the type I salts
hydrogen-bonding scheme stabilizes the three-dimensional
is rather predictable because this is nothing more than
structure of the hydrated crystal. Comparison of the chain-
dissociation of salt molecules in aqueous environment.
packing feature between the two polymorphs indicates that
Although any reasonable mechanism for the water-re-
drastic rearrangement of chains appears to be necessary on
moving action has not currently been proposed, the
transformation from the hydrated to the anhydrous crys-
phenomena may intimately be related to the stereochemical
tal, obviously involving breaking and subsequent forma-
strain inherent in the chitosan chain and the crystal lattice
tion of intermolecular hydrogen bonds. This may be a
force. The strict twofold helical conformation observed
possible reason for the very high annealing temperature of
among most of h-(1!4)-linked chains is resulted from the
240jC that is required for preparing the anhydrous poly-
crystal lattice force to achieve a reasonable chain packing.
morph of chitosan having a high molecular weight (e.g.,
Such chains, partly stabilized by O(3)UO(5) intramolecu-
viscosity average degree of polymerization: 10,800) [25].
lar hydrogen bond, are slightly strained as an expense for
Such an excessive heating causes unfavorable thermal
the crystal packing. As obvious from Fig. 6c, the chitosan
decomposition of the chitosan sample, in particular, on
chains are loosely packed in the type II salt crystals, where
the surface, and consequently provides a less inert sample
the chains are relaxed into the less-extended helix. In
of anhydrous chitosan. Therefore as an alternative ap-
addition, there seems to be no direct hydrogen bonding
proach to prepare the anhydrous or annealed chitosan
between the chains in the type II crystal. This facilitates
reformation of the intermolecular hydrogen-bonding
scheme for the anhydrous crystal as a result of releasing
anions as well as waters.
In the crystals of chitosantransition metal complexes
where the free amino groups of chitosan molecule coordi-
nate metal ions, such as cadmium, zinc, cupric, nickel,
cobalt, and mercury ions, the backbone chitosan molecule
always retains the most abundant twofold helical confor-
mation [38]. Improvements [39] of crystallinity of nine
chitosanmetal salts complexes revealed that all the crys-
tals were orthorhombic, and that the unit cell parameters
(a- and b-axis lengths) depended on the counterions of the
metal salt (such as SO42, Cl, AcO, and NO3) and not
the metal ions. In each chitosanmetal complex, a metal
ion is coordinated with an amino group of D-gluosamine
dimer residues. Based on these ndings a pendant model
was proposed for the coordination mode of the chitosan
transition metal complexes [39] which is conict with the
bridge model proposed earlier, where four amino groups
of chitosan chains coordinate one metal ion [40].
Figure 6 Crystalline transformations of chitosan. The ab

planes of (a) hydrated (Tendon) and (b) anhydrous 2. Polygalactosamine
(Annealed) chitosans, and that of (c) the type II salt. Another polysaccharide consisting of amino sugar
Molecules with gray color are up-chain, while the others extracted from the culture uid of Paecilomyces sp. I-1 is
are down-chain. poly[(1!4)-a-D-galactosamine], which is a linear polymer
108 Yui and Ogawa
of a-(1!4)-linked 2-amino-2-deoxy-D-galactose [41]. This

polysaccharide was found to have a occulating action
similar to that of chitosan on suspended soil in aqueous
solution [42]. The congurational dierence between D-
glucosamine (glucose) and D-galactosamine (galactose) lies
only in the geometrical position of the hydroxyl group with
respect to the pyranose ring at C(4)-equatorial for the
former and axial for the latter, while those of C(1) hydroxyl
groups are of the opposite congurations to the respective
C(4) hydroxyl congurations when these sugars are poly-
merized with (1!4)-linkage. Despite the signicant dier-
ence in the linkage structure between the two (1!4)-linked
glycans, x-ray analysis indicated that the poly[(1!4)-a-D-
galactosamine] forms the twofold helical (zigzag) confor-
mation similar to its glucose counterpart, e.g., cellulose,
but with a somewhat kinked structure (Fig. 7) [43].
B. (1!3)-B-D-Glucans
Some polysaccharides are expected to work as medicines.
The most topical polysaccharide medicine is a branched
(1!3)-B-D-glucan, such as lentinan, schizophyllan, or
scleroglucan, which exhibits an anticancer activity. Their
chemical structures are almost the same: a main chain
consisting of (1!3)-linked B-D-glucopyranosyl units along
which there are side chains of single B-D-glucopyranosyl
units attached by (1!6)-linkage to every three glucose
residues of the backbone glucan chain [44]:
h-D-Glcp
1
#
6
Poly!3-h-D-Glcp-1!3-h-D-Glcp-1!3-D-Glep-1!
(1!3)-h-D-Glucan has been of interest not only for the

food industries as a food additive, as it forms a strong gel
[45] in the presence of water, but also for basic research
because of its unique conformation, a triple helix. This
glucan has been studied by Marchessault et al. [4,18,46] and Figure 8 Projection of the triple helix of (1!3)-h-D-glucan
Bluhm and Sarko [47]. All of their x-ray results indicated in the anhydrous polymorph on the xz (top) and xy (bottom)
planes. Dashed lines are intrahelix hydrogen bonds. Hydro-
gen atoms are omitted. (From Ref. 46.)
that this glucan constructs triple-helical conformations in

the crystal. Two polymorphs have been found for the
triplex, anhydrous and hydrated, which transform revers-
ibly from one to the other by changing relative humidity;
20% RH is the boundary. Fig. 8 shows the molecular
structure of the glucan in the crystal of the anhydrous
polymorph [46]. The intramolecular hydrogen bond stabi-
lizing the 6/1 helix of each strand was not detected; the
original distance between O(5) and O(4) atoms, 0.318 nm,
was possibly indicative of hydrogen-bonding formation.
Figure 7 Twofold poly[(1!4)-a-D-galactosamine] helix, As shown in Fig. 8 (bottom), the three strands of the triplex
projected perpendicular (top) and parallel (bottom) to the are linked together through triads of strong interstrand
chain axis. The striped balls are nitrogen atoms. All the hydrogen bonds between the O(2) hydroxyls (distance:
hydrogen atoms are omitted. (From Ref. 43.) 0.272 nm) stabilizing the triplex structure. All the O(6)
hydroxymethyl groups of the glucose residues are outside backbone glucan. In the case of the hydrated polymorph
of the cylinder of the triple-helical structure. Therefore in (Fig. 9), the crystal structure and the triplex are very similar
the case of the branched (1!3)-h-D-glucan, the glucose to those of the anhydrous polymorph, and hydration
residues of the side group attached at the O(6) position of simply expands the unit cell, permitting the water to enter
every three glucose residues of the backbone (1!3)-h-D- the intertriplex spaces [18].
glucan may be located further outside of the cylinder. This It has been found that (1!3)-h-D-glucan exhibits
suggests that the side group of the branched (1!3)-h-D- single-stranded sixfold or sevenfold helical conformations
glucan may not disturb the triple-helix formation of the in crystals [48,49]. Okuyama et al. [50] proposed the single
Figure 9 Top: Stereo views of the triplex of (1!3)-h-D-glucan in the hydrated polymorph. Bottom: Projection of the crystal
structure in the ab plane. Hydrogen atoms are not shown and water molecules are indicated by lled circles. Hydrogen bonds are
drawn with dashed lines. The O(6) atoms of all 18 residues are numbered. (From Ref. 18.)
110 Yui and Ogawa
right-handed sixfold helical conformation of the glucan

(Fig. 10) in the highly hydrated crystal, in which water
content was more than 70 wt.% in the unit cell. This single
helix is considered to be stabilized with a weak intramo-
lecular hydrogen bond between O(5) and O(4) of the next
glucose residue (distance: 0.314 nm) [50]. Therefore this
helix may be stabilized by the water molecules present in
the unit cell.
After nding the triplexes of (1!3)-h-D-glucan, rela-
tively similar x-ray ber diagrams of the branched (1!3)-
h-D-glucan, scleroglucan, were obtained, suggesting that
the backbone chain formed a triple helix similar to that of
(1!3)-h-D-glucan [51]. In fact, an x-ray analysis of schizo-
phyllan revealed the triple-stranded right-handed, 6/1,
helical conformation as shown in Fig. 11 [52]. All the side
glucose residues are located outside of the triplex of the
backbone glucan chain, indicating that the side chain does
not disturb the backbone triplex formation. In water
solution, the polysaccharide also forms a triple-stranded
helix [53]. Interestingly, the antitumor activity is considered
to require the triple-helical conformation of schizophyllan
[54].
Single crystal structure analysis of oligosaccharides
provides the unambiguous knowledge of the conforma-
tions of glycosidic bonds (U, W) and exocyclic groups that
may be applicable for further interpretation for the Figure 11 Molecular structure of schizophyllan, projected
corresponding polysaccharide structures. As shown in perpendicular (top) and parallel (bottom) to the chain axis.
Fig. 12, Okuyama and Noguchi [55] suggested, by survey- (From Ref. 52.)
ing the crystal structures of the (1!3)-h-linked oligosac-
charides, that the UW conformations of the acetyl
oligosaccharide (compounds AF) were found in the same whose structure has been determined by Noguchi et al.
potential well as those of the polysaccharides (compounds [56]. On the other hand, the conformations of the non-
IIII [50]). It should be noted that this group of acetyl substituted and nonacetylated disaccharides (compounds
oligomers also involves the acetyl trisaccharide with (1!6) L and M, respectively) belong to a dierent potential well
branching acetyl glucose residue (compound F, Fig. 13) which corresponded to a considerably large, left-handed
helix chain (n=1417). Obviously, such a large helix
should be unstable as a regular conformation, unless the
chain includes a small molecule inside the helix. In the
disaccharide crystal, the intermolecular packing force
appears to be more dominant in determining the glycosidic
conformations.
C. (1!3)-a-D-Glycans
1. (1!3)-a-D-Glucans
Streptococcus mutans, a bacteria isolated from human
saliva, produces a water-insoluble a-D-glucan from su-
crose. This glucan forms dental plaque and, consequently,
contributes to dental caries. The chemical structure of the
glucan consists of a backbone (1!3)-linked a-D-glucan
chain with side chains of a-D-glucose residues attached on
the O(6) position of the backbone chain. The insolubility
property is attributable mainly to the linear, (1!3)-linked
backbone chain, whereas the (1!6)-linked side chains are
related to the adhesion of the D-glucan to the surface of
teeth, hydroxyapatite [57]. Another, but noncariogenic,
water-insoluble a-D-glucan is produced by Streptococcus
Figure 10 Molecular and crystal structure of the single helix salivarius which is also isolated from human saliva and has
of (1!3)-h-D-glucan. (From Ref. 50.) a similar backbone chain, (1!3)-a-D-glucan, but the side
chains attached on O(6) of the backbone are longer than

those of the mutans glucan. They consist of D-glucose
residues linked by (1!3)-, (1!4)-, and (1!6)-linkages
[58]. The molecular and crystal structures of the backbone
(1!3)-a-D-glucan, which was prepared from the salivarius
glucan, in its dry form were determined by x-ray analysis
(Fig. 14) [3,59]. The chain conformation of the glucan is
nearly completely extended, and it is a twofold helix (i.e., a
zigzag structure). An intramolecular O(2)UO(4) hydrogen
bond stabilizes the conformation, and extensive intermo-
lecular hydrogen bonds stabilize the sheetlike structure,
with an alternating polarity of chain directions (antiparal-
lel fashion) within the sheet. In addition to this dry form of
(1!3)-a-D-glucan, two hydrated polymorphs have been
reported by Jelsma and Kreger [60,61]. They obtained ber
patterns of these polymorphs using a (1!3)-a-D-glucan
from a fungus, Laetiporus sulphureus (Bull ex. Fr.) Murrill.
Although these ber patterns have not been analyzed
completely, it is clear that the glucan conformation in each
Figure 12 Distribution of the glycosidic conformations (A,

C) of (1!3)-linked oligosaccharides and (1!3)-h-linked
polysaccharides; A=O(5)UC(1)UO(1)UC(3) and C=C(1)U
O(1)UC(3)UC(2). Broken and solid lines denote iso-n and
iso-h contours, respectively. Iso-energy contours, shown in
dotted lines, are drawn at interval of 1 kcal/mol above the
absolute minimum. L: h-laminaribiose; M: methyl-h-lami-
naribioside; A: methyl hepta-O-acetyl-a-laminaribioside; B:
octa-O-acetyl-h-laminaribiose; C: octa-O-acetyl-a-laminari-
biose; D: methyl hepta-O-acetyl-a-laminaribioside; E: methyl
hepta-O-acetyl-1-thio-h-laminaribioside; F: the compound
given Fig. 15; I: curdlan form I; II: curdlan form II; III:
curdlan form III; Ac: curdlan triacetate. (From Ref. 55.)
Figure 14 Projections of the structure of (1!3)-a-D-glucan

Figure 13 Chemical structure of (2,3,4,6-tetra-O-acetyl-h-D- on ac plane (top) and ab plane (bottom) in the dry form. All
glucopyranosyl)(1!3)-[2,3,4,6-tetra-O-acetyl-h- D-gluco- hydrogen atoms are omitted, and hydrogen bonds are shown
pyranosyl)(1!6)]-(2,4-di-O-acetyl-h- D-glucopyranosyl) as dashed lines. The atoms of the asymmetric unit are num-
(1!3)-1,2,4,6-tetra-O-acetyl-h-D-glucopyranose. bered. (From Ref. 59.)
112 Yui and Ogawa
crystal is an extended twofold helix similar to that of the helix similar to that of (1!3)-a-D-glucan: both structures
streptococcal (1!3)-a-D-glucan. Viscosity measurements are stabilized by the O(2)UO(4) intramolecular hydrogen
and the polymorphic behavior of (1!3)-a-D-glucan from bonds. These common features support the hypothesis that
these three origins (i.e., two bacterial and one fungal the chain conformation in polysaccharides tends to be
glucans) suggested that the backbone glucan produced by governed more by its type of linkage rather than by the
S. mutans has the lowest length (molecular weight), and residue. In the crystalline unit cell, the mannan chains pack
unlike other two glucans, the glucans are always crystal- with antiparallel polarity and are connected by interchain
lized in the dry form at any relative humidity from 0% to hydrogen bonds that form an innite, zigzag sheet. There
100%; in other words, the zigzag sheet of the backbone are 16 water molecules in the unit cell, embedded between
(1!3)-a-D-glucan of the S. mutans glucan is the most the sheets. It should be noted that the regenerated ber
stable. This may be one of the reasons why this a-glucan samples of both (1!3)-linked a-D-glucan and a-D-mannan
can stick to the surface of teeth [62]. were obtained by the similar procedure: both involve
annealing the stretched lms in the presence of water. Only
2. (1!3)-a-D-Mannan the mannan crystal comprises water molecules, as shown in
(1!3)-a-D-Mannans are found as backbone chains of the projections of Fig. 15, and exhibits the loosely packed
branched heteropolysaccharides in the fruit bodies of some appearance of molecular chains. No direct interaction
edible mushrooms, such as Auricularia auricula-judae exists between the chains.
(Kikurage) [63] and Tremella fuciformis Berk (Shirokikur-
age) [64]. The congurational dierence between D-glucose
and D-mannose is only in the disposition of the hydroxyl D. Food Additives
group at C(2)-equatorial for the former and axial for the Polysaccharide gums have been used in the food industry as
latter. Because the position of the hydroxyl group at C(2) is thickeners, stabilizers, suspending materials, gelling
not related to the glycosidic linkage structure, (1!3)-a-D- agents, emulsiers, lubricants, lms, and so forth. Xanthan
mannan is expected to have a similar conformation with and gellan are topical materials in both research and
(1!3)-a-D-glucan. Fig. 15 shows the molecular and crystal industrial purposes. All the polysaccharides introduced
structure of (1!3)-a-D-mannan [65]. As expected, the herewith have the ability to form gels.
chain conformation of the mannan is an extended twofold
1. Xanthan
Xanthan, produced by a bacteria, Xanthomonas campest-
ris, is an acidic polysaccharide and is a heteropolymer
composed of the following large chemical repeating unit:
Poly!4-h-D-Glcp-1!4-h-D-Glcp-1!
3
z
1
h-D-Manp-1!4-h-D-GlcpA-1!2-a-D-Manp-6-OAc
6 4
H3 C-C-COOH
Xanthan is commercially important because of the follow-
ing reasons: it dissolves in cold water, the aqueous solution
shows a thixotropy, and its viscosity is insensitive to
variation of temperature. Based on a ber pattern of high
quality, Okuyama et al. [66] determined xanthan confor-
mation in the crystal. As shown in Fig. 16, the xanthan
chains are aligned with an antiparallel right-handed ve-
fold (5/1) double helix which is stabilized by four intramo-
lecular bonds and one intermolecular hydrogen bond [66].
2. Gellan
Gellan gum is an extracellular polysaccharide derived from
Figure 15 Projections of the structure of (1!3)-a-D- Pseudomonas elodea. The alkali-treated gellan has been
mannan on ab plane. All hydrogen atoms are omitted and widely utilized in food industry and biotechnology because
hydrogen bonds are shown as dashed lines (top). Stereo views it forms a transparent gel which is heat-resistant and its gel
of the structure on bc plane (bottom). Open circles are water strength is less dependent on pH in comparison with other
molecules. (From Ref. 59.) polysaccharide gels. It is a linear anionic heteropolysac-
ylate group (Fig. 17) [68]. Gellan salts with the monovalent
cation, such as lithium or potassium, form sti gels and
that with divalent cation, Ca2+, make a more rigid gel.
Based on their x-ray analysis of gellan potassium salt,
Chandrasekaran and Thailambal [69] revealed that in the
crystal of the potassium salt, double helixpotassium
waterpotassiumdouble helix interactions promote the
aggregation of molecules and subsequent gelation. Fur-
thermore, they extrapolated these results by computer
modeling to the calcium salt and revealed that Ca2+ ions
intervene in direct and strong double helixcalciumdouble
helix interactions [69].
The native gellan has L-glycerate groups at C(2) on all
the (1!3)-linked h-D-glucose residues in the backbone
chain and O-acetyl groups at O(6) on half of them [70]. It
forms a weak gel in water, but after treatment with alkali,
the gum forms a rigid gel. Before the precise chemical
Figure 16 The 5/1 antiparallel double helix of xanthan

viewed perpendicular to the helix axis. (From Ref. 66.)
charide composed of the following tetrasaccharide repeat-

ing unit containing a D-glucuronic acid:
Poly! 3 h D Glcp 1
! 4 h D GlcpA 1
! 4 h D Glcp 1
! 4 a L Rhamp 1 !
Fiber diraction analyses [67,68] revealed that the two left- Figure 17 Side view of the double helix of gellan in stereo
handed, threefold helical chains are organized in parallel showing the OHO hydrogen bonds within the molecule.
fashion in an intertwined double helix and that the duplex Intrachain H-bonds are indicated by thin, dashed lines and
is stabilized by interchain hydrogen bonds at each carbox- interchain H-bonds by thick, dashed lines. (From Ref. 68.)
114 Yui and Ogawa
structure of native gellan was found, the acetic ester had

been thought to disturb the sti gel formation of native
gellan. However, Kuo et al. [70] found that the glyceric
ester was the major cause of the dierence in gelation,
rather than the acetic ester. This assertion was supported
by a modeling calculation for native gellan potassium salt
[69].
3. Beijeran
Recently, an acidic polysaccharide was found, showing a
gelation property similar to gellan. The polysaccharide,
designated beijeran, was excreted by the newly isolated
bacteria Azotobacter beijerinckii YNM 1 and is expected to
have potential applications in the food and cosmetic
industries [71,72]. Beijeran is a linear polymer consisting
of (1!3)-linked D-galacturonic acid, L-rhamnose, and D-
glucose residues. Although all the glucose residues in the
chain are O-acetylated at C6, the backbone chain is built up
by the following trisaccharide sequence [71,72]:
Poly! 3 a D GalpA 1
! 3 h L Rhamp 1 Figure 18 Interhelical interactions of beigeran sodium salt

in the unit cell. The sodium ions are shown as lled circles
! 3 a D Glcp 1 ! and water molecules are numbered. Dashed lines are hydro-
gen bonds. (From Ref. 75.)
The chemical structure is dierent and simpler, but beijeran
has a similar function to gellan; that is, the alkali-treated
beijeran forms a strong gel when reacted with calcium ion.
However, the gelation mechanism appears to be dierent. mannan, and both linear and branched glucomannans are
The alkali treatment causes deacetylation of the O-acetyl present. Among these glucomannans, KGM has the high-
groups, and the deacetylated beijeran forms gel in water in est molecular weight (degree of polymerization: 6000) [76]
the presence of a divalent metal ion, such as Ca2+ and and the highest glucose content (mannose/glucose ra-
Zn2+, rather than a monovalent cation [71,72]. A well- tio=1.6) [77]. It is a linear copolymer where h-(1!4)-
dened x-ray ber diraction patterns of both calcium and linked mannose sequences of the chain are at most pen-
sodium salts of beijeran have been obtained [73,74], and a tameric in length, and these short segments are connected
complete analysis of the latter was done by Bian et al. [75] with only D-glucose or cellobiose units through h-(1!4)
recently. As shown in Fig. 18, the beijeran molecule forms linkage. A well-dened ber diagram of KGM was
the extended twofold helix with the trisaccharide unit as a obtained by the acetylationdeacetylation procedure de-
symmetric unit. The beijeran chain spirals around the scribed in the case of (1!3)-a-D-glucan in Sample Prep-
molecular axis with a right-handed twist. Two beijeran aration [78]. The diagram was very similar to that of the
chains are nestled tightly in the monoclinic unit cell of mannan II (hydrated) polymorph of (1!4)-h-D-mannan
dimensions a=1.272, b=1.141, c (ber axis)=2.462 nm, [78,79]. A similar result was obtained by an electron
and c=123.7j in an antiparallel fashion. They are diraction study on a single crystal of KGM [80]. Based
connected at their carboxylate groups by sodiumwater on the x-ray ber diagram, the crystal and molecular
sodium bridges. As seen in Fig. 19, there is no room for any structure of KGM was proposed as shown in Fig. 20 [79].
guest molecule to sit in or pass through this polymer sheet. The chain conformation of KGM is a twofold helix
All the sodium ions and water molecules are embedded stabilized by intramolecular O(3)UO(5) hydrogen bonds,
between the sheets and none elsewhere. Altogether, they with the O(6) rotational position at gt [30]. The adjacent
glue the sheets to form a well-knitted network. This could KGM chains are packed in an antiparallel fashion, and
explain beijerans abilities for water holding and formation intermolecular hydrogen bonds occur exclusively between
of oxygen-impervious lms. chains and water molecules, establishing the three-dimen-
sional hydrogen-bond network in the crystal structure. The
glucose residues replace mannose without changing the
4. Konjac Glucomannan dimensions of the mannan-type unit cell, referred as an
Konjac glucomannan (KGM) is a major component of isomorphous replacement, although some disorder ap-
konjac our, a traditional food in Japan, produced from pears possible. The local formation of alternating gggt
the tubers of Amorphophallus konjac C. Koch [76], and it O(6) rotational position [30] may describe the disorder re-
also forms a sti gel. Glucomannans, copolymers of D- gion due to the glucose-rich part of KGM chain in the
mannose and D-glucose, have sequences of h-(1!4)-linked crystal.
Figure 19 Packing arrangement of helices in the monoclinic lattice. Seven unit cells shown in the c-axis projection highlight the
formation of sheets along the short diagonal. Water molecules (numbered) and sodium ions (lled circles) hold the sheets
together by hydrogen bonds and ionic interactions. (From Ref. 75.)
IV. RECENT PROGRESS IN CRYSTAL computer modeling. Sarko and Muggli [82] and Gardner
STRUCTURE STUDIES ON CELLULOSE and Blackwell [83] independently proposed the crystal
ALLOMORPHS structure models of the native Valonia cellulose I, and
Woodcoock and Sarko [84] reported the Ramie cellulose
Cellulose, a linear (1!4)-linked h-D-glucopyranose resi- I structure. The same two groups, Stipanovic and Sarko
due, is the major structural component of all plant cell [85] and Kolpak and Blackwell [86], subsequently estab-
walls and the largest biomass on earth; more than 104 tons lished the crystal structures of cellulose II by analyzing
of cellulose are produced in every year. It exists as a highly the x-ray diagrams of rayon bers. These studies reached
crystalline microbril in all higher plants and some bacte- the same conclusions with regard to chain directionality in
ria, fungi, and algae. The crystal structures of the cellulose the cellulose allomorphs. Whereas the cellulose chains are
microbrils as well as those of the manmade bers have aligned in the same direction in the cellulose I structure, or
been the subject of the intensive diraction studies for parallel chain, they are arranged with alternative direc-
almost a century. The native crystalline form, originally tions in the cellulose II structure, or antiparallel chain.
referred as cellulose I, converts to the second crystalline Thus this readily explains the irreversible transition from
form or cellulose II by regeneration or mercerization of cellulose I to cellulose II which accompanies the signicant
native cellulose bers. The other crystalline forms known rearrangement of the chain polarity from the parallel chain
as cellulose III and IV are also derived from both cellulose I to the antiparallel chain.
and II by treatment with liquid NH3 and successive heating
in glycerol to 260jC. The realistic molecular packing A. Cellulose I
scheme of cellulose I was rst proposed more than 70 years
ago [81]. The detailed crystal structures of these cellulose The three cellulose I models [8284] exhibit similar struc-
allomorphs with atomic resolution have been reported tural features with regard to the molecular conformation
since the mid-1970s, which was promoted by the develop- and the chain-packing arrangement. The twofold helix con-
ment of the ber diraction technique combined with the formation is stabilized by intramolecular O(3)HUO(5)
116 Yui and Ogawa
carbons, whose intensity patterns varied among the several

cellulose samples. These results suggest that the native
cellulose crystals consist of the two crystalline forms,
designated Ia and Ih, and that their relative amounts
depend on the cellulose origin; while Valonia and Aceto-
bacter celluloses are rich in cellulose Ia, cellulose Ih may be
major constituent in cotton and ramie celluloses [90,91].
This crystalline scheme of the native cellulose was further
studied by electron microdiraction analysis [9193].
Sugiyama et al. classied the diraction diagram of some
algal celluloses into two distinct crystalline phases. The
diagram of a major phase representing the Ia crystalline
form is indexed with the one-chain triclinic unit cell, and
the rest is interpreted as the two-chain monoclinic unit
cell of the Ih phase. As obvious from the unit cell dimen-
sions, the Ih crystalline form was once considered as the
conventional cellulose I form. The Ia phase is transformed
into the Ih phase by the alkaline hydrothermal treatment,
suggesting that the latter has more stable form. The two-
phase system of the cellulose microbrils allows a full
indexation of their ber diraction diagrams, instead of
adopting the eight-chain unit cell.
This nding of the cellulose I allomorphism has pro-
moted the further attempts to dene the subclass struc-
tures. French et al. [9496] studied possible chain-packing
schemes of the cellulose allomorphs using the molecular
Figure 20 Projections of the structure of konjac gluco- modeling techniques on the basis of the established crystal
mannan on ab plane (top) and bc plane (bottom). All formations such as the unit cell geometry and the space
hydrogen atoms are omitted and hydrogen bonds are shown group symmetry. These studies indicated that the models
as dashed lines. Dotted circles are water molecules. (From having the parallel-up chains and a tg orientation of the
Ref. 79.) hydroxymethyl group are most likely in both the cellulose
Ia and Ih allomorphs [95,96]. The structure of the cellulose
Ih model is essentially identical to that of the conventional
cellulose I, and it is more stable than the Ia model by 0.4
hydrogen bond over the glycosidic linkage. All hydroxy- kcal/mol of cellobiose units calculated from the potential
methyl groups adopt a tg orientation [30]. The two chains energy calculations [96]. Heiner et al. [97] proposed more
of the parallel polarity pass through the monoclinic unit cell reasonable amount of 2.1 kcal/mol based on the molec-
with the P21 symmetry. The intermolecular O(2)UO(6) ular dynamics calculations. The latter value explains a
hydrogen bonds connect adjacent chains to form the chain complete conversion from Ia to Ih on a hydrothermal
sheet. A major dierence among the three models is the treatment. Recently, a more primary approach to predict
chain directionality with respect to the c-axis; while the the native cellulose structures was presented by Vietor et al.
Gardner and Blackwell model corresponds to the parallel- [98], where the prediction was made by the chain pairing
up structure, the two others correspond to the parallel- procedure without any crystal information. In the study,
down structure [87]. the two best chain-packing models were found to be the
It has been well known that the native celluloses triclinic- and monoclinic-type arrangements having the
slightly dier among their origins. There are three reec- unit cell dimensions and the symmetry close to those
tions in the Valonia x-ray diraction data that cannot be reported for either the cellulose Ia or Ih allomorphs. The
indexed with the two-chain monoclinic unit cell. A possible molecular modeling studies also suggested that the conver-
interpretation of this is to double the ab dimensions to the sion of the cellulose allomorphs could be reasonably
eight-chain monoclinic unit cell [88], which, however, explained by slipping of the chain sheets with respect to
introduces formidably complicated chain-packing schemes the neighboring chain sheet along the ber axis [96,98].
and variation of a chain conformation within the unit cell. Finkenstadt and Millane [99] reanalyzed the two sets
In the previous studies based on the two-chain unit cell, of the x-ray diraction data provided by Sarko and Muggli
therefore, the cellulose I structures were determined as an [82] and Gardner and Blackwell [83] as a representative
approximation to the Valonia crystal [81,82]. VanderHart of the cellulose Ih data. In order to obtain a denitive
and Atalla [89,90] proposed much clearer understanding of structure, this structure analysis was carried out more ex-
a crystalline system of cellulose I based on the result of haustively than the preceding studies, where the geome-
high-resolution solid-state 13C NMR measurements. The tries of the two residues in the asymmetric unit were varied
multiplicities were shown in resonance for C4, C6, and C1 independently while assuming both the parallel-up and
parallel-down models. These conditions had not been

considered previously due to the limited computing re-
sources available in those days [82,83]. This work showed
that while some structure features such as a hydrogen-
bonding scheme and a chain conformation are essentially
the same with those of the previous models, both the two
x-ray diraction data denitively prefer the parallel-up
model. The projections of the nal cellulose Ih model are
depicted in Fig. 21.
Figure 22 A schematic representation of the hydrogen

bonds in the origin (top) and center (bottom) sheets of
cellulose Ih. Only the oxygen atoms involved in hydrogen
bonding are labeled with clarity. (From Ref. 100.)
Recently, for the rst time, Nishiyama et al. [100]

reported a set of the full atomic coordinates of cellulose
Ih, using the combined synchrotron x-ray and neutron
diraction analyses with a resolution of better than 1 A. In
this study, the Finkenstadt and Millane models were sub-
jected to the fully optimized structure renement including
restrained renement of bond lengths and angles to deter-
mine the C and O atom positions against the x-ray dirac-
tion data. The neutron diraction data were obtained from
the deuterated cellulose Ih sample (D-cellulose-Ih) as well
as from the ordinary sample (H-cellulose-Ih). The former
was prepared by intracrystalline deuteration of the cellu-
lose Ih microcrystals for replacing the six independent
hydroxyl hydrogen atoms in the asymmetric unit. The
deuterium atom locations were identied examining the
Fourier dierence synthesis derived from the electron
density maps between the D-cellulose-Ih and the H-cellu-
lose-Ih. The dierence density peaks could be denitively
identied with possible D-O(3) positions, but D-O(2) and
D-O(6) positions were less well dened. These results
provided the two alternative patterns in deuterium posi-
tions for each of D-O(2) and D-O(6) atoms. Such ambigu-
ities in positioning of some deuterium atoms yielded the
two types of hydrogen-bonding schemes, designated A and
B, as displayed in Fig. 22. In the hydrogen-bonding net-
work A, which corresponds to the major one, both the
origin and center chains involve the intramolecular
O(3)UDUO(5) and O(2)UDUO(6) hydrogen bonds and
Figure 21 Views of the revised structure of cellulose Ih (a) the origin chain forms an additional O(2)UDUO(1) bond.
obliquely to and (b) along the c-axis. Thin lines show hydro- The intermolecular O(6)UDUO(3) hydrogen bonds con-
gen bonds. The hydrogen atoms are excluded from part (a) nect the same type of chains to construct the chain sheets
for clarity. (From Ref. 99.) and the center chains are further bound through the
118 Yui and Ogawa
O(6)UDUO(2) bond. On the other hand, the hydrogen- respectively. The corner chains construct the chain sheet
bonding network B, consisting of the deuterium atoms with connected by the interchain O(2)UO(6) hydrogen bond
lower occupancies, indicates that both the origin and center and their O(2) atoms also involve the O(2)UO(2) bond in
chains involve the same intramolecular hydrogen bonds of the 110 plane with the neighboring center chain. A sheet
O(3)UDUO(5), O(6)UDUO(2), and O(6)UDUO(1). A formation also occurs in the center chains along the 020
sheet formation only occurs in the center chains stabilized plane, consisting of the interchain O(3)UO(6) hydrogen
by the intermolecular O(2)UDUO(6) hydrogen bond, bond. The crystal structure model of Stipanovic and Sarko
whereas no hydrogen bond is present among the origin [85] exhibits an additional interchain intersheet O(6)UO(3)
chains. There is no intersheet OUHUO hydrogen bond in hydrogen bond. Thus as suggested by more intensive
both the hydrogen-bonding networks, which is in accord hydrogen-bonding scheme, the cellulose II crystal is ener-
with the previous structure analyses [8284]. The chain getically more stable allomorph than the cellulose I crystal.
sheets therefore are held together by hydrophobic inter- The next objective in exploring the cellulose II struc-
actions and weak CUHUO hydrogen bonds. ture was to study the single crystal structure of h-D-
cellotetraose hemihydrate as a model crystal for cellulose
B. Cellulose II II [101103]. There are some resemblances between the
cellotetraose hemihydrate and the cellulose II crystals. The
The two cellulose II models proposed by Stipanovic and unit cell of h-D-cellotetraose hemihydrate contains two
Sarko [85] and Kolpak and Blackwell [86] are virtually independent molecules, which are arranged with antipar-
identical, and an apparent dierence in the chain arrange- allel polarity. In fact, one can draw the subcell inside the
ment is caused by the denition of chain positions. As is the unit cell, which is nearly identical to the unit cell of cellulose
same with the cellulose I chain, the two chains of the II [102]. Likewise, the intermolecular hydrogen bonds are
twofold helix conformation are packed with an antiparallel formed between the antiparallel molecules as well as be-
fashion in the monoclinic unit cell with the P21 symmetry. tween the parallel molecules, and they correspond to the
Both the up corner and down center chains form the intersheet and intrasheet hydrogen bonds in the cellulose II
intramolecular O(3)UO(5) hydrogen bond, and, according structure, respectively. Notable features in h-D-cellote-
to the denition of Kolpak and Blackwell, the center chain traose molecules are that the orientations of all hydroxy-
only involves an extra hydrogen bond at O(6)UO(2), as a methyl groups are gt, and that one molecule is more
result of the hybrid orientations of the hydroxymethyl sterically strained than the other as indicated by the
groups being gt and tg for the corner and center chains, puckering parameters of the D-glucopyranoses. The same
Figure 23 Views of the revised structure of cellulose II (bold line) superimposed over that of Kolpak and Blackwell model (thin
line). The short OO distances that correspond to hydrogen bonds are given by dashed lines. (A) Projection in ab plane. (B)
Projection of the 13 molecules parallel to the c-axis. (C) Projection of molecules 4 and 5 parallel to the c-axis. (From Ref. 104.)
hand, according to the results of Raymond et al. [104],

preference was given to the all-gt model, which accompa-
nies signicant strain on the D-glucopyranoses of the center
chain. Fig. 23 compares the crystal structure proposed by
Raymond et al. with that of the original Kolpak and
Blackwell model.
A complete scheme of the cellulose II structure was
dened by the neutron diraction analysis [105] and later
complemented by the synchrotron x-ray diraction analy-
sis [106]. In the former study, Langan et al. [105] prepared
the deuterated cellulose II bers, designated the D-cellu-
lose-II, by mercerization of ax bers in NaOD/D2O
solution. The two neutron diraction data with 1.2 A
resolution were collected from the D-cellulose-II and the
ordinary cellulose II ber or H-cellulose-II. The phasing
model of cellulose II was established using the published
x-ray diraction data of Kolpak and Blackwell [86]. As has
been observed in the h-D-cellotetraose hemihydrate crystal
structures [102,103], the all-gt model is preferred over the
gt/tg hybrid model and the D-glucopyranose geometries
of the center chain are conformationally strained. The
Fourier dierence maps calculated from the neutron dif-
fraction data of the D- and H-cellulose II locate the
deuterium positions on the phasing models, which reveals
details of hydrogen bonding scheme shown in Fig. 24. The
two intramolecular hydrogen bonds, O(3)UDUO(5) and
O(3)UDUO(6), compose the three-center bond where the
former plays the role of the major component in both the
origin and center chains. The intermolecular and intrasheet
hydrogen bonds are also formed between O(2) and O(6)
atoms in both the chain sheets, but they exhibit the oppo-
site donoracceptor pattern of a deuterium atom. As for
the intermolecular and intersheet hydrogen bonds, O(6) of
the origin chain donates the deuterium atom to three
acceptors of the center chain, forming the four-centered
bonding scheme which consists of the O(6)UDUO(6),
O(6)UDUO(3), and O(6)UDUO(5) bonds in this strength-
ening order. Another major bond of this type is the
O(2)UDUO(2) bond from the center chain to the origin
chain. The following high-resolution synchrotron x-ray
diraction study with 1 A resolution indicated better t-
Figure 24 A schematic representation of the hydrogen bonds ting of the diraction data to the all-gt model than that of
in cellulose II. Only the oxygen atoms involved in hydrogen the Kolpack and Blackwell data [106]. The report also sug-
bonding are labeled with clarity. (From Ref. 105.) gested that the four-centered hydrogen bond on O(6) of
the origin chain is a statistical eect along with the disorder
of the hydroxymethyl conformation of the origin chain of
two groups engaged in the structure analysis of h-D- which degree is estimated to be about 10%.
cellotetraose hemihydrate successively attempted to rean-
alyze the cellulose II structure [102,104] using the published
x-ray diraction data [85,86]. Both the two groups estab-
REFERENCES
lished the initial molecular chain structures based on their
respective h-D-cellotetraose structures [102,104] and, as for 1. Rees, D.A.; Scott, W.E. Polysaccharide conformation. Pt.
one group, partly on the methyl h-D-cellotrioside structure VI. Computer model-building for linear and branched
[104]; each of two independent molecules in the cellodextrin pyranoglycans. Correlations with biological function.
crystals was applied to either origin or center chain of the Preliminary assessment of inter-residue forces in aqueous
solution. Further interpretation of optical rotation in terms
cellulose II structure. Gessler et al. [102] concluded that of chain conformation. J. Chem. Soc. B, 1971; 469.
both the gt/tg hybrid model as observed in the original 2. Atkins, E.D.T.; Phelps, C.F.; Sheehan, J.K. The con-
cellulose II structures and the all-gt model based on the formation of the mucopolysaccharides, hyaluronates.
single crystal structure are equally likely. On the other Biochem. J. 1972, 128, 1255.
120 Yui and Ogawa
3. Ogawa, K.; Misaki, A.; Oka, S.; Okamura, K. X-ray dif- 24. Sakurai, K.; Shibano, T.; Kimura, K.; Takahashi, T.
fraction data for (1!3)-a-D-glucan. Carbohydr. Res. 1979, Crystal structure of chitosan. II. Molecular packing in unit
75, C13. cell of crystal. Seni Gakkaishi (Tokyo) 1985, 41, T-361.
4. Marchessault, R.H.; Deslandes, Y.; Ogawa, K.; Sundar- 25. Ogawa, K. Eect of heating an aqueous suspension of
arajan, P.R. X-ray diraction data for h-(1!3)-D-glucan. chitosan on the crystallinity and polymorphs. Agric. Biol.
Can. J. Chem. 1977, 55, 300. Chem., Tokyo 1991, 55, 2375.
5. Millane, R.P.; Arnott, S. Background removal in x-ray 26. Okuyama, K.; Noguchi, K.; Miyazawa, T.; Yui, T.;
diraction patterns. J. Appl. Crystallogr. 1985, 18, 419. Ogawa, K. Molecular and crystal structure of hydrated
6. Millane, R.P.; Arnott, S. Digital processing of x-ray chitosan. Macromolecules 1997, 30, 5849.
diraction patterns from oriented bers. J. Macromol. 27. Ogawa, K.; Hirano, S.; Miyanishi, T.; Yui, T.; Watanabe,
Sci. Phys. 1985, B24, 193. T. A new polymorph of chitosan. Macromolecules 1984,
7. Millane, R.P. Relating reection boundaries in x-ray ber 17, 973.
diraction patterns to specimen morphology and their use 28. Cairns, P.; Miles, M.J.; Morris, V.J.; Ridout, M.J.;
for intensity measurement. J. Macromol. Sci. Phys. 1989, Brownsey, G.J.; Winter, W.T. X-ray bre diraction
B28, 149. studies of chitosan and chitosan gels. Carbohydr. Res.
8. Okuyama, K.; Obata, Y.; Noguchi, K.; Kusaba, T.; Ito, Y.; 1992, 235, 23.
Ohno, S. Single helical structure of curdlan triacetate. 29. Ogawa, K.; Inukai, S. X-ray diraction study of sulfuric,
Biopolymers 1996, 38, 557. nitric, and halogen acid salts of chitosan. Carbohydr. Res.
9. Okuyama, K.; Obata, Y. Structure analysis of polybenza- 1984, 160, 425.
mide using imaging plate as x-ray detectors. Polym. Prepr., 30. This notation denes a hydroxymethyl conformation: gg,
ACS 1992, 33, 280. gauche to C5O5 and gauche to C4C5; gt, gauche to C5
10. Obata, Y.; Okuyama, K. Structural analysis of brous O5 and trans to C4C5; tg, trans to C5O5 and trans to C4
polymers by using an imaging plateA comparison C5.
with the conventional photographic method. Kobunshi 31. Kawada, J.; Yui, T.; Okuyama, K.; Ogawa, K. Crystalline
Ronbunshu (Tokyo) 1992, 49, 297. in Japanese. behavior of chitosan organic acid salts. Biosci. Biotechnol.
11. Obata, Y.; Okuyama, K. Data processing system for x-ray Biochem. 2001, 65, 2542.
ber diraction patterns obtained by using an imaging 32. Muzzarelli, R.A.A. Chitin; Pergamon Press: Oxford, 1977.
plate. Kobunshi Ronbunshu (Tokyo) 1994, 51, 297. in 33. Ogawa, K.; Nakata, K.; Yamamoto, A.; Nitta, Y.; Yui, T.
Japanese. X-ray study of chitosan L- and D-ascorbates. Chem. Mater.
12. Smith, P.J.C.; Arnott, S. LALS: A linked-atom least- 1996, 8, 2349.
squares reciprocal-space renement system incorporating 34. Yamamoto, A.; Kawada, J.; Yui, T.; Ogawa, K. Con-
stereochemical restraints to supplement sparse diraction formational behavior of chitosan in the acetate salt. Biosci.
data. Acta Crystallogr. 1978, A34, 3. Biotechnol. Biochem. 1997, 61, 1230.
13. Zugenmaier, P.; Sarko, A. The variable virtual bond 35. Kawada, J.; Yui, T.; Abe, Y.; Ogawa, K. Crystalline
modeling technique for solving polymer crystal structures. features of chitosanL- and D-lactic acid salts. Biosci.
In Fiber Diraction Methods; French, A.D., Gardner, Biotechnol. Biochem. 1998, 62, 700.
K.-C.H., Eds.; ACS Symposium Series, 141; American 36. Kawada, J.; Abe, Y.; Yui, T.; Okuyama, K.; Ogawa, K.
Chemical Society: Washington, DC, 1980; 225 pp. Crystalline transformation of chitosan from hydrated to
14. Millane, R.P.; Narasaiah, T.V. A comparison of the linked- anhydrous polymorph via chitosan monocarboxylic acid
atom squares and PS79 systems for determining polymer salts. J. Carbohydr. Chem. 1999, 18, 559.
structures from x-ray bre diraction data. Polymer 1989, 37. Okuyama, K.; Noguchi, K.; Kanenari, M.; Egawa, T.;
60, 1763. Osawa, K.; Ogawa, K. Structural diversity of chitosan and
15. Arnott, S.; Scott, W.E. Accurate x-ray diraction analysis its complexes. Carbohydr. Polym. 2000, 41, 237.
of brous polysaccharide containing pyranose rings. Part I. 38. Ogawa, K.; Oka, K.; Miyanishi, T.; Hirano, S. X-ray
The linked-atom approach. J. Chem. Soc., Perkin Trans. II Diraction Study on ChitosanMetal Complexes; In
1972; 324. Chitin, Chitosan, and Related Enzymes; Zikakis, J.P., Ed.;
16. Arnott, S. Twenty years hard labor as a ber diractionist. Academic Press, INC: Orlando, 1984; 327 pp.
In Fiber Diraction Methods; French, A.D., Gardner, 39. Ogawa, K.; Oka, K.; Yui, T. X-ray study of chitosan
K.-C.H., Eds.; ACS Symposium Series, 141; American transition metal complexes. Chem. Mater. 1993, 5, 726.
Chemical Society: Washington, DC, 1980; 1 pp. 40. Schlick, S. Binding sites of Cu2+ in chitin and chitosan. An
17. Fraser, R.D.B.; Macrae, T.P.; Suzuki, E. An improved electron spin resonance study. Macromolecules 1986, 19,
method for calculating the contribution of solvent to the 192.
x-ray diraction pattern of biological molecules. J. Appl. 41. Takagi, H.; Kadowaki, K. Flocculant production by
Crystallogr. 1978, 11, 693. Paecilomyces sp. taxonomic studies and culture conditions
18. Chuah, C.T.; Sarko, A.; Deslandes, Y.; Marchessault, R.H. for production. Agric. Biol. Chem., Tokyo 1985, 49, 3151.
Triple-helical crystalline structure of curdlan and para- 42. Takagi, H.; Kadowaki, K. Purication and chemical
mylon. Macromolecules 1983, 16, 1375. properties of a occulant produced by Paecilomyces.
19. Gardner, K.H.; Blackwell, J. Renement of the structure of Agric. Biol. Chem., Tokyo 1985, 49, 3159.
h-chitin. Biopolymers 1975, 14, 1581. 43. Ogawa, K.; Tanaka, F.; Tamura, J.; Kadowaki, K.;
20. Minke, R.; Blackwell, J. The structure of a-chitin. J. Mol. Okamura, K. Structure of a poly[(1!4)-a-D-galactos-
Biol. 1978, 120, 167. amine anhydride] studied by x-ray diraction coupled with
21. Roberts, G.A.F. Chitin Chemistry; The Macmillan Press conformational analysis. Macromolecules 1987, 20, 1172.
Ltd.: Houndmills, Basingstoke, Hampshire, 1992. 44. Bohn, J.A.; BeMiller, J.N. (1!3)-h-D-Glucans as bio-
22. Clark, G.L.; Smith, A.F. X-ray diraction studies of chitin, logical response modiers: a review of structurefunctional
chitosan, and derivatives. J. Phys. Chem. 1937, 40, 863. activity relationships. Carbohydr. Polym. 1995, 28, 3.
23. Yui, T.; Imada, K.; Okuyama, K.; Obata, Y.; Suzuki, K.; 45. Harada, T.; Harada, A. Curdlan and Succinoglycan. In
Ogawa, K. Molecular and crystal structure of the anhy- Polysaccharides in Medical Applications; Dumitriu, S., Ed.;
drous form of chitosan. Macromolecules 1994, 27, 7601. Marcel Dekker, Inc.: New York, 1996; 21 pp.
46. Deslandes, Y.; Marchessault, R.H.; Sarko, A. Triple- berk, and the growing culture of its yeast-like cells. Agric.
helical structure of (1!3)-h-D-glucan. Macromolecules Biol. Chem., Tokyo 1979, 43, 1659.
1980, 13, 1466. 65. Yui, T.; Ogawa, K.; Sarko, A. Molecular and crystal
47. Bluhm, T.L.; Sarko, A. The triple helical structure of structure of the regenerated form of (1!3)-a-D-mannan.
lentinan, a linear h-(1!3)-D-glucan. Can. J. Chem. 1977, Carbohydr. Res. 1992, 229, 57.
55, 293. 66. Okuyama, K.; Arnott, S.; Moorhouse, R.; Walkinshaw,
48. Kasai, N.; Harada, T. Ultrastructure of curdlan. In Fiber M.D.; Atkins, E.D.T.; Wolf-Ullish, CH. Fiber diraction
Diraction Methods; French, A.D., Gardner, K.-C.H., studies of bacterial polysaccharides. In Fiber Diraction
Eds.; ACS Symposium Series, 141; American Chemical Methods; French, A.D., Gardner, K.-C.H., Eds.; ACS
Society: Washington, DC, 1980; 363 pp. Symposium Series, 141; American Chemical Society:
49. Fulton, W.S.; Atkins, E.D.T. The gelling mechanism and Washington, DC, 1980; 411 pp.
relationship to molecular structure of microbial polysac- 67. Upstill, C.; Atkins, E.D.T.; Attwool, P.T. Helical con-
charide curdlan. In Fiber Diraction Methods; French, formation of gellan gum. Int. J. Biol. Macromol. 1986, 8,
A.D., Gardner, K.-C.H., Eds.; ACS Symposium Series, 275.
141; American Chemical Society: Washington, DC, 1980, 68. Chandrasekaran, R.; Millane, R.P.; Arnott, S.; Atkins,
385 pp. E.D.T. The crystal structure of gellan. Carbohydr. Res.
50. Okuyama, K.; Otsubo, A.; Fukuzawa, Y.; Ozawa, M.; 1988, 175, 1.
Harada, T.; Kasai, N. Single-helical structure of native 69. Chandrasekaran, R.; Thailambal, V.G. A new generation
curdlan and its aggregation state. J. Carbohydr. Chem. of gel-forming polysaccharides. In Computer Modeling of
1991, 10, 645. Carbohydrate Molecules; French, A.D. Brady, J.W., Eds.;
51. Bluhm, T.L.; Deslandes, Y.; Marchessault, R.H.; Perez, S.; ACS Symposium Series, 430; American Chemical Society:
Rinaudo, M. Solid-state and solution conformation of Washington, DC, 1990; 300 pp.
scleroglucan. Carbohydr. Res. 1982, 100, 117. 70. Kuo, M.-S.; Mort, A.J.; Dell, A. Identication and location
52. Takahashi, Y.; Kobatake, T.; Suzuki, H. Triple helical of L-glycerate, an unusual acyl substituent in gellan gum.
structure of schizophyllan. Rep. Prog. Polym. Phys. Jpn. Carbohydr. Res. 1986, 156, 173.
1984, 26, 767. 71. Ooiso, Y.; Okumiya, T.; Kawashima, K.; Sone, Y.; Kakuta,
53. Norisuye, T.; Yanaki, T.; Fujita, H. Triple helix of a M.; Misaki, A. Beijeran, a New Acidic Polysaccharide Pro-
Schizophyllum commune polysaccharide in aqueous solu- duced by Azotobacter beijerinckii YNM1: Structure and
tion. J. Polym. Sci., Polym. Phys. Ed. 1980, 18, 547. Rheological Properties, Abstract of XVIIth Japanese Car-
54. Norisuye, T. Triple-stranded helical structure of schizo- bohydrate Symposium, 1995, Kyoto, p. 89.
phyllan and its antitumor activity of aqueous solution. 72. Misaki, A.; Ooiso, Y.; Kakuta, M.; Sone, Y.; Ogawa, K.
Makromol. Chem., (Suppl.) 1985, 14, 105. Structure and Functional Properties of Beijeran, a New
55. Okuyama, K.; Noguchi, K. Structural studies on poly- Exopolysaccharide of Azotobacter beijerinckii, Abstract
saccharides by using structural information of the related of XVII International Carbohydrate Symposium, 1996,
oligosaccharides. Kobunshi (Tokyo) 1994, 43, 848. in Milano, p. 651.
Japanese. 73. Ogawa, K.; Yui, T.; Nakata, K.; Nitta, Y.; Kakuta, M.;
56. Noguchi, K.; Kobayashi, E.; Okuyama, K.; Kitamura, S.; Misaki, A. Chain conformation of deacetylated beijeran
Takeo,K.;Ohno,S.(2,3,4,6-tetra-O-acetyl-h-D-Glucopyra- calcium salt. Biosci. Biotechnol. Biochem., Tokyo 1996, 60,
nosyl)(1!3)-[2,3,4,6-tetra-O-acetyl-h-D-glucopyrano- 551.
syl](1!6)]-(2,4-di-O-acetyl-h-D-glucopyranosyl)(1!3)- 74. Ogawa, K.; Yui, T.; Nakata, K.; Kakuta, M.; Misaki, A.
1,2,4,6-tetra-O-acetyl-h-D-glucopyranose. Carbohydr. X-ray study of beijeran sodium salt, a new galacturonic
Res. 1994, 258, 35. acid-containing exo-polysaccharide. Carbohydr. Res.
57. Ebisu, S.; Misaki, A.; Kato, K.; Kotani, S. The structure of 1997, 300, 41.
water-insoluble glucans of cariogenic Streptococcus mu- 75. Bian, W.; Chandrasekaran, R.; Ogawa, K. X-ray structure
tans, formed in the absence and presence of dextranase. analysis of the sodium salt of beijeran. Carbohydr. Res.
Carbohydr. Res. 1974, 38, 374. 2002, 337, 305.
58. Tsumuraya, Y.; Misaki, A. Structure of the water-insoluble 76. Kishida, N.; Okimasu, S.; Kamata, T. Molecular weight
a-D-glucan of Streptococcus salivarius HHT. Carbohydr. and intrinsic viscosity of konjac gluco-mannan. Agric. Biol.
Res. 1979, 74, 217. Chem., Tokyo 1978, 42, 1645.
59. Ogawa, K.; Okamura, K.; Sarko, A. Molecular and crystal 77. Kato, K.; Matsuda, K. Studies on the chemical structure of
structure of the regenerated form of (1!3)-a-D-glucan. Int. konjac mannan, pt. I. Isolation and characterization of
J. Biol. Macromol. 1981, 3, 31. oligosaccharides from the partial acid hydrolyzate of the
60. Jelsma, J.; Kreger, D.R. Polymorphism in crystalline mannan. Agric. Biol. Chem., Tokyo 1969, 33, 1446.
(1!3)-a-D-glucan from fungal cell-walls. Carbohydr. 78. Ogawa, K.; Yui, T.; Mizuno, T. X-ray diraction study of
Res. 1979, 71, 51. glucomannans and their acetates. Agric. Biol. Chem.,
61. Jelsma, J. Ultrastructure of Glucans in Fungal Cell Walls, Tokyo 1991, 55, 2015.
Thesis, University of Groningen, 1979. 79. Yui, T.; Ogawa, K.; Sarko, A. Molecular and crystal
62. Ogawa, K.; Yui, T.; Okamura, K.; Misaki, A. Crystalline structure of konjac glucomannan in the mannan II
features of streptococcal (1!3)-a-D-glucans of human polymorphic form. Carbohydr. Res. 1992, 229, 41.
saliva. Biosci. Biotechnol. Biochem., Tokyo 1994, 58, 80. Chanzy, H.D.; Grosrenaud, A.; Joseleau, J.P.; Dube, M.;
1326. Marchessault, R.H. Crystallization behavior of gluco-
63. Sone, Y.; Kakuta, M.; Misaki, A. Isolation and character- mannan. Biopolymers 1982, 21, 301.
ization of polysaccharides of Kikurage, fruiting body 81. Meyer, K.H.; Misch, L. Position des Atomes dans le
of Auricularia auricula-judae. Agric. Biol. Chem., Tokyo Nouveau Modele Spatial de la Cellulose. Helv. Chim. Acta
1978, 42, 417. 1937, 20, 232.
64. Kakuta, M.; Sone, Y.; Umeda, T.; Misaki, A. Comparative 82. Sarko, A.; Muggli, R. Packing analysis of carbohydrates
structural studies on acidic heteropolysaccharides isolated and polysaccharides. III. Valonia cellulose and cellulose II.
from Shirokikurage, fruit body of Tremella fuciformis Macromolecules 1974, 7, 486.
122 Yui and Ogawa
83. Gardner, K.H.; Blackwell, J. The structure of native calculations of cellulose Ia crystal structure. Macromol.
cellulose. Biopolymers 1974, 13, 1975. Theory Simul. 1994, 3, 185.
84. Woodcoock, C.; Sarko, A. Packing analysis of carbohy- 96. Aabloo, A.; French, A.D.; Mikesaar, R.; Prestin, A.J.
drate and polysaccharides. 11. Molecular and Crystal Studies of crystalline native cellulose using potential energy
structure of native ramie cellulose. Macromolecules 1980, calculations. Cellulose 1994, 1, 161.
13, 1183. 97. Heiner, A.P.; Sugiyama, J.; Teleman, O. Crystalline
85. Stipanovic, A.J.; Sarko, A. Packing analysis of carbohy- cellulose Ia and Ih studied by molecular dynamics
drate and polysaccharides. 6. Molecular and crystal simulation. Carbohydr. Res. 1995, 273, 207.
structure of regenerated cellulose II. Macromolecules 98. Vietor, R.J.; Mazeau, K.; Lakin, M.; Perez, S. A priori
1976, 9, 851. crystal structure prediction of native cellulose. Biopoly-
86. Kolpak, F.J.; Blackwell, J. Determination of the structure mers 2000, 54, 342.
of cellulose II. Macromolecules 1976, 9, 273. 99. Finkenstadt, V.L.; Millane, R.P. Crystal structure of
87. The chain direction is dened as up when the z coordi- Valonia cellulose Ih. Macromolecules 1998, 31, 7776.
nate of O5 is greater than that of C6, and it is dened as 100. Nishiyama, Y.; Langan, P.; Chanzy, H. Crystal structure
down otherwise. and hydrogen-bonding system in cellulose Ih form
88. Honjo, G. Examination of cellulose ber by the low- synchrotron x-ray and neutron ber diraction. J. Am.
temperature specimen method of electron diraction and Chem. Soc. 2002, 124, 9074.
electron microscopy. Nature 1958, 181, 326. 101. Gessler, K.; Krauss, N.; Steiner, T.; Sandman, C.; Betzel,
89. Attala, R.; VanderHart, D.L. Native cellulose: a composite C.; Saeger, W. Crystal structure of b-D-cellotetraose
of two distinct crystalline forms. Science 1984, 223, 282. hemihydrate with implications for the structure of cellulose
90. VanderHart, D.L.; Attala, R. Studies of microstructures in II. Science 1994, 266, 1027.
native cellulose using solid-state 13CNMR. Macromole- 102. Gessler, K.; Krauss, N.; Steiner, T.; Sandman, C.; Sarko,
cules 1984, 17, 1465. A.; Saeger, W. h-D-Cellotetraose hemihydrate as a
91. Sugiyama, J.; Okano, T.; Yamamoto, H.; Horii, F. structural model for cellulose II. An x-ray diraction
Transformation of Valonia cellulose crystals by an alka- study. J. Am. Chem. Soc. 1995, 117, 11397.
line hydrothermal treatment. Macromolecules 1990, 23, 103. Raymond, S.; Heyraud, A.; Tran Qui, D.; Kvick, A.;
3196. Chanzy, H. Crystal and molecular structure of h-D-
92. Sugiyama, J.; Persson, J.; Chanzy, H. Combined infrared cellotetraose hemihydrate as a model of cellulose II.
and electron diraction study of the polymorphism of Macromolecules 1995, 28, 2096.
native celluloses. Macromolecules 1990, 24, 2461. 104. Raymond, S.; Kvick, A.; Chanzy, H. The structure of
93. Sugiyama, J.; Vuong, R.; Chanzy, H. Electron diraction cellulose II: A revisit. Macromolecules 1995, 28, 8422.
study on the two crystalline phases occurring in native 105. Langan, P.; Nishiyama, Y.; Chanzy, H. A revised structure
cellulose form algal cell wall. Macromolecules 1991, 24, and hydrogen-bonding scheme in cellulose II form a neu-
4168. tron ber diraction analysis. J. Am. Chem. Soc. 1999, 121,
94. French, A.D.; Miller, D.P.; Aabloo, A. Miniature crystal 9940.
models of cellulose polymorphs and other carbohydrates. 106. Langan, P.; Nishiyama, Y.; Chanzy, H. X-ray structure of
Int. J. Biol. Macromol. 1993, 15, 30. mercerized cellulose II at 1 A resolution. Biomacromole-
95. Aabloo, A.; French, A.D. Preliminary potential energy cules 2001, 2, 410.
5
Recent Developments in Spectroscopic and Chemical
Characterization of Cellulose
Rajai H. Atalla
USDA Forest Service and University of Wisconsin, Madison, Wisconsin, U.S.A.
Akira Isogai
Graduate School of Agricultural and Life Science, University of Tokyo, Tokyo, Japan
I. INTRODUCTION II. STRUCTURES

This chapter represents a summary review and an update of The beginning of the last three decades of studies on the
an earlier discussion of the phenomenology of cellulose, structure of cellulose was marked by the reintroduction of
together with an overview of recent developments in the unit cell models based on parallel alignment of the cellu-
chemistry of cellulose. Rather than attempting to integrate lose molecular chains [2,3], not unlike those abandoned by
the discussions, they will be presented in two separate Meyer and Misch [4] in the 1930s, but also incorporating
sections. Part A, by R.H. Atalla, deals with the states of bending of the glycosidic linkage to allow the intramolec-
aggregation of celluloses and key structural issues, particu- ular hydrogen bond, as suggested by Hermans [5]. The new
larly those with questions of structure still outstanding. models were not consistent with each other, however,
Part B, by A. Isogai, presents an overview of recent develop- apart from the fact that both were based on parallel
ments in the chemistry of cellulose, both basic and applied. alignment of the cellulose chains. As French [6] pointed
To minimize the possibility of confusion, the references and out, they were also not strongly preferred over an anti-
gures are numbered consecutively and separately for each parallel structure. In the analysis by French [6], it was
of the sections. recognized that the source of the inconsistency was not so
much that the dierent laboratories were using dierent
computational approaches as it was that the dierent dif-
Part A fractometric data sets were gathered from dierent sam-
Spectroscopic Characterization ples and represented dierent intensities for the same
reections. All of these studies were undertaken before
In Part A, we will be concerned with delineating the fron- the variability of the crystalline forms of native celluloses
tiers of our understanding of cellulose, particularly with was revealed through the high-resolution solid-state 13C
respect to its native forms. The presentation is also relevant NMR investigations.
to the industrial utilization of cellulose, because it ad- The new crystallographic models also remained in
dresses the nature of the native forms of many of the feed- question because the analyses on which they were based
stocks used, as well as the eects of processes of isolation incorporated a level of symmetry in the unit cell that was
on structure and reactivity. The evolution of the historical inconsistent with some of the diractometeric data. Some
perspective is included in an earlier report [1] and in of the reections that are consistently observed in electron
references cited therein. diraction patterns and are disallowed by the selection
123
124 Atalla and Isogai
rules for the space group P21 [3] were ignored in these III. NEW SPECTROSCOPIC METHODS
crystallographic analyses. In addition to the disallowed
reections in the electron diraction patterns that placed A. Raman Spectroscopy
the crystallographic models in question, new spectral
evidence was developed pointing to the need for further Both Raman and infrared spectroscopy provide informa-
renement of the structural models, particularly for nation about chemical functionality, molecular conforma-
tive celluloses. The models derived from the crystallo- tion, and hydrogen bonding. Raman spectroscopy,
graphic studies could not rationalize many features of the however, has some important advantages in the study of
spectral data known to be quite sensitive to structural biological materials. The key advantage arises from the
variations. dierent bases for activity of molecular vibrations in the
On the other hand, electron microscopic studies based Raman and infrared spectra. That is, whereas activity in
on new staining techniques, specic to the reducing end the infrared region requires nite transition moments
groups of the polysaccharides, conrmed the parallel involving the permanent dipoles of the bonds undergoing
alignment of molecular chains within the microbrils in vibrations, activity in the Raman spectrum requires nite
native celluloses. These ndings were further conrmed transition moments involving the polarizabilities of the
by the manifestation, at the electron microscopic level, of bonds. Thus in infrared spectroscopy, the exchange of
the action of cellulases specic to the nonreducing end energy between the molecules and the exciting eld is
group; they were clearly active at only one end of each dependent on the presence of an oscillating permanent
microbril. The remaining questions at the time, therefore, dipole. In Raman spectroscopy, in contrast, the exciting
were concerned with the degree to which the symmetry of eld induces a dipole moment in the molecule and the
space group P21 is consistent with the other structure- induced moment then becomes the basis for exchange of
sensitive observations. energy with the exciting eld. It is useful in this context to
It is well to revisit the issue of levels of structure at this view bonds in terms of Paulings classication [7] along a
point and clarify the levels at which the dierent investi- scale between the two extremes of polar and covalent.
gative methods are most sensitive. The crystallographic Bonds that are highly polar and possess relatively high
models, which represent coordinates of the atoms in the dipole moments and reduced polarizabilities tend, when
unit cell, represent the most complete possible specication they undergo vibrational transitions, to result in bands that
of structure because they include primary, secondary, and are intense in the infrared and relatively weak in Raman
tertiary structures. And indeed, crystallographic studies of spectra. Conversely, bonds that are primarily covalent in
the monosaccharides and of related structures provide the character and have relatively low permanent dipoles and
basis for considerable information concerning bond high polarizabilities generally result in bands that are
lengths and bond angles, as well as conformations in intense in the Raman spectra, but are relatively weak in
saccharide structures. However, for polymeric systems, the infrared. This is perhaps best illustrated by the fact
the diractometric data are far more limited than for a that O2 and N2, which are homonuclear and without per-
single crystal of a low-molecular weight compound, so that manent dipoles, have very intense Raman spectra although
diraction data from a polymer must be complemented by they are inactive in infrared absorption, while H2O, with a
information from other structure-sensitive methods. An high permanent dipole moment, is a very strong absorber
acceptable model must rationalize not only the diracto- in the infrared but a very weak Raman scatterer. With
metric data, which for cellulose are quite limited in com- respect to cellulose, the OH groups of cellulose and those
parison to the number of coordinates that must be specied of adsorbed water are dominant in many of the spectral
in a denition of the unit cell, but it must also be such that features in infrared spectra. In contrast, the skeletal CC
they can be reconciled with information derived from other bonds and the CH bonds dominate the Raman spectra.
experimental measurements known to be sensitive to dif- A further simplication in the Raman spectra results from
ferent levels of structure. the circumstance that the selection rules forbidding activ-
The new spectral evidence that must be rationalized by ity of overtone and combination bands are more rigidly
any acceptable structure came from two methodologies adhered to than is the case in infrared spectra so that
that are most sensitive to structure at the secondary and the bands observed in Raman spectra are usually con-
tertiary levels. These are Raman spectroscopy and solid- ned to the fundamental modes of the molecules under
state 13C nuclear magnetic resonance (NMR) spectros- investigation [8].
copy, both of which were applied to cellulosic samples In the context of studies on the structure of cellulose,
for the rst time during the mid-1970s. The exploration of the key advantage of Raman spectroscopy is the degree of
spectra measurable by these two methods can provide its sensitivity to the skeletal vibrations of the cellulose
signicant information concerning both secondary and molecule, with the mode of packing in the lattice having
tertiary structures in the solid state. Because the spectral only secondary eects. This sensitivity is a consequence of
features observed are also sensitive to molecular environ- the reality that most of the skeletal bonds are CC bonds
ment, they are inuenced by the degree of symmetry of the and CO bonds, both of which have relatively high polar-
aggregated state. Hence they provide another avenue for izabilities and, hence, high Raman scattering coecients.
exploration of the applicability of the symmetry of space The minimal contribution of packing eects arises from the
group P21 to the structures of the solid state. low Raman scattering coecients of the highly polar OH
Developments in Characterization of Cellulose 125
groups, which are the functionalities that are most directly drates are predominantly made up of CC and CO bonds,
involved in intermolecular associations. The result is that which possess similar reduced masses and vibrational force
intramolecular variations such as changes in internal coor- constants and, hence, have very similar vibrational fre-
dinates have a signicantly greater inuence on the Raman quencies. In consequence, a high degree of coupling occurs
spectra than do variations in intermolecular associations. between the vibrations, with the result that very few of the
Finally and very signicantly, as the studies of the vibrational modes are localized within specic bonds or
celluloses progressed, it became clear that the most dra- functional groups. Thus, the traditional group frequency
matic dierences between the spectra associated with dif- approach common in the assignment of infrared and
ferent states of aggregation of cellulose occurred in the Raman spectra is of very limited use, except in the case
region between 200 and 700 cm 1, which is generally of vibrations localized in the bonds of hydrogen atoms
inaccessible with most infrared spectrometers. bonded to much heavier atoms such as O or C. On the
These considerations were paramount in the rst other hand, the normal coordinate analyses allow identi-
detailed examination and comparison of the Raman spec- cation of the degree to which the vibrations of each of the
tra of celluloses I and II [9]; the spectra are shown in Fig. 1. internal coordinates contributes to each of the observed
It was concluded that the dierences between the spectra, bands. Because the coupling of the vibrations is very
particularly in the low-frequency region, could not be sensitive to changes in the bond angles and in the dihedral
accounted for in terms of chains possessing the same angles associated with the bonds the vibrations of which
conformation, but packed in dierent ways in the dierent are coupled, the normal coordinate analyses allow a de-
lattices. As noted earlier, that had become the accepted tailed and systematic exploration of the eects of dier-
rationalization of the dierences between celluloses I and ences in skeletal conformations on the bands associated
II, as developed from diractometric studies of these two with particular vibrations.
most common allomorphs. The analyses of the Raman With respect to the question concerning the confor-
spectra led to the proposal that two dierent stable mations of celluloses I and II, it is useful to rst consider
conformations of the cellulose chains occur in the dierent some of the pertinent information developed from the
allomorphs. normal coordinate analyses, particularly with respect to
To establish a basis for assessing the dierences be- the classes of molecular motions associated with the
tween celluloses I and II, Atalla and coworkers undertook dierent spectral features. The region below 1500 cm 1
an extensive series of studies of model compounds of was the primary focus of the early exploration because the
increasing complexity [1018]. The model systems investi- intense bands clustered at about 2900 cm 1 can be iden-
gated included the 1,5-anhydropentitiols, the pentitols and tied with the CH stretching vibrations and the region
erythritol, the pentoses, the inositols, the hexoses, and the beyond 3000 cm 1 is clearly associated with the OH
cellobiose. The studies included comprehensive normal stretching vibrations. In addition to the CH and OH
coordinate analyses of the molecular vibrations of each stretching vibrations, the internal deformation of the
of the groups of model compounds based on complemen- methylene group on C6 is the only vibration that closely
tary infrared and Raman spectra. The objective of these approximates a group or local mode in the usual sense
analyses was to establish the degree to which the dierent implicit in discussions of assignments of vibrational spec-
classes of vibrational motions contribute to the spectral tra; the HCH bending vibration usually occurs above 1450
features in the dierent regions of the spectrum. Such a cm 1. In all other bands at frequencies below 1450 cm 1,
comprehensive approach was necessary because the skele- the normal coordinate analysis indicated that the vibra-
tal bond systems occurring in the structures of carbohy- tions are so highly coupled that, in most instances, no
single internal coordinate contributes more than 20% of
the potential energy change associated with any particular
frequency, although in a few instances contributions were
as high as 40%. Thus, as noted above, the traditional
group frequency approach to assignment of vibrational
spectra, which is based on the concept of local modes,
is generally not applicable in this region in the spectra
of saccharides. Instead, it is necessary to focus on the
classes of internal motions that are associated with the
dierent frequency ranges and to interpret the spectra in
terms of the inuence that variations in the internal co-
ordinates can have on the coupling between dierent types
of vibrational deformations.
For analysis of the spectra of celluloses, it is possible to
classify the groups of features in the dierent spectral
regions in terms of the types of internal deformations that
make their maximum contributions to bands in those
Figure 1 Raman spectra of high-crystallinity celluloses I regions. The bands between 1200 and 1450 cm 1 are
and II. attributable to modes involving considerable coupling
between methine bending, methylene rocking and wag- surfaces were found to possess multiple minima. When
ging, and COH in-plane bending motions; these are angle the additional constraint of a repeat length of approxi-
bending coordinates involving one bond to a hydrogen mately 0.515 nm per anhydroglucose unit was added, two
atom and the other to a heavy atom. Signicant contri- minima representing both left-handed and right-handed
butions from ring stretching begin below 1200 cm 1 and departures from the twofold helix appeared to be likely loci
these modes, together with CO stretching motions, of the stable conformations. It was noted in this context
dominate between 950 and 1150 cm 1. Below 950 cm 1, that these two minima were close to the positions of the
angle bending coordinates involving heavy atoms only dihedral angles of the glycosidic linkages in cellobiose and
(i.e., CCC, COC, OCC, OCO) begin to contribute, al- methyl h cellobioside, respectively, as these were deter-
though ring and CO stretches and the external bending mined from crystallographic studies [24,25].
modes of the methylene group may be major components Next, inquiry was made into the degree to which
as well. The region between 400 and 700 cm 1 is domi- changes in the dihedral angles about the bonds in the
nated by the heavy atom bending, both CO and ring glycosidic linkage could inuence the modes of vibration
modes, although some ring stretching coordinates still responsible for the spectral features in the dierent regions
make minor contributions. In some instances OH out- of the spectra. Two approaches were adopted for this
of-plane bending motions may make minor contributions purpose. The rst was based on examining the Raman
in this region as well. Between 300 and 400 cm 1, the ring spectra of polysaccharide polymers and oligomers that
torsions make some contributions, and below 300 cm 1, were known to occur in dierent conformations. The
they generally dominate. second was a theoretical one based on an adaptation of
In addition to the above generalized categorization the matrix perturbation treatment used by Wilson et al. [26]
concerning modes that occur in one or another of the to discuss the eects of isotopic substitution on infrared
model compound systems used in the normal coordinate and Raman spectra.
analyses, the spectrum of cellulose will have contributions The polysaccharide systems chosen for investigation
because of modes centered at the glycosidic linkage. Com- were among those most closely related to cellulose in the
putations based on the cellodextrins indicate that these sense that they are the a-1,4-linked polymers and oligomers
modes are strongly coupled with modes involving similar of anhydroglucose. They included amylose and two of its
coordinates in the adjacent anhydroglucose rings. The cyclic oligomers, with primary emphasis on the latter, the
contributions of the dierent classes of internal coordi- a- and h-Schardinger dextrins, often also known as cyclo-
nates to the dierent bands are presented in greater detail hexa- and cyclohepta-amylose. The structures of the two
elsewhere [19]. oligomers dier in that the values of the dihedral angles
As noted above and shown in Fig. 1, dierences about the bonds of the glycosidic linkages have to change
between the Raman spectra of celluloses I and II are quite to accommodate the dierent number of monomer units.
signicant particularly in the region of the skeletal bending Comparison of the Raman spectra of the cyclic dextrins
modes of vibration. In the region above 800 cm 1, the showed that the dierences between them were quite minor
dierences are most obvious with respect to the relative in the regions above 800 cm 1, but they were quite signif-
intensities of the bands and the broadening of some of the icant in the lower frequency region dominated by the
bands upon conversion from cellulose I to cellulose II. In skeletal bending and torsional modes. The dierences were
the region below 700 cm 1, in contrast, the main features similar in kind and distribution to the dierences between
are quite dierent in the two spectra; these dierences are celluloses I and II. It was also noted that in earlier studies of
even more evident in the spectra of single bers, which will the Raman spectra of amylose [27], it had been observed
be presented later. that forms Va and Vh, which are very similar in conforma-
In the analyses of the spectra of model compounds, tion but had dierent levels of hydration, had almost
changes of the magnitude indicated in Fig. 1 were exclu- identical spectra. In contrast, form B, which is known to
sively associated with the occurrence of dierences in have a distinctly dierent helix period, was found to have a
conformations. It seemed very probable therefore that spectrum that diers from those of forms Va and Vh in a
the dierences between the spectra of celluloses I and II manner approximating the dierences between the two
reect a change in molecular conformation accompanying cyclic oligodextrins. Taken together, the observations of
the transition from one form to the other. As the basic ring the Raman spectra of the amyloses support the interpreta-
structure is not expected to change [19], it would appear tion of the dierences between the Raman spectra of
that variations in the dihedral angles at the glycosidic celluloses I and II as pointing to dierences in the chain
linkages provide the only opportunity for conformational conformations localized at the glycosidic linkages.
dierences. Because of the controversy surrounding simi- In the theoretical analysis, the method of Wilson et al.
lar conclusions based on crystallographic studies carried [26] was adapted to explore the consequences of varia-
out in the early 1960s [21,22], a number of experimental tions in the dihedral angles about the bonds in the glyco-
and theoretical avenues for validating this interpretation sidic linkage; this approach is discussed in greater detail
were pursued. elsewhere [1]. These considerations led to the conclusion
The rst consideration was whether a multiplicity of that skeletal bending and torsional modes are altered to a
stable conformations is consistent with the results of greater degree than the skeletal stretching modes when the
conformational energy calculations that were available at dihedral angles associated with the glycosidic linkage
the time [20,23]. In both studies, the potential energy undergo variations. When translated to spectral features
in the Raman spectra, these observations point to major presenting dierent categories of information concerning
dierences in the low frequency region below 700 cm 1, the conformation of the anhydrocellobiose unit as a func-
and minor ones in the ngerprint region between 900 and tion of the values of the two dihedral angles about the
1500 cm 1. These are indeed precisely the types of dier- bonds in the glycosidic linkage. w is dened as the dihedral
ences observed in comparisons of the spectra of celluloses I angle about the bond between C4 and the glycosidic
and II. linkage oxygen and / as the dihedral angle about the bond
One nal consideration that was addressed is the between C1 and the glycosidic linkage oxygen. The paral-
possibility that rotations of the primary alcohol group at lel lines indicated by n=3(L), 2, and 3(R) represent values
C6 could account for the spectral dierences seen in the of the dihedral angles that are consistent with a left-
spectra of celluloses I and II and in the spectra of the handed threefold helical conformation, a twofold helical
amyloses. The normal coordinate analyses of the hexoses conformation, and a right-handed threefold helical con-
showed that rotations about the C5C6 bond can result in formation, respectively; a twofold helical conformation
minor variations in the region below 600 cm 1, but that the inherently does not have a handedness to it. The dashed
major impact of such rotations is expected in the spectral contours represent conformations that have the indicated
region above 700 cm 1 [16,17]. With all of the above repeat period per anhydroglucose unit; the innermost
considerations in mind, it became clear that the only represents a period of 5.25 A corresponding to 10.5 A
plausible rationalization of the dierences between the per anhydrocellobiose unit. The two dotted lines indicate
Raman spectra of celluloses I and II had to be based on conformations corresponding to values of 2.5 and 2.8 A
the possibility that dierences between the skeletal confor the distance between the two oxygen atoms anchoring
formations were the key. the intramolecular hydrogen bond between the C3 hy-
The rst eort to rationalize dierences in conforma- droxyl group of one anhydroglucose unit and the ring
tion was based on the results of the conformational energy oxygen of the adjacent unit; the values bracket the range
mappings that were available at the time [20,23]. The key wherein hydrogen bonds are regarded as strong. The two
points derived from those analyses, which have been domains dened by solid lines on either side of the twofold
conrmed by more recent studies [28,29], were that the helix line (n=2) represent the potential energy minima
two energy minima associated with variations in the dihe- calculated by Rees and Skerret for the dierent conforma-
dral angles of the glycosidic linkage correspond to relations of cellobiose. Finally, the points marked by J and W
tively small left-handed and right-handed departures from represent the structures of cellobiose determined by Chu
glycosidic linkage conformations that are consistent with and Jeries [24] and the structure of methyl h cellobioside
twofold helical symmetry. The minima also represented determined by Ham and Williams [25]. The key point to be
values of the dihedral angles that were very similar to those kept in mind in relation to this diagram is that structures
reported for cellobiose and methyl h cellobioside on the along the twofold helix line and with a repeat period of
basis of crystallographic analyses [24,25]. The relationship 10.3 A per anhydrocellobiose unit possess an unacceptable
between the dierent conformations is represented in degree of overlap between the van der Waals radii of the
Fig. 2, which was adapted by Atalla [30] from a diagram protons on either side of the glycosidic linkage.
rst presented by Rees and Skerret [20]. It is a w// map Consideration of these issues together with the results
of the Raman spectral observations led to exploration of
the possibility that small departures from the twofold helix
structures may be small enough that the conformation was
still approximated by a twofold helix. Some plausible
alternatives were explored. One was motivated by the
comparisons of the Raman spectra of cellulose II and of
cellobiose in the OH stretching region [30]. The latter
showed a single sharp band superimposed on a broader
background, and the band was identied with the OH
stretching vibration of the isolated intramolecular hydro-
gen bond revealed in the crystal structure [24]; it occurs
between the hydroxyl group on C3 of the reducing anhy-
droglucose unit and the ring oxygen of the nonreducing
unit. The spectrum of cellulose II revealed two such sharp
bands in the same region; similar bands were observed in
the spectra of the cello-oligodextrins [18]. As the frequency
Figure 2 W/U map ( ) loci of structures with at which such bands occur is very sensitive to the distance
constant anhydroglucose repeat periods; (: : : : : :) loci of between the oxygen atoms anchoring the hydrogen bond,
structures of constant intramolecular hydrogen bond (OO) it appeared that the structure of cellulose II must incor-
distances; ( _____ ) contours of potential energy minima porate intramolecular hydrogen bonds with two distinct
based on nonbonded interactions in cellobiose; W, h- values of the OO distance. This led to the proposal that
methylcellobiose, n=2, the twofold helix line; n=3 the successive units in the structure are not equivalent, and
threefold helix lines; (R) right-handed, (L) left-handed. The that, as a consequence, alternating glycosidic linkages
MeyerMisch structure is at W=180, U=0. have dierent sets of dihedral angles dening their coor-
dinates [30]. Thus, the dimeric anhydrocellobiose was

regarded as the repeat unit of physical structure rather
than the anhydroglucose unit. These conclusions, based
on the Raman spectra in the OH region, were conrmed
when the solid-state 13C NMR spectra became available
[31] as splittings were observed in the resonances associ-
ated with C1 and C4, which anchor the glycosidic linkage.
The occurrence of these splittings is indicative of the
presence of nonequivalent glycosidic linkages within the
structure; the NMR spectra will be considered in greater
detail in a subsequent section.
Upon further reection, it was recognized that when
alternating glycosidic linkages are admitted as an option,
and when anhydrocellobiose is viewed as the repeat unit of
structure, the alternating glycosidic linkages need not have
the same sense of departure from the twofold helix. That is,
it was now possible to consider structures wherein the
nonequivalent glycosidic linkages are alternating left-
handed and right-handed departures from the twofold
helix. Such structures would be ribbonlike and could
appear to approximate the twofold helix. The proposal
incorporating the alternating glycosidic linkage has the
advantage that it can be reconciled with much of the
diractometeric data. If the departure from twofold helical
symmetry is relatively small, it may account for the weak-
ness of the reections that are disallowed by the selection
rules of space group P21.
Based on the considerations outlined, the model that Figure 3 Structure of h-cellobiose and h-methylcellobioside.
was adopted as a basis for continuing explorations of the
spectra of cellulose was based on the proposal that the
glycosidic linkages alternated between small left-handed
and right-handed departures from the twofold helical con- explanation would also be consistent with the observation
formation. Thus, dierences between the conformations of that the two HCH bending bands in the Raman spectra of
celluloses I and II now had to be understood in terms of native celluloses collapse into a single band upon mercer-
dierences in the internal organization of the anhydrocel- ization, suggesting a nonequivalence of the two primary
lobiose units that were the basic units of structure [32,33]. alcohol groups in native cellulose and a shift closer to
In search of a rationalization of the changes in the equivalence upon mercerization. It is also consistent with
internal organization of the cellobiose unit associated with the greater splitting of the resonances associated with C1
the transition from cellulose I to cellulose II, Atalla drew on and C4 seen in the solid-state 13C NMR spectra of cellulose
an analogy with the structures of cellobiose and methyl h II to be discussed in the following section. While the evi-
cellobioside, which are represented in Fig. 3. The methyl h dence supporting this proposal is strong, it is not conclusive
cellobioside, which has values of the dihedral angles and, thus, awaits further conrmation. Atalla also intro-
corresponding to a right-handed departure from the two- duced the terms kI and kII to designate the conformations
fold helix, also has a bifurcated intramolecular hydrogen corresponding to celluloses I and II; the term k0 was
bond in which the proton from the C3 hydroxyl group introduced to describe cellulose in a disordered state [34].
appears to be located between the ring oxygen and the
primary alcohol oxygen at C6 of the adjacent unit. This B. Solid-State 13C NMR Spectra and the Two
bifurcation is in part responsible for the absence of a sharp Forms of Native Cellulose Ia and Ih
OH band in the OH region of the spectrum of the methyl h
cellobioside. Atalla suggested that such bifurcated intra- Although applied to cellulose later than Raman spectros-
molecular hydrogen bonds may occur in connection with copy, high-resolution solid-state 13C NMR has provided
every other glycosidic linkage in a molecule of native perhaps the most signicant new insights regarding the
cellulose; these bifurcated hydrogen bonds would be asso- structures of cellulose, particularly in its native state. The
ciated with those glycosidic linkages that have values of the development of high-resolution solid-state NMR spectros-
dihedral angles representing right-handed departures from copy and its application to polymeric materials grew from
the twofold helix in a manner not unlike those in methyl h complementary application of a number of procedures that
cellobioside. The action of mercerizing agents was seen as had been developed in NMR spectroscopy. The rst is
resulting in the disruption of the bifurcated OH bonds, proton carbon cross-polarization (CP) that is used to
thus, allowing the glycosidic linkages to relax to slightly enhance sensitivity to the low abundance 13C nucleus. This
greater departure from the twofold helix [22,23]. Such an was combined with high-power proton decoupling to
the analysis and interpretation of the information con-

tained within the spectra.
The rst applications of the new technique to cellulose
[31,35] demonstrated resolution of multiple resonances for
some of the chemically equivalent carbons in the anhydro-
glucose units. It became clear that rationalization of the
spectra that were observed would provide valuable addi-
tional information concerning the structure of the cellu-
loses investigated. The rst step in such a rationalization
was the assignment of the resonances that appear in the
spectra. The assignments, which have been discussed in a
number of reports [31,3540], were based on comparisons
Figure 4 13C CP-MAS spectrum of cotton linters. The with solution spectra of cello-oligosaccharides and of a
horizontal bars indicate the spectral ranges of the corre- low-DP cellulose [41]. They are indicated in Fig. 4, which
sponding carbon sites in the anhydroglucose monomer unit shows a spectrum of cotton linters [42]. Beginning at the
of cellulose. upeld part of the spectrum, the region between 60 and 70
ppm is assigned to C6 of the primary alcohol group. The
next cluster of resonances, between 70 and 81 ppm, is
attributed to C2, C3, and C5, the ring carbons other than
eliminate the strong dipolar interaction between the 13C those anchoring the glycosidic linkage. The region between
nuclei and neighboring protons. Finally, the angular de- 81 and 93 ppm is associated with C4, and that between 102
pendence of the chemical shift, or chemical shift anisot- and 108 ppm with C1, the anomeric carbon.
ropy, is overcome by spinning the sample about an axis at a In one of the rst reports on the application of the
special angle to the direction of the magnetic eld, com- technique to studies of dierent celluloses, the splittings of
monly referred to as the magic angle, the procedure the resonances of C4 and C1 in the spectrum of cellulose II
denoted by (MAS). The combined application of these (Fig. 5) were regarded as conrmation of the occurrence of
procedures, usually designated by (CP/MAS), results in nonequivalent glycosidic linkages that had earlier been
the acquisition of spectra that contain isotropic chemical proposed on the basis of the comparison of the Raman
shift information analogous to that obtained from liquid- spectra of cellulose II and of cellobiose in the OH stretch-
state 13C NMR with proton decoupling. ing region [31]. These splittings were also observed in the
In summary, the most important characteristic of the CP/MAS spectra of the cello-oligodextrins, which crystal-
spectra acquired using the (CP/MAS) 13C NMR technique lize in a lattice very similar to that of cellulose II. In that
is that, if they are acquired under optimal conditions, they context the splittings were attributed to the occurrence of
can have sucient resolution so that chemically equivalent
carbons occurring in magnetically nonequivalent sites can
be distinguished. In the present context, the corresponding
carbons in dierent anhydroglucose units would be
regarded as chemically equivalent. If they are not also
symmetrically equivalent, that is, if they occur in dierent
environments or if the anhydroglucose rings possess dif-
ferent conformations, within the rings, or at the glycosidic
linkage, or at the primary alcohol group, the carbons will
not have magnetically equivalent environments and will,
therefore, result in distinctive resonances in the NMR
spectrum. The fundamental challenge in the application
of this method is to achieve a level of resolution sucient to
distinguish nonequivalences between chemically equiva-
lent carbons, because the magnetic nonequivalence can
result in variations in the chemical shift that are small
relative to the shifts determined by the primary chemical
bonding pattern.
Another important feature of the (CP/MAS) 13C
NMR technique is that, for a system such as cellulose,
which consists of rather rigid hydrogen-bonded molecules
and in which all carbons have directly bonded protons, the
relative intensities of the resonances are expected to corre- Figure 5 CP/MAS 13C spectrum of high-crystallinity cel-
spond to the proportion of the particular carbons giving lulose II recorded at relatively low resolution. Chemical shifts
rise to them. Thus, the intensities arising from each of the are shown in parts per million relative to Me4Si. Assignment
six carbons in the anhydroglucose ring are expected to be of the C-1, C-4, and C-6 resonances are based on pertinent
equal. This is an important characteristic that is central to liquid-state spectra.
nonequivalent cellulose molecules in the same unit cell [38]. uted to chains at the surfaces of the brils and, on the basis of
However, such an interpretation leaves open the question these comparisons, it was concluded that approximately
as to why the resonances for carbons 2, 3, and 5, do not 50% of the wing is contributed by cellulose chains in each of
display similar splittings. If the splittings were indeed due the two types of less-ordered environments described in the
to nonequivalent molecules, it would be anticipated that preceding paragraph. Although the upeld wing of C4 is the
those carbons nearest to the boundaries of the molecule basis of this allocation of intensities, it can be assumed that
would be the most aected. The carbons anchoring the the relative contributions are similar for the upeld wing of
glycosidic linkage, that is C1 and C4, are the ones most C6 and for the component that appears to underlie the
removed from adjacent molecules, yet they also display the sharper resonances at C1. It is also expected that these
greatest splittings. domains contribute to the total intensity of the C2, C3,
Interpretation of the spectra of native celluloses pre- and C5 cluster between 70 and 81 ppm.
sented an even more challenging task. In the spectrum of The sharper resonances in the C6 and C4 regions,
cotton linters (Fig. 4), the two resonance regions associated centered at 66 and 90 ppm, respectively, each appear to
with C6 and C4 include sharper resonances overlapping consist of more than one resonance line, although the
broader upeld wings. After excluding the possibility that resolution is not sucient to distinguish the components
the broader wings could arise entirely from molecular well. The C6 resonance seems to include at least two com-
mobility [35,36], the wings were attributed to cellulose ponents while the C4 resonance appears to include three
chains in two categories of environment. The rst includes closely spaced component lines. These multiplicities were
all chains located at the surfaces of cellulose microbrils, interpreted as arising from carbons in cellulose molecules
which, because of their occurrence at the boundary, are less within the interior of crystalline domains and are therefore
constrained with respect to the conformations they can taken as evidence of the occurrence of chemically equiva-
adopt. The surfaces are regarded as regions of limited two- lent carbons in dierent magnetic environments within the
dimensional order. The importance of this category of crystalline domains.
order had earlier been demonstrated in a study of dierent The region between 102 and 108 ppm, attributed to
native celluloses undertaken by Earl and VanderHart [36]. C1, also reveals multiplicity and sharp resonance features.
The celluloses had natural bril diameters varying between Here, however, the shoulder is very limited. It appears that
3.5 and 20 nm, and it was shown that the areas of the the resonances associated with the two categories of disor-
upeld wings of C4 and C6 declined as the surface-to- dered domains described above lie underneath the sharp
volume ratio declined. The second category of environ- resonances associated with the interior of the crystalline
ments contributing to the upeld wings is that of chains in domains. It can be concluded that, in most instances, the
regions within which the incoherence of order is not limited dispersion of frequencies associated with the disorder is
to two dimensions. Here, the dispersion of the frequencies small relative to the shift associated with the character of
at which resonances occur may arise from conformational the anomeric carbon C1, while that is not the case for the
dierences, variations in bond geometries, changes in shifts associated with C4 and C6. One possible rationali-
hydrogen bonding patterns, and nonuniformities in neigh- zation may be that, because of the anomeric eect, the
boring chain environments. These possibilities arise be- internal coordinates surrounding C1 are much less exible
cause in such regions the molecular chains are free to adopt within the range of possible conformational variations
a wider range of conformations than the ordering in a than are the other internal coordinates.
crystal lattice or its boundaries would allow. In search of a rationalization of the splittings observed
Although the obvious upeld wings of the C4 and C6 in the sharp resonances, (CP/MAS) 13C NMR spectra of a
resonances are the most direct evidence for the cellulose wide variety of samples of cellulose I were recorded. Some
chains in less-ordered environments, it is expected that the of these are shown in Fig. 6. They include ramie bers (a),
chains in these environments make similar contributions to cotton linters (b), hydrocellulose prepared from cotton
the resonance regions associated with the other carbons. In linters by acid hydrolysis (c), a low-DP regenerated cellu-
the region of C1, the contribution appears to be primarily lose I (d), cellulose from Acetobacter xylinum (e), and
underneath the sharper resonances, although a small com- cellulose from the cell wall of Valonia ventricosa (f), an
ponent appears to extend toward 104 ppm. Similarly, it is alga. While similar observations were reported in a number
expected that the contribution from chains in the less- of studies [31,32,3640], their implications with respect to
ordered environments underlie the sharper resonances of structure were more fully developed in the work of Van-
the C2, C3, and C5 cluster. derHart and Atalla [42,43], which provides the basis for the
The relative contributions of the two categories of following discussion.
environment to the intensities of the upeld wings were All of the spectra shown in Fig. 6(AF) are of cellu-
assessed in a careful analysis of the C4 wing [42]. It was loses that occur in relatively pure form in their native states
demonstrated that part of the wing could be correlated with and require relatively mild isolation procedures. The most
the range of the C4 resonance in amorphous cellulose striking feature in these spectra, when viewed together, is
prepared by ball milling. It was therefore assigned to cellu- the variation in the patterns of the multiplets at C1, C4, and
lose chains occurring in the second type of environment, that C6. These resonances, which are viewed as arising from
is, domains wherein the incoherence of order is extended in chains in the interior of crystalline domains, appear to be
all three dimensions. The other part of the wing was attrib- unique to the particular celluloses; among the native forms
Figure 6 13C CP-MAS spectra of several cellulose I samples: (a) ramie; (b) cotton linters; (c) hydrocellulose from cotton linters;
(d) a low-DP regenerated cellulose I; (e) Acetobacter xylinum cellulose; (f ) Valonia renrricosa cellulose. Note the varied ne
structure particularly at C-1 and C-4. Signal-to-noise variation due to limited amount of some samples. In that instance more
polyethylene was added so the side band intensity increased. No line broadening or resolution enhancement techniques were
applied in the acquisition of the spectra (after VanderHart and Atalla).
they appear to be distinctive of the source species. The rst position are shown as spectra (b) and (c) in Fig. 7, and were
attempt to rationalize the spectra was in terms of informa- designated as the Ia and Ih forms of native cellulose; this
tion that they might provide concerning the unit cell of the designation was chosen in order to avoid the possibility of
structure of cellulose I. However, it soon became obvious confusion with the IA and IB forms that had earlier been
that such a rationalization was not possible because the dened in terms of dierences in the appearance of the O
relative intensities within the multiplets were not constant, H bands in dierent types of native celluloses [44,45].
nor were they in ratios of small whole numbers as would be Spectrum (A) was acquired from a high-crystallinity sam-
the case if the same unit cell prevailed throughout the ple of cellulose II and is included so as to distinguish the
crystalline domains. The conclusion was that the multiplic- heterogeniety of crystalline forms occuring in the dierent
ities were evidence of site heterogeniety within the crystal- forms of cellulose I from the long-known polymorphic
line domains and that therefore native celluloses must be variation of the crystallinity of cellulose.
composites of more than one crystalline form. Spectra b and c in Fig. 7 were in fact derived from
Further rationalization of the spectra required a care- appropriate linear combinations of the spectra of the low-
ful analysis of the mutliplets at C1, C4, and C6, and the DP cellulose I (d) and of the A. xylinum cellulose (e) in Fig.
variations of the relative intensities of the lines within each 6. Although they represent the best approximations to the
multiplet among the spectra of the dierent celluloses. In two forms of cellulose postulated, they cannot be regarded
addition to excluding a single crystal form on the basis of as representative of the pure forms as they do not ade-
the considerations noted above, it was also possible to quately reect the component of the cellulose at the
exclude the possibility of three dierent forms with each surfaces of the crystalline domains. Spectrum 7B does have
contributing a line to the more complex multiplets. Thus, a some intensity in the upeld wings of C4 and C6, but
decomposition of the spectra on the basis of two distinct spectrum 7C has very little evidence of such wings. There is
crystalline forms was pursued. The results of the decom- very little question, however, that the sharp components of
associated with dierent morphologies could contribute

distinctive spectral features. Exploration of the spectra of
higher plant celluloses with dierent native morphologies
revealed very little dierence in the essential features of the
spectra, even after the samples had been subjected to acid
hydrolysis. Furthermore, it was concluded that the Ia
component of higher plant celluloses was suciently low
that some question was raised as to whether it occurs at all
in these higher plant celluloses. In this context, it was also
concluded that without the Ia component in higher plant
celluloses, the lineshapes of the Ih form at C4 could only be
reconciled with a unit cell possessing more than four
anhydroglucose residues per unit cell.
Attention was then directed to analysis of the spectra
of algal celluloses, wherein the Ia component is the dom-
inant one. Relaxation experiments conrmed that the
essential spectral features identied with the two crystalline
forms of cellulose were characteristic of the core crystalline
domains; when measurements were conducted such that
magnetization of the surface domains was rst allowed to
undergo relaxation, very little change in the spectral fea-
tures was observed. The relaxation experiments suggested
that domains consisting of both the Ia and Ih forms have
equal average proximity to the surface. One possible
interpretation of these observations, that the two forms
are very intimately mixed, was ruled out at that time on the
basis of hydrolysis experiments the results of which are now
Figure 7 Comparison of the 13C CP-MAS spectrum (a) of a in question.
low-DP cellulose II sample and the spectra (b) and (c) cor- Two groups of modifying experiments were carried
responding, respectively, to the two proposed crystalline out with the algal celluloses. In the rst, the algal cellu-
forms of cellulose I, namely Ia and Ih. Spectra (b) and (c) loses were subjected to severe mechanical action in a
were obtained by taking linear combinations of the low-DP Waring blender. In the second, the algal celluloses were
and Acetobacter cellulose spectra. Discontinuities in spectra subjected to acid hydrolysis, in 4 N HCl for 44 h at 100jC.
(b) and (c) occur where the polyethylene sidebands would have While the mechanical action resulted in some reduction in
appeared. The Ia spectrum still contains a signicant amount the proportion of the Ia form, the acid hydrolysis resulted
of non-Ia resonances as shown by the visible C-4 and C-6 in a dramatic reduction, sucient indeed to make the
upeld wings. Multiplicities of the C-1, C-4, and C-6 narrower spectra seem like those of the higher plants, except that
resonances ought to indicate unit cell inequivalences. the resolution of the spectral lines was much enhanced
relative to that observed in the spectra of even the purest
higher plant celluloses. The samples subjected to hydro-
lysis, wherein the recovery varied between 12% and 22%,
spectra 7B and 7C include the key features in the spectra of were examined by electron microscopy and shown to have
the Ia and Ih forms. It is of interest to note here that among lateral dimensions not unlike those of the original sam-
the distinct resonances of the Ia form at C1, C4, and C6, ples. These observations were interpreted to imply that the
only the one at C4 appears to be split, while for the Ih form Ia form is more susceptible to hydrolysis than the Ih form.
all three resonances associated with these carbons show An earlier study of the eect of hydrolysis, under similar
splitting, with the one at C1 the most pronounced. conditions but for only 4 h, had been carried out with
In an eort to further validate the proposal that the Ia cellulose from Rhizoclonium heiroglyphicum with no dis-
and Ih forms were the primary constituents of native cernible eect on the spectra [47]. The dierence in
celluloses, VanderHart and Atalla [46] undertook another duration of the hydrolysis may well have been the key
extensive study to exclude the possibility that experimental factor. Both of these observations and their interpreta-
artifacts contributed to the key spectral features assigned to tions had been presented, however, before it was recog-
the two forms. A number of possible sources of distinctive nized that exposure of celluloses with relatively high
spectral features were explored. The rst was the question contents of the Ia form to elevated temperatures can
whether surface layers associated with crystalline domains result in its conversion to the Ih form [48]. When the
within particular morphological features in the native possibility that the Ia content of the algal cellulose had
celluloses could give rise to features other than those of been converted to the Ih form is taken into account, the
the core crystalline domains. The second was whether results of the relaxation experiments of VanderHart and
variations in the anisotropic bulk magnetic susceptibility Atalla cited above can be reinterpreted as indicating
standing the dierences between them and their relation-

ship to each other within the morphology of native
cellulosic tissues. A number of complementary approaches
were pursued by dierent investigators in the search for
answers. Some were based on further application of solid-
state 13C NMR to the study of dierent celluloses as well as
to celluloses that had been subjected to dierent modifying
treatments. Others were based on application of Raman
and infrared spectroscopy to new classes of cellulosic
samples. Still, others were based on renement of electron
microscopic and electron diractometric methods. Results
of these investigations will be presented in summary.
A. Raman and Infrared Spectra

The categorization of native celluloses into the IA and IB
group by Howsmon and Sisson [44] and Blackwell and
Figure 8 Alternative candidates for the spectra of cellulose
Ia (top) and Ih (bottom) derived from linear combinations of Marchessault [45] on the basis of the appearance of the
the spectra of Ia-rich Cladophera glomerata, before and after OH stretching region of their infrared spectra suggested
acid hydrolysis, which resulted in a Ih-rich cellulose. that the hydrogen bonding patterns within the crystalline
domains may be part of the key to the dierences between
the two forms of native cellulose. This was, in fact,
conrmed in the course of more detailed investigations
intimate mixing of the Ia and Ih forms within the crystal-
of the Raman spectra carried out on single oriented bers
line domains of the algal celluloses.
of native celluloses [49] and in a comprehensive study of
VanderHart and Atalla also took advantage of the
the infrared spectra of a number of celluloses of the two
spectra derived from the acid hydrolyzed samples of the
forms [50]. The Raman spectral investigations were part
algal cellulose to generate more highly resolved represent-
of a broader study directed primarily at rationalizing the
ative spectra of the Ia and Ih forms. These are shown in
bands associated with the skeletal vibrational motions
Fig. 8, where it is clear that even in the spectrum represent-
and at exploring the dierences between celluloses I and
ative of the Ia form the upeld wings of the C4 and C6
II [49]. They diered from earlier Raman spectral studies
resonances are reduced to a minimum. With the comple-
in that the spectra were recorded with a Raman micro-
tion of this study by VanderHart and Atalla, most of the
probe on which individual bers could be mounted for
questions about the possibility that the spectral features
spectral investigation. With this system, it was also pos-
were the results of artifacts were put to rest, and the
sible to explore the variation of the intensity of the bands
hypothesis that all native celluloses belong to one or to a
as the polarization of the exciting laser beam was rotated
combination of these forms was generally accepted.
relative to the axis of the bers.
With the above resolution of the questions concern-
The observed spectra are shown in Figs. 9 and 10, each
ing the nature of native celluloses in mind, it was possible
of which includes six spectra. Fig. 9 shows the region
to classify these celluloses with respect to the relative
between 250 and 1500 cm 1, while Fig. 10 shows the region
amounts of the Ia and Ih forms occurring in the celluloses
above 2600 cm 1; the region between 1500 and 2600 cm 1
produced by particular species. It emerged in these early
does not contain any spectral features. The spectra in Figs.
studies that the celluloses from more primitive organisms
9 and 10 are of native and mercerized ramie bers and of
such as V. ventricosa and A. xylinum are predominantly of
native V. ventricosa, and they are recorded with both
the Ia form, while those from higher plants such as cotton
parallel and perpendicular polarization of the exciting laser
and ramie are predominantly of the Ih form. As noted
beam. Those identied as 0j spectra were recorded with the
earlier, the nomenclature chosen was intended to avoid
polarization of the electric vector of the exciting laser beam
confusion with the IA and IB forms previously used to clas-
parallel to the direction of the ber axes, while those
sify the celluloses on the basis of their infrared spectra in
identied as 90j spectra were recorded with the polariza-
the OH stretching region. In relation to that classication,
tion of the electric vector of the laser perpendicular to the
the NMR spectra suggest that the IA group has the Ia form
ber axes. The ramie bers are known to have the molec-
as its dominant component, while the IB group is predom-
ular chains parallel to the ber axes; the V. ventricosa bers
inantly of the Ih form.
were prepared by drawing the cell wall so as to align the
microbrils within it.
IV. FURTHER STUDIES OF STRUCTURES A number of features in the spectra are noteworthy
IN CELLULOSE with respect to earlier discussions. The rst is a compar-
ison of the spectra of V. ventricosa and ramie. It is clear
With the wide acceptance of the proposal of the two that, apart from a broadening of the bands in the ramie
crystalline forms (Ia and Ih) came the challenge of under- spectra, because of the smaller lateral dimensions of the
the polarization of the exciting radiation is parallel to the

chain direction. The sensitivity of the Raman spectra to the
orientation of an intramolecular vibrational motion is also
illustrated in the intensity of the methine CH stretching
band at about 2889 cm 1. It is most intense with the electric
vector of the exciting radiation at 90j to the chain axis, an
orientation that is parallel to that of the methine CH
bonds of the pyranose rings.
Finally, in light of the discussion of the nonequiva-
lence of adjacent anhydroglucose units and the cor-
responding nonequivalence of alternating glycosidic
linkages, the OH region in the 0j spectrum of the mercer-
ized ramie is of particular interest. It shows the two distinct
sharp bands that provide evidence of the presence of
isolated nonequivalent intramolecular hydrogen bonds in
agreement with the alternating glycosidic linkages along
the chain; the hydrogen bonds are oriented parallel to the
chain direction. This alternation clearly stands out most
distinctly in cellulose II. These distinct bands cannot be
attributed to nonequivalent chains as the dierence in fre-
quency implies a dierence in the OO distances between
the oxygen atoms anchoring the hydrogen bond, as well as
Figure 9 Comparison of the Raman spectra from Valonia,

ramie, and mercerized ramie (low-frequency region). Spectra
were recorded with the electric vector at both 0j and 90j.
crystalline domains, the spectra are very similar except in

the OH stretching region. This was interpreted as evidence
that the chain conformations in both the Ia and Ih forms
are the same, but that the hydrogen bonding patterns
between the chains are dierent within the two forms. This
interpretation is more clearly demonstrated in a compari-
son of the spectra of V. ventricosa and Halocynthia to be
presented below.
The second feature worthy of note is the dramatic
dierence between the spectra of native (cellulose I) and
mercerized (cellulose II) ramie bers, particularly in the
low-frequency region, which is inaccessible to most infra-
red spectrometers. This was taken as further conrmation
that the conformations of cellulose I and cellulose II must
dier suciently to result in signicant alteration of the
coupling patterns between the internal vibrational modes
of the pyranose rings in the molecular chains. It is also
interesting, in this connection, to compare the intensities of
the band at 1098 cm 1 in the spectra of the two forms of
ramie. The band is clearly less intense in the spectrum of the
mercerized sample, suggesting that a conformational
change, which reduces the coupling of the skeletal motions, Figure 10 Comparison of the Raman spectra from Valonia,
has occurred. The 1098-cm 1 band is the strongest skeletal ramie, and mercerized ramie (high-frequency region). Spectra
band and it is the most intense feature in the spectrum when were recorded with the electric vector at both 0j and 90j.
a dierence in the dihedral angles w and / of the associated appears to be polarized perpendicular to the bril direc-
glycosidic linkages. Nonequivalent chains would have tion, while the one at 750 cm 1 is not polarized. It was also
dierent periods in the chain direction if they were to observed that upon transformation of the Ia-rich celluloses
possess twofold helical symmetry. to the Ih form through annealing, the corresponding bands
The infrared spectral studies of the Ia and Ih forms changed accordingly. The authors concurred with the
were carried out by Sugiyama et al. [50] on a number of interpretation of the dierences between the two forms
dierent native celluloses of both forms. Furthermore, it suggested by Wiley and Atalla, and concluded that the Ia-
included examination of a number of Ia-rich celluloses that to-Ih transformation primarily corresponded to a rear-
were converted to the Ih form through the annealing rangement of the hydrogen bond system within the struc-
process rst reported by Yamamoto et al. [51]. To com- tures and that the two structures appeared to have very
plement the infrared spectra, Sugiyama et al. [52] recorded similar conformations. The infrared spectral studies by
electron diraction patterns for the samples, which allowed Sugiyama et al. are particularly interesting because they
classication of the celluloses through comparison with the included the spectra of both Valonia and Halocynthia, the
diraction patterns acquired in an earlier electron dirac- Raman spectra of which have been investigated at high
tometeric study to be discussed in greater detail in a resolution [53].
subsequent section. The Raman spectra of Valonia macrophysa and Hal-
The key nding emerging from the examination of the ocynthia (tunicate) celluloses obtained by Atalla et al. [53]
infrared spectra of the dierent forms was that the only are shown in Fig. 11. These particular spectra are of interest
dierences noted were in bands clearly associated with the because V. macrophysa is known to be predominantly the
OH group. This was also true of the changes observed upon Ia form while Halocynthia is predominantly of the Ih form.
conversion of the Ia form to the Ih form through annealing. Comparison of their spectra can be more rigorous than was
The bands associated with both the dierences in native possible in the earlier work of Wiley and Atalla [49] because
forms and with the eects of transformation were observed the lateral dimensions of the brils of both forms are of the
in both the OH stretching region above 3000 cm 1 and the order of 20 nm, with the result that their spectra show equal
OH out-of-plane bending region between 650 and 800 resolution of the bands in all regions of the spectrum. It is
cm 1. It was reported that spectra of the Ia form had to be noted that their spectra are essentially identical in all
distinctive bands at 3240 and 750 cm 1, while the spectra of the regions associated with skeletal vibrations of all
of the Ih form had distinctive bands at 3270 and 710 cm 1. types as well as regions associated with most of the
Furthermore, it was observed that the band at 3240 cm 1 vibrations involving CH bonds, whether in the bending
appears to be polarized parallel to the direction of the bril or stretching regions. Indeed, the primary dierences be-
orientation while the band at 3270 cm 1 is not polarized. tween the two spectra are in the broad complex bands
Among the low-frequency bands, the one at 710 cm 1 occuring in the OH stretching region, and these dierences
Figure 11 Raman spectra of Ia-rich Valonia and Ih-rich Haleynthia celluloses. Because both have brils of large lateral
dimensions, the spectra of both are well resolved and provide a better basis comparison of the spectra of Ia-rich and Ih-rich
celluloses.
are not unlike those noted in the earlier Raman spectral spectral data because of the dierences in the level of
studies described above. In addition, the weak band at resolution between the spectra of ramie and Valonia cellu-
about 840 cm 1 in the spectrum of V. macrophysa has no loses, the spectra shown in Fig. 11 are of suciently high
corresponding band in the spectrum of Halocynthia; this is resolution and suciently similar that the comparisons can
the band attributed to the out-of-plane bending vibrations indeed be made with condence.
of hydrogen-bonded OH groups. There are also minor The key issue is that when crystal structures possess
dierences in the relative intensities of the methylene CH more than one molecule per unit cell, and the molecules
stretches and HCH bending vibrations, but these are the have the same vibrational frequencies, the vibrational
natural consequences of dierent hydrogen bonding pat- modes of the unit cell become degenerate. Under these
terns for the hydroxyl group at C6. Comparison of the two circumstances, couplings will arise between equivalent
spectra reinforces the interpretation presented earlier, on modes in the dierent molecules, and it is generally ob-
the basis of spectra in Figs. 9 and 10, concluding that the served that such couplings result in splittings of the bands
only dierence between the Ia and Ih forms is in the pattern associated with key vibrational modes. The type of cou-
of hydrogen bonding. Thus, the Raman spectral compar- pling that is relevant in the case of cellulose is that described
ison of the two forms is entirely consistent with that as correlation eld splitting [54]. This eect arises because,
reported for the infrared spectra of these highly crystalline as a result of the coupling, the vibrations of a particular
celluloses. It must be kept in mind, of course, that the bands mode in the two molecules will now occur at two frequen-
associated with OH group vibrations are not expected to cies that are dierent from those of the isolated molecule;
coincide in the Raman and infrared spectra; because of the one of the two new frequencies will have the modes in the
dierent bases for activity in the two dierent spectral two dierent molecules in phase with each other, while the
approaches to measurement of vibrational frequencies, other will have the modes out of phase with each other.
Raman active vibrational modes are frequently silent in Such correlation eld eects result in doublets with a
the infrared and vice versa. This, of course, is true for the splitting of 1015 cm 1 in some modes of crystalline poly-
skeletal bands as well. ethylenes having two chains per unit cell. Because no
In view of the considerable variation observed in the evidence of such splittings occurs in the Halocynthia spec-
Raman spectra of celluloses as a result of changes in trum shown in Fig. 11, it must be concluded that the Ih
molecular conformations, there can be little question that form cannot have more than one molecule per unit cell.
the spectra in Fig. 11 indicate that the conformations of the Nor can it be suggested that the two molecules in a
cellulose molecules in Valonia and Halocynthia are essen- monoclinic unit cell are nonequivalent and may have
tially identical. It is also important to note that the Raman modes that are at dierent frequencies, because the skeletal
spectra of the celluloses from V. macrophysa and V. bands in the Halocynthia spectrum are essentially identical
ventricosa, both of which have been used in dierent studies to those in the Valonia spectrum. Furthermore, this simi-
as representatives of the Ia form, are eectively indistin- larity was also reported in the infrared spectra observed by
guishable in all regions of the spectra. This is also true of Sugiyama et al. (cited earlier). Thus, it is clear that the
the Raman spectra of celluloses from the algae Cladophera vibrational spectra, both Raman and infrared, point to the
glomerata and Rhizoclonium heirglyphicum, which have conclusion that both the Ia and Ih forms have only one
also been used in many studies as representative of cellu- molecule per unit cell. This conclusion of course raises the
loses that are predominantly of the Ia form. question as to why the crystallographic data have been
In summary, the Raman and infrared spectral studies viewed for so long as pointing to a two-chain unit cell with
undertaken after the discovery of the composite nature of the symmetry of space group P21. This is an issue that is
native celluloses point to the conclusion that the only best addressed after the results of the electron diracto-
dierence between the two forms is in the pattern of metric studies have been described in greater detail.
hydrogen bonding between chains that possess identical
conformations. Yet electron microscopic and electron B. Solid-State 13
C NMR Spectra
diractometric studies, to be described in greater detail
in a following section, have led to conclusions that the two It is not surprising that the methodology that rst provided
forms represent two crystalline phases with dierent crys- the basis for understanding the composite nature of native
tal habits [52]. It is therefore important to consider what celluloses in terms of the Ia and Ih duality has continued to
information may be developed from the vibrational spec- be the one most often used for seeking deeper understand-
tra with regard to this question. ing of the dierences between native celluloses derived
The key conclusion drawn from the electron dirac- from dierent biological sources. This has been facilitated
tometeric data was that the Ia form represents a triclinic by the greater availability of solid-state 13C NMR spec-
phase with one chain per unit cell, while the Ih form trometer systems and by the relative simplicity of the
represents a monoclinic phase with two chains per unit procedures for acquiring the spectra from cellulosic sam-
cell. Furthermore, the symmetry of the monoclinic phase ples. The studies undertaken on the basis of further exam-
appeared to be that of space group P21. It has been recently ination of the solid-state 13C NMR Spectra of celluloses are
recognized [53] that such a proposal is not consistent with in a number of categories. The rst group is focused on
the vibrational spectra. While it was not possible to have further examination of the spectra of dierent native
full condence in this conclusion based on the earlier celluloses, in part aided by mathematical procedures for
deconvolution of the spectra or for resolution enhance- on deconvolution of the spectral features into combina-
ment. Another group relies on exploring the spectral tions of Lorentzian functions centered at the assigned shifts
manifestations of native celluloses that have been modied for the particular resonances. It is to be noted that the use
in dierent ways. Yet, a third approach is based on of Lorentzian functions, which can be justied at a funda-
investigation of celluloses subjected to dierent but well- mental level in the case of spectra from molecules in
known procedures for inducing structural transformations solution, has no basis in any fundamental understanding
in the solid aggregated state of cellulose. of the phenomenology of acquisition of the solid-state 13C
The group at the Kyoto University Institute for Chem- NMR spectra. However, because deconvolution into Lor-
ical Research carried out important studies that were entzians has been found to be a useful tool in assessing the
complementary to those undertaken by VanderHart and spectral features in the spectra of cellulose, its use has
Atalla [42,43,45]. More recently, a number of other groups continued. The qualications that must be kept in mind
have made contributions. As a number of questions con- when it is used have recently been addressed by Vander-
cerning the nature of the Ia and Ih forms remain outstand- Hart and Campbell [56].
ing, it is useful to begin with an overview of the ndings In the early studies by Horii et al., all of the upeld
of dierent groups in this respect. These will then make wing of the C4 resonance was attributed to molecules in
it possible to view results of studies by using other methods noncrystalline domains. On this basis, the lineshape anal-
in a clearer perspective. ysis of the C4 resonance of dierent native celluloses did
The early studies by the Kyoto University group have not seem consistent with the model proposed by Vander-
been well summarized in a report that addresses the key Hart and Atalla [42] with respect to the composite nature of
points that were the focus of their investigation [55]. In a native celluloses. In later studies, when Horii et al. took
careful analysis of the chemical shifts of the C1, C4, and note of the fact that, in the study by VanderHart and
C6 carbons in the (CP/MAS) spectra of monosaccharides Atalla, approximately half of the upeld wing of C4 in the
and disaccharides for which crystallographic structures spectra of higher plant celluloses was attributed to the
were available, Horii et al. recognized a correlation surface molecules of crystalline domains, Horii et al. [57]
between the chemical shifts and the dihedral angles de- indicated that their results conrm the proposal of Van-
ned by the bonds associated with these particular car- derHart and Atalla. It is to be noted that in their early
bons. In particular, with respect to C6, they demonstrated reports in this area, Horii et al. used the designations Ib and
a correlation between the chemical shift of the C6 reso- Ia to designate the dierent groups of celluloses in which
nance and the value of the dihedral angle v dening the the Ia and Ih forms were dominant. However, in their more
orientation of the OH group at C6 relative to the C4C5 recent studies, they have adopted the Ia and Ih designations
bond in the pyranose ring. This correlation is of value in that are designed to avoid the confusion with the categories
the interpretation of the solid-state 13C NMR spectra with rst introduced by Howsmon and Sisson discussed earlier.
respect to structure as well as discussion of the implica- In pursuit of further understanding of the Ia and Ih
tions of splittings of the C6 resonances observed in some duality, Horii et al. explored the eects of transformative
of the spectra. treatments on the solid-state 13C NMR spectra. The rst
Of even greater interest, in light of the discussions of group of studies was directed at the eects of annealing,
deviations from twofold screw axis symmetry in some of rst in saturated steam [48], and later in aqueous alkaline
the structures, it was observed that the chemical shifts of C1 solutions (0.1 N NaOH) selected to avoid hydrolytic
and C4 are correlated with the dihedral angles about the decomposition of the cellulose [58,59]. In summary, the
glycosidic linkage. In particular, there was a correlation key ndings were that the celluloses wherein the Ia form is
between the shift of C1 and the dihedral angle / about the dominant are substantially transformed into the Ih form
C1O bond, and a correlation between the shift of C4 and when conditions are established so as to allow the trans-
the dihedral angle w about the OC4 bond. As the spectra formation to be complete. The cellulose representative of
published in the earliest studies did not have sucient the Ia form that was used for these studies was V. macro-
resolution to reveal the splittings of the resonances of C1 physa. The eects of the annealing treatment are demon-
and C4, the possibility of occurrence of nonequivalent strated in Fig. 12, which shows the progression in the
glycosidic linkages was not addressed at that time. degree of conversion as the temperature of treatment is
In addition to the analysis of the correlation between increased. Each of the treatments was for 30 min in the
the chemical shifts and the dihedral angles, the Kyoto aqueous alkaline solution. These results, of course, point
group investigated the distribution of cellulosic matter to the susceptibility of the Ia form to conversion to the Ih
between crystalline and noncrystalline domains on the form, suggesting that the latter is the more stable form.
basis of measurements of the relaxation of magnetization To test this hypothesis, a sample of tunicate cellulose,
associated with the dierent features of the spectra. By which had earlier been shown to be of the Ih form by
measurement of the values of the spin lattice relaxation Belton et al. [60], was also annealed in an aqueous alkaline
times T1(C) associated with the dierent spectral features, solution at 260jC; it showed little change as a result of
they developed a quantication of the degree of crystallin- the annealing [59].
ity in the dierent celluloses. They also undertook analysis Additional studies by the Kyoto group relied on the
of the lineshapes of the dierent resonances, particularly solid-state 13C NMR to explore the eects of dierent
that of the C4 resonance. The lineshape analysis was based variables on the structure of cellulose [61]. It is in order,
ously such as the monitoring of the value of T1(C)

associated with the dierent spectral features. While these
procedures incorporate a signicant degree of empiricism,
they have facilitated rationalization of the spectral fea-
tures of a number of native celluloses and are therefore
valuable contributions to the repertoire of methods avail-
able for interpreting the 13C NMR spectra of native
celluloses. It must be noted, however, that the application
of these methods has been complemented in the work of
Newman and Hemingson, by a considerable degree of
awareness of the complexity of the structures of both
native and processed celluloses, so that their application
by others needs to be approached with this awareness in
mind. This work is described in the publications of New-
man et al. [6267], and has been presented in an overview
elsewhere [1].
A dierent approach to mathematical analysis of the
solid state 13C NMR spectra of celluloses was introduced
by the group at the Swedish Forest Products Laboratory
(STFI) [68]. They took advantage of statistical multivariate
data analysis techniques that had been adapted for use with
spectroscopic methods. Principal component analysis
(PCA) was used to derive a suitable set of subspectra from
the CP/MAS spectra of a set of well-characterized cellu-
losic samples. The relative amounts of the Ia and Ih forms
and the crystallinity index for these well-characterized
samples were dened in terms of the integrals of specic
features in the spectra. These were then used to derive
subspectra of the principal components, which in turn were
used as the basis for a partial least squares analysis of the
experimental spectra. Once the subspectra of the principal
components are validated, by relating their features to the
known measures of variability, they become the basis for
Figure 12 50-MHz CP/MAS 13C NMR spectra of Valonia analysis of the spectra of other cellulosic samples that were
cellulose annealed at dierent temperatures in 0.1 N NaOH not included in the initial analysis. Here, again, the inter-
solution: (a) original; (b) 220jC; (c) 240jC; (d) 260jC. ested reader can refer to the original publications [6871] or
the overview presented earlier [1].
in the present context, to briey note the results of one C. Electron Microscopic Studies
study in which celluloses from A. xylinum cultures were
investigated. One of the variables explored was the tem- The use of electron microscopy in the study of celluloses,
perature of the culture; it was observed that lower temper- particularly in their native state, has resulted in important
atures favored the formation of the Ia form at the expense advances beginning with investigations that were under-
of the Ih form. This nding raises a fundamental question taken at the time of the introduction of the earliest electron
regarding the possibility that the variation of the balance microscopes. The early work has been ably reviewed by a
between the two forms is, in part, an adaptive response to number of authors [72,73]. Of particular note among these
changes in the environment. is the coverage of the subject in the treatise by Preston [74].
We follow with a commentary on the manifestations in The earliest and most signicant observations, from a
the solid-state 13C NMR spectra of a broader category of structural perspective, were those by Hieta et al. [75], in
structural changes induced by dierent treatments known which they applied a staining method incorporating a
to alter the states of aggregation of cellulose. In selecting chemistry that requires the presence of reducing end
the investigations to be noted in our discussion, we will groups. They observed that when whole microbrils of
focus on studies that provide insight into the variations of Valonia were viewed, only one end of each microbril was
the states of aggregation with the history of particular stained. This clearly indicated that the molecular chains
celluloses, both with respect to source and with respect to were parallel as the reducing ends of the cellulose chains
processes of isolation and transformation. occurred together at one end of the brils. Had the
In 1990, Newman and Hemingson [62] began to structure been one with an antiparallel arrangement of
combine some additional methods of processing the 13C cellulose chains, it would have been expected that the
NMR spectral data with those that had been used previ- reducing end groups would occur with equal frequency at
both ends of the microbril with the result that both ends cellulose chains than could possibly arise from the individ-
would be equally stained. ual membrane complexes associated with the biogenesis
The conclusions of Hieta et al. were independently of cellulose.
conrmed by another method introduced by Chanzy and Later studies by Sugiyama et al. were based on elec-
Henrissat [76], wherein the microbrils were subjected to tron diraction and were directed at addressing questions
the action of a cellobiohydrolase that is specic in its concerning the nature of the dierences between the Ia and
action on the nonreducing ends of the cellulose chains. Ih forms of cellulose. In a landmark study [82], electron
They observed a clear narrowing of the tips of the microdiraction patterns were recorded from V. macrophysa in
brils to a triangular form at only one end of each micro- both its native state, wherein the Ia and Ih forms occur in
bril. In this instance, the action was at the nonreducing their natural relative proportions, and after annealing
ends, but the observations were equally convincing evi- using the process rst reported by Yamamoto et al. [51],
dence that the chains are aligned in a parallel arrangement which converts the Ia form into the Ih form. The native
in these microbrils. material, which is predominantly the Ia form, was shown
These early studies were focused on microbrils from to produce a complex electron diraction pattern similar
algal celluloses that, because of their larger lateral dimen- to that which had earlier led Honjo and Watanabe [83] to
sions, could be more easily visualized in detail. More propose an eight-chain unit cell. In sharp contrast, the
recently, the technique of specic staining of reducing annealed sample, which is essentially all of the Ih form,
end groups was adapted for application to cotton micro- produced a more simple and symmetric pattern that could
brils by Maurer and Fengel [77]. In addition to applica- be indexed in terms of a two-chain monoclinic unit cell. The
tion of the technique to examination of native cellulose, observed patterns are shown in Fig. 13. Figure 13(a) shows
Maurer and Fengel applied the method to examination of the diraction pattern of the native forms, while Fig. 13(b)
microbrils of mercerized cellulose (cellulose II), for which shows how the diraction pattern is transformed upon
they also observed staining at only one end of the micro- annealing. It is the latter that is identied with the Ih form
brils. This last observation, which indicates a parallel and which has been interpreted to indicate a monoclinic
chain structure in cellulose II, is very much in contrast to unit cell. Figure 13(c) and (d) are schematic representations
the crystallographic models that point to an antiparallel of the spots in the diraction diagrams (a) and (b) and show
structure for this form of cellulose. It reinforces the view more clearly how the diraction pattern is transformed by
that the structure of cellulose II still has many uncertainties annealing; the spots marked with arrows are the ones that
associated with it, in spite of the many theoretical analyses disappear upon annealing. Upon separating the diraction
that have attempted to rationalize the antiparallel form. pattern of the Ih form from the original pattern, it was
In yet another important set of investigations by possible to identify the components of the original pattern
Sugiyama et al. [7880], reported at approximately the that could be attributed to the Ia form, and it was found to
same time, it was demonstrated that lattice images could correspond to a triclinic unit cell.
be recorded from the microbrils of V. macrophysa. The In this rst report concerning the dierences between
rst images captured were based on lateral observation the diraction patterns of the Ia and Ih forms, the
of the microbrils [78,79]. Later, the techniques were positioning of the chains within the monoclinic unit cell
rened to allow the acquisition of lattice images of cross associated with the Ih form was left open. Two possibil-
sections of microbrils [80]. The signicance of these ities were regarded as consistent with the diraction
observations was that it was now possible to demonstrate patterns, the rst with the twofold screw axes coincident
conclusively that the microbrils are uniform in formation, with the molecular chains, the second with the twofold
and that there is no evidence that they are composed of screw axes between the chains. Both possibilities were
smaller subunits aggregating together to form the individ- consistent with the occurrence of nonequivalent anhydro-
ual microbrils that are observed in the electron micro- glucose units. The triclinic unit cell associated with the Ia
graphs. Thus, the observations resolved some of the form was also viewed as consistent with two possibilities:
questions that had arisen earlier concerning the interpre- the rst, a two-chain unit cell and the second, an eight-
tation of electron micrographs of native celluloses [74,81]; chain unit cell similar to the one rst proposed by Honjo
the ndings of Sugiyama et al. were the rst direct evidence and Watanabe [83].
that the approximately 2020-nm cross sections were not In a later study by Sugiyama et al. [84], the possibilities
composed of distinguishable smaller subunits. It should be were narrowed. It was stated that the monoclinic unit cell
noted, however, that the electron diraction processes corresponding to the Ih form was viewed as one wherein the
responsible for formation of the lattice images are domi- chains were coincident with the twofold screw axes. It was
nated by the organization of the heavy atoms in the also indicated that the pattern of the triclinic unit cell
molecular chains and would be insensitive to any nonuni- corresponding to the Ia form appeared consistent with a
formity in the hydrogen bonding patterns within the unit cell with only one chain per unit cell. In both instances,
interior of the 20 20-nm brils. The homogeneity of the the rationale for these determinations was not presented.
microbrils revealed in the lattice images is an issue that Another interesting group of observations reported in
needs to be revisited in the context of discussions of the second electron diraction study by Sugiyama et al. [84]
biogenesis, for in each instance, the homogeneous crystal- were interpreted as evidence of the occurrence of the two
line domains clearly include a much larger number of forms of cellulose in separate domains within the same
Figure 13 Typical electron diraction patterns of V. macrophysa before (a) and after (b) annealing. In the schematic
representations of the patterns, the spots marked with arrows correspond to the reections that disappear during the annealing
treatment (after Sugiyama).
microbrils. It was reported that the subsets of reections and, given the short duration of the exposures, result in an
associated with the two dierent forms could be separately unintended editing of the diraction patterns. Thus, only
observed, or in combination along the length of an indi- those diraction spots that are intense enough to be
vidual microbril within domains separated by 50 Am from observed at a particular angle will be detected, while
each other. This set of observations was interpreted to weaker ones go unseen. For example, if the lattice structure
indicate an alternation between the Ia and Ih forms along rst suggested by Honjo and Watanabe [82] is the true one
the length of an individual microbril. Such an interpreta- characteristic of the algal celluloses, diraction patterns
tion of course raises questions concerning the processes of observed at dierent angles would result in dierent
biogenesis, particularly because the relative proportions of degrees of enhancement of the dierent subsets of the total
the two forms of cellulose has been found to be invariant diraction pattern. This would also be true if the three-
for a particular species as long as the procedures used for dimensional organization of the chains is more appropri-
isolation of the cellulose do not incorporate exposure to ately viewed as a superlattice. Indeed, it is possible that the
conditions that can result in transformations of the Ia form lattice structure rst proposed by Honjo and Watanabe
into the Ih form. represents the unit cell of such a supelattice.
The observation of dierent domains producing dif- Such an interpretation of these observations is consist-
ferent diraction patterns along the same microbril can be ent with earlier observations by Roche and Chanzy [85],
envisioned as arising in two ways. The rst is the possibility wherein an electron microscopic image of microbrils of
that the microbril that was used to acquire the diraction algal celluloses was formed by use of a technique based on
patterns had a limited amount of curvature or twist to it so diraction contrast. It resulted in images of the algal
that the angle between the electron beam and the unit cell microbrils that had alternating dark and bright domains
axes was not constant. This could result in dierences of that appeared to be of the order of 50 nm in length. This
relative intensities of diraction spots from dierent planes suggests that the Bragg angle associated with a particular
set of reections is not likely to be coherently ordered and premises introduced at the outset, rather than con-
relative to the electron beam in domains that are more than clusions that can provide denitive answers to questions
50 nm in length. Given that the coherence of orientation under exploration. As alluded to earlier, the analysis by
relative to the electron beam was not found to extend Rees and Skerret [20] was one of the rst computational
beyond 50 nm, it would appear unlikely that it would eorts to explore the constraints on the freedom of
remain invariant over a distance of 50 Am. variation inherent in the structure of cellobiose. It relied
An alternative interpretation of the observation is on a potential function that is focused on van der Waals
that the alternation of the Ia and Ih forms is real, as interactions to establish the degree to which domains
proposed by Sugiyama et al. [84], and it reects an within w// space may be excluded by hard sphere overlap.
assembly process that is not yet suciently well under- The key nding was that approximately 95% of w// space
stood. It has been suggested that mechanical stress can was indeed excluded from accessibility on the basis of
facilitate the transformation of the Ia form into the Ih hard sphere overlaps that were unacceptable in the sense
form and that the formation of the Ih form may arise from that they required particular atoms associated with the
mechanical deformations of the brils in the course of region of the glycosidic linkage to be signicantly closer to
deposition; as they emerge from the plasma membrane each other than the sum of the van der Waals radii. And
they are required, in most instances, to be bent to be upon mapping the energy associated with allowable con-
parallel to the plane of the cell wall. If this is indeed the formations, they found that the two regions indicated by
source of the reported alternation of the Ia and Ih forms the solid line contours in Fig. 2 represented energy
along the microbril, it would raise questions concerning minima close to the conformations dened by the twofold
the uniqueness of the balance between the Ia and Ih forms helical constraint. The boundaries of the acceptable re-
that seems to be characteristic of particular species. gion are not very far removed from the domains within
the contours; the region between the two domains along
the twofold helix line (n=2) was not excluded by hard
V. COMPUTATIONAL MODELING sphere overlap, but it did represent a saddle point in the
potential energy surface.
The computational modeling has found particular favor The next group of computational studies did incor-
in the analyses of large molecules of biological origin. porate hydrogen-bonding energies as well the van der
And, of course, cellulose and its oligomers have attracted Waals interactions. Whether they exhibited the double
some attention in this arena. It is valuable to review minima, and the degree to which the double minima were
briey some of the eorts directed at advancing the pronounced, depended in large measure on the relative
understanding of cellulose because, in addition to provid- weighting given to the dierent types of nonbonded
ing insights regarding the contributions of dierent classes interactions. In many, particularly those relying on the
of interactions, they illustrate the reality that the results of potential energy functions incorporated in the linked
analyses can often be the consequences of assumptions atom least squares (LALS) programs, the weighting was
Figure 14 Perspective drawing of the three-dimensional shape of the mirror image of the conformational energy well for the
full angular range of U and W. The volume was constructed using the following scheme: V(U,W)? 15 kcal 1 mol 1; Vp(U,W)?
1/5 kcal 1; Vp(U,W)= (V(U,W) 15), with V being the energy expressed relative to the minimum. Proceeding from top to bottom
of the three-dimensional shape, note the very low energy region (the arrows point towards the conformations observed for
crystalline cellobiose and methyl-h-D-cellobioside). The 510 kcal 1 mol 1 energy contours correspond to the light gray region
of the volume.
based on tting the potential functions to optimize the as either IIII or IIIII to indicate both the source material
match between computed structures for small molecules and the form that will be recovered if the cellulose is boiled
for which the crystal structures were known from crystal- in water. In the case of native celluloses, the transformation
lographic studies. The results were that some showed very to the IIII form and back to the I form, also has the unusual
shallow minima o the twofold helix line [23]; the two- eect of converting those which have the Ia form dominant,
fold helical structures were then rationalized on the basis such as those from algal sources, into forms in which the Ih
that the departure represented a small dierence in the form is dominant. This eect, rst reported by Chanzy et
energies that were regarded as within the error of the al. [87], is accompanied by the partitioning of the algal
computation. When the criterion for quality of t is chosen microbrils into smaller ones that are closer in lateral
as a global minimum of the potential energy, without dimensions to those characteristic of higher plants. The
attention to the fact that it may incorporate unacceptable solid-state 13C NMR spectra also then appear more like
hard sphere overlaps, the results of the computational those of the higher plants. No such changes have been
analysis can be misleading. reported for native celluloses in which the Ih form is
Later studies did not incorporate disproportionate dominant. These behaviors by cellulose III point to a
weighting of the dierent types of nonbonded interactions memory eect with respect to the secondary and tertiary
[28,29], and the result is perhaps best illustrated in the structures of cellulose that remains very much a mystery at
mapping of the potential energy for cellobiose shown in the present time.
Fig. 14 taken from the study of cello-oligomers by Henris- Cellulose IV is most often described as the high-
sat et al. [29]. In this instance, for purposes of visualization, temperature cellulose because it can be prepared by expos-
the w// map presents the mirror image of the potential ing the source cellulose to temperatures in the vicinity of
energy surface computed for cellobiose. While well more 260jC while it is immersed in glycerol. In this preparation,
than two minima are shown in this mapping, it is to be it is reported to depend in structure on whether it is
noted that only the two corresponding to the crystal prepared from cellulose I or cellulose II; hence the frequent
structures of cellobiose and methyl-h-cellobioside, marked designation as IVI or IVII. When prepared from cellulose I,
by arrows, are within the boundaries established in the it is rst converted to the IIII form prior to the treatment at
analysis by Rees and Skerret described earlier. The other high temperature in glycerol. When prepared from cellu-
minima correspond to conformations that are more favor- lose II, it can be directly produced from the II form or via
able to hydrogen bonding, but with relatively high energies the IIIII form as an intermediate. However, in the case of
associated with the van der Waals interactions pointing to cellulose IV, there are no known procedures that allow
severe hard sphere overlap. restoration to the original form; the use of the dierent
designations reects some dierences in the diraction
patterns observed from the two dierent forms. Further-
VI. POLYMORPHY IN CELLULOSE more, most of its reported preparations from native forms
of cellulose have been from higher plant celluloses wherein
One of the discoveries growing out of the early diracto- the Ih form is dominant and the lateral dimensions of the
metric studies of cellulose was that it can occur in a number native microbrils are quite small; it is not at all clear that
of allomorphic forms in the solid state, each producing treatment of microbrils of larger lateral dimensions such
distinctive X-ray diractometric patterns [86]. In addition as those of Valonia or those of Halocynthia will result in
to the cellulose II form, which has been extensively dis- such changes.
cussed, two other forms are well recognized: cellulose III In addition to its preparation by heating at 260jC in
and cellulose IV. It is of interest to consider them briey glycerol, cellulose IV has been recovered when cellulose is
because they reect the capacity of cellulose to aggregate in regenerated from solution at elevated temperatures. This
a wide variety of secondary and tertiary structures, and has been observed with solutions in phosphoric acid regen-
because some of the higher plant celluloses produce dif- erated in boiling water or in ethylene glycol or glycerol at
fraction patterns that are not unlike those of cellulose IV. temperatures above 100jC [88]. It has also been observed
Furthermore, they reect the tendency for some of the upon regeneration from the dimethylsulfoxideparafor-
celluloses to retain some memory of their earlier states of maldehyde solvent system at the elevated temperatures
aggregation in a manner not yet understood. [88]. In yet another exploration of high temperature eects
Cellulose III is of little interest from a biological on the aggregation of cellulose, it was found that when
perspective except to the extent that its behavior may reveal amorphous celluloses are prepared under anhydrous con-
some of the interesting characteristics of the native cellu- ditions and then induced to crystallize by exposure to
loses from which it can be prepared. It can be prepared water, the exposure at elevated temperatures resulted in
from either native cellulose or from cellulose II by treat- the formation of cellulose IV rather than cellulose II, which
ment with anhydrous liquid ammonia at temperatures near is the form usually obtained upon crystallization at room
30jC. It produces distinctive X-ray patterns, Raman temperature [89].
spectra, and solid-state 13C NMR spectra. Its most inter- Samples of cellulose IV obtained through regeneration
esting characteristic is that it can be restored to the original from solution were shown to have Raman spectra that
form by treatment in boiling water. Because of this char- could be represented as linear combinations of the spectra
acteristic, it is common to designate samples of cellulose III of celluloses I and II, suggesting that it may be a mixed
lattice in which molecules with two dierent secondary cesses begin with a heterogeneous reaction system, which
structures coexist. This possibility is consistent with the may or may not eventually evolve into a homogeneous
earlier conclusion that both cellulose I and cellulose II have system as the reaction progresses. Thus, in all chemical
ribbonlike structures that depart, to a limited degree, from investigations that begin with cellulose as one of the
a twofold helix but in dierent ways. It is not at all ingredients, issues associated with heterogeneous reaction
implausible that molecules so similar in shape could coexist systems arise. Understandably, the one that has been
in the same lattice. dominant in most investigations is the question of the
One of the complications in interpreting observations accessibility of the cellulose.
of the occurrence of cellulose IV is that its X-ray diraction A variety of methods have been developed to relate
powder pattern is very similar to that of cellulose I. The 020 accessibility to microstructure. Almost all of them begin
reection is nearly identical to that of cellulose I and the with the premise that the cellulose can be regarded as
110 and the 110 reections collapse into a single reection having a crystalline fraction and a disordered or amor-
approximately midway between those of cellulose I. As a phous fraction. It is then assumed that the amorphous or
result, many of the less well-ordered native celluloses disordered fraction is accessible while the crystalline frac-
produce X-ray patterns that could equally well be inter- tion is not. In some instances, the portion of the crystalline
preted as indicating cellulose I, but with inadequate reso- domains that is at their surface is regarded as accessible
lution of the 110 and 110 reections, or as cellulose IV. and it is therefore included as part of the disordered frac-
They are usually characterized as indicating cellulose I tion. In other instances, the particular chemistry is thought
because they represent celluloses derived from native sour- to occur only in the disordered fraction and the surfaces
ces. Indeed, when cellulose IV was rst observed, it was of crystalline domains are not included. The dierent ap-
thought to be a less-ordered form of cellulose I. proaches have been reviewed by Bertoniere and Zeronian
The close relationship between cellulose IV and the [91], who regard the dierent approaches as alternative
native state is also reected in reports of its observation in methods for measuring the degree of crystallinity or the
the native state of primary cell wall celluloses. These were crystalline fraction in the particular celluloses.
observations based on electron diraction studies of iso- A number of dierent chemical and physical ap-
lated primary cell wall celluloses [90]. proaches are described by Bertoniere and Zeronian. The
rst is based on acid hydrolysis acids followed by quanti-
cation of the weight loss due to dissolution of glucose,
VII. CHEMICAL IMPLICATIONS OF STRUCTURE cellobiose, and the soluble oligomers [92]. This method is
thought to incorporate some error in the quantication of
It was noted earlier that an acceptable t to the diracto- the crystalline domains because the chain cleavage upon
metric data is not the ultimate objective of structural hydrolysis can facilitate crystallization of chain molecules
studies. Rather, it is the development of a model that that had been kept in disorder as a result of entanglement
possesses a signicant measure of validity and usefulness with other molecules. Another method is based on moni-
as the basis for organizing, explaining, and predicting the toring the degree of formylation of cellulose when reacted
results of experimental observations. In the sections above, with formic acid to form the ester [93]. In this method, the
the new and evolving conceptual framework for describing progress of the reaction with cellulose is compared with a
the structures of cellulose was described in relation to similar reaction with starch, which provides a measure of
spectral observations. It is important also to consider the the possibility of formylation in a homogeneous system
degree to which the structural information that has been wherein the issue of accessibility does not arise.
developed above may be useful as the basis for advancing In another method developed by Rowland and his
the understanding the response of celluloses to chemical coworkers [9497], accessible hydroxyl groups are tagged
reagents and to enzyme systems. It is useful rst to review through reaction of the particular cellulose with N,N-
briey past works directed at rationalizing the responses to diethylaziridinium chloride to a produce diethylamineether
such agents. (DEAE)cellulose. This is then hydrolyzed, subjected to
The vast majority of studies of the chemistry of enzyme action to remove the untagged glucose, silylated,
cellulose have been directed at the preparation of cellulose and subjected to chromatographic analysis. This method
derivatives with varying degrees of substitution depending has the added advantage that it can be used to explore the
on the desired product. Sometimes the goal is to prepare a relative reactivity of the dierent hydroxyl groups. It is
cellulose derivative that possesses properties that signi- usually observed that the secondary hydroxyl group on C2
cantly dier from those of the native form; some deriva- is the most reactive and the one at C3, the least reactive,
tives are water-soluble, others are thermoplastic, and with the primary hydroxyl at C6 having a reactivity
others still are used as intermediates in processes for the approaching that of the group on C2 under some condi-
regeneration of cellulose in the form of lms or bers. At tions. Here, of course, steric eects are also factors in these
other times, the objective is to introduce relatively small substitution reactions.
amounts of substitution to modify the properties of the Among the physical methods discussed by Bertoniere
cellulosic substrate without it losing its macroscopic iden- and Zeronian [91] are ones based on sorption and on
tity or form such as ber or microcrystalline powder or solvent exclusion. One of the earliest studies relying on
regenerated lament or lm. All such modication pro- the use of sorption as a measure of accessibility was the
classical study by Mann and Marrinan [98], in which are of two types. The rst is dierences in the steric
deuterium exchange with the protons was monitored. environment of the glycosidic linkage, particularly with
The cellulose was exposed to D2O vapor for a period respect to activity of the C6 group as a steric hindrance to,
sucient to attain equilibrium and then the degree of or as a potential promoter of, proton transfer reactions,
exchange was measured by observation of the infrared depending on its orientation relative to the adjacent glyco-
spectra. Comparison of the band associated with the OD sidic linkage. The second type of dierence is electronic in
stretching vibration with those associated with the OH nature and involves readjustment of the hybridization of
stretching vibration provided the measure of the relative the bonding orbitals at the oxygen in the linkage. It is
amounts of accessible and inaccessible hydroxyl groups. worthwhile to examine the potential contribution of each
Another approach to monitoring availability to adsorbed of these eects.
molecules is measurement of moisture regain upon condi- The steric environment emerges most simply from
tioning under well-dened conditions as described by examination of scale models of the cellodextrins. They
Zeronian and coworkers [99]. reveal that when C6 is positioned in a manner approximat-
The method of solvent exclusion has been used to ing the structure in h-methylcellobioside, the methylene
explore issues of accessibility on a somewhat larger scale. hydrogens are so disposed that they signicantly contribute
An approach pioneered by Stone and Scallan [100] and to the creation of a hydrophobic protective environment
Stone et al. [101] relied on static measurement using a series for the adjacent glycosidic linkage. If, however, rotation
of oligomeric sugars and dextrans of increasing size to about the C5C6 bond is allowed, the primary hydroxyl
establish the distribution of pore sizes in dierent prepa- group can come into proximity with the linkage and
rations of a variety of native celluloses. provide a potential path for more rapid proton transfer.
While methods for characterizing celluloses on the If, as suggested earlier on the basis of spectral data, the
basis of their accessibility have been useful, they do not orientation of some C6 groups in native cellulose is locked
provide a basis for understanding the level of structure at in by its participation in a bifurcated hydrogen bond to the
which the response of a particular cellulose is determined. hydroxyl group on C3, it may contribute to the higher
This follows from the rather simple categorization of the degree of resistance to hydrolytic action. Access to the
substrate cellulose into ordered and disordered fractions linkage oxygen would be through a relatively narrow solid
corresponding to the fractions thought to be crystalline and angle, barely large enough to permit entry of the hydro-
those that are not. This classication does not allow nium ions that are the primary carriers of protons in acidic
discrimination between eects that have their origin at media [102]. If, on the other hand, the C6 group has
the level of secondary structure and those that arise from greater freedom to rotate, as is likely to be the case in
the nature of the tertiary structure. Thus, in terms of cellulose II, the hindrance due to the methylene hydrogens
chemical reactions, this approach does not facilitate sepa- can be reduced and, in some orientations, the oxygen of
ration of steric eects that follow from the conformation of the primary hydroxyl group may provide a tunneling path
the molecule as it is approached by a reacting species, from for transfer of protons from hydronium ions to the glyco-
eects of accessibility, which is inherently a consequence of sidic linkage. This would result in greater susceptibility of
the tertiary structure. nonnative celluloses to hydrolytic attack.
The possibility of advancing the understanding of the The hypothesis concerning steric eects in acid hydro-
chemical implications of structure is best illustrated in the lysis has as its corollary the proposal that the role of the C1
context of hydrolytic reactions. Among the patterns that component in cellulase enzyme system complexes is to
emerge fairly early in any examination of the published disrupt the engagement of the C6 oxygen in the bifurcated
literature on acid hydrolysis and on enzymatic degradation intramolecular hydrogen bond and thus permit rotation of
of cellulose are the many similarities in the response to the the C6 group into a position more favorable to hydrolytic
two classes of hydrolytic agents. In both instances, a rapid attack.
initial conversion to glucose and cellodextrins is followed The key role of C6 in stabilizing the native cellulose
by a period of relatively slower conversion, the rate of structures is supported by ndings concerning the mecha-
conversion in the second period depending on the prior nism of action of the dimethylsulfoxideparaformaldehyde
history of the cellulosic substrate. In general, the nonnative solvent system for cellulose, which is quite eective in
polymorphic forms are degraded more rapidly during this solubilizing even the most crystalline of celluloses. The
second phase. In addition, it is found that the most crucial step in the mechanism that has been established for
crystalline or highly ordered of the native celluloses are this system is substitution of a methylol group on the
particularly resistant to attack, with the most highly crys- primary hydroxyl at the C6 carbon [103,104].
talline regions converted much more slowly than any of the The eect of conformation on the electronic structure
other forms of cellulose. of the linkage is also likely to be a factor with respect to its
The relationship of the patterns of hydrolytic suscep- susceptibility to hydrolytic attack. Although there is no
tibility to the range of conformational variation discussed basis for anticipating the directions of this eect at this
above can be interpreted in terms of contrast between the time, it is well to consider it from a qualitative perspective.
states of the glycosidic linkage in cellobiose and h-methyl- First, it is clear that the hybrids of oxygen orbitals involved
cellobioside. The dierences between the states that are in the bonds to carbon must be nonequivalent because the
likely to contribute to the dierences in observed reactivity bond distances dier to a signicant degree [24,25]. The
angle of approximately 116j imposed on the linkage is native forms, and more recently for the Ih form. The Ia
likely to result in greater dierences between the bonding form is thought to possess a triclinic unit cell structure.
orbitals and the lone pair orbitals than might be expected in Some important questions remain regarding the degree to
a typical glycosidic linkage that is free from strain. Among which these are adequate representations of the organiza-
themselves, the lone pair orbitals are likely to be dierent tion of the crystalline domains in native celluloses. The
because of their dierent disposition with respect to the majority of crystallographic studies also point to parallel
ring oxygen adjacent to C1 in the linkage; the dierences alignment of the cellulose chains in the native celluloses,
may be small and subtle, but they are no less real. Given and this conclusion has been conrmed by electron micro-
these many inuences on the nature of the hybridization at graphic observations. Also, for cellulose II, the structures
the oxygen in the linkage, it seems most unlikely that they derived from the X-ray diraction data suggest a ribbon-
would remain unaltered by changes in the dihedral angles like secondary structure approximating twofold helical
of the magnitude of the dierence between cellobiose and organization and, in this instance, antiparallel alignment
h-methylcellobioside. Hence a dierence in electronic char- of the chains, although the antiparallel proposal has been
acter must be expected. contradicted by recent electron micrographic observations.
At present, it is not possible to estimate the magnitude The unit cell organization of space group P21, with two
of the eects discussed nor to speculate concerning the chains per unit cell, has also been suggested for cellulose II,
direction of the change in relative reactivity of the glyco- although the degree of condence is even less than that with
sidic linkage in the two dierent conformations. Yet it is respect to the structures of cellulose I.
clear that dierences can be anticipated and they may be The early Raman spectroscopic studies clearly could
viewed, within limits of course, as altering the chemical not be reconciled with the premise that both cellulose I
identity of the glycosidic linkage as its conformation and cellulose II possess twofold helical conformations as
changes. It remains for future studies to dene the dier- the crystallographic studies had suggested. The Raman
ences more precisely. spectra, together with some corresponding infrared spec-
The points raised with regard to the inuence of tra, also pointed to the probability that the repeat unit
conformation on factors that determine the pathways of the structure of crystalline celluloses is anhydrocello-
for chemical reaction have not been specic subjects of biose, so that alternating nonequivalent glycosidic link-
investigation because methods for characterizing second- ages occur within each chain. To preserve the ribbonlike
ary structure as apart from the tertiary structure have not structural approximation, the dierent conformations of
been available. It has also been true that suitable concep- celluloses I and II were rationalized as alternate left- and
tual frameworks have not been available for developing the right-handed departures from the twofold helical struc-
questions beyond the levels of the orderdisorder duality. ture, with those in cellulose II representing somewhat
With the development of the approaches outlined above larger departures from the twofold helical conformation
for exploring and distinguishing between matters of sec- than those in cellulose I.
ondary structure and those of tertiary structure it is quite The introduction of high-resolution solid-state 13C
likely that in the years ahead, it will be possible to achieve a NMR spectral analyses into the study of celluloses resulted
higher level of organization of information concerning the in the resolution of one of the fundamental mysteries in the
chemistry of cellulose. variability of native celluloses by establishing that all native
With respect to questions of tertiary structure, the key celluloses are composites of two forms. These were identi-
issue introduced by the new structural information, and ed as the Ia and Ih forms to distinguish them from the IA
one that has not been explored at all at this time, is whether and IB categories that had been introduced more than three
the dierent hydrogen bonding patterns associated with the decades earlier to classify the celluloses produced by algae
Ia/Ih duality have associated with the dierences between and bacteria from those produced by higher plants. The
the reactivity of the hydroxyl groups involved. It is not clear correspondence between the two classications is that
at this time how experiments exploring such eects might those in the IA category have the Ia from as the dominant
be carried out so as to separate issues associated with the component, while those in the IB category are predomi-
dierences between the hydrogen bonding patterns from nantly of the Ih form. The nature of the dierence between
issues associated with dierences in accessibility. the Ia and Ih forms remains the subject of serious inquiry.
Recognition of the Ia/Ih duality has facilitated a signicant
amount of additional research seeking to establish the
VIII. CELLULOSE STRUCTURES IN SUMMARY balance between the two forms in a wide range of higher
plant celluloses.
From crystallographic studies, based on both X-ray and In later studies, the Raman spectra and corresponding
electron diraction measurements, it can be concluded that infrared spectra indicated that the primary dierences
the secondary structures of native celluloses are ribbonlike between the Ia and Ih forms of native cellulose were in
conformations approximating twofold helical structures. the pattern of hydrogen bonding. Furthermore, the Raman
Their organization into crystallographic unit cells remains spectra of the two forms raise questions as to whether the
uncertain, however. The monoclinic space group P21, with structures can possess more than one molecule per unit cell
two chains per unit cell, has been proposed for both the because there is no evidence of any correlation eld split-
earlier studies prior to the discovery of the Ia/Ih duality in tings of any of the bands in the spectra of the two forms.
Electron microscopic studies relying on agents that have been generally regarded as measures of disorder
act selectively either at the reducing or at the nonreducing when, in fact, they are more appropriately regarded as
end groups of the cellulose chains have provided convinc- indicators of the nonlinear organization in a biological
ing evidence that cellulose chains are aligned parallel to structure. Another issue arising at the microscale is asso-
one another in native cellulose. More recently, similar ciated with the occurrence of signicant fractions of the
evidence has been presented supporting the view that the cellulose molecules at the surface of the microbrils of most
alignment of the chains is also parallel in cellulose II. native celluloses, particularly in the case of higher plants. It
Other electron microscopic studies using the methods of is the question as to whether the microbrillar structure can
lattice imaging have been used to demonstrate that the be viewed as a separate phase in the traditional sense and
highly ordered microbrils derived from algal celluloses whether criteria developed for the stability of homoge-
represent homogeneous lattice structures with respect to neous phases in the context of classical thermodynamics
the diraction planes dened by the organization of their can have meaning when applied to native cellulosic struc-
heavy atoms. tures. These issues arise in relation to discussions of native
Electron diraction studies carried out on algal cellu- celluloses and their biogenesis.
loses after the discovery of the Ia/Ih duality have been
interpreted to indicate that the two forms may alternate
along the length of individual microbrils. These observa- Part B
tions can also be interpreted as manifestations of the slow Chemical Characterization
twisting about the long axis that has been observed in other
studies of similar algal celluloses. I. INTRODUCTION
The possibility of the coexistence of the Ia and Ih forms
within a superlattice structure has been suggested in the Cellulosic materials have been used in various elds from
context of studies intended to mimic the conditions of commodities to industrial materials after mechanical and
biogenesis. These will be examined in greater detail in chemical modications. Fig. 15 illustrates the chemical
relation to the discussions of native celluloses and of their structure of cellulose in terms of chemical modications
biogenesis. [105]. The h-1,4-glycoside bonds and other functional
Our discussion of structure has focused so far on issues groups such as carboxyls and aldehydes present in most
of structure at the nanoscale level, identied as cellulosic material as minor groups are also possible sites
corresponding to domains that are of the order of 2 nm for chemical modications.
in dimension. Organization at the microscale level, dened
as the range between 2 and 50 nm, requires consideration of II. SOLVENTS
a number of issues that have not been adequately dealt with
in the literature on structures of cellulose. These include the Table 1 summarizes the representative solvents or solvent
well-recognized departures from a linear lattice, which systems of cellulose. The xanthate system has been used for
Figure 15 Positions in cellulose structure for chemical reactions.

Table 1 Conventional and New Cellulose Solvent Systems

Category Solvent Remarks
Acid >52% H2SO4 Partial hydrolysis

>85% H3PO4 Partial hydrolysis
Alkali 6% LiOH Needs pretreatments of cellulose
69% NaOH Needs pretreatments of cellulose
Alkaline metal Cu(NH3)4(OH)2 [cuoxam] Cuprammonium rayon production
system Cu(H2NCH2CH2NH2)2(OH)2 [cuen] Standard solvent for DPv measurement
complex Co(H2NCH2CH2NH2)2(OH)2
Ni(NH3)6(OH)2
Cd(H2NCH2CH2NH2)3(OH)2 [cadoxen] Transparent solution
Zn(H2NCH2CH2NH2)3(OH)2
Fe/3(tartaric acid)/3NaOH [EWNN] Relatively stable
Alkaline CS2/NaOH Dissolves cellulose, forming
xanthogenate Viscose rayon production system
derivatives
Inorganic salt >64% ZnCl2 Dissolves cellulose by heating at 100jC
>50% Ca(SCN)2 Dissolves cellulose by heating at 100jC
Organic solvent Cl3CHO/DMF Dissolves cellulose, forming chloral
systems hemiacetals at all celluloseOH
(CH2O)x/DMSO Dissolves cellulose, forming (poly)-
methylol hemiacetals at celluloseOH
N2O4/DMF, N2O4/DMSO Dissolves cellulose, forming nitrite ester
at
all celluloseOH
LiCl/DMAc, LiCl/DMI Stable; needs pretreatments of cellulose
SO2/amine/DMSO Unstable; gives stable amorphous
regenerated cellulose
CH3NH2/DMSO Dissolves cellulose, forming complex
CF3COOH (triuoroacetic acid: TFA) Dissolves cellulose, forming TFA ester
at
C6OH; volatile solvent
(Bu)4N+F ?3H2O/DMSO Dissolves cellulose with DP<650
ca. 80% N-methylmorpholine-N-oxide/H2O Uses for lyocell production
Dissolves cellulose by heating at 90jC
Others NH4SCN/NH3/water Forms mesophase states
N2H4 Explosive
DMF: N,N-dimethylformamide, DMAc: N,N-dimethylacetamide, DMI: N,N-dimethylimidazolidinone, DMSO: dimethylsulfoxide, Bu: butyl.
a long time to make viscose rayon and sponge. However, Cellulose hydroxyls form unstable derivatives or com-
the rayon production, where H2S is emitted more or less plexes with the solvent components in the solution states
during the recovery system of spent liquor, is shrinking (Fig. 16). These nonaqueous cellulose solvents were used to
because of environmental issues. On the other hand, cellu- prepare cellulose derivatives under homogeneous condi-
lose is soluble in aqueous NaOH alone under limited tions mainly to control degree of substitutions (DS) and
conditions. Microcrystalline cellulose with DP 200300 is distribution of substituents. The LiCl/DMAc system dis-
soluble in 69% NaOH by freezing and defrosting proce- solves cellulose by one of the following two procedures: (1)
dures [106]. Pretreatments of cellulose such as steam ex- solvent exchange of cellulose soaked in water to DMAc
plosion are necessary for complete dissolution in the through ethanol, followed by stirring in 8% LiCl/DMAc at
aqueous NaOH for normal bleached wood pulp and cotton room temperature, (2) heating of cellulose/DMAc suspen-
linters with DP of more than 500 [107]. sion at ca. 165jC for 30 min, and LiCl is added to the
Many nonaqueous cellulose solvent systems have been cellulose suspension at about 100jC in the course of cool-
developed in the last three decades. Solvent systems consist ing to adjust to 8% LiCl/DMAc [109]. Because cellulose
of reagent(s) reactive to cellulose hydroxyl groups and a solutions in LiCl/DMAc are fairly stable and powderlike
polar aprotic solvent such as dimethylsulfoxide (DMSO) LiCl is easy to handle, these solvent systems were exten-
or N,N-dimethylacetamide (DMAc). Triuoroacetic acid sively studied as reaction media for homogeneous deriva-
is the only volatile solvent to dissolve cellulose [108]. tizations of cellulose. Furthermore, the LiCl/DMAc
added to the solution before spinning in order to avoid

strong coloration and oxidative degradation. It is possi-
ble to insert an air-gap stage in between spinning nozzles
and the water bath for regenerationhence this system
is called drywet spinning process. Drawing can be
achieved in the air-gap stage, resulting in a higher degree
of crystallinity as well as a higher degree of orientation of
crystals in the regenerated cellulose ber thereby prepared
(Table 2) [111].
III. DERIVATIZATION
Because cellulose has three dierent hydroxyl groups at C2,
C3, and C6 in the anhydroglucose unit (Fig. 1), various
chemical reactions can possibly apply. Factors inuencing
the characteristics of cellulose derivatives are as follows: (1)
chemical structures of substituents introduced, (2) degree
of substitution (DS: the amount of substituents per anhy-
droglucose unit), (3) distribution of substituents, (4) degree
of polymerization and its distribution, (5) purities includ-
ing the presence of chromophores, and others.
A. Esterification
Typical cellulose esters examined so far are depicted in
Fig. 17. Cellulose triacetate (DS>2.9) is prepared by heat-
ing cellulose suspended in a mixture of acetic anhydride/
acetic acid/H2SO4 around 60jC. Cellulose triacetate is
soluble in chlorinated hydrocarbons such as dichloro-
methane. Cast lms of cellulose triacetate have optically
characteristic properties of no polarization of penetrated
Figure 16 Dissolving states of cellulose in nonaqueous light, and thus have been used as lm bases for photograph
cellulose solvents. and supporting lms for liquid crystal display. Cellulose
diacetate (DS 2.32.5) is consecutively prepared from the
cellulose triacetate solution by diluting with water and
solvent system has been used as an eluent in size-exclusion heating. Cellulose diacetate lms have been used for ultra-
chromatographic analyses using a multiangle laser-light ltration to purify tap water partly in place of chlorination.
scattering (SEC-MALLS) detector for molecular mass and When pulps having lower a-cellulose content are used,
molecular mass distribution of cellulose samples [110]. some acetone-insoluble gel fractions originating from
N-Methylmorpholine-N-oxide (NMMO), containing hemicellulose are formed from both softwood and hard-
ca. 20% water, can dissolve cellulose by melting of NMMO wood bleached kraft pulps [112]. Thus, bleached sulte
at about 90jC, and has recently been used to make regen- pulp or at least bleached prehydrolyzed kraft pulp pre-
erated cellulose ber (lyocell) at industrial level. NMMO pared from softwood is acceptable as the pulp resources
is a kind of oxidant, and thus some antioxidant must be at this point. Therefore, the possibility of using normal
Table 2 Crystallinity Index and Degree of Orientation of Crystals of Regenerated Cellulose Fibers Calculated from their X-ray
Diraction Patterns
Degree of orientation of
Cellulose solvent Crystal structure Crystallinity index (%) crystals (%)
NaOH/CS2/water Cellulose II 24 85
(viscose rayon)
Aqueous Cu(NH3)4(OH)2 Cellulose II 41 90
(cuprammonium rayon)
69% aqueous NaOH Cellulose II 46 75
80% NMMO/water Cellulose II 46 91
(lyocell)
zation media at industrial level. The multicomponent

solvent systems, the high boiling point of the solvent such
as DMAc and DMSO, the necessity of pretreatments
including complete drying of cellulose, and the solvent-
exchanging processes for dissolution are the challenges that
would render practical applications of the nonaqueous
solvent systems to derivatizations a dicult prospect.
B. Etherification
Fig. 18 illustrates representative cellulose ethers. In most
cases in industries, cellulose ethers are produced via al-
kalicellulose (cellulose swollen with, e.g., 18% aqueous
NaOH) by reacting with etherifying reagents around
60jC in the presence of i-propanol or i-butanol, where
etherications proceed heterogeneously to the swollen
alkalicellulose without dissolution.
Carboxymethylcellulose sodium salt (CMC), methyl-
cellulose (MC), hydroxyethylcellulose (HEC), and
hydroxypropylcellulose (HPC) are typical water-soluble
cellulose ethers manufactured at the industrial level, and
primarily used as thickeners in various elds. Commercial
CMC has DS values in the range of 0.61.2. HEC and
HPC are produced from alkalicellulose by reacting with
ethylene oxide and propylene oxide, respectively. In the
case of these cellulose ethers, additional substitution also
occurs in hydroxyl groups of the introduced substituents,
such as grafting, with increasing the amount of substitu-
ents, and thus molecular substitution (MS) in place of DS is
used for these cellulose ethers. Water-soluble cellulose
ethers in solution states have been characterized by SEC-
MALLS, and the presence of coagulation among cellulose
ether molecules in water under particular conditions has
been reported [113]. When a small amount of long alkyl
chains (C12C24) are introduced into HEC, viscosity of the
aqueous solution extremely increases by the formation
of hydrophobic interactions among HEC molecules in
water [114].
Nonaqueous media oer an advantageous method to
prepare cellulose ethers with high DS, which generally
cannot be achieved by the conventional aqueous alkalicel-
lulose system. About 30 kinds of cellulose ethers with DS 3
were prepared by one-step reactions with powdered NaOH
and etherifying reagents using the cellulose solution in SO2/
Figure 17 Typical cellulose esters. diethylamine/DMSO [115]. The triphenylmethyl (trytyl)
group can be selectively introduced at C6 primary hydroxyl
of cellulose by homogeneous reaction with pyridine in 8%
bleached kraft pulp produced from eucalyptus without any LiCl/DMAc. This tritylcellulose was used as an intermedi-
spinning problems is one of the signicant themes for ate for further conversion to some regioselective cellulose
cellulose diacetate production. Furthermore, one-step pro- ethers and esters such as 2,3-di-O-methylcellulose and 6-O-
duction of cellulose diacetate without going through the methylcellulose (Fig. 19) [116].
cellulose triacetate stage is also required.
Various cellulose esters such as acetate, tosylate ( p-
toluenesulfonate), sinnamoylate, and uorine-containing IV. OXIDATION
substituents were prepared with pyridine as a base under
homogeneous and nonaqueous conditions using, for ex- There are several methods to modify the chemical structure
ample, 8% LiCl/DMAc. Although interesting results were of cellulose by oxidation. Periodate oxidation of cellulose
obtained at the laboratory level, none of these nonaqueous suspended in water and N2O4 oxidation of cellulose sus-
cellulose solvent systems has been applied to the derivati- pended in chloroform are well-known conventional meth-
Figure 18 Typical cellulose ethers.
ods to convert cellulose to dialdehyde cellulose and C6- oxidation is applied to native celluloses, only small
carboxy cellulose, respectively. Generally, however, some amounts of carboxyl groups are introduced into the brous
side reactions including depolymerization are inevitable celluloses. When regenerated or mercerized, cellulose sus-
during the oxidation process, which makes it dicult to pended in water is used as the starting material; on the
achieve regioselective oxidation completely. other hand, water-soluble products are obtained quantita-
2,2,6,6-Tetramethylpiperidine-1-oxy radical (TEM- tively at room temperature within 1 h (mostly within 20
PO) is a water-soluble and commercially available radical min). NMR analyses revealed that these water-soluble
reagent. When sodium hypochlorite is used as a co-oxidant oxidized products have homogeneous chemical structures,
in the presence of catalytic amounts of NaBr and TEMPO h-1,4-linked polyglucuronic acid (Fig. 20) [118]. Thus,
in water, C6 primary hydroxyl groups of polysaccharides selective oxidation at C6 primary hydroxyl groups of
dissolved in water at pH 1011 can be selectively converted cellulose can be achieved by TEMPO-mediated oxidation
to carboxyl groups [117]. When this TEMPO-mediated in aqueous media when regenerated or mercerized cellulose
Figure 19 Preparation scheme of regioselectively substituted methylcelluloses via tritylcellulose.
is used (Fig. 21). However, some depolymerization of main specic circumstances, during use. The quality of cellulosic
chains are inevitable. This new water-soluble polyglucu- materials is reduced through degradation by means of these
ronic acid is degradable to glucuronic acid and hexenuronic outside stimuli. On the other hand, if these degradations
acid residues by commercial crude cellulase [119]. can be well controlled, useful cellulose-related compounds
can be obtained.
V. DEGRADATION A. Acid Hydrolysis

Cellulosic materials undergo numerous and sometimes Acid hydrolysis of cellulosic biomass can be used to
harsh stimuli during manufacturing processes and, under produce glucose, which is then converted to ethanol by
fermentation. Susceptibility of cellulosic materials to acid

hydrolysis is remarkably dierent between their ordered
and disordered regions. When native cellulosic materials
such as cotton linters and bleached chemical wood pulps
are heated in a dilute acid, hydrolysis of disordered regions
in cellulose microbrils precedes that of ordered regions to
form the so-called microcrystalline cellulose with DP
200300. A part of glucose once formed from cellulose by
acid hydrolysis is further degraded to hydroxymethylfur-
fural, levulinic acid, formic acid, and others during acid
hydrolysis (Fig. 22).
B. Enzymatic Degradation
Cellulase hydrolyzes cellulose under mild conditions com-
pared with inorganic or organic acid. Generally, cellulases
such as cellobiohydrolase II (CBH II) consist of core and
cellulose-binding domains and a linker, which binds the
Figure 20 13C NMR spectra of cellulose oligomer (DP 7) in two domains. The core domain contains an active center to
DMSO and cellouronic acid Na salt (h-1,4-linked polyglu- hydrolyze cellulose in catalytic manner and the subsites,
curonic acid) in D2O. Cellouronic acid Na salt was prepared which interact with cellulose chain close to the active
from viscose rayon by the TEMPO-mediated oxidation. center. The cellulose-binding domains consist of amino
Figure 21 TEMPO-mediated oxidation of C6 primary hydroxyl group of cellulose to carboxyl group.

Figure 22 Some degradation products of cellulose.
Figure 23 Classication of cellulase into processive and nonprocessive types.

Figure 24 Enzymatic synthesis of cellulose from h-cellobiosyl uoride in acetonitrile/aqueous buer mixture.
acids having aromatic rings such as tyrosin or triptophan, the basis of hydrolysis patterns of cellulose chains (Fig. 23)
and these aromatic rings of the cellulose-binding domains [121].
attach to hydrophobic plains of cellulose chains through
van der Waals force. The active center of the core domains
of cellulase can then attack the cellulose chain. The subsites C. Thermal Degradation
of the core domains can give some mechanical stress to the
cellulose chain, and one of glucose residues of the cellulose When cellulose is analyzed by thermogravimetry under
chain compulsorily have the unstable boat form. Thus, nitrogen atmosphere, thermal decomposition starts at
remarkable reduction of activation energy to hydrolyze 200270jC, depending on the purity of cellulose samples.
cellulose can be achieved [120]. The temperature of ignition in the air is in the range of 390
Various types of cellulase have been reported so far, 420jC, and the maximum ame temperature reaches
and they have been, for a long time, classied into two 800jC or more. When cellulose is heated at temperatures
typesexo- and endocellulasesdepending on whether or exceeding 100jC under reduced pressure, levoglucosan
not the cellulase can recognize the reducing ends of cellu- (Fig. 22) is obtained in the maximum yield of 70%. When
lose chains. Cellobiohydrolase (CBH) and endoglucanase thermal carbonization is applied to cellulosic materials
(EG) are then further categorized into two types. However, under inactive gas atmosphere, the yields of carbon are
recent studies revealed that there are no exo-type cellulases, lower than the theoretical value (44%) because a part of
and that all cellulases are included in the endo-type. On cellulose is converted to levoglucosan. Yields of carbon can
the other hand, the following classication are now well be increased by adding hydrochloric acid, which enhances
accepted: the processive and nonprocessive cellulases on dewatering rather than the formation of levoglucosan
Figure 25 Chemical syntheses of cellulose.

[122]. When cellulose microcrystals, which are obtained reactions and conformational restrictions for polymeriza-
from crystalline native celluloses by acid hydrolysis, are tion of carbohydrate monomers [128].
carbonized under suitable conditions, carbon nano-lods
are obtained [123].
Microwave treatment is one of the heating methods
used, in which more homogeneous heating can be REFERENCES
achieved, compared with the thermal conduction-type 1. Atalla, R.H. In Comprehensive Natural Products Chemis-
heating. Saccharication of lignocellulosics has been stud- try, Carbohydrates; Pinto, B.M., Ed.; Elsevier: London,
ied from this aspect, and the maximum yield of glucose 1999; Vol. 3, 529598.
was reported to be 81% based on theoretical value [124]. 2. Gardner, K.H.; Blackwell, J. Biopolymers 1974, 13, 1975.
Two of the thermal treatments, explosion and laser irra- 3. Hebert, J.J.; Muller, L.L. J. Appl. Polym. Sci. 1974, 18,
3373.
diation treatments, were employed to obtain glucose and
4. Meyer, K.H.; Misch, L. Helv. Chim. Acta 1937, 20, 232.
levoglucosan. Supercritical water treatment is also one of 5. Hermans, P.H. Physics and Chemistry of Cellulose Fibers;
the heating methods in the presence of acid. Dissociation Elsevier: New York, 1949.
of water can increase under supercritical conditions, and 6. French, A. In The Structures of Celluloses, ACS sympo-
thus water behaves like an acid catalyst to cellulose. sium Series 340; Atalla, R.H., Ed.; American Chemical
Noncatalytic hydrolysis of cellulose can, therefore, be Society: Washington, DC, 1987; 15 pp.
achieved by supercritical water treatments. The maximum 7. Pauling, L. The Nature of the Chemical Bond, 3rd Ed.;
Cornell University Press: Ithaca, NY, 1960; 65 pp.
yield of glucose was reported to be in the range of 3248% 8. Woodward, L.A. Introduction to the Theory of Molecular
[125]. Vibrations and Vibrational Spectroscopy; Oxford Univer-
sity Press: Oxford, 1972; 344 pp.
9. Atalla, R.H. Appl. Polym. Symp. 1976, 28, 659.
10. Pitzner, L.J. The Vibrational Spectra of the 1,5-anhydro-
pentitols, Doctoral dissertation; The Institute of Paper
VI. CHEMICAL AND ENZYMATIC SYNTHESES Chemistry: Appleton, WI, 1973.
OF CELLULOSE 11. Pitzner, L.J.; Atalla, R.H. Spectrochim. Acta 1975, 31A,
911.
Although cellulose is one of the naturally occurring poly- 12. Watson, G.M. The Vibrational Spectra of the Pentitols and
saccharides, articial synthesis of cellulose from monomers Erythritol, Doctoral Dissertation; The Institute of Paper
or dimers has been of scientic interest for a long time. Chemistry: Appleton, WI, 1974.
Syntheses of cellulose have recently been succeeded by the 13. Edwards, S.L. The Vibrational Spectra of the Pentose
Sugars, Doctoral Dissertation; The Institute of Paper
following two dierent principles.
Chemistry: Appleton, WI, 1976.
Cellulase hydrolyzes the h-1,4-glucoside bonds of 14. Williams, R.M. The Vibrational Spectra of the Inositols,
cellulose, and this enzymatic hydrolysis is essentially re- Doctoral Dissertation; The Institute of Paper Chemistry:
versible. Kobayashi et al. [126] successfully carried out Appleton, WI, 1977.
cellulose synthesis by using the reversible reaction of 15. Williams, R.M.; Atalla, R.H. J. Phys. Chem. 1984, 88, 508.
cellulase, where a particular substrate, h-cellobiosyl uo- 16. Wells, H.A. The Vibrational Spectra of Glucose, Galactose
ride, was used in an aqueous buer containing acetonitrile and Mannose, Doctoral Dissertation; The Institute of Paper
chemistry: Appleton, WI, 1977.
(Fig. 24). The origin and purity of cellulase and combina- 17. Wells, H.A.; Atalla, R.H. J. Mol. Struct. 1990, 224, 385.
tion of the solvent systems inuenced the yields of synthe- 18. Carlson, K.P. The Vibrational Spectra of the Cellodextrins,
sized cellulose and its crystal structure. Cellulase from Doctoral Dissertation; The Institute of Paper Chemistry:
Trichoderima viride gave the highest yield, ca. 54%, of Appleton, WI, 1978.
water-insoluble, low-molecular weight cellulose (DP 22), 19. Wiley, J.H.; Atalla, R.H. Carbohydr. Res. 1987, 160, 113.
whereas h-glucosidase yielded no cellulose. Generally, the 20. Rees, D.A.; Skerret, R.J. Carbohydr. Res. 1968, 7, 334.
21. Petipas, T.; Oberlin, M.; Mering, J. J. Polym. Sci. C 1963, 2,
enzymatically synthesized cellulose has the crystal struc- 423.
ture of cellulose II, which is the same as that of regenerated 22. Norman, M. Text. Res. J. 1963, 33, 711.
or mercerized cellulose. On the other hand, cellulose bear- 23. Sarko, A.; Muggli, R. Macromoles 1974, 7, 486.
ing the crystal structure of cellulose I, which is the same as 24. Chu, S.S.C.; Jerey, G.A. Acta. Crystallogr. 1968, B24,
that of native cellulose, was synthesized in vitro by using a 830.
highly puried cellulase component [127]. 25. Ham, J.T.; Williams, D.G. Acta. Crystallogr. 1970, B29,
Cellulose oligomers and low-DP cellulose have been 1373.
26. Wilson, E.B., Jr.; Decius, J.C.; Cross, P.c. Molecular
prepared by the following three routes and the successive Vibrations; McGraw-Hill: New York, 1955; 188 pp.
elimination of the protecting groups at hydroxyl groups: 27. Nelson, M.L.; OConnor, R.T. J. Appl. Polym. Sci. 1964, 8,
(1) linear synthetic reaction using the imidate method from 1311.
allyl 2,3,6-tri-O-benzyl-4-O-p-methoxybenzyl-h-D-gluco- 28. Melberg, S.; Rasmussen, K. Carbohydr. Res. 1979, 71, 25.
pyranoside, (2) conuent-type reaction between cellote- 29. Henrissat, B.; Perez, S.; Tvaroska, I.; Winters, w.T. In The
traose and cellooctaose derivatives using the imidate Structures of Celluloses, ACS symposium Series 340;
Atalla, R.H., Ed.; American Chemical Society: Washing-
method, and (3) cationic ring-opening polymerization of ton, DC, 1987; 38 pp.
glucose 1,2,4-orthoester (Fig. 25). These studies revealed 30. Atalla, R.H. Adv. Chem. Ser. 1979, 181, 55.
the roles of specic protecting groups at particular hy- 31. Atalla, R.H.; Gast, J.C.; Sindorf, D.W.; Bartuska, V.J.;
droxyl positions of the starting materials in regioselective Maciel, G.E. J. Am. Chem. Soc. 1980, 102, 3249.
32. Atalla, R.H. Proceedings of the International Symposium 63. Newman, R.H.; Hemmingson, J.A. Cellulose 1995, 2, 95.
on Wood and Pulping Chemistry, SPCI Rept. No 38, 64. Newman, R.H. J. Wood Chem. Technol. 1994, 14, 451.
Stockholm 1981, 1, 57. 65. Newman, R.H.; Ha, M.A.; Melton, L.D. J. Agric. Food
33. Atalla, R.H. In Structure, Function and Biosynthesis of Chem. 1994, 42, 1402.
Plant Cell Walls; Dugger, W.M., Bartinicki-Garcia, S., 66. Newman, R.H.; Davies, L.M.; Harris, P.J. Plant Physiol.
Eds.; American Society of Plant Physiologists: Rockville, 1996, 111, 474.
MD, 1984. 381 pp. 67. Newman, R.H. Cellulose 1997, 4, 269.
34. Atalla, R.H. J. Appl. Pol. Sci., Appl. Pol. Symp. 1983, 37, 68. Lenholm, H.; Larsson, T.; Iversen, T. Carbohydr. Res.
295. 1994, 261, 119.
35. Earl, W.L.; VanderHart, D.L. J. Am. Chem. Soc. 1980, 69. Lennholm, H.; Larsson, T.; Iversen, T. Carbohydr. Res.
102, 3251. 1994, 261, 119.
36. Earl, W.L.; VanderHart, D.L. Macromoles 1981, 14, 570. 70. Larsson, T.; Westermark, U.; Iversen, T. Carbohydr. Res.
37. D.L. VanderHart, Deductions about the morphology of 1995, 278, 339.
wet and wet beaten cellulose from solid state 13C NMR, 71. Larsson, T.; Wickholm, K.; Iversen, T. Carbohydr. Res.
NBSIR 82-2534. 1997, 302, 19.
38. Fyfe, C.A.; Dudley, R.L.; Stephenson, P.J.; Deslandes, Y.; 72. Hock, C.W. In Cellulose and Cellulose Derivatives, Pt. I;
Hamer, G.K.; Marchessault, R.H. J. Am. Chem. Soc. 1983, Ott, E., Spurlin, H.M., Grain, M.W., Eds.; Interscience:
105, 2469. New York, 1954; 347 pp.
39. Horii, F.; Hirai, A.; Kitamaru, R. Polym. Bull. 1982, 8, 163. 73. Morehead, F.F. In Cellulose and Cellulose Derivatives, Pt.
40. Maciel, G.E.; Kolodziejski, W.L.; Bertran, M.S.; Dale, IV; Bikales, N.M., Segal, L., Eds.; Wiley-Interscience: New
B.R. Macromoles 1982, 15, 686. York, 1971; 213 pp.
41. Gast, J.C.; Atalla, R.H.; McKelvey, R.D. Carbohydr. Res. 74. Preston, R.D. The Physical Biology of Plant Cell Walls;
1980, 84, 137. Chapman & Hall: London, 1974.
42. VanderHart, D.L.; Atalla, R.H. Macromoles 1984, 17, 75. Hieta, K.; Kuga, S.; Usuda, M. Biopolymers 1984, 23, 1807.
1465. 76. Chanzy, H.; Henrissat, B. FEBS Lett. 1985, 184, 285.
43. Atalla, R.H.; VanderHart, D.L. Science 1984, 223, 283. 77. Maurer, A.; Fengel, D. Holz als Roh- und Werksto 1992,
44. Howsmon, J.A.; Sisson, W.A. In Cellulose and Cellulose 50, 493.
Derivatives, Pt. I; Ott, E., Spurlin, H.M., Graine, M.W., 78. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N.
Eds.; Interscience: New York, 1954; 237 pp. Mokuzai Gakkaishi 1984, 30, 98.
45. Blackwell, J.; Marchessault, H. In Cellulose and Cellulose 79. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N.
Derivatives, Pt. IV; Bikales, N.M., Segal, L., Eds.; Wiley- Mokuzai Gakkaishi 1985, 31, 61.
Interscience: New York, 1971; 1 pp. 80. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N. Planta
46. VanderHart, D.L.; Atalla, R.H. In The Structures of 1985, 166, 161.
Celluloses, ACS Symposium Series 340; Atalla, R.H., Ed.; 81. Frey-Wyssling, A. The Plant Cell Wall; Gebruder Borntr-
American Chemical Society: Washington, DC, 1987; 88 pp. ager: Berlin, 1976.
47. Atalla, R.H.; Whtimore, R.E.; VanderHart, D.L. Biopoly- 82. Sugiyama, J.; Okano, T.; Yamamoto, H.; Horii, F.
mers 1985, 24, 421. Macromoles 1990, 23, 3196.
48. Horii, F.; Yamamoto, H.; Kitamaru, R.; Tanahashi, M.; 83. Honjo, G.; Watanabe, M. Nature 1958, 181, 326.
Higuchi, T. Macromoles 1987, 20, 2946. 84. Sugiyama, J.; Vuong, R.; Chanzy, H. Macromoles 1991,
49. Wiley, J.H.; Atalla, R.H. In The Structures of Celluloses, 24, 4168.
ACS Symposium Series 340; Atalla, R.H., Ed.; American 85. Roche, E.; Chanzy, H. J. Biol. Macromol. 1981, 3, 201.
Chemical Society: Washington, DC, 1987; 151 pp. 86. Ellefsen, 0.; Tonessen, B.A. In Cellulose and Cellulose
50. Sugiyama, J.; Presson, J.; Chanzy, H. Macromoles 1990, Derivatives, Pt. IV; Bikales, N.M., Segal, L., Eds.; Wiley-
24, 4168. Interscience: New York, 1971; 151 pp.
51. Yamamoto, H.; Horii, F.; Odani, H. Macromolecules 87. Chanzy, H.; Henrissat, B.; Vincendon, M.; Tanner, S.;
1989, 22, 4130. Belton, P.S. Carbohydr. Res. 1987, 160, 1.
52. Sugiyama, J.; Okano, T.; Yamamoto, H.; Horii, F. 88. Atalla, R.H.; Dimick, B.E.; Nagel, S.C. In Cellulose Chem-
Macromoles 1990, 23, 3196. istry and Technology; Arthur, J.C. Jr., Ed.; ACS Symp.
53. Atalla, R.H.; Hackney, J.; Agarwal, U.P.; Isogai, A. Int. J. Series No. 40, American Chemical Society: Washington,
Biol. Macromol. in press. DC, 1977; 30 pp.
54. Bower, D.I.; Maddams, W.F. The Vibrational Spectroscopy 89. Atalla, R.H.; Ellis, J.D.; Schroeder, L.R. J. Wood Chem.
of Polymers; Cambridge University Press: Cambridge, UK, Technol. 1984, 4, 465.
1989. 90. Chanzy, H.; Imada, K.; Mollard, A.; Vuong, R.; Barnoud,
55. Horri, F.; Hirai, A.; Kitamaru, R. The Structures of Cel- F. Protoplazma 1979, 100, 303.
luloses, ACS Symposium Series 340; Atalla, R.H., Ed.; 91. Bertoniere, N.R.; Zeronian, S.H. In The Structures of Cel-
American Chemical Society: Washington, DC, 1987; luloses, ACS Symposium Series 340; Atalla, R.H., Ed.;
119 pp. American Chemical Society: Washington, DC, 1987;
56. VanderHart, D.L.; Campbell, G.C. J. Magn. Reson. in 255 pp.
press. 92. Rowland, S.P.; Roberts, E.J. J. Pol. Sci. A-1 1972, 10, 2447.
57. Yamamoto, H.; Horii, F. Macromoles 1993, 26, 1313. 93. Nickerson, R.F. Text. Res. J. 1951, 21, 195.
58. Yamamoto, H.; Horii, F.; Odani, H. Macromoles 1989, 22, 94. Rowland, S.P.; Roberts, E.J.; Wade, C.P. Text. Res. J.
4130. 1969, 39, 530.
59. Yamamoto, H.; Horii, F. Macromoles 1993, 26, 1313. 95. Rowland, S.P. In Modied Cellulosics; Rowell, R.M.,
60. Belton, P.S.; Tanner, S.F.; Cartier, N.; Chanzy, H. Young, R.A., Eds.; Academic Press: New York, 1978; 147 pp.
Macromoles 1989, 22, 1615. 96. Rowland, S.P.; Roberts, E.J.; Bose, J.L. J. Pol. Sci. A-1
61. Yamamoto, H.; Horii, F. Cellulose 1994, 1, 57. 1971, 9, 1431.
62. Newman, R.H.; Hemmingson, J.A. Holzforschung 1990, 97. Rowland, S.P.; Roberts, E.J.; Bose, J.L.; Wade, C.P. J. Pol.
44, 351. Sci. A-1 1971, 9, 1623.
98. Mann, J.; Marinan, H.J. J. Polym. Sci. 1958, 32, 357. 116. Kondo, T.; Gray, D.G. The preparation of O-methyl-
99. Zeronian, S.H.; Coole, M.L.; Alger, K.W.; Chandler, J.M. cellulosese and O-ethylcelluloses having controlled distri-
J. Appl. Pol. Sci., Appl. Pol. Symp. 1983, 37, 1053. bution of substituents. Carbohydr. Res. 1991, 220, 173
100. Stone, J.E.; Scallan, A.M. Pulp Pap. Mag. Can. 1968, 69, 69. 183.
101. Stone, J.E.; Treiber, E.; Abrahamson, E. TAPPI 1969, 28, 117. de Nooy, A.E.J.; Besemer, A.C.; Bekkum, H. Highly
139. selective nitroxyl radical-mediated oxidation of primary
102. Bell, R.P. The Proton in Chemistry; Cornell University alcohol groups in water-soluble glucans. Carbohydr. Res.
Press: Ithaca, NY, 1973. 1995, 269, 8998.
103. Johnson, D.C.; Nicholson, M.D.; Haigh, F.C. Appl. 118. Isogai, A.; Kato, Y. Preparation of polyuronic acid from
Polym. Symp. 1976, 28, 931. cellulose by TEMPO-mediated oxidation. Cellulose 1998,
104. Johnson, D.C.; Nicholoson, M.D. Cellul. Chem. Technol. 5, 153164.
1977, 11, 349. 119. Kato, Y.; Habu, N.; Yamaguchi, J.; Kobayashi, Y.N.;
105. Isogai, A. Chemical modication of cellulose. In Wood Shibata, I.; Isogai, A.; Samejima, M. Biodegradation of h-
and Cellulosic Chemistry; Hon, D.N.-S., Shiraishi, N., Eds.; 1,4-linked polyglucuronic acid (cellouronic acid). Cellulose
Mercer Dekker: New York, 2000, 599625. 2002, 9, 7581.
106. Isogai, A.; Atalla, R.H. Dissolution of cellulose in aqueous 120. Henrissat, B. Cellulases and their interaction with cellulose.
NaOH solutions. Cellulose 1998, 5, 309319. Cellulose 1994, 1, 169196.
107. Kamide, K.; Okajima, K.; Kowsaka, K. Dissolution of nat- 121. Davies, G.; Henrissat, B. Structures and mechanisms of
ural cellulose into aqueous alkali solutions: Role of super- glycosyl hydrolases. Structure 1995, 3, 853859.
molecular structure of cellulose. Polym. J. 1992, 24, 7186. 122. Kim, D.Y.; Nishiyama, Y.; Wada, M.; Kuga, S. High-yield
108. Isogai, A.; Atalla, R.H. Preparation of cellulosechitosan carbonization of cellulose by sulfuric acid impregnation.
polymer blends. Carbohydr. Polym. 1992, 19, 2528. Cellulose 2001, 8, 2933.
109. Turbak, A.F., El-Kafrawy, A., Snyder, F.W., Jr., 123. Kuga, S.; Kim, D.Y.; Nishiyama, Y.; Brown, R.M. Nano-
Auerbach, A.B., Solvent system for cellulose, U.S. Pat., brillar carbon from native cellulose. Mol. Cryst. Liq.
4302252 (1981). Cryst. 2002, 387, 237243.
110. Schult, T.; Hjerde, T.; Optun, O.I.; Kleppe, P.J.; Moe, S. 124. Azuma, J.; Asai, T.; Isaka, M.; Koshijima, T. Microwave
Characterization of cellulose by SEC-MALLS. Cellulose irradiation of lingocellulosic materials. 5. Eects of micro-
2002, 9, 149158. wave irradiation on enzymatic susceptibility of crystalline
111. Okajima, K.; Yamane, C. Cellul. Commun. 1997, 4, 712. cellulose. J. Ferment. Technol. 1985, 63, 529536.
112. Saka, S.; Takahashi, K.; Matsumura, H. Eects of solvent 125. Saka, S.; Ueno, T. Chemical conversion of various cellu-
addition to acetylation medium on cellulose triacetate pre- loses to glucose and its derivatives in supercritical water.
pared from low-grade hardwood dissolving pulp. J. Appl. Cellulose 1999, 6, 177191.
Polym. Sci. 1998, 69, 14451449. 126. Kobayashi, S.; Kashiwa, K.; Kawasaki, T.; Shoda, S.
113. Jumel, K.; Harding, S.E.; Mitchell, J.R.; To, K.-M.; Novel method for polysaccharide synthesis using an
Hayter, I.; OMullane, J.E.; Ward-Smith, S. Carbohydr. enzymeThe rst in vitro synthesis of cellulose via a non-
Polym. 1996, 29, 105109. biosynthetic path utilizing cellulase as catalyst. J. Am.
114. Tanaka, R.; Meadows, J.; Phillips, G.O.; Williams, P.A. Chem. Soc. 1991, 113, 30793084.
Viscometric and spectroscopic studies on the solution be- 127. Lee, J.H.; Brown, R.M.; Kuga, S.; Shoda, S.; Kobayashi, S.
havior of hydrophobically modied cellulosic polymers. Assembly of synthetic cellulose I. Proc. Natl. Acad. Sci. U.
Carbohydr. Polym. 1990, 12, 443459. S. A. 1994, 91, 9195.
115. Isogai, A.; Ishizu, A.; Nakano, J. Preparation of tri-O- 128. Nakatsubo, F.; Kamitakahara, H.; Hori, M. Cationic ring-
alkylcelluloses by the use of a non-aqueous cellulose sol- opening polymerization of 3,6-di-O-benzyl-alpha-D-glu-
vent and their physical characteristics. J. Appl. Polym. Sci. cose 1,2,4-orthopivalate and the rst chemical synthesis of
1986, 31, 341352. cellulose. J. Am. Chem. Soc. 1996, 118, 16771681.
6
Two-Dimensional Fourier Transform Infrared Spectroscopy
Applied to Cellulose and Paper
Lennart Salmen and Margaretha Akerholm
STFI (Swedish Pulp and Paper Research Institute), Stockholm, Sweden
Barbara Hinterstoisser
BOKU-University of Natural Resources and Applied Life Sciences, Vienna, Austria
I. INFRARED SPECTROSCOPY FOR WOOD cellulose I, cellulose II, and amorphous cellulose using
AND CELLULOSE RESEARCH polarized IR radiation.
A further important investigation was carried out on
An infrared spectrum contains the complete information mercerized cellulose and wood polysaccharides by Liang
about the molecular assembly of a material or a substance. and Marchessault [6,7,2325], who estimated the crystal-
This fact made infrared (IR) spectroscopy a widely used linity of cellulose and assigned specic cellulose-, lignin-,
analytical tool especially, but not exclusively, for identify- and hemicellulose-deriving bands by IR spectrometry.
ing and characterizing the molecular structure of organic When Liang and Marchessault [6] in 1959 wrote about
compounds. IR spectroscopy has therefore a long tradition native celluloses, a large part of their publication ques-
in organic chemistry and was, for a substantial length of tioned the possibilities of hydrogen bonding. They found a
time, among the main spectroscopic tools for elucidating strong parallel OH stretching band vibrating in-phase with
the chemical structure of puried substances. IR spectros- all C3 hydroxyl groups (3350 cm1). They attributed it to
copy has also for a long time been a useful analytical an intramolecular hydrogen bond between the hydroxyl
method for wood and cellulose chemistry. Even in the group on the C3 atom of one glucose residue and the ring
1940s, it was used to investigate the native structure of oxygen of the next residue, a bond which is subsequently
lignin [1,2]. Over the years, it was developed and used for referred to as O3H. . .O5V in the text. Referring to their IR
determining the composition of lignin, cellulose, and hemi- data, they also discussed other possibilities of hydrogen
celluloses in wood and pulps, for studying derivatives and bonding, for example, between the hydroxyl group of the
model compounds of these polymers, as well as for study- C6 and the bridge oxygen of the glycosidic linkage. Fur-
ing changes caused by heat or chemical treatment as used thermore, there was a proposal that the C6 hydroxyl group
in various processes (e.g., Refs. [320]). In Fig. 1, an might be bonded either to the oxygen of the C2 of the next
overview of the band assignments of an IR spectrum of glucose ring, bifurcated between the bridge oxygen and the
cellulose is shown as a guide to accompany the text in oxygen of the C2 of the next ring, or that the hydroxyl
this chapter. group at the C2 might be bonded to the C6 oxygen. It was
Although the IR spectra of cellulose contain several suggested that intermolecular hydrogen bonds might exist
overlapping bands making them dicult to interpret, IR between the C6 hydroxyl group of one chain and the bridge
spectroscopy has been used for more than 50 years as an oxygen of the neighboring chain. The weaker bands at 3405
accompanying technique for wood and cellulose chem- and 3305 cm1 in the IR spectra of bacterial celluloses were
istry to estimate the crystallinity of cellulose; some impor- ascribed to intermolecular hydrogen bonds. It was pro-
tant work was carried out by Mann and Marrinan [21,22]. posed that a fourth band found at 3245 cm1 was also
These IR studies were based on the reaction of cellulose derived from intermolecular hydrogen bonds. This band at
with heavy water. Tsuboi [5] early on assigned a number of 3245 cm1 was found only in bacterial and algae celluloses
bands for cellulose and worked out dierences between but not for cotton and ramie.
159
160 Salmen et al.
Figure 1 The static FT-IR spectrum of a spruce dissolving pulp cellulose together with an overview of the band assignments.
In 1963, Smith et al. [26] again used infrared spec- of the polymer chains. Their investigations were based on
troscopy to determine the degree of crystallinity of cellu- the fact that the dichroism of the crystalline OH stretching
lose using deuterated samples. Nelson and OConnor [27] vibrations is related to the molecular orientation as a result
compared highly crystalline cellulose samples of dierent of the extent of stretching of the cellulose bers. In the
lattice types (I, II, and III) and amorphous cellulose, following years, several papers were published dealing with
focusing on the spectral region between 850 and 1500 determinations of the degree of crystallinity (e.g., Refs.
cm1. They questioned the use of bands at 1420, 893 [3133]).
897, and 1111 cm1 for estimating crystallinity for mixed While in the old days, samples in a solid or liquid
lattice types as done by other authors. The stabilizing state were measured by dispersive working instruments,
function of intramolecular hydrogen bonds was discussed the 1980s brought a renaissance of IR spectroscopy
as well. Tritium exchange was another attempt to obtain through the introduction of the Fourier transform (FT)
more knowledge of crystallinity and the accessibility of technique. While the former dispersive instruments
hydrogen bonds using IR spectroscopy as a detection contained a monochromator which provided one well-
system [28]. Ranby concluded that in dry cellulose, less dened wavelength range after the other to which the
than 1% of the hydroxyl groups exist as free groups and sample was exposed, FT-IR instruments irradiate the
that in the crystalline regions, all hydroxyl groups are sample with an interference wave produced in an interfer-
involved in bonding. The possible applications of IR ometer. The interferogram recorded by the detector is
spectroscopy to the qualitative and quantitative testing of related to the spectrum through its Fourier transform.
pulp and paper were reviewed in 1965 by Jayme and The fact that the radiation reaching the detector contains
Rohmann [29]. all wavelengths at one time gives the so-called multiplex (or
Several papers have dealt with trials to unravel the Fellgett) advantage. It allows spectra of the same signal-to-
broad OH region, which hides all the structure-relevant noise ratio (SNR) to be measured much faster on an FT-IR
information relating to hydrogen bonding. An overview of spectrometer than on a traditional dispersive instrument.
band assignments carried out up to 1972 was given by Another outstanding advantage is given by the optical
Dechant et al. [30]. Siesler et al. [11] described a technique throughput (Jacquinot advantage), which is greater for
for the measurement of the plane-polarized IR spectra of an interferometer than for a monochromator. The inven-
deuterated cellulosic bers by frustrated multiple internal tion of the Fourier transform infrared (FT-IR) technique
reectance (FMIR). Their aim was to investigate regener- opened up new possibilities in instrumentation and in the
ated cellulose bers according to the molecular orientation coupling of analytical systems as well as in the application
2D FT-IR Spectroscopy Applied to Cellulose and Paper 161
of specic software facilities. This includes, for example, II. TWO-DIMENSIONAL FOURIER TRANSFORM
the possibility of mathematical processing of the spectra, INFRARED SPECTROSCOPY
such as dierentiation to give the second derivative [34],
and deconvolution of spectra [16,18,35], all of which In polymer research, IR spectroscopy has been and is still
provide better resolution of the absorption bands. The applied to identify and determine chemical composition,
deconvolution of the range of the OH stretching vibrations including end groups and chain branching of the polymers.
gives detailed evidence of crystallinity, crystal modica- The conguration and conformation, as well as steric and
tion, and degree of substitution of cellulose and cellulose geometric isomerism, is a further area that, to some extent,
derivatives [36]. However, neither deconvolution nor dif- can be unraveled [40,62]. To obtain such information on
ferentiation increases the instrumental resolution; they are polymers is of great importance since the mechanical
merely methods for resolving overlapping bands by com- properties, such as strength, ductility, and glass transition
putation [34,37]. temperature, are a result of the organization and orienta-
Progress in FT-IR spectroscopy led to investigations tion of the polymer chains and of the molecular structure in
not only of molecular structures, but also of pattern general. IR spectroscopy, even in the 1970s, was a proven
recognition in complex systems, as well as qualitative and technique which allowed statements about structural
quantitative assessment of components as pure substances changes relating to deformation through external stress
or within complex matrices like wood [13,38,39]. Addition- [11]. All these measurements had, however, been based on
ally, several sophisticated techniques, such as, for example, static studies.
attenuated total reection (ATR), photoacoustic, and dif- Time-resolved IR spectroscopy (TRS) was proposed
fuse reection FT-IR (DRIFT), and instrument couplings in the 1980s as a new tool for dynamic studies to investigate
between FT-IR and gas chromatography (GC), high-per- the deformation behavior of polymers [63]. Burchell and
formance liquid chromatography (HPLC), or thermogra- Hsu [63] connected a computer-controlled hydraulic
vimetric analyzers (TGA) provided further possibilities for stretching device, providing a predened periodic oscilla-
solving analytical questions [4043]. This also resulted in tory strain to an FT-IR spectrometer working in rapid
new application possibilities for wood and pulping chem- scanning mode. An important point was that the applied
istry [41]. A third point to be mentioned is that an extremely strain had to be far below the yield point, namely, in the
high wave number precision is obtained by the FT-IR linear region of the stressstrain diagram. In general, the
technique. One of the promising (new) techniques was idea was that if the directions of the dipole transition
FT-IR microspectroscopy, which made possible the inves- moments with respect to the chain axis were known,
tigation of very small samples with diameters of about 10 stress-induced changes in orientation, conformation, and
50 Am [44]. Ludwig and Fengel [45] used FT-IR microscopy packing could be measured. Therefore it was necessary to
to study cellulose nitrate bers of dierent degrees of establish and calibrate the changes in the FT-IR spectra
substitution. (e.g., frequency, intensity, vibrational bandwidth, and
In the 1990s, focus was drawn to the allomorphic dichroism) as a function of the strain amplitude and the
forms of native cellulose, namely, cellulose Ia and cellulose strain rate. This led to the possibility of directly assigning
Ih. FT-IR spectroscopy, besides electron diraction stud- the molecular dipole transition moments involved in stress-
ies, electron microscopy, and CP/MAS 13C-NMR spec- induced intramolecular changes via the obtained changes
troscopy, was one of the techniques used [17,4649]. The in the absorption bands. Furthermore, direction-depen-
IR bands between 400 and 800 cm1, as well as the region dent orientational, conformational, and packing changes
of the OH stretching vibration, turned out to be of interest could also be observed. FT-IR spectroscopy now provided
in investigating the polymorphism of native cellulose. a tool for not only static, but also dynamic measurements.
Absorption bands near 3240 and 750 cm1 were assigned In the following years, time-resolved IR spectroscopy
to the triclinic cellulose Ia phase. The bands near 3270 and became more and more recognized as a promising tech-
710 cm1 were assigned to the monoclinic cellulose Ih nique for polymer research [64,65]. Lasch et al. [66] studied
phase. The crystallinity of cellulose remained of great polymer deformations by slowly stretching oriented poly-
interest in investigations of higher plants [42, 50], algae mer lms while IR-spectrometrically studying changes in
[51], and cell wall formation [52], as well as for pulp and the macromolecular structure. In these experiments, the
paper research [53,54]. Associated with the allomorphic stretcher, which provided the stress to the polymer sample,
forms, the hydrogen bonding pattern also remained a topic was run continuously and not synchronized to the inter-
of interest [55]. ferometer. Therefore all the data were collected and all
With the development of a combination of dynamic interferograms with identical phases had to be co-added.
mechanical analysis (DMA) and IR spectroscopy, new The combination of IR spectroscopy and mechanical
possibilities were seen for assigning and interpreting spec- (so-called rheo-optical) measurements led to more detailed
tra of biopolymers like cellulose. Starting in the late 1990s, data and therefore to a better understanding of the mech-
this technique was applied to investigate cellulose, in anisms involved in polymer deformation. Noda et al. [67]
particular, the hydrogen bonding, the allomorph compo- constructed a system designed to detect dynamic IR linear
sition, the stretching behavior, and the interactions within dichroism (DIRLD) in polymer samples undergoing small
the wood polymer network also including hemicelluloses oscillatory strains using a dispersive IR instrument. Small
and lignin [20,5661]. amplitude oscillatory strain was applied to thin lms of
162 Salmen et al.
quency, x, to the sample, the dynamic signal A(m,t) from

the sample can be written as
t Amsinxt
Am; bm 2
with A(m) being the amplitude and b being the phase loss
angle.
As the reorientations of the dipole transition moments
are directionally dependent, the resulting directional ab-
sorbances represent the time-dependent reorientations of
Figure 2 Schematic drawing of a dynamic FT-IR experiment. the submolecular groups, which are strongly inuenced by
the intermolecular and intramolecular interactions. The
phase dierence is due to the rate-dependent nature of the
reorientation process of the dierent submolecular groups.
As a result of this, the rheo-optical response can be
polyethylene mounted in a stretching device. The polar- expressed by the rate-independent in-phase and the
ization of the IR beam was altered at a very high frequency rate-dependent out-of-phase portion of the response,
between parallel and perpendicular directions (i.e., with the rst representing the storage modulus and the second
respect to the stretching). Dichroic dierences between the the loss modulus of the sample. The storage modulus
absorbances parallel and perpendicular to the stretch stands for the ability of a polymer to elastically store the
direction as small as 5105 with a time resolution of absorbed mechanical energy as potential energy, whereas
better than 14 Asec were detected. the loss modulus represents the ability of the material to
The development of the dynamic IR linear dichroism dissipate the absorbed energy.
technique turned out to be fast and sensitive enough to The dynamic response given in Eq. (2) can therefore be
detect molecular level relaxations of strain-induced orien- expressed as the sum of two terms, which are orthogonal to
tations in polymers, although these orientations occur and each other:
relax very rapidly. t AVm sin xt AWmcos xt
Polymers such as isotactic polypropylene served as Am; 3
model compounds in the development of the new tech- AV(m) represents the dynamic responses that are in-phase
nique. In 1987, Noda et al. [68] compared Fourier trans- with respect to the applied external perturbation. AW(m)
form and dispersive spectroscopy in the characterization represents the dynamic responses that are 90j out-of-phase
of polymers in polarizationmodulation IR techniques. with the applied perturbation (cp. Fig. 3).
By that time, the dispersive instrument had been given The in-phase spectrum is derived from reorientations
preference. of the dipole transition moments occurring simultaneously
In 1991, Palmer et al. [69] introduced FT-IR spec- with the applied strain. The response of the in-phase
troscopy to perform such dynamic experiments. A tech- spectrum is proportional to the applied perturbation.
nical improvementthe step scan interferometry The out-of-phase (quadrature) spectrum shows signals of
allowed advantage to be taken of FT-IR as well as of submolecular constituents which are reorienting with a
the two-dimensional correlation and coupling to dynamic phase delay of p/2that means perpendicular to the
mechanical analysis (DMA). As the step-scanning decou- applied perturbation.
ples the spectral multiplexing from the time domain, the
time dependence of the sample response to the external
perturbation was shown to be regained quite easily [70].
Again, an external sinusoidal small amplitude strain is
applied to a sample that is irradiated with polarized IR
light (Fig. 2).
The time-dependent, dynamic IR absorbance of the
strained sample measured at a wave number, m, can be seen
as the combination of two components: a quasistatic [A(m)]
and a dynamic one [A(m,t)].
t
Am; t Am Am; 1
The dynamic response obtained as a dynamic varia-

tion of the IR signals [A(m,t)] can be expressed in terms of
two parameters. The rst is a phenomenological coecient
relating the amplitude of the oscillatory strain to the IR
response [A(m)] and the second representing the phase loss
angle (b) between the strain and the IR response. As the Figure 3 Connection of magnitude and phase spectra to in-
sinusoidal external perturbation is applied at a xed fre- phase and out-of-phase spectra.
The in-phase and out-of-phase spectra can be tion for changes occurring with 90j phase dierence [74].
expressed as follows: Peaks located on the 2-D spectra provide information
about interactions among the dierent functional groups

AVm Amcos bm associated with the IR bands. A further advantage of this
4 new data handling was that overlapping bands could be

AWm Amsin bm resolved.
The in-phase and out-of-phase spectra are related to the In 2-D IR, the dynamic IR cross-correlation function
magnitude and phase spectra (see Fig. 3) mathematically X(s) is dened for a pair of dynamic IR signals measured at
as follows: two dierent wave numbers [A(m1,t) and A(m2,t)] as,
q
Xs Um1 ; m2 cos xt Wm1 ; m2 sin xt 6

Am AVm2 AWm2
5 where U(m1,m2) and W(m1,m2) are the real and imaginary
bm arctanAWm=AVm components, respectively.
An additional method for analyzing the spectra was The synchronous and asynchronous correlation inten-
introduced by the two-dimensional infrared correlation sities of the dynamic spectrum are given by:
analysis [7173]. Again, time-resolved detection of IR 1
signals in response to an external perturbation, e.g., me- Um1 ; m2 Am
1 Am2 cosbm1 bm2
chanical strain, was the basis. In contrast to the former 2
correlation analysis, the dynamic variation of the IR 1
AVm1 AVm2 AWm1 AWm2 7
signals was analyzed yielding new spectra which were 2
dened as functions of two independent wave number 1
Wm1 ; m2 Am
1 Am2 sinbm1 bm2
axes. For a pair of dynamic IR signals measured at two 2
dierent wave numbers, the dynamic IR cross-correlation
functions were dened, giving synchronous and asynchro- 1
AWm1 AVm2 AVm1 AWm2 8
nous spectra. The synchronous correlation intensity char- 2
acterizes the degree of coherence between the dynamic Fig. 4 gives an example of a synchronous spectrum in
uctuations of IR signals measured at two dierent wave the wave number region 36002500 cm1 of an oriented
numbers. The asynchronous spectra represent the correla- cellulose sheet irradiated with IR light polarized parallel to
Figure 4 Synchronous 2-D FT-IR spectrum of an oriented cellulose sheet irradiated with IR light polarized parallel to the
stretching direction. Autopeaks on the diagonal are marked as (.) and cross-peaks as (5).
164 Salmen et al.
the stretching direction. The 2-D IR correlation spectrum is synchronized and therefore that a strong chemical inter-
in fact three-dimensional, with independent wave numbers action is lacking. Functional groups that are located in
on the x- and y-axes and the correlation intensities on the z- dierent chemical environments can exhibit dierent dy-
axis. In a synchronous spectrum, peaks appear for pairs of namic responses to the external perturbation. The signs of
bands with identical dynamic behavior. The peaks on the the cross-peaks give information on the relative rates of
diagonal, the so-called autopeaks, indicate which transition the response of the involved dipoles. Fig. 5 gives an
dipoles and thus which submolecular components have an example of the asynchronous correlation map of the OH
orientational response to the applied sinusoidal strain. Since region of an oriented cellulose sheet irradiated with IR
the intensity change of each band is correlated with itself, a light polarized parallel to the stretching direction. The
series of positive maxima along the diagonal show up. static spectrum consists of a broadband, obviously derived
The o-diagonal peaks (cross-peaks) appear whenever from the overlapping of several bands. This can clearly be
the corresponding dipole transition moments reorient in- observed in the 2-D plot where several dierent cross-
phase (simultaneously) with each other. Therefore a pair of peaks, also with a dierent sign, appear. Because of the
intense cross-peaks indicates the existence of a strong mathematics, equivalent peaks with opposite signs appear
synchronous correlation between the two bands. If the on the dierent sides of the diagonal. A closer discussion
cross-peak appears to be positive in sign, the two corres- of these features for the cellulose bands in question will
ponding dipole transition moments reorient parallel to also follow in Two-Dimensional FT-IR Spectroscopy
each other. Negative peaks appear if the reorientations Applied to Cellulose of this chapter.
are mutually perpendicular. Further details of the cellulose In the years following the development of this 2-D FT-
spectrum will be discussed under Two-Dimensional FT- IR technique, investigations on several synthetic polymers
IR Spectroscopy Applied to Cellulose of this chapter. such as polyurethane [75], polyethylene [70,76], polypropyl-
The asynchronous correlation function (an example ene [77,78], polymer lms and liquid crystals [79], epoxy
for the wave number region 35003200 cm1 is drawn in resins [80,81], polymer blends [8284], melt crystallized
Fig. 5) gives information about the degree of indepen- nylon [85], acetylene terminated polyisoimide prepolymer
dence of reorientational behavior of corresponding dipole [86], duroquinone [87], and inorganic NaCl crystals [88]
transition moments. No peaks appear on the diagonal. have been performed. The method has also successfully
Cross-peaks are produced if the transition dipoles reorient been applied to some biological materials, for instance, to
out-of-phase with each other, meaning that they are not study protein conformations in human skin [89], keratin in
Figure 5 Asynchronous 2-D FT-IR spectrum of an oriented cellulose sheet irradiated with IR light polarized parallel to the
stretching direction. Cross-peaks are marked as (0).
human hair [89,90], broin in silk [91], and arabinoxylans

[92,93]. The technique was introduced to cellulose research
by Hinterstoisser and Salmen [5658]. Akerholm and
Salmen [59,60] extended the investigations to the wood
polymer network to determine which wood polymers were
connected to each other and the nature of the interactions.
Furthermore, the rst studies on pulps were carried out [61].
III. TWO-DIMENSIONAL FOURIER

TRANSFORM INFRARED SPECTROSCOPY
APPLIED TO CELLULOSE
A. Orientation Aspects
In dynamic IR spectroscopy, thin lms or sheets of a
material are stretched. Even with a lm of random polymer
chain orientation, the small static and dynamic strains put
on the sample will shift the random distribution toward a
small orientation in the direction of the stretch. This
orientation, together with the fact that both the dipoles
and the polarized light have a direction, means that the
spectral response to the straining will dier in dierent
polarization modes. With a preoriented sample, this dif-
ference will be enhanced. The eects of sample orientation
on the spectral result of 2-D FT-IR measurements have
Figure 6 Dynamic in-phase FT-IR spectra showing the result
been studied for isotactic polypropylene samples of dier- of a sheet stretched parallel to the ber axis, perpendicular to
ent orientation [78]. It was found that for this material, the the ber axis, as well as a non-oriented sheet during 0j (upper
spectral result depended strongly on the pretreatment of spectra) and 90j (lower spectra) polarization modes.
the sample, as well as on the polarization of the infrared
radiation used. The spectral features occurring in dynamic
FT-IR spectra originate from absorption changes, frequen- loaded parallel to the ber axis and is related to the
cy shifts, and changes in band shape as a result of the normalization at 1165 and 1169 cm1, respectively. In the
applied dynamic strain of the samples. These stress-in- case of the perpendicular orientation, there are few cellulose
duced changes have been shown to vary with morpholog- chains oriented in the stretching direction, capable of taking
ical, thermal, and stress history of the samples. up the load. This load situation results in a low dynamic
With an oriented lm or sheet, the specimen may also intensity of the skeletal vibration/COC band. Thus in this
be stretched in dierent directions. Such eects are illus- case, the overall deformation by bending and shearing of
trated in Fig. 6, where the dynamic in-phase spectra for the bers would show up as a larger change in the
spruce cellulose sheets with dierent ber orientations are O3H. . .O5V intramolecular hydrogen bond deformation.
shown for both 0j polarization and 90j polarization [56]. Thus when it comes to a general characterization of
Comparing the spectra from the dierent orientations the strain distribution within cellulose in a ber network
reveals no great dierence in band positions between them. material like paper, the ber direction toward the straining
The main eects observed for the dierent ber directions direction is not the most important factor in the experi-
were the dierences in relative intensity between absorption mental arrangement for dynamic FT-IR studies since the
bands. For these cellulose samples, it can be seen that the same peaks for cellulose appeared for all the ber direc-
intensity of the peak for the nonoriented sample is always tions examined. The highest resolution has, though, been
in-between the intensities of the other two loading modes. obtained using oriented samples mounted so that the
There is a strong correlation between the deformation stretch is applied in parallel to the ber direction.
of the cellulose skeleton including the glycosidic bond, the In Fig. 7, the dynamic in-phase spectra of spruce
band at 1165/1169 cm1, and the band at 1435 cm1. This cellulose ber sheets stretched parallel to the ber direction
can be seen as the peak at 1435 cm1 having the same for light polarized at 0j and 90j to the strain direction [56]
intensity for all dierent ber orientations after normal- are shown. The dynamic spectra recorded at 0j polarization
ization of the spectra at 1165/1169 cm1 (skeletal stretching are dominated by changes in vibrations aligned parallel to
vibrations including the COC bridge stretching). The the straining direction, while spectra recorded at 90j polar-
intensity of the O3H. . .O5V intramolecular hydrogen vi- ization are dominated by perpendicularly aligned vibrations.
bration is the highest in both the 90j (3332 cm1) and 0j In general, more peaks appear in the in-phase spectra of the
(3329 cm1) polarization modes for the sheet loaded per- 90j polarization than at 0j, both in the OH region and in the
pendicular to the ber axis and the lowest for the sheet ngerprint region. For both polarizations, the most intense
166 Salmen et al.
ditions, the understanding of how dierent entities of the

molecule contribute is essential. This applies in particular
to a system such as that of cellulose in which there are
possibilities for hydrogen bonding in several dierent
positions. In fact, cellulose is a very highly coupled system
in terms of IR vibrations. This is demonstrated via the
synchronous 2-D FT-IR spectrum in Fig. 8. As an example,
the close correlation between the O3H. . .O5V intramolec-
ular hydrogen and a skeletal stretching band can be
observed in the 2-D spectrum as a signicant cross-peak
between 3329 and 1169 cm1, the latter often assigned in
the literature to the COC stretching [16,97,98]. This band,
though, is not a real local mode vibration and therefore
cannot be solely stated to be a COC stretching mode, as
the glycosidic linkage is part of a whole sequence of eight
coplanar CC or CO bonds and these bonds are very highly
coupled in their vibrations. The O3 bond, which is the last
in the sequence, is precisely the one that is involved in the
intramolecular O3H. . .O5V hydrogen bond [94].
The relatively high intensity of the O3H. . .O5V intra-
molecular hydrogen vibration, the high intensity of the
skeletal vibrations including the COC bridge stretching,
and the high intensity of the CO and ring stretching in
general (Fig. 7) all point to their importance in the loading
of the cellulose chain. This fact, in combination with the
Figure 7 Dynamic in-phase (thick line) and out-of-phase close coupling observed in the 2-D FT-IR spectrum (Fig. 8)
(thin line) FT-IR spectra of cellulose. The bers of the [56], supports the calculations of Tashiro and Kobayashi
cellulose sheets were oriented parallel to the stretching [97], who found that the strain energy in cellulose is mainly
direction. The polarization plane of the incident beam was distributed via deformation of the glucose rings (f30%),
0j (upper spectra) and 90j (lower spectra) to the strain bending of the ether linkages connecting the adjacent rings
direction. The most intense bands are marked. (f20%), and deformation of O3H. . .O5V hydrogen bonds
(f20%). In this respect, the O2VH. . .O6 intramolecular
hydrogen bond seems to play a minor role.
peaks are found between 1200 and 1050 cm1 (CO, CC, and
skeletal stretching vibrations). At 0j polarization to the
direction of the straining, the two most intense peaks are C. Hydrogen Bonding
at 1169 cm1, corresponding to skeletal stretching vibrations
1. General Remarks
including the COC bridge stretching [24,94], and at 3329
cm1, corresponding to the O3H. . .O5V intramolecular hy- Hydrogen bonds are of special interest when dealing with
drogen vibration [6,95,96]. In the 90j experiment, the 1064 macromolecules, particularly biomacromolecules. The
cm1 band, in the CO valence vibration region, is the most reason is evident: hydrogen bonds are known to be the
intense peak. In this polarization mode, there are also peaks most important cohesive forces involved in the organiza-
for skeletal stretching including the COC stretching, tion of the three-dimensional structure and the mode of
positioned at 1165 cm1, and for the O3H. . .O5V intramo- recognition and association of biological molecules. Fur-
lecular hydrogen vibration at 3332 cm1. Between 1200 and thermore, they are known to be stronger and more direc-
1500 cm1, CH2 deformation vibrations and COH in-plane tional than van der Waals forces [99]. It was during the
bending motions are expected. More signals appear in the 1930s that hydrogen bonds were introduced as an impor-
90j than in the 0j polarization experiment for this area, but tant principle in structural chemistry [100,101]. It was then
the intensity is less than that for the lower wave numbers. As that the interest in research into this incredibly important
the peak at 1462 cm1, probably from a CH2 bending issue that hydrogen in special cases has the ability to
vibration [97], represents an orthogonal vibration in relation function as a bridge atoma function being of basic
to the backbone, its appearance in only the 90j polarization importance for lifebegan.
experiment is anticipated. The strength of hydrogen bonds ranges from some-
thing close to that of covalent bonds down to a primarily
electrostatic interaction, depending on the other partners
B. Load Distribution involved. On the one hand, the hydrogen atom within the
functional group might be covalently bound to a more
In understanding the strength development of polymeric electronegative atom. On the other hand, the hydrogen
material and the way this is aected under various con- atom might face as its nearest neighbor another electro-
Figure 8 Synchronous 2-D FT-IR spectrum of oriented cellulose sheets stretched parallel to the ber orientation, irradiated with
IR light polarized parallel to the stretching direction and the ber direction (0j polarization mode). The cross-peaks can be seen
o the diagonal. The cross-peak between the band of the COC and the O3H. . .O5V hydrogen bond is marked.
negative atom. This belongs to another functional group an alcohol diluted in CCl4, or CS2, for instance, appears
which serves as electronegative acceptor within the hy- at about 3600 cm1. The corresponding OH stretching
drogen bonding. As the electron of the hydrogen atom is vibration of a pure, and therefore very much associated,
used for the closure of the covalent bond to the more alcohol is a broadband around 3300 cm1 [102].
electronegative atom, the hydrogen becomes more or less Weak hydrogen bonds are long-range interactions,
descreened. This results in a dipole with a positive charge falling o with r1. The rst neighbor hydrogen bond
at the H-end of the linkage. If the nearest neighbor is interactions are still signicant at distances as great as
carrying excess electron density, a hydrogen bond to this 0.35 nm from the hydrogen atom. Hydrogen bonds,
neighbor is built. The strength of the hydrogen bond therefore, appear to have group properties. This means
depends mainly on the electron anity of the partners that they depend not only on their nearest neighbor atoms
involved. From this point of view, it is easy to under- involved in the bridge building, but also on the sequential
stand that a variety of hydrogen bonds exist. These vary nature of the total pattern of bonding. In general, the
both in bond energy and in their structural features. Very stretching and bending force constants of hydrogen bonds
strong hydrogen bonds such as that in FH. . .F and are about 15 times smaller than for covalent bonds.
OH. . .O are of minor importance in biological systems. However, bond length and angles depend a lot on the
More important in these are the normal or weak chemical environment.
hydrogen bonds. These are often two, three, or four Hydrogen bonds can be easily deformed by other
centered bonds, with a bond length ranging from 0.15 intermolecular interactions, such as other hydrogen bonds
to 0.3 nm. They are weakly directional, the bond energy or van der Waals forces [99]. Intramolecular hydrogen
is lower than 20 kJ/mol, and the IR vibration frequencies bonds dier from intermolecular ones as they are not af-
are found above 2000 cm1 [99]. Hydrogen bonds lead to fected by solvents because they are less accessible [28,102].
a remarkable red shift (shift toward longer wavelengths) Consequently, hydrogen bonds are a complex issue to
of the OH stretching vibration bands in IR spectra, as study. Dierences particularly in bond length, energy, and
the bonding between the electronegative oxygen and the angles imply that they also dier in their vibrational
hydrogen atom is weakened through the bridge building. behavior, making IR spectroscopy a useful tool for such
The OH stretching vibration frequency, for example, of investigations.
168 Salmen et al.
2. Hydrogen Bonding in Cellulose

There is no question about the general concept that cellu-
lose is made up of glucose molecules forming poly-h(1,4)-
D-glucose chains. On the other hand, many researchers
have put much eort into answering questions relating to
its three-dimensional structure, questions undoubtedly
concerned with hydrogen bonds, and the matter is not
yet resolved. In fact, intramolecular as well as intermolec- Figure 9 Schematic drawing of cellulose molecules including
ular hydrogen bonds play a major role in the interactions the hydrogen bonds (----): intramolecular hydrogen bonds
within and between the cellulose molecules and, therefore, O3H. . .O5V and O2VH. . .O6; intermolecular hydrogen bond
also inuence the strength properties of the cellulosic O6H. . .O3.
material. Intramolecular hydrogen bonds have an impor-
tant stabilizing function within cellulose chains. The inter-
molecular hydrogen bonds are also of importance as they
play a key role in the formation of crystalline structures and cation of the predicted vibrational energies of the dierent
the association of microbrils. bondings is at hand.
The physical properties of dry cellulose, for example, The dynamic 2-D FT-IR technique has, in this respect,
the ber-to-ber linkage in paper, are mainly a function of been found useful in providing answers to the remaining
the OH groups and their ability to build hydrogen bonds. questions relating to hydrogen bonding [5658]. Fig. 10
In dry cellulose, practically all OH groups are involved to shows an example of the OH stretching vibration region in
some extent in hydrogen bonding. The proton donor group both static and dynamic 2-D FT-IR spectra of native
is an OH group bonded to another hydroxyl or oxygen spruce cellulose. These spectra were produced from orient-
group, functioning as proton acceptor. Some of these ed sheets made of a spruce dissolving pulp. Comparing the
hydrogen bonds can be destroyed by water leading to dynamic in-phase and out-of-phase IR spectra, the rst fact
new hydrogen bond formation between the OH or O to emerge is that the response of the out-of-phase spectrum
groups of the cellulose and the water molecules. The is two to three times less intense than that of the in-phase
accessibility of dierent hydrogen bonds is selective in spectrum. This indicates that the dynamic behavior of the
character, the intramolecular bonds being more resistant cellulose is nearly exclusively elastic.
than the intermolecular ones [28]. The second and even more obvious feature is that the
Studies of hydrogen bonding are an intrinsic part of in-phase IR spectra consist of dierent distinct bands,
cellulose research. Since the 1950s, IR spectroscopy has while the static spectra consist of an unstructured broad-
been one of the favored tools for investigating hydrogen band. The higher resolution provided by the dynamic
bonds, although the broadband around 30003700 cm1 measurement allows experimental visualization of the
causes diculties in spectral interpretation because it bands belonging to dierent dipole transition moments.
includes all the specic OH stretching vibration bands of There are also dierences between the spectra mea-
interest in one large peak. Several attempts have been made sured in the parallel polarization mode and those measured
to unravel this hump by using deuterium exchange (for in the perpendicular mode. The main band in the 0j
example, Refs. [11,21,103]), as well as mathematical pro- polarization mode experiment appears, in fact, as a split,
cessing such as deconvolution and dierentiation [34,95]. or a so-called bipolar, band. This indicates that the tran-
As investigations proceeded over the years, specic fre- sition dipole moments of the components involved reorient
quencies were assigned to dierent hydrogen bonds. This in dierent directions with respect to the polarization axis
became even more important with the discovery that there when subjected to the oscillatory strain. One average dipole
existed dierent allomorphs of native cellulose (cp. Crys- is moving toward the polarization axis, which is in this case
tal Structure), which opened up further discussions relat- parallel to the strain axis, and the other away from it. The
ing to hydrogen bonding. dominating signal is placed at 3329 cm1 with an opposite
In the generally accepted structure of native cellu- peak at 3372 cm1. A bipolar dynamic band has its
lose (Fig. 9), intramolecular hydrogen bonds of types fundamental energy in-between the two bands. Further
O3H. . .O5V and O2VH. . .O6 are present on both sides of details are examined and discussed in connection with 2-D
the chain [104]. This is related to a nearly trans gauche (tg) correlation spectra.
orientation of the hydroxymethyl group [105]. The bond In Fig. 11, the synchronous correlation spectrum of
length of the O3H. . .O5V hydrogen bond is reported to be the OH region of spruce cellulose is shown. The spectrum
0.275 nm and the length of the O2VH. . .O6 is reported to be is symmetric with respect to the diagonal line as the
0.287 nm. An intermolecular hydrogen bond, O6H. . .O3, is synchronous correlation intensities characterize the degree
formed with a bond length estimated to be 0.279 nm of coherence between the dynamic uctuations of IR
[104,105]. It is generally known and accepted that these signals measured at two dierent wave numbers. As men-
hydrogen bonds play an important role in determining the tioned above, the autopeaks indicate which functional
conformational and mechanical properties of cellulosic groups change dynamically as a result of the applied
materials [19,55,104106]. However, no satisfactory veri- sinusoidal strain.
Figure 10 OH-stretching vibration region of FT-IR spectra: static spectra (----), in-phase and out-of-phase dynamic spectra of
native spruce cellulose stretched parallel to ber orientation: (A) light polarized parallel and (B) light polarized perpendicular to
the stretching direction.
The observed autopeaks in the synchronous 2-D spec- moments reorienting in the stretching direction give a
trum in the OH range thus represent the perturbation- stronger contribution to the spectrum, and it is the hydro-
induced local reorientation of the hydrogen bonds. The gen bonds involved that are more likely to be stretched and
main peaks are at 3329, 3372, and 3472 cm1. As the changed in energy during straining. The high intensity of
cellulose molecule reacts quite elastically, the peaks ob- the O3H. . .O5V intramolecular hydrogen bond vibration,
served are equal to the ones observed in the in-phase and its domination in the 0j polarization mode, clearly
spectra. The synchronous plot of the oriented sheets shows indicate its importance in the loading of the cellulose chain
strong positive cross-peaks, correlating the main maxima as a kind of second bridge between adjacent glucose
with each other. Obviously, these dipole transition molecules beneath the main, covalent COC bridge.
moments reorient in phase with each other, showing a high As the intramolecular hydrogen bonds O3H. . .O5V
degree of coupling. and O2VH. . .O6 are expected to be stabilizers in the cellu-
Negative cross-peaks (shaded in the gure) between
3329 cm1 and the other peaks are clearly evident. The
appearance of negative cross-peaks indicates that the
reorientation direction of the one transition dipole moment
is orthogonal to that of the other. Therefore it can be
concluded that the transition dipole moment of the 3329
cm1 band is perpendicular to that of the 3372 and 3472
cm1 ones. It is therefore clear that although the two sets of
absorption bands are synchronized to each other, they
must have dierent origins. According to the literature,
the frequencies for the O3H. . .O5V intramolecular hydro-
gen bond can be found between 3340 and 3375 cm1
[6,28,97]the peaks of the in-phase spectra are almost
within this wave number region.
Examining again the in-phase spectra shown in Fig.
10, it can be seen that in the 90j polarization mode (Fig.
10B), a corresponding peak appears at 3332 cm1, but this
one is not split. A band at 3375 cm1 is, however, unidi-
rectional with the 3332 cm1 in the 90j polarization mode.
It appears as well in the region predicted for the O3H. . .O5V Figure 11 Synchronous 2-D FT-IR spectrum of the OH-
intramolecular hydrogen bond leading to the question of stretching vibration region of native spruce cellulose
whether a bifurcated hydrogen bond might exist, as Atalla stretched parallel to the ber orientation and irradiated with
[19,94] has suggested for h-methylcellobioside. In the 0j light polarized parallel to the stretching direction. Negative
polarization experiments (Fig. 10A), transition dipole cross-peaks are shown shaded.
170 Salmen et al.
lose molecule, they should both be found as well-recogniz-

able bands in the dynamic spectra. As the bond length of
the O2VH. . .O6 intramolecular hydrogen bond [104,105] is
longer than that of the O3H. . .O5V hydrogen bond, the
associated band will appear at a higher frequency, namely,
between 3410 and 3460 cm1 [6,107]. Only small bands
were found in this range in the 2-D spectrum, emphasizing
that the O2VH. . .O6 intramolecular hydrogen bond obvi-
ously plays a minor role in the load distribution [56].
In general, it is clear that more signals appear in the
90j polarization mode. For well-ordered samples of spruce
cellulose, the band at 3267 cm1 is the most intense in this
polarization direction. There is also a shoulder in the in-
phase spectrum at 3232 cm1. These two bands are close to
the characteristic cellulose I h and cellulose Ia bands
assigned to 3270 and 3240 cm1, respectively [17]. Both
of them are in the suggested wave number area for the
O6H. . .O3 intermolecular hydrogen bond between 3230
and 3310 cm1 [6,11,17,95]. This assignment might be Figure 13 Synchronous 2-D FT-IR spectrum of oriented
questioned as the O. . .O distance is slightly greater than cellulose sheets. IR light polarized perpendicular to the
that of O3H. . .O5V intramolecular bond, and therefore the stretching and to ber orientation.
band is expected to occur at higher frequencies [94], but
according to Gardner and Blackwell [104], as well as by
Okamura [105], the distance is quite close to the O3H. . .O5V Fig. 12. Several cross-peaks can be found on the plot. Two
bond length. In the 90j polarization mode, the response in cross-peaks near the diagonal establish a correlation
the dynamic spectrum is preferentially from orthogonal square at 3332 cm1 giving two distinguishable bands at
vibrations in relation to the stretching and the main 3332 and 3314 cm1. Another cross-peak is found at 3362
direction of the cellulose chains. Hence the intermolecular cm1. Only nonsynchronized responses appear in the
hydrogen bonds, connecting adjacent molecular chains, asynchronous spectrum. There is thus no cross-peak be-
are likely to provide the main signal in the 90j polarization tween bands at 3372 and 3329 cm1, again underlining a
mode. The allomorph-characteristic bands of cellulose I high coupling of these two. Specic assignments of the
can also be seen in the 0j in-phase spectrum, but they are dierent new bands cannot be given at this stage.
not the main bands in this case. Figs. 13 and 14 show the 2-D plots for the experiments
The asynchronous spectrum of the orientated spruce carried out on the oriented sheets in the 90j polarization
cellulose samples irradiated with IR light polarized parallel mode. In the synchronous spectrum (Fig. 13), again,
to the stretching and to the ber orientation is shown in autopeaks appear (3267, 3332, 3372, and 3440 cm1)
Figure 12 Asynchronous 2-D FT-IR spectrum of oriented Figure 14 Asynchronous 2-D FT-IR spectrum of oriented
cellulose sheets, IR light polarized parallel to stretching and cellulose sheets. IR light polarized perpendicular to the
to ber orientation. stretching and to the ber orientation.
coupled to one another via positive cross-peaks. The

reorientation directions of the dipole transition moments
are obviously the same for these bands.
Interestingly, in the asynchronous spectrum (Fig. 14),
a cross-peak is found between the bands of 3267 and 3232
cm1, which is between the bands assigned to cellulose Ih
and Ia, respectively, clearly distinguishing these two signals.
D. Crystal Structure
Native cellulose is a composite of two dierent crystalline
forms, cellulose Ia and cellulose Ih [108]. These two allo-
morphs are suggested to dier in their secondary structures
which is in the conformation around the glucosidic linkage
and the C5C6 bond, the hydrogen bonding patterns, or the
molecular packing [54]. Although dierences in IR spectra
of dierent native celluloses were revealed almost 50 years
Figure 16 Dynamic FT-IR intensity of 3240 cm1 plotted
ago [109], the highly overlapping bands in the IR spectra of against relative cellulose Ia content in cellulose mixtures of
biopolymers have meant that the possibilities of studying cotton linters and a Cladophora cellulose.
such structural dierences using conventional IR spectros-
copy have been reduced. The 2-D IR technique is, however,
sensitive to the secondary structure since dierences in the
molecular environment for a transition dipole result in pulping processes not only dissolve the lignin from the
dierent responses when the polymers are dynamically wood structure, but also aect the hemicellulose and
strained during the experiment. This opens up new possi- cellulose structures. Such dierences in cellulose structure
bilities for IR spectroscopy because 2-D IR spectroscopy is between dierently pulped bers have been demonstrated
not only sensitive to chemical composition, but also to by NMR spectroscopy [110113]. FT-IR spectroscopy has
structural conformations such as the secondary structure also been used to determine the allomorphic composition
of the polymers. of cellulose I from dierent origins. Sassi et al. [114] used
During pulping of wood, the bers are exposed to high the deconvoluted bands of the OH stretching vibrations,
temperatures under alkaline or acidic conditions. The whereas Imai and Sugiyama [49] determined the ratio of the
Figure 15 Comparison of dynamic and static FT-IR spectra of one cellulose mixture for two areas where characteristic peaks of
cellulose Ia and cellulose Ih are to be found. Thick line=static spectrum and thin line=dynamic spectrum.
172 Salmen et al.
The bands characteristic of cellulose Ia and cellulose Ih

in the OH region are reported to be found at 3240 and 3270
cm1, respectively [17]. These bands are marked in the
static spectrum of Fig. 14. It can be seen that in the dynamic
in-phase spectrum, the peak maxima dier from these. This
is a result of the change in the energy of the vibration as a
result of the straining of the polymer (the so-called split/
bipolar dynamic peaks [78]). The peak-to-valley intensity
of the characteristic cellulose Ia peak at about 3240 cm1
may be used for quantitative purposes as illustrated in Fig.
16 for mixtures of pure celluloses, cotton linters, and a
Cladophora cellulose. The correlation was, however, linear
only for the mixed samples (1842% cellulose Ia). For
chemical pulps, the resolution in this region compared to
the pure celluloses is too low for quantitative evaluation.
In the low wave number region, the characteristic Ia
peak (750 cm1) is also slightly shifted in the dynamic
Figure 17 Dynamic FT-IR intensity of 756 cm1 plotted spectrum (Fig. 15, right). The static peak characteristic of
against relative cellulose Ia content of cellulose mixtures of cellulose Ih at 710 cm1 could also be found in the dynamic
cotton linters and a Cladophora cellulose. spectrum, and additionally, the dynamic spectrum con-
tains a third peak at 725 cm1. In this wave number region,
800700 cm1 (OH out-of-plane bending [19]), a correla-
absorption coecients between 750 and 710 cm1. Yama- tion to the allomorphic composition is only linear for the
moto et al. [115] also used the 750 and 710 cm1 peaks, but 710 cm1 peak. The characteristic cellulose Ia peak at 756
made lineshape analyses of the spectra and then correlat- cm1 only shows a linear correlation between peak inten-
ed the ratios to the mass fraction determined by 13C NMR. sity and allomorphic composition for cellulose Ih-rich
All these studies were, however, carried out on highly samples. When cellulose Ia contents are above about
ordered materials such as Cladophora, Valonia, and bacte- 30%, some kind of saturation point is reached (Fig. 17).
rial celluloses, whose FT-IR spectra are higher in resolu- The intensity of the peak at 725 cm1, which only
tion compared to the spectra of wood celluloses. With the appears in the dynamic spectrum and not in the static one,
2-D FT-IR method, characteristic IR peaks of cellulose Ia is independent of cellulose allomorphic composition point-
and cellulose Ih may also be studied in pulp samples ing to its relationship to some feature other than the
[61,116]. The higher resolution of the dynamic compared allomorphic composition or the crystallinity in general
to the static IR spectrum with regard to the characteristic (Fig. 18).
peaks of celluloses Ia and Ih [17] is shown in Fig. 15 for a The peak at about 710 cm1 (characteristic of cellulose
mixture of two dierent native celluloses, a cotton linters Ih) has a linear correlation with the relative cellulose Ih
Ih-rich cellulose and a Cladophora (algae) Ia-rich cellulose. content as seen in Fig. 19. This correlation was used as a
Figure 18 Dynamic FT-IR intensity of 725 cm1 plotted against relative cellulose Ia content of cellulose mixtures of cotton
linters and a Cladophora cellulose.
Figure 19 Dynamic FT-IR intensity of 710 cm1 plotted against relative cellulose Ih content of cellulose mixtures of cotton
linters and a Cladophora cellulose.
basis for estimating cellulose allomorphic composition in cellulose. However, the 710-cm1 peak also diers in
pulps [116]. Moreover, this peak also shifts linearly in wave intensity between dierent chemical pulps (Fig. 20). The
number position from 706 cm1 in the pure cotton linters 710-cm1 peak may therefore be used together with the
sample to 714 cm1 in the pure Cladophora sample. The correlation from the cotton/Cladophora mixtures (Fig. 19)
dierent positions of this band have also been reported for to estimate the relative cellulose Ih content of these chem-
other native celluloses [17]. ical pulps (Table 1).
For wood pulps, the dominating peak in this low-wave As seen from Table 1, the birch kraft pulp has a higher
number region is the 725-cm1 peak, indicating that struc- content of cellulose Ih than the two corresponding soft-
tural dierences exist in relation to cotton and Cladophora wood kraft pulps studied. This is most likely related to the
fact that birch wood is more enriched in the cellulose Ih
form, while softwoods are more enriched in the cellulose
Ia form [117]. The holocellulose and the acid sulte pulp
have the lowest amounts of cellulose Ih, whereas the
dissolving pulp has the highest amount. The higher
amount of cellulose Ih in chemical pulps compared to
holocellulose has also been demonstrated with NMR
measurements [111,112]. The reason for the altered allo-
morphic composition is that the monoclinic cellulose Ih
form is more thermodynamically stable, and therefore
Table 1 Estimations of relative content of cellulose Ih in

pulps
Estimated relative content

Pulp sample of cellulose Ih (%)
Dissolving pulp 79 (F2.0)

Bleached softwood
Kraft 49 (F2.8)
Softwood kraft liner 50 (F5.7)
Birch kraft 54 (F0.9)
Acid sulte pulp 41 (F0.9)
Holocellulose 43 (F0.4)
Figure 20 Dynamic in-phase FT-IR spectra at 0j polar-
ization for dierent chemical pulps. The spectra are plotted Numbers in parentheses are the standard deviations based on three
at dierent osets. measurements.
174 Salmen et al.
conversion from the triclinic cellulose Ia to cellulose Ih

occurs at the high temperature and alkalinity of the kraft
process. The acid sulte pulping process does not appear
to aect the allomorphic conversion in the same way, as
also revealed by NMR [113]. The reason for this could be
that this transformation is aected both by the tempera-
ture as well as the medium, where an alkaline medium is
favorable for the transformation [46]. Another factor
worth considering is the structural hindrance to transfor-
mation that the lignin might provide during pulping. In
acid sulte pulping, the lignin is in a rather unswelled state
and softens subsequently, in contrast to its behavior in the
alkaline kraft process [118]. It is suggested that this
reduces the diusion rates in the ber wall and might also
reduce the chance for cellulose to structurally rearrange.
Figure 22 Static FT-IR spectrum of a spruce holocellulose.
Characteristic vibration bands from the dierent polysac-
IV. TWO-DIMENSIONAL FOURIER charides in wood pulps are marked in the diagram.
TRANSFORM INFRARED SPECTROSCOPY
APPLIED TO PULPS
A. Hemicellulose Interaction 1. Characteristic Vibrations
Two-dimensional IR spectroscopy is particularly useful for In using 2-D IR spectroscopy to study interactions between
studies of interactions between dierent polymers in com- polymers in a composite, it is a prerequisite that absorption
posite materials. Although there have been numerous peaks for the dierent polymers are distinguishable. This
studies on synthetic composites (see Infrared Spectros- was, in fact, thought to be an obstacle in the interpretation
copy for Wood and Cellulose Research of this chapter), of dynamic spectra in such a study of polysaccharide
only a few studies have dealt with interactions within com- interactions in onion cell walls [119]. Since the dierent
posites of biopolymers, such as wet onion cell walls [119], polysaccharides in the wood cell wall are all built up with
composites of Acetobacter cellulose [93], and dierent pulp sugar units, they also have very similar IR spectra. How-
bers [5961]. For a native composite such as the wood cell ever, the glucomannan (or more correctly, O-acetyl-galac-
wall, such studies of these interactions could contribute toglucomannan) has characteristic vibrations due to the
much toward the understanding of the ultrastructural equatorially aligned hydrogen in the mannose unit. These
arrangement of the polymers within this biologically con- vibrations can be found at 810 and 870 cm1. Furthermore,
structed material. it is the vibration characteristics of carboxylic acids (1735,
1600, and 1245 cm1) from the 4-O-methyl-a-D-glucopy-
ranosyl uronic acid side group of xylan [arabino-(4-O-
methylglucurono)xylan in softwoods and O-acetyl-(4-O-
methylglucurono)xylan in hardwoods] that distinguish the
xylan spectrum from the cellulose spectrum. These char-
acteristics have been demonstrated in a multivariate data
analysis on alkali-extracted holocelluloses [59] (Fig. 21).
It is dicult to spectrally distinguish between cellulose
and glucomannan. A high correlation between the cellulose
content in holocelluloses and the bands at 1110, 1315, 1335,
and 1430 cm1 has, though, been demonstrated. These
bands are sensitive to cellulose crystallinity [27,120] and are
therefore more distinct in the spectrum of the more ordered
cellulose compared to glucomannan. In Fig. 22, the char-
acteristic vibration bands of the three main polysacchar-
ides within wood pulps are marked in a static FT-IR
spectrum of a spruce holocellulose sample.
2. Holocellulose
The production of holocellulose by treatment with sodium
Figure 21 Weights from partial least squares (PLS) analyses chlorite under acidic conditions is known to remove the
of extracted holocelluloses. Positive peaks show high con- lignin without causing major changes to the native struc-
tribution from the polymer to the absorption in this area. ture of the wood cell wall. Therefore such a sample can be
Figure 25 Model of the cell wall structure of a latewood

softwood ber (tracheid). The lines in the dierent cell wall
layers represent the organization of the cellulose brils in the
Figure 23 Dynamic in-phase (thin line) and out-of-phase dierent layers. Observe the dominance of the S2 layer with
(thick line) FT-IR spectra of spruce holocellulose sheets the brils aligned almost parallel to the ber axis. (From Ref.
strained in the ber direction. The spectra are recorded at 90j 138.)
polarization.
used as a reference for the polymer interactions in the tions. The glass transition of the dry wood polysaccharides
native wood ber if lignin-free samples are required. rstly occurs at temperatures over 180jC, implying that
In Fig. 23, the two components of the dynamic spec- they are all in their glassy state at room temperature under
trum of a spruce holocellulose are shown [59]. These dry conditions [118]. Under these conditions, it is in the in-
measurements have been performed at room temperature phase dynamic spectrum that the information will be found
in dry air. The weak out-of-phase (viscous) signal points to since the out-of-phase spectrum mainly consists of noise.
a very elastic response of the sample under these condi- Therefore in the following discussion, concerning holocel-
Figure 24 Comparison of dynamic in-phase FT-IR spectra at 0j polarization for a spruce cellulose (dissolving pulp free of
hemicelluloses) (thin line) and a holocellulose (cellulose with 33% hemicelluloses) (thick line). The sheets are strained in the ber
direction.
176 Salmen et al.
Figure 26 Comparison of dynamic in-phase FT-IR spectra at 90j polarization for spruce cellulose (thin line) and holocellulose
(thick line). The sheets are strained in the ber direction.
lulose and chemical pulps, only the in-phase spectra (elastic

response) are shown in the gures.
The dominance of cellulose brils for the load-carry-
ing ability in pulp bers is clearly evident from the com-
parison of the dynamic in-phase IR spectra in 0j
polarization of a spruce cellulose (dissolving pulp with
>98% cellulose) and a spruce holocellulose containing
33% hemicellulose as seen in Fig. 24. These ber sheets
are strained in the ber direction. Although the holocellu-
lose is composed of over one-third hemicellulose, the
dynamic spectrum looks very similar to that of the pure
cellulose. There are no dynamic peaks in the holocellulose
spectrum characteristic of xylan or glucomannan, which,
however, can be found in the static IR spectrum (Fig. 22).
Since 2-D IR spectroscopy only shows changes in intensity
as an eect of external perturbation (straining), only the
polymers involved in the stress transfer will contribute to
the dynamic spectrum.
The structure of the wood cell wall is built up of
dierent layers (Fig. 25). In the dominant S2 layer, which
makes up 7585% of the cell wall, the cellulose is arranged
in brils oriented mainly in the direction of the ber axis
[121]. These cellulose brils, which are embedded in a
matrix of lignin and hemicellulose, act as reinforcement
and therefore take most of the load in the ber direction. It
is therefore not surprising that there are only the dynamic
Figure 27 Comparison of dynamic in-phase FT-IR spectra signals from cellulose in the holocellulose spectrum. The
at 90j polarization for spruce cellulose (thin line) and dierences seen between the two in-phase spectra in Fig. 24
holocellulose (thick line). Characteristic vibrations of gluco- are mainly related to the dierences in cellulose structure
mannan due to the orthogonal directed hydrogen of the originating from changes occurring during pulping as
mannose ring are marked at 810 and 870 cm1 in the gure. discussed in Crystal Structure.
90j and not at 0j polarization is due to the fact that the

characteristic vibrations of glucomannan are oriented per-
pendicular to the backbone of the polymer chain, and the
fact that the backbone of glucomannan is oriented parallel
to the cellulose chains [59].
As described earlier, the synchronous and asynchro-
nous 2-D spectra are useful for studying interactions be-
tween the polymers in a composite. In Fig. 28, an o-
diagonal area (dierent wave numbers on x- and y-axes)
of the synchronous 2-D spectrum from a 90j polarization
measurement of a spruce holocellulose is shown. The area
chosen is where characteristic bands of cellulose and gluco-
mannan appear. The o-diagonal cross-peaks represent
similar time-dependent movements of the molecular
groups. The appearance of cross-peaks between all the
characteristic bands of cellulose and glucomannan reveals
that the two polymers move synchronously as a result of the
Figure 28 An o-diagonal part of the synchronous 2D applied strain. Furthermore, this suggests a strong interac-
spectrum at 90j polarization of the spruce holocellulose. tion between the two polysaccharides inside the wood cell
Characteristic peaks of cellulose and glucomannan are listed wall. Thus a strong coupling between the glucomannan
beside the gure and cross-peaks between the two polymers aligned in parallel with the cellulose chains is anticipated.
are marked with arrows in the gure.
3. Softwood Pulps
If similar sheets of bers of spruce cellulose and spruce When wood bers are used for paper production, they are
holocellulose are studied in the same loading mode, but rst exposed to a pulping process involving high temper-
with 90j polarization instead, the spectra look dierent atures and chemical reagents in order to remove the lignin.
(Fig. 26). In this case, vibrations oriented perpendicular to As mentioned earlier, during this process, other wood
the straining direction are more enhanced. In the 90j polymers, the cellulose and the hemicelluloses, are also
polarization spectra, dierences between the holocellulose aected. Akerholm and Salmen [61] used 2-D FT-IR
and cellulose can be seen between 800 and 900 cm1 spectroscopy to investigate how dierent pulping processes
(enhancement in Fig. 27). As mentioned earlier, this area aected the mechanical interaction between the cellulose
contains absorption peaks characteristic of glucomannan. and the hemicelluloses. In Figs. 29 and 30, dynamic in-
The appearance of dynamic peaks at 810 and 870 cm1 phase spectra at 0j and 90j polarization of three dierent
shows that glucomannan is also taking part in the load chemical softwood pulps are plotted together with the
transfer in the ber direction. The fact that they appear at corresponding spectra of holocellulose.
Figure 29 Dynamic in-phase FT-IR spectra at 0j polarization for holocellulose and three dierent chemical softwood pulps.
The sheets are strained in the ber direction.
178 Salmen et al.
Figure 30 Dynamic in-phase FT-IR spectra at 90j polarization for holocellulose and three dierent chemical softwood pulps.
The sheets are strained in the ber direction.
Evidently, the dynamic FT-IR spectra are surprisingly still unaected by the applied strain in the ber direction.
similar in the ngerprint region for these pulps, and no This is probably also a consequence of the native con-
large dierence could be seen in the spectra between struction of the wood cell wall. The cellulose-reinforced
holocellulose pulp and the dierent chemical pulps of composite structure is not reorganized very much during
softwood. As for the holocellulose, only dynamic peaks the pulping processes.
from cellulose were found in the 0j polarization mode
(Fig. 29). At 90j polarization, with the stretching parallel
to the ber direction (Fig. 30), dynamic peaks appeared 4. Interaction Between Fibers
both for cellulose and glucomannan for all samples. No As mentioned earlier in Orientation Aspects, the strain-
dynamic signals from xylan were observed. This means ing of a sheet of bers perpendicular to the ber direction
that the interactions between glucomannan and cellulose results in loading conditions that are dierent from the
that were established for the wood structure remained loading of the sheet in the ber direction. Because of the
after the dierent pulping processes, while the xylan was bending and shearing of the bers in this loading mode,
Figure 31 Dynamic in-phase FT-IR spectra at 0j polarization of a kraft liner (thick line) and an acid sulte pulp (thin line);
bers stretched perpendicular to ber direction.
tions. In this case, there are also dynamic signals from the
xylan for some of the pulps, showing that xylan is more
important for the interber bonding than for the mechan-
ical properties along the ber axis. In Fig. 31, it can be
seen that for a kraft liner pulp, a pulp containing 24%
hemicellulose, a stronger dynamic response is obtained for
the xylan (the band at 1730 cm1) than for the acid sulte
pulp of comparable chemical composition. This is proba-
bly due to the resorption of xylan on the ber surfaces
during kraft pulping in contrast to what occurs in sulte
pulping [122].
5. Hardwood Pulps
Hardwoods dier chemically from softwoods mainly in
that they usually have a higher content of xylan in relation
to the glucomannan. Other types of hemicellulose might
also be present and the hardwood xylan contains acetyl
groups but not arabinose substituents as softwood xylan.
For a birch pulp, the content of glucomannan is very small,
while much more xylan is present compared to softwood
Figure 32 Dynamic in-phase FT-IR spectra of a birch kraft pulp bers. Despite the large dierence in chemical com-
pulp (thin line) compared to a bleached softwood kraft pulp position, the dynamic spectra of birch bers [61] are very
(thick line). The bers are strained in the ber direction and similar to those of softwood bers (see Fig. 32). The main
the upper spectra are recorded at 0j polarization, whereas response in the dynamic spectra originates from the cellu-
the lower spectra are recorded at 90j polarization. lose. No signals originating from xylan (the principle
hemicellulose in birch) could be found in this case when
the sheets were strained in the ber direction. Again, this
the straining of the structure reects more the interactions demonstrates the importance of the cellulose brils in
in the transverse direction of the bers and the interactions determining the longitudinal ber properties.
in the bonds between bers. The dynamic spectra of sheets
of pulp bers are also dierent in these loading circum-
stances. When comparing spectra from 0j and 90j polar- B. Lignin Interaction
ization modes, the orientational eect, which occurs for
bers loaded in the longitudinal direction, is not seen. In The mechanical properties of the cell wall lignin are
the rst direction, there are dynamic signals from both especially important in determining the properties of me-
cellulose and glucomannan in both the polarization direc- chanical pulp bers. Even if almost all the lignin is removed
Figure 33 Static FT-IR spectrum for a mechanical pulp (thin line) plotted together with the corresponding spectrum for
holocellulose (thick line).
180 Salmen et al.
from the chemically pulped and bleached bers, a consid- ber direction contain dierent lignin-characteristic peaks
eration of the viscoelastic properties of the cell wall lignin is (see Fig. 35). In the 90j polarization experiments, the
still important for understanding the chemical pulping spectra mainly show changes perpendicular to the strain-
processes [118]. ing direction. When most materials are strained, there will
The vibrational spectrum of lignin diers from the also be a dimensional change perpendicular to the strain-
spectrum of the wood polysaccharides in several wave ing direction (the Poison eect). For a ber-reinforced
number regions due to its aromatic structure [123,124]. composite, these transverse changes appear mainly in the
This is evident from Fig. 33, which compares the static matrix material. As seen in this polarization direction, the
FT-IR spectrum of a holocellulose sheet with that of a TMP spectra are considerably dierent from the dynamic
lignin-rich thermomechanical pulp (TMP) sheet. Lignin, spectra of lignin-free pulps (compare Figs. 23 and 35). For
however, also has absorption bands in the areas where TMP, there is a large contribution from the viscous
characteristic peaks of xylan and glucomannan are found. component (out of phase) of the dynamic spectrum with
Applying 2-D FT-IR spectroscopy [60] to a lignin- a number of peaks that refer to vibrations in the lignin
containing pulp (Fig. 34) shows that in the 0j polarization macromolecule [60].
mode, the spectra are very similar to those for lignin-free As for the polysaccharides, the lignin is well below its
pulps (compare Fig. 34 with the spectra in Fig. 29). From glass transition temperature under the measurement con-
the lignin characteristic vibrations, only a very weak ditions and ought to be elastic in nature [118]. Secondary
negative signal at 1504 cm1 is seen in the dynamic in- transitions in the lignin also exist [125], which ought to give
phase spectrum. This is not surprising though if the wood an increased damping of the material. Such secondary
ber is compared with a ber-reinforced composite, here transitions could be the reason for the high phase angles
with cellulose as the reinforcement. In the ber direction, of the lignin vibrations. Mechanical spectra of spruce pulp
the cellulose brils are the load-bearing elements. Since the bers have actually been shown to have a slightly higher
dynamic spectra only show changes in spectral intensity, damping in bers containing more than 10% lignin com-
and the cellulose is the strained polymer, the dynamic pared to lignin-free bers [126]. In lignin, one secondary
spectra of the 0j polarization mode for all cellulose-rein- transition is attributed to the rotation of the methoxyl
forced materials ought to be very similar. Irrespective of group [125], and most of the peaks marked in the out-of-
whether the matrix material consists only of hemicelluloses phase spectrum in Fig. 35 are connected to the methoxyl
or a mixture of hemicelluloses and lignin, it might be group as dierent CH or CO vibrations. Why the aromatic
expected that there would only be signals originating from vibration at about 1510 cm1 is aected and not the
the cellulose brils when the matrix material is strained in aromatic vibration at 1600 cm1 still has to be explained.
the ber direction. The results above show that the lignin is contributing to the
When detecting the signals at 90j polarization, the viscoelastic properties of the ber as a matrix material
dynamic FT-IR spectra of a TMP sheet strained in the capable of moving independently of the cellulose brils.
Figure 34 Dynamic FT-IR spectra of lignin-rich mechanical pulp (TMP) ber sheets recorded at 0j polarization. The thin line
represents the elastic part (in-phase), while the thick line represents the viscous part (out-of-phase) of the dynamic spectrum. The
TMP sheets were strained in the ber direction.
Figure 35 Dynamic FT-IR spectra of lignin-rich mechanical pulp (TMP) ber sheets recorded at 90j polarization. The thin line
represents the elastic part (in-phase), while the thick line represents the viscous part (out-of-phase) of the dynamic spectrum. The
TMP sheets were strained in the ber direction.
As there is a time-delayed response in the sinusoidally wood ber wall [60]. The results show that the phenyl
strained sheets of TMP bers, the full advantage of the 2-D propane units are preferably oriented in the direction of
FT-IR technique can be explored. Compared to mechan- the ber axis. This ordered structure could be the reason for
ical spectroscopy, dynamic 2-D FT-IR spectroscopy is the dierent dynamic behavior in the 0j and 90j polariza-
much more sensitive in detecting specic time-delayed tion measurements of the TMP.
responses. With 2-D FT-IR spectroscopy, the time delay
of each separate molecular vibration in the polymer mix-
ture can be displayed as shown in Fig. 35. The dierent time C. Moisture Effects
delays of the dierent molecular vibrations enhance the
All of the wood polymers are hygroscopic and the absorbed
resolution of the IR spectra (compare Figs. 33 and 35).
moisture has a profound inuence on the properties of
Another advantage is the use of 2-D IR correlation spectra
wood bers which are seen as dimensional changes, swell-
(Eqs. 7 and 8 of this chapter), which spread the spectral
result over three dimensions which therefore facilitates
interpretation. In Fig. 36, the synchronous 2-D IR corre-
lation spectrum of a TMP ber sheet (90j polarization) is
shown for the 15501300 cm1 region. As described earlier,
on the o-diagonal, there are cross-peaks between all
synchronized vibrations. For instance, there are cross-
peaks between the aromatic vibration at 1508 cm1 and
the lignin-characteristic vibrations at 1430, 1373, 1339, and
1315 cm1. There are also cross-peaks between cellulose
bands showing an elastic response such as the ones at 1462
and 1319 cm1.
In Fig. 37, the corresponding asynchronous 2-D IR
correlation spectrum is shown. This spectrum shows high
correlation intensity (cross-peaks) for vibrations with dif-
ferent time-dependent behavior and consequently there are
no peaks on the diagonal. In this case, there are, for
instance, cross-peaks between the aromatic 1508-cm1
band and bands with an elastic response such as the
cellulose bands at 1462, 1377, and 1319 cm1.
It has recently been shown by polarized FT-IR mea- Figure 36 Synchronous 2-D FT-IR spectrum of a mechan-
surements that lignin has an ordered structure within the ical pulp ber sheet measured in the 90j polarization direction.
182 Salmen et al.
the viscous component can be seen for the spectra mea-

sured under humid conditions, especially in the low-wave
number region. This indicates that the polymers are closer
to a transition point and more time-dependent responses
are shown. The spectra illustrated were recorded at 90j
polarization and are more indicative of the response of the
matrix material; this increased viscoelasticity must there-
fore be attributed to the hemicelluloses within the ber
material. The dynamic spectra at 0j polarization which
reect the cellulose straining (Hemicellulose interac-
tion) show no increase in the viscous component for
the holocellulose measured at 90%RH and 40jC. In the
90j polarization mode, the high response of the out-of-
phase spectrum can mainly be seen for the characteristic
peaks of glucomannan at 810 and 870 cm1, in the area
between 1040 and 1080 cm1, and also, to some extent, at
some of the higher wave numbers. In the 1040- and 1080-
Figure 37 Asynchronous 2-D FT-IR spectrum of a mechan-
cm1 area, characteristic peaks of galactose, side groups
ical pulp ber sheet measured in the 90j polarization direction.
of glucomannan, have been reported [119,132]. There are,
however, several strong cellulose bands in this region also,
consisting of highly coupled motions dominated by CC
ing, and eects on its mechanical properties. As water acts and CO stretching with small contribution from HCC,
as a plasticizer, there is a drastic lowering of the softening HCO, and skeletal atom bending [98]. From the PLS
temperature for the wood polymers under humid condi- analysis of alkali extracted holocelluloses (Fig. 21) de-
tions [125]. This eect is of particular relevance when scribed earlier in this chapter, it can be seen that both
selecting a temperature for the mechanical pulping process xylan and glucomannan contribute strongly to the ab-
or by introducing moisture and/or heat during calendering. sorption in this area. Although a softening of the xylan
Eects of moisture on the elastic modulus have been and the glucomannan is probable under the high humidity
determined both for paper [127] and for extracted hemi- conditions used, there are no viscous signals from the
celluloses [128] and lignin [129]. Close to their softening
points, polymers clearly exhibit a viscoelastic behavior
which is why mechanical spectroscopy provides a good
tool for characterizing the inuence of dierent compo-
nents on the mechanical properties [125]. This also applies
for wood bers [130]. Since 2-D FT-IR spectroscopy
provides the elastic and viscous component of each molec-
ular vibration, this method provides a means of revealing
the behavior of the components, and even the molecular
groups, in their contribution to the softening of a compos-
ite material.
In general, IR studies under moist conditions can be
problematic because of the many broad absorption bands
associated with water and the changes in these as moisture
levels around the sample are changed. To overcome this,
the use of a mechanism to precisely control the environ-
ment around the sample is necessary. By placing the
polymer stretcher in a heated moisture chamber (Manning
Applied Technologies Inc., Troy, ID, U.S.A.) connected
to an accurate humidity generator, it is possible to per-
form 2-D FT-IR experiments under humid conditions. As
far as wood polymers are concerned, the use of temper-
atures up to 40jC and high humidities in measurements
would result in viscous contributions mainly from the
hemicelluloses, which ought to be softened in this region Figure 38 Dynamic FT-IR spectra of spruce holocellulose
[128,131]. Fig. 38 shows the dynamic 2-D FT-IR spectra recorded at 90j polarization. The thin lines represent in-phase
of a spruce holocellulose measured at room temperature spectra and the thick lines represent out-of-phase spectra. The
and 0% relative humidity (RH) compared to similar upper spectra are recorded at 0%RH and room temperature,
measurements at 40jC and 90%RH. A small increase of whereas the lower spectra are recorded at 90%RH and 40jC.
characteristic xylan vibrations at 1600 and 1730 cm1. increase in phase angle from 15j to 37j going from 0%RH,
These signals originate from side groups of the polymer 25jC to 90%RH, 40jC. The corresponding values for the
chain and may not be aected to the same extent as the CTMP spectra in Fig. 39 are from 28j to 35j. The CTMP
main chain when the bers are subjected to loading. obviously has a higher viscous response even in the dry
The region between 1150 and 1270 cm1 contains both state as was discussed earlier in this section in which
skeletal stretching vibrations as well as methine bending measurements on TMP were discussed. There is, however,
and is sensitive to the orientation of the glycosidic linkage a lower increase in viscosity as a result of the changed
[98]. This region is also aected by changes in the environ- environment for the lignin-rich pulp compared to the
ment, but here it is mainly the in-phase spectrum that holocellulose. There is also a considerable dierence in
shows the changes. The peak at 1169 cm1 in the in-phase the plasticizing eect that water has on the lignin compo-
spectrum recorded under dry conditions is split into one nent as compared to the hemicelluloses. Because of its
peak at 1150 cm1 and one peak at 1184 cm1 in the same cross-linked structure, the glass transition temperature
spectrum recorded under humid conditions. The induced (Tg) of lignin can only be reduced to about 75jC through
softening of the hemicelluloses might result in movements absorption of moisture [133], while the Tg of the hemi-
of polymer segments and thereby result in spectral changes celluloses may be reduced to room temperature [118].
in this area. In the in-phase spectrum of the CTMP recorded under
In Fig. 39, the dynamic spectra for the dierent moist conditions (Fig. 39), a broad signal with its maxi-
environments, 0%RH and 90%RH, are also compared mum at 1630 cm1 can be seen, which is absent from the
for a chemithermomechanical pulp (CTMP). The 0%RH spectrum measured under dry conditions. This signal
spectrum indicates a greater degree of viscoelasticity than probably originates from a deformation vibration of mo-
the corresponding spectrum of the holocellulose. The lecularly adsorbed water [134]. It has been shown that the
degree of viscoelasticity may be quantied from the phase moisture content of paper increases under mechanical load
angle of the spectra, where an elastic material has a phase under humid conditions, both by measurements using an
angle of 0j and a viscous material has a phase angle of 90j. analytical balance [135] and by NIR measurements [136].
From the dynamic IR spectra, a mean phase angle can be The appearance of this peak in the in-phase spectrum also
calculated for a selected wave number range using the shows the instantaneous change of adsorbed water as a
absolute values of the in-phase and out-of-phase spectra. result of the small dynamic strain applied. The fact that this
For the holocellulose spectra shown in Fig. 38, there is an peak is not as apparent in the spectrum of holocellulose
under humid conditions (Fig. 38) could be a consequence
of the greater number of charged groups in the CTMP
capable of holding a higher volume of water. This eect has
also been shown for a birch pulp with a large number of
charged groups [137].
V. FUTURE DEVELOPMENTS
From the studies described, it is clear that 2-D FT-IR
represents a powerful tool for increasing our knowledge of
cellulose structure and the dynamic behavior of isolated
cellulose, as well as of the wood polymers in bers. One of
the key points is that it provides a possibility for studying
the interactions within biopolymers. 2-D FT-IR is a tool
for elucidating interactions in biological assembles at the
molecular level. There is still a lack of systematic funda-
mental work on, for instance, dierent cellulose poly-
morphs as well as dierent types of hemicelluloses that
could provide a more exact assignment of dierent dynam-
ic IR bands. One of the main problems associated with
transmittance 2-D FT-IR is the prerequisite for thin sheets
in the equipment. A development toward a reectance
application could hence be of great benet, although
mostly surface-type behavior would be observed.
Figure 39 Dynamic FT-IR spectra of a spruce chemither-
momechanical pulp recorded at 90j polarization. The thin
lines represent in-phase spectra and the thick lines represent REFERENCES
out-of-phase spectra. The upper spectra are recorded at
0%RH and room temperature, whereas the lower spectra are 1. Jones, E.J. The infrared spectrum of spruce native lignin.
recorded at 90%RH and 40jC. J. Am. Chem. Soc. 1948, 70, 1984.
184 Salmen et al.
2. Lange, P.W. Ultraviolettabsorption av fast lignin. Sven. 23. Marchessault, R.H.; Liang, C.Y. Infrared spectra of
Papp.tidn. 1945, 48, 241. crystalline polysaccharides. III. Mercerized cellulose. J.
3. Freudenberg, K.; Siebert, W.; Heimberger, W.; Kraft, R. Polym. Sci. 1960, 18, 71.
Ultrarotspektren von Lignin und ligninahnlichen Stoen. 24. Liang, C.Y.; Marchessault, R.H. Infrared spectra of
Ber. Dtsche. Chem. Ges. 1950, 83, 533. crystalline polysaccharides. II. Native celluloses in the
4. Tschalmer, H.; Kratzl, K.; Leutner, R.; Steininger, A.; region from 640 to 1700 cm1. J. Polym. Sci. 1959, 39, 269.
Kisser, J. Die Ultrarotspektren mikroskopischer Holzsch- 25. Liang, C.Y.; Basset, K.H.; McGinnes, E.A.; Marche-
nitte und einiger Modellsubstanzen. Mikrosk./Zent.bl. ssault, R.H. Infrared spectra of crystalline polysacchar-
Mikrosk. Forsch. Method. 1953, 8, 238. ides; VII. Thin wood sections. Tappi 1960, 43, 1017.
5. Tsuboi, M. Infrared spectrum and crystal structure of 26. Smith, J.K.; Kitchen, W.J.; Mutton, D.B. Structural study
cellulose. J. Polym. Sci. 1957, 25, 159. of cellulosic bers. J. Polym. Sci. C 1963, 2, 499.
6. Liang, C.Y.; Marchessault, R.H. Infrared spectra of 27. Nelson, M.L.; OConnor, R.T. Relation of certain infra-
crystalline polysaccharides. I. Hydrogen bonds in native red bands to cellulose crystallinity and crystal lattice type.
celluloses. J. Polym. Sci. 1959, 37, 385. Part II. A new infrared ratio for estimation of crystallinity
7. Marchessault, R.H. Application of infra-red spectroscopy in cellulose I and II. J. Appl. Polym. Sci. 1964, 8, 1325.
to cellulose and wood polysaccharides. Pure Appl. Chem. 28. Ranby, B. Kristallinitat, Accessibilitat und Wassersto-
1962, 5, 107. brucken-Bindungen in Cellulose und Holz. das Papier.
8. Sandermann, W.; Augustin, H. Chemische Untersuchun- 1964, 18, 593.
gen uber die thermische Zersetzung von Holz-dritte 29. Jayme, G.; Rohmann, E.M. Uber die Anwendung der IR-
Mitteilung: Chemische Untersuchung des Zersetzungsa- Spektroskopie bei Zellsto-und Papieruntersuchungen.
blaufes. Holz Roh-Werkst. 1964, 22, 377. das Papier. 1965, 19, 719.
9. Sarkanen, K.V.; Chang, H.-M.; Ericsson, B. Species 30. Dechant, J., Danz, R., Kimmer, W., Schmolke, R., Eds.;
variations in lignins. I. Infrared spectra of guaiacyl and Ultrarotspektroskopische Untersuchungen an Polymeren.
syringyl models. Tappi 1967, 50, 572. Akademie-Verlag: Berlin, 1972.
10. Hergert, H.L. Infrared spectra. In Lignins. Occurrence, 31. Sarko, A. What is the crystalline structure of cellulose?
Formation, Structure and Reactions; Sarkanen, K.V. Tappi 1978, 61, 59.
Ludwig, C.H., Eds.; New York: John Wiley & Sons, 1971. 32. Schultz, T.P.; McGinnis, G.D.; Bertran, M.S. Estimation
11. Siesler, H.; Krassig, H.; Grass, F.; Kratzl, K.; Derkosch, of cellulose crystallinity using Fourier transform-infrared
J. Strukturuntersuchungen an Cellulosefasern verschiede- spectroscopy and dynamic thermogravimetry. J. Wood
nenen Verstreckungsgrades mittels IR-Reexionsspek- Chem. Technol. 1985, 5, 543.
troskopie und Deuteriumaustausch. Angew. Makromol. 33. Yamamoto, H.; Horii, F.; Odani, H. Structural changes of
Chem. 1975, 42, 139. native cellulose crystals induced by annealing in aqueous
12. Pecina, H. Zur Aussagefahigkeit von Infrarot-Spektrog- alkaline and acidic solutions at high temperature. Macro-
rammen uber chemische Strukturveranderungen des molecules 1989, 22, 4130.
Holzes mit dem Beispiel thermischer Behandlung. Holz- 34. Michell, A.J. Second-derivative FT-IR spectra of native
technologie 1982, 23, 78. celluloses. Carbohydr. Res. 1990, 197, 53.
13. Schultz, T.P.; Glasser, W.G. Quantitative structural 35. Fengel, D. Characterization of cellulose by deconvoluting
analysis of lignin by diuse reectance Fourier transform the OH valency range in FTIR spectra. Holzforschung
infrared spectrometry. Holzforschung 1986, 40, 37. 1992, 46, 283.
14. Bourgois, J.; Guyonnet, R. Characterization and analysis 36. Fengel, D. The application of FTIR spectroscopy in
of torried wood. Wood Sci. Technol. 1988, 22, 143. cellulose research. In Cellulosics: Chemical, Biochemical
15. Michell, A.J. FTIR spectra of cellulosesUse of second and Material Aspects; Kennedy, J.F., Phillips, G.O.,
derivative mode. In Cellulosics: Chemical, Biochemical and Williams, P.A., Eds.; Ellis Horwood: New York, 1993.
Material Aspects; Kennedy, J.F., Phillips, G.O., Williams, 37. Kauppinen, J.K.; Moatt, D.J.; Mantsch, H.H.; Camer-
P.A., Eds.; Ellis Horwood: New York, 1993. on, D.G. Fourier self-deconvolution: A method for
16. Fengel, D.; Ludwig, M. Moglichkeiten und Grenzen der resolving intrinsically overlapped bands. Appl. Spectrosc.
FTIR-Spektroskopie bei der Charakterisierung von Cel- 1981, 35, 271.
lulose. das Papier. 1991, 45, 45. 38. Gellerstedt, G.; Josefsson, T. The use of FT-spectroscopy
17. Sugiyama, J.; Persson, J.; Chanzy, H. Combined infrared and chemometrics for analysis of wood components. Proc.
and electron diraction study of the polymorphism of 3rd European Workshop on Lignocellulosics and Pulp:
native cellulose. Macromolecules 1991, 24, 2461. Advances in Totally Chlorine Free Bleaching Chemistry
18. Faix, O. Fourier transform infrared spectroscopy. In Structure and Reactivity of Wood Components; Stock-
Methods in Lignin Chemistry; Lin, S.Y., Dence, C.W., Eds.; holm: STFI, 1994.
Springer-Verlag: New York, 1992. 39. Wegener, G.; Strobel, C. Bestimmung der phenolischen
19. Atalla, R.H. Celluloses. In Carbohydrates and their Deriv- Hydroxylgruppen in Ligninen und Ligninfraktionen
atives Including Tannins, Cellulose, and Related Lignin; durch Aminolyse und FTIR-Spektroskopie. Holz Roh-
Pinto, B.M., Eds.; Elsevier: Oxford, 1999. Werkst. 1992, 50, 417.
20. Akerholm, M. Dynamic FT-IR spectroscopy applied to 40. Griths, P.R.; Haseth, J.A.D. Fourier Transform Infrared
studies on wood polymers, Licentiate thesis, Department Spectrometry; Elving, P.J., Koltho, J.D.W.I.M., Series
of Pulp and Paper Chemistry and Technology, Stock- eds.; John Wiley & Sons: New York, 1986.
holm, Royal Institute of Technology 2001. 41. Michell, A.J. Infra-red spectroscopy transformedNew
21. Mann, J.; Marrinan, H.J. The reaction between cellulose applications in wood and pulping chemistry. Appita 1988,
and heavy water. Part 3. Quantitative study by infra-red 41, 375.
spectroscopy. J. Chem. Soc., Faraday Trans. I 1956, 52, 42. Hulleman, S.H.D.; Hazendonk, J.M.V.; Dam, J.E.G.V.
492. Determination of crystallinity in native cellulose from
22. Mann, J.; Marrinan, H.J. Crystalline modications of higher plants with diuse reectance Fourier transform
cellulose. Part II. A study with plane-polarized infrared infrared spectroscopy. Carbohydr. Res. 1994, 261, 163.
radiation. J. Polym. Sci. 1958, 32, 357. 43. Bouchard, J.; Douek, M. Structural and concentration
eects on the diuse reectance FTIR spectra of cellulose, stretched polypropylene lms. J. Polym. Sci., Polym. Lett.
lignin and pulp. J. Wood Chem. Technol. 1993, 13, 481. 1982, 20, 445.
44. Faix, O.; Bottcher, J.H. Anwendung von FTIR-und 65. Bretzla, R.S.; Wool, R.P. Frequency shifting and
Mikro-FTIR-spektroskopischen Methoden fur quantita- asymmetry in infrared bands of stressed polymers.
tive Analysen in der Holzchemie., AIF-Projekt 7979 Macromolecules 1983, 16, 1907.
Mikro-FTIR. Bundesforschungsanstalt fur Forst- und 66. Lasch, J.E.; Burchell, D.J.; Masoaka, T.; Hsu, S.L.
Holzwirtschaft, Hamburg, 1993. Deformation studies of polymers by time-resolved Fourier
45. Ludwig, M.; Fengel, D. Micro-FTIR studies on cellulose transform infrared spectroscopy. III: A new approach.
nitrate bres of dierent degrees of substitution. Acta Appl. Spectrosc. 1984, 38, 351.
Polym. 1992, 43, 261. 67. Noda, I.; Dowrey, A.E.; Marcott, C. Dynamic infrared
46. Debzi, E.M.; Chanzy, H.; Sugiyama, J.; Tekely, P.; linear dichroism of polymer lms under oscillatory
Excoer, G. The Ia!I h transformation of highly deformation. J. Polym. Sci., Polym. Lett. Ed. 1983, 21, 99.
crystalline cellulose by annealing in various mediums. 68. Noda, I.; Dowrey, A.E.; Marcott, C. Characterization of
Macromolecules 1991, 24, 6816. polymers using polarizationmodulation infrared techni-
47. Wada, M.; Sugijama, J.; Okano, T. Native celluloses on ques: Dynamic infrared linear dichroism (DIRLD)
the basis of two crystalline phase (Ia/Ih) system. J. Appl. spectroscopy. In Characterization of Polymers; Ishida,
Polym. Sci. 1993, 49, 1491. H., Ed.; Plenum Press: New York, 1987; 3359.
48. Nishiyama, Y.; Isogai, A.; Okano, T.; Muller, M.; 69. Palmer, R.A.; Manning, C.J.; Chao, J.L.; Noda, I.;
Chanzy, H. Intercrystalline deuteration of native cellu- Dowrey, A.E.; Marcott, C. Application of step-scan inter-
lose. Macromolecules 1999, 32, 2078. ferometry to two-dimensional Fourier transform infrared
49. Imai, T.; Sugiyama, J. Nanodomains of Ia and Ih cellulose (2D FT-IR) correlation spectroscopy. Appl. Spectrosc.
in algal microbrils. Macromolecules 1998, 31, 6275. 1991, 45, 12.
50. Stewart, D. Fourier transform infrared microspectroscopy 70. Gregoriou, V.G.; Noda, I.; Dowrey, A.E.; Marcott, C.;
of plant tissues. Appl. Spectrosc. 1996, 50, 357. Chao, J.L. Dynamic rheo-optical characterization of a low-
51. Wada, M.; Okano, T.; Sugijama, J. Allomorphs of native density polyethylene/perdeuterated high-density polyethy-
cellulose I evaluated by two equatorial d-spacing. J. Wood lene blend by two dimensional step-scan FTIR spectro-
Sci. 2001, 47, 124. scopy. J. Polym. Sci., Part B, Polym. Phys. 1993, 31, 1769.
52. Kataoka, Y.; Kondo, T. FT-IR microscopic analysis of 71. Noda, I.; Dowrey, A.E.; Marcott, C. A spectrometer for
changing cellulose crystalline structure during wood cell measuring time-resolved infrared linear dichroism induced
wall formation. Macromolecules 1998, 31, 760. by a small-amplitude oscillatory strain. Appl. Spectrosc.
53. Evans, R.; Newman, R.H.; Roick, U.C.; Suckling, I.D.; 1988, 42, 203.
Wallis, A.F.A. Changes in cellulose crystallinity during 72. Noda, I.; Dowrey, A.E.; Marcott, C. Two-dimensional
kraft pulping. Comparison of infrared, x-ray diraction infrared (2D IR) spectroscopy. A new tool for interpreting
and solid state NMR results. Holzforschung 1995, 49, 498. infrared spectra. Mikrochim. Acta (Wien) 1988, 1, 101.
54. Horii, F. Structure of cellulose: Recent developments in 73. Noda, I. Two-dimensional infrared spectroscopy. J. Am.
its characterization. In Wood and Cellulosic Chemistry; Chem. Soc. 1989, 111, 8116.
Hon, D.N.-S., Shiraishi, N., Eds.; New York: Marcel 74. Noda, I. Two-dimensional infrared (2D IR) spectroscopy:
Dekker, 2001; 83107. Theory and applications. Appl. Spectrosc. 1990, 44, 550.
55. Kondo, T. Hydrogen bonds in cellulose and cellulose 75. Siesler, H.W. The characterization of polymer deforma-
derivatives. In Polysaccharides. Structural Diversity and tion by rheo-optical Fourier transform infrared spectro-
Functional Versatility; Dumitriu, S., Ed.; Marcel Dekker, scopy. Makromol. Chem., Macromol. Symp. 1992, 53, 89.
Inc: New York, 1998; 131172. 76. Singhal, A.; Fina, L.J. Dynamic two-dimensional infra-
56. Hinterstoisser, B.; Akerholm, M.; Salmen, L. Eect of red spectroscopy of the crystal-amorphous interphase
ber orientation in dynamic FTIR study on native region in low density polyethylene. Polymer 1996, 37,
cellulose. Carbohydr. Res. 2001, 334, 27. 2335.
57. Hinterstoisser, B.; Salmen, L. Two-dimensional step-scan 77. Budevska, B.O.; Manning, C.J.; Grith, P.R. Compar-
FTIR: A tool to unravel the OH-valency-range of the ison of two-dimensional power and phase spectra
spectrum of cellulose I. Cellulose 1999, 6, 1. generated from sample modulation step scan FT-IR
58. Hinterstoisser, B.; Salmen, L. Application of dynamic 2-D experiments. Appl. Spectrosc. 1994, 48, 1556.
FTIR to cellulose. Vibr. Spectrosc. 2000, 22, 111. 78. Budevska, B.O.; Manning, C.J.; Griths, P.R.; Roginski,
59. Akerholm, M.; Salmen, L. Interactions between wood R.T. Step-scan Fourier transform infrared study on the
polymers studied by dynamic FT-IR spectroscopy. eect of dynamic strain on isotactic polypropylene. Appl.
Polymer 2001, 42, 963. Spectrosc. 1993, 47, 1843.
60. Akerholm, M.; Salmen, L. The oriented structure of lignin 79. Gregoriou, V.G.; Palmer, R.A. Dynamic FT-IR spectro-
and its viscoelastic properties studied by static and dynamic scopy of polymer lms and liquid crystals. Proc. 11th
FT-IR spectroscopy. Holzforschung 2003, 57, 459. European Symposium on Polymer Spectroscopy (ESOPS-
61. Akerholm, M.; Salmen, L. Dynamic FTIR spectroscopy 11), Macromolecular Symposia (MSYMEC), Polymer
for carbohydrate analysis of wood pulps. J. Pulp Pap Sci. Spectroscopy. Hutig & Wepf Verlag: Zug, 1995; 75.
2002, 28, 245. 80. Scherzer, T. Rheo-optical FTIR spectroscopy of amine
62. Chalmers, J.M.; Everall, N.J. The role of vibrational cured epoxy resins. J. Molec. Struct. 1995, 348, 465.
spectroscopymicroscopy techniques in polymer charac- 81. Scherzer, T. FTi.r.-rheo-optical characterization of the
terization. Macromol. Symp. 1995, 94, 33. molecular orientation behaviour of amine cured epoxy
63. Burchell, D.J.; Hsu, S.L. Deformation studies of polymers resins during cyclic deformation. Polymer. 1996, 37, 5807.
by time-resolved Fourier transform IR spectroscopy. 82. Turner, P.H. Identication of a polymer lm laminate by
In Polymer Characterization: Spectroscopic, Chromato- FT-Raman microscopy. Bruker-Rep. 1994, 140, 36.
graphic, and Physical Instrumental Methods; Carver, C.D., 83. Hayes, C.; Mendes, E.; Bokobza, L.; Boue, F.; Monnerie,
Ed.; ACS: Washington, 1983; 533540. L. Analysis of orientational relaxation in binary blends of
64. Fately, W.G.; Koenig, J.L. Time-resolved spectroscopy of long and short polystyrene chains by Fourier transform
186 Salmen et al.
infrared dichroism and small-angle neutron scattering. 107. Sugiyama, J.; Vuong, R.; Chanzy, H. Electron diraction
Macromol. Symp. 1995, 94, 227. study on the two crystalline phases occurring in native
84. Eklind, H.; Maurer, F.H.J. The eect of interfacil cellulose from algal cell wall. Macromolecules 1991, 24,
interactions on the rheo-optical behaviour of compatibi- 4168.
lized polystyrene/low-density polyethylene/styreneethyl- 108. Atalla, R.H.; VanderHart, D.L. Native cellulose: A
enebutylenestyrene copolymer blends. Polymer. 1996, composite of two distinct crystalline forms. Science 1984,
37, 4465. 223, 283.
85. Singhal, A.; Fina, L.J. Dynamic two-dimensional infrared 109. Marrinan, H.J.; Mann, J. Infrared spectra of the crystal-
spectroscopy. Part I: Melt-crystallized nylon 11. Appl. line modications of cellulose. J. Polym. Sci. 1956, XXI,
Spectrosc. 1995, 49, 1073. 301.
86. Huang, W.X.; Wunder, S.L. A dynamic FT-IR method 110. Lennholm, H.; Wallbacks, L.; Iversen, T. A 13C-CP/
for determining the Cuinr temperature ranges of an MAS-NMR-spectroscopic study of the eect of labora-
acetylene-terminated polyisoimide prepolymer. J. Appl. tory kraft cooking on cellulose structure. Nord. Pulp Pap.
Polym. Sci. 1996, 59, 511. Res. J. 1995, 1, 46.
87. Sun, H.; Frei, H. Time-resolved step-scan Fourier trans- 111. Maunu, S.; Liitia, T.; Kauliomaki, S.; Hortling, B.;
form infrared spectroscopy of triplet excited duroquinone Sundquist, J. 13C CPMAS NMR investigation of cellulose
in a zeolite. J. Phys. Chem. B 1997, 101, 205. polymorphs in dierent pulps. Cellulose 2000, 7, 147.
88. Woody, D.L. Two dimensional infrared resolution of shear 112. Hult, E.-L.; Larsson, P.T.; Iversen, T. A comparative CP/
bands formed during low velocity impacts of NaCl crystals MAS 13C NMR study of cellulose structure in spruce
using fast infrared detectors. J. Appl. Phys. 1992, 72, 783. wood and kraft pulp. Cellulose 2000, 7, 35.
89. Noda, I.; Dowrey, A.E.; Marcott, C. Two-dimensional 113. Hult, E.-L.; Larsson, P.T.; Iversen, T. A comparative CP/
infrared (2D IR) spectroscopy. In Modern Polymer Spectro- MAS 13C-NMR study of the supermolecular structure of
scopy; Zerbi, G., Ed.; Wiley-VCH: Weinheim, 1999; 132. polysaccharides in sulphite and kraft pulps. Holzfor-
90. Meier, R.J. 2D infrared spectroscopy. Spectrosc. Eur. schung 2002, 56, 179.
1993, 5, 28. 114. Sassi, J.-F.; Tekely, P.; Chanzy, H. Relative susceptibility
91. Sonoyama, M.; Miyazawa, M.; Katagigi, G.; Ishida, H. of the Ia and Ib phases of cellulose towards acetylation.
Dynamic FT-IR spectroscopic studies of silk broin lms. Cellulose 2000, 7, 119.
Appl. Spectrosc. 1997, 51, 545. 115. Yamamoto, H.; Horii, F.; Hirai, A. In situ crystallization
92. Kacurakova, M.; Eberingerova, A.; Hirsch, J.; Hromad- of bacterial cellulose II. Inuences of dierent polymeric
kova, Z. Infrared study of arabinoxylans. J. Sci. Food additives on the formation of cellulose Ia and Ib at the
Agric. 1994, 66, 423. early stage of incubation. Cellulose 1996, 3, 229.
93. Kacurakova, M.; Smith, A.C.; Gidley, M.J.; Wilson, R.H. 116. Akerholm, M.; Hinterstoisser, B.; Salmen, L. Character-
Molecular interactions in bacterial cellulose composites ization of the crystalline structure of cellulose using static
studied by 1D FT-IR and dynamic 2D FT-IR spectro- and dynamic FT-IR spectroscopy. Carbohydr. Res. 2004,
scopy. Carbohydr. Res. 2002, 337, 1145. 339, 569.
94. Atalla, R.H. Personal communication (2001). 117. Newman, R.H. Crystalline forms of cellulose in soft-
95. Fengel, D. Inuence of water on the OH valency range in woods and hardwoods. J. Wood Chem. Technol. 1994,
deconvoluted FTIR spectra of cellulose. Holzforschung 14, 451.
1993, 47, 103. 118. Back, E.L.; Salmen, N.L. Glass transitions of wood
96. Ivanova, N.V.; Korolenko, E.A.; Korolik, E.V.; Zban- components hold implications for molding and pulping
kov, R.G. Mathematical processing of IR-spectra of processes. Tappi 1982, 65, 107.
cellulose. Z. Prikl. Spektrosk. 1989, 51, 301. 119. Wilson, R.H.; Smith, A.C.; Kacurakova, M.; Saunders,
97. Tashiro, K.; Kobayashi, M. Theoretical evaluation of three- P.K.; Wellner, N.; Waldron, W. The mechanical properties
dimensional elastic constants of native and regenerated and molecular dynamics of plant cell wall polysaccharides
celluloses: Role of hydrogen bonds. Polymer 1991, 32, 1516. studied by Fourier-transform infrared spectroscopy. Plant
98. Wiley, J.H.; Atalla, R.H. Band assignment in the Raman Physiol. 2000, 124, 397.
spectra of celluloses. Carbohydr. Res. 1987, 160, 113. 120. Nelson, M.L.; OConnor, R.T. Relation of certain infra-
99. Jeery, G.A., Saenger, W., Eds.; Hydrogen Bonding in red bands to cellulose crystallinity and crystal lattice type.
Biological Structures; Springer Verlag: Berlin, 1994. Part I. Spectra of lattice types I, II, III and of amorphous
100. Huggins, M.L. Hydrogen bonding in organic compounds. cellulose. J. Appl. Polym. Sci. 1964, 8, 1311.
J. Org. Chem. 1936, 1, 407. 121. Thomas, R.J. Wood: formation and morphology. In
101. Pauling, L., Ed. The Nature of Chemical Bond; Cornell Wood Structure and Composition; Lewin, M. Goldstein,
Univ. Press: Ithaca, NY, 1939. I.S., Eds.; Marcel Dekker: New York, 1991; 747.
102. Moore, W.J., Hummel, D.O., Eds. Physikalische Chemie; 122. Yllner, S.; Enstrom, B. Studies of the adsorption of xylan
Walter deGruyter: Berlin, 1974. on cellulose bres during the sulphate cook, part 1. Sven.
103. Fengel, D. Structural changes of cellulose and their eects Papp.tidn. 1956, 59, 229.
of the OH/CH2 valency vibration range in FTIR spectra. 123. Faix, O. Classication of lignins from dierent botanical
In Cellulose and Cellulose Derivatives: Physico-chemical origins by FTIR spectroscopy. Holzforschung 1991, 45,
Aspects and Industrial Applications; Kennedy, J.F., Phil- 21.
lips, G.O., Williams, P.A., Piculell, L., Eds.; Woodhead 124. Agarwal, U.P.; Ralph, S.A. FT-Raman spectroscopy of
Publ. Ltd.: Cambridge, 1995; 7584. wood: Identifying contributions of lignin and carbohy-
104. Gardner, K.H.; Blackwell, J. The structure of native drate polymers in the spectrum of black spruce (Picea
cellulose. Biopolymers 1974, 13, 1975. mariana). Appl. Spectrosc. 1997, 51, 1648.
105. Okamura, K. Structure of cellulose. In Wood and Cel- 125. Salmen, L.; Hagen, R. Viscoelastic properties. In Hand-
lulosic Chemistry; Hon, D.N.-S., Shiraishi, N., Eds.; book of physical testing of paper; Mark, R.E. Habeger,
Marcel Dekker: New York, 1991; 89112. C.C., Borch, J., Lyne, M.B., Eds; Marcel Dekker: New
106. OSullivan, A.C. Cellulose: The structure slowly unravels. York, 2001; 77113.
Cellulose 1997, 4, 173. 126. Kolseth, P.; Ehrnrooth, E.M.L. Mechanical softening of
single wood pulp bers. In Paper: Structure and Proper- and Polymer Technology, Stockholm, Royal Institute of
ties; Bristow, J.A., Kolseth, P., Eds.; Marcel Dekker: New Technology, 2002.
York, 1986; 2750. 133. Salmen, L. Viscoelastic properties of in situ lignin under
127. Salmen, N.L.; Back, E.L. Moisture-dependent thermal water-saturated conditions. J. Mater. Sci. 1984, 19, 3090.
softening of paper, evaluated by its elastic modulus. Tappi 134. Kalutskaya, E.P.; Gusev, S.S. An infrared spectroscopic
J. 1980, 63, 117. investigation of the hydration of cellulose. Polym. Sci.
128. Cousins, W.J. Youngs modulus of hemicellulose as USSR 1981, 22, 550.
related to moisture content. Wood Sci. Technol. 1978, 135. Kubat, J.; Nyborg, L. Inuence of mechanical stress on
12, 161. the sorption equilibrium of paper. Sven. Papp.tidn. 1962,
129. Cousins, W.J. Elastic modulus of lignin as related to 65, 698.
moisture content. Wood Sci. Technol. 1976, 10, 9. 136. Olsson, A.-M.; Salmen, L. Molecular mechanisms in-
130. Salmen, L.; Olsson, A.-M. Interaction between hemi- volved in creep phenomena of paper. J. Appl. Polym. Sci.
celluloses, lignin and cellulose: Structureproperty rela- 2001, 79, 1590.
tionships. J. Pulp Pap Sci. 1998, 24, 99. 137. Akerholm, M.; Salmen, L. Softening of wood polymers
131. Olsson, A.-M.; Salmen, L. Humidity and temperature induced by moisture studied by dynamic FT-IR spectro-
aecting hemicellulose softening in wood. In Wood Water scopy. J. Appl. Polym. Sci. 2004. Submitted for publication.
Relations; Homeyer, P., Ed.; Tekst og Tryck: Copenha- 138. Brandstrom, J. Morphology of Norway spruce tracheids
gen, 1997; 269280. with emphasis on cell wall organisation, Ph.D. thesis, De-
132. Bjarnestad, S. Characterization of the carbohydrate compartment of Wood Science, Uppsala, Swedish University
position of pulp bers, Licentiate, Department of Fibre of Agricultural Sciences, 2002.
7
Light Scattering from Polysaccharides
Walther Burchard
Institute of Macromolecular Chemistry, University of Freiburg, Germany
I. INTRODUCTION The impressing success in unraveling the protein

structures results from the fact that single crystallites of
Carbohydrate polymers or polysaccharides establish the suciently large size could be grown. This permitted a
main biomass of annually renewable sources, far above the detailed structure analysis in three-dimensional space via x-
two other groups of biopolymers, nucleic acids and pro- ray diraction techniques. Further information, for in-
teins. In view of this fact the research on these products stance on complexes of enzymes, could be gained also by
appears strongly focused on application in food industry, directly viewing the structures by electron microscopy and,
agriculture, and papermaking, but are otherwise much more recently, by atomic force microscopy. None of these
neglected compared to the two other types of biological techniques can be applied to macromolecules in solution
macromolecules. Two main reasons can be made responsi- where the particles are in continuous Brownian motion. In
ble. The often highly irregular primary structures made addition, segmental mobility with respect to the particle
these samples inadequate for control of biological process- center of mass has to be taken into account. Only for very
es, and this renders little interest to traditional biochemists large particles can this Brownian motion be directly ob-
and biophysicists. Furthermore, the overwhelming number served in light microscopes, preferably when the particles
of hydroxyl groups per chain with their capability of have been tagged with a uorescing chromophore.
hydrogen binding and the seemingly chaotic forms of The application of scattering techniques partially
branching causes a complexity in behavior that had an leads out of this dilemma. Roughly speaking, the size
appalling eect on common polymer scientists. Indeed, of the chosen wavelength operates here as a ruler for
experience gained from synthetic polymers often seems measuring particle sizes. Often, the dimensions of poly-
not applicable to polysaccharides. One striking example is saccharides lie in the range of visible light, and this
represented by the highly branched amylopectin in starch allows us to extract information not only on the mean
which is semicrystalline, whereas crystallization of synthet- average radius of an equivalent sphere but also on the
ic polymers is strongly prevented by the presence of shape of the macromolecules and on some details of the
branches. Because of this discrepancy in behavior, tradi- internal structure and segmental motions. Nonetheless,
tional polymer scientists kept away from the study of Brownian motion cannot be circumvented. Therefore
polysaccharides. much information is lost when the scattering signals are
The apparently contradicting properties toward the collected between fairly large time intervals, because then
well-established rules in polymer science are in fact based only average overall orientations and all internal uctua-
on supramolecular structures formed during the process of tions are recorded. Great progress was achieved when in
biosynthesis. These structures are kinetically controlled the late 1960s instrumental requirements were developed
and will, in most cases, not represent the thermodynamic for the observation of scattered light in very short time
equilibrium structure. Once this supramolecular structure intervals [15]. This development now resulted in the
is broken up, a more disordered conformation will occur possibility of carrying out two types of light scattering,
and a return to the original ordered biological structure will the static (SLS) and dynamic light scattering (DLS) [6,7];
not be feasible. New aggregation structures may result, often the latter is also called photon correlation spectros-
possibly some with a certain order but still of unknown copy or quasi-elastic light scattering and the former
functional properties for application in nonfood industries. sometimes integrated light scattering. The possibilities
189
190 Burchard
of these two techniques are shortly summarized as fol- with q the magnitude of the scattering vector which
lows [810]. is related to the scattering angle h, the refractive
In SLS the scattering volume is large compared to index n0 of the solution, and the wavelength k0 of
the dimensions of the dissolved particles and the the light in vacuum.
time of recording the scattering signal is chosen
suciently long (about 12 sec). Under these q 4pn0 =k0 sinh=2 3
conditions a reliable time average over all internal
relation and diusive processes is obtained which Both light scattering techniques are well-established
gives smooth signals by averaging over many methods in the eld of synthetic polymers and in colloid
particles in space. Despite the average overall science, but are less applied to polysaccharides, probably
particle orientations, the data give information on because of the complexity in behavior of these materials.
the molar mass, the radius of gyration, and the To tackle this complexity a comprehensive knowledge of
interparticle interaction. In ideal cases also the normal linear chain behavior in dilute solution is required,
shape of the particles and their internal structure which can also be observed with some polysaccharides if a
can be estimated. special treatment in preparing the solution is carefully
In DLS the scattering volume is made small applied. Sometimes strongly polydisperse samples have
(<102 mm3), and the time interval of intensity to be separated into a number of fractions of lower
recording is made variable, ranging over 911 polydispersity before a consistent interpretation can be
decimals from 107 to 104 sec. Short intervals of made. Preparative fractionation is cumbersome and often
107 sec have become applicable with modern not satisfying. However, analytical results from fractions
photomultipliers. Because of concentration uctu- can be obtained from size exclusion chromatography
ations, as a result of Brownian motion, strong (SEC) if combined with a multiangle light scattering and
intensity uctuations in time of the scattered light a viscosity detector. In fact, this now well-developed
are observed. These uctuations are not fully technique represents a very ecient third method of light
random but are correlated. This correlation scattering. In the near future it will probably become a
becomes evident when a time correlation function main equipment for analytic characterization. All three
is built up from these scattering intensities. To this techniques will be discussed. The combination of all three
end the scattering intensities are collected in n techniques is needed for a comprehensive analysis of
channels and the scattering intensity of the rst complex materials.
channel is multiplied with that from the jth Two main architecture types are observed with poly-
channel, where j is running over all n1 pairs. saccharides which deviate in behavior from linear exible
This process is repeated about 105 times until a chains. These are a pronounced chain stiness of linear
good average over these products is obtained. chains and long-chain branching. Both phenotypes can
Expressed by an equation the nonnormalized time be analyzed in dilute solutions [1113]. Problems arise
correlation function G2(t) is obtained as given by when rigidity exists in the main chain or in the attached
branches. Considerable problems are encountered when
semidilute solutions are studied, because in such cases
X
m
strong repulsive interparticle interactions modify the
G2 t u < it 0it > Ai Aij
1 scattering behavior at nite concentration [14]. These
i1
contributions from interactions have to be separated
6
mc10 ; j 1; 2; : : : n before drawing conclusions. The scattering behavior
becomes even more complex when marked association
in which Ai and Ai+j denote the registered photons commences. The onset of such association is easily rec-
in the channels i and i+j, m is the number of ognized, but the question as to what structure is formed
repetitions and n the number of channels. These still remains to be answered.
channels correspond to increasing time intervals, This review is organized in four sections. In Section 2
covering the mentioned time range of up to 104 sec. basic relationships of light scattering are outlined for the
The angular brackets hi denote the time average, mentioned techniques of SLS, DLS, and SEC in combi-
which is approximated by the sum over m repeti- nation with multiangle light scattering (MALS). Special
tions. Fig. 1a shows with a simulated example the features will be demonstrated, already at this point, with
correlated uctuations of photons for 500 time examples from selected polysaccharides dissolved in the
intervals and in the right side of Fig. 1b the regime of dilute solutions. The next rather extended
resulting correlation function. In simple cases this section (Section 3) deals with the behavior of the many
function decays exponentially with a decay con- types of polysaccharides in the dilute solution regime.
stant that contains the translational diusion co- The review nishes in Section 4 with a summary and
ecient D general conclusion. For reasons of space and time the
intriguing behavior of semidilute and concentrated sol-
2 utions, with the striking association phenomena, is not
G2 t A1 e2Dq 2 included.
Light Scattering from Polysaccharides 191
Figure 1 (a) Computer simulation of scattering intensity uctuations, from which the time correlation function is constructed as
G2(t)=<AiAi+n>, where Ai denotes the number of photons within a certain time interval and n denotes the channel number. (b)
The derived intensity time correlation function (left) and the eld time correlation function g1(t)=[ G2(t)/G2(l)1]1/2 in a
logarithmic plot against the channel number which determines the delay t=Dtn (right). Dt is the time interval of collecting
photons in a channel.
II. BASIC RELATIONSHIPS light of the same wavelength as the primary beam light,
where each atom, hit by the light, becomes an emitter of
A. Static Light Scattering. Some General Remarks scattered light (Huygens principle). The basic quantitative
theory of light scattering goes back to Lord Rayleigh
Light scattering arises from the excitations of the electrons who applied the Maxwell theory of light. Later in his con-
in the outermost shells of atoms by a monochromatic siderations on Brownian motion, Einstein discovered that,
primary beam, which causes a periodic vibration of the in addition to these regular vibrations of the polarizabil-
polarizability. This vibration in turn causes an emission of ity, a thermal uctuation has to be superimposed. Other-
192 Burchard
wise, all scattered light will cancel each other, and only data complex. Therefore Debye [16,17] suggested to use
the transmitted and the reected light survive. With this the reciprocal scattering intensity which nally leads to the
modication of Lord Rayleighs theory the scattering Debye equation
intensity can be expressed by an equation that contains
three important contributions which are (1) the mean Kc 1
2A2 c 3A3 c2 : : : 9
square density uctuations <Dq2>; (2) the mean square Rh Mw Ph
concentration uctuations <Dc2>; and (3) an angular
dependent function P(h) [15]. Thus the total scattering
intensity is given by B. The Particle Scattering Factor P(q)
1. Origin of the Angular Dependence
Rtotal
h K V < Dq2 > K < Dc2 > Ph 4
The basic Eqs. 6 and 7 show that the scattering intensity
where Rh is the so-called Rayleigh ratio which is the depends on two physically very dierent factors. The rst
normalized scattering intensity one, (Bp/Bc)RT1, is a thermodynamic function which
gives information on how strongly the individual macro-
ih 2 molecules or colloid particles repel each other. The other
Rh r 5
I0 factor, P(h), is a function that describes the size of the
i(h) and I0 are the scattering and primary beam light particle, in ideal case also the shape of the particle and to
intensities, respectively, and r is the distance of a detector some extent the internal structure. This factor is denomi-
from the scattering volume. The angular brackets in Eq. 4 nated as particle scattering factor. The angular dependence
denote the thermodynamic equilibrium average, per- of the scattering intensity was already observed in 1869 by
formed with the aid of Gibbs free energy. K V and K are Tyndall and is often called the Mie-eect [18]. Mie repre-
optical contrast factors to be discussed later. Experience sented a general light scattering theory [19] that includes
with macromolecules and colloids in solution revealed that the Rayleigh theory as a special limiting case. The origin of
even at very low concentrations the eect of concentration this angular dependence is easily understood on the basis of
uctuation exceeds the density uctuations by orders in the following Fig. 2.
magnitude. The latter almost completely arise from the According to Huygens principle each atom, hit by
density uctuations of the solvent. Thus the rst term in light, will become the origin of a scattered light wave.
Eq. 4 represents the scattering from the solvent and may Macromolecules consist of repeating units that are cova-
be subtracted. The mean square concentration uctua- lently bound to each other. The scattering from the atoms
tions can be expressed in terms of the chemical potential which establish the repeating unit may be contracted such
Dl1 or equivalently by the osmotic pressure p which - that each repeating unit now represents a scattering ele-
nally leads Eq. 4 to [16] ment in the sense of Huygens principle. Fig. 2 shows what is
to be expected of the scattering from a macromolecule that
Rh uRtotal
h Rsolvent
h KcRTBc=BpPh 6 schematically is represented by a branched structure. Let us
consider the two scattering units o and j, then we notice that
The contrast factor K strongly depends on the refractive the path of the light that hits the element j is longer than
index increment Bn/Bc and is for vertically polarized that arising from element o. Therefore there will be a phase
primary beam light given as dierence between the two paths which depends on the
distance roj and the magnitude of the scattering angle h, i.e.,
4p2 2 Bn 2 the angle between the two unit vectors s0 and s in the
K n 7
k40
0
Bc direction of the primary beam and the scattered light.
The refractive index increment Bn/Bc has to be measured

separately with a special dierential refractometer, and n0
is the refractive index of the solvent.
Eq (6) contains the osmotic compressibility, and
therefore a strong scattering arises if a small change in
the osmotic pressure causes large uctuations in the con-
centration, i.e., large deviations from the average concen-
tration. This occurs, for instance, near a critical point of
phase separation [17]. In most application of light scatter-
ing the systems are far away from such critical behavior. In
these cases the osmotic pressure can be expanded in a
power series which gives
Figure 2 Scattering of light from a branched particle with
p c dimensions larger than k0/20. s0 and s are unit vectors in the
A 2 c2 A 3 c3 : : : 8 direction of the primary beam and the scattered ray
RT M
originating from the scattering elements 0 and j. The value
If this is inserted in Eq. 6, a power series is obtained in the of the scattering vector is q=(2p/k)js0 sj=(4p/k)sin(h/2).
denominator, which makes interpretation of scattering Note, qrj has no dimension.
Performing the calculation on the basis of these two vectors this total scattering intensity is normalized at q=0 to unity
one obtains for the phase dierence which nally gives
uol qrol 10 n
Rh 1 X n X
sinqrlk
Phu 2 13
with Rh0 n l1 k1 qrlk
q 4pn0 =k0 sinh=2 11
The angular brackets hi indicate that for segmental
The phase dierence causes a destruction of the scat- motion the distance between the two scattering elements
tering intensity by interference that becomes signicant are not xed but can undergo uctuations. In this case the
when qrol is larger than 0.2. The scattering intensity from average value of this function has to be used.
such a pair is given by the sum of the two individual
centers plus the sum of two interfering light scattering
2. Behavior of the Particle Scattering Factors
passes, i.e.,
of Selected Examples
Rol h 21 expiqrol 12a The evaluation of the double sum in Eq. 13 can become a
serious problem for complex structures and has to be
However, this relationship holds only for a pair that is solved numerically on a computer. In such cases it is always
xed in space, a condition that is not fullled for a particle helpful to look for the limit of small scattering angles (or
in solution. Because of the thermally induced Brownian small q-values). In this limit the sin(qrlk)/(qrlk) can be
motion, even a rigid particle undergoes rotation such that expanded in a Tailor series which gives
in an ensemble of many particles, all orientations are, on " #
average, realized. This average can be easily performed 1 2 1 X n X n
2
with the exponential function in Eq. 12a and leads to Ph 1 q < rlk > : : : 14a
3 2n2 l1 k1

sinqrol
Rol h 2 1 < > or because in the Debye Eq. 9 the reciprocal particle
qrol 12b scattering factor is required, one has

2 1 expqroj =6 " #
1 1 2 1 X n X n
2
where the < > denotes the average over length uctua- 1 q < rlk > : : : 14b
Ph 3 2n2 l1 k1
tions. This average can be calculated if the uctuations
follow a Gaussian distribution and yields exp(qroj/6). where the higher-order terms in q2 are not considered.
Fig. 3 shows the scattering intensity from such a two- This result is remarkable, because the expression in the
center particle which represents a dumbbell. In addition, squared brackets is known as the mean square radius of
the corresponding curves for a short exible chain with gyration <S2>, which in a simplied manner is denoted
three and four beads is shown. as R2g . Thus in a plot of 1/P(h) against q2 the initial slope
If the particle consists of n scattering elements, which is (1/3)R2g , or in other words, without knowing the details
may represent the number of repeat units or degree of of the particle structure the radius of gyration of the par-
polymerization, the total scattering intensity is the sum ticle can be determined. Moreover, the product qRg turned
over all scattering pairs. Fig. 3 also contains the angular out to be a universal scaling function by which particles
dependencies from exible chains with three and four units. of the same architecture but dierent sizes can be univer-
As we are interested here only in the angular dependence, sally described. (Note: qRg has no dimension.)
The radius of gyration is commonly described by the
sum of all distances of the n scattering elements in the
particle from the center of mass. As the center of mass
in general is not positioned on one of the scattering ele-
ments it was useful to express this position in terms of the
scattering elements which results in the abovementioned
double sum, i.e., [20]
X
n n X
X n
R2g 1=n < r2j;CM > 1=2n2 < r2lk > 15
j1 l1 k1
Fig. 4 schematically explains the denition of Rg and the

meaning of the double sum.
In quite a few examples analytical solutions of the dou-
ble sums in Eq. 13 were possible and some of them can be
used as a useful frame for orientation. Characteristic lim-
Figure 3 Particle scattering curves for short exible chains iting functions are those for hard sphere of uniform density
which contain two (dumbbell), three, or four beads (mono- [22], thin rigid rods [22], and in between of these the
meric units), respectively. random coil of exible linear chains [23]. Table 1 gives a
194 Burchard
tween neighbor particles becomes larger and larger, and

in the limit of zero concentration no interaction from
another particle is possible. Thus the particle scattering
factor represents the structure of individual particles.
However, at zero concentration also the scattering inten-
sity becomes zero. To nd this limiting function a reliable
procedure of extrapolation has to be applied.
Mostly applied is a plot that in 1948 was suggested
by Zimm [24]. In such Zimm plot the left side of Eq. 9 is
plotted against q2+kc, where q2=(4pn0/k)2sin2(h/2) is
related to the scattering angle h, c is the concentration in
g/mL, and k is a constant whose value can be deliberately
chosen. It has the eect that the scattering curves from the
Figure 4 (a) Conformational chain parameters of a macro- dierent concentrations will occur well separated from
molecule. CM: center of mass, si and sj are vectors from the each other. The Zimm plot was derived from the particle
center of mass to the monomeric units i and j. R denotes the scattering factor of polydisperse linear and exible chains
end-to-end distance. The radius of gyration is given by Eq. which has the particularly simple form of [24]
(15) where rj rCM=sj. (b). In addition to the geometric
parameters which dene the radius of gyration, the hydro- 1=Pq 1 1=3u2 with u qRg 16
dynamic radius is determined by hydrodynamic interactions.
This interaction impedes particle draining by the solvent. The Fig. 5 shows as an example the Zimm plot from a
draining is decreased with increasing segment density. A core pullulan in water [25]. Pullulan is a linear glucan with a
of the particle remains impenetrable for the solvent when the trisaccharide repeating unit of -[1,4)-aGlc (1,4)-aGlc (1,6)-
particle moves through the liquid. Simply speaking, this core aGlc-]n. Because of the exible a(1,6) linkage the chain
determines the hydrodynamic radius. adopts the conformation of a random coil. The Zimm plot
has two limiting curves which are obtained after linear
extrapolation of the angular dependence from each con-
centration toward q2=0, and the other after extrapolating
list of particle scattering factors for various architectures. the data of each q value at the various concentrations
All are expressed in terms of the universal parameter qRg. toward c=0. In an ideal case of low experimental errors
both curves intersect the ordinate at the same point, which
3. Zimm Plots gives the reciprocal weight average molar mass 1/Mw. The
The particle scattering factor represents the angular de- curve c=0 represents the angular dependence at zero
pendence of the scattering intensity in the limit of zero concentration whose slope is determined by the mean
concentration. At continuous dilution the distance be- square radius of gyration R2g=3 (slope/intercept)c=0. The
Table 1 Particle Scattering Factors From Some Relevant Models
Model Particle scattering factor Comments

a
Coil, polydisperse 1 u=qRg
Pq
1 1=3u2
1
DebyeBueche identical Pq u=qRg
with hyperbranched 1 1=6u2 2
chains with C=0 1 1=3Cu2
Hyperbranched structure Pq u=qRg
C=1, random coil 1 1=61 Cu2 2
C=0, DebyeBueche 1
Rigid rod, innitely Pq arctgu u=qRg
thin polydisperse u
2
3
Hard sphere, monodisperse P0 q cosX XsinX u=qRg=(0.6)0.5X
X3 X=qR=(5/3)0.5u
l
1
Hard sphere, polydisperseb Pq r=R8 P0 qrexpr=R3 dr u=qRg=1.0078X
2 0 X=qR
a
Polydisperse means a most probable molar mass distribution with Mw/Mn=2.
b
Note: the mass fraction w(M) and the molar mass M of individual species are both proportional to the cube of
sphere radius r, i.e., M~r3.
One now realizes that the globular sphere structure

causes an exponential upturn and the thin rod a slight
downturn, whereas the coil of exible linear chains gives
exactly a straight line. Thus we can expect to nd parti-
cle scattering factors from globular and branched struc-
tures in the region between random coil and hard sphere,
whereas semiexible chains may be found in the region
between exible coils and rigid rods. Such behavior is
often fullled with polysaccharides, but this assignment
is still rather uncertain, and a more detailed procedure is
required for a more denite structure formation.
4. Modied Zimm Plots

(Berry and Guinier Modications)
One signicant problem has to be discussed at more detail
which is the accurate determination of molar mass and
radius of gyration. The problem becomes stringent when
the angular dependence displays a pronounced upturn. In
Figure 5 Berry modication of a Zimm plot from a pullulan the attempt to nd a linear initial slope the extrapolation in
in water. (From Ref. [25].) (By permission of ACS.) a Zimm plot often leads to a negative value. This result is
observed in particular for particles of very large molar mass
because then the value of 1/Mw is already very near the
other initial slope of the curve q2=0 gives the second virial origin. Clearly, in this case the Zimm plot is an inappro-
coecient A2=(1/2)(slope)q=0.
Not in all cases does the Zimm plot result in a system
of parallel straight lines. Figs. 6 and 7 give two marked
examples. In Fig. 6 a weak downturn of the angular
dependencies is obtained. The scattering curves resulted
from an exo-polysaccharide (EPS) expired by Rhizobium
trifolii bacteria (strain TA1-EPS) [26]. It is a comblike
macromolecule whose primary structure is shown in Fig.
6 underneath the Zimm plot. It has a regular repeat unit
which consists of four sugars in the backbone and four
sugars as a side chain attached to the C6 position of the
anhydro glucose unit. As will be discussed later this poly-
saccharide has a strong tendency to form a well-dened
supramolecular structure. Contrary to Fig. 6 the Zimm plot
in Fig. 7 exhibits a pronounced upturn of the angular
dependencies from the various concentrations. The scatter-
ing data were recorded from a glycogen of a rat liver [27],
immediately after the death of the animal inhibiting all
activities of enzyme. Fig. 7 shows an electron micrograph
[28] of the structure underneath the Zimm plot. These
particles had a very large molar particle weight of Mw=300
106 g/mol but a comparatively small radius of gyration
Rg=156 nm. The spherical rosette-like structure indicates a
well-dened supramolecular structure via aggregation.
The question is, why do we get in some cases a slight
downturn but in another one a pronounced upturn? To
receive some insight it is useful to compare the particle
scattering factor from geometrically opposite architec-
ture, which are hard spheres of homogeneous density, thin
rigid rods, and, in between, the random coil from linear
exible chains. Mostly, the particles in an ensemble do not
have the same size but obey a most probable distribution
of the molar mass for which the polydispersity index is Figure 6 Rhizobium trifolii, strain TA1-EPS (a) repeating
Mw/Mn=2. The size distribution requires the calculation unit, (b) Zimm plot in 0.1 N NaCl aqueous solution. The
of the average over this distribution [24]. Fig. 8 shows the slight bending of the angular dependence towards the x axis
plots of the reciprocal particle scattering factors of the indicates chain stiness. The polysaccharide forms a double
three mentioned structures. helix. (From Ref. [26].) (By permission of ACS.)
196 Burchard
Even if the Guinier-modied Zimm plot does not give a

linear angular dependence the plot remains advantageous
for an estimation of very large particle weights in the order
of 109 g/mol. Such high molar masses are obtained for
branched samples near the critical point of gelation or for
highly associated polysaccharides. In all other plots no
reliable distinction would be possible between 107 and 1011
g/mol, whereas in the Guinier modication the error is
mostly not larger than 50% which, of course, is still large.
Polynomial ts of the fourth or fth order may be required
and, in addition, also a truncation of the q-range.
C. Dynamic Light Scattering

1. Some Properties of Time Correlation Functions
in Dynamic Light Scattering
Some general remarks on DLS were already made in
Section 1. Fig. 1b showed that the intensity time correlation
function (TCF) decayed to a baseline which is just the
square of the SLS (in the present case not normalized to a
Rayleigh ratio), i.e., G2(t!l)=<i(q)>2. In other words,
there is no longer correlation if the delay time between the
scattering intensities at time t is equal to zero and a very
large time later. On the contrary, for extremely short delay
times between the two intensities the motion is fully
correlated, and the average of the squared scattering
intensity G2(t=0)=<i(q)2> is obtained.
Figure 7 Glycogen from rat liver (a) Zimm plot in water; It is convenient to normalize the measured TCF with
(b) EM photograph, negatively stained. (From Refs. [27,28].) respect to the baseline. In general, this intensity TCF,
g2(t)uG2(t)/A, is dicult to interpret by well-developed
theories. In most cases of common application the intensity
priate method. One can try to use a quadratic t of the
curve, but even better is a plot of (Kc/Rh)1/2 against q2+kc,
a modication of Zimms procedure that was rst sug-
gested by Berry [29]. This technique has two advantages,
rst the intercept is now (1/Mw)1/2 and suciently away
from the origin, and second, a linearization over a fairly
large initial range is obtained. In both cases a signicant
improvement in accuracy is obtained although mostly a
weak bending toward the x axis at large q2 is obtained. In
this case a quadratic t of the curves becomes necessary.
In the Berry plot the radius of gyration is now given as
R2g =6 (slope/intercept)c=0. Sometimes a weak upturn
curvature in the Berry plot still remains. Such behavior is
found mainly with globular colloid particles and this was
already known to Guinier [30,31] in his studies of x-ray
small angle scattering. He found that the particle scat-
tering factor can often be reliably described by a single ex-
ponential approximation P(h)cexp[(1/3)u2] or 1/P(h)=
exp[(1/3)u2]. This approximation suggests the logarithmic
modication of Zimms suggestion, i.e., a plot of ln(Kc/Rh)
against q2+kc. In fact, a good linearization is obtained,
for instance with the Zimm plot of Fig. 7 as is demon- Figure 8 Reciprocal particle scattering factors for hard
strated by Fig. 9. Applying the ln(Kc/Rh) to the Debye spheres, polydisperse random coils of exible chains, and
Eq. 13 one nds in the limit of c=0 polydisperse thin rigid rods (polydispersity index Mw/
Mn=2). In the region between hard sphere and exible
linear chains the behavior indicates globular structures, in the
lnKc=Rh ln1=Mw 1=3u2 17 region between coil and rigid rods sti chains are found.
Figure 9 Guinier-modied Zimm plot of the same data as shown in Figure 7. The linearity in the angular dependence is in
agreement with the spherical shape of the glycogen aggregate as shown by the EM photograph in Figure 7. (From Ref. [27].)
TCF can be expressed by the square of the electric eld Dapp(q)uC1/q2 can be dened which for particle sizes
TCF, Rgq<2 is approximated by [34]
< E*0Et > Dapp q C1 =q2 Dc 1 CRg q2 : : : 22
g1 tu 18
< E*E > where C is a characteristic coecient that is determined by
g2 t 1 bg21 t 19 the slowest internal (segmental) motion with respect to the
center of mass.
which is called the Siegert relationship [7,32] in which bc1 The index c at the diusion coecient indicates a
is a coherence factor that depends on the quality of dependence on concentration. It is obtained after extrap-
instrumental setup. E(t) represents the electric scattering olation of Dapp(q) against q2!0. Dc represents solely
wave at time t, and the denominator is the average static translational mobility and contains no contributions from
scattering intensity, <E*E>=<i>. For simple mono- segmental motions, but it is inuenced by interactions
disperse particles, e.g., hard spheres, or particles and among the particles at a nite concentration. This mutual
macromolecules with small dimensions against the wave- diusion coecient is not the self-diusion coecient. The
length, the eld TCT is easily derived from the Brownian concentration dependence is mostly well described by a
motion or the well-known dierential equation of transla- linear relationship
tional diusion and is given by Dc D0 1 kD c 23
where D0 is now the translational, self-diusion coecient.
g1 t expCt expDq2 t 20 Mostly two separate extrapolations have to be performed,
i.e., (1) an extrapolation toward zero scattering angle and
i.e., the eld TCF decays as a single exponential with a (2) extrapolation of these data to zero concentration.
decay constant C, which is denoted as the rst cumulant. In
these cases the diusion coecient is determined from the 2. Hydrodynamic Radius and Rho-Parameter
ratio of C/q2=D. Applying the StokesEinstein relationship to the self-dif-
However, most samples in practical application are fusion coecient a hydrodynamically eective sphere ra-
polydisperse, and, in addition, some structures have a dius Rh is obtained
certain segmental mobility. Now the TCF no longer decays kT
as a single exponential, but a deviation toward longer delay D0 24
6pg0 Rh
times becomes noticeable (Fig. 1b, right). Still the initial
part at short delay times can be well expressed by a single In general, the hydrodynamic radius Rh diers from
exponential. This suggested an approximation by the so- the radius of gyration Rg as a result of hydrodynamic
called cumulant expansion [33] interactions among the segments in the particle in addition
to the geometrically dened connectivity, which determines
C2 2 C3 3 the radius of gyration. Macromolecules and polymer
ln g1 t C0 C1 t t t microgels are highly swollen, and solvent can penetrate
2! 3! 21
C4 4 : : : when the particle is moved through a viscous medium. This
t penetration is strongly impeded at short distances between
4!
two segments [35]. The streaming lines in a laminar ow
where C1=C. Often, the rst cumulant is not strictly q2 start to overlap. Schematically, the eect is shown in Fig.
dependent. In this case an apparent diusion coecient 4b. If the distance becomes less than a critical value the
198 Burchard
Table 2 List of q=Rg/Rh Parameters for Various Model Table 3 Parameters, which for Large Particles, can be
Architectures Obtained from a Combination of Static and Dynamic Light
Scattering (Static and Dynamic Zimm Plots)
Architecture q
Static light scattering
Homogeneous (hard) spheres 0.778 Intercept Mw
Random coil, linear chains Slope of q2 dependence, c=0 (1/3) R g2/Mw
monodisperse Slope of c dependence, q=0 2A2/Mw
Q-solvent 1.504 Dynamic light scattering
Good solvent 1.78 Intercept D
Random coil, linear chains, Slope of q2 dependence, c=0 DR g2C
polydisperse Slope of c dependence, q=0 kD=2A2Mwkf
Q-solvent 1.73 Combinations
Good solvent 2.05 Hydrodynamic radius Rh=kT/(6pg0D0)
Regular stars, uniform arm length q-parameter q=Rg/Rh
Q-condition f = 4 1.333
Q-condition f H1 1.079 C is dened by the largest internal relaxation time with respect to the
Regular stars, polydisperse arm length center of mass, kD is a constant that describes the concentration
Q-condition f = 4 1.534 dependence of Dc=D0(1+kDc), and kf is the corresponding constant
Q-condition f H1 1.225 of the friction coecient fc=f0(1+kfc).
Dendrimers n>10 shells 0.977
(soft sphere model)
Randomly branched (A3-monomers) 1.732 dependence essentially cancels, and a parameter is obtained
Q-conditions that is indicative for the segment density. The ratio [34,37]
Hyperbranched (AB2monomers), DPH10 1.225
Cyclic chains, Q-solvent 1.253 q Rg =Rh 25
Rigid rings (N>3) ~(1/p) lnN
proved to be a valuable characteristic parameter which
Rigid rods ~[(2/3) lnN]1/2
allowed us to draw conclusions whether a particle is a
N is the number of small beads in a string. loosely coiled linear chain or of a more compact structure.
Table 2 gives a list of theoretically derived q-parameters
for some typical molecular architectures.
solvent becomes trapped. The laminar ow takes the core
as an impenetrable obstacle and surpasses it without 3. Dynamic Zimm Plot
penetration. Simply speaking, the radius of this core can Instead of performing the two extrapolations in separate
be considered as the hydrodynamic radius [36]. With this graphs the data can also be represented by one graph.
picture in mind a particle of high segment density will have Combining Eq. 22 with Eq. 23 one notices a very similar
a larger hydrodynamic radius than that of a low segment two-parameter dependence of the apparent diusion coef-
density but of the same radius of gyration. cient to that of the Debye Eq. 9. This fact suggests a
Both radii, Rg and Rh, depend on the molar mass of the construction similar to that of Zimm for SLS data and
particle. We can expect very similar behavior and therefore which may be called a dynamic Zimm plot [8,38]. With
when the ratio of both radii is formed, the molar mass modern DLS equipment both the SLS and DLS can be
measured simultaneously at the same concentration and
the same scattering angle. Figure 10 shows such an example
that represents the counterpart to Fig. 6. Thus in ideal cases
six characteristic molecular parameters can be obtained
from the combination of SLS and DLS data, which are
collected in Table 3.
One important comment to Fig. 10 is needed. The
dynamic Zimm plot in this gure displays strong deviations
from a linear behavior toward a nonlinear increase in the
angular dependence, as was to be expected from Eq. 22.
These deviations are eected by the spectrum of internal
segment relaxations.
III. DILUTE SOLUTION PROPERTIES

Figure 10 Dynamic Zimm plot from R. trifolii, with the same
OF POLYSACCHARIDES
sample as measuered by static light scattering and shown in A. Grouping Into Various Classes
Figure 6. The intercept on the ordinate gives the translational
diusion coecient. The angular dependence arises from The number of dierent polysaccharides appears illimit-
segmental motions. (From Ref. [26].) (By permission of ACS.) able because of the large number of monosaccharides and
are cellulose and amylose, the linear part of starch. In both

cases the anhydro glucose is the repeat unit, but in cellulose
the units are linked via the h(1,4)- and in amylose via the
a(1,4)-glycosidic bonds. The dierence appears to be small
but the inuence on the polymer conformation is excep-
tionally large. Figure 11 shows sections from these linear
chains. Neglecting for a moment the existence of the three
free hydroxyl groups per anhydro glucose unit (AGU) the
h(1,4)-glycosidic bond favors a stretched conformation
whereas the a(1,4)-glycosidic bond gives preference to a
helical chain. The strictly stereoregular primary structure
and the presence of the many OH-groups give impetus to
very stable and well-organized supramolecular structures
which make these abundant renewable polysaccharides
water insoluble. In both cases crystalline structures are
formed which are stabilized by a regular net of hydrogen
bonds. In native cellulose Ia;h crystalline modications are
observed where the two modications Ia and Ih dier only
Figure 11 Conformational dierence between cellulose and slightly [3943]. In the semicrystalline starch granule am-
amylose. ylose is amorphous [44,45], but on leaching in hot water it
very quickly undergoes a liquidsolid phase transition to a
B-type crystalline modication of double helices [46]. The
the dierent kinds of linkages. The variety of conceivable very dierent solution properties of cellulose and amylose
homopolysaccharides composed of only one sugar type are now discussed separately.
and the same linkage is comprehensible but increases
drastrically when a certain heterogeneity in the type of 1. Cellulose
linkage is present. Characterization of polysaccharides The poor solubility of cellulose in common solvents has
becomes immensely dicult, if heterogeneity in composi- caused many problems in industrial application. Only
tion and branching occurs. In these cases application of
light scattering alone cannot lead to a satisfactory structure
elucidation. Combination with spectroscopy, enzymatic
degradation techniques, and other physicalchemical
methods is imperative. Nonetheless, despite the apparent
limitations with light scattering some general conclusions
can be drawn for each class.
The following treatment starts with the strictly stereo-
regular homopolymers. The next class of increased com-
plexity is presented by microbial polysaccharides which are
composed of repeat units built up of two to eight mono-
saccharides in a well-dened sequence. Finally, examples
from plant and animal polysaccharides are considered
which show the full complexity of heterogeneity. Many
polysaccharides are of industrial importance, but because
of the often limited solubility and a glass transition tem-
perature above decomposition, these samples are trans-
formed into derivatives. In general, the introduction of
substituents cannot be deterministically performed by
common chemical reactions. The reactions are statistical
processes but mostly not random. Here again the full
complexity is obtained. Nonetheless, signicant progress
could be achieved in the last decade. These derivatives are
only mentioned in passing and not discussed in detail.
B. Homopolysaccharides
Homopolysaccharides are the simplest form of polysac- Figure 12 Zimm plot from a linters cellulose dissolved in
charides. In a strict sense they consist of one anhydro sugar Cd-tren. The chemical structure of the cadmium complex and
type as a repeat unit and are connected only via one type of its coordinative binding to the OH-groups in the C2 and C3
linkage. In the vegetable kingdom such homopolysaccha- positions are shown above the plot. (From Ref. [50].) (By
rides are rare, and so far only two types are known. These permission of ACS.)
200 Burchard
possible with the common cellulose membranes.

For cuoxam we used the value of Gralen [55], who
measured dn/dc in a slowly spinning ultracentri-
fuge. For the other solvents we used a linear
dependence of dn/dc on the refractive index of the
solvent, which is mostly observed with other
polymers. This dependence is the same for all
glucans and is well documented [48]. Thus the
required dn/dc was obtained from this dependence
and the measured solvent refractive index. The
degrees of polymerization were checked with
carbanilate derivatives prepared with some se-
lected samples of linters cellulose. The DPs in the
metal complex solvents were signicantly larger
than those from the derivatives and, as a result, the
Figure 13 Molar mass dependence of the radius of gyration complex stability constants were higher. Appa-
Rg (upper curve) and hydrodynamic radius Rh (lower curve) rently, the coordinately bound complexes behave
of native celluloses (lled symbols) in Cd-tren at 20jC. Pulp like partially covalently bound substituents. The
celluloses (open symbols). (From Ref. [50].) (By permission factors are listed in Table 4.
of ACS.) 2. The metal complex solvents are mostly colored
and the scattering intensity required a correction.
This was done by measuring the transmission T at
the wavelength of the used laser for an optical path
aqueous metal complexes were found to dissolve cellulose
that equals the scattering cell diameter. The
to the macromolecular level [47]. Best known are the two
corrected scattering intensity is given as
copper complexes cuoxam and cuen which are deeply blue
Rh,corr.=Rh,measured/T. The reduction of scattering
colored and thus appeared inappropriate for light scatter-
intensity was for cuoxam in a tolerable range but
ing [4850]. Later, Henley [51] applied the cadmium com-
was rather large for Ni-tren which caused a large
plex cadoxen which is colorless, and within a certain range
experimental error. For Cuen the blue color was
of Cd content is capable of completely dissolving cellulose
too deep as to perform reliable measurements.
of low degrees of polymerization, DP<1200. The results
3. Cellulose is often quoted as a sti chain. The
were dicult to reproduce and show signicant inuence
exponent in molar mass dependence of the radii of
on the preparation method. These results will not be
gyration was found to be in the range for a solvent
discussed further.
of medium quality and gave no indication for
On the basis of a discussion with Dr. T. Carstens from
deviations from exible Gaussian coils. However,
Rhodia AG in 1989, Seger and Burchard [48] successfully
the size of the radii was much larger than observed
applied light scattering to the cuoxam solution and proved
with synthetic polymers of the same degree of
molecular solubility. These experiments were slightly im-
polymerization. Only to a certain extent is this
proved by Saalwachter et al. [50] who also used two other
caused by the bond length of the AGU l=0.515
metal complexes, Ni-tren and Cd-tren, which were
nm, which is about twice as large as the
designed by Klufers [53,54] and his research group. The
corresponding bond length l=0.25 of a typical
suggestion of these solvents was developed on the basis of
vinyl polymer.
fundamental investigations on low molar mass model-
4. The large coil dimensions permitted a quantitative
saccharides. The structure of the Cd-tren is shown in
analysis for the stiness. To this end the angular
Fig. 12 together with a Zimm plot from a bacterial cel-
dependence of the scattering intensity was tted
lulose (the Ni-tren complex is obtained by replacing Cd by
Ni). Cd-tren is a colorless solvent but Ni-tren is even more
deeply blue colored than cuoxam. Both new solvents have Table 4 Refractive Index n0, Refractive Index Increment
the advantage in that the side reaction of cuoxam to form dn/dc, Viscosity g0, and Correction Factor f by which the
cross-links is prevented by the inert back of the tren- Measured Molar Mass Has to be Divided to Receive the Same
complex [tren=tris(2-aminoethyl)amine]. The two new Degree of Polymerization as was Found from the Cellulose
solvents were capable of dissolving native cellulose up to Tricarbanilate Derivative
the highest available DP(9400) and even bacterial cellulose
dn/dc f correction
of similar chain length [50].
Solvent n0 (mL/g) g0 (cPa) factor
Figure 12 shows a typical Zimm plot, and Fig. 13 the
molar mass dependencies of Rg and Rh of cellulose in Cd- Cd-tren 1.347 0.233 1.384 1.47 F 0.23
tren. At this point some special diculties have to be Ni-tren 1.373 0.199 2.284 1.35 F 0.21
mentioned. Cuoxam 1.375 0.198 2.090 1.62 F 0,16
1. The refractive index increment could not be The factor f indicates how much of the metal on average is bound to
directly measured as no equilibrium dialysis was the chain.
with the approximation of the Koyama [113]

theory for semiexible chains in which the Kuhn
segment length is the leading parameter. The
results are most instructively demonstrated in a
CasassaHoltzer presentation [5759] in which
(qRh/Kcp) is plotted against qRg. For a random
coil of exible chains the scattering curve passes
through a maximum and decreases proportionally
to 1/q toward zero. Sti chains behave at small qRg
values like a exible coil but at large qRg short-
chain sections are probed, which displays rodlike
behavior. This conformation is described by the
Kuhn segment length lK. The rodlike behavior is
characterized by a constant plateau in the Casas-
saHoltzer plot whose magnitude is the linear
mass density ML=(M/L), where M is the molar
mass and L the contour length of the chain. This
magnitude can be estimated from the mass and
dimensions of a repeating unit which for the AGU
is ML=M0/l=162/0.514=312.6 g/(mol nm). Fig.
14 shows the CasassaHoltzer plots of the scat-
tering intensities from nine native and bacterial
celluloses [50]. The data for the Kuhn segment
lengths of cellulose in the mentioned three solvents
are listed in Table 5. The Kuhn segment lengths are Figure 15 Schematic graph of cellulose micellar aggregate
lK=14.8F1.0 nm, about seven times larger than structure in NMMNO. The dense objects of parallel-aligned
that, for instance, of polystyrene with lK=2 nm. chains correspond to an individual fringed micelle. At small
values of qRgb1 the whole particle is probed in light
This means a Kuhn segment in cellulose has the
scattering; at large qRg>1 smaller lengths and distances
length of about 29 AGUs, whereas for polystyrene
within the object are probed.
it corresponds to a length of 9.8 monomer units.
Thus the rotation around the bond length is not as
much hindered as was anticipated from the
absolute value of the Kuhn segment length.
In previous studies a certain association of chains was
Recently, a number of new solvents for cellulose are
always found which increased with the lithium content
found and applied in cellulose characterization and indus-
[62]. Only recently was it discovered that molecularly dis-
trial processing. Most relevant for characterization is the
persed cellulose could be obtained rst after dissolving the
dimethyl acetamide/lithium chloride (DMA/LiCl) [59,61].
material in DMA/LiCl with 89% Li content followed by a
dilution with pure DMA until a 0.9% Li content was
reached [63,65]. This solvent was successfully applied to
size exclusion chromatography (SEC) in combination with
a refractive increment (RI) and a MALS detector [6365]
More important in industrial application is the N-
methyl morpholine N-oxide monohydrate (NMMNO)
[6669]. Native and pulp cellulose can be dissolved and
processed for ber spinning. Detailed studies by light
scattering by Roeder and Morgenstern [69] revealed a
strong side-by-side alignment which tentatively was inter-
preted by aggregates of fringed micelles which are sche-
matically depicted in Fig. 15. The Zimm plot exhibited an
anomalous angular dependence, which was interpreted by
the presence of large and smaller aggregated cellulose
clusters. A typical Kratky plot is shown in Fig. 16. Partial
dissolution of these clusters became possible in mixtures of
NMMNO with diethylenetriamine which permitted light
scattering measurement also at room temperature [70].
Similar aggregated structures were also found for cellulose
Figure 14 CasassaHoltzer plots from six bacterial (BC) in several salt melts as discovered by Fischer et al. [7173]
celluloses, two linters (Blin), and one microcristalline These clusters may be considered as incompletely dissolved
(AviCel) cellulose. (From Ref. [50].) (By permission of ACS.) cellulose fragments. Evidently, the dissolution power is not
202 Burchard
neutralization with HCl [82]. The material is also soluble in

boiling water and could be kept at room temperature for a
certain time and used for light scattering measurements
[76,81]. The time until onset of aggregation and precipita-
tion strongly depends on the amylose chain length and the
width of the size distribution.
Extensive light scattering and viscosity studies in
various solvents were made by Everett and Foster [82],
Banks and Greenwood [83], Cowie [84], and by Burchard et
al. [76,81], Kadoma et al. [85], Ring et al. [86], and Rollings
[87]. Dissolved in aqueous 0.33 N KCl, Q-solvent behavior
was found by Banks and Greenwood [83], and Burchard
measured the unperturbed dimensions in a mixture of
DMSO/acetone=56.2/43.8 (v/v) [88]. The unperturbed
dimensions were found considerably smaller than those
for cellulose in the metal complexing solvents and gave for
amylose a Kuhn segment length of only lK=1.792.13 nm
[82,88]. The bond length in the a(1,4)-linked amylose is
lamylose=0.44 nm, smaller than lcellulose=0.515 nm, but
even when this fact is taken into account one has only
about 45 anhydro-glucose units per Kuhn segment in
Figure 16 Kratky plots from four dierent celluloses in amylose, whereas the Kuhn segment length of cellulose in
NMMNO in comparison to random coils (upper curve), to the metal complexing solvents contains about 29 units.
DebyeBueche (middle curve), and Guinier approximations Thus amorphous amylose in solution behaves as a exible
(low curve). The DebyeBueche curve corresponds to hyper- coil with an apparent rotational hindrance that is weaker
branched macromolecules, the Guinier to globular struc- than for polystyrene. This conclusion is based on a chain in
tures. Inset: scheme of scattering curve analysis where the which the Kuhn segment length is assumed to behave as a
system was assumed to be composed of two components. straight rigid rod. However, amylose is known for its
(From Ref. [68].) (By permission of Zellcheming/Hulig.) tendency of single-helix formation. Assuming the confor-
mation of a sixfold helix with a pitch height of 1.06 nm we
nd for the bond length of the AGU in the direction of the
helix lpitch=0.177 which increases the number of repeat
strong enough to fully break up the semicrystalline struc-
units per Kuhn segment to 10.712.0. This is 1/3 of the
ture of solid cellulose.
cellulose value. Monte Carlo simulations by Brant [8191]
indicate that even this value is an underestimation. Actu-
2. Amylose ally, left-handed and right-handed, so-called wobbled
Amylose is probably amorphously imbedded in the semi- helices are transitionally formed resulting in a seemingly
crystalline starch granules and can be extracted by a random conformation. Over a certain contour length, for
leaching process of starch in hot (6070jC) aqueous sus- instance, a left-handed helix is formed that at a certain
pension [44,45]. The main problem with amylose arises point can change to a right-handed helix. This change in
from the strong tendency to double-helix formation which handedness causes coiling backward and results in a small
usually proceeds fairly quickly. Once double helices are average dimension, despite the much larger instantaneous
formed, a quick liquid to solid phase transition occurs and Kuhn segment length of helix sections. Brant estimated
ends in a semicrystalline precipitate [44,46]. This precipi- about 40 repeat units per such a helix section. The ndings
tate is almost insoluble in all conventional solvents. Sur- of Brant agree in a way with a model by Szeitli [92] who,
prisingly, amylose is not soluble either in most of the metal several years ago, suggested that amylose is built up of
complexing solvents for cellulose. An exception is the iron broken helical chains. Brant took care of the dynamics in
sodiumtartrate complex which dissolves molecularly cellu- solution and remaining exibility of the helices.
lose and amylose [74]. The existence of fairly sti helical segments has a
The double-helix formation can be prevented by add- signicant eect on the solution properties because it forms
ing n-butanol to the hot leached suspension whereupon the the basis for the astounding eect of retrogradation which
n-butanol becomes included in the channel of a single helix is connected with double-helix formation followed by
[7580]. This complex precipitates on cooling. Dry amor- crystallization. Pfannemuller et al. [93,94] studied this
phous amylose is obtained by replacing the butanol and process as a function of chain length where narrowly
water traces with methanol and ethyl ether in a hot mortar distributed synthetic amylose was used. From turbidity
[76,81]. To prevent double-helix formation this process has and CD measurements she found a marked increase of this
to be quick and must be followed by extensive drying under tendency with the chain length with a sharp maximum at a
vacuum in an exsiccator. This amorphous amylose is DP 80. Beyond this value the retrogradation slowed down
soluble in alkaline media and remains in solution after again, and above DP 850 the samples remained in aqueous
solution for a suciently long time for carrying out light

scattering measurements. This was done by Burchard et al.
[76,81] who found an uncommon DP dependence of the
second virial coecient as shown in Fig. 17. A pronounced
maximum was found around DP 4200, which indicates
optimum solubility. According to the FloryHuggins the-
ory the driving force for dissolution is the entropy of
dilution which strongly decreases for sti chains [95]. In
the end a rigid rod becomes fully insoluble. With this
theory in mind the decrease of A2 for shorter chains is
probably caused by the sti helical segments whose inu-
ence increasingly becomes eective and nally dominates
and causes phase separation toward retrogradation. The
decrease for larger chain length was interpreted as a result
of intramolecular association which, according to theory,
will cause a stronger decrease of A2 than commonly
observed for exible chains [81].
This conclusion was drawn from the angular depen-
dence of SLS as a function of time for several synthetic and
natural amyloses. Figure 18a gives an example for a synthet-
ic (i.e., monodisperse) amylose of DP 2950 that was com-
pared with the ndings from natural amylose of DP 2730 as
shown in Fig. 18b [76,81]. A common statistical aggrega-
tion is observed for natural amylose with the broad most
probable molar mass distribution. The randomness arises
from the presence of short and quickly aggregating chains
in coexistence with longer, slowly aggregating ones. Very Figure 18 Time-dependent aggregation of (a) synthetic and
likely the short chains rst become attached to the longer (b) native amyloses of approximately the same DP and
ones and these attached chains will then cause cross-linking concentration in water. The striking dierences arise from
and gel formation that nally rearranges into a higher the polydispersity and the stronger aggregation tendency of
order and crystallization. This picture is supported by the short chains in the polydisperse system. The inset in (a)
fact that the long monodisperse synthetic amylose re- shows the time dependence of molar mass increase.
mained in solution for more than 4 weeks. Only in the rst
few hours was a weak aggregation observed by the de-

viation of the inverse scattering intensity from the straight
line. No further change took place for weeks. This behavior
is typical for a ringchain competition.
Remarkable behavior of natural amylose, obtained by
leaching from rice starch, is reported by Rollings and
discussed by Roger [96]. Results of Rg as a function of
Mw are shown in Fig. 19. In contrast to the good agreement
of the data from Kadoma et al. [85] and Burchard et al. [81],
Rollings found in the low molar mass regime up to
Mw=3 105 g/mol an exponent of about 1.0 and a sharp
transition to a power law behavior with exponent less than
0.34. These data strongly suggest individual helices in the
low molar mass regime which is followed by a pronounced
side-by-side alignment of these helices in the high molar
mass regime. Such lateral alignment does not much change
the radius of gyration as the bundle thickness still remains
small compared to the wavelength of visible light. Evident-
Figure 17 DP dependence of the second virial coecient for ly, Rollings was not suciently successful to prevent the
synthetic and leached native amylose in water at 20jC. The double-helix formation when preparing dry and amor-
pronounced maximum indicates optimized solubility at phous amylose.
DPc4200. At DP<800 solubility could no longer be Several years later a detailed study of the aggregation
achieved. Inset: models for (a) lateral association at low DP; and gel formation of synthetic amylose as a function of
(b) intramolecular hydrogen bonding for high DP, weak concentration was made by Gidley and Bulpin [97], but this
association. (From Ref. [76].) (By permission of Wiley/Starch.) study was conned to rheology and to an admiring analysis
204 Burchard
special polysaccharides are involved in the process of cell

recognition. Three types of bacterial polysaccharides are to
be distinguished. These are (1) the surface or O-specic
neutral lipopolysaccharides (LPS) from Gram-positive
bacteria; (2) the capsular acid LPS (K-antigens); and (3)
exopolysaccharides (EPS) which are expired by the bacte-
rium into the surrounding aqueous medium. The O-specic
and acid LPS have antigenic properties, the EPS form
slimes. Figure 20 shows an electron microscopic photo-
graph from a microslice through a capsular Klebsiella
aerogenes bacterium of serotype K8 [100] that belongs to
the Gram-negative bacteria. In Gram-positive bacteria the
outer lipid double layer is missing. Coupled at the reducing
end the LPS contain a lipid that is integrated into the
double lipid layer of the cell. The integration into the outer
membrane of the bacterium is schematically depicted
underneath the EM-photograph in Fig. 20. The lipid-free
polysaccharide is obtained by splitting o the lipid chain in
aqueous phenol. The lipid is not water soluble and can be
separated by gel ltration. Details of the general structure
Figure 19 Molar mass dependence of the radius of gyration of O-antigens and K-polysaccharides and their binding to
of amylose from various laboratories. Squares with dot, the phospholipids are given, for example, by Jann and Jann
synthetic amylose in water81, diamonds synthetic amylose in [101103] and Sutherland [104].
0.33 KCl96. open circles amylose in 0.33 KCl87. The lled All polysaccharides from bacterial surfaces and also
circles represent the data from the size exlusion chromatog- the EPS are mostly characterized by identical repeat units.
raphy. The initial part at low Mw indicate rigid rod behavior,
the nal part with the low exponent is characteristic for a side-
by-side alignment.
of NMR spectra. Light scattering data were not reported

and evidently were not feasible because of the strongly
developed turbidity. The problems of light scattering with
occurring turbidity. A detailed study of the hydrodynamic
radii was carried out by Roger [96].
DMSO is a good solvent for amylose. Even retro-
graded amylose can be dissolved if the dispersions are
submersed a special heat treatment [98]. Common light
scattering measurements were made by Cowie [84] in
various mixtures of DMSO with water which, however,
gave molar masses twice as large as found by other
techniques. The deviation is not yet fully understood and
may be a result of solvent polymer complexation where on
average just two chains are involved, but it could also be a
calibration error.
DMSO was later extensively used by Radosta et al.
[99] in a combination of common SLS and DLS and size
exclusion chromatography in combination with a multi-
angle light scattering detector. Also, the concentration
dependence toward semidilute solutions was studied. The
results will be discussed briey later in connection with the
ndings from starch.
C. Microbial Polysaccharides of Well-Defined

Repeating Units Figure 20 Crossection through a Klebsiella aerogenes
bacterium, and a schematical section through the bacterium
Polysaccharides with primary structure of next higher membrane. The chemical structure of the K8 capsular
complexity are produced by bacteria. Bacteria can be polysaccharide is shown above and the photograph. Un-
infectious or noninfectious to mammalians but in all cases published Ph.D. data from Wolf [100].
The repeat unit contains a well-dened sequence of mary structure and the conformation of the repeating
several sugars. In some cases a homoglucan is formed, unit. These structures were recognized as deterministic
e.g., by h(1,3) glycosid bonds in curdlan, but the number groups for specic antigenic behavior. Specic activity of
of dierent sugars in the backbone can amount up to degrading enzymes was used to determine the primary
four members in a strictly determined sequence. In structure of these repeat units. In addition, application of
addition, the sugars can be branched keeping up to 16 the highly developed NMR analysis became most e-
sugars in the side chain. The main types of monosac- cient. Information on the local and macromolecular
charides are glucose (Glc), galactose (Gal), mannose conformation was mostly extracted from the abovemen-
(Man), fucose (Fuc), and rhamnose (Rha), linked at tioned crystalline structure and electron microscopy.
various positions of the sugar ring, but occasionally all Interest in bacterial polysaccharides again grew by the
other monosaccarides occur. These saccharides can be in observation that the local conformation of the repeating
the acid form or are stereoregularly derivatized by N- unit often determines a well-organized macromolecular
acetylamino- or pyruvate groups. Despite the apparent conformation in solution. Furthermore, fermentation of
complexity in the primary structure most of these poly- EPS in a large scale became possible and this fact extended
saccharides could be crystallized by a technique similar the interest of these polysaccharides from immunology to
to that applied to DNA. A highly concentrated solution industrial application. Now, it became feasible to apply
is prepared that forms a disordered gel. Such a gel is light scattering for a detailed study. Some polysaccharides
loaded with a weak weight and put in a constant of this type will be discussed rst. Because of the complex
temperature cabin for a controlled slow drying primary structure similar disagreeable complexity was
[105,106]. The weight causes creep ow that induces expected also for the light scattering pattern. Surprisingly,
orientation of the chains along the stress direction. This this is not observed, or more precisely, other diculties
liquid crystalline stage slowly (within weeks) transforms than anticipated occurred. As the size of the repeat unit is
into a partially crystalline sample. Atkins and his co- small compared to the applied wavelength, the complex
workers noticed a favorite eect of annealing at elevated repeat unit behaves as nonstructured small bead, and the
temperatures under a controlled humidity atmosphere polysaccharide as a string of such beads.
[106]. From the x-ray diraction pattern detailed infor-
mation on the helix structure is obtained. The knowledge
of these data is imperative for the interpretation of the 1. Xanthan
scattering experiments. The capability of these polysac- The discussion may be started with xanthan. This polysac-
charides to adopt a helical conformation is a result of the charide is synthesized by the bacterium Xantomonas cam-
strictly stereoregular primary structure and a striking pestris. Figure 21 shows the primary structure. The polymer
chain stiness. For a long time the complexity of the consists of a cellulose backbone with a cellobiose as
primary structure has kept physical chemists away from repeating unit. In each of the rst anhydro glucose units
performing light scattering. Another reason arose from a side chain is attached via a(1,3)-links which consists of
the requirement of fairly large amounts of samples for three sugars carrying at the chain end a pyruvate group. A
light scattering measurements (at least 50 mg) which are h(1,3)-linked mannose is acetylated in the C6 position, the
laborious to prepare in particular for capsule polysac- second unit is a a(1,3)-linked glucuronic acid, and the last is
charide from invasive and infectious bacteria. In these the h(1,4)-linked (4,6)-substituted mannose. Xanthan is a
cases immunologists were mainly interested in the pri- polyelectrolyte but of a fairly low charge density. The eect
Figure 21 Repeating unit in xanthan.

206 Burchard
of Coulomb interactions is already fully screened in aque- length could be estimated from the pitch height of the
ous 0.1 N NaCl solutions. helical structure that was obtained from x-ray diraction
Light scattering measurements from dilute solutions experiments with semicrystalline lms, and this was com-
were performed by Brant and Paradossi [107], Coviello et pared with the data obtained from the plateau height at
al. [108], and Ross-Murphy et al. [109]. Properties of large q values. Approximately a two times larger value than
semidilute solution in the presence of Al2(SO4)3 and the estimated from a single helix was found which supported
thermally induced gelation were studied by Rodd et al. the predicted double helix, also in solution. The same
[110]. Electron microscopy measurements by Holtzwarth procedure was applied by Coviello et al. [108] to natural
[111] and Stokke et al. [112] indicated a double helical xanthan (X), the pyruvate free xanthan (PFX), and to the
structure that would induce remarkable rigidity. Further- deacetylated xanthan (AFX). The motivation of this study
more, evidence for special aggregation structures was was to nd out how much inuence the pyruvate and acetyl
obtained [112]. The question arose whether such organized groups have on the double-helix conformation. Initially,
structure will persist in solution, and whether the double the work was encumbered with diculties of measuring the
helix is still a rigid rod. The rst accurate measurements correct molar mass. Extremely large values were found
were made by Paradossi and Brant [107]. From the molar when the polysaccharide was dissolved in cold water con-
mass dependence of degraded xanthan fractions they containing 0.1 N NaCl. The obviously existing aggregation
cluded sti chain behavior but with signicant deviations could be prevented by two essential precautions. First, the
from that of a rod. The angular dependence was not fermented crude xanthan had to be carefully transformed
analyzed, because a suitable theory for the angular depen- into sodium salt form. Traces of bivalent or trivalent salts
dence of semiexible chains was not yet available. Schmidt cause a weak cross-linking. Second, the solutions had to be
et al. [11] remembered the approximation by Koyama [113] submersed using a short heating procedure at about 120jC
for the particle scattering factor that was derived from a and had to be measured immediately after cooling to room
stochastic theory by Daniels [114] which is an extension of temperature. In all three samples a chain multiplicity of
the KratkyPorod wormlike chain [115]. The Koyama somewhat less than two was found indicating that the
theory combined the exactly known second and fourth stability of the double helix is not noticeably aected by
moments of the molecule radius (<R2> and <R4>) with changing the substitution. Table 5 gives a list of the
the behavior of rods at large qRg values and derived what structure parameters of these xanthans together with those
may be called a hybrid theory. Schmidt et al. noticed that from various other microbial polysaccharides.
the results for chains of dierent chain stiness are most
informatively represented in a plot of qRh/(pKc) which they
denoted as CasassaHoltzer plot [11,56,57]. A t of exper- 2. Exopolysaccharides from Pseudomonas Elodea
imental data with this theory allowed an estimation of the (Gellan) and Rhizobia Leguminosarum
Kuhn segment length, the mass per unit length, and the Soon after the industrial fermentation of xanthan a large
number of Kuhn segments per chain. The mass per unit number of other EPS were discovered and fermented [116].
Table 5 Kuhn Segment Length lK, Characteristic Ratio Cl=lK/l and Number of Laterally Aligned Strands of Some Linear
Polysaccharides, Compared with Those From Other Biological and Synthetic Polymers
Polymer lK (nm) Cl nstrands Ref. Comments
Schizophyllan 390 757 3 143,145 triple helix

Gellan 322 625 2 26,127 double helix
Xanthan 255 494 2 108 double helix
Rhizobium TA-1 152 314 3 26,121 triple helix
exopolysaccharide
L-Carrageenan 470 5.7 158 c=0.88 mg/mL, 20jC
300 3.1 158 c=4.8 mg/mL, 20jC
Tamarind, guar, 9.1 36.9 1 234,235 this paper random coil
locust bean
Hyaluronic acid 17.4 6 1 246 polyelectrolyte random coil
Cellulose in Cd-tren 14.8 28.7 1 49 random coil
Amylose in 0.1 KCl 2.05F0.15 11.4F0.7 1 89,90 V-helix, 6 units/turn
40 91 Monte Carlo simulation
Collagen 269 38 3 triple helix
Polybenzyl-glutamate 190 499 1 a-helix
DNA c80 235 2 double helix
Cellulose-tri-carbanilate 22 42.7 1 240 random coil
Polystyrene 2 9.8 1 polymer handbook random coil
L is the bond length, Cl equals the number of sugar units per kuhn segment. The persistence length lp is half of the kuhn segment length lK=2lp.
Figure 22 Repeating unit in gellan.
The industrial interest was mainly focused on samples of a nation of optical activity and light scattering measurements
high viscosity which is coupled to chain stiness. In par- was applied which gave evidence for the formation of
ticular, samples are desired whose rigidity is insensitive to ordered structures. The results from these samples may be
salts and elevated temperatures. Besides xanthan, only discussed at some length, because they disclose a signicant
gellan has gained industrial relevant application [116 principle of the bacterial polysaccharide behavior. The
118] as a thickener, and because of the high tendency to static and dynamic Zimm plots of the EPS TA-1 from
form thermoreversible gels [119]. The primary structure of Rhizobium trifolii are already shown in Figs. 6 and 10,
gellan is given in Fig. 22. It presents the simplest form in a respectively. The curvature in the angular distribution was
class of other polysaccharides of the same backbone repeat mentioned as an indication for chain stiness. Application
unit (i.e., welan, rhamsan, S85, and S-195) [119], but with of the CasassaHoltzer plot conrmed this supposition.
attached side chains of dierent sugar components at well- The structural data from three Rhizobia polysaccharides are
dened positions of the backbone [116120]. This feature given in Table 5 [121]. In all cases the crystalline structure
appears to be a widely spread principle in nature and results was known. This permitted the calculation of the number of
from mutations to make the bacteria resistant against strands that determines the linear mass density ML=(M/L)
bacteriophage attack. of the rodlike Kuhn segment and its length. The data gave
Table 6 gives an example for members of a class from evidence for double helices, and in one case possibly for a
leguminosome bacteria, which cause infection of the plant triple helix. The values are mostly somewhat smaller than
roots that leads to the well-known symbiosis for nitrogen two, which may be considered to lie in the range of
binding of the leguminoses. The behavior of the samples experimental error, but very likely has a physically well-
was studied as a function of the side chain length by the supported reason. This point became clear when the sample
Italian group of Crescenzi [121] and other coworkers from 127, K87, with the longest side chain of seven sugars, was
the United Kingdom and Germany [26]. Mainly a combi- studied. In this case no ordered structure was found.
Table 6 Primary Structure of Member from the Family of Rhizobium Exopolysaccharides

208 Burchard
DLS experiments also revealed a deviation from ex-

ible chain behavior [26,125127]. The curvature in the
angular dependence as shown in Fig. 10 arises from
segmental motions. For large structures, with contour
lengths in the order of the wavelength, the inuence of
the translational diusion is strongly diminished already at
low values of qRg via interference between scattered rays
emerging from dierent sites in the particle. The internal
segment motions start to dominate. In the limit of qRg>4
only these segmental motions contribute. In this limit the
ZimmRouse segmental relaxation spectrum is eective for
exible chains, which results in a q3 dependence of the rst
cumulant C that is given as [126]
C ! C*qRg 3 25
Figure 23 Angular dependence of the normalized apparent in which C* slightly increases under the inuence of
diusion coecient Dapp(qRg)/D=C/( q2D) of gellan. Sym- exclude volume eects in the chain. Such q3 dependence
bols: experimental data of Coviello et al. [125], full line: was not found with the sti double helices, rather an
according to theory [127]. At qRg>2 power law behavior is exponent of 2.8 was estimated. Similar behavior was found
obtained with an exponent of 0.56 in contrast to exible for all double helical chains mentioned in this contribu-
chains (dotted line) where the exponent is 1.0 [126]. (by tion. For explanation a conjecture was made that the
permission of ACS.) relaxation spectrum of sti chains deviates from that of
the ZimmRouse spring bead model. Indeed, bending
modes of rigid rods make a signicant contribution. The
The reason for the behavior of both is based on the corresponding spectrum was calculated 10 years later by
entropy of mixing. This congurational entropy of a fully Harnau et al. [127], and with this the time correlation
exible chain in a solvent was calculated by Flory and function of DLS was derived. A very satisfactory descrip-
Huggins on the basis of a lattice model and was recognized tion with the same data as found from SLS was now
as the main driving force to dissolution. This entropy is possible, (Fig. 23). The angular dependence of the nor-
drastically reduced for sti chains, and eventually for fully malized apparent diusion coecient could be approxi-
rigid rods it leads to a nematic liquid crystalline structure mated by D(q)/D(q=0)f(qRg)0.8. Another detailed study
[95]. If exible chains are attached to such a rod, the was made with the R. leguminosarum 8002 by Coviello et
entropy of mixing is again increased and will keep this al. [125]. Further, EPS were studied by Ding et al. [128],
hairy rod in solution [122,123]. Apparently in the EPS Yang et al. [129], and Zhang et al. [130,131].
127, K87 Rhizobium polysaccharide, with its extraordinari-
ly long side chain, the entropic dissolution force is already 3. Surface Polysaccharides from Mammalian
so strong that it destabilized a double-helix formation. Invasive Bacteria
Similar entropic contributions are eective also in double
helices. Zimm and Bragg [124] showed in their theory of As already mentioned, there exist a vast number of invasive
helixcoil transition that the chain ends of a helix neces- bacteria, where the polysaccharide primary structure from
sarily must exist in a disordered form. A helical turn can
only be stabilized by hydrogen bonds to the ordered helical
section; but toward the free chain end there does not exist
any possibility for a hydrogen bond.
The dangling exible chain ends increase the solubility
via the entropy of mixing. It also reduces for a double helix
the number of aligned strands slightly below two. The
double helices are characterized by a very long Kuhn
segment length which is about 10 times larger than for
cellulose and cellulose derivatives. It is even twice as large
as for DNA double helices. The increased rigidity of double
helices compared to single-stranded chains or helices could
be expected; nonetheless, these structures are not rigid
rods. The remaining exibility is probably based on some
imperfections, e.g., small loops or breaks caused by mis-
matching of the two strands. In light scattering these
defects are not detectable because of the long wavelength
of the used light, and they do not cause a reduction of the Figure 24 Zimm plot from E. coli K 29. Unpublished data
observed mass per unit length. [133]. Inset: repeating unit.
Table 7 List of Repeat Units of Bacterial Polysaccharides Used for Light Scattering
Measurements
E. coli
K 29 R2
ja1; 2
-Man-a1; 3G1c-h1; 3G1cUA--h1; 3Ga1-a1; 2n
Klebsiella
K5 Pyr 2-0Ac
j1; 4 j
-Man-h1; 4G1cUA-h1; 4G1c-b1; 3n
K8 G1cUA
ja1; 4
-Gal-b1; 3G1cUA-a1; 3G1c-h1; 3n
K 11 R1
ja1; 4
-Glc-b1; 3G1cUA-h1; 3Gal-a1; 3n
K 56 Pyr Rha
j4; 6 ja1; 2
-Gal-h1; 3Gal-h1; 3Gal-b1; 3-a1; 3Gal-b1; 3n
Neisseria meningitides
Group B -[NacNeuA-(1-8)]n
3-O-Ac
j
Group C -NacNeuA-1 82
For molecular parameters see Table 8.

R1=-Gal-(4,6)-Pyr; R2=-Man-b(1,2)Glc-(4,6)-Pyr.
the bacterium cell wall has decisive signicance for infec- 7.65 105 g/mol and radii of gyration of 26.3 to 41.4 nm
tion. These bacteria are catalogued by (1) the type of were measured. For details the original papers may be
bacterium (e.g., Salmonella, Escherichia coli, Klebsiella, consulted (Table 8).
etc.), (2) a serotype number, and (3) the O-antigenic and
K- capsular antigenic surface polysaccharide types. Table 7
gives our examples of K-antigens from Klebsiella [132] and 4. Bacterial Cellulose and Triple Helix
one K-antigens from E. coli [133] bacteria. These polysac- Forming b(1,3) Glucans
charides are the very rare cases, known to the author, where The EPS with only one type of sugar as a repeat unit were
light scattering was used for characterization. Figure 24 only briey mentioned so far. The most important repre-
gives an example. A main problem arose from the diculty sentative in this class is the bacterial cellulose fermented by
in performing reproducible extractions of the polysaccha- Acetobacter xyllium bacteria. Dierent strains are known
ride. Comparison of various preparations gave a very and cause some variations in the maximum DP that can be
satisfactory reproducibility of the composition, but the reached, but the samples show no dierence in light
samples still deviated appreciably in their molar mass, the scattering and no deviating behavior from that of native
molecular dimensions (Rg), and the intrinsic viscosity. plant cellulose [50].
(Table 8). Other examples of antigens from Neisseria Another example is curdlan, a h(1,3) glucan that is
meningitides, two infectious and one without immunologic synthesized by dierent strains, e.g., Alcaligenes facalis var.
response, were studied by light scattering in a group around myxogenes [138]. This polysaccharide exists as a triple
Chu et al. [134137]. Molar masses between 1.8 105 and helix, which is believed to be responsible for the antitumor
210 Burchard
Table 8 Molar Mass and Radius of Gyration of Some

Capsular Polysaccharides 5. Pullulan
3
10 Mw The formation of single- to triple-helix formation is quite a
Type of bacterium (g/mol) Rg (nm) Ref. general rule for the strictly regular primary structure of the
repeat unit, but there is one striking exception. This is
E. coli pullulan processed by Pullulans pullularia, an a-glucan
K 29 (1) 4700 114.0 133 with a trisaccharide repeating unit of [a(1,4)Glc
K 29 (2) 4700 79.4 133 a(1,4)Glca(1,6)Glc-]. The two rst glycosidic bonds are
those of amylose and would predetermine helix formation,
Klebsiella but the a(1,6) glcycosidic bond introduces a break [147
K5 1290 30.5 132 149]. This bond has a much higher freedom for rotation
K 8 (1) 1090 36.2 132 and thus favors random coil formation. Fortunately,
K 8 (2) 1130 31.6 132
samples also of a fairly narrow molar mass distribution
K 8 (3) 1060 55.3 132
are processed, and these two properties render pullulan as
K 11 (1) 40 22.3 133
ideal water-soluble samples for SEC calibration and as
K 11 (2) 2000 126.5 133
K56 (1) 67 21.0 132 reference for comparison with branched polysaccharides.
K 56 (2) 175 41.5 132 Careful light scattering measurements were carried out by
Kato et al. [150] and by Nordmeier [25]. The molar mass
Neisseria dependencies of Rg, Rh, the q-parameter, and the second
meningitides virial coecient A2 were measured and evaluated in con-
Group B 183 26.3 134137 text with current theories.
Group C (1) 646 34.5 134137
Group C (2) 765 41.4 134137
D. Microbial Polysaccharides of Higher
Heterogeneity
activity [139,140]. It is no longer water-soluble but forms a 1. Exopolysaccharides From Red Algae
gel after a special heat treatment [138,139]. However, the The next higher complexity is found with the polysaccha-
product is soluble in strong alkali and will allow light rides from red algae. In this class we nd the series of
scattering measurements. Such experiments are in progress carrageenans and agarose. They are typical (AB)n alter-
and will soon shed more light into this structure in solution. nating copolymers and are built up of a [h(1,3)-Gal-
Another type of triple-helix-forming EPS based on h(1,4)-3,6-anhydro-Gal-] repeating unit in the backbone
h(1,3) glycosidic glucans is schizophyllan produced by but dier in the extent of sulfonation and in the anhydro
Schizophillum commune. The backbone is the same as in galactose unit. The E-carrageenan has one SO3-substituent
curdlan, but the repeat unit is a h(1,3)-linked trisaccharide
with one h(1,6)-linked glucose unit on every third residue.
The light scattering behavior was intensely studied by
Norisuye et al. [141144], both in 0.01 N NaOH aqueous
solution and in DMSO. The triple helix as found in the
aqueous medium became denatured in DMSO and disinte-
grated into single chains of much higher exibility [145].
Measurements in pure water gave no reliably reproducible
results because of a strong tendency to association by
lateral alignment, which could not be broken up even after
a heating step to the boiling temperature of water. This
problem is common to all stretched regular polysacchar-
ides and will later be discussed in detail in the context of the
results of Section 3.5. The proposed triple-helix structure is
shown in Fig. 25. In their studies on thermally reversible
gelation of schizophyllan, induced by sorbitol, Fuchs et al.
[145,146] repeated some measurements with two industri-
ally available samples. The analysis was made on the basis
of static and dynamic Zimm plots and the corresponding
CasassaHoltzer plot. The data from measurements in 0.01
N NaOH and DMSO are given in Table 9. A chain
multiplicity about 15% larger than three was obtained
which indicates a weak side-by-side association of the triple
helices. The values for the Kuhn segment length are given Figure 25 Repeating unit of Schizophillum and a model of
in Table 5. the suggested triple helix.
Table 9 Kuhn Segment Length lK, Contour Length L, Polydispersity Ratio Mw/Mn, Radius of Gyration Rg, Linear Mass
Density ML, and Number of Aligned Strands n of Two Commercial Schizophyllan Samples, Measured in 0.01 N NaOH and
Water at 20jC
Schizophyllan 1
Solvent lK (nm) L (nm) Mw/Mn Rg (nm) ML (g mol1 nm1) na

0.01 N NaOH 171 F 20 551 F 30 1.91 F 0.1 127 F 3 1770 F 200 2.47 F 0.28
H2O 199 F 20 664 F 35 1.91 F 0.1 157 F 3 2710 F 200 3.78 F 0.28
Schizophyllan 2
0.01 N NaOH 208 F 20 730 F 20 1.43 F 0.1 159 F 4 2670 F 100 3.23 F 0.12
a
Based on ML=2150 g/(mol nm) determined by Kashiwagi et al. [143] for the triple helix.
Source: From Refs. 145 and 146.
in the A-unit and two SO3 groups in the B-unit, but no 3,6 Carrageenans have played an important role in un-
anhydro-ring. With this high degree of sulfonation the derstanding how a thermal reversible gel is obtained. It is
polysaccharide shows a typical behavior of a strong poly- the outstanding merit of Rees [151] who suggested a
electrolyte. The L-carrageenan probably presents an inter- sensible model and proved that in all gel-forming poly-
mediate stage between E- and n-carrageenans with only one saccharides bundles of laterally aligned chains are formed
SO3-substituent in both units and with 3,6-anhydro ring in over a certain segment length, and these junction zones
the B-unit. The latter makes this unit more hydrophobic. replace the point-like cross-links in chemical or permanent
The n-carrageenan represents the nal stage in the biosyn- networks. A tough and occasionally bitter discussion
thesis and carries only one SO3 group in the A-unit and in started on how these bundles are formed and what the
the B-unit the 3,6 anhydro-ring. In agarose also the A-unit structure is [152155]. Two facts were known to Rees which
no longer carries the SO3 group in the C4 position of the were not denied by his opponents. These are (1) the crys-
ring (Fig. 26). Thus agarose is the most hydrophobic talline structure as found by Anderson et al. [156] and
polysaccharide with the strongest tendency to gel forma- Arnott et al. [157] gave clear evidence for a double helix for
tion that melts at about 40jC. n-carrageenan has a good both the agarose and the n-carrageenan. (2) Furthermore,
capability of gel formation which, however, depends the gel could preferentially be cleaved by chemical means
strongly on the kind of counterion. Potassium ions induce into double-stranded segments [158,159]. The bonds that
gel formation stronger than the corresponding sodium are sensitive to hydrolysis were recognized as those where
ions. In the highly charged E-carrageenan the hydropho- the 3,6-anhydro-galactose was broken and substituted in
bicity is weak and the repulsion due to the high charge the C6 position by a SO3-group as shown for the E-car-
density fully prevents gelation. The L-carrageenan forms a rageenan. However, the postulated double-helix formation
clear gel at lower temperatures, whereas the n-carrageenan in solution as prerequisite for gelation was violently re-
results in turbid gels. Therefore the L-carrageenean would jected, and doubts were expressed whether such double
make these gels more appropriate for a light scattering helices will be a sucient basis for the observed strong gels.
study. A disadvantage is the diculty to completely re- Rees modied his model [152,153] by assuming that the
move n-carrageenan. double-helix formation is only the rst step that is followed
Figure 26 Repeating units of three types of carrageenan and of agarose.

212 Burchard
tions. In the low concentration limit the curves represent a

transitional behavior in which nonassociated and already
highly associated chains coexist. At higher concentrations
a clear tendency toward a better dened structure is no-
ticed, and this could be analyzed in greater detail.
The deviation of the angular dependence from a
straight line at large q values gave indications for chain
stiness. This supposition was supported when the exper-
imental data were represented in a CasassaHoltzer plot.
As already outlined in the discussion of xanthan, the well-
dened plateau in Fig. 28a indicates sti rod behavior in
the large q-regime and the height of the plateau gives the
linear mass density ML. The gure shows a change in the
bundle thickness with temperature. It is also inuenced by
the applied concentration. Surprisingly, the lateral associ-
ation of chains was very large at low concentration but
decreased at higher concentration and approached a con-
Figure 27 Temperature dependence of molar mass of stant plateau at concentrations c>0.8% (w/v). At small q
segmented L-carrageenan measured in 0.1 N KCl and 0.1 N values the curves in Fig. 28a increase with decreasing q
TMACl (trimethylammonium cloride). (From Ref. [158].)
by further side-by-side alignment of these rods. Also, this

modication was not accepted by his opponents [154,155]
who argued that for topological reasons double-helix for-
mation cannot be a fast process or may even be completely
prevented in long chains by the constraints of entangle-
ments. This disagreement could not be convincingly dis-
solved until recently when Bongaerts and coworkers
[160,161] combined a number of dierent techniques which
gave evidence for association of single helices.
Light scattering could answer only some of the ques-
tionable points, but a double-stranded chain section prin-
cipally cannot be discriminated to arise from a double helix
or side-by-side alignment. In 1982 Rees suggested to the
present author to solve the questions by light scattering
from carrageenan at high temperatures, where it is in the
sol state, and at lower temperatures until a gel is formed.
He suggested to use the L-carrageenan, as the gels remain
clear whereas the corresponding gel from n-carrageenan
becomes turbid and makes light scattering measurements
not feasible. Unfortunately, as was found out later, traces
of n-carrageenan could not be completely removed from
the main L-components, but the n-carrageenan components
had a signicant inuence. Nonetheless, ter Meer [158]
could prove several points of the dispute. First, the seg-
mented carrageenan permitted a reversible orderdisorder
transition to single chains of DP=25F5 which on cooling
aligned again and gave twice the molar mass. Figure 27
shows the temperature dependence of the DP measured in
0.1 N KCl and 0.25 N trimethyl ammonium chloride
(TMACl). A slight shift toward higher temperatures and
a weaker transition to single chains were found with Figure 28 Nonnormalized (a) CasassaHoltzer plot from
TMACl as salt solution. L-carrageenan at two dierent temperatures and c=2.34 mg/
A very dierent scattering behavior was found with mL. (b) Normalized CasassaHoltzer plot for concentrations
the native L-carrageenans. The Zimm plots in the dilute of c=0.88 mg/mL (upper curve) and c=4.8 mg/mL (lower
regime (at 26.4jC in 0.1 N KCl solutions) disclosed di- curve). The lines in the normalized plots correspond to the
culties which are typical for many polysaccharide solu- Koyama theory of semiexible chains. (From Ref. [158].)
nm) were obtained. The latter is by a factor 2.7 lower than

calculated from the data of the repeating unit. Let us
assume that this value refers to single-chain sections, then
we still have to keep in mind that actually only a fraction f
of all repeating units will be in this nonassociated form.
This leads to the conclusion ML,SANS = fML,single chain
with a fraction f = 0.63 representing repeating units that
are not bound to another chain in the bundle, but may still
be incorporated in the bundle. These imperfections have
only little inuence on the value of the bundle structure as
Figure 29 Model suggestion for structure formation of determined by light scattering. The unbound repeating
carrageenan aggregates. (From Ref. [158].) units in their majority still belong to segments which with
both ends are incorporated in the bundle. The bundle is not
represented by a homogeneous cylinder, which would be
completely rigid, but it contains imperfections, probably in
values but nally at q=0 have to go to zero. Thus a the form of small loops. These imperfections permit bend-
maximum has to be passed whose height increases with ing motions and conned the bundle length to a nite
the number of Kuhn segments [11,12]. The position of the value. Figure 30 shows the direct comparison of light
maximum depends on the length of a Kuhn segment and is scattering with SANS results. One notices a fairly large
shifted toward smaller q values for large Kuhn lengths. The gap between these two experiments of about one decade in
maximum appeared below the detection limit, and a direct the q value, and secondly, the onset of a transition of the
determination of the Kuhn segment length was not possi- SANS data toward the light scattering structure. The
ble. However, ter Meer also measured the radius of gyra- important full region of transition could not be measured
tion, and with these data the Kuhn segment length could be because the wavelength of the neutrons was too short and
calculated. The molecular parameters, i.e., contour length the wavelength of visible light too long.
L, Kuhn segment length lK, and number of Kuhn segments The heterogeneity of the present L-carrageenan struc-
NK per contour length, could be inserted in the Koyama tures could be seen clearly in DLS. Figure 31 shows as an
theory [113] on the scattering from semiexible chains, and example time correlation functions at a scattering angle of
the result could be compared with the measurements. This h=90j for ve concentrations at a temperature of 15.7jC.
is shown for two selected concentrations at room temper- Two relaxation modes are detectable. The fast motion
ature in Fig. 28b. The excellent agreement is remarkable. remained almost independent of concentration, whereas
The scattering data allowed the following conclusions. the slow motion was slowed down as the concentration was
The contour length increases with concentration.

The number of laterally aligned chains is high at
low concentration but decreases toward a constant
value at a certain concentration.
At high temperature (sol state) and at high con-
centrations the number of laterally aligned chains
never reaches the value of single chains. The ex-
perimentally determined value is close to two.
At very low concentration the number of aligned
chains strongly increased from about two to about
six chains.
From these data the model of Fig. 29 was suggested.

Because of the long wavelength of visible light, details of
the bundles and the corresponding ne structure cannot
be determined by light scattering. Still it remained an im-
portant question whether the chains in the bundle are
smoothly aligned to a homogeneous cylinder or whether
numerous imperfections in the form of loops are present.
In the present case, small angle neutron scattering (SANS)
experiments with the same samples shed some light into the
question. The wavelength of neutrons is about 1500 times Figure 30 Comparison of the reciprocal angular depen-
shorter than that of the red HeNe laser light. Therefore the dence from light scattering with that from small angle neu-
thickness of the sti chains and the linear mass density tron scattering (SANS). A transition from SANS to light
could be measured. A chain cross-section diameter of 0.7 scattering behavior seems to occur in the intermediate
0.9 nm and a linear mass density of ML,SANS=185 g/(mol q-regime (see text). (From Ref. [158].)
214 Burchard
saccharides dier signicantly in behavior from macro-

molecules with DP>100 and will not be discussed here.
High molar mass fructans are synthesized by a number
of bacteria. Pure samples were recently obtained by enzy-
matic polymerization with fructosyl transferase, cloned
from Streptococcus mutans with E. coli bacteria [164].
Two limiting types of fructans, inulin and levan, are
conceivable. In the inulin type the fructose units are linked
mainly by h(2,1) bonds but in the levan type mainly h(2,6)
bonds are formed. Figure 33 shows the structures. A higher
exibility may be expected for the inulin polymers than for
levan. In inulin the backbone consists of a (CCO)
repeat and the furanose unit is a side group of a polyeth-
ylene glycol chain. In levan the furanose is part of the
backbone, and the repeat unit is, with the rigid furanosyl
ring (CCO ring in the backbone), appreciably longer. In
both idealized cases the polysaccharides would represent
Figure 31 Time correlation functions of dynamic light scat- linear chains. However, commonly in inulin h(2,6) and in
tering from L-carrageenan as a function of concentration. Two the levan h(1,2) bonds occur in addition and cause long-
modes occurred when the concentration was increased. The fast chain branching.
mode corresponds to the motion of individual chains, the slow Extensive work was invested by Stivala and his co-
mode indicates the motion of aggregates. (From Ref. [158].)
workers [165168] in the characterization of levans from
Streptococcus salivarius, Bazillus subtilis, Aerobacter leva-
nicum, and Bazillus vugatus. Very high molar masses in the
increased. The fast motion may be assigned to the mobility range from 25 106 to about 100 106 g/mol were found.
of individual chains, but the slow motion is certainly A detailed characterization by SLS and viscometry was
related to motion of bundles and its relaxation time will made from S. salivarius levans, both in H2O/0.1 M NaCl
be the lifetime of a chain being bound to the bundle. After and in DMSO/0.1 M NaCl. Very high molar masses
that time a single chain can freely move for a short time Mw=(20 73) 106 g/mol but with fairly small radii of
until it becomes captured again by another bundle. These gyration Rg=(40.7129) nm were found and a strikingly
two modes and their temperature dependence were further strange molar mass dependence of the intrinsic viscosity.
analyzed but will not be discussed. The Zimm plot of a low molar mass sample gave a set of
The complex behavior of the carrageenans in aqueous parallel straight lines for the angular dependence in agree-
KCl solutions is considerably simplied if NaCl is used as ment with exible coil behavior. No Zimm plot was shown
salt. Even for the chemically much better dened n-carra- for the sample of Mw=73 106 g/mol and Rg=129 nm. In
geenan the strong tendency to gel formation could be
suppressed and accurate light scattering measurements
could be obtained. Recently, Cuppo and Reynaers
[161,162] could extend the scattering measurements toward
very dilute solutions and proved a dissociation of the
double strands into single chains. Figure 32 shows one of
their results. Together with other types of experiments they
now got strong evidence for association of strands without
forming intertwined double helices.
Recently, another light scattering study on the kinetics
of aggregate structure formation with n-carrageenan in 0.1
M KCl was carried out [163]. Despite the much better
developed equipment, used with great expertise, the
authors were not successful in nding new facts that would
allow conclusions exceeding those given by ter Meer 20
years ago. The kinetics of aggregate structure formation
was studied by Meunier et al. [163].
2. Fructans of the Inulin and Levan Type

Fructans are polysaccharides on the basis of fructofurano-
sides. Fructans are widely spread in the vegetable kingdom
but are mostly oligomeric in size with degree of polymer- Figure 32 Zimm plots of L-carrageenan on 0.02 N NaCl
ization smaller than DP 60. In addition, these fructans are (squares) and 0.09 N NACl (circles). (From Ref. [162].) (By
often highly branched. Like other oligomers, such oligo- permission of Wiley-VCH.)
Figure 33 Fructans of the inulin and levan type. In the h(2,1)-linked fructan of inulin the ring is a side group to the backbone, in
the h(2,6)-linked fructan of levan it is incorporated in the backbone. Both types of fructans are partially branched via h(2,6) and
h(2,1) linkages, respectively.
this case a more detailed analysis would have been possible than 0.589, to be expected for exible coils in a good
and would give us more insight into the structure in solvent, and may indicate chain stiness. The observed
solution. curvature may be an artifact, and the value may actually be
Another rather comprehensive study was made by a little smaller and in agreement with coil behavior. The
Heyer et al. [164] and Wol et al. [169] who focused on molar mass dependencies of the radius of gyration and the
the inulin-type fructans. A careful analysis of h(2,6) link- intrinsic viscosity from the levan and inulin types are
ages in addition to the h(2,1) linkages of inulin was made shown in Fig. 36 and compared with data from Kitamura
and gave 5.66.9% branching points [169]. SLS and DLS et al. [170]. In the high molar mass region the data give
and viscometry were applied, and a further check was made evidence for impenetrable sphere behavior which is in
from the interaction among the macromolecules at con- agreement with the radii of gyration of the furanosyl
centrations slightly below the overlap concentration c*.
Despite the high molar mass of 71 106 and 27 106 g/mol
small radii of gyration of Rg=48 and 30 nm were obtained
for S. mutans and Aspergillus sydowi inulins, respectively.
Note, for levan of Mw=70 106 g/mol, Rg=129 nm was
found. This marked dierence in the inulin gives evidence
to the abovementioned higher chain stiness of levan over
that of inulin. Already for the comparatively small radii of
gyration a weak upturn was obtained, and the scattering
data were analyzed from Guinier plots. A typical Guinier
plot is shown in Fig. 34. A good determination of Mw, Rg,
and the second virial coecient A2 was possible where the
last parameter indicated water at 33jC as a good solvent.
The authors also carried out SEC measurements in
online combination with MALS and determined the molar
mass dependence of the radius of gyration. The results are
shown in Fig. 35. A very weak increase of the radii with
molar mass was obtained for the enzymatically synthesized
S. mutans inulins. The exponent in the molar mass depen-
dence is even lower than 0.33 for a homogeneous sphere. Figure 34 Guinier plot from Aspergillus sydowi inulin in
For the Asperagus sydowi the exponent of 0.66 was higher water. (From Ref. [164].) (By permission of Elsevier.)
216 Burchard
coil, and hard sphere, the functions are known from theory
[171,172]. Because of insetting turbidity it was not possible
to extend the measurements to rather high concentration
but the data in the accessible regime, nonetheless, point to
hard-sphere behavior.
3. Dextran
Dextran is another widely used polysaccharide of similar
complexity as the fructans. In this glucan the anhydro-
glucose units are mainly linked via a(1,6) glycosidic bonds.
The polysaccharide is branched via a(1,3) and a(1,4) link-
ages and to a weak extent also via a(1,2) linkages [173175].
Most common is the dextran synthesized by the bacterium
Leuconostoc mesenteroides strain B 512(F) with an overall
degree of branching of about 5%. The average structure is
shown in Fig. 37. Dierent strains of this bacterium
Figure 35 Molar mass dependence of the radius of gyration produce samples that dier in the extent of branching
from three inulins. The curves were obtained from size and in the primary structure of the backbone. Most
exclusion chromatography in online combination with multi- spectacular were the ndings by Misaki et al. [176] with a
angle light scattering. (From Ref. [169].) (By permission of dextran of L. mesenteroides B-1355. This polysaccharide
Elsevier-Polymer.) contains about 35% a(1,3) linkages and was nally ana-
lyzed to have an alternating a(1,3)a(1,6) backbone in
which on average every 10th unit carries an a(1,6)-linked
transferase inulins, but for the A. sydowi inulin a structural long-chain branch of the same alternating a(1,6)a(1,3)
change occurred for Mw>3 107 g/mol. The sphere behav- primary structure. No light scattering measurements from
ior cannot be explained solely by the 5% branching but these dextrans are known.
seems to present a collapse due to intramolecular interac- All physicalchemical investigations so far were solely
tions, still keeping a well-swollen state of V/Vdry=16.3. made with the L. mesenteroides strain B 512. The rst
The transition in the structure of A. sydowi inulin at results were probably obtained by Wales et al. [177] and
Mw=3 107 g/mol may be a result of aggregation of chains Senti et al. [178] in a rather comprehensive manner. The
rather than an intramolecular collapse. The decrease of [g] measurements were later repeated with the now much
for Mw<2 105 g/mol demonstrates open random coil advanced light scattering techniques by Kuge et al. [179],
behavior of linear exible chains which may be partially Nordmeier [25], Hanselmann and Burchard [180], and
drained. nally by Ioan et al. [181,182]. Good agreement among
In order to get more information on inulins the the various research groups was found, but in most cases
interchain interaction (Mw/RT)(Bp/Bc)uMw/Mapp(c) was the authors conned themselves to special aspects, e.g., the
measured as a function of concentration [169], where p is radius of gyration, intrinsic viscosity, and the extent of
the osmotic pressure, and the mentioned interaction func-
tion is the reduced osmotic modulus or inverse osmotic
compressibility. This function again is structure dependent,
and for some limiting structures, e.g., rigid rod, exible
Figure 36 Molar mass dependence of the intrinsic viscosity Figure 37 Chemical structure of dextran. R1, R2, and R3 are
of levans [170] (lled symbols) and inulins [169] (open positions for branching with frequencies less than 5% for
symbols). Leuconostoc mesenteroides B 512.
branching. Only in the studies by Nordmeier [25] and Ioan

et al. [181] were the shape of the dextrans, the hydrody-
namic interaction, and the thermodynamic interaction of
the macromolecules examined in greater detail (Fig. 38).
The analysis of the global structure parameters Rg and
[g] in terms of the branching density was made on the basis
of the ZimmStockmayer theory [183], StockmayerFix-
man [184], and the ZimmKilb theories [185]. Two so-
called shrinking or contraction factors g and gV were
dened by these authors [184,185], which are given by
R2g;branch
gu 26
R2g;linear
gbranch
g Vu 27
glin
Figure 39 Relationship between the contraction factors g V
where both ratios have to be taken at the same molar mass.
and g obtained from online SEC/MALS/viscosity measure-
Zimm and Kilb suggested a relationship g V=g0.5, which
ments. (From Ref. [181].) (By permission of ACS.)
later was empirically extended by Kurata et al. [186] to
g V gb 28
this change is caused by a change in the FoxFlory draining
Experiments with randomly branched polymers gave evi- function U in
dence to b=0.6, but for star-branched molecules the
exponent steadily increased with the number of arms per R3g
macromolecule from b=0.5 to 1.5 [171]. It was shown that g U 29
M
which increases with the segment density. An exponential
correlation between the two quantities g and g V was indeed
observed also for the dextrans but with a much higher value
of b=0.71. [181c]. Figure 39 shows the relationship from
SEC/MALS measurements in combination with online
viscometry, where pullulan was chosen as linear reference.
As the intrinsic viscosity, radius of gyration, and the
molar mass were simultaneously measured, the U param-
eter could be determined as a function of the g contraction
values. A power law was found as shown in Fig. 40. The
dashed line was drawn from the following relationships
[171,181c]
Ubranch 3=2
gV g g0:71 30
Ulinear
or
Ubranch Ulinear g0:79 30V

where the exponent b=0.71 was the experimentally deter-
mined value (Fig. 39) and the other relationship immedi-
ately follows from the ratio of the FoxFlory [187]
equations for linear and branched macromolecules. Evi-
dently, the hydrodynamic properties of the branched dex-
Figure 38 (A) Molar mass dependencies for the radius of trans approach impenetrable sphere behavior as the
gyration and the hydrodynamic radius. (B) The correspond- branching density increased, but hard-sphere behavior
ing depencence of the U-parameter. The straight line in (B) was not reached. At this point the behavior of the fructans
corresponds to pullulan; the dotted lines are theoretical may be recalled which approached hard-sphere behavior
curves for 1% to 5% branching. Squares are results by much closer.
Nordmeier [25]; the other symbols correspond to data from The samples investigated by Ioan et al. [181,182]
(A) [181]. (By permission of ACS.) had molar masses below 2.5 106 g/mol and radii below
218 Burchard
several monosaccharide types are incorporated in the

chain. Examples in the mentioned order are hemicelluloses
of the xylan type, galactomannans and xyloglucans, amy-
lopectin, pectins, and hemicelluloses of the glucomannan
and arabinogalactan types. Undoubtedly, this irregularity
in primary structure has signicant functional meaning in
biological systems, which is largely not yet known. All
these polysaccharides have gained much industrial interest
and were always considered under aspects of possible
application, for instance, as thickeners. The relevance of
the biological function remained mostly neglected.
Because of the complexity in primary structure the
characterization, in particular by light scattering, proved to
be involved and often immensely dicult. Plants have to
endure heat and cold, rain and snow, but under all these
Figure 40 Dependence of the Flory-parameter U on the
contraction factor g [181b]. DS1...DS5 are samples from strains the biological function has to sustain. Resistance
SIGMA. (By permission of ACS.) against these strains was achieved in nature by evolution of
environmentally persistent supramolecular structures. In
the temperature range of life, these polysaccharides are not
soluble in common solvents. Even at elevated temperatures
Rg=47 nm which were too small for a specic molecular below 100jC the materials may dissolve visibly but often
shape analysis on the basis of a Kratky plot. However, not to the macromolecular level. Reluctantly, scientists
Nordmeier [25] could get hold of four other samples of realized that they entered, with the characterization of
much higher molar masses up to 100 106 g/mol and radii plant polysaccharides, a little explored eld, placed in a
up to 170 nm, supplied by Pharmaceutical Company transition regime intermediate of macromolecular and
Zdravlje in Leskovac, Yugoslavia. The Kratky plots from colloid science. Light scattering techniques are good tools,
the three highest molar masses are shown in Fig. 41. A but consistent conclusions have only been possible when
check with the generalized hyperbranched model [188] was several methods were combined, e.g., common rheology,
not possible, but a very satisfactory approximation was rheo-optics, SANS, and, of course, the various spectro-
possible with the soft sphere model [189], which in scopic techniques.
modern terminology corresponds to generalized den-
drimers of various shells or generations. Fig. 41 shows
theoretical curves for various shells. Interestingly, compact
sphere properties are approached as the molar mass in-
creased, i.e., with increasing number of branches. This
result is in good agreement with the conclusions drawn
by Ioan et al. from the hydrodynamics. Nonetheless,
conclusions from the good t should cautiously be drawn,
because the dendrimers are absolutely uniform in size and
primary structure. According to SEC/MALS all dextrans,
even those of fractions, showed a fairly broad molar mass
distribution. Furthermore, enzymatic degradation un-
doubtedly revealed statistical branching where even the
functionality f of the branching unit varies between f=3
and 5, whereas the dendrimer model was calculated for
constant f=3.
E. Polysaccharides from Eukaryotic Cells

1. Some General Remarks
The highest complexity is found with polysaccharides from
higher developed plants and animals whose cells possess a
nucleus. With the exception of cellulose and amylose no
other plant polysaccharide is known so far that has a Figure 41 Kratky plot from the three highest molar mass
perfectly dened primary structure and absolutely regular dextrans measured by Nordmeier [25]. The theoretical curves
sterical conguration. Irregularity can be introduced by were drawn with the soft sphere model [190] (generalized
irregular substitution along the chain, by short- and long- dendrimer). The model is shown as inset. (By permission of
chain branching and by copolymer composition where ACS.)
2. Starch
The specic outline may be opened with starch. At rst
sight this material appears to be a disadvantageous starting
point, because of the amount of amylose which varies from
species to species. However, the molar mass of amylose is
small compared to the branched amylopectin [44,45,190],
and therefore many similarities are found among the
various sources of starch. Most striking is the well-known
microstructure of starch granules which for all species is
almost identical [191,192]. A semicrystalline pattern is
encountered with alternating crystalline and amorphous
shells. The crystalline shell in turn has a laminar ne
structure in which the crystalline areas are interrupted by
a thin layer of noncrystalline lower density. Figure 42 shows
an enlarged section through such a lamella. In all cases the
thickness of this ne structure is about 9 nm [191,192].
Unexpected to polymer scientists, the branched amylopec-
tin forms the crystalline morphology in apparent contra- Figure 43 Molar mass dependence of various starches,
diction to ndings with synthetic polymers. In general, dissolved in DMSO/H2O=9/1 after heating the solvent at
branching inhibits crystallization. This counterevidence 80jC for 1 hr. (From Ref. [197].) The solid line refers to
gives indication for a supramolecular structure that was synthetic amylose, the dashed one to polyclopene amylose
formed under the kinetic conditions of biosynthesis and (Mw/Mn = 2.)
does not represent the equilibrium state of lowest energy.
The supramolecular structure of the granules is highly
stabilized and is not soluble in cold water. For a long time
A certain improvement toward molecular solubility
this supramolecular structure was assumed to be broken up
was achieved after keeping the dispersion in DMSO at
during the gelatinization process in hot water, whereupon
80jC for about 1 hr. Somewhat smaller molar particle
the crystalline structure disappears. Early light scattering
weights were obtained, but with unsatisfactory reproduc-
measurement from amylopectin, dissolved in boiling water,
ibility. An essential improvement was achieved in 1994
or under the action of hydrogen breaking agents, gave the
when an autoclaving procedure in water at temperatures
astounding result of almost the same high molar mass in
between 145jC and 165jC was applied [98,99], but each
the range of Mw=400 500 106 g/mol and radii of gyra-
starch species required a dierent optimized temperature
tion Rg=400 480 nm [44,193195], independent of the
and application time. These conditions were determined in
source and the amount of amylose in the starch. Measure-
numerous experiments mainly by Radosta et al. [99]. The
ments from starches dissolved in DMSO/H2O=90/10 at
optimization was time consuming, as a sharp ridge between
room temperature revealed a certain variation in the molar
disaggregation and thermal degradation of the covalently
mass for a number of various starches, but almost no
bound polysaccharide had to be observed.
change in the radius of gyration [196,197]. The invariance
Later this procedure was also extensively used by
was interpreted by strongly stabilized aggregates. Figure 43
Aberle et al. [198] and applied to starches of various
shows the mentioned dependence.
amylose contents. Figure 44 shows the result of the average
molar mass as a function of the amylose content. The well-
developed, only slightly nonlinear, dependence nally led
to the amylopectin molar mass of about 80 106 g/mol, a
value that is in good agreement with the early ndings by
Witnauer et al. [199]. The observed correlation has a simple
reason. Assuming molecular dispersion of the starch com-
ponents, then amylopectin and amylose should be present
as individual macromolecules. In light scattering the weight
average of both component molar masses is measured
which is given by the relationship [198] (Table 10).
Mw;starch 1 pMw;AP pMw;AM

31
i1 pMw;AP
The fraction of amylose p was determined from the iodine
binding capacity and the subscripts AP and AM indicate
Figure 42 Schematic cut through a crytalline laminar layer amylopectin and amylose, respectively. The simplied
in starch. (From Ref. [191].) approximation on the right side of the equation results
220 Burchard
Figure 45 Berry plot from one of the acid degraded dextrins

(LD) in alcohols measured in 0.5 N NaOH.
out in the Freiburg laboratory. In her Ph.D. work Galinsky

[200] took up an idea that was realized by Fox and Robyt
[201] who prepared well-dened Naegeli starches [202] of
dierent molar masses by acid degradation of starches in
methanol and higher aliphatic alcohols. During the whole
process the starch remained in a crystalline-only slightly
swollen state. Galinsky and Burchard [200c] wondered
whether the acid degradation would proceed predominant-
ly in the noncrystalline layer where the branching points
are supposed to be. The measurements were carried out in
Figure 44 Molar mass of starches with dierent amylose
0.5 N NaOH solutions. Figure 45 gives an example of the
content p. The radius of gyration (lower part of the Figure)
Berry plot, and Fig. 46 shows the molar mass dependence
remained within experimental errors unchanged. This behav-
of the Rg and Rh. According to a suggestion by Flory [203]
ior agrees with the molar mass of amylopectin, which was
obtained by application of Eq. 31. (From Ref. [198].) and the more detailed calculation on the molar mass by
Erlander and French [204], amylopectin could be consid-
ered as a special example of hyperbranched macromole-
from the huge dierence in the molar mass which for cules. The angular dependence of scattered light from these
amylose is about 100 times smaller than for amylopectin. macromolecules was calculated by Burchard [206] in 1972
Table 10 reveals the unexpected result of approximately the without the eect of excluded volume interactions. It
same amylopectin molar mass in all starches measured in characteristically deviates from that of linear and random-
this study. Fig. 44 shows the dependence of molar mass on ly branched macromolecules. Figure 47 shows Kratky plots
the amylose content of these amylopectins in water. of the samples and the t of these curves by the equation
A molar mass of (76.9 F 11.7) 106 g/mol is unusually
high for synthetic macromolecules and doubts remained. 1 C=3qRg 2
qRg 2 Pq qRg 2 32
For this reason a series of other experiments were carried 1 1 C=6qRg 2 2
Table 10 Weight Average Molar Mass Mw of Starches and Radius of Gyration Rg from Various Species of Starch
Starch Amylopectin Amylose
Mw 106 (g/mol) Rgz (nm) p Map 106 (g/mol) RAp (nm) MAm 106 (g/mol) RAm (nm)
Waxy maize 76.9 234 0.00 76.9 234.0
Maize 88.0 213 0.22 112.2 213.6 2.09 19.4
Amylomaize 16.7 231 0.76 68.9 237.6 2.45 60.1
Wrinkled pea 30.5 235 0.63 77.9 241.5 2.62 38.1
Smooth pea 33.5 165 0.42 53.8 170.8 5.45 27.6
Potato 51.0 222 0.24 60.9 224.3 19.6 31.8
After curve analysis according to Eqs. 31 and 32 the molar mass and radius of gyration of amylopectin, MAp and Rg,Ap, and of amylose, MAm,
and RgAm, were derived. p denotes the amylose content according to iodine binding capacity.
Figure 47 Kratky plots from the various degraded starches.

The dotted lines are ts with Eq. 32 of general theory of
hyperbranched macromolecules [204,206], where C is the only
t parameter that depends on the number of branches per
macromolecule. C=1 represents linear chains and C=0 the
limiting case of a very large number of branches per macro-
molecule. The curve C=0 is identical with the DebyeBueche
approximation. (From Ref. [205].) (By permission of ACS.)
from waxy maize where the dierent curves refer to the

same starch treated at 175jC for three dierent times. The
Figure 46 Molar mass dependence of the radius of gyration
angular dependencies from various slices in the range of
and the hydrodynamic radius from nine dierent degraded
starch species. The q=Rg/Rh parameter gives clear indication Rg=150 220 nm were plotted in the form of a normalized
for hyperbranching (ABC model). The dotted lines corre- Kratky plot. An excellent universal curve was obtained
spond to theory. (From Ref. [206].) (By permission of ACS.) (Fig. 49), which indicates self-similarity of the amylopectin
fractions in the indicated size region. A C=0.023 param-
eter was estimated in this case which is slightly lower than
0.115 obtained by Galinsky and Burchard for the highest
in which C depends on the number of branches per fraction LD 13.
molecule and is a t parameter. This equation includes Starch is also soluble in the aqueous sodium iron
the limit C=1 of linear chains and C=0 of the Debye tartrate complex (FeTNa). A similar dependence was
Bueche [205] approximation which, in the hyperbranching observed [210]. However, the curve was shifted somewhat
theory, corresponds to an innite number of branching to higher apparent molar masses which was recognized as
points per macromolecule in the limit of innitely large
macromolecules. The curves are very satisfactorily de-
scribed by Eq. 32 but with a lower branching density of
1.2% instead of about 4.5%. This underestimation results
from the presence of excluded volume interaction which
shifts the asymptotic part of the curves to higher values. A
detailed calculation was made by Ganazzoli [207]. In the
limit of no degradation a molar mass of Mwc108 g/mol
was obtained.
The scattering behavior of amylopectin was cross-
checked with scattering curves from sedimentation eld
ow fractionation [208,209]. Starches of dierent origin
were autoclaved without particular precaution to degrada-
tion [208]. The sedimentation eld toward the wall of a thin
channel was produced by rotating the channel in a centri-
fuge. After a steady-state gradient was achieved the sample
was ushed out and detected by a refractive index and a
MALS detector. The angular dependencies from the dif- Figure 48 Molar mass dependence of the radius of gyration
ferent slices were analyzed in terms of radius of gyration from autoclaved starches. A fractal dimension of df = 2.39
and shape of the macromolecules. Figure 48 shows the was obtained from the slope. (From Ref. [208].) (By
molar mass dependence of the measured radii of gyration permission of ACS.)
222 Burchard
on whether the degree of methylation is high or low [213]

and is decisively inuenced by the presence of calcium ions
or other multivalent metal ions. In addition, the sugars of
the hairy region can associate in a random manner.
This random association is the main reason for the
often irreproducible determination of molar mass, the
molecular dimensions, and viscosity behavior. SLS has
not been very successful. Even after ltration a solution
through a 0.22-Am lter, radii of gyration larger than 500
nm were found, indicating a dissociation under shear and
reassociation after stress release. Signicant improvement
was achieved by Berth et al. [217] who collected a number
of fractions by preparative GPC fractionation. Reliable
Zimm plots could be obtained for fractions with
Mw<17 106 g/mol. The results were conrmed by Har-
ding et al. [218] by ultracentrifugation. Chapman et al.
Figure 49 Kratky plots from the angular dependencies of [219], who applied SLS and DLS, attained further improve-
the scattered light determined from SEC/MALS for r radii of
gyration. Each of the ve curves is the average from 10
neighboring slices. The data gave one universal curve which
represents particles of the same fractal dimension df = 2.32,
u = qRg (From Ref. [208].) (By permission of ACS.)
an eect of partially bound FeTNa complex to the OH-

groups of the starch. Similar eect was obtained with
cellulose and amylose which are both soluble in this iron
complexing solvent.
In the course of time the starch research group from
the Fraunhofer Institute of Applied Polymer Research in
Golm, Germany [99] improved the process for dissolution
in DMSO by a special treatment. This procedure is now
applied in routine investigations by SEC/MALS measure-
ments in DMSO at 75jC [211]. These data conrmed the
high molar mass of amylopectin and, in addition, gave a
good separation for the amylose in starch.
3. Pectin
The commercially available pectin, which commonly is
used as a gelling polysaccharide for jam, is considered a
a(1,4)-galacturonan whose acidic groups are largely meth-
ylated. The degree of methylation depends on the plant
where it was extracted from. Often, a simple linear chain is
assumed. However, native pectin has a far more complex
primary structure and contains neutral sugars, e.g., rham-
nose, galactose, xylose, and arabinose [212]. This fact has
made characterization of pectin immensely dicult. In
fact, the gelling quality strongly depends on a successful
hydrolysis to remove the content of neutral sugars.
In common understanding this polysaccharide is con-
sidered as a heterogeneous linear chain with rhamnose in the
backbone that is interrupted by short segments, the galac-
turonan chain. The rhamnose causes a kink in the regular
chain conformation and prevents crystallization. It is also
the unit to which the highly branched hairy region is
anchored [214216]. Figure 50 shows some details. The gal-
acturonan segments have a strong tendency to align laterally Figure 50 (a) Model of heterogeneity in pectin, (b) detailed
side-by-side. Several models have been suggested depending structure of the hairy region. (From Refs. [214216].)
et al. [220,221] reached a state by which a satisfying set of

data could be obtained. Figure 51 shows one of his latest
results of the molar mass dependencies of the intrinsic vis-
cosity. The samples were heated to about 130jC for 2.5,
4.0, and 6.0 min, and under those circumstances degra-
dation occurred. No power law behavior was found, or
in other words, the fractions are not self-similar to each
other. Beyond a certain molar mass the intrinsic viscosity
shows a weaker increase with molar mass, and in one case
it even passes through a maximum and then decreases
again. Clearly, a closer packing with increasing particle
weight was obtained. Recalling the FloryFox [187] rela-
tionship between intrinsic viscosity, radius of gyration, and
molar mass
R3g
g U 33
M
one recognizes a decrease in the intrinsic viscosity if the
volume of the particle increases less than the molar mass.
The eect is slightly compensated by the fact that with
increasing segment density the draining parameter U
increases (compare Fig. 40 for dextran). Disregarding the
aggregation regimes the three curves can be nicely tted by
Figure 51 Molar mass dependence of the intrinsic viscosity one common curve which has a high exponent of 1.58 in the
from orange pectins heated at 130jC for 2.5, 4, and 6 min. low Mw range, which gradually decreases to a value of 0.52
Online SEC/MALS/Viscos detection. The initial part of the for the highest molar mass as is shown in Fig. 51. This
three runs could be well tted by one common curve. The feature is characteristic for sti chains, and the result
corresponding slopes are shown below. Short chains exhibit qualitatively agrees with the ndings of Axelos [222] who
sti chain behavior. For very long chains Gaussian coil from SANS measurements concluded signicant chain
behavior is approached [220,221]. The bending of the 6-min stiness. Similar curvature in the molar mass dependence
curve toward smaller intrinsic viscosities is caused by
of apple pectins was observed by Berth et al. [217]. See
association which is connected with an increase of the segment
Fig. 52 and the critical comments by Berth [223].
denisity. (By permission of Elsevier.)
4. Hemicellulose of Xylan Type
Hemicelluloses are accompanying materials to cellulose in
ment by careful isolation and hydrolysis of apple pectin wood. Three main types are known which dier in the type
from the middle lamella of apple cells. A good Zimm plot of monosaccharides in the polysaccharide [39,224]. In
from this pectin was reported with Mwc800,000 g/mol. hardwood from deciduous trees partially substituted
A breakthrough was obtained with the now highly xylans establish the main fraction. h-Xylose is similar to
developed SEC technique combined with MALS (Wyatt h-glucose but has no primary alcohol group in the C5
Technology, Santa Barbara, U.S.A.) and online viscome- position. In xylan these saccharides are linked via h(1,4)
try. In a nonfatiguing endeavor over the years Fishman glycosidic bonds in the same way as the glucose in cellulose.
Figure 52 Molar mass dependence of the intrinsic viscosity from apple pectin fractions. The curvature at high molar mass
indicates association which is connected with an increase of segment density. (From Ref. [223].)
224 Burchard
renewable sources of biodegradable polymers. Repeated

interest was shown in these materials but did not gain
industrial application so far. These xylans are much strong-
ly substituted in the C3 or C2 position by arabino-furanose
(Arf) via a(1,2)- and a(1,3) bonds. In some of these
pentosans a xylose unit is doubly substituted in both the
C2 and C3 positions (Fig. 53). These doubly substituted
xyloses essentially determine water solubility. Much eort
was invested in particular by Ebringerova [226,229233] in
cooperation with a number of other laboratories.
Two examples may be shown and discussed here. In
the rst case an arabinoxylan was carefully extracted with
DMSO from beech wood. This sample was repeatedly
measured in DMSO within 4 years. Soon after isolation a
molar mass was obtained [233] (Fig. 54), which later proved
to be about two times larger than that of the single chain. In
the course of time, when the sample was stored as powdery
material, increasingly larger molar masses were found.
Figure 53 Chemical structure of three dierent xylans. When the sixfold chain mass was reached a steep increase
in the radius of gyration occurred. After that stage the
particle weight further increased in time but the radius of
gyration increased only weakly. The whole feature was
Irregularly, every 10th xylose unit is substituted by an interpreted by a side-by-side alignment of chains, the align-
a(1,2)-linked 4-O-methylglucuronic acid, and the back- ment led to bundle formation in which the exibility of the
bone also contains in the C2 position of the xylose unit chains is strongly reduced. This causes an increase in the
varying amounts of acetyl groups. In coniferous wood the Kuhn segment length and a drastic expansion of the aggre-
xylan in addition is decorated by one a(1,3)-bound arabi- gated particle, in particular when about ve to seven chains
nofuranose, but contains no acetyl substituents. The sugar come together. Once this bundle was formed further lateral
composition is Xyl/GlcUA/Arf=5/1/0.6. Acetyl groups association took place which did not cause further increase
signicantly improve the solubility in water and DMSO. in chain stiness. The thickness of the bundles still re-
The glucomannan from coniferous trees is one of the
rare cases where the backbone is a random copolymer of
mannose and glucose in a ratio of Man/Glc=3/1. Every
15th mannose unit carries a(1,6)-linked galactose as a side
chain. The chain is weakly acetylated in the C2 or C3
positions of glucose or mannose. Finally, the arabinoga-
lactan from larch wood has a backbone of h(1.3)-galactose
units, and each galactose unit carries in the C6 position a
short chain of h(1-6)-linked galactose which at the end
carries a(1,3) arabinofuranose. The structures are shown
in Fig. 53.
As already mentioned the solubility of xylans and
other hemicelluloses in water depends on the degree of
acetylation. These acetyl groups are easily cleaved. Under
commonly rough isolation conditions almost all acetyl
groups are removed. The bare xylan is insoluble in almost
all known solvents and appears inapplicable to industrial
use. The presence of hemicelluloses in paper signicantly
deteriorates the quality. Therefore much eort in paper-
making is invested to remove these hemicelluloses mostly
via specic enzymatic degradation. The molar mass of Figure 54 Change of molar mass, radius of gyration, and
carefully isolated hemicelluloses was found in the range shape of an arabioxylan as a result of ageing. Three domains
between DPn=60 and 200 [224227], but considerably are recognized which refer (a) to random coil, (b) bundle
larger weight average degrees of polymerization, up to formation combined with drastic stiening of the objects,
DPwc600 [228], were observed for arabino xylans. Often, and (c) lateral aggregation with weak increase of the radius of
only the number average was measured. gyration but pronounced increase in molar mass. These
Xylans are found also in annual plants, in particular in aggregates dissolve in typical metal complexing solvents for
straw, corn cobs, and from hulls and bran of grain. These cellulose, but only to bundles of six laterally aligned chains.
polysaccharides present a considerable amount of annually (From Ref. [231].) (By permission of Elsevier.)
mained small compared to the wavelength of light and thus similar manner the intrinsic viscosity whose average is
had no inuence on the radius of gyration. The aging eect closer to the number than to the weight average. In
was not understood in the beginning but appears to be the ultracentrifugation the large aggregates are not suciently
result of gradual cleavage of acetyl or arabino groups. well recorded, because the concentration signal is too weak
In another approach hemicelluloses from various for detection, and in SEC experiments this high molar mass
sources were transformed into the carbanilates by reaction fraction is often just ltered o.
with phenyl isocyanate in hot pyridine [228]. All free OH- Recent results by Ebringerova et al. [233] gave clear
groups reacted but the uronic acid groups remained intact. evidence for this interpretation. The intrinsic viscosity
These samples were soluble in THF and were measured by from fractions which were measured by SLS showed only
light scattering. Figure 55 shows the dependence of the radii in the low molar mass region power law behavior. At
of gyration on the degree of polymerization. The seemingly large molar mass the viscosity deected from the straight
chaotic results were tentatively interpreted as follows. line and eventually even decreased again. This behavior
There are several samples which more or less obey power corresponds to that of pectin observed by Fishman et al.
law behavior. These are considered as double-stranded [221] and Berth [223]. It is a clear indication of the forma-
chains. Taking these as reference and assuming mainly tion of compact structures. Complexation with cell wall
side-by side association one can estimate the number of proteins was excluded by treatment with protonase, and
laterally aligned chains. The result is shown by the inset to only aggregation of the AGX chains appears to be the
Fig. 55. Most frequently dimerization was found, followed right interpretation.
by aggregation to a bundle of six chains in a few cases. This
lateral association is probably caused by the uronic acid
groups which can form strong hydrogen bonds in organic 5. Polysaccharides as Thickeners
solvents. Double-stranded chains already gain a signicant Some polysaccharides develop an unusual increase in the
rigidity which reduces the entropy of mixing that will lead viscosity even at very low concentrations. These polysac-
to further association. charides are of high interest mainly in food application but
Light scattering measurements of hemicellulose in increasingly also in other industrial branches. The mecha-
water or aqueous NaOH mostly give extremely high molar nism is not yet fully understood. Two types are known. One
masses and radii of gyration, at variance to the ndings by is represented by the class of galactomannans in which the
osmosis, ultracentrifugation [225], and SEC in combina- mannan backbone is decorated by galactose units. The
tion with viscosity measurements [224,226]. This discrep- other class has a cellulose backbone which in natural
ancy can be understood by the large polydispersity. products is partially substituted by short chains containing
Actually, only a very small number of large aggregates is a single xylose or a dimer of xylose h(1,2)-linked galactose.
present, and most of the material consists of single chains. Even more ecient thickeners are obtained with various
However, because of the very large molar mass in light industrially available neutral cellulose ethers. Conceivable
scattering this small fraction of particles dominates the mechanisms for thickening eects are discussed at the end
scattering behavior. Nonetheless, the mean moiety of low of this section. Two types of structure are shown in Fig. 56.
molar mass determines the osmotic pressure, and in a In all cases no reliable scattering results could be
obtained for samples dispersed in cold water. Results from
the galactomannans at room temperatures in water were
never published. Somewhat more successful were light
scattering measurements by Lang et al. [234236] from
tamarind seed polysaccharides. The data uctuated for
each newly prepared solution, but he received a common
and reasonable molar mass dependence for the radius of
gyration, the hydrodynamic radius, and the intrinsic vis-
cosity. His data will be compared with recent results
obtained by Picout et al. [237239] with the galacto-
mannans from guar, locust bean, and tara gums. The
authors applied an autoclaving procedure similar to that
already described for starch. The solutions were heated to
130jC for a certain time and then measured at room
temperature. An additional pressure was applied at 90jC
and the magnitude of pressure was varied. For reasons of
Figure 55 Dependence of the radius of gyration from comparison the data from the galactomannans and the
various xylan carbanilates on the degree of backbone polym- tamarind xyloglucans were plotted against the degree of the
erization. AGX=arabino-glucuronoxylan, AX=arabin- backbone. The result is shown in Fig. 57. Picout et al.
oxylan, X=beechwood xylan (Lenzing). The straight line [237,238] plotted their data as a function of the molar mass
corresponds to dimeric chains. With this reference a multi- and found for the three galactomannans dierent depen-
plicity of side-by-side aggregation was be estimated as given dencies. When the backbone degree of polymerization is
by the inset. (From Ref. [228].) chosen one common dependence for guar, locust bean, and
226 Burchard
Figure 56 Average composition of galactomannans (above) and of tamarind seed polysaccharide (below).
tamarind is obtained within experimental error, but not for

the tara gum. The curve is shifted to larger degrees of
polymerization approximately by a factor 2. In the low
molar mass regime the galactomannans follow power law
behavior with an exponent of 0.575, but the curve attens
for DP>4000. The slope of 0.61 for the tara gum data is
somewhat higher and indicates an increased chain rigidity.
Possibly two tara gum chains have formed a complex by
side-to-side alignment. This association does not aect the
radius of gyration but causes a marked decrease in the
segmental exibility.
The fairly well-established curve for the galacto-
mannans and tamarind allows us to draw further conclu-
sions. Figure 58 shows a plot of R2g/DP as a function of
Figure 57 Plot of the radius of gyration from galacto- DP1/2. For a Gaussian chain a constant plateau is expected,
mannans and tamarind seed polysaccharide as a function of and for chains in a good solvent R2g/DP increases with DP1/
2
the backbone degree of polymerization With the exception of due to excluded volume interaction. In the low molar
the tara gum a common behavior is found. The kind around mass region the increase is proportional to DP1/2 which is
DMw,backbonec4000 indicates the inset of side-by-side followed by a weaker nonlinear increase. At a chain length
association. (From Refs. [237,238].) (By permission of ACS.) of one Kuhn segment the excluded volume is no longer
macromolecules is positive and large. Because of this high

positive change in entropy of mixing, the entropy repre-
sents the main contribution to molecular dissolution. If the
macromolecular chain has sites of repulsive interaction
with water this is responded by stabilization and further
increase of clusters which takes place in the vicinity of these
sites. This represents a marked further decrease of the
entropy [241] which will drive together the hydrophobic
segments of the chain. Association will be the outcome and
nally gelation. Very likely it is a combination of cooper-
ative hydrogen bonding for bundle formation of chains and
of cooperative hydrophobic cluster formation of water
molecules which forms the basis for the astounding thick-
Figure 58 Plot of R2g/DPw,backbone vs. DP1/2 w,backbone to
ening behavior.
eliminate the inuence of excluded volume, which vanishes at
DPw,backbone!0. The decrease of the curve for DP1/2 w,backbone >
60 indicates the formation of more condense structures. Open 6. Hyaluronic Acid (Hyaluronan)
circles: tamarind seed polysaccharide; circles with dot: guar
and locust bean. Straight line: initial part of the curve. So far, only polysaccharides from plants and bacteria have
been discussed. Oligo- and polysaccharides are widespread
also in the tissue of vertebrates. But in contrast to plant
eective, and the unperturbed dimensions are obtained. polysaccharides these carbohydrates are mostly covalently
The evaluation gave a characteristic ratio of Cl = 36.9 F bound to proteins to form glycoproteins, also called muco-
2.1 and a Kuhn segment length of lK = 19.1 F 1.2 nm. This polysaccharides or mucins. Prominent representatives are
value is considerably larger than lK = 6.8 F 1.6 nm which the proteoglycans, in which chondroitin sulfates are cova-
was estimated by Picout et al. [237,238] from the intrinsic lently attached as long polysaccharide side chains to a
viscosity data. In fact, application of the BurchardFix- protein stem to form brush-like combpolymers. These
manStockmayer procedure to sti chain molecules leads polysaccharide side chains are highly sulfated and are
to a signicant underestimation of the Kuhn segment strong polyelectrolytes. The separation from the protein
length. Application of a method by Hearst gave backbone is dicult, and because the chain is fairly low
lK=18 F 0.8 nm (see Refs. [237,238]), in good agreement (Mw<5 104 g/mol) these samples have not been of special
with the present evaluation. It may be mentioned, for interest for detailed light scattering studies. There are in
cellulose tricarbanilate in dioxan lK=22 nm was found fact only a few examples of pure polysaccharides in the
[240] from neutron small angle scattering. tissue of vertebrates. One example is heparin with at least
These structural parameters form an important basis four types of sugars in the backbone. Some segments have a
for the understanding of the outstanding thickening prop- well-dened primary structure which enables complex
erty. Picout and his coworkers [237,238] adopted the view formation with the protein antithrombin, but other sec-
that high chain exibility is a necessary requisite for this tions of the chain have a rather random copolymer com-
behavior. This opinion is at variance with experience with position. The complex formation plays a signicant role in
exible synthetic macromolecules which in no case exhibit controlling the blood clotting process; the function of the
a remarkable thickening eect. Two requirements appear segments with random composition is not yet fully clear.
to be substantial. First, thickening is promoted by a high Probably because of this heterogeneity this polysaccharide
tendency of chain association, which increases with con- was not much studied by light scattering.
centration. Second, the overlap concentration should be Another example is hyaluronic acid, a linear polysac-
low, because then the mentioned association will set in charide of high molar mass up to Mw=7 106 g/mol. The
already at very low concentration. Small overlap concen- chain is a typical alternation (AB)n copolymer of the type
trations in turn are correlated to sti macromolecules. -(h(1,3)GlcUA)h(1,4)GlcNac-)n, where GlcUA denotes
The type of interaction that forms associates in water glucuronic acid and GlcNac N-acetylglucosamine, i.e.,
is not yet known. Certainly, hydrogen bonding has a the repeating unit is a dimer which is h(1,3)-linked to form
signicant eect on the stability of the aggregates, or on a chain. The polysaccharide is found in variable amounts in
the lifetime of these clustered chains. This interaction is all tissues and is especially abundant in early embryos. The
eective only over short distances and would require isolation of pure material is often dicult and insucient
concentrations above the overlap concentration. The same because of the high tendency of keeping proteins attached.
arguments also hold for dipolar and van der Waals inter- Fairly pure hyaluronic acid was isolated from the umbilical
actions. These interactions cannot be taken as the driving cord, bovine vitreous humor, and rooster combs. Later, by
force. The key for this force is probably found in the screening Streptococcus zooepidemicus the polysaccharide
peculiar properties of water. Water is a structured liquid could be produced at a large scale by fermentation of these
and exists as quickly uctuating clusters which on average bacteria. The isolation of the polysaccharide from the
contain 25 water molecules [241]. Such liquid as a solvent broth still remained laborious and is outlined in detail by
reduces the entropy of mixing which for nonpolar synthetic Berriaud et al. [242].
228 Burchard
The polysaccharide has three important features. (1)

Hyaluronic acid is a polyelectrolyte, however, of a low
charge density. (2) Hydrogen bonds are possible between
the OH-group in the C3 position of the glucuronic acid and
the ring oxygen of the N-acetyl glucosamine, which would
cause chain stiening. In addition, between the NH and the
carboxylic group of the glucuronic acid a further hydrogen
bond mediated via one water molecule appears conceivable
[243]. (3) The same sort of hydrogen bonds without the
interplay of water is possible between hyaluronic acid and
proteins (compare the helix stabilization in synthetic poly-
peptides). Keeping these facts in mind, the source of the
sample from which it originates, the purication, and the
applied technique for characterization will have a signi-
cant inuence on the obtained result.
Three studies will be discussed which all appear being
carried out with great care. The earliest was made by
Cleland starting in 1969 and ending with a summary in
1984 [244]. The highly puried material was prepared from
rooster comb by Pharmacia, Uppsala. The two other
studies by Fouisac at al. [245] and by Ghosh et al. [246]
were made with bacterial hyaluronates prepared by fer-
mentation of S. zooepidemicus and were products of
A.R.D. Co., Paris, and of Sigma, respectively. As already
mentioned, hyaluronic acid is a polyelectrolyte, and this
property has especially attracted the three mentioned Figure 59 Dependence of the radius of gyration and the
groups. One issue was the evaluation of chain stiness hydrodynamic radius from bacterial hyaluronic acid on
sodium hydroxide molarity. Below: the q=Rg/Rh parameter
under conditions where all coulombs interaction were
together with results for some models. (From Ref. [248].)
screened. Cleland [244] made his studies in 0.25 N NaCl,
Fouissac et al. [245] for three salt concentrations (0.01,
0.06, and 0.6 N NaCl), and Ghosh et al. [246] in NaCl and
NaOH in the molarity range of 0.010.8. Remarkable NaOH. In contrast to the data obtained in NaCl the radius
dierences in the nal results were obtained. of gyration decreased by a factor of two, but the hydrody-
Cleland applied a large number of various techniques namic radius remained almost unchanged. The authors
including SANS and viscosity and found a persistence interpreted this behavior as being caused by the break of
length of lp=5.0 F 0.05 nm [244]. Fouissac et al. [245] hydrogen bonds between two adjacent units in the dimer,
found a better t of their light scattering data with lp=8 and this causes exibilization. Their data can be further
nm, but from the viscosity behavior lp=4.5 nm when the analyzed as is shown in Fig. 59, where the original data are
YamakawaFujii [247] theory is applied. Finally, Ghosh et redrawn in the same scale for both types of radii, and, in
al. [248] established a relationship on the basis of the addition, the q=Rg/Rh parameter was calculated. Com-
OdijkHouward theory [249] paring these data with those from various models, it
becomes clear that typical coil behavior is valid at low
L L ionic strength, but this coil does not adopt the typical Q-
R2g lp0 lp C0:5
s 8:7 307=Cs0:5 34
3 3 solvent behavior when the NaOH molarity was increased.
where L is the contour length and Cs the ionic strength. The The q-parameter decreased further close to a value of unity
contour length was estimated from the molar mass divided (Table 3). This decrease resembles highly branched prop-
by the mass per unit length L=Mw/ML with ML=400 g/ erties and gives rise to speculations. Possibly, the well-
(mol nm). Thus in the limit of full screening lp0=8.7 nm organized hydrogen bonds between neighbored sugar units
was obtained. The dierence between the early work by are replaced by random and probably uctuating interac-
Cleland [244] and those of the two other laboratories may tions. This will cause a decrease of the geometrical radii,
have its basis in the dierent source. The isolation from a i.e., Rg,. But the hydrodynamic radius, in addition, is
rooster comb and purication is certainly involved with the determined by the hydrodynamic interaction, i.e., the
source from a vertebrate, and traces of proteins may still be segment density. The two eects balance each other in part
kept associated, but there may also be another reason. The and keep the hydrodynamic radius almost unchanged.
agreement among the groups around Reed et al. [248] and
Rinaudo [242] is very satisfactory. Reed, however, brought IV. CONCLUSION
another aspect in discussion when he analyzed their data
measured in NaOH. The research on polysaccharides is highly specialized.
The authors measured both the radius of gyration and Often, the laboratories are exclusively concerned with
the hydrodynamic radius in a range from 102 to 1.0 M one type of polysaccharides, for instance, with cellulose
or starch, pectins or thickeners from other sources, micro- primary structure as cellulose but seems not to t this
bial polysaccharides or antigens. All these laboratories supposition. However, this chain has a dierent chain
have outstanding experts in their own eld but do not topology with a predetermined tendency to helix formation
know much of the properties of other polysaccharides. This with six repeat units per turn. Because of inherent exibility
review took account of the need for people who just wish to the helix is not stable in water but needs another partner for
get information on recent development for their own stabilization. This could be various alcohols which in the
specialized subject. However, a simple listing of the prop- channel of amylose helix form an inclusion complex.
erties from the various polysaccharides would be unsatis- Otherwise, an amylose chain takes another amylose chain
factory without pointing out interrelationships. To and forms a double helix. Such double-helix formation
accentuate these interrelationships right from the begin- causes an immense stiening of the chain which is consid-
ning, the present article tried to bring some order in this list erably stronger than observed with the DNA double helix
by discussing the polysaccharides according to increasing and favors further side-by-side alignment. The process goes
complexity in primary structure. Such procedure will not on until a crystalline precipitate is obtained. Interestingly,
be sucient to gain a wider view and deep insight into the crystallization is prevented at low concentration by intra-
properties which are uncommon with synthetic polymers. chain hydrogen bonding if the amylose chain is suciently
These dierences in commodity polymers are considered long (see inset of Fig. 17). The reason for crystallization is
important because they contain the basis for the develop- based on absolute regularity and the long cooperative
ment of new materials which could be prepared only with length of the double helices.
great diculties by chemical synthesis. Despite the limiting The question arises as to how crystallization could be
possibilities of light scattering techniques an attempt is now prevented. Biological materials are always synthesized by
made to extract some principles common to many poly- enzymes which work precisely step by step in the same
saccharides. First, a list of keywords is given which will manner and necessarily would yield perfect regularity.
then be commented on. There are two possibilities to prevent crystallization, and
Some striking properties of polysaccharides both are realized in nature. The one is the introduction of
Large number of OH-groups kinks in the regular chain, i.e., the chemical structure is
Native supramolecular structures modied by sections of one or more repeating units in a
Chain stiness nonregular manner. In most algae polysaccharides such
Aggregation kinks are created by another enzyme which becomes
Double- and triple-helix formation eective after the chain synthesis. In other cases, e.g.,
Bundle formation pectin, another kind of sugar with a dierent type of
Coexistence of linear chains and dense clusters glycosidic bonds is inserted. The other possibility com-
Prevention of crystallization prises irregular short- or long-chain branching. Long regular
sections still tend to align and the process goes on until
The ample OH-groups are the basis for hydrogen either gelation takes place or a micellar aggregation is
bonding. The relevance of these interactions is manifested obtained. These aggregates contain many dangling chains
in the semicrystalline structure of native cellulose. A regu- and cause, in the language of colloid science, sterical
lar net of intrachain and interchain hydrogen bonds is the stabilization. A polymer scientist will keep the view that
reason for the high stability against environmental strain, the exibility of dangling chains increases the entropy of
for the rigidity of the cell wall and the high tensile strength mixing which again stabilizes the particle in solution. These
of bers. In a somewhat less regular manner the hydrogen particles are not as dense as the crystalline domains in
bonds are also present in regenerated cellulose. The native cellulose, because several fringed micelles aggregate fur-
solid cellulose is not perfectly crystalline but shows a rather ther, now in an irregular manner (Fig. 15). It is a great merit
organized supramolecular structure, which was given the of D.A. Rees [151] when he recognized the bundle forma-
name fringed micellar crystalline structure in which tion as the essential junction zones for association and gel
short crystalline sections are interrupted by highly oriented formation. Similar behavior is not found with commodity
but not crystalline domains. Very likely this peculiar polymers because of the irregular mostly a-tactic primary
feature is responsible for the mostly incomplete dissolution structure which impedes lateral alignment.
of cellulose. The dissolution process starts with the amor-
phous domains, but the morphology of the crystalline Now we can state a rst important principle:
domains is not fully disintegrated and survives in the form Stretched chains favor lateral alignment and can
of bundles. The simplest structure would be single-fringed lead to crystallization, gelation, or only cluster
micelles, i.e., a bundle with a number of dangling chains formation. Crystallization can be prevented by
[250], but scattering experiments revealed larger, rather any sort of irregularity in particular when these
dense aggregates of such fringed micelles (Fig. 15). irregularities heavily perturb the smooth spatial
Fringed micellar aggregation is most pronounced with conformation of the chain.
cellulose and its derivatives (which could not be included in
this review). This type of aggregation is also observed with The next question is, which prerequisites will induce
many other polysaccharides, e.g., hemicelluloses, pectins, stretched conformations? Here the results from microbial
and carrageenans. Evidently, there is a common principle polysaccharides and of the antigenic polysaccharides from
behind this behavior. Amylose has a similar stereoregular capsules of infectious bacteria give an answer. All these
230 Burchard
polysaccharides are constructed, at least over a certain As a last point the striking eciency of thickeners has
segment length, by the same repeating unit and form under to be understood. A strong increase of the viscosity is
common environmental conditions well-ordered, optically known for long if the concentration exceeds the coil
active supramolecular structures. These are mostly single, overlap concentration c*. However, the astounding in-
double, or triple helices. This observation was a little crease of viscosity with common thickeners occurs already
unexpected for the antigens from capsular polysaccharides at very low concentrations apparently below c*. Two
with their often very complex repeating unit. Topological requisites appear to be necessary for this behavior. The
conditions given by the special type of glycosidic bonds one is a high chain stiness which already causes a high
may predetermine a helix or another type of supramolec- viscosity below c*, and the other is a strong tendency to
ular structure, but there must also exist special interactions association already below the overlap concentration. The
to stabilize the structures. These interactions are dicult to association tendency became clear when a heating step to
analyze and remained largely not well-known. 130j was applied. Molecular dispersity could be achieved,
and the high temperature gives indication of the break of
This allows us to formulate a second principle. The
hydrogen bonds. But only hydrogen bonds can x a
prerequisite for lateral alignment is a stretched structure. The type of interaction that drives the polysac-
conformation. For cellulose this condition is al- charides together still remains unknown. Possibly the
ready given by the h(1,4)-glycosidic bond. In other hydrophobic eect [241] plays an important role, but more
cases a transition to a supramolecular structure eort has to be invested before the basis for thickening can
chain must have occurred with the single chain conclusively be disclosed.
before bundle formation can become eective. The
enormous stiness of the formed objects is under-
standable, because in a bundle of aligned chains the ACKNOWLEDGMENTS
segmental motion is seriously restricted.
The author wishes to close this review with an excuse to
Highly branched samples cannot aggregate in this his colleagues. He was not able to discuss all types of
manner and would not be expected to form aggregates at polysaccharides, as there were too many. In particular,
all. The many dangling chains should give these macro- friends will nd this gap disappointing. I regret this fact
molecules sucient sterical stabilization. Nonetheless, ag- myself. However, it is a great pleasure for me to express
gregation occurs in a manner that has never been my warmest thanks to all of them who helped me with
considered as a theoretical possibility. The aggregation their comments and discussions, often without knowing
phenomenon is connected with the peculiarity of a weak that I was consulting them.
increase in the radius of gyration while the molar mass
changes by orders in magnitude. Power law behavior is
often found with exponents of 0.15<m<0.25 [250]. Mostly, REFERENCES
a fractal dimension df=1/m is determined from this expo-
1. Cumins, H.Z.; Carlson, F.D.; Woods, T.J. Translational
nent [251,252], but gives in these cases df=6.7 4.0. Values
and rotational diusion constants of tobacco mosaic virus
of df>3 have no physical meaning. The characteristic self- from Rayleigh linewidths. Biophys. J. 1969, 9, 516.
similarity of synthetic polymers [253] is not obeyed by these 2. Fujime, S. Quasi-elastic light scattering from solutions of
aggregated polysaccharides. Another reason must be re- macromolecules. I. Doppler broadening of light scattered
sponsible for the small exponents. Obviously, a more and from solutions of tobacco mosaic virus particles. J. Phys.
more condensed aggregate structure is obtained as the Soc (Japan) 1970, 29, 416.
aggregation proceeds. The mechanism for this process is 3. Schaefer, D.W.; Benedek, G.B.; Schoeld, J.D.G.;
Bradford, E. Spectrum of light quasi-elastically scattered
not understood by current theories. from tobacco mosaic virus. J. Chem. Phys. 1971, 55,
Another question is, why do large aggregate clusters 3884.
coexist with nonassociated, freely moving single macromo- 4. King, T.A.; Knox, A.; McAdam, J.D.G. Translational
lecules? In random associating processes a broad continuous and rotational diusion of tobacco mosaic virus from
size distribution is predicted similar to cross-linking reaction polarized and depolarized light scattering. Biopolymers
with synthetic polymers [254]. This is not found. The bimod- 1973, 12, 1917.
5. Pecora, R. Polarized dynamic light scattering as a probe
al distribution reminds of micelle formation. However, there of macromolecular intramolecular motions. Scattering
is a signicant dierence between the two aggregation Techniques Applied to Supramolecular and Non-Equili-
mechanisms. Common soap micelles are in a thermodynam- brium Systems; Chen, S.-H., Chu, B., Nossal, R., Eds.;
ic equilibrium, i.e., chains from a micelle can leave the micelle Plenum Press: New York, 1981.
and other chains may enter the micelle instead [255]. In 6. Pecora, R. Doppler shifts in light scattering from pure
contrast, polysaccharide aggregates are metastable and do liquids and polymer solutions. J. Chem. Phys. 1904, 40,
not dissociate even at the lowest concentration applied in 1604.
6a. Pecora, R. Spectral distribution of light scatted by mono-
common light scattering. It remained unclear whether in disperse rigid rods. J. Chem. Phys. 1968, 48, 4126.
SEC experiments a dissociation occurs, because of the much 7. Berne, B.J.; Pecora, R. Dynamic Light Scattering; Wiley:
lower concentration, and the eect of high shear, or whether New York, 1967.
the large aggregates are just ltered away. 8. Bantle, S.; Schmidt, M.; Burchard, W. Simultaneous
static and dynamic light scattering. Macromolecules 1982, 33. Koppel, D.E. Analysis of macromolecular polydispersity
15, 1604. in intensity correlation spectroscopy: The method of
9. Burchard, W. Light scattering techniques. In Physical cumulants. J. Chem. Phys. 1972, 57, 4814.
Techniques for the Study of Food Biopolymers; Ross- 34. Burchard, W.; Schmidt, M.; Stockmayer, W.H. Informa-
Murphy, S.B., Ed.; Blackie Academic & Professional: tion on polydispersity and branching from combined
London, 1994. quasi-elastic light scattering. Macromolecule 1980, 13,
10. Burchard, W. Combined static and dynamic light 1265.
scattering. In Light Scattering, Principles and Develop- 35. Yamakawa, H. Modern Theory of Polymer Solutions;
ments; Brown, W., Ed.; Clarendon Press: Oxford, 1996. Harper & Row: New York, 1971. Chap. 6.
11. Schmidt, M.; Paradossi, G.; Burchard, W. Remarks on 36. Kirkwood, J.G. The general theory of irreversible
the determination of chain stiness from static light processes in solutions of macromolecules. J. Polym. Sci.
scattering experiments. Makromol. Chem. Rapid Com- 1954, 12, 1.
mun. 1985, 6, 767. 37. Akcasu, A.Z.; Benmouna, M. Concentration eects on the
12. Denkinger, P.; Burchard, W. Determination of chain dynamic structure factor. Macromolecules 1978, 11, 1193.
stiness and polydispersity from static light scattering. J. 38. Burchard, W.; Richtering, W. Dynamic light scattering
Polym. Sci. Phys. Ed. 1991, 29, 589. from polymer solutions. Prog. Colloid Polym. Sci. 1989,
13. Burchard, W. Solution properties of branched macro- 80, 151.
molecules. Adv. Polym. Sci. 1999, 143, 113. 39. Fengel, D.; Wegener, G. WoodChemistry, Ultrastruc-
14. Burchard, W. Structure formation of polysaccharides in ture, Reactions; De Gruyter: Berlin-New York, 1984.
concentrated solutions. Biomacromolecules 2001, 2, 342. 40. OSulivan, A.C. Cellulose, the structure slowly unravels.
15. Born, M. Optik; Springer-Verlag: Berlin-Heidelberg, Cellulose 1997, 4, 173.
1965. reprint. 41. Van der Hart, D.L.; Atalla, R.H. Studies of micro-
16. Debye, P. Molecular weight determination by light scat- structure in native cellulose using solid state 13C NMR.
tering. J. Chem. Phys. 1947, 51, 18. Macromolecules 1984, 17, 1465.
17. Debye, P. Angular dissimmetry of the critical opalescence 42. Horii, F.; Yamamoto, H.; Kitamura, R.; Tanahashi, T.;
in liquid mixtures. J. Chem. Phys. 1959, 31, 680. Higuchi, T. Transformation of native cellulose crystals
18. van der Hulst, H.C. Light Scattering by Small Molecules; induced by saturated steam at high temperatures. Macro-
Wiley: New York, 1957. molecules 1987, 20, 2946.
19. Mie, G. Be` trage zur Optik truber Medien, speziell 43. Yamamoto, H.; Horii, H. CP/MAS 13C Analysis of the
kolloidaler Metallosungen. Ann. Phys. 1908, 25, 377. crystal transformation induced for Valonia cellulose by
20. Zimm, B.H.; Stockmayer, W.H. The dimension of chain annealing at high temperatures. Macromolecules 1993,
molecules containing branches and rings. J. Chem. Phys. 26, 1313.
1949, 17, 1301. 44. Banks, W.; Greenwood, C.T. Starch and its components;
21. Rayleigh, D.W. The incidence of light upon a transparent University Press: Edinburg, 1975.
sphere of dimensions comparable with the wavelength. 45. Guilbot, A.; Mercier, C. In Starch, The Polysaccharides;
Proc. R. Soc. (London) 1910, A84, 25. Aspinall, G.O., Ed.; Academic Press: Orlando-New York,
22. Neugebauer, T. Calculation of scattering of light by solu- 1985; Vol. 3, 209282.
tions of open-chain compounds. Ann. Phys. 1943, 42, 509. 46. Wu, H.H.; Sarko, A. The crystal structure of A-, B- and C-
23. Debye, P. Angular dissimmetry of scattering and shape of polymorphs of amylose and starch. Starch/Starke 1978,
particles. In Light Scattering From Dilute Polymer Solu- 30, 78.
tions; McIntyre, D., Gornick, F., Eds.; Gordon & Breach: 46a. Wu, H.H.; Sarko, A. The double helical molecular struc-
New York, 1964. ture of crystalline B-amylose. Carbohydr. Res. 1978,
24. Zimm, B.H. Apparatus and methods for measurement 61, 7.
and interpretation of the angular variation of light 47. Klemm, D.; Philipp, B.; Heinze, T.; Heinze, U.; Wagen-
scattering. J. Chem. Phys. 1948, 16, 1099. knecht, W. Comprehensive Cellulose Chemistry; Wiley-
25. Nordmeier, E. Static and dynamic light scattering VCH: Weinheim, 1998; Vol. 2, Chap. 4.3.
solution behavior of pullulan and dextran in comparison. 48. Seger, B.; Burchard, W. Structure of cellulose in cuoxam.
J. Phys. Chem. 1993, 21, 5770. Macromol. Symp. 1994, 83, 291.
26. Dentini, M.; Coviello, T.; Burchard, W.; Crescenzi, V. 49. Burchard, W.; Habermann, N.; Klufers, P.; Seger, B.;
Light scattering from gellan and from the extracellular Wilhelm, U. Cellulose in Schweizers reagent: A stable
polysaccharide of Rhizobium trifolii (strain TA-1) in the polymer complex with high chain stiness. Angew. Chem.
ordered state. Macromolecules 1988, 21, 3312. 1994, 106, 936.
27. Burchard, W.; Keppler, D.; Decker, K. Uber Grosse, 50. Saalwachter, K.; Burchard, W.; Klufers, P.; Kettenbach,
Form und Partikelgewicht von nativem Glykogen aus G.; Duarmaa, S.; Klemm, D. Cellulose solutions in water
Rattenleber. Makromol. Chem. 1968, 155, 250. containing metal complexes. Macromolecules 2000, 33,
28. Rosati, G. Enzyme treatment of glycogen particles in rat 4094.
liver and muscle. J. Ultrastruct. Res. 1967, 18, 444. 51. Henley, D. Arkiv kemi 1961, 18, 327.
29. Berry, G.C. Thermodynamic and conformational proper- 52. Jayme, G. Cellulose and Cellulose Derivatives; Bikales,
ties of polystyrene: I. Light scattering studies on dilute M.M., Segal, L., Eds.; Wiley: New York, 1971.
solutions of linear polystyrene. J. Chem. Phys. 1966, 44, 53. Burger, J.; Kettenbach, G.; Klufers, P. Coordination
4550. equilibria in transient metal based cellulose solvents.
30. Guinier, A.; Fournet, G. Small Angle Scattering of X-rays; Macromol. Symp. 1995, 99, 113.
Wiley: New York, 1955; 25 pp. 54. Kettenbach, G.; Klufers, P.; Mayer, P. Deprotonation
31. Guinier, A. La diraction des rayons X aux tre`s petits patterns of disaccharides in coordinating cellulose sol-
angles; Appication a` letude de phenome`nes ultramicro- vents: A status report. Macromol. Symp. 1997, 120, 291.
scopique. Ann. Phys. 1938, 12, 166. 55. Gralen, N. Sedimentation and diusion measurements on
32. Siegert, A.J.F. Report 465 of th MIT Rad. Laboratory cellulose and cellulose derivatives, Ph.D. Thesis, Uppsala,
1943. 1944.
232 Burchard
56. Casassa, E.F. Light scattering from very long rod-like weight amylose by dimethyl sulfoxide dispersion. J.
particles and an application to polymerized brinogens. Polym. Sci. 1960, 46, 65.
J. Chem. Phys. 1955, 23, 596. 78. Whistler, R.L.; Hilbert, G.E. Separation of amylose and
57. Holtzer, A. Interpretation of the angular distribution of amylopectin by certain nitroparans. J. Am. Chem. Soc.
the light scattered by a polydisperse system of rods. 1945, 67, 1161.
J. Polym. Sci. 1955, 17, 432. 79. French, D.; Plley, A.O.; Whelan, W.J. Starch fractiona-
58. McCormick, C.L.; Lichatowich, D.K. Homogeneous tion by hydrophobic complex formation. Starch/Starke
solution reaction of cellulose, chitin and other poly- 1963, 15, 349.
saccharides to produce controlled-activity pesticide sys- 80. See Ref. 44, 141 pp.
tems. J. Polym. Sci. Polym. Lett. 1979, 17, 479. 81. Husemann, E.; Pfannemuller, B.; Burchard, W. Streu-
59. Dawsey, T.R.; McCormick, C.L. The lithium chloride/ lichtmessungen und Viskositatsmessungen an wassrigen
dimethylacetamide solvent for cellulose. J. Macromol. Amyloselosungen. Makromol. Chem. 1963, 59 (1), 16.
Sci. Rev. Macromol. Chem. Phys. 1990, C30, 405. 82. Everett, W.W.; Foster, J.F. The conformation of amylose
60. Terbojewich, M.; Cosami, A.; Conio, G.; Ciferri, A.; in solution. J. Am. Chem. Soc. 1959, 81, 3464.
Bianchi, E. Mesophase formation and chain rigidity in 83. Banks, W.; Greenwood, C.T. Hydrodynamic properties
cellulose derivatives: 3. Aggregation of cellulose in N,N- and dimensions of linear potato amylose molecules in
dimethylacetamide-lithium chloride. Macromolecules dilute aqueous salt solutions. Makromol. Chem. 1963, 67,
1984, 18, 640. 49.
61. Schelosky, N.; Roder, T.; Baldinger, T. Molamassenver- 84. Cowie, J.M.G. Studies on amylose and its derivatives:
teilung cellulosischer Produkte mittels Grossenaussch- Part1. Molecular size and conguration of amylose mole-
kusschromatigraphie in DMAc/LiCl. Das Papier 1999, cules in various solvents. Makromol. Chem. 1961, 42, 230.
53, 728. 85. Kadoma, M.; Node, H.; Kamata, T. Conformation of
62. Ekmanis, J.L. Gel permeation chromatographic analysis amylose in water. Light scattering and sedimentation-
of cellulose in N,N-dimethylacetamide/lithium chloride. equilibrium measurements. Biopolymers 1978, 17, 985.
Polym. Notes Waters 1980, 1. 86. Ring, S.G.; JAnson, K.J.; Morris, V. Static and dynamic
63. Roder, T.; Morgenstern, B.; Glatter, O. Solutions of light scattering studies of amylose solutions. Macro-
cellulose in N,N-dimethylacetamide/lithium chloride. molecules 1985, 18, 182.
Polymer 2001, 42, 6765. 87. Rollings, J.E. In Laser Light Scattering in Biochemistry;
64. Johnson, D.L. Brit. Pat. No. 1 144 048, 1969. Harding, S.E., Ed.; The Royal Society of Chemistry:
65. McCorsley, C.C. US Pat. No. 4 144 980, 1979. Cambridge, 1992.
66. Chanzy, H.; Dube, M.; Marchessault, R.H. Crystalliza- 88. Burchard, W. Viskositatsverhalten von Amylose in ver-
tion of cellulose with N-methylmorpholine N-oxide: A schiedenen Losungsmitteln. Makromol. Chem. 1963, 64,
new method of texturing cellulose. J. Polym. Sci. Polym. 16.
Lett. 1979, 17, 219. 89. Brant, D.A.; Burton, B.A. The conformational statistics
67. Gagnaire, D.; Mancier; Voncendon, M. Cellulose organic of pullulan and related glucans. In Solution Properties of
solutions. A nuclear magnetic resonance investigation. J. Polysaccharides; Bant, D.A., Ed.; ACS Symp. Ser. 1980,
Polym. Sci. Polym. Chem. 1980, 18, 13. 150, 81.
68. Morgensterm, B.; Roder, T. Investigations on structures 90. Goebel, K.D.; Brant, D.A. Conguration of amylose and
in the system cellulose/N-morpholine-N-oxide monohy- derivatives in aqueous solutions. Experimental results.
drate by means of light scattering measurements. Das Macromolecules 1970, 3, 634.
Papier 1998, 52, 713. 91. Brant, D.A.; Goebel, K.D. General treatment of the
69. Roder, T.; Morgenstern, B. The inuence of activation of congurational statistics of polysaccharides. Macromo-
the solution state of cellulose, dissolved in N-methylmor- lecules 1975, 8, 522.
pholin-N-oxide-monohydrate, Polymer 1999, 40, 4143. 92. Holo, J.; Szeitli, J. Die Struktur der Amylosemolekule in
70. Drechsler, U.; Radista, S.; Vorwerg, W. Characterization wassriger Losung. Starch/Starke 1958, 10, 49.
of cellulose in solvent mixtures with N-methylmorpholin- 93. Pfannemuller, B.; Mayerhofer, R.; Schulz, R.C. Con-
N-oxide by static light scattering. Macromol. Chem. Phys. formation of amylose in aqueous solution: Optical
2000, 201, 2023. rotatory dispersion and circular dichroism of amylose-
71. Fischer, S.; Voigt, W.; Fischer, K. The behavior of iodine complexes and dependence on chain length of
cellulose in hydrated melts of the composition LiX.bul. retrogradation of amylose. Biopolymers 1971, 10, 243.
nH2O (X = I, NO3 , CH3COO, ClO4 ). Cellulose 1999, 94. Pfannemuller, B.; Ziegast, G. Properties of aqueous amy-
S3, 1. lose-iodine solutions. Solution properties of polysaccha-
72. Fischer, S.; Leipner, H.; Brendler, E.; Voigt, W.; Fischer, rides. Brant, D.A., Ed.; ACS Symp. Ser. 1981, 150, 529.
K. Molten inorganic salt hydrates as cellulose solvents. 95. Flory, P.J. Phase equilibria in solutions of rodlike
ACS Symp. Ser 1999, 737, 143. particles. Proc. R. Soc 1956, A234, 73.
73. Leipner, H.; Fischer, S.; Brendler, E.; Voigt, W. Structure 95a. Flory, P.J. Statistical thermodynamics of mixtures of
changes of cellulose dissolved in molten salts hydrates. rodlike particles: 1. Theory for polydisperse systems; 2.
Macromol. Chem. Phys. 2000, 201, 2041. Ternary systems; 3. Most probable distributions; 4. Pois-
74. Seger, B.; Asberle, T.; Burchard, W. Solution behavior of son distribution. Macromolecules 1978, 11, 1119; 1121;
cellulose and amylose in iron-sodiumtartrate (FeTNa). 1126:1141.
Carbohydr. Polym. 1996, 31, 105. 96. Roger, P. Comportement hydrodynamique de la`minose
75. Schoch, T.J. Fractionation of starch by selective precip- en solutions diluces, Ph.D. Thesis, Universite de Nantes,
itation with butanol. J. Am. Chem. Soc. 1941, 64, 2957. 1999.
Cereal Chem. 1941, 18, 121. 97. Gidley, M.J.; Bulpin, P.V. Aggregation of amylose in
76. Husemann, E.; Burchard, E.; Pfannemuller, B.; Werner, aqueous systems. Macromolecules 1989, 22, 341.
R. Uber die Bestimmung des Molekukargewichts von 97a. Gidley, M.J. Molecular mechanism underlying amylose
Amylosen. Starch/Starke 1961, 13, 196. aggregation and gelation. Macromolecules 1989, 22, 351.
77. Killion, J.P.; Foster, J.F. Isolation of high molecular 97b. Clark, A.H.; Gidley, M.J.; Richardson, R.H.; Ross-
Murphy, S.B. Rheological studies of aqueous amylose 119. Crescenzi, V.; Dentini, M. Solution conformation of the
gels: The eect of chain length and concentration on gel polysaccharide gelan. In Gums and Stabilizers for the Food
modulus. Macromolecules 1989, 22, 346. Industry. 4; Philipps, G.O., Wedlock, D.J., Williams,
98. Aberle, T.; Burchard, W.; Vorwerg, W.; Radosta, S. P.A., Eds.; IRL Press: Oxford, 1988.
Conformational contributions of amylose and amylopec- 120a. Janson, P.J.; Lindberg, B.; Sandford, P.A. Structure
tin to the structure properties from various starches. studies of gellan gums. An extracellular polysaccharide
Starch/Starke 1994, 46, 329. elaborated by Pseudomonas elodea. Carbohydr. Res
99. Radosta, S.; Haberer, M.; Vorwerg, W. Molecular 1983, 124, 135.
characterization of amylose and starch in dimethyl 120b. ONeill, M.A.; Selvedon, R.; Morris, V.R. Structure of
sulfoxide. Biomacromolecules 2001, 2, 970. the acidic extracellular gelling polysaccharide produced
100. Wolf, Ch.; Elsasser-Beile, U.; Stirm, S.; Dutton, G.S. by Pseudomonas elodea. Carbohydr. Res. 1983, 124, 123.
Conformational structure of bacterial polysaccharides: II. 120c. Chowdhury, T.A.; Lindberg, B.; Lindquist, U. Structural
Optical activity of some bacterial capsular polysacchar- studies of an extracellular polysaccharide (S-198) elabo-
ides. Biopolymers 1978, 17, 731. rated by Alcaligens ATCC 31853. Carbohydr. Res. 1987,
101. Jann, K.; Jann, B. Structure and biosynthesis of O- 161, 127.
antigens. In Handbook of endotoxins; Rietschel, E.T., Ed.; 121. Coviello, T.; Burchard, W.; Dentini, M.; Crescenzi, V.;
Elsevier: Amsterdam, 1984; Vol. 1, 138 pp. Morris, V.J. Solution properties of exocellular polysac-
102. Jann, K.; Jann, B. Polysaccharide antigens of Escherichia charides of Rhizobium leguminosarum. Static and dynamic
coli. Rev. Infect. Dis. 1987,9, 517. light scattering characterization in dilute solution. J.
103. Jann, K.; Jann, B. The K-antigens of Escherichia coli. Polym. Sci., Part B 1995, 33, 1833.
Progr. Allergy 1983, 33, 53. 122. Wegner, G. Photochemistry and photophysics of nano-
104. Sutherland, I.W. Surface Carbohydrates of the Prokary- components prepared from rod-like macromolecules by
otic Cell; Academic Press: New York, 1977. LB-technique. Mol .Cryst. Liq. Cryst. 1992, 216, 7.
105. Atkins, E.D.T.; Nieduszynski, I.A.; Porker, K.D.; Smol- 123. Ballau, M. Phase equilibria in rod-like systems
ko, E.E. Structural components of algic acid I and II. The with exible chain molecules. Macromolecules 1986, 19,
crystalline structure of polyh-D-mannuronic acid. Results 1366.
of X-ray diraction and polarized infrared studies. 124. Zimm, B.H.; Bragg, J.K. Theory of phase transition
Biopolymers 1973, 12, 865, 874. between helix and random coil in polypeptide chains. J.
106. Atkins, E.D.T.; Isaac, D.H.; Nieduszynski, I.A.; Phelps, Chem. Phys. 1959, 31, 526.
C.F.; Sheehan, J.K. Polyuronides: Their molecular 125. Coviello, T.; Burchard, W.; Geissler, E.; Maier, D. Static
architecture. Polymer 1974, 15, 263. and dynamic light scattering by a thermoreversible gel
107. Paradossi, G.; Brant, D.A. Light scattering study of a from Rhizobium leguminosarum 8002 exopolysaccharide.
series of xanthan fractions in aqueous solution. Macro- Macromolecules 1997, 30, 2008.
molecules 1982, 15, 874. 126. Han, C.C.; Akcasu, A.Z. Dynamic light scattering of
108. Coviello, T.; Kajiwara, K.; Burchard, W.; Dentini, M.; dilute polymer solutions in the non-asymptotic region.
Crescenzi, V. Static and dynamic light scattering from Macromolecules 19801, 14, 1080.
native and modied xanthans in dilute solution. Macro- 127. Harmau, L.; Winkler, R.G.; Reinecker, P. Remarks on
molecules 1986, 19, 2826. the interpretation of dynamic light scattering from gellan
109. Ross-Murphy, S.B.; Morris, V.J.; Morris, E.R. Molecular in dilute solution. Macromolecules 1997, 30, 6974.
viscoelasticity of xanthan polysaccharide. Faraday Symp. 128. Ding, Q.; Labelle, M.; Yang, B.Y.; Montgomery, R. Phys-
Chem. Soc. 1983, 18, 115. icochemical studies of extracellular polysaccharides of
110. Rodd, A.B.; Dunstan, D.E.; Boger, D.V.; Schmidt, J.; Erwinia chrysanthemi spp, Carbohydr. Polym. 1993,
Burchard, W. Heterodyne and non-ergodic approach to 51, 333.
dynamic light scattering of polymer gels. Aqueous 129. Yang, B.Y.; Ding, Q.; Montgomery, R. Extracellular
xanthan in the presence of metal ions (aluminum III). polysaccharides of a bacterium associated with a fungal
Macromolecules 2001, 34, 3339. canker disease of Eucalyptus sp. Carbohydr. Res. 2002,
111. Holtzwarth, G. Molecular weight of xanthan polysac- 337, 731.
charide. Cabohydr. Res. 1978, 66, 173. 130. Xu, X.; Zhang, L.; Nakamura, Y.; Norisuye, T. Chain
112. Stokke, B.T.; Smidsrod, O.; Elgsaeter, A. Electron stiness of heteropolysaccharide from Aeromonas gum in
microscopy of native xanthan and xanthan exposed to dilute solution by dynamic light scattering. Biopolymers
low ionic strength. Biopolymers 1989, 28, 617. 1998.
113. Koyama, R. Light scattering of sti chain polymers. 131. Zhang, K.; Xu, X.; Iijima, G.; Tsuchiya, H. Aggregation
J. Phys. Soc. Jpn. 1973, 34, 1029. of Aeromonas gum in aqueous solution. Polym. J. 1999,
114. Daniels, H.E. The statistical theory of sti chains. Proc. 31, 150.
R. Soc. Edinburg 1952, 63, 290. 132. Wolf, Ch. Konformationsuntersuchungen an bakteriellen
115. Kratky, O.; Porod, G. Diuse small angle scattering of Heteropolysacvhariden der Kapseln von Klebsiella K5,
X-rays and sphere clusters: Cellulose application. J. K8 and K56. Unpublished data from the Ph. D. Thesis,
Colloid Sci. 1949, 4, 35. University of Freiburg, 1977.
116. Crescenzi, V.; Dentini, M.; Coviello, T. In Recent 133. Eschwey, A. Untersuchungen zur Uberstruktur bakteriel-
Developments in Industrial Polysaccharides; Stivala, S.S., ler Kapselpolysacchaide: Unpublished data from the
Crescenzi, V., Dea, T.M.M., Eds.; Gordon Breach: New Ph.D. Thesis, University of Freiburg, 1976.
York, 1987; 68 pp. 134. Chen, F.C.; Tscharnuter, W.; Schmidt, D.; Chu, B.
117. Kang, K.S.; Veeder, G.T.; US Patent 4,326,053, 1982. Measurement of diusion coecients of Meningococcal
118. Chapman, H.D.; Chilvers, G.R.; Miles, M.J.; Morris, V.J. polysaccharides. Optical mixing spectroscopy. A prelimi-
Studies on gelation of microbial polysaccharides XM16 nary characterization on the aggregation of group C
and gelan gum. In Gums and Stabilizers for the Food polysaccharide. Biopolymers 1974, 13, 2281.
Industry. 4.; Phillips, G.O., Wedlock, D.J., Williams, P.A., 135. Fudjime, S.; Moro, K.; Chu, B.; Liu, T.Y. Character-
Eds.; IRL Press: Oxford, 1988; 147 pp. ization of diusion coecients of Meningococcal poly-
234 Burchard
saccharides by optical mixing spectroscopy. Eect of saccharide sulphates: double helix models for k- and l-
temperature, EDTA and ionic strength on the aggregation carrageenans. J. Mol. Biol 1969, 45, 85.
of group C polysaccharide. Biopolymers 1977, 16, 945. 157. Arnott, S.; Scott, W.E.; Rees, D.A.; McNab, C.G.A.
136. Gulari, E.; Chu, B.; Liu, T.Y. Characterization of L-carrageenan: Molecular structure and packing of poly-
Meningococcal polysaccharides by light scattering. Mo- saccharide double helices in oriented bers of divalent
lecular weight distribution of group B Meningococcal cation salts. J. Mol. Biol. 1974, 90, 253.
polysaccharide in dilute solution. Biopolymers 1979, 18, 158. ter Meer, H.-U. Strukturelle Untersuchungen der ther-
2943. moreversiblen Gelierung von Iota-Carrageenenan.
137. DiNapoli, A.; Chu, B.; Liu, T.Y. Light scattering Ph.D. Thesis University o Freiburg, 1984.
spectroscopy of Meningococcal polysaccharides. In Sol- 159. Hjerdje, T.; Smidsroed, O.; Sokke, B.T.; Christensen, B.E.
ution Properties of Polysaccharides; Brant, D.A., Ed.; Acid hydrolysis of n-carrageenan in the disordered and
ACS Symp. Ser. 1981, 150, 173. ordered conformations; characterization of partially hydro-
138. Harada, T.; Masada, M.; Fudjimori, K.; Maeda, I. Agric. lyzed sample and single stranded oligomers released from
Biol. Chem. 1960, 30, 196. the ordered structure. Macromolecules 1998, 31, 1842.
139. Saito, H.; Ohki, T.; Sasaki, T. A 13C NMR study of gel- 160. Bongaerts, K.; Reynaers, H.; Zernetti, F.; Paoletti, S. (a)
forming (1,3)h-d-glucans. Molecular weight dependence On the molar mass of n-carrageenan in the course of
of helical conformation. Biochemistry 1978, 16, 908; and conformational transition from disordered to the funda-
of the presence of junction zones for association of mental ordered form. Macromolecules 1999, 32, 675; (b)
primary molecules. Macromolecules 1978, 11, 1244. Equilibrium and nonequilibrium association processes of
140. Sasaki, T., et al. Cancer Res. 1978, 38, 379. n-carrageenan in aqueous salt solutions. Macromolecules
141. Norisuye, T.; Yanaki, T.; Fujita, H. Triple helix of 1999, 32, 683.
Schizophyllan commune polysaccharide in aqueous sol- 161. Bongaerts, K.; Paoletti, S.; Denef, B.; Varneste, K.;
ution. J. Polym. Sci. 1980, 18, 547. Cuppo, F.; Reynaers, H. Light scattering investigation of
142. Norisuye, T.; Yanaki, T.; Fujita, H. Triple helix of L-carrageenan aqueous solutions. Concentration depend-
Schizophyllan commune polysaccharide in aqueous solu- ence of association. Macromolecules 2000, 33, 8709.
tion: 3. Hydrodynamic properties in water. Macromole- 162. Cuppo, F.; Renaers, H. Light scattering investigation of
cules 1980, 13, 1462. association phenomena in saline carrageenan solution.
143. Kashiwagi, Y.; Norisuye, T.; Fujita, H. Triple helix of Macromol. Symp. 2002, 190, 65.
Schizophyllan commune polysaccharide in dilute solution. 163. Meunier, V.; Nicolai, T.; Durand, D. Structure and
Light scattering and viscosity in dilute aqueous sodium kinetics of aggregating n-carrageenan studied by light
hydroxide. Macromolecules 1981, 14, 1220. scattering. Macromolecules 2000, 33, 2497.
144. Kitamura, S. A dierential scanning calorimetric study of 164. Heyer, A.G.; Schroeer, B.; Radosta, S.; Wol, D.; Czapla,
the conformational transition of Schizophyllan commune S.; Springer, J. Structure of the enzymatically synthesized
in mixture of water and DMSO. Biopolymers 1989, 28, fructan inulin. Carbohydr. Res. 1998, 313, 165.
639. 165. Bahary, W.S.; Stivala, S.S.; Newbrun, E.; Ehrlich, J.
145. Fuchs, T.; Richtering, W.; Burchard, W. Thermorever- Levans: III. A light scattering study of Streptococcus
sible gelation of a polysaccharide with immunological salivarius levan in dimethyl sulfoxide. Biopolymers 1975,
activity. Rheology and dynamic light scattering. Macro- 14, 2467.
mol. Symp. 1995, 99, 227. 166. Garg, S.K.; Stivala, S.S. Assessment of branching in
146. Fuchs, T. Thermoreversile Gelierung von Schizophyllan, polymers from small angle X-ray scattering. J. Polym. Sci.
Ph.D. Thesis University of Freiburg, 1996. 1978, 16, 1419.
147. Brant, D.A. Conformational theory applied to polysac- 167. Stivala, S.S.; Zweig, J.E.; Ehrlich, J. Dilute solution
charide structure. Q. Rev. Biophys. 1976, 9, 527. properties of Streptococcus salivarius levan and its hydro-
148. Brant, D.A. In The Biochemistry of Plants; Preiss, J., Ed.; lysates. ACS Symp. Ser. 1981, 150, 101.
Academic Press: New York, 1980; Vol. 3, Chap. 11. 168. Sivala, S.S.; Zweig, J.E. Physicochemical parameters of
149. Brant, D.A.; Burton, B. The congurational statistics of partially hydrolyzed S. salivarius levan fractions. Biopol-
pullulan and some related glucans. ACS Symp. Ser. 1981, ymers 1981, 20, 605.
150, 81. 169. Wol, D.; Czapla, S.; Heyer, AS.G.; Radosta, S.;
150. Kato, T.; Katsuki, T.; Takahashi, A. Static and dynamic Mischnick, P.; Springer, J. Globular shape of high molar
solution properties of pullulan in a dilute solution. mass inulin revealed by static light scattering and
Macromolecules 1984, 17, 1726. viscometry. Polymer 2000, 41, 8009.
151. Rees, D.A. Structure, conformation and mechanism in 170. Kitamura, S.; Hirano, T.; Takeo, K.; Mimura, M.;
the formation of polysaccharide gels and networks. Adv. Kajiwara, K.; Stokke, B.T.; Harada, T. Conformation
Carbohydr. Chem. Biochem. 1969, 24, 267. of (2->1)-beta-D-fructan in aqueous solution. Int. J. Biol.
152. Morris, E.R.; Rees, D.A. Principles of biopolymer Macromol. 1994, 16, 313.
gelation. Possible models for mucus gel structure. Brit. 171. Burchard, W. Solution properties of branched macro-
Med. Bull. 1978, 34, 49. molecules. Adv. Polym. Sci. 1999, 143, 113.
153. Morris, E.R.; Rees, D.A.; Robinson, G.J. Cation-specic 172. Burchard, W. Light scattering from reacting polymer
aggregation of carrageenan helices: Domain model of systems. Associating polymers in a good solvent. Macro-
polymer gel structure. J. Mol. Biol 1980, 138, 349. mol. Symp. 1990, 39, 179.
154. Smidsroed, O.; Andersen, I.L.; Grasdalen, H.; Larsen, B.; 173. Cleve, J.W.; Schaefer, W.C.; Rist, C.E. The structure of
Painter, T. Evidence for a salt-promoted freeze-out of NRRL-B512 dextran. Methylation studies. J. Am. Chem.
linkage conformation in carrageenans as pre-requisite for Soc. 1956, 78, 4435.
gel-formation. Carbohydr. Res. 1980, 80, C11. 174. Kenne, L.; Lindberg, B. Bacterial polysaccharides. In The
155. Paoletti, S.; Smidsroed, O.; Grasdalen, H. Thermody- Polysaccharides; Aspinall, G.O., Ed.; Academic Press:
namic stability of the ordered conformation of carra- London, 1983; Vol. 2, 346 pp.
geenan polyelectrolyte. Biopolymers 1984, 23, 1771. 175. Sandford, P.A.; Baird, J. Industrial Utilization of Poly-
156. Andersen, N.S.; Campbell, J.W.; Harding, M.M.; Rees, saccharides. G.O. Aspinall Ed. The Polysaccharides. Aca-
D.A.; Samuel, J.W. X-ray diraction studies of poly- demic Press, London, 1983; Vol. 2, 474 pp.
176. Misaki, A.; Toril, M.; Sawai, T.; Goldstein, I.J. Structure 198. Aberle, T.; Burchard, W. Inuence of amylose content in
of the dextran of Leuconostoc mesenteroides N-1355. starches on molar mass and aggregated structure in semi-
Carbohydr. Res. 1980, 84, 273. dilute solution. Biopolym. Sci. & New Food Appl. 1999,
177. Wales, M.; Marshall, P.A.; Weissberg, S.G. Intrinsic 91, 247.
viscosity molecular weight relationship of dextran. J. 199. Witnauer, L.P.; Senti, F.R.; Stern, M.D. Light scattering
Polym. Sci. 1953, 10, 229. of potato amylopectin. J. Polym. Sci. 1955, 16, 1.
178. Senti, F.Z.; Hellmann, N.N.; Ludwig, N.H.; Babarck, 200. Gaklinsky, G.; Burchard, W. Starch fractions as exam-
G.E.; Tobin, R.; Glass, C.A.; Lamberts, B.L. Viscosity, ples for non-randomly branched macromolecules; (a)
sedimentation and light scattering properties of fractions Dimensional properties. Macromolecules 1995 28,
on an acid-hydrolyzed dextran. J. Polym. Sci. 1955, 17, 2363; (b) Angular dependence in static light scattering.
527. Macromolecules 1997, 30, 4445; (c) Angular depen-
179. Kuge, T.; Kobayashi, K.; Kitamura, S.; Tanahashi, H. dence of dynamic light scattering. Macromolecules
Degrees of long-chain branching in dextran. Carbohydr. 1997, 30, 6966.
Res. 1987, 160, 205. 201. Fox, J.D.; Robyt, J.F. Modication of starch granules by
180. Hanselmann, R.; Burchard, W. Characterization of hydrolysis with hydrochloric acid in various alcohols, and
DEAE-dextran by means of light scattering and combined the formation of new kind of limit dextrins. Carbohydr.
size exclusion chromatography/low angle laser light Res. 1992, 227, 163.
scattering/viscometry. Makromol. Chem. 1995, 196, 2259. 202. Kainnuma, K.; French, D. Nageli amylodextrin and its
181. Ioan, C.E.; Aberle, T.; Burchard, W. (a) Structure proper- relationship to starch granule structure: 1. Preparation
ties of dextran; Dilute solution. Macromolecules 2000, 33, and properties of amylodextrins from various starch
5730; (b) Shrinking factors of individual clusters. Macro- types. Biopolymers 1971, 10, 1673.
molecules 2001, 34, 3765. 203. Flory, P.J. Principle of Polymer Chemistry; Cornell
182. Ioan, C.E.; Aberle, T.; Burchard, W. Light scattering and University Press: Ithaca, N.Y., 1953.
viscosity behavior of dextran in semidilute solution. 204. Erlander, S.R.; French, D. A statistical model for
Macromolecules 2001, 34, 326. amylopectin and glycogen. The condensation of A-R-
183. Zimm, B.H.; Stockmayer, W.H. The dimensions of chain Bf-1 units. J. Polym. Sci. 1956, 20, 7.
molecules containing branches and rings. J. Chem. Phys. 205. Debye, P.; Bueche, A.M. Scattering by an inhomogeneous
1949, 17, 1301. solid. Scattering measurements used to obtain the average
184. Stockmayer, W.H.; Fixman, M. Dilute solutions of square of uctuation in refractive index and electron
branched polymers. Ann. N. Y. Acad. Sci. 1953, 12, 1. density, and a correlation function which measures the
185. Zimm, B.H.; Kilb, R.W. Dynamics of branched polymer degree of correlation between two uctuations as a function
molecules in dilute solution. J. Polym. Sci. 1959, 37, 19. of this distance separation. J. Appl. Phys. 1949, 20, 518.
186. Kurata, M.; Abe, M.; Iwawa, M.; Matsushima, M. 206. Burchard, W. Angular distribution in Rayleigh scattering
Randomly branched polymers: I. Hydrodynamic proper- from branched polycondensates of amylopectine and
ties. Polym. J. 1972, 3, 729. glycogen type. Macromolecules 1972, 5, 604.
187. Flory, P.J.; Fox, T.G. Treatment of intrinsic viscosity. J. 207. Ganazzoli, F. Linear, branched and hyperbranched
Am. Chem Soc. 1951, 73, 1904. macromolecules in dilute solution. Macromol. Symp.
188. Burchard, W. Angular distribution of Rayleigh scattering 2002, 190, 55. and further literature therein.
from branched polycondensates of the amylopectin and 208. Hanselmann, R.; Burchard, W.; Ehret, M.; Widmer,
glycogen type. Macromolecules 1972, 5, 604. H.M. Structure properties of fractionated starch polymers
189. Burchard, W.; Kajiwara, K.; Nerger, D. Static and and their dependence on the dissolution process. Macro-
dynamic scattering behavior of regularly branched chains. molecules 1996, 29, 3277.
A model of soft sphere microgels. J. Polym. Sci. 1982, 20, 209. Giddings, J.C. Non equilibrium theory of eld ow
157. fractionation. J. Chem. Phys. 1968, 49, 81.
190. Whistler, R.L.; BeMiller, J.N.; Paschall, E.F. Starch: 210. Seger, B.; Aberle, T.; Burchard, W. Solution behavior of
Chemistry and Technology, 2nd Ed.; Academic Press: cellulose and amylose in sodium iron tartrate (FeTNa).
Orlando, 1981. Carbohydr. Polym. 1996, 31, 105.
191. Jenkins, P.J.; Cameron, R.E.; Donald, A.M.; Brass, W.; 211. Radosta, S.; Vorwerg, W. Personal communication.
Derbyshire, G.E.; Mant, G.E.; Ryan, A.J. In situ 212. Cook, G.M.W.; Stoddart, R.W. Surface Carbohydrate of
simultaneous small and wide angle X-ray scattering: A the Eukaryotic Cell; Academic Press: London, 1973; 177
new technique to study starch gelatinization. Polym. Sci., 179. Chap. 4.
Phys. Ed. 1994, 32, 1579. 213. Rees, D.A.; Welsh, E.J. Secondary and tertiary structure
192. Jenkins, P.J.; Donald, A.M. The inuence of amylose on of polysaccharides in solution and in gels. Angew. Chem.
starch granule structure. Int. J. Biol. Macromol. 1995, 17, Int. Ed. 1977, 16, 214.
315. 214. Voragen, F.G.J.; Pilnik, W. Pectin degrading enzymes in
193. Banks, W.; Geddews, R.; Greenwood, C.T.; Jones, I.G. fruit and vegetable processing. Biocatalysis in Agricultural
Physicochemical studies on starches: Molecular size and Biotechnology; Whitaker, J., Sonnet, P., Eds.; J. Am.
shape of amylopectin. Starch/Starke 1972, 24, 245. Chem. Soc. 1989; 93.
194. Stacy, C.J.; Foster, J.F. A light scattering study of corn 215. Will, F.; Dietrich, H. Isolation, purication and charac-
amylopectin and its beta-amylase limit dextrins. J. Polym. terization of neutral polysaccharides from extracted apple
Sci. 1956, 20, 76. juices. Carbohydr. Polym. 1992, 18, 109.
195. Erlander, S.R.; French, D. Starch granules and validity of 216. Saulnier, L.; Brillouet, J.M.; Joselean, P.J. Structure
light scattering results on amylopectin. J. Am. Chem. Soc. studies of pectic substances from the pulp of grape berries.
1958, 80, 4413. Carbohydr. Res. 1988, 182, 63.
196. Andres-Koksal, K. Starke in Losung: Eine Struktur im 217. Berth, G.; Dautzenberg, H.; Lexow, D.; Gother, G. The
Bereich zwischen makromolekularem und kolloidalem determination of the molecular weight distribution of
Verhalten, Ph.D. Thesis University of Freiburg, 1996. pectin by calibrated GPC: Part 1. Calibration by light
197. Kirsten andres-Koksal, K.; Burchard, W.; Vorwerg, W. scattering and membrane osmometry. Carbohydr. Polym.
unpublished data. 1990, 12, 39.
236 Burchard
218. Harding, S.E.; Berth, G.; Ball, A.; Mitchell, J.R.; Garcia 235. Lang, P.R. Assoziationsverhalten und thermoreversible
de la Torre, J. The molecular weight distribution and Gelierung von Tamarind Samen Polysaccharid (TSP).
conformation of citrus pectins in solution studied by Ph.D. Thesis, University of Freiburg, 1991.
hydrodynamics. Carbohydr. Polym. 1991, 16, 1. 236. Lang, P.R.; Burchard, W. Structure and aggregation
219. Chapman, H.D.; Morris, V.J.; Selvendran, R.R.; ONeil, behavior of tamarind seed polysaccharide in aqueous
M.A. Static and dynamic light scattering studies of pectic solution. Macromol. Chem. 1993, 194, 3157.
polysaccharides from the middle lamellae and primary cell 237. Picout, D.A.; Ross-Murphy, S.B.; Errington, N.; Har-
walls of cider apples. Carbohydr. Res. 1987, 165, 53. ding, S.E. Pressure cell assisted solution characterization
220. Fishman, M.L.; Chan, H.K.; Hoogland, P.; Ayad, K. of polysaccharides: 1. Guar gum. Biomacromolecules
Characterization of pectin, ash extracted from orange 2001, 2, 1301.
albedo by microwave heating under pressure. Carbohydr. 238. Picout, D.A.; Ross-Murphy, S.B.; Jumel, K.; Harding,
Res. 2000, 323, 126. S.E. Pressure cell assisted solution characterization of
221. Fishman, M.L.; Chan, H.K.; Con, D.R.; Hotchkin, T., polysaccharide: 2. Locust bean gum and tara gum.
Jr. A comparison of lime and orange pectin which were Biomacromolecules 2002, 3, 761.
rapidly extracted from albedo. In Advances in Pectinase 239. Wang, Q.; Ellis, P.R.; Ross-Murphy, S.B.; Burchard, W.
Research; Voragen, A.G.J., Scholz, H.A., Vissert, R., Solution characteristics of xyloglucan extracted from
Eds.; Kluwer Press, 2003. in press. Detarium senegalis Gmelin. Carbohydr. Polym. 1997, 33,
222. Axelos, M.AS.V. Ion complexation of biopolymers: 115.
Macromolecular structure and viscoelastic properties of 240. Gupta, A.K.; Cotton, J.P.; Marchal, E.; Burchard, W.;
gels. Makromol. Chem., Macromol. Symp. 1990, 39, 323. Benoit, H. Persistence length of cellulose-tricarbanilate by
223. Berth, G. Methodical aspects of characterization of small angle neutron scattering. Polymer 1976, 17, 363.
alginate and pectate by light scattering and viscometry 241. Tanford, C. The Hydrophobic Eect; Wiley: New York,
coupled with GPC. Carbohydr. Polym. 1992, 19, 1. 1973.
224. Jacob, A.; Dahlman, O. Characterization of the molar 242. Berriaud, N.; Milas, M.; Rinaudo, M. Characterization
mass of hemicelluloses from wood and pulps, employing and properties of hyaluronic acid (hyaluronan). Dumitriu
size exclusion chromatography and matrix-assisted laser S., Ed. Polysaccharides.
desorption ionization time of ight mass spectroscopy 243. Heatley, F.; Scott, J.E. A water molecule participates in
(MALDI-TOF). Biomacromolecules 2001, 2, 894. the secondary structure of hyaluran. Biochem. J. 1980,
225. LeBel, R.G.; Goring, D.A.; Timel, A.I. Solution 254, 489.
properties of birch xylan: I. Measurement of molecular 244. Cleland, R.L. Viscometry and sedimentation equilibrium
weight. J. Polym. Sci., Part C, Polym. Symp. 1963, 2, 9; of partially hydrolyzed hyaluronate: Comparison with
Solution properties of birch xylan: II. Fractionation theoretical models of wormlike chains. Biopolymers 1984,
and conguration. J. Polym. Sci., Part C, Polym. Symp. 23, 647.
1963, 2, 29. 245. Fouissac, E.; Milas, M.; Rinaudo, M.; Borsali, R.
226. Eeremeeva, T.E.; Khinoverova, O.E. Exclusion liquid Inuence of the ionic strength on the dimensions of
chromatography of 4-O-methylglucuronoxylan in dipolar sodium hualuronate. Macromolecules 1992, 25, 5613.
aprotic solvents. Cellul. Chem. Technol. 1990, 24, 439. 246. Ghosh, S.; Kobal, I.; Zanette, D.; Reed, W.E. Conforma-
227. Ebringerova, A.; Eeremeeva, T.E.; Khinoverova, O.E.; tional contraction and hydrolysis of hyaluronate in
Ershov, B.G. 4-O-Methyl-D-glucorono-D-xylan degrada- sodium hydroxide solutions. Macromolecules 1993, 26,
tion by g-irradiation: Part 1Dose eects on the macro- 4685.
molecular properties. Carbohydr. Polym. 1991, 15, 255. 247. Yamakawa, H.; Fujii, M. Intrinsic viscosity of wormlike
228. Merz, P.C. Losungsverhalten von Xylancarbabilaten. Di- chains. Determination of the shift factor. Macromolecules
ploma Thesis, University of Freiburg, 1991. 1974, 7, 128.
229. Ebringerova, A. Xylans of industrial and biomedical 248. Ghosh, S.; Li, X.; Reed, C.E.; Reed, W.F. Apparent
importance. In Biotechnology and Genetic Engineering; persistence length and diusion behavior of high molec-
Harding, S.E., Ed.; Interapt Ltd.: England, 1994; Vol. 16, ular weight hualuronate. Biopolymers 1990, 30, 1101.
325 pp. 249. Odijk, T.; Houwart, A.C. On the theory of the excluded
230. Ebringerova, A.; Heinze, T. Xylan and xylan derivatives. volume eect of a polyelectrolyte in a 1-1 electrolyte
Biopolymers with valuable properties: 1. Natural occur- solution. J. Polym. Sci., Polym. Phys. 1978, 16, 627.
ring xylans, structure, isolation procedures and proper- 250. Schulz, L.; Burchard, W.; Donges, R. Evidence of
ties. Macromol. Rapid. Commun. 2001, 21, 542. supramolecular structures of cellulose derivatives in
231. Ebringerova, A.; Hromadkova, Z.; Burchard, W.; Dole- solution. Cellulose Derivatives, Modication, Character-
ga, R.; Vorwerg, W. Solution properties of water- ization and Nanostructures; Heinze, T.J., Glasser, W.G.,
insoluble rye-bran arabinoxylan. Carbohydr. Polym. Eds.; ACS Symp. Ser. 1998, 688, 218.
1994, 24, 181. 251. Stauer, D. Introduction to Percolation Theory; London:
232. Ebringerova, A.; Hromadkova, Z.; Berth, G. Structure Taylor and Francis, 1985.
and molecular properties of water-soluble arabinoxylan 252. Daoud, M.; Martin, J.E. Fractal properties of polymers.
protein complexes isolated from rye-bran. Carbohydr. In The Fractal Approach to Heterogeneous Chemistry;
Res. 1994, 264, 97. Avnir, D., Ed.; Wiley: New York, 1992.
233. Dhami, R.; Harding, S.E.; Elizabeth, N.J.; Ebringerova, 253. Stanley, H.E. Introduction to Phase Transition and
A. Hydrodynamic characterization of the molar mass and Critical Phenomena; Clarendon Press: Oxford, 1971;
gross conformation of corn cob heteroxylan AGX. 176178.
Carbohydr. Polym. 1995, 28, 113. 254. Stockmayer, W.H. Theory of molecular size distribution
234. Gidley, M.J.; Lillford, P.J.; Rowland, D.W.; Lang, P.R.; and gel formation in branched-chain polymers. J. Chem.
Dentini, M.; Crescenzi, V.; Edwards, M.; Famitti, C.; Phys. 1943, 11, 45;. J. Chem. Phys. 1944, 12, 125.
Reid, J.S. Structure and solution properties of tamarind- 255. Israelachvili, J. Intermolecular and Surface Forces;
seed polysaccharide. Carbohydr. Res. 1991, 214, 299. Academic Press: London, 1992. Chap. 16.7.
8
Advances in Characterization of Polysaccharides in Aqueous
Solution and Gel State
Marguerite Rinaudo
Centre de Recherches sur les Macromolecules Vegetales (CERMAV), CNRS, and Joseph Fourier University,
Grenoble, France
Abstract state (xanthan or cellulose is well discussed in the litera-

ture) [1,2]. The stereoregularity of these polymers as well as
The aim of this chapter is to describe the main techniques intrachain H bonds lead to local stiness of the chains,
well adapted to characterize polysaccharides, especially which have to be modeled by the semirigid chain model (or
water-soluble polysaccharides; the main characteristics of worm-like chain model) characterized by a persistence
polyelectrolytes are recalled and it is shown how the ionic length [3]. This stereoregularity is the basis of the cooper-
behavior of the polymers can be used to establish the nature ative helix-coil transition depending on the thermody-
of the conformation; in this case, it is necessary to combine namic conditions (temperature, pH, ionic concentration,
thermodynamic characteristics with optical rotation (or nature of the counterions. . .). As we will show later,
DSC, CD) and molar mass determinations. The semirigid helix-coil transition is often also related to the cooperative
characterization of the polysaccharide chains depending gelsol transitions in polysaccharide systems. The origins
on their conformation is introduced; the persistence length of polysaccharides are mainly plants, animals, fungi, or
Lp, which characterizes the local stiness of the chain, is bacteria as shown in Table 1. The polymers are neutral (as
determined by steric exclusion chromatography in appli- cellulose, amylose, curdlan, scleroglucan, agarose. . .) or
cation of the worm-like model and is validated by molec- ionic (xanthan, gellan, alginate. . .) and become polyelec-
ular modeling. trolytes; this chapter will be devoted to water-soluble ionic
Rheology of polysaccharides is of great interest on polysaccharide characterization but the main character-
fundamental and applied points of view; the solutions even istics of neutral polysaccharides (stiness, H bonds, and
at low polymer concentration are non-Newtonian due to cooperativity) are obtained with the same approach as for
the stiness of the polysaccharides, which depresses the ionic polymers in the presence of external salt (the example
critical overlap concentration. The main relations relating of galactomannan is clear). The choice of solvent to be used
viscosity to molar mass, polymer concentration, and shear is the most important factor; for ionic systems, water is the
rate are given; then, the ow and dynamic measurements best solvent, the solubility being promoted by electrostatic
are described. repulsions; for neutral polymers, water often gives inter-
Finally, the mechanisms of physical gelation are acting systems (solubility is usually low), and DMSO or a
recalled and characterization of the solgel transition H-bond breaker is a better solvent in this case. The
is introduced. problem of aggregation in polysaccharide solutions is
perhaps the most important point to solve before going
further in the characterization.
I. INTRODUCTION
Polysaccharides are natural polymers based on sugar unit
repeating units; they are rich in -OH functional groups that II. POLYELECTROLYTE CHARACTERIZATION
form H bonds; in stereoregular polysaccharides (especially AND CONFORMATIONAL TRANSITION
in bacterial polysaccharides), an H-bond network is
formed as intrachain stabilizing helical conformations or When a polymer has ionic groups regularly xed on a
interchain as may be found in physical gels or in the solid polymeric chain, it is called a polyelectrolyte; the parameter
237
238 Rinaudo
Table 1 List of Some Natural Polysaccharides
Origins Polymers Main properties
Plant and Pectins Thickening, gelation

wood hemicelluloses
cell walls
Seeds and Galactomannans, Thickening, gelation
roots glucomannans
Seaweeds Carrageenans, Thickening, gelation,
alginates, agar stabilizing, suspending,
emulsication
Animal Hyaluronan, Thickening,
chitin, lm forming
chondroitins
Bacteria Xanthan, Thickening,
hyaluronan, pseudoplasticizer,
succinoglycan, gelation Figure 1 Helix-coil transition for a polyelectrolyte. b, the
gellan distance between two charged groups depends on the
Fungi Curdlan, Gelation, thickening conformation. Helical conformation is stabilized by decrease
scleroglucan, of temperature or increase of the external salt concentration.
schizophyllan
that controls the thermodynamic properties is the charge two charges, gives information on the conformation of the
parameter k, which is proportional to the linear charge chain. It also allows to conclude if it is a single helical chain,
density and introduced in the polyelectrolyte theories from a double helix on itself, or a double helix (or a helical dimer)
Katchalsky et al. [4] and later by Manning [5]. It is made of two chains if these experiments are combined with
expressed as: complementary experimental determinations such as opti-
cal rotation, which indicates that an ordered conformation
k m=h e2 =DkT 1 is formed in given conditions and of molar mass determi-
where m is the number of ionic charges along a chain with a nation (one or two chains are associated). An example is
contour length h; e is the electronic charge; D is the given in Table 2 for xanthan; here we concluded that from
dielectric constant taken as that of water (78); and kT is the native state, xanthan is a single helix in the ordered
the Boltzmann term; k is also written as k = (m / h) Q, with conformation stabilized by calcium counterions [6]; in
Q being the Bjerrum length (i.e., 7.2 A at 25jC in aqueous Table 3, the results obtained for gellan are given on both
solution). sides of the conformational transition [7], allowing to
Then, k is directly imposed by the distance b between conrm the formation of a double helix. A double helix
two ionic sites projected on the axis of the chain (or length was also demonstrated with n-carrageenan [8].
of a monomeric unit if each monomer has a charge); the Dierential scanning calorimetry (DSC), circular di-
ionic sites are mainly -COO, -NH+ chroism (CD), and NMR are convenient methods to
3 , -SO3 . The polyelec-
trolyte behavior is established as soon as the number of demonstrate the existence of an helical conformation (at
charges (or degree of polymerization) is larger than 15. least the existence of an ordered conformation in given
In addition, b is directly related to the conformation of conditions) and to determine the thermodynamic condi-
the polymer; single-chain helix formation reduces b (Fig. 1). tions for the helix-coil transition [912].
This helical conformation is stabilized by H bonds.
One of the most useful experiments to obtain infor-
mation on conformation is the determination of activity Table 2 Activity Coecient of Counterions on Xanthan at
coecient of counterions obtained by potentiometry (or 25jC in the Absence of External Salt
conductimetry). Its theoretical value is directly related to
the charge parameter and the valency of the counterion. ccalc. ccalc. cexp. cexp.
The main relations are: Xanthan (Na+) (Ca2+) (Na+) (Ca2+)
kc = 1.03 0.587 0.293 0.650

c1 f2c1 for monovalent counterions 2
kh = 1.13 0.535 0.267 0.295a
and k2h = 2.26 0.267 0.134
a
c2 f4c1 for divalent counterions 3 Optical rotation indicates the presence of an ordered conformation
(helix); the molar mass is the same on both sides of the conforma-
Then, the determination of the activity coecient of coun- tional transition.
terions, inversely proportional to b, the distance between Source: Ref. 6.
Advances in Characterization of Polysaccharides in Aqueous Solution and Gel State 239
Table 3 Activity Coecient of Counterions on Gellan concentrations covered) [1721]; qmax increases when poly-
on Both Sides of the Conformational Transition mer concentration increases; it indicates that a preferential
distance exists in solution in absence (or at low concentra-
ccalc. cexp.
tion) of external salt. The position of the peak qmax varies as
Gelan deacylateda (Na+) (Na+)
a function of C1/2 (C being the polymer concentration).
kc = 0.37 0.831 0.765 This peak is suppressed in excess of salt when Cs is larger
k2h = 0.75 0.687 0.675 than the value corresponding to Cp/Cs f2 or 2k (when k is
larger than 1); Cp is the polymer concentration expressed in
a
Molar mass is doubled in the helical conformation. equiv./L. The position of the viscosity peak was shown to
Source: Ref. 7. be independent on the molar mass of the polymer [14].
An original behavior also concerns the ionic selectivity
in some polysaccharides; the ionic selectivity is related to
An original behavior in salt-free solution is observed ion pair formation; the ionic selectivity occurs in the range
by viscometry or light scattering and neutron scattering. A of a charge parameter of 1; it was demonstrated from
peak is observed in the reduced viscosity plotted as a ultrasound experiments on carrageenans [22] and alginate
function of polymer concentration as shown in Fig. 2; but also on carboxymethylcelluloses (CMC) [23]. But for
these results are obtained on the sodium salt form of a k < 1, it was shown even in the coiled conformation for K+
short sodium polygalacturonate; rst, each curve passes and Na+ forms of n-carrageenan [22]: at the same time, it
through a maximum, the position of which depends on the
external salt concentration; it was demonstrated that the
maximum is located at Cp = 2Cs or 2 kCs (when k is larger
than 1). The major contribution to the viscosity in this case
is related to electroviscous eects as the intrinsic viscosity
calculated for the sti chain of equal length should be equal
to 155 mL/g [1316]. On the right side of the peak,
interchain electrostatic interaction are established between
the chains in solution when, on the left side, chains are
progressively diluted and controlled by the external salt
concentration, which remains constant and high in com-
parison with the polymer concentration. Corresponding to
the right side of the peak, in the same conditions, a peak is
observed in light or neutron scattering (the q vector neces-
sary to evidence this peak depends on the range of polymer
Figure 2 Variation of the reduced viscosity of sodium

polygalacturonate (Mw = 25,500 g/mol) as a function of the
polymer concentration at dierent NaCl concentrations. (.)
5 104 M; (o) 2 104 M; (+) 1 104 M; () 5 105 Figure 3 Schematic representation of the purication tech-
M; (4) 1 105 M. (From Ref. 13. Copyright 2003.) nique proposed. (From Ref. 24. Copyright 2003.)
240 Rinaudo
is found that K+ formed more ion pairs and also favored

the double-helix formation compared to Na+.
From all these data, it comes that to characterize a
polyelectrolyte in aqueous solution, it is necessary to isolate
the chains by screening the long-range electrostatic repul-
sions; this situation is obtained in presence of 0.05 M, or
better 0.1 M, monovalent external salt (NaCl, NaNO3. . .).
But, another very important step in the study of a poly-
saccharide is its purication as proposed in Fig. 3 adapted
to ionic polysaccharides [24].
III. SEMIRIGID CHAIN CHARACTERIZATION

AND CONFORMATION
It was recently observed that polysaccharides originally
behave in an original manner in two aspects related with
the viscometric behavior:
the MarkHouvink exponent is always higher than 0.6
(usually obtained for exible synthetic polymers in
a good solvent) going to 1;
the viscosity is only weakly inuenced by the external
salt concentration for ionic polysaccharides.
These two evidences were especially demonstrated on
xanthan; this polysaccharide is produced by the bacteria
Xanthomonas campestris and was the rst one to be devel-
oped for industrial applications (for tertiary oil recovery).
An important result is given in Fig. 4 [25].
Concerning xanthan, it was established that it adopts a
helical conformation in the presence of external salt; this
conformation is stabilized by H bonds [1] and destablized
Figure 5 Conformational transition of xanthan evidenced

by optical rotation. (a) Specic optical rotation as a function
of temperature T for xanthan (Cp = 0.66 g/L) in (a) H2O; (b)
5.6 mM NaCl; (c) 10.8 mM NaCl; (d) 15.8 mM NaCl; (e) 28
mM NaCl; (f) 0.1 M NaCl. (From Ref. 26. Copyright 2003).
(b) Specic optical rotation as a function of the total degree
of dissociation (at) at dierent temperatures from 10jC to
70jC; the transition occurs at a value (at)M, decreasing when
the temperature increases. (From Ref. 27. Copyright 2003.)
by electrostatic repulsions between ionic groups along the

chain. So a helix-coil transition is induced by an increase of
the temperature (characterized by the thermal transition
Tm at a given salt concentration) and/or a decrease of the
ionic concentration; the stability of the helical conforma-
tion increase when the degree of neutralization of the
carboxylic groups decreases (Fig. 5) [26,27]. This variation
is represented by a usual phase diagram in which the coil
conformation is separated from the helical conformation in
the linear representation of log (CT) as a function of Tm1.
From this, a relation between Tm and the salt concentration
Figure 4 Inuence of the ionic concentration on the
was established [28]:
intrinsic viscositymolar mass relationship for xanthan ()

and sodium polystyrene sulfonate (- - - -). The values are Tm1 K1 2:16 103 0:5 103 log CT 4
obtained at 102 M NaCl ionic concentration (l) and
extrapolated at innite ionic concentration (l). (From Ref. Under helical conformation, the local stiness of the chain
25. Copyright 2003.) is much larger than in the disordered conformation. An
evidence of the change in chain mobility is also obtained

from liquid high-resolution NMR experiment: in the heli-
cal conformation, no signal corresponding to the substitu-
ents (-CH3 from pyruvate and acetyl groups, -CH2- from
succinyl groups) appears in the spectrum; when tempera-
ture increases, signals progressively appear following the
conformational transition. This transition is also con-
rmed by optical rotation; some results were described
for succinoglycan [12]. Dierential scanning calorimetry
gives the enthalpy of conformational change (associated
with the helix-coil transition); the calorimetric peak is
located at Tm and the amplitude gives DH (J/mol) charac-
terizing the stability of the helical conformation. In Table 4,
few data obtained by calorimetry are given for some
polysaccharides.
Then, it was proposed to adopt a worm-like chain
model to interpret the behavior of such polysaccharides;
the treatment is valid for neutral polysaccharides as well as
ionic ones; in this last case, it is necessary to take into
account the role of electrostatic eects on (1) the local Figure 6 3-D plot of the radius of gyration vs. the salt
stiness characterized by an intrinsic persistence length Lp concentration C1/2 (NaNO3) and the molar mass as log(M)
s
added with an electrostatic contribution Le and (2) on the obtained by SEC. The calculated values including the
excluded volume eect, the electrostatic contribution here electrostatic excluded volume and the electrostatic persist-
is predominant especially for low Lp [3,2933]. ence length (U) are compared with the experimental values
The proposed treatment allows to predict the dimen- (the points). (Reproduced from Ref. 3. Copyright 2003.)
sions of the chain (and particularly the radius of gyration
Rg) and the intrinsic viscosity ([g]); it was previously
published and used for analysis of dierent polysaccha- This value is obtained experimentally from steric
rides (for details, see Ref. 3) (Fig. 6); this gure shows that exclusion chromatography (SEC) experiments using multi-
in a wide range of salt concentrations and molar masses, detection equipment in which three detectors are in line:
the agreement is very good. dierential refractometer, multiangle light scattering de-
The main relation is the following: tector, and a viscometer as discussed in the following. The
R2g LLt =3 with Lt Lp Le 5 data obtained allow to obtain Lp when the ionic concen-
when the contour length, L, is large compared with Lt;

otherwise, it is necessary to used the development proposed Table 5 Intrinsic Persistence Length (Lp) for Some
by Benoit and Doty [34]. Polysaccharides
The local stiness of the polysaccharides allows to
interpret the high viscosity obtained at a given molar mass Lp
compared to exible polymer. The Lp value, obtained in Polysaccharides (nm) References
salt excess, is characteristic of the local structure of the a
polysaccharides and can be predicted from molecular Hyaluronan 7.58 7.5 29, 35
modeling as shown recently [3538]. Xanthan
helical conformation 46 40, 65
coiled conformation 5
Gellan
Table 4 Enthalpy of Helix-Coil Transition for Some
helical conformation 72 7
Polysaccharides
coiled conformation 6.1 7
Polysaccharides DH References Galactomannan 9.3 9.6a 36
(M/G = 1)
n-carrageenan (K+) 12 kJ/disaccharide 10 Alginates
17 kJ/disaccharide 63 rich in guluronic acid 9 30
L-carrageenan 20.6 kJ/disaccharide 63 rich in mannuronic acid 4 30
Native gellan 18.6 J/g 55, 57 Succinoglycan
Deacylated gellan 9.6 J/g 55, 57 helical conformation 35 30, 39
Succinoglycan 30 kJ/repeat unit 9 coiled conformation 5
Xanthan 13.9 J/g 64 Chitosan
DA <10% 11 9a 38a, 38b
For ionic polymers, Tm depends on the ionic concentration; the DA > 60% 15 12.5a 38a, 38b
values of DH given correspond to the complete conformational
a
transition. Predicted values from molecular modeling.
242 Rinaudo
tration is known (it is the concentration of solvent, i.e., 0.1 data gives the average Mw, Mn, [g], and the weight distri-
M in our work, which gives a negligible value for Le, bution wi(Mi) or the cumulative distribution function
around 5 A). Few experimental values of Lp are given (Fig. 8); two successive analyses are compared, which show
in Table 5. the good reproducibility of this technique. In addition, one
obtains the MarkHouwink parameters relating the intrin-
sic viscosity and the molar mass and those relating the
IV. STERIC EXCLUSION CHROMATOGRAPHY radius of gyration and the molar mass (Fig. 9).
ANALYSIS In addition, the curve Rg(M) allows to determine
the intrinsic persistence length when the ionic concen-
Steric exclusion chromatography is one of the most useful tration is known to be able to take into account the role
techniques to characterize a polysaccharide in solution. We of the electrostatic contributions [Eq. (5)]. In Fig. 10,
are using a SEC equipment from Waters (Alliance the experimental and calculated representations of
GPCV2000) associated with a MALLS from Wyatt cy. Rg(M) are given for the two conformations of xanthan
(Dawn DSP-F). Then we have three detectors in line: a (native form as extracted from the broth without any
viscometer giving a signal proportional to Ci[g]i, a dier- heating and the renatured structure after conformational
ential refractometer giving Ci, and a multiangle light change and helical conformation reformation). Some val-
scattering detector giving CiMi (for each eluted species i). ues of Lp were given in Table 5. The value found for
Two columns Shodex OH Pack with dierent porosities are succinoglycan was recently conrmed by atomic force
used in series. An example of chromatogram with the three microscopy [39].
traces and the representation log(M) as a function of the Another series of experiments allow to conrm the
elution volume are given in Fig. 7. The analysis of these existence of two dierent conformations for xanthan; the
Figure 7 Steric exclusion chromatography: (a) Chromatogram obtained on a sample of hyaluronan at 30jC showing the three
signals from the three detectors on line (LS = light scattering; AUX 1 = dierential refractometer; AUX 2 = viscometer).
Elution with 0.1 M NaNO3. (b) Calibration curve representing log M (Ve).
Figure 8 Molar mass distribution on a hyaluronan sample. (a) Integral or cumulative distribution function showing the good
reproducibility of the experiments. (b) Dierential distribution function [weight fraction wi (Mi )] for the same sample.
data are shown in Fig. 11 [40] and corresponds to the to form H bonds); in this case, light scattering signal is
following equilibrium: perturbed by the aggregates and the molar masses are
overestimated; also, the intrinsic viscosity is modied by
native xanthan ! denatured xanthan X renatured xanthan
the chain association; it may increase or decrease in
relation with the aggregate structure. In the same view,
single helix irreversible coil reversible double helix
the viscosity of a polysaccharide can be predicted from
The persistence length was recently calculated from Lp: Lp allows to calculate [g] for given values of Cs and
molecular modeling; this prediction can be drawn as M and a given polymer; then the zero shear rate-specic
soon as the chemical structure is perfectly known; it viscosity is calculated and compared with the experimen-
was proposed on hyaluronan, chitosan with dierent tal data [41]. This point is discussed later with the
degrees of acetylation, and galactomannan with dierent rheology of polysaccharides.
M/G ratios [3538]. The techniques developed show that
for neutral, anionic, and cationic polysaccharides, there is
a good agreement between experimental results and the V. RHEOLOGY OF POLYSACCHARIDE
prediction. The main question in fact concerns the ex- SOLUTION
perimental data published in the literature: the values of A. Flow Behavior
Lp deduced from Rg or [g] data are considered as wrong
due to the presence of aggregates or interactions between Two types of experiments are usually performed in direct
chains in solution in relation with the conditions of relation with the fundamental properties of the systems.
polymer purication, choice of the solvent, chemical Polysaccharides are usually good thickeners, which means
structure of the polysaccharides (rich in -OH and able that, when dissolved in a solvent, they increase its viscosity.
244 Rinaudo
Figure 9 Examples of SEC analysis on the same hyaluronan sample: (a) Relation Rg (M ) with Rg in nm and a slope of 0.57. (b)
Relation [g] (M) with a slope of 0.75. These slope agree with the semirigid character of the chain.
Figure 10 Radius of gyration (in A) as a function of the molar mass for a xanthan sample in the two states: (5) native form and
(o) renatured form after thermal treatment. Filled symbols are the theoretical values from the model proposed allowing the
determination of Lp: Native state Lp = 460 A and renatured state Lp = 1600 A. (From Ref. 40. Copyright 2003.)
This eect is reected by the viscosity, which has to be

examined as a function of the polymer concentration,
molar mass, stiness of the polymer, temperature, shear
rate adopted, and the composition of the solvent (ionic
concentration in aqueous medium, nature of the ions,
pH. . .). The rst experiment concerns the ow experi-
ments; an example obtained for xanthan at the same
concentration but at dierent molar masses is given in
Fig. 12. Due to their characteristics, the ow curves show a
non-Newtonian character (characterized by a critical shear
rate c c over which the viscosity decreases when the shear
rate increases). The rst part of the curve is called the
Newtonian plateau and it is the domain where the viscosity
has to be taken to determine the intrinsic viscosity. The
critical shear rate c c decreases when the molar mass
increases or when the polymer concentration increases
[40]; an interesting result concerns the inuence of the
Figure 11 Weight fraction as a function of the molecular
shear rate on the relationship between viscosity and molar
weight of a sample of partially depolymerized xanthan by mass (Fig. 13); this type of curve has to be taken into
sonication in State 1 = native xanthan (a) before and (b) account when a mechanism of depolymerization is studied,
after heating over the temperature of conformational change, by example.
and in the State 2 = renaturated xanthan (a) before and (b) Following the Huggins relation, the reduced viscosity
after heating over the temperature of conformational change. is linear with C in the dilute domain:
The results demonstrated that the native state is a single-
ordered chain when the renatured state is a double helix
gsp =C g k Vg2 C 6
(corresponding to an intramolecular structure change having
the same molar mass) with a higher stiness. (From Ref. 40.
Copyright 2003.) with kV being the Huggins constant and 0.3 < kV < 0.7.
Figure 12 Flow curves representing the relative viscosity obtained on xanthan at 2 g/L but dierent molar masses as a function
of the shear rate. (From Ref. 42. Copyright 2003.)
246 Rinaudo
Figure 13 Inuence of the shear rate on the viscositymolar mass relationship at a polymer concentration of 2 g/L in 101 M
NaCl. (From Ref. 42. Copyright 2003.)
Over C* (when C[g] > 1), the following development galactomannan increases when the yield in galactose
as a function of the fundamental parameter C[g] (the increases; at the same time, the interchain interactions
overlap parameter) has to be adopted at zero shear rate decreases as well as the slope n in the semidilute regime
[42,43]: [4749]. Table 6 gives few values of the slope n for die-
rent galactomannans.
gsp gC k VgC 2 BgCn 7 So deviation from the predicted curve relating the
with the Huggins constant around 0.4 and B = 2 102 specic viscosity and the overlap parameter [Eq. (8)]
with n f 4 [39] or indicates the existence of interchain interactions (i.e., a
h i lack of solubility). At the same time, the Newtonian
gsp gC 1 k1 gC k2 gC2 k3 gC3 8 plateau usually disappears in the low c range values.
with k1 = 0.4, k2 = k12/2!, k3 = k13/3!

In the semidilute regime, the chains overlap and
B. Dynamic Measurements
entanglements are progressively formed giving a larger The second series of experiments concerns the dynamic
dependency of the specic viscosity with C[g] [44,45]. measurements. A sinusoidal deformation is imposed to
Exponent n is predicted to increase to a value from 3.4 to the solution in a large range of frequencies, and the re-
4. It is also the regime in which the viscosity seriously sponse is a complex modulus decomposed in an in-phase
decreases with the shear rate (slope p in loglog plot) due to response ( GV reecting the elastic character) and out-phase
disentanglements and which characterizes the viscoelastic response ( GU reecting the viscous response). This study
character. The critical c value (c crit), which separates the
Newtonian and non-Newtonian regimes, is also displaced
to lower shear rate when the polymer concentration
increases. The inuence of C[g] on c crit and on the slope
( p) of the viscosity curve in the non-Newtonian regime Table 6 Values of the Exponent of Viscosity Relating
were previously discussed [44,46]. Specic Viscosity with the Polymer Concentration in
As soon as loose interactions exist in solution, as LogLog Plot for Few Galactomannans
frequently with polysaccharides in aqueous medium, the M/G ratios 3.55 4 2 1.1 1.56
limit slope n is larger than 3.44 and progressively n values 6.4 6.65 4.5 4.2 5.1
increases with the increase of the interactions. It is well
demonstrated with galactomannans: the solubility of Source: Ref. 48.
value of GV(xo) increases. For a perfect solution of linear

polymer, shifts along the two axes allow to obtain a
master curve (all the points are on the same reference
curve). This was clearly obtained for xanthan [50] and
for hyaluronan [44].
In the presence of chemical cross-link as we have in
Hylan (loosely cross-linked hyaluronan), the behavior is
clearly modied as shown in Fig. 15.
VI. MECHANISM OF GELATION AND GEL

BEHAVIOR CHARACTERIZATION
Polysaccharides in many cases give gelsusually physical
and reversible gels; their formation is based on the ther-
modynamic characteristics of the systems in relation to
their chemical structure. The dierent mechanisms were
previously discussed [51].
Figure 14 Dynamic rheology of a hyaluronan solution (Cp It can be recognized that
= 20 g/L) in 0.1 M NaCl at 37jC; GV, GU and the complex ionic gels are stabilized by cooperative interaction
viscosity jg*j as a function of the frequency. with calcium counterions as in pectins with low degree
of methylation and in alginates. The model proposed is
called the egg-box model; the gels can be destroyed in the
presence of excess of monovalent counterions (ion-
allows the analysis of the viscoelastic behavior of a poly- exchange mechanism) and addition of complexing agents
meric solution. (oxalate, citrate). The role of the nature of the saccharidic
An example is given in Fig. 14. For a solution, at unit and its conguration are very specic: in alginates,
low frequency, GU is larger than GV, but over a critical mannuronic acid is not involved in the calcium complex
frequency xo, GV becomes larger than GU corresponding but guluronic acid is complexed as well as galacturonic acid
to the presence of entanglements, i.e., transitory crossin pectins. Ability to form gel can be obtained from
link points. xo is displaced to lower frequency when the viscosity or light scattering experiments in dilute solution.
polymer concentration increases; at the same time, the A percolation point is observed for progressive addition of
Figure 16 Gel point determination on a pectin (2 g/L) with

Figure 15 Dynamic rheology of a hyaluronan solution (Cp a 30% degree of esterication at 25jC in the presence of
= 10 g/L) containing a fraction of chemically cross-linked Ca2+ counterions (5 103 M CaCl2). PE is obtained by
hyaluronan in 0.1 M NaCl at 37jC. The elastic modulus GV is enzymic partial hydrolysis giving a blockwise distribution of
larger than the viscous modulus GU in all the range of the carboxylic groups; PH is obtained by alkaline hydrolysis
frequency covered. The complex viscosity continuously giving a random type distribution of the carboxylic groups.
decreases as a function of the frequency. (From Ref. 52. Copyright 2003.)
248 Rinaudo
divalent counterions in the polymeric solution as shown in sitions exists for the solgel transition and for the me-
Fig. 16. [52]; this gure shows the role of the carboxylic sites chanical rigidity of the gels [56,57]. In addition, in the case
distribution: blockwise obtained by pectinesterase hydro- of n-carrageenans, the anion is demonstrated to have a
lysis and random obtained by alkaline hydrolysis. It is specicity: iodine stabilized the double helix but prevent
important to mention that Mg never induces chain associ- gelation [58].
ation nor gelation [53]. a third type of gel concerns thermoinducible gels that
H bond stabilized gels as found in highly methylated form on temperature increase. It is the case of amphiphilic
pectins, carrageenans, and gellan. They formed at low polymer and it is well known on methylcellulose. These
temperature and melt on heating. A mechanism was polymers present a low critical temperature (LCST)
proposed based on a two-step process: double helices are around 30jC [59].
formed that associate to give connected aggregates. A All these physical gels are based on junction zones
schematic representation is given in Fig. 17 [54,55]. This involving cooperative interactions; in alginate, it needs
is clearly shown also on gellan: Dierential scanning blocks of guluronic acid; in low methoxy pectins, blocks
calorimetry gives one peak on cooling but two on heating; of galacturonic acid; in methylcellulose, blocks of trime-
these two peaks successively correspond to the melting of thylglucose. In gellan, carrageenan, or XM6, gel results of
isolated helices and the melting of the more stable aggre- the aggregation of sti double helices [7,56,57]. These gels
gates of helices [55]. Induction of gel is related to the are also often more rigid than that obtained by chemical
nature of the counterions: for gellan and n-carrageenan, cross-linking of exible synthetic polymers; the swelling
K+ is known to promote gelation better than Na+ and and deswelling are also very dierent from chemically
Li+; the phase diagram obtained for n-carrageenan is cross-linked gels [60].
given in Fig. 18: gel with a hysteresis in temperature Physical gels can be characterized by compression test;
appears at a lower KCl salt concentration than in presence a piece of gel is molded from a solution; then, it is deformed
of NaCl. For gellan and n-carrageenan, it was shown that in a tensile test machine (Instrom machine series 4301). The
the same ionic selectivity observed for the helix-coil tran- Young modulus was obtained on carrageenan, gellan,
alginate gels [51]. But, when the polymer concentration is
low, or when the gel is too soft, it can be tested in a dynamic
machine in the same way as solution. We are using a AR
1000 equipment from TA Instruments. The rheological
behavior is very characteristic: GV and GU are quasi inde-
pendent on the frequency and GV is larger than GU [55]
(Fig. 19).
This gure shows that the physical properties of
the system strongly depends on the temperature and it
gives the characteristic behavior on both sides of the gelsol
transition.
It was shown that the ionic selectivity characterizing
the ability to induce helical conformation and gel forma-
tion is also recognized to play a role on the elastic modulus;
few results obtained on gellan are given in Table 7.
VII. ROLE OF THE CHEMICAL STRUCTURE ON

THE PROPERTIES
With polysaccharides extracted from natural sources, such
as hemicelluloses, pectins, galactomannans, gluco-
mannans, etc. the chemical structure is often irregular
and dicult to establish. Less-cooperative properties than
those previously described are observed. In absence of
divalent counterions, all pectins are not ordered; they just
behave as coiled polymer. Their viscosity is related to the
molar mass with a moderate persistence length; loose
interchain interactions often exist as described for galac-
tomannans. In these polymers, the distribution of the
ionic groups (in pectins) or galactose side chains (in
galactomannans), the existence of blockwise or random
Figure 17 Mechanism of gelation proposed for n-carra- distribution, and the length of the blocks are all very
geenan and gellan. important and make the dierence from one sample to
Figure 18 Variation of the inverse of the helix-coil temperature of transition (Tm) as a function of the total ionic concentration
(CT) in log scale for n-carrageenan under the K+ form and the Na+ form. C* is the critical salt concentration over which gelation
occurs. (From Ref. 54a. Copyright 2003.)
another. In the case of pectins, the dierence in hydrolysis The existence of blocks of specic groups induces a
of methoxy groups in alkaline conditions (random distri- tendency to form strong cooperative interaction; on the
bution of -COOH groups) or with pectin esterase (block- other hand, a regular distribution will give more homoge-
wise distribution) was discussed [52]. neous systems (or better solution) [41].
For chemical derivatives of starch, cellulose, chito- An important structural feature is the nature and
san, etc. the same situation exists. It was well discussed position of carbohydrate or noncarbohydrate substituents
in the case of methylcelluloses where the comparison of especially in bacterial polysaccharides. The role of acetyl
industrial samples (obtained by a heterogeneous reaction and L-glyceric substituents was clearly recently demon-
giving a blockwise distribution of trimethylated glucose strated on gellan [55,57]; after deacylation of the native
units) and laboratory samples prepared in homogeneous gellan in alkaline conditions, it forms strong gels able to
conditions having the same average degree of substitu- compete with agarose or gelatin. The L-glyceric groups
tion but a random distribution of highly methylated stabilize the double helix and the acetyl groups located
units (Fig. 16) [61]. outside the double helix prevent their aggregation and gel
250 Rinaudo
formation. This is just an example; it was also shown Table 7 Values for the Elastic Modulus GVa (Pa) of (a)
recently on K54-type bacterial polysaccharide [62]. Gellan as a Function of Polymer Concentration and Nature
of the Counterion and (b) On L-Carrageenan in 0.1 M Salt
Concentration at 25jC
VIII. CONCLUSION
(a)
The objective of this chapter was to give the main proper-
ties and characteristics of water-soluble polysaccharides; Polymer 3 5 8 13
we proposed to use the polyelectrolyte characteristics to concentration (g/L)
determine the conformation of these polymers; it is espe- Nature of 0.44
cially important with stereoregular bacterial polysaccha- the counterion
rides where helical conformations are frequent. The Li
techniques adopted to determine the valuable parameters Na 0.7 1.63 4.43 11.9
have been described; it is clear that SEC with three detec- K 1.94 5.77 11.7 31.4
tors in line is a very powerful technique. In addition,
rheology is essential to test the behavior in connection with (b) [63]
the applications as thickeners or gelling polymers.
NaI TMACl LiCl NaCl KCl
Polysaccharides also give physical gels stabilized by GV (Pa) 1.77 4.22 2.20 3.43 4.81
dierent types of cooperative interactions. These gels are
a
often rigid and behave dierently from synthetic cross- At 20jC, 1.12 Hz; Cp = 4 g/L; salt concentration 0.1 M.
linked polymers; the enthalpic contribution is large in these
physical gels. Ionic selectivity in these systems is very
important to control the ability to induce helix formation
and to form gels as well as their stereoregularity. It is mechanical properties of the gels in the case of gellan and
shown that the selectivity is also connected with the carrageenan. The dierent aspects of these systems need to
be examined to better understand their behavior and their
domains of applications.
REFERENCES
1. Moorhouse, R.; Walkinshaw, M.D.; Arnott, S. Xanthan
gummolecular conformation and interaction. In Extrac-
ellular Microbial Polysaccharides; Sandford, P.A. Laskin,
A., Eds.; ACS Symp. Ser. 1977, 45, 90102.
2. Nishiyama, Y.; Langtan, P.; Chanzy, H. Crystal structure
and hydrogen-bonding system in cellulose I-beta from
Synchrotron X-ray and neutron ber diraction. J. Am.
Chem. Soc. 2002, 124, 9074.
3. Reed, W. Light-scattering results on polyelectrolyte con-
formations, diusion and interparticle interactions and
correlations. In Macro-Ion Characterization from Dilute
Solutions to Complex Fluids; Schmitz, K.S., Ed.; ACS
Symp. Ser. 1994, 548, 297.
4. Katchalsky, A.; Alexandrowicz, Z.; Kedem, O. Polyelec-
trolyte Solutions. In Chemical Physics of Ionic Solutions;
Conway, B.E., Barradas, R.G., Eds.; Wiley & Son: New
York, 1966.
5. Manning, G.S. Limiting laws for equilibrium and transport
properties of polyelectrolyte solution. In Polyelectrolytes;
Selegny, E., Mandel, M., Strauss, P., Eds.; D. Reidel
Publishing Company: Dordrecht, 1972.
6. Rinaudo, M.; Milas, M. Polyelectrolyte behaviour of
bacterial polysaccharide from Xanthomonas campestris.
Comparison with carboxymethylcellulose. Biopolymers
1978, 17, 2663.
7. Milas, M.; Shi, X.; Rinaudo, M. On the physicochemical
properties of gellan gum. Biopolymers 1990, 30, 451.
8. Rochas, C.; Rinaudo, M. Activity coecients of counter-
ions and conformation in kappa-carrageenan systems.
Figure 19 Rheology of deacylated gellan (C = 10 g/L) in Biopolymers 1980, 19, 1675.
0.1 M NaCl (a) at 20jC where a gel is formed and (b) at 80jC 9. Boutebba, A.; Milas, M.; Rinaudo, M. Orderdisorder.
in the sol state (over the gelsol transition). (From Ref. 55. Conformational transition in succinoglycan: calorimetric
Copyright 2003.) measurements. Biopolymers 1997, 42, 811.
10. Rochas, C.; Rinaudo, M. Calorimetric determination of tration, molecular weight, and temperature viscosity
the conformational transition of kappa-carrageenan. dependences of hyaluronates, a wormlike polyelectrolyte.
Carbohydr. Res. 1982, 105, 227. Macromolecules 1993, 26, 6945.
11. Milas, M.; Rinaudo, M. Conformational investigation on 30. Rinaudo, M. Wormlike chain behaviour of some bacterial
the bacterial polysaccharide xanthan. Carbohydr. Res. polysaccharides. In Macromolecules; Kahovec, J., Ed.;
1979, 76, 189. VSP, 1992;207219.
12. Gravanis, G.; Milas, M.; Rinaudo, M.; Clarke-Sturman, 31. Fouissac, E.; Milas, M.; Rinaudo, M.; Borsali, R. Inuence
A.J. Conformational transition and polyelectrolyte behav- of the ionic strength on the dimensions of sodium
iour of a succinoglycan polysaccharide. Int. J. Biol. hyaluronate. Macromolecules 1992, 25, 5613.
Macromol. 1990, 12, 195. 32. Rinaudo, M.; Roure, I.; Milas, M. Use of steric exclusion
13. Malovikova, A.; Milas, M.; Rinaudo, M.; Borsali, R. chromatography to characterize hyaluronan, a semi-rigid
Viscosimetric behavior of Na-polygalacturonate in the polysaccharide. Int. J. Polym. Anal. Charact. 1999, 5,
presence of low salt content. In Macro-Ion Characterization 277.
from Dilute Solutions to Complex Fluids; Schmitz, K.S., 33. Rinaudo, M. On the abnormal exponents ag and aD in
Ed.; ACS Symp. Ser. 1994, 548, 315321. Mark Houwink type equations for wormlike chain
14. Roure, I.; Rinaudo, M.; Milas, M. Viscometric behavior of polysaccharides. Polym. Bull. 1992, 27, 585.
dilute polyelectrolytes. Role of electrostatic interactions. 34. Benoit, H.; Doty, P. Light scattering from non-Gaussian
Ber. Bunsenges. Phys. Chem. 1996, 100, 703. chains. J. Phys. Chem. 1953, 57, 958.
15. Rinaudo, M.; Roure, I.; Milas, M.; Malovikova, A. 35. Haxaire, K.; Braccini, I.; Milas, M.; Rinaudo, M.; Perez, S.
Electrostatic interactions in aqueous solutions of ionic Conformational behavior of hyaluronan in relation to its
polysaccharides. Int. J. Polym. Anal. Charact. 1997, 4, physical properties as probed by molecular modeling.
57. Glycobiology 2000, 10, 587.
16. Roure, I.; Rinaudo, M.; Milas, M.; Frollini, E. Viscometric 36. Petkowicz, C.L.O.; Reicher, F.; Mazeau, K. Conforma-
behaviour of polyelectrolytes in the presence of low salt tional analysis of galactomannans: from oligomeric seg-
concentration. Polymer 1998, 39, 5441. ments to polymeric chains. Carbohydr. Polym. 1998, 37,
17. Nierlich, M.; Williams, C.E.; Boue, F.; Cotton, J.P.; 25.
Daoud, M.; Farnoux, B.; Jannink, G.; Picot, C.; Moan, 37. Petkowicz, C.L.O.; Rinaudo, M.; Milas, M.; Mazeau, K.;
M.; Wol, C.; Rinaudo, M.; DE Gennes, P. Small angle Bresolin, T.; Reicher, F.; Ganter, J.L.M.S. Conformation
neutron scattering by semi-dilute solutions of polyelectro- of galactomanan, experimental and modeling approaches.
lyte. J. Phys. 1979, 40, 701. Food Hydrocoll. 1999, 13, 263.
18. Milas, M.; Lindner, P.; Rinaudo, M.; Borsali, R. Inuence 38a. Mazeau, K.; Perez, S.; Rinaudo, M. Predicted inuence of
of the shear rate on the small-angle neutron scattering N-acetyl group content on the conformational extension of
pattern of polyelectrolyte solutions: the xanthan example. chitin and chitosan chains. J. Carbohydr. Chem. 2001, 19,
Macromolecules 1996, 29, 473. 1269.
19. Milas, M.; Rinaudo, M.; Duplessix, R.; Borsali, R.; 38b. Brugnerotto, J.; Desbrie`res, J.; Roberts, G.; Rinaudo, M.
Lindner, P. Small angle neutron scattering from polyelec- Characterization of chitosan by steric exclusion chroma-
trolyte solutions: from disordered to ordered xanthan tography. Polymer 2001, 42, 9921.
chain conformation. Macromolecules 1995, 28, 3119. 39. Balnois, E.; Stoll, S.; Wilkinson, K.J.; Bue, J.; Rinaudo,
20. Morn, I.; Reed, W.F.; Rinaudo, M.; Borsali, R. Further M.; Milas, M. Conformations of succinoglycan as observed
evidence of liquid-like correlations in polyelectrolyte by atomic force microscopy. Macromolecules 2000, 33,
solutions. J. Phys. II 1994, 4, 1001. 7440.
21. Borsali, R.; Rinaudo, M.; Noirez, L. Light scattering and 40. Chazeau, L.; Milas, M.; Rinaudo, M. Conformations of
small angle neutron scattering from polyelectrolyte sol- xanthan in solution: analysis by steric exclusion chroma-
utions: the succinoglycan. Macromolecules 1995, 28, 1085. tography. Int. J. Polym. Anal. Charact. 1995, 2, 21.
22. Rinaudo, M.; Rochas, C.; Michels, B. Etude par absorp- 41. Rinaudo, M. Relation between the molecular structure of
tion ultrasonore de la xation selective du potassium sur le some polysaccharides and original properties in sol and gel
carraghenane. J. Chim. Phys. 1983, 80, 305. states. Food Hydrocoll. 2001, 15, 433.
23. Zana, R.; Tondre, C.; Rinaudo, M.; Milas, M. Etude 42. Milas, M.; Rinaudo, M.; Tinland, B. The viscosity
ultrasonore de la xation sur site des ions alcalins de dependence on concentration, molecular weight and shear
densites de charge variable. J. Chim. Phys. 1971, 68, 1258. rate of xanthan solutions. Polym. Bull. 1985, 14, 157.
24. Rinaudo, M. Polysaccharide characterization in relation 43. Kwei, T.K.; Nakazawa, M.; Matsuoka, S.; Cowman,
with some original properties. J. Appl. Polym. Sci.: Appl. M.K.; Okamoto, Y. The concentration dependence of
Polym. Symp. 1993, 52, 11. solution viscosities of rigid rod polymers. Macromolecules
25. Tinland, B.; Rinaudo, M. Dependence of the stiness of the 2000, 33, 235.
xanthan chain on the external salt concentration. Macro- 44. Milas, M.; Rinaudo, M.; Roure, I.; Al-Assaf, S.; Phillips,
molecules 1989, 22, 1863. G.O.; Williams, P.A. Comparative rheological behavior of
26. Milas, M.; Rinaudo, M. Properties of xanthan gum in hyaluronan from bacterial and animal sources with cross-
aqueous solutions: role of the conformational transition. linked hyaluronan (hylan) in aqueous solution. Biopol-
Carbohydr. Res. 1986, 158, 191. ymers 2001, 59, 191.
27. Milas, M.; Rinaudo, M. Investigation on conformational 45. Milas, M.; Rinaudo, M.; Roure, I.; Al-Assaf, S.; Phillips,
properties of xanthan in aqueous solutions. In Solution G.O.; Williams, P.A. Rheological behaviour of hyalur-
Properties of Polysaccharides; Brant, D., Ed.; ACS Symp. onan, healon and hylan in aqueous solution. In Hyalur-
Ser. 1981, 150, 2530. onan, vol. 1: Chemical, Biochemical and Biological Aspects;
28. Rinaudo, M.; Milas, M.; Bresolin, T.; Ganter, J. Physical Kennedy, J.F., Phillips, G.O., Williams, P.A., Hascall,
properties of xanthan galactomannan, their mixtures in V.C., Eds.; Woodhead Pub.: Cambridge, U.K., 2002, 181
aqueous solutions. Macromol. Chem. Phys., Macromol. 193.
Symp. 1999, 140, 115. 46. Berriaud, N.; Milas, M.; Rinaudo, M. Characterization
29. Fouissac, E.; Milas, M.; Rinaudo, M. Shear-rate, concen- and properties of hyaluronic acid (hyaluronan). In Poly-
252 Rinaudo
saccharides: Structural Diversity and Functional Versatility; transition of native, modied gellan. Int. J. Biol. Macro-
Dumitriu, S., Ed.; Dekker: New York, U.S.A., 1998, 313 mol. 1999, 26, 109.
334. 56. Milas, M.; Rinaudo, M. The gellan solgel transition.
47. Ganter, J.; Milas, M.; Rinaudo, M. Study of solution Carbohydr. Polym. 1996, 30, 177.
properties of galactomannan from the seeds of Mimosa 57. Rinaudo, M.; Milas, M. Gellan gum, a bacterial gelling
scabrella. Carbohydr. Polym. 1992, 17, 171. polymer. In Novel Macromolecules in Food Systems;
48. Kapoor, V.P.; Milas, M.; Taravel, F.R.; Rinaudo, M. Doxastakis, G., Kiosseoglou, V., Eds.; Elsevier: Amster-
Rheological properties of seed galactomannan from Cassia dam, The Netherlands, 2000, 239263.
nodosa buch.-hem. Carbohydr. Polym. 1994, 25, 79. 58. Ciancia, M.; Milas, M.; Rinaudo, M. On the specic role of
49. Kapoor, V.P.; Taravel, F.R.; Joseleau, J.P.; Milas, M.; coions and counterions on kappa-carrageenan conforma-
Chanzy, H.; Rinaudo, M. Cassia spectabilis DC seed tion. Int. J. Biol. Macromol. 1997, 20, 35.
galactomannan: structural crystallographical and rheolog- 59. Hirrien, M.; Chevillard, C.; Desbrie`res, J.; Axelos, M.A.;
ical studies. Carbohydr. Res. 1998, 306, 231. Rinaudo, M. Thermogelation of methylcelluloses: new
50. Milas, M.; Rinaudo, M.; Knipper, M.; Schuppiser, J.L. evidence for understanding the gelation mechanism.
Flow and viscoelastic properties of xanthan gum solutions. Polymer 1998, 39, 6251.
Macromolecules 1990, 23, 2506. 60. Rinaudo, M.; Landry, S. On the volume change on non
51. Rinaudo, M. Gelation of polysaccharides. J. Intell. Mater. covalent gels in solventnon solvent mixtures. Polym. Bull.
Syst. Struct. 1993, 4, 210. 1987, 17, 563565.
52. Thibault, J.F.; Rinaudo, M. Gelation of pectinic acids in 61. Desbrieres, J.; Hirrien, M.; Rinaudo, M. A calorimetric
the presence of calcium counterions. Br. Polym. J. 1985, 17, study of methylcellulose gelation. Carbohydr. Polym. 1998,
181. 37, 145.
53. Malovikova, A.; Rinaudo, M.; Milas, M. Comparative 62. Guetta, O.; Milas, M.; Rinaudo, M. Structure and proper-
interactions of magnesium and calcium counterions with ties of a bacterial polysaccharide from a Klebsiella strain
polygalacturonic acid. Biopolymers 1994, 34, 1059. (ATCC 12657). Biomacromolecules. in press.
54. (a) Rochas, C.; Rinaudo, M. Mechanism of gel formation 63. Rinaudo, M.; Desbrie`res, J.; Piallat, F. unpublished data.
in kappa-carrageenan. Biopolymers 1981, 23, 735. 64. Bresolin, T.; Milas, M.; Rinaudo, M.; Ganter, J. Xanthan
(b) Rinaudo, M.; Rochas, C. Investigations on aqueous galactomannan interactions as related to xanthan con-
solution properties of n-carrageenans. In Solution Proper- formations. Int. J. Biol. Macromol. 1998, 23, 263.
ties of Polysaccharides; Brant, D., Ed.; ACS Symp. Ser. 65. Tinland, B.; Maret, G.; Rinaudo, M. Reptation in semi-
1984, 150, 367378. dilute solutions of wormlike polymers. Macromolecules
55. Mazen, F.; Milas, M.; Rinaudo, M. Conformational 1990, 23, 596.
9
Conformational and Dynamics Aspects of Polysaccharide
Gels by High-Resolution Solid-State NMR
Hazime Saito
Himeji Institute of Technology, Kamigori, Hyogo, Japan and
Center for Quantum Life Sciences, Hiroshima University, Higashi-Hiroshima, Japan
I. INTRODUCTION of either magic angle spinning or proton decoupling.

Further, existence of conformational heterogeneity among
Many polysaccharides of higher molecular weight are such domains in polysaccharide gels may hamper their in
known to have specic ability to be able to form soft, situ characterization by such X-ray diraction, optical, or
elastic, or brittle gels depending on their type of primary viscoelasticity measurements, which are sensitive to char-
and secondary structures. Network structures for such gels acterization of either solid-like or liquid-like domains,
are obviously formed by physical aggregation or self- respectively, together with requirement for special sample
assembly of polysaccharide chains and subsequently swol- preparations suitable for respective measurements, al-
len by diluents such as water or a variety of organic though many of previous pictures for such gels have been
solvents, although no additional chemical cross-links were obtained by a rather simple manner based on these
deliberately introduced as in the case of synthetic network measurements [7,8].
polymers. Such gel-forming ability is therefore one of the In this connection, it should be recognized that an
most important properties of polysaccharides in view of attempt to anneal gel preparations of curdlan, a linear (1
biomedical applications, food processing, etc. 3)-h-D-glucan from Alcaligenes faecalis, at higher tem-
It appears that gel network consists of at least two perature for the purpose of achieving better crystalline
regions, swollen polymer chains of highly mobile liquid- sample for X-ray diraction studies would result in an in-
like domains and rigid solid-like domains from cross- evitable conformational change as manifested from a
linked domains and their vicinity, depending on the conversion from one polymorph to the other [9], although
respective correlation times for uctuation motions as the resulting triple helix form may be considered as a suit-
illustrated in Fig. 1, in spite of highly heterogeneous nature able model for the cross-links or solid-like domains. How-
from conformational and dynamic point of view. This ever, the revealed triple-helix conformation from the
picture has been revealed by 13C nuclear magnetic reso- annealed curdlan [1012] turns out to be not sucient to
nance (NMR) studies either by high-resolution solution or explain why highly exible swollen polymer chains are
solid-state NMR methods [16]. However, this picture present in the resilient gel of this polysaccharide to yield
may be too much simplied in some cases, if gel networks well-resolved, high-resolution NMR signals from the liq-
were considered as highly heterogeneous assembly in spite uid-like domain [13].
of its simple solid-like appearance. In such cases, the solid-
like domain should be classied into several types of
regions with dierent manner of uctuation motions
undergoing with a variety of correlation times or motional II. NUCLEAR MAGNETIC RESONANCE
frequencies. In some instances, it should be taken into APPROACHES TO CHARACTERIZE
account that high-resolution solid-state 13C NMR signals CONFORMATION AND DYNAMICS
would completely disappear as a result of failure of OF GEL NETWORKS
attempted peak-narrowing process for high-resolution
solid-state NMR, when such motional frequencies in the This is the reason why high-resolution solution and solid-
order of 104105 Hz, if any, were interfered with frequency state NMR approach is especially suitable for in situ
253
254 Saito
backbone motions from gel samples were interfered with

frequencies of either magic angle spinning or proton
decoupling [16,17]. In fact, we realized that considerable
proportions of 13C NMR signals were suppressed for
hydrated cereal seed storage protein, C-hordein, and high
molecular weight (HMW) glutenin subunits and their
model peptides [18,19] because of the presence of swollen
peptide chains.
High-power proton decoupling as well as magic angle
spinning has been utilized as the most ecient means to
yield narrowed 13C NMR signals with modest linewidth,
(1/pT2C )S, instead of extremely broadened signals from
solid samples as dened by 1/pT2 in Fig. 1. However,
Figure 1 Schematic representation of the 13C spinlattice such narrowed 13C NMR linewidths would be inevitably
relaxation times (T1), spinspin relaxation times (T2), 1H
deteriorated when incoherent frequency of random mo-
spinlattice relaxation times in the rotating frame (T1q) as a
tion is interfered with either coherent frequency of the
function of the correlation times of local uctuations. 13C
proton decoupling or magic angle spinning. This hap-
NMR signals from the domains undergoing incoherent
uctuation motions with the correlation times in the order of pens when the following second or third terms could be
104 to 105 sec (indicated by the gray color) could be lost due dominant instead of the rst term (1/pT2C )S of the static
to failure of attempted peak-narrowing due to interference of component [16,17].
frequency with proton decoupling or magic angle spinning.
1=pT2C 1=pT2C S 1=pT2C M C M
DD 1=pT2 CS 1
characterization of a number of polysaccharide gels as

where (1/T2C ) M C M
DD and (1/T2 ) CS are the transverse
viewed from the conformation and dynamics aspect, be-
components due to the uctuation of dipolar and chem-
cause the present NMR approach is the sole means to be
ical shift interactions, respectively. The latter two terms
able to reveal conformation and dynamics of such hetero-
are given as a function of the correlation time sc by
geneous materials. In such case, it is preferable to utilize
13
C nuclei as NMR probes over proton NMR signals X
because of better spectral dispersion over 200 ppm in the 1=T2C M
DD 4c2I c2S h2 =15r6 II 1
2
former as compared with that of ca. 10 ppm in the latter, sc =1 x2I s2c
although sensitivity of signal detection in the latter is much
better than that of the former.
1=T2C M 2 2 2 2 2
CS x0 d g =45sc =1 4xr sc
3
A. Dynamics 2sc =1 x2r s2c
The most important aspect of characterization of gels Here cI and cS are the gyromagnetic ratios of I and S
seems to clarify their backbone dynamics together with nuclei, respectively, and r is the internuclear distance
conformations as viewed from their highly heterogeneous between spins I and S. xo and xI are the carbon resonance
nature. As a rst step to this end, backbone dynamics of gel frequency and the amplitude of the proton decoupling
network can be very conveniently characterized by means RF eld, respectively. xr is the rate of spinner rotation. d
of simple comparative high-resolution 13C NMR measure- is the chemical shift anisotropy and g is the asymmetric
ments by cross-polarization-magic angle spinning (CP- parameter of the chemical shift tensor. The contribution of
MAS) and dipolar decoupled-magic angle spinning the second and third terms is dominant for methyl and
(DD-MAS) techniques, which are suitable for recording carbonyl or methine carbons with larger chemical shift
spectra mainly from the solid-like and liquid-like domains, anisotropy with frequency of 105 or 104 Hz as far as proton-
respectively, depending on the correlation times of back- decoupling frequency and spinning rate is in the order of
bone motions, as schematically illustrated in Fig. 1 [16]. In 50 kHz (xI) or 4 kHz (xr), respectively (see the gray-
principle, the relative proportions of the former and latter colored zone in Fig. 1). The presence of this kind of
could be simply evaluated by comparison of their 13C interference has been recognized in the cases of 13C NMR
NMR peak intensities taking into account of the correla- spectra of synthetic polymers at a temperature above the
tion times of the respective domains. This kind of the two- glass transition temperature [20] and also for a number of
step model was successfully applied to studies of brillation biological systems [21,22] including crystalline peptides
kinetics of human calcitonin, a thyroid peptide hormone [23], collagen brils [24], and also membrane proteins
with tendency to form amyloid brils in concentrated [21,22,25]. This consideration suggests a possibility that a
solution [14,15]. In contrast, NMR observation of gel considerable proportion of 13C NMR signals from poly-
samples is not so simple as expected, because peak-narrow- saccharide chains could not be detected by both CP-MAS
ing procedure to achieve high-resolution 13C NMR signals and DD-MAS NMR techniques as far as swollen polysac-
from the solid-like domain fails when any frequency of charide chains undergo slow motions with frequencies in
Conformational and Dynamics Aspects of Polysaccharide Gels 255
Table 1 13C Spinlattice Relaxation Times of Starch Gel arising from the cross-linked region of starch gel is crys-
(33%) by DD-MAS and CP-MAS NMR Methods talline portion as obtained in the solid state. On the
contrary, the T1 values from the liquid-like domain arose
C-1 C-2 C-3 C-4 C-5 C-6 from exible molecular chains taking random coil con-
Liquid-like 0.36 0.32 0.30 0.29 0.29 0.16 formation, although their mobility may be restricted to
domaina some extent due to the presence of the cross-links. How-
Solid-like 9.2 11.9 11.9 11.8 11.9 2.1 ever, it was found that this approach is not always suc-
domainb cessful in distinguishing the liquid- and solid-like domains
a
of (1!3)-h-D-glucans in which low-frequency motions,
b
By DD-MAS. instead of the high-frequency motions sensitive to the 13C
By CP-MAS. T1 values of laboratory frame, are also dominant, as will
Source: Ref. 4.
be discussed later.
the order of 104 to 105 Hz. This situation turned out to be B. Conformational Characterization
rather serious for recording 13C NMR spectra of carra-
geenan as will be discussed later. To reveal gelation mechanism of polysaccharide gels,
Characterization of high-frequency motions present in which occurs as a result of physical association of individ-
swollen polymer chains with correlation times shorter than ual chains adopting an ordered conformation through
108 sec in the liquid-like domain is feasible by measure- either formation of cross-links or junction zones by multi-
ments of relaxation parameters such as the spin-lattice ple-stranded helices or aggregation of single- or multiple-
relaxation times (T1), spinspin relaxation times (T2), and stranded helices or both. This is in contrast to the case of
nuclear Overhauser eect (NOE). Surprisingly, it turned chemically cross-linked gels in which the conformational
out that greater changes in the linewidths (1 / pT2) between feature of gels and sols is unchanged as disordered [26]. In
the gel state (about 150 Hz) and sol state (14 Hz), for such case, conformational characterization of polysaccha-
instances, were noted from the liquid-like domain of the rides is feasible with reference to the conformation-depen-
gels from chemically cross-linked synthetic polymers [26] dent displacement of 13C NMR signals as demonstrated for
and curdlan [27], whereas no signicant changes were a number of peptides, polypeptides, and proteins [28,29].
observed as viewed from the T1 and NOE values. This is As in the case of two torsion angles (/ and u) dening local
because the T2 values tend to be aected by the slow motion conformation in the peptide unit, the secondary structure
of long correlation times, while the T1 and NOE values are of an individual polysaccharide is dened by a similar set of
mainly determined by fast motions. This means that fast torsion angles (/ and u) about the glycosidic linkages, as
backbone motions of swollen polymer chains visible from illustrated for linear (1!3)-h-D-glucan (I) such as curdlan
13
C NMR signals by solution or DD-MAS spectra are and (1!4)-a-D-glucan (II) such as amylose (Fig. 2). There-
indierent from the presence or absence of such cross-links. fore it is expected that the 13C chemical shifts of carbons at
For this reason, segmental motions of backbones of swol- the glycosidic linkages of these polysaccharides are primar-
len polymer chains were well described by isotropic ily displaced in line with their particular conformations
motions with correlation times of log-v2 distribution in- unless otherwise they take disordered conformation in
stead of treatment of single correlation times [26,27]. In DMSO or aqueous solution at alkaline pH. In fact, Saito
such case, it is recommended to utilize NMR spectrometer et al. [30] rst suggested a correlation of the 13C chemical
capable for signal detection with high-power proton decou- shifts the C-1 and C-4 carbons of (1!4)-a-D-glucans with
pling and magic angle spinning designed for solid state 13C
NMR to remove residual dipolar interactions.
It is natural to expect, on the basis of the schematic
representation in Fig. 1, that the above-mentioned 13C T1
values can be also utilized as a convenient means to
distinguish dynamic feature of a variety of gel samples
between the liquid-like and solid-like domains as recorded
by DD-MAS and CP-MAS NMR, respectively. In harmo-
ny with this expectation, we found that 13C T1 values of
starch gel (33%) as summarized in Table 1 [4] are very
promising in view of their signicant dierences in one
order of magnitude. This is because the 13C T1 values of the
liquid-like domains are in the vicinity of the T1 minimum,
sC f108 sec (x0sc = 1), whereas those of the solid-like
domain are in the lower temperature side of the T1 mini-
mum, sC > 108 sec. In fact, it is pointed out that the latter
values are very close to those obtained for a variety of Figure 2 Chemical structures of (1!3)-h-D-glucan (I) and
(1!3)-h-D-glucans in the solid state [9]. Therefore this (1!4)-a-D-glucan (II) together with the torsion angles at the
nding is consistent with a view that the solid-like domain glycosidic linkages.
256 Saito
their torsion angles / and u, respectively, although some to form an elastic gel when its aqueous suspension is heated
variations of such relations were later proposed [31,32]. to a temperature above 54jC [36,37] and also suitable as
Further, distinction of the single and triple helices of reference material for gel studies in view of commercial
(1!3)-h-D-glucan is made possible by a careful examina- availability (Wako Pure Chemical, Osaka, Japan). It takes
tion of the 13C chemical shifts at carbons not always close three kinds of distinctly dierent conformations (or poly-
to the glycosidic linkages [33]. morphs) depending on changing the manner of sample
In practice, it is not always straightforward to be able preparation as manifested from the 13C CP-MAS NMR
to determine such torsion angles at the glycosidic linkages spectra illustrated in Fig. 3 [33]. It was shown that anhy-
by a ber X-ray diraction study, because the experimental drous sample (a), as received from a commercial source or
data points may not be sucient to arrive at the nal lyophilized from DMSO solution, is readily converted to
structure. Especially, distinguishing between multiple- hydrated form (b) by placing it in a desiccator at relative
stranded helices and nested single helices is of one of the humidity at 95% overnight. The 13C chemical shifts of
most dicult problems in interpreting ber diraction [34]. hydrated form (b) turned out to be identical to those
Thus it is a major advantage to record 13C NMR spectra of observed in the elastic gels obtained by solution NMR
these polysaccharides to reveal the secondary structure, [1,13] or DD-MAS spectra [2,46]. Therefore the hydrated
because structural information is equally available from form turns out to take a single helix and the anhydrous
noncrystalline samples. sample is thought to take a single chain form, because this
is the dehydrated form of the sample (b) and conversion
between the anhydrous and hydrated forms is reversible at
III. DISTINCTION OF SINGLE/MULTIPLE ambient temperature. The C-3 13C NMR peak of the
CHAINS BY MUTUAL CONVERSION anhydrous sample (single chain form; 89.8 ppm) is dis-
AMONG POLYMORPHS placed downeld by 2.253.3 ppm from that of the hydrat-
A. (1!
!3)-h
h -D-Glucans [3537] ed sample (single helix; 87.3 ppm), together with the
narrowed line widths. The annealed sample (c) was pre-
A number of linear and branched (1!3)-h-D-glucans have pared by heating an aqueous suspension of curdlan at a
been isolated from a variety of cell walls of plants, bacteria, temperature above 150jC, followed by slow cooling [9,33]
fungi, or reserved polysaccharides. In particular, curdlan is and its secondary structure turned out to be the triple-helix
a bacterial linear (1!3)-h-D-glucan of high molecular form with reference to the data of X-ray diraction [1012].
weight isolated from A. faecalis and is unique in its ability The triple helix thus obtained can be distinguished from the
13
Figure 3 C CP-MAS NMR spectra of curdlan in anhydrous (a), hydrate (b), and annealed state (c). (From Ref. 33.)
single helix by closer examination of the C-5 13C chemical hydration/dehydration process (C). In contrast, it is em-
shifts (75.8 ppm for the former and 77.5 ppm for the latter) phasized that annealing the elastic gel sample at higher
and the peak-separation between the C-5 and C-2 carbons temperature (B) is required for a linear (1!3)-h-D-glucan
(2.0 and 3.2 ppm, respectively). In a similar manner, to achieve full conversion from the single helix to triple
laminaran, a linear low molecular weight glucan from helix form [9,33], although this process is very simple for
seaweed [9, 39], and lentinan, schizophyllan, screloglucan, branched (1!3)-h-D-glucans (hydration).
HA-h-glucan, etc. (branched glucans from fungi) take the The present approach for the distinction of the single-
triple helix conformation with reference to the data of the chain/multiple-stranded chains utilizing the cycles of mu-
conformation-dependent 13C chemical shifts [38], although tual conversions among several conformations has proved
their conformational characterizations by X-ray dirac- to be a very useful and promising means and can be readily
tion are not always easy because of very low crystallinity. extended to various types of polysaccharide systems, in-
Nevertheless, storage (1!3)-h-D-glucan, paramylon gran- cluding (1!3)-h-D-xylan and (1!4)-a-D-glucan. In fact,
ule from Euglena gracilis, is believed to take the triple (1!3)-h-D-xylan takes similar kinds of three types of
helical form as judged from X-ray diraction study [12]. conformations [41], because the xylose residue is an analog
However, our 13C NMR data showed that this polysac- of the glucose residue in which the hydroxymethyl group at
charide contains several other conformations including the the C-5 position is removed from the glucose residue. It has
single helix conformations to achieve the highly crystalline been shown by X-ray diraction [42, 43] that both (1!3)-
state [9]. It is emphasized that the triple helix of curdlan was h-D-glucans and -xylan take very similar triple helices,
achieved with expense of thermal depolymerization of because the hydroxymethyl group in the former does not
chain from DP (degree of polymerization) of 3980 to 106 play an important role in stabilization of the triple helix.
at 180jC [40]. Therefore high molecular weight is essential Nevertheless, the triple helix in the latter is found to be
for a linear (1!3)-h-D-glucan to exhibit gel-forming abil- much more stable than the single helix form because of
ity, because such thermally depolymerized preparation is its hydrophobic property, as compared with that of the
not anymore capable of forming elastic gel. former. Accordingly, conversion of the triple helix to the
These ndings indicate that any given preparations of single chain for the latter is not simply achieved by lyoph-
(1!3)-h-D-glucans do not always take the most energeti- ilization by DMSO solution as in the former [39].
cally stable form such as multiple-stranded helical forms Besides the above-mentioned 13C NMR approach, it is
(triple helix). Instead, their conformations are mainly worthwhile to point out that there are a variety of host-
determined by respective individual sample history such defense biological systems responsible for distinction be-
as source of isolation, solubilization by alkaline or DMSO tween the single-chain/single-helix and triple-helix forms
solution, heat treatment, depolymerization, etc. Distinc- for (1!3)-h-D-glucan because of their widespread distri-
tion between the single and multiple-helices is very easily butions in nature, especially in fungi. This may be the
performed by the current 13C NMR approach, if involved reason why the single helix conformation of (1!3)-h-D-
individual polymorphic structures can be identied in view glucan is sensitive to activation of the coagulation system
of the sample history and mutual conversions among them of horseshoe crab amebocyte lysate (LAL) and antitumor
are manipulated by specic physical treatments under a activity [40,44,45], while the triple helix is not. It is probable
controlled manner. Fig. 4 summarizes how these three that the rst step in these biological responses is recogni-
types of conformations of (1!3)-h-D-glucans are mutually tion of a secondary structure of (1!3)-h-D-glucans as
converted by several types of physical treatments such as mentioned above, as manifested from the potency of these
hydration, dehydration (lyophilization from aqueous or polysaccharides, for instance, for activation of the coagu-
DMSO solution), or annealing at an elevated temperature. lation factor G from LAL against the concentration of the
In particular, mutual conversion between the triple helix D-glucan. The potency of the triple helical curdlan is very
and single chain forms is straightforward for branched low but increased over 100-fold upon treatment with a
(1!3)-h-D-glucans by reversible dehydration and hydra- NaOH solution, which leads to a complete or partial
tion process (A). In addition, conversion between the single conversion from the triple- to the single-helix form [44].
chain and single helix is readily made possible by reversible Laminaran, taking a random coil form in aqueous solu-
tion, turns out to be ineective for the activation of the
factor G of LAL because of random coil form in aqueous
media. In particular, the bioassay for antitumor activity in
mice may be involved in more complicated process than the
above-mentioned LAL system but is still consistent with
the data mentioned above [40,44,45]. This means that their
biological responses are initiated by recognition of the
single-helical conformations. Nevertheless, it turned out
that (1!3)-h-D-xylan did not exhibit any such activity even
if it takes the similar secondary structure like single helix. It
Figure 4 Conversion diagram for (1!3)-h-D-glucans and is probable that the hydroxymethyl group at the C-5
-xylan by a variety of physical treatments. (From Refs. 9, 33, carbon may play an important role in exhibiting such a
and 41.) biological activity [41].
258 Saito
B. (1!
!4)-a
a-D-Glucans
It has been shown that amylose and starches exhibit the
following polymorphs as revealed by X-ray diraction
study: V, A, B, and C forms [46,47]. The V form exists as
complexes with small organic molecules and has in com-
mon a left-handed, single six-residue helix, whereas the A
and B forms are found for cereal and tuber starches,
respectively. The latter two forms are readily distinguished Figure 6 Conversion diagram of amylose by various
by 13C CP-MAS NMR spectra, because the C-1 peaks are physical treatments. (From Ref. 51.)
split into a triplet and a doublet for the A and B forms,
respectively [4850]. In contrast, the C-1 and C-4 signals of
the V form give rise to single lines and are displaced
downeld from those of the B form (45 ppm for the C-4 of amorphous amylose of low molecular weight (DP 17)
peak; see Fig. 5) [51]. It is emphasized that utilization of the results in complete conversion to the B type form by
cycle for mutual conversion as demonstrated for (1!3)-h- hydration (A) [51]. In addition, Senti and Witnauer [55]
D-glucan is also useful for (1!4)-a-D-glucan as illustrated previously demonstrated that the B form of amylose can be
in Fig. 6. It is noteworthy that the 13C NMR spectra of both obtained from the V form by hydration (B). Consistent
A and B forms of starch and amylose are substantially with this view, we noticed that the V form amylose of high
distorted by drying to give a spectral prole of amorphous molecular weight (DP 1000) complexed with DMSO was
form [5254] (A). In contrast, it was shown that hydration converted to the B form by humidication by 96% relative
Figure 5 13C NMR spectra of amylose lm (DP 1000). (A) anhydrous, (B) hydrated, (C) hydrated iodine complex, and (D)
anhydrous iodine complex. (From Ref. 51.)
humidity for 12 hr, as illustrated for Fig. 5A and B, IV. NETWORK STRUCTURES, GELATION
although dehydration of B form by lyophilization from MEHANISM, AND DYNAMIC FEATURE
DMSO resulted in V form (B). A similar conversion from B
to V form was obtained for amylose of low molecular It is now possible to clarify the network structures with
weight (DP 17). However, it turned out that this sort of elastic, brittle, or soft properties and also gelation mecha-
conversion is incomplete (50%) for amylose of intermedi- nism for a variety of polysaccharides on the basis of the
ate molecular weight (DP 100). Further, conversion from aforementioned arguments available from 13C NMR meas-
the amorphous structure to V form was facilitated by urements. Dynamic feature of such gels can be related with
lyophilization from DMSO solution (C). the revealed network structures as viewed from various
The B form was initially considered as a single-helical types of spin-relaxation processes.
conformation [56] because the conversion of V to B amy-
lose takes place on humidication [55]. Later, the structure A. (1!
!3)-h
h -D-Glucans
was rened as a right-handed double helix in an antiparallel
fashion [57]. However, the handedness of the double helix As demonstrated in Fig. 7, we have recorded 13C NMR
was recently revised as a left-handed one [58]. Nevertheless, spectra of elastic curdlan gel by a variety of 13C NMR
it is hardly likely that simple humidication of amylose in a methods including broad-band decoupling by a solution
desiccator causes such an unfolding/folding process lead- NMR spectrometer (B), DD-MAS (C), and CP-MAS
ing from the single stranded helix (V form) to double- methods (D) with expectation to be able to detect dierent
13
stranded helix (B form). In this connection, it should be C NMR spectra from dynamically heterogeneous
pointed out here that complete dissolution in aqueous domains such as liquid-like and solid-like domains, with
solution, which is made possible by thermal depolymeriza- reference to the 13C CP-MAS NMR measurements on
tion during annealing at high temperature, is an essential hydrated starting powder sample (A) [59]. Nevertheless,
requirement, to achieve conversion from the single-helix to we found that conformations of these distinct domains, as
the triple-helix forms of a linear (1!3)-h-D-glucan as viewed from their 13C NMR signals, exhibit identical
mentioned above [9,36,37,40]. single-helix conformation. The amount of the triple-helical
Figure 7 13C NMR spectra of curdlan gels recorded by a variety of experimental conditions. (A and D) CP-MAS NMR
technique. (B) Conventional solution NMR. (C) DD-MAS NMR. (From Ref. 59.)
260 Saito
13
Figure 8 C CP-MAS NMR spectra of anhydrous (A) and hydrated (B) lentinan in the solid state and gel state (C). (From
Ref. 59.)
chain as a potential candidate for the cross-links is nom- clinically used in Japan as an antitumor polysaccharide.
inal (ca. 10% at most), if any, as far as heating temperature This kind of a readiness to the conversion from the single-
is kept below 80jC (low-set gel) [59]. This amount is helix to the triple-helix forms (see the conversion diagram
sucient to form elastic gels, because similar elastic gels in Fig. 4) seems to be common for a variety of branched
are formed by the presence of cross-linking agent of 0.1 glucans such as schizophyllan, HA-h-glucan, etc. and may
5% in the cases of chemically cross-linked synthetic poly- be ascribed to less hydrophobic character of the polymer
mers [1,26]. Instead, it appears that the increased gel chain as compared with that of linear (1!3)-h-D-glucan
strength caused by heating the gel at a temperature be- and -xylan. As a result, all of the 13C NMR signals were
tween 80jC and 120jC (high-set gel) [36,37] is well completely suppressed at neutral pH as recorded by a
explained in terms of formation of additional cross-links conventional solution NMR spectrometer, although 13C
arising from hydrophobic association of the single-helical NMR signals are made visible under the condition of
chains, in parallel with the reduced peak intensities of the NaOH > 0.06 M [1,2,6062]. This means that gelation of
single-helical chain visible by solution NMR signals as well the branched glucans proceeds from partial association
as development of turbidity [1]. of the triple-helical chains. This network structure is far
In contrast, we found that the 13C NMR signals from formation of an elastic gel because of the absence of
characteristic of the triple-helix form are dominant in the rather exible single-helical chains and seems to be consist-
13
C NMR of a brittle gel sample from a branched (1!3)- ent with formation of brittle gel structure. It appears that
h-D-glucan, lentinan, from an edible mushroom from the reason why all of the 13C NMR signals of branched
Lentinus edodes [Fig. 8]. This polysaccharide is currently (1!3)-h-D-glucan disappear from solution NMR spec-
Table 2 The Observed TCH and T1q of Linear and Branched (1-3)-h-D-glucans
C-1 C-2 C-3 C-4 C-5 C-6

TCH T1q TCH T1q TCH T1q TCH T1q TCH T1q TCH T1q
(Asec) (msec) (Asec) (msec) (Asec) (msec) (Asec) (msec) (Asec) (msec) (Asec) (msec)
Curdlan 128 17.9 145 19.2 138 16.6 110 14.0 137 17.0 82.2 22.9
Schizophyllan 67.3 3.32 56.4 4.09 62.8 4.18 32.0 5.69 53.1 4.30 22.1 10.0
HA-h-glucan 56.5 5.04 47.8 5.34 35.4 5.38 50.7 5.80 64.4 6.27 53.0 7.28
Source: Ref. 4.
trometer [6062] can be explained in terms of insucient amount of cross-linking agent, say 0.15%, is sucient
averaging of the CH dipolar and/or chemical shift anisot- to form elastic gels, as manifested from 13C NMR studies of
ropy interactions due to the presence of a slow tumbling chemically cross-linked gels of synthetic polymers [1,26].
motion of the sti, rod-like molecules of the triple helix. In This means that the amylose gel does not necessarily
this connection, Norisue et al. [63,64] showed that the triple consist entirely of molecular chains arising from the dou-
helix of schizophyllan is stier than that of native collagen ble-helical structure, if any. Instead, it appears that the
triple helix, for the persistent chain length of the former solid-like domains of the amylose gel might be ascribed to
(200 F 30 nm) is larger than that of the collagen triple helix the presence of phase-separated aggregates of the B-type
(130 nm). In fact, the preservation of the chemical shift single-helical chains as cross-links. This view seems to be in
anisotropy as large as 50140 ppm in native collagen makes
impossible the observation of the 13C NMR spectra by
conventional spectrometer [65].
As demonstrated above, 13C T1 values are not always
sensitive to distinguishing the signals between the liquid-
like and solid-like domains. In such case, it is expected that
13
C-resolved 1H spin-lattice relaxation times in the rotating
frame (T1q) can be utilized as an alternative means because
these parameters are very sensitive to the presence of low-
frequency motions such as 105 sec (see the diagram in
Fig. 1). 13C-resolved 1H spin-lattice relaxation time in the
rotating frame (T1q) and cross polarization time (TCH)
were very easily evaluated by tting the variation of an
experimental peak-intensity I(s) as recorded by varying
contact times s [66].
Is I0 =TCH exps=T1q
4
exps=TCH =1=TCH 1=T1q
It is clearly seen from the Table 2 that the TCH and T1q
values of curdlan taking the single-helix conformation are
signicantly longer than those of schizophyllan and HA-h-
glucan taking the triple helix conformation in the gel state
[4]. This means that the single-helical curdlan is able to
aord low-frequency motions in the solid-like domains in
the gel state (at high temperature side of the T1q minimum;
see Fig. 1), whereas the triple helical glucans are not.
B. (1!
!4)-a
a-D-Glucans
It was shown that amylose gel contains two kinds of 13C
NMR signals: the B-type signals from motionally restricted
regions as recorded by CP-MAS NMR method and the
signals identical to those found in aqueous solution [67].
The latter signals could be ascribed to exible molecular
chains adopting random coil conformation (liquid-like
domain). The former peaks, on the other hand, can be
ascribed to the solid-like domain as cross-links, either
double-helical junction-zones [67] or aggregated species
of single-helical chains [59]. This view is consistent with
the data of the 13C spinlattice relaxation times of the
laboratory frame, as mentioned already (see Table 1 as well
as arguments in Sec. IIA). So far, two dierent views have
been proposed for gelation mechanism of amylose gels.
Miles et al. [68] have suggested that amylose gels are
formed upon cooling molecularly entangled solutions as
a result of phase separation of the polymer-rich phase, Figure 9 13C CP-MAS NMR spectra of potato starch (A)
whereas Wu and Sarko [57] proposed that gelation occurs and its gel (BE). Freshly prepared gel (33%, B), after 3 days
through cross-linking by double-helical junction zones. At (33%, C); freshly prepared gel (17%, D) and after 3 days
this point, it is worthwhile to recall that only a small (17%, E). (From Ref. 4.)
262 Saito
favor of the gelation mechanism of Miles [68]. To support multiple-stranded helices and nested single helices are very
this view, we recorded 13C CP-MAS NMR spectra of dicult by ber X-ray diraction alone [34]. In accordance
freshly prepared starch gel and its retrograded samples with this line, we recorded 13C NMR spectra of dried
after 3 days in a refrigerator, as illustrated in Fig. 9. agarose lm and its hydrated preparation cast from N,N-
Obviously, the 13C NMR signals characteristic of the B dimethylacetamide solution at 80jC under anhydrous con-
form chains were noted and their peak-intensities were sig- ditions with expectation to increase proportion of single
nicantly increased by retrogradation. This nding is con- chain [74]. It was found that the 13C NMR spectrum thus
sistent with the previous data that retrogradation results in obtained is identical to that obtained from gel sample.
crystallization as detected by X-ray diraction [68]. Therefore it is more likely that the network structure of
agarose gel consists mainly of the single chain.
C. Agarose and Carrageenans Therefore it is probable that the 13C NMR signals
visible by either solution or DD-MAS NMR signals may
Agarose (III) (Fig. 10) is an alternating copolymer of 3- be ascribed to the liquid-like domain taking random coil
linked h-D-galactopyranose and 4-linked 3, 6-anhydro-a- conformation. In fact, Usov [71] showed that these peaks
L-galactopyranose residues [69]. The well-documented are resonated at 81.9 and 77.0 ppm in aqueous solution.
network model arises from the junction zones consisting Undoubtedly, this network structure is consistent with
of aggregated double-helical chains. However, it seems to formation of either elastic or soft gels. In particular, the
be very dicult to explain why the intense 13C NMR sig- spectral patterns as well as peak intensities from both CP-
nals are visible from the liquid-like domain by a conven- MAS and DD-MAS NMR spectra, resonated at 7782
tional NMR spectrometer [70] or DD-MAS experiment ppm ascribable to the C-3 and C-4 carbons for (1!3)- and
(top trace in Fig. 10), in addition to the intense 13C NMR (1!4)-linked galactosyl residues, respectively, dier sig-
signals available from the solid-like domain by CP-MAS nicantly depending on their respective conformations.
NMR method (bottom trace in Fig. 10). Therefore it In addition, n- (IV) and L-carrageenans (V) are ionic
appears that the well-documented network model for alternating copolymers of 3-linked h-D-galactose-4-sulfate
agarose gel is too much exaggerated and only a small and 4-linked 3,6-anhydro-a-galactose (in kappa) or its 2-
proportion of such double-helical junction zone, if any, sulfate (in iota) residues (Fig. 11) and known to form
is sucient for gelation, as pointed out already in the cases thermally reversible gels at sucient concentration (0.5
of the chemically cross-linked synthetic polymers [26], 4.0% w/v) in the presence of a variety of cations [75,76].
curdlan [27], and amylose [4]. In addition, several workers The solgel transition of L-carrageenan was proposed as a
questioned the validity of the double-helical junction- random coil-double-helix transition, because its well-re-
zones for agarose gel and proposed an alternative model solved 13C NMR signals at temperature at 80jC by solu-
of gel network containing extended single helices tion NMR disappeared completely at 15jC, together with
[34,72,73]. This is mainly because distinguishing between adoption of conformationally rigid double-helical form
Figure 10 13C NMR spectra of agarose gel. Liquid-like domain by DD-MAS (top) and solid-like domain by CP-MAS
measurements (bottom). Broad signal at 110 ppm is from probe assembly. (From Ref. 4.)
ment with Dowex 50W-XB ion exchange resin, followed

by addition of NaOH at pH 7.0, as illustrated in the
bottom trace of Fig. 12. Inevitably, 13C NMR signal from
the solid-like domain recorded by CP-MAS NMR turned
out to be less intense (middle trace, in Fig. 12). In par-
ticular, the 13C NMR peaks at the two lowermost regions
of the liquid-like domain at 104.5 and 96.6 ppm [C-1 peaks
of (1!3)-linked h-D-galactosyl and (1!4)-linked 3,6-
anhydro-a-L-galactosyl residues, respectively] as recorded
by DD-MAS NMR are signicantly displaced downeld
as compared with those recorded by CP-MAS NMR and
also corresponding peaks in the solid state. Further, it was
found that 13C NMR signals of soft gel from L-carrageen-
an (as received) were unavailable from the solid-like
domain by CP-MAS NMR method, although intense
13
C NMR signals were recorded from the liquid-like
domain by DD-MAS NMR (spectra not shown). This
Figure 11 Chemical structures of agarose (III) and n- and
may be caused either by a smaller proportion of the cross-
L- carrageenans (IV and V, respectively).
linked regions of associated single helices or double-helical
junction-zones, if any, or suppressed 13C NMR signals by
interference of motional frequency with proton decou-
[75] as judged by measurements of optical rotation [77]. pling or magic angle spinning. In any case, it should be
Such suppressed 13C NMR signals could be recorded in always taken into account of a possibility that substantial
many instances by observation of either CP-MAS or DD- amount of signals could be lost in the swollen gel samples
MAS or both. The domain model has been also proposed
to account for carrageenan gelation to involve intermolec-
ular double-helix formation of a limited number of chains
and further association by cation-mediated helixhelix
aggregation depending on the type of cations to develop
a cohesive network [76].
However, instead of the double helices or intermolec-
ular association of any kind mentioned above, Smidsrd et
al. [78] proposed an alternative view for gel formation of
carrageenans as salt-promoted freezing-out of linkage
conformations. They showed that, upon cooling the solu-
tion of oligomeric L-carrageenan in the presence of lithium
iodide from 90jC to 25jC, three broad signals originating
from C-1 of the two monomeric units of and from C-3 of
the D-galactopyranosyl residues appear at lower elds
besides the narrowed peaks from a random coil chain, as
a result of formation of an ordered conformation in which
the rotameric forms of the glycosidic linkages are frozen.
Therefore gel formation may be seen as a two-step process,
the rst involving an intramolecular conformational
change and the second involving a decrease in solubility
that is ion-dependent.
For a brittle gel sample (5% w/v) of n-carrageenan as
received from Sigma, there appear no 13C NMR signals
from exible portions taking random coil conformation
as recorded by DD-MAS NMR technique, in contrast
to the case of agarose gel, although intense signals from
the solid-like domain were readily available from CP-
MAS method (spectra not shown). It turns out that 13C
chemical shifts of the gel samples are very close to those of
starting powder within the experimental error as a result
of taking similar conformations. In contrast, 13C NMR Figure 12 13C CP-MAS NMR spectra of n-carrageenan in
signals from the liquid-like domain were available by DD- the solid (top) and gel (middle) and DD-MAS NMR spec-
MAS NMR for resilient gel of n-carrageenan in the ab- trum of gel (bottom) (Saito, H.; Nishino, M.; Yamaguchi, S.;
sence of cations other than Na+ ion, prepared by treat- Zhang, Q.; Watanabe, T., unpublished).
264 Saito
when polysaccharide backbone undergoes uctuation 7. Clark, A.H.; Ross-Murphy, S.B. Structural and mechan-
motions with the time scale mentioned in Fig. 1, as far ical property of biopolymer gels. In Advances in Polymer
Science; Dusek, K., Ed.; Springer-Verlag: Berlin, 1987,
as 13C NMR spectra were recorded by high-resolution
57192.
solid-state NMR spectroscopy. 8. Morris, V.J. Gelation of polysaccharides. In Functional
Properties of Food Macromolecules; Mitchell, J.R., Led-
ward, D.A., Eds.; Elsevier: London, 1986; 121170.
V. CONCLUDING REMARKS 9. Saito, H.; Tabeta, R.; Yokoi, M.; Erata, T. High-resolution
solid-state 13C NMR spectra of secondary structure of
We demonstrated here that conformation and dynamics of linear (1!3)-h-D-glucan: Conformational elucidation of
polysaccharide gels are very conveniently studied by high- noncrystalline and crystalline forms by conformation-
resolution solid-state 13C NMR spectroscopy. The major dependent 13C chemical shifts. Bull. Chem. Soc. Jpn. 1987,
advantage of the NMR approach is that the liquid-like and 60, 4259.
10. Marchessault, R.H.; Deslandes, Y.; Ogawa, K.; Sundarajan,
solid-like domains can be examined separately by DD- P.R. X-ray diraction data for (1!3)-h-D-glucan. Can. J.
MAS and CP-MAS NMR techniques, respectively, al- Chem. 1977, 55, 300.
though expected 13C NMR signals might be suppressed 11. Deslandes, Y.; Marchessault, R.H.; Sarko, A. Triple-helical
when uctuation frequencies of swollen backbone involv- structure of (1!3)-h-D-glucan. Macromolecules 1980, 13,
ing cross-linked region are very close to frequency of either 1466.
proton-decoupling or magic angle spinning. In addition, a 12. Chuah, C.T.; Sarko, A.; Deslandes, Y.; Marchessault, R.H.
Triple-helical crystalline structure of curdlan and para-
systematic examination of conversion diagram among
mylon. Macromolecules 1983, 16, 1375.
polymorphs by a series of a variety of physical treatments 13. Saito, H.; Ohki, T.; Sasaki, T. A 13C nuclear magnetic
is especially useful to clarify whether the polysaccharide resonance study of gel-forming (1!3)-h-D-glucan. Evidence
chain under consideration is taking either single or multiple of the presence of single-helical conformation in a resilient
chains or both in the solid and gel state. gel of a curdlan-type polysaccharide 13140 from Alcaligenes
faecalis var. myxogenes IFO 13140. Biochemistry 1977, 16,
908.
14. Kamihira, M.; Naito, A.; Tuzi, S.; Nosaka, A.Y.; Saito, H.
ACKNOWLEDGMENTS
Conformational transitions and brillation mechanism of
The author is indebted to his collaborators who joined in human calcitonin as studied by high resolution solid state
13
C NMR. Protein Sci. 2000, 9, 867.
the articles cited herein. He also wishes to thank Dr. 15. Kamihira, M.; Oshiro, Y.; Tuzi, S.; Nosaka, A.Y.; Saito, H.;
Satoru Yamaguchi, Misato Nishino, and Kazutoshi Naito, A. Eect of electrostatic interaction on bril
Yamamoto of Himeji Institute of Technology, Professor formation of human calcitonin as studied by high resolution
Tokuko Watanabe and Q. Zhang of Tokyo University of solid state 13C NMR. J. Biol. Chem. 2003, 278, 2859.
Fisheries, and Professor Tomoki Erata of Hokkaido 16. Suwelack, D.; Rothwell, W.P.; Waugh, J.S. Slow molecular
University, for collaborate work, discussion, and help for motion detected in the NMR spectra of rotating solids. J.
Chem. Phys. 1980, 73, 2559.
preparation of this manuscript.
17. Rothwell, W.P.; Waugh, J.S. Transverse relaxation of
dipolar coupled spin system under Rf irradiation: Detecting
motions in solid. J. Chem. Phys. 1981, 74, 2721.
REFERENCES 18. Gil, A.M.; Masui, K.; Naito, A.; Tatham, A.S.; Belton, P.S.;
Saito, H. A 13C NMR study on the conformational and
1. Saito, H. Conformation, dynamics, and gelation mechanism dynamical properties of a cereal seed storage protein, C-
of gel-state (1!3)-h-D-glucans revealed by C-13 NMR. ACS hordein, and its model peptides. Biopolymers 1996, 41, 289.
Symp. Ser. 1981, 150, 125. 19. Alberti, E.; Gilbert, S.M.; Tatham, A.S.; Shewry, P.R.;
2. Saito, H. Conformation and dynamics of (1!3)-h-D- Naito, A.; Okuda, K.; Saito, H.; Gil, A.M. Study of wheat
glucans in the solid and gel state. High-resolution solid- high molecular weight 1D 5 Subunit by 13C and 1H solid-
state 13C NMR spectroscopic study. ACS Symp. Ser. 1992, state NMR. II. Roles of nonrepetitive terminal domains and
489, 296. length of repetitive domain. Biopolymers (Biospectroscopy)
3. Saito, H. Conformational characterization of polysacchar- 2002, 65, 158.
ides as studied by high-resolution 13C solid-state NMR. 20. Lyerla, J.R. High-resolution NMR of glassy amorphous
Annu. Rep. NMR Spectrosc. 1995, 31, 157. polymers. In High Resolution NMR Spectroscopy of Syn-
4. Saito, H.; Shimizu, H.; Sakagami, T.; Tuzi, S.; Naito, A. thetic Polymers in Bulk; Komoroski, R.A., Ed.; VCH Pub-
Conformation and dynamics of polysaccharide gels as lishers: Deereld Beach, Florida, 1986; 63119.
studied by high resolution solid state NMR. In Magnetic 21. Saito, H.; Tuzi, S.; Yamaguchi, S.; Tanio, M.; Naito, A.
Resonance in Food Science; Belton, P.S. Delgadillo, I., Conformation and backbone dynamics of bacteriorhodop-
Gil, A.M., Webb, G.A., Eds.; Special Publication No. 157, sin revealed by 13C NMR. Biochim. Biophys. Acta 2002,
Royal Society of Chemistry: London, 1995; 227271. 1460, 39.
5. Saito, H. Polysaccharide solid state NMR. In Encyclope- 22. Saito, H.; Tuzi, S.; Tanio, M.; Naito, A. Dynamic aspects
dia of Nuclear Magnetic Resonance; Grant, D.M. Harris, of membrane proteins and membrane-associated peptides
R.K., Eds.; John-Wiley and Sons: New York, 1996, 3740 as revealed by 13C NMR: Lessons from bacteriorhodopsin
3745. as an intact protein. Annu. Rep. NMR Spectrosc. 2002, 47,
6. Saito, H.; Tuzi, S.; Naito, A. Polysaccharides and biological 39.
systems. In Solid State NMR of Polymers: Studies in Physi- 23. Kamihira, M.; Naito, A.; Nishimura, K.; Tuzi, S.; Saito, H.
cal and Theoretical Chemistry; Ando, I., Asakura, T., Eds.; High-resolution solid-state 13C and 15N NMR study on
Elsevier Science B.V.: Amsterdam, 1998; Vol. 84, 891921. crystalline Leu- and Met-enkephalins: Distinction of poly-
morphs, backbone dynamics, and local conformational conformational change and conformational characteriza-
rearrangements induced by dehydration or freezing of tion by spin-relaxation measurements. Bull. Chem. Soc. Jpn.
motions of bound solvent molecules. J. Phys. Chem. B 1989, 62, 392.
1998, 102, 2826. 40. Aketagawa, J.; Tanaka, S.; Tamura, H.; Shibata, Y.; Saito,
24. Saito, H.; Yokoi, M. NMR study on collagens in the solid H. Activation of limulus coagulation factor G by several
state: Hydration/ dehydration-induced conformational (1!3)-h-D-glucans: Comparison of the potency of glucans
change of collagen and detection of internal motions. J. with identical degree of polymerization but dierent
Biochem. (Tokyo) 1992, 111, 376. conformations. J. Biochem. (Tokyo) 1993, 113, 683.
25. Yamaguchi, S.; Tuzi, S.; Yonebayashi, K.; Naito, A.; 41. Saito, H.; Yamada, J.; Yoshioka, Y.; Shibata, Y.; Erata, T.
Needleman, R.; Lanyi, J.K.; Saito, H. Surface dynamics of Evidence of three distinct conformations-single chain, single
bacteriorhodopsin as revealed by 13C NMR studies on helix, and triple helix-of (1!3)-h-D-xylan in the solid and
[13C]Ala-labeled proteins: Detection of millisecond or intact frond of green algae as studied by 13C-NMR
microsecond motions in interhelical loops and C-terminal spectroscopy. Biopolymers 1991, 31, 933940.
a-helix. J. Biochem. (Tokyo) 2001, 129, 373. 42. Atkins, E.D.T.; Parker, K.D.; Preston, R.D. Proc. R. Soc.
26. Yokota, K.; Abe, A.; Hosaka, S.; Sakai, I.; Saito, H. A 13C 1969, B173, 209221.
nuclear magnetic resonance study of covalently cross-linked 43. Atkins, E.D.T.; Parker, K.D. The helical structure of a h-D-
gels. Eect of chemical composition, degree of cross-linking, 1,3 xylan. J. Polym. Sci. 1977, C28, 69.
and temperature to chain mobility. Macromolecules 1978, 44. Saito, H.; Yoshioka, Y.; Uehara, N.; Aketagawa, J.;
11, 95. Tanaka, S.; Shibata, Y. Relationship between conformation
27. Saito, H.; Miyata, E.; Sasaki, T. A 13C nuclear magnetic and biological response for (1!3)-h-D glucans in the
resonance study of gel-forming (1!3)-h-D-glucans: Molec- activation of coagulation factor G from limulus amebocyte
ular-weight dependence of helical conformation and of the lysate and host-mediated antitumor activity. Demonstration
presence of junction zones for association of primary of single-helix conformation as a stimulant. Carbohydr. Res.
molecules. Macromolecules 1978, 11, 1244. 1991, 217, 181.
28. Saito, H. Conformation-dependent 13C chemical shifts: A 45. Yoshioka, Y.; Uehara, N.; Saito, H. Conformation-depend-
new means of conformational characterization as obtained ent change in antitumor activity of linear and branched
by high-resolution solid-state NMR. Magn. Reson. Chem. (1!3)-h-D-glucans on the basis of conformational elucida-
1986, 24, 835. tion by Carbon-13 nuclear magnetic resonance spectro-
29. Saito, H.; Ando, I. High-resolution solid-state NMR studies scopy. Chem. Pharm. Bull. 1992, 40, 1221.
on synthetic and biological macromolecules. Annu. Rep. 46. Sarko, A.; Zugenmaier, P. Crystal structures of amylose and
NMR Spectrosc. 1989, 21, 209. its derivatives. A review. ACS Symp. Ser. 1980, 141, 459.
30. Saito, H.; Izumi, G.; Mamizuka, T.; Suzuki, S.; Tabeta, R. A 47. French, D., Wistler, R.L., Pascall, E.D., Eds.; Starch:
13
C cross polarization-magic angle spinning (CP-MAS) Chemistry and Technology, 2nd ed. Academic Press: New
N.M.R. study of crystalline cyclohexa-amylose inclusion York, 1984.
complexes. Conformation-dependent 13C chemical shifts are 48. Veregin, R.P.; Fyfe, C.A.; Marchessault, R.H.; Taylor,
related to the dihedral angles of glycosidic linkages. J. Chem. M.G. Characterization of the crystalline A and B starch
Soc., Chem. Commun. 1982; 1386. polymorphs and investigation of starch crystallization by
31. Ripmeester, J.A. NMR studies on solid cyclohexaamylose high-resolution 13C CP/MAS NMR. Macromolecules 1986,
inclusion compounds. J. Incl. Phenom. 1986, 4, 129. 19, 1030.
32. Veregin, P.; Fyfe, C.A.; Marchessault, R.H.; Taylor, M.G. 49. Gidley, M.J.; Bociek, S.M. Molecular organization in
Carbohydr. Res. 1987, 160, 41. starches: A 13C CP/MAS NMR study. J. Am. Chem. Soc.
33. Saito, H.; Yokoi, M.; Yoshioka, Y. Eect of hydration on 1985, 107, 7040.
conformational change or stabilization of (1!3)-h-D- 50. Horii, F.; Yamamoto, H.; Hirai, A.; Kitamaru, R.
glucans of various chain lengths in the solid state as studied Structural study of amylose polymorphs by cross-polar-
by high-resolution solid-state 13C NMR spectroscopy. ization magic angle spinning 13C NMR spectroscopy.
Macromolecules 1989, 22, 3892. Carbohydr. Res. 1987, 160, 29.
34. Ford, S.A.; Atkins, E.D.T. New X-ray diraction results 51. Saito, H.; Yamada, J.; Yukumoto, T.; Yajima, H.; Endo, R.
from agarose: Extended single helix structures and Conformational stability of V-amyloses and their hydration-
implications for gelation mechanism. Biopolymers 1989, induced conversion to B-form as studied by high-resolution
28, 1345. solid-state 13C NMR spectroscopy. Bull. Chem. Soc. Jpn.
35. Stone, A.; Clarke, A.E. Chemistry and Biology of (1!3)-h- 1991, 64, 3528.
D-Glucans; La Trobe University Press: Victoria, Australia, 52. Veregin, R.P.; Fyfe, C.A.; Marchessault, R.H. Investigation
1992. of the crystalline V amylose complexes by high-resolution
13
36. Harada, T. Production, properties and application of C CP/MAS NMR spectroscopy. Macromolecules 1987,
curdlan, in exocellular microbial polysaccharide. ACS 20, 3007.
Symp. Ser. 1977, 45, 265. 53. Gidley, M.J.; Bociek, S.M. 13C CP/MAS NMR studies of
37. Harada, T.; Harada, A. Curdlan and succinoglycan. In Poly- amylose inclusion complexes, cyclodextrins, and the amor-
saccharides in Medical Applications; Dumitriu, S., Ed.; Mar- phous phase of starch granules: Relationships between
cel Dekker: New York, 1996; 2158. glycosidic linkage conformation and solid-state 13C chem-
38. Saito, H.; Tabeta, R.; Yoshioka, Y.; Hara, C.; Kiho, T.; ical shifts. J. Am. Chem. Soc. 1988, 110, 3820.
Ukai, S. A high-resolution solid-state 13C NMR study of the 54. Horii, F.; Hirai, A.; Kitamaru, R. CP/MAS 13C NMR
secondary structure of branched (1!3)-h-D-glucans from spectroscopy of hydrated amyloses using a magic angle
fungi: Evidence of two kinds of conformers, curdlan-type spinning rotor with an O-ring seal. Macromolecules 1986,
single helix and laminaran-type triple helix forms, as 19, 930.
manifested from the conformation-dependent 13C chemical 55. Senti, F.R.; Witnauer, L.P. Structure of alkali amylose. J.
shifts. Bull. Chem. Soc. Jpn. 1987, 60, 4267. Am. Chem. Soc. 1948, 70, 1438.
39. Saito, H.; Yokoi, M. High-resolution 13C NMR study of 56. Blackwell, J.; Sarko, A.; Marchessault, H. Chain conforma-
(1!3)-h-D-glucans in the solid state: DMSO-induced tion in B-amylose. J. Mol. Biol. 1969, 42, 379.
266 Saito
57. Wu, H.C.H.; Sarko, A. The double-helical molecular struc- 67. Gidley, M.J. Molecular mechanisms underlying amylose
ture of crystalline B-amylose. Carbohydr. Res. 1978, 61, 7. aggregation and gelation. Macromolecules 1989, 22, 351.
58. Imberty, A.; Perez, S. A revisit to the three-dimensional 68. Miles, M.J.; Morris, V.J.; Ring, S.G. Carbohydr. Res. 1985,
structure of B-type starch. Biopolymers 1988, 27, 1205. 135, 257.
59. Saito, H.; Yoshioka, Y.; Yokoi, M.; Yamada, J. Distinct 69. Arnott, S.; Fulmer, A.; Scott, W.E.; Dea, I.C.M.; Moor-
gelation mechanism between linear and branched (1!3)-h- house, R.; Rees, D.A. The agarose double helix and its
13
D-glucans as revealed by high-resolution solid-state C function in agarose gel structure. J. Mol. Biol. 1974, 90, 269.
NMR. Biopolymers 1990, 29, 1689. 70. Nicolaisen, F.M.; Meyland, I.; Schaumburg, K. 13C NMR
60. Saito, H.; Ohki, T.; Takasuka, N.; Sasaki, T. A 13C NMR spectra at 67.9 MHz of aqueous agarose solutions and gels.
spectral study on a gel-forming (1!3)-h-D-glucan (Len- Acta Chem. Scand. 1980, B34, 579.
tinan) from Lentinus edodes, and its acid-degraded fractions. 71. Usov, A.L. NMR spectroscopy of red seaweed polysacchar-
Structure, and dependence of conformation on the molec- ides: Agars, carrageenans, and xylans. Bot. Mar. 1984, 27,
ular weight. Carbohydr. Res. 1977, 58, 293. 189.
61. Saito, H.; Ohki, T.; Yoshioka, Y.; Fukuoka, F. A 13C 72. Letherby, M.R.; Young, D.A. The Gelation of Agarose. J.
nuclear magnetic resonance study of a gel-forming branched Chem. Soc. Faraday Trans. I 1981, 77, 1953.
(1!3)-h-D-glucan, A3, from Pleurotusbostreatus (Fr.) Quel: 73. Norton, I.T.; Goodall, D.M.; Austin, K.R.J.; Morris, E.R.;
Determination of side-chains and conformation of the Rees, D.A. Dynamics of molecular organization in agarose
polymer-chain in relation to gel-structure. FEBS Lett. sulphate. Biopolymers 1986, 25, 1009.
1976, 68, 15. 74. Saito, H.; Yokoi, M.; Yamada, J. Hydration-dehydration-
62. Saito, H.; Ohki, T.; Sasaki, T. A 13C nuclear magnetic induced conformational changes of agarose, and kappa- and
resonance study of polysaccharide gels. Molecular architec- iota-carrageenans as studied by high-resolution solid-state
13
ture in the gels consisting of fungal, branched (1!3)-h-D- C nuclear magnetic resonance spectroscopy. Carbohydr.
glucans (lentinan and schizophyllan) as manifested by Res. 1990, 199, 1.
conformational changes induced by sodium hydroxide. 75. Arnott, S.; Scott, W.E.; Rees, D.A.; McNab, C.G.A.
Carbohydr. Res. 1979, 74, 227. L-Carrageenan: Molecular structure and packing of poly-
63. Yanaki, T.; Norisue, T.; Fujita, H. Triple helix of saccharide double helices in oriented bres of divalent cation
Schizophyllum commune polysaccharide in dilute solution salts. J. Mol. Biol. 1974, 90, 253.
3. Hydrodynamic properties in water. Macromolecules 76. Morris, E.R.; Rees, D.A.; Robinson, G. Cation-specic
1980, 13, 1462. aggregation of carrageenan helices: Domain model of
64. Kashiwagi, Y.; Norisue, T.; Fujita, H. Triple helix of polymer gel structure. J. Mol. Biol. 1980, 138, 349.
Schizophyllum commune polysaccharide in dilute solution 4. 77. Bryce, T.A.; McKinnon, A.A.; Morris, E.R.; Rees, D.A.;
Light scattering and viscosity in dilute aqueous sodium Thom, D. Chain conformations in the solgel transitions for
hydroxide. Macromolecules 1981, 14, 1220. polysaccharide systems and their characterization by spec-
65. Sarkar, S.K.; Sullivan, C.E.; Torchia, D.A. Nanosecond troscopic methods. Faraday Discuss. Chem. Soc. 1974, 221,
evaluation of the molecular backbone of collagen in hard 57221.
and soft tissue: A Carbon-13 nuclear magnetic relaxation 78. Smidsrd, O.; Andresen, I.-I.; Grasdalen, H.; Larsen, B.;
study. Biochemistry 1985, 24, 2348. Painter, T. Evidence for a salt-promoted freezing-out
66. Mehring, M. High Resolution NMR Spectroscopy in Solids; of linkage conformations in carrageenans as a pre-
Springer: New York, 1983. requisite for gel-formation. Carbohydr. Res. 1980, 80, C11.
10
Correlating Structural and Functional Properties
of Lignocellulosics and Paper by Fluorescence
Spectroscopy and Chemometrics
Emmanouil S. Avgerinos, Evaggeli Billa, and Emmanuel G. Koukios
National Technical University of Athens, Athens, Greece
Summary spectrometry in conjunction to chemometrics to provide

valuable information on aging and storing of these paper
With this paper, we present the work on the multivariate materials, including the use of multivariate models to
analysis of uorescence spectral and chemical data of derive useful chemical information.
lignocellulosic ber materials processes, showing the po-
tential of the uorescence spectroscopy through this
chemometric technique for the study of lignocellulosics
and the usefulness of this tool for studying the ber I. INTRODUCTION AND BACKGROUND
materials and their production method. For this, uores-
cence spectral data were correlated with physical and The main objective of the work reported here was to apply
chemical properties through partial least squares (PLS) multivariate, uorescence spectra-based analysis in order
models, whereas principal component analysis (PCA) was to lead to a new, nondestructive method. More specically,
employed as a multivariate method aiming at determining the uorescence of plant bers was investigated by means
the main variation in a multidimensional data set by of mathematical and statistical methods in order to extract
creating new linear combinations of the raw data. Exam- reliable and relevant information from chemical data. The
ples of this tool are the results of some selected applica- uorescence phenomenon is a form of energy transfer
tion of this characterization method. In these examples, mediated in each case by a specic electron arrangement
we have studied paper pulps from wheat straw and sweet/ which, upon receiving excitation light of a specic wave-
ber sorghum stalks, pulped with aqueous ethanol solu- length, transforms this energy to another rather specic
tion under acid- or alkali-catalyzed conditions and emission band of a higher wavelength. Fluorescence spec-
bleached with hydrogen peroxide aqueous solutions in troscopic methods are cheap, rapid, sensitive, specic, and
an alkali environment during various processing times. nondestructive. Lignocellulosics exhibit autouorescence
The results from the study of these examples have indi- permitting to be analyzed without any treatment. More-
cated that the uorescence emission spectra of solid paper over, the spectrum depends on the treatment to which the
pulps and their black liquors could provide vital infor- sample has been subjected. According to this approach
mation about both the origin of the pulp sample and the (short name: auence), whole uorescence emission spec-
kind of chemical treatment applied. Additional research tra in combination with principal components analysis
ndings include the existence of good correlation between (PCA) were used. Moreover, the presence of good statisti-
the uorescence data and the chemical and physical data cal correlations between the uorescence data and the
(kappa number, brightness, cellulose, hemicellulose, and chemical properties of the samples was shown by means
lignin contents) of the pulps through partial least squares of partial least squares (PLS) regression.
(PLS) models. We also studied the evaluation of phenom- This uorescence method provides a data matrix (X)
ena related to paper aging, using samples of naturally (uorescence) for each sample. The speed and noninvasive
aging paper from various Greek archives. The results properties of spectroscopic techniques make them poten-
obtained have veried the power of the use of uorescence tial on-line or at-line methods and hence very useful for
267
268 Avgerinos et al.
process monitoring and control. Fluorescence spectrosco- The main rationales for developing and using multi-
py is similar to UV/VIS spectroscopy in that it measures way methods are the following:
electronic transitions, but it is detecting emitted rather than
1. The instrumental development makes it possible
transmitted light. This results in fewer interferences, as not
to obtain information that more adequately
all samples having a UV/VIS spectrum exhibit uores-
describes the intrinsic multivariate and complex
cence. Fluorescence is detected as the light emitted follow-
reality. Along with the development on the ins-
ing excitation by monochromatic light. This is similar to
trumental side, development on the data analytical
Raman spectroscopy, but contrary to the latter method,
side is natural and benecial. Multiway analysis is
which measures vibrational transitions, uorescence mon-
one such data analytical development.
itors electronic transitions. The eects are competitive, but
2. Some multiway model structures are unique. No
usually uorescence is much stronger than the Raman
additional constraints, like orthogonality, are
eect. As the emission spectrum is highly dependent on
necessary to identify the model. This implicitly
the excitation wavelength, both an excitation and an
means that it is possible to calibrate for analytes
emission spectrum can be obtained. Thus the advantages
in samples of unknown constitution, i.e., estimate
of uorescence spectroscopy are the absence of interfer-
the concentration of analytes in a sample where
ences and that higher-order data can be obtained from it.
unknown interferents are present.
Fluorescence spectroscopy is widely used as an analytical
3. Another aspect of uniqueness is what can be
technique in many elds of science including chemistry,
termed computer chromatography. In analogy to
biology, biochemistry, medicine, environmental science,
ordinary chromatography, it is possible in some
and food science [1]. The simplest way to measure uores-
cases to separate the constituents of a set of
cence is with instruments recording a response at a preset
samples mathematically, thereby alleviating the
excitation wavelength and emission wavelength. Multivar-
use of chromatography and cutting down the
iate data analysis techniques dene the use of specic
consumption of chemicals and time. Curve reso-
statistical and mathematical methods developed for deal-
lution has been extensively studied in chemo-
ing with the multivariate data [2].
metrics but has seldom taken advantage of the
The inclusion of the vast quantity of data supplied by
multiway methodology.
the computerized measurement in the measurement system
4. While uniqueness as a concept has long been the
is dened as multivariate data techniques. This denition
driving force for the use of multiway methods, it is
covers both the data acquisition technique for the inspected
also fruitful to simply view the multiway models as
object and the multivariate statistical and mathematical
natural structural bases for certain types of data,
analyses performed on these data. The latter subject is
e.g., in sensory analysis, spectral analysis, etc. The
becoming known as chemometrics in the chemical sciences.
mere fact that the models are appropriate as a
This study introduces such new multivariate techniques in
structural basis for the data implies that using
the area of lignocellulosics.
multiway methods should provide models that are
In traditional chemical analysis, one starts by dening
parsimonious, thus robust and interpretable, and
the hundreds of chemical substances involved in a process.
hence give better predictions and better possibil-
For chemometrics to be successful, access to a full chain of
ities for exploring the data.
interdisciplinary resources including, for example, analyt-
5. Better structural models increase the robustness
ical chemical analysis, spectroscopy, mathematics, com-
and increase the noise reduction, provide simpler
puter programming, and information technologies is
models using fewer parameters, give more inter-
required by the researcher. Every link in this chain has to
pretable models because of parsimony and cor-
have basic understanding of multivariate data analysis in
respondence between the nature of the data and
order to contribute optimally to the solution to the prob-
the model, and give better predictions in many
lem since issues like repeatability, variation, validation,
cases [37].
and data quality are of fundamental importance to the
exploratory multivariate data analysis. Undoubtedly, multiway analysis can be benecially
Chemometrics, being a juxtaposition of chemo (Latin: used in analytical chemistry for developing fast and cheap
chemistry) and metrics (Greek: measure), is the common calibration methods for a variety of chemical applications.
denominator of all possible tools applied to make rational Such methods may well reduce the cost and use of
analysis of chemical measurements. Using the term chemo- additional chemicals while providing more robust and
metrics serves an important purpose: it makes clear that the accurate estimations. Besides pure chemical and spectral
whole of the problem is to be observed, analyzed, and data, multiway problems are often encountered in other
interpreted in a direct or indirect chemical context. For the areas. Examples are analysis of quantitative structure
engaged practitioner, chemometrics oers signicant new activity relationships for designing medical drugs or
possibilities in the approach toward multivariate problems assessing environmental and health eects, batch and
that perfectly complements the classical scientic method- continuous process analysis, electronic noise data, con-
ology, thus providing a technology in the sense that it is a sumer analysis, sensory analysis, image analysis, blind
holistic pragmatic solution combining strategies and tools source separation in, e.g., telecommunication and speech
in the very center of the application. recognition systems, time series analysis, signal processing
Correlating Lignocellulosics and Paper 269
for example related to medical instruments or process

analysis, etc.
An application of this approach is the use of uores-
cence spectra and physico/chemical characteristics of lig-
nocellulosic materials correlating structural and functional
properties of lignocellulosics and paper by uorescence
spectroscopy and chemometrics for evaluation and/or
study of processes. Fig. 1 presents graphically this concept.
So using this methodology approach, we could de-
scribe the complex multivariate information in the data in
simple graphic displays without interference of a priori
knowledge and let the evaluation be based on plots and
graphs. The samples data from spectroscopic instruments
are typically so complicated that direct interpretation is
sometimes impossible. That is why chemometric methods
need to be applied for the data to be analyzed eectively.
The most used chemometric methods are principal
component analysis (PCA) for decomposition and inter- Figure 2 Principal component analysis represents the core
pretation of large data set and partial least squares (PLS) idea of condensing large amounts of data to a few representa-
regression for linear regression of multivariate data. Prin- tive parameters (principal components or latent factors).
cipal component analysis represents the core idea of con-
densing large amounts of data to a few representative
parameters ( principal components or latent factors) which
capture the levels of, and dierences between, objects and Partial least square regression (PLS) is used to make a
variables in the data under investigation. Patterns and model correlating X and y where X contains the uores-
clusters in the parameters are easily represented in the form cence spectra and y (s1) is a vector containing the
of scatter plots in the Euclidean plane with an exploratory property of interest [4,5] (Fig. 2).
choice of dierent principal components as axes. By na- The fast, precise, and nondestructive spectroscopic
ture, PCA implies that the world is under indirect obser- methods in combination with chemometrics are suitable
vation as variations in data are caused by principal for process analysis and optimization leading to improved
components in the sense that these are hidden and under- productivity, eciency, and product quality. The uores-
lying instead of manifest and directly observable [3]. cence and multiway approach has the potential to provide
In the work described, PCA was used to form a general direct chemical ngerprinting of a range of natural mole-
view of the data sets and to discover unknown trends. cules in a variety of biological matrices. The end quality of
ber materials that we studied reects both the quality of
the raw ingredients and the actions of the processing unit
operations. Some of the traditional methods are quite time
consuming and all the methods are destructive. Today, it is
possible to replace most of these inconvenient methods by
instrumental, rapid, and nondestructive techniques such as
uorescence spectroscopy [69].
II. METHODS: THE AFFLUENCE APPROACH

This method was applied in both solid and liquor samples.
As raw materials, we used several samples from dierent
ber plants and their product. The raw materials were
wheat straw (Triticum durum sp.) and sorghum (Sorghum
bicolor {L.} Moench) stems. Wheat straw and sweet
sorghum pulps were prepared after treatment with (1)
aqueous ethanol with the addition of H2SO4 (acid cata-
lyzed) for 1.5 and 3 hr and (2) aqueous ethanol with the
addition of NaOH (alkali catalyzed) for 0.5, 1, and 2 hr.
Figure 1 This gure presents graphically the use of uo- The pulping treatment was performed according to Papa-
rescence spectra and physico/chemical characteristics of theophanous et al. [10]. Kappa number was determined
lignocellulosic materials correlating structural and functional according to Berjings [11] and the brightness according
properties of lignocellulosics and paper by uorescence Tappi Standard T457. The pulps were bleached with H2O2
spectroscopy and chemometrics for evaluation and/or study under the following conditions: pulp consistency 12%,
of processes. H2O2 4%, NaOH 2%, Na2SiO3 4%, DTPA 2%, and
temperature 75jC, for 1, 2, and 3 hr. Also, we applied the wavelength, was removed from the spectral emission
auence method on samples of old paper. So naturally region; so, from the 3004 initial wavelength variables, we
aging papers from the Historical Archives Division of considered only 1354 or 1479 (according to the samples)
National Bank of Greece (NBG) and the National Library (Fig. 4a and b), and in some cases, the modication V=ln
of Greece (NLG) were collected. The samples from NBG V was used (Fig. 4c).
covered a period of 70 years (18691940) and those from So after the preview treatment of spectra, the multi-
NLG the years 15941965. variate analysis was applied to a transformed data of an
Fluorescence spectra were recorded on a Perkin Elmer s1479 matrix (s: number of samples and 1479 variables).
LS 50B Luminescence Spectrometer connected with a PC. Using these inputs, principal component analysis (PCA)
Autouorescence emission spectra were recorded at exci- nds the main variation in a multidimensional data set by
tation wavelengths of 450, 400, 350, and 280 nm, whereas creating new linear combinations of the raw data [12]. In
the emission was measured in the region of 275650 nm matrix form, we have X=TPV (X is the analyzed data
with intervals of 0.5 nm (751 data points, in total, matrix with dimensions sw, T is the score matrix with
4751=3004 data points). Excitation and emission mono- dimensions smin(s,w), and P is the loading matrix with
chromator slit widths were 10 nm. The measurement dimensions wmin(s,w). Only a signicant number of
started from the highest and nished with the lowest principal components, f, equal to the chemical rank of
excitation wavelength in order to minimize the photode- the X matrix, is relevant in describing the information in X.
composition of the sample. The spectrum of each sample Each component (each new variable) is a linear combina-
was taken twice. In all measurements, the temperature was tion of the original measurements. This projection of data
24F1jC. Spectral data were converted to ASCII les by a is continued by composing additional, orthogonal princi-
program furnished by Perkin Elmer (FL Data Manager, pal components, until all latent structures of the data are
version 3.50). Each of the spectra is the average of the two described. In this way, PCA provides an approximation of
measurements, and for this chemometrics analysis (PCA the data matrix (e.g., near-infrared spectra of a number of
and PLC), the ASCII les were introduced to and pro- samples) in terms of the product of two low-dimensional
ceeded by the UNSCRAMBLER v 7.0 software [12]. matrices T (scores) and PV (loadings). These two matrices
The spectral shapes of the dierent samples are similar capture the systematic variation of the data matrix:
with some common high peaks corresponding to the X=TPV+E, and leave the unsystematic variation in the
Rayleigh scatter (Fig. 3). Rayleigh scatter, which gives a residual matrix (E). Plots of the columns of T (score plots)
large contribution to the emission spectrum at the emis- provide a picture of the sample concentrations of the prin-
sion wavelength corresponding to the actual excitation cipal components, while plots of the rows of PV (loading
Figure 3 Whole emission spectra of NLG samples as introduced to the UNSCRAMBLER program. Emission variables 1751
correspond to kex=450 nm, variables 7521502 correspond to kex=400 nm, variables 15022253 correspond to kex=350 nm, and
variables 22533004 correspond to kex=280 nm.
Figure 4 (a and b) Concatenated emission spectra of pulp samples after the removal of Rayleigh peaks. (c) Concatenated
emission spectra of NBG samples after V=ln V modication.
Figure 4 Continued.
plots) depict the variable contribution to the principal from these models have pairwise maximal covariance. That
components. is, components are found in X and Y simultaneously and
Partial least square regression (PLS) is used to make a such that the scores in the X and Y spaces have maximal
model correlating X and y where X contains the uores- covariance. Since the covariance is the product of the
cence spectra and y (s1) is a vector containing the correlation between the scores and the variance of each
property of interest (Fig. 5). In chemometrics, PLS regres- score, these three measures are collectively maximized.
sion is a widely used approach for obtaining multivariate Maximizing the variation of the score vectors ensures that
regression models. The main dierence between PCR and the model is real and not due to small random variation.
PLS is that in PLS, the independent data are modeled such Maximizing the correlation (the linear relationship)
that variation specically relevant for predicting the de- ensures that it is possible to predict the Y score from the
pendent variables is emphasized. Rather than decompos- X score, thus optimizing the predictive ability of the model.
ing the independent data into a least squares bilinear Using measured spectral data to predict important
model, a model of the dependent and a model of the quality parameters ( y) involves ecient multivariate re-
independent data are obtained such that the score vectors gression techniques. The multivariate calibration task is to
Figure 5 Partial least square (PLS) regression is used to

make a model correlating X and y where X contains the Figure 6 Principal component analysis score plot of PC4 vs.
uorescence spectra and y (s1) is a vector containing the PC2 for the 48 paper pulp samples explaining 1% and 4% of
property of interest. the total variance, respectively.
Figure 7 Partial least square model for the yield of pulping using the uorescence data of pulps (correlation=0.985).
build a relationship between the spectra or landscapes (x/ samples (full cross-validation) or segments of samples
X) and the reference parameter ( y) for all the samples in a (segmented cross-validation) kept out of the calibration
given data set. The purpose of the relationship is to predict alternately, until all samples have been kept out once. The
the ys from the x/Xs in the future and to interpret the samples kept out are then used for validation, and the
relationships between x/X and y. In this work, the multi- average of the validation results is calculated. Such meth-
variate regressions were performed also by partial least ods will at their best provide a robust consistent estimation
squares regression (PLSR) [13], which is a predictive of the prediction error. The measure of model performance
regression method based on estimated latent variables is usually given by the correlation coecient (R), which is
describing the relations between X and y (corresponding the correlation between the measured reference ( y) and the
reference measurements of the sample set). The strategy of predicted reference ( y), and by the prediction error root
PLSR is to reduce the dimension of the X and y space by
creating linear combinations of the original variables.
These new (latent) variables or components are statistically
independent and ideally carry all relevant information. The
reference variable to be predicted is used actively in deter-
mining these components, and a linear regression model is
dened as y=Xb+E where b is the corresponding vector
of regression coecients and E is their residuals (model
errors, noise, etc.).
By chemometric validation, it is possible to obtain as
realistic performance of the models as possible with the
available data. In our case, the model performance was
validated by a test set or cross-validation depending on the
data set. Test-set validation requires two data sets which
are similar with respect to their ability to cover future
sample variations and sampling conditions. One of the data
sets is used for calibration, while the other is used for
validation. Test-set validation requires sucient samples in
order to span the existing variation in both sets. It may
often occur that it is not possible to collect enough samples
for producing usable calibration and test sets. In the Figure 8 Score plot (PC1PC2) of the uorescence data of
absence of a test set, it is necessary to apply cross-valida- paper pulp (160jC and 200jC: temperature of treatment;
tion, where several subcalibrations are made with single 60% and 70% EtOH/water ratio in organosolv method).
based on multivariate analysis of the uorescence emission

spectra of solid and black liquor samples. Correlation and
prediction of kappa number as well as cellulose, hemicel-
lulose, and lignin content of corresponding pulps were
performed using the model that was developed. Finally,
the application of multivariate analysis on old paper
samples is presented at the end of this part.
A. Delignification Processes
Fig. 6 presents the two-dimensional representation of PC2
vs. PC4 explaining that the 5% of the total variance
revealed the presence of three clusters. The one included
samples of alkali-catalyzed organosolv wheat straw pulp
samples (bleached and unbleached), the other one included
the alkali-catalyzed sweet sorghum samples, and the third
one included the acid-catalyzed organosolv wheat straw
and sweet sorghum samples (W: wheat straw; S: sweet
Figure 9 Tridimensional score plot (PC1PC2PC3) of the sorghum; F: ber sorghum) and the two dierent processes
uorescence data of paper pulp black liquor samples. W: that we followed (O: acid treatment; A: alkali treatment).
wheat straw; S: sweet sorghum; F: ber sorghum; O: acid The yield of pulping according to multivariate anal-
treatment; A: alkali treatment. ysis of uorescence emissions of black liquors is presented
in Fig. 7.
In Fig. 8, we could observe the clusters that present
mean square error of cross-validation (RMSECV) or root the process that produced the corresponding solid, as the
mean square error of prediction (RMSEP). samples that were treated at 200jC are altogether below
the line AF when the others treated at 160jC are above
it. Also, at the same gure, we could distinguish that the
III. RESULTS treated samples with the same EtOH ratio (70:1) are
concentrated in the drawn circle (Fig. 8). For these solid
In this part, we present the results from the application of samples of pulps, the variance explained by the rst four
auence method correlating the data from the pulping principal components (PCs) resulting from the (PCA)
and bleaching processes of lignocellulosics raw materials was 100%.
Figure 10 Partial least square model of the predicted vs. measured values for the kappa number of organosolv wheat straw
samples. R=0.94 for fth PC.
model of the predicted vs. measured values for the kappa

number of organosolv wheat straw pulps (Fig. 10).
Next, Fig. 11 presents the result from our study of
correlations between uorescence data and the 31P NMR
structural information (data like the COOH) on residual
kraft lignin of paper samples.
In addition, the correlation coecient becomes 0.95
when we correlate the uorescence data with the brightness
of the alkali-catalyzed organosolv wheat straw pulp sam-
ples (Fig. 12).
As a next step, we constructed models correlating the
uorescence data and quantitative properties like the con-
centration of cellulose, lignin, and hemicellulose content of
the solid samples (Figs. 1315). Fig. 13 shows a PLS model
Figure 11 Partial least square model for the formation and/
for the cellulose concentration ranged from 42% to 65%
or elimination of carboxylic acids during the treatment of
residual kraft lignin (RKL) with dierent charges of chlorine
for the wheat straw pulps with the correlation coecient
dioxide, dimethyldioxirane, alkaline hydrogen peroxide, and R=0.96.
oxygen. R=0.938.
C. Paper Aging
Finally, we examined the case of a multivariate calibration
B. Product Characterization model built between the uorescence emission spectra and
the age (be counted using the year of print for the printed
For the black liquor samples and the multivariate chemo- materials) and alkaline reserve of samples of old archival
metric analysis, we could observe also the clusters that papers [1417].
present the process that produced the corresponding liquor It was noted that the uorescence intensity of the two
(Fig. 9). In this gure, we see the tridimensional score plot resources of samples is dierent as well as the emission
(PC1PC2PC3) of the uorescence data of paper pulp according to the year of the sample. In order to see whether
black liquor samples, and we could detect the groups our samples could be classied according to their alkaline
clusters where each one represents the three dierent raw reserve, which is a measure of paper deterioration, a PCA
materials. The correlation coecient becomes 0.94 for PLS model of 70 samples (matrix X: 701354) was built using
Figure 12 Partial least square model of the predicted vs. measured values for the brightness (B/R475) of the bleached and alkali-
catalyzed organosolv wheat straw samples. R=0.95 for the second PC.
Figure 13 Partial least square model for the cellulose content of the uorescence data of paper pulp.
Figure 14 Partial least square model of the predicted vs. measured values for the lignin content of wheat straw samples.
R=0.917 for the fourth PC.
Figure 15 Partial least square model of the predicted vs. measured values for the hemicellulose content of alkali-catalyzed
organosolv wheat straw samples. R=0.882.
test-set validation. The score plot of PC2 vs. PC1, explain- others are on the left. The alkaline reserve is a measure of
ing 96% of the total variance, is shown in Fig. 16 and the progress of the deterioration of the paper, so the T01
demonstrates the presence of two clusters according to the T04 with the low alkaline reserve are the samples that cover
age of the samples. We observe the presence of two clusters the years 15941780 and T05T09 for years 18371965.
according to the alkaline reserve. The samples with the low In Fig. 17, a PLS model for year of print (conse-
alkaline reserve are on the right part of the diagram and the quently the age of the paper) for all the samples is
Figure 16 Principal component analysis score plot of principal component 1 (PC1) vs. PC2 of the NLG samples explaining 91%
and 5% of the total variance, respectively. Samples T01T04 for years 15941780 and T05T09 for years 18371965.
Figure 17 Partial least square model of the predicted vs. measured values for the age of the samples.
presented. The correlation coecient (R=0.96) was very years. If we consider the fact that the year of print is z2
good. Considering the number of PCs that we used for the years after the year of production, we realize that we have
PLS model, we achieved the best explanation of Y variance an accepted result trying to estimate the age and the
(year) about 93.4%. This means that the more complicated production year of a paper particularity when we study
the model is, the better is the prediction of the model. We samples of nature-aged paper that were produced 100200
could also see that the model gives an overestimated value years before.
of the year of the sample when we study samples from 1869 In a second example, the score plot of PC2 vs. PC1,
to 1900 and an underestimation for samples from 1900 to explaining 98% of the total variance, is shown in Fig. 18
1940. Also, the value of RAMSEP=5.92 years means that and demonstrates the presence of two clusters according
we could predict the year of print with an error about 5.92 to the origin of the samples. In this case, we use for our
Figure 18 Principal component analysis score plot of PC2 vs. PC1 of NLG samples (from rooms E and S) explains the origin of
the samples.
Figure 19 Partial least square model of the predicted vs. measured values for alkali reserve of the samples from NLG for the
third PC.
analysis the uorescence data from samples that were losics. The uorescence data derived from archival old
storing for the same period into two dierent storing papers were correlated with the chemical properties and
rooms. Studying the result of this PCA at Fig. 18, we the age of these samples and veried the power of the use of
observe the plain separation of two areas of scores. The one uorescence spectrometry in conjunction to chemometrics
that involves the samples from room is marked as E and the to provide valuable information on aging and storing of
others are marked as S. The last three gures present the these materials. This study could drive us to develop the
results for three dierent correlation studies. Next, Fig. 19 new procedure of qualication of phenomena related to
presents the PLS model for the predicted vs. measured aging. Overall, the auence approach allows qualitative
values of alkaline reserve of samples according to the and quantitative information to be obtained from complex
spectra with a correlation factor 0.754. spectral data [1820].
REFERENCES
IV. CONCLUDING REMARKS
1. Munck, L. Practical experiences in the development of
From the above study of various pulps using the uores- uorescence analyses in an applied food research laboratory.
cence spectral data, the results indicated that the uores- Fluorescence Analysis in Foods (Lars Munck); Longman
cence emission spectra of solid paper pulps through Scientic Technical: UK, 1989; 132.
2. Lundquist, K.; Josefsson, B.; Nyquist, G. Analysis of lignin
principal component analysis can give useful qualitative products by uorescence spectroscopy. Holzforschung 1978,
information about the origin of the pulp samples as well as 32 (1), 2732.
the condition of the treatment. Moreover, the existence of a 3. Noergaard, L. A multivariate chemometric approach to
good correlation between the uorescence data and the uorescence spectroscopy. Talanta 1995, 42, 13051324.
chemical characteristics of the pulps, like the cellulose, 4. Andersson, C. Exploratory Multivariate Data Analysis with
hemicellulose, and lignin content as well as the kappa Applications in Food Technology, Thesis, 2000.
5. Baunsgaard, D. Analysis of colour impurities in sugar pro-
number, shows the quantitative use of PLS models. These cessing using uorescence spectroscopy and chemometric,
ndings corroborating our previous results strengthen our Thesis, 2000.
claim on the potential of uorescence through chemo- 6. Bro, R. Multi-way analysis in the food industry models,
metrics as process analytical method applied to lignocellu- algorithms applications, Thesis.
7. Brndum J., On-line Evaluation of Meat Quality Using Samples, La Conservation: Une Science en Evolution, bilans
Multivariate Data Techniques, Thesis, 1999. et Perspectives, Actes des Deuxiemes Journees Internatio-
8. Hansen, W. Spectroscopic and chemometric exploration of nales DEtudes de LArsag, Paris, 1997.
food quality Spectroscopic Analyses on Dairy Products, 16. Drenth, P.J.D. Why Choosing to Preserve, International
Thesis, 1998. Conference.
9. Pedersen, D. Early prediction of meat quality, Thesis. 17. Zou, X.; Gurnagul, N.; Uesaka, T.; Bouchard, J. Accel-
10. Papatheophanous, M.G.; Billa, E.; Koullas, D.P.; Mon- erated ageing of papers of pur cellulose: mechanism of
ties, B.; Koukios, E.G. Two-stage acid-catalyzed fraction- cellulose degradation and paper embrittlement. Polym.
ation of lignocellulosic biomass in aqueous ethanol Degrad. Stab. 1994, 43, 393.
systems at low temperatures. Bioresour. Technol. 1995, 18. Billa, E.; Pastou, A.; Monties, B.; Romero, J.; Koukios,
54, 305310. E.G. Multivariate chemometric analysis of the uorescence
11. Berjings, V. Pulp Pap. Mag. Can., 1966; 206208. spectra of eucalyptus wood. Ind. Crops Prod. 2000, 11, 187
12. Esbensen, K.; Schonkopf, S.; Midtgaard, T. Multivariate 196.
Analysis in Practice; Camo AS: Trondheim, Norway, 1994. 19. Billa, E.; Koukios, E.G. A new, uorescence based, multi-
13. Martens, H.; Ns, T. Multivariate Calibration; Wiley: variate chemometrics approach for the characterization of
Chichester, 1989. lignocellulosics. Proceedings 9th International Symposium
14. Anders, M.; Bredereck, K.; Haberditzl, A. Mechanisms of on Wood and Pulping chemistry, Montreal, Canada; 1997;
paper ageing and non-aqueous paper deacidication com- 14. T1.
bined with paper strengthening. 11th Triennieal meeting 20. Billa, E.; Argyropoulos, D.S.; Koukios, E.G. Recent
Dinburg, Scotland, 1996; Vol. II. Advances in Residual Kraft Lignins Characterization Com-
15. Begin, P.; Iraci, J.; Grattan, D.; Kaminska, E.; Woods, D.; bining 31P NMR and Fluorescence Spectroscopy by Chemo-
Zou, X.; Gurnagul, N.; Dechatelets, S. The Impact of Lignin metrics, Progress in Analytical Methodologies Applied to
on Paper Permanence Part I: A Comprehensive Study of the Lignocellulosic Materials; TAPPI Press: Atlanta, USA,
Ageing Behaviour of Handsheets and Commercial paper 1999, Chapter 5.
11
Computer Modeling of PolysaccharidePolysaccharide
Interactions
Francois R. Taravel and Karim Mazeau
Grenoble, France
Igor Tvaroska
Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia
I. INTRODUCTION quence, molecular weight, anomeric conguration, linkage

position, and charge density. Additional variability may
Carbohydrate polymers derive from natures capacity to arise from environmental changes such as ionic strength
convert carbohydrate molecules into polyacetals by several and degree of hydration. Also, whereas in crystals most
biochemical pathways [1]. Carbohydrate components of molecules usually adopt one single conformation, in solu-
macromolecules such as glycoproteins, glycopeptides and tion, however, there are certain dynamic variations of the
glycolipids, have been shown to be implicated in biological preferred conformations. In general, the resulting mixture
recognition through carbohydrate-mediated information of available conformations undergoes fast interconversion,
transfer [24]. and most experimental techniques show only results that
Polysaccharidepolysaccharide interactions play an are time-averaged over all conformations [10].
important role in the control of the architecture of animal Polysaccharides are well known to possess a high
and plant cells. Throughout development, the constitutive tendency to associate. This association is usually caused
bers are somehow manipulated into precise directions by the abundant hydroxyl or amino groups present in a
within the supporting tissues, so as to be best adapted to macromolecule and which easily undergo hydrogen bond-
their chemical, mechanical, and specic functions and ing. Polysaccharides oer an exceptional ratio of hydroxyl
properties [59]. Without this control, many animals and groups per saccharide residues. Such hydrogen-bonding
plants would collapse. In general, the macromolecules potential has to be taken into account when considering
involved have very long chains, which may self-assemble interactions, either in association with neighboring carbo-
or may follow various cellular mechanisms to form directed hydrate polymers or surrounding water molecules [1113].
assemblies. In general, their intrachain backbone bonds are For polysaccharides forming three-dimensional networks
covalent, whereas the lateral interactions between chains under specic conditions (gel structure), these interactions
are by interchain hydrogen bonds. Fibrous composites are hydrogen bonding, dipole and ionic interactions, and
occur most commonly as parallel bers, orthogonal or solvent partition eects [14]. Individually, these interac-
helicoidal plywood embedded in a matrix whose chemistry tions are so weak that conformational stability is achieved
is dominated by polysaccharides and proteins. only when a large number of them is simultaneously
The biological, chemical, or physical properties of favorable (cooperativity). Changes in such sequences pre-
carbohydrates are largely determined by what is exposed vent, in general, the continuation of ordered associations.
to the outer surface. Most interactions with other mole- In many applications, particularly in food systems,
cules will occur as a consequence of the almost innite industrial polysaccharides are used in combination with
array of chemical structures and conformations that can be other polymers (including proteins). Thus, the combina-
generated for polysaccharides. In reality, the primary tion of aqueous glycan solutions provides a very eective
structures of polysaccharides vary in composition, se- means of obtaining systems with new and unique proper-
281
282 Taravel et al.
ties. Such polymer combinations may either be compatible, motions. The literature on the structure of biomolecules
leading to synergistic interactions and property enhance- shows an increasing eort to overcome these problems
ments, or incompatible and result in precipitation or phase- [2326]. The goal must be to generate the ensemble of
separation phenomena. However, both compatible and structures that is consistent with the data, taking into
incompatible systems oer advantages that are exploitable account considerations concerning the frequency with
in dierent areas (composite materials, biological process- which transitions between the dierent structures occur
es, food, and nonfood industries). and the relative time scales of internal and overall molec-
Several attempts have been made to construct new ular motions.
functional materials based on stimuli-responsive polymer From an approach combining experimental and com-
solution and gel systems. These systems undergo isother- putational data, useful information can be extracted
mal phase transitions by external stimulation, such as concerning how crystalline arrangements are formed,
photons, temperature, ionic strength, pH, electric, or mag- how two polymer chains pack, and how a polysaccharide
netic eld, etc. The conformation of these polymers gov- chain is going to interact with other macromolecular
erns their various physicochemical properties. When the chains. Whereas only a few of these arrangements would
conformation, under stimuli, changes, a concomitant correspond to chain pairing capable of generating ecient
change happens in the polymer properties [1517] and packing, the other ones represent situations that could
many industrial applications could proceed. occur in the amorphous state or at the surface of polymeric
Many water-soluble polysaccharides are ionic and materials. Another application extends to low-symmetry
present original electrostatic properties depending not only systems, such as gels, in which chainchain interactions
on the structure of the polymer but also on the ionic may promote the formation of the so-called junction
concentration. Electrostatic interactions between polyion zones. Furthermore, a methodology is needed to investi-
and counterions and the ionic selectivity have been studied gate all interaction and aggregation phenomena in low-
leading, in some cases, to gel formation. The role of the ordered polymeric assemblies [27,28].
charge density of the polymer, of the molar mass, and of the The present chapter is concerned with computer mod-
chemical structure were examined as well as the thermo- eling of polysaccharidepolysaccharide interactions. It is
dynamic conditions (ionic strength, temperature, etc). divided into three parts. The rst part sketches the theo-
Their intrinsic persistence length reecting their stiness retical background of molecular modeling of saccharides
characterizes the ionic polysaccharides. Their stereoregu- and provides a view of experimental and theoretical meth-
larity favors the stabilization of helical conformation and odologies. The reliability of theoretical methods is dis-
cooperative interactions. Rigid hydrophilic physical gels cussed, and the usefulness of molecular modeling is
are also formed depending on their chemical structure [18]. considered. The second part attempts a brief survey of
Polyion complexes are formed by the reaction of a dierent procedures applied to structure modeling of oli-
polyelectrolyte with an oppositely charged polyelectrolyte gosaccharides. This is followed by applications to the
in aqueous solution. Polyion complexes have numerous structure characterization of individual polysaccharide
applications such as membranes, antistatic coatings, and chains. Emphasis is brought here on issues that relate to
microcapsules. This is the case of cationic chitosan and the accuracy and eciency of the calculations. The third
anionic gellan gum with carboxyl functional groups [19,20] part deals with modeling of interactions between polysac-
or poly (a,L-glutamic acid) [21] for diverse medical, agri- charide molecules and molecules of increasing complexity.
cultural and ber industrial applications. Mixtures of Starting with docking problems, and in particular, inter-
hyaluronatealginate exhibit physicochemical properties actions of Congo Red or benzophenone with cellulose, the
for surgical applications [22]. nal section covers some insights into polysaccharide
In both the industrial application and biological func- polysaccharide interactions in the dierent phases (solid,
tions, the three-dimensional characteristics of carbo- amorphous, and solution). Intermolecular binding be-
hydrates are essential. Stereochemistry plays also an tween dierent mixtures of polysaccharides involving ga-
important role in determining the properties of polysaccha- lactomannans or glucomannans with n-carrageenan or
rides. Among the methods for studying molecular shape, xanthan is given. The nature of the molecular mechanisms
single-crystal diraction experiments for the solid-state explaining the synergistic interactions between these bio-
and nuclear magnetic resonance (NMR) spectroscopy in polymers have been focused.
solution provide increasing detailed conformational in-
formation. However, when ordered single structures are
dicult or impossible to obtain, the diraction data deter- II. METHODS
mined for brous structures are always of low resolution A. Experimental Methods
and must be supplemented by computational methods.
NMR in solution also necessitates the combination with An excellent review of the methods used to characterize
molecular modeling for appropriate interpretation of mixed polysaccharide systems and their value has been
structural information contained in, for example, cou- already published [29].
pling constants and nuclear Overhauser eects. In fact, More recently, new insights have been drawn by
to deal properly with the latter, one also has to be aware improved biophysical methodologies. Spectroscopic tech-
of the complications arising from the existence of internal niques for characterizing structure and dynamics have been
PolysaccharidePolysaccharide Interactions 283
rened, increasingly more sensitive for probing the ener- obtained from X-ray diraction) could be NMR spectros-
getic of molecular interactions. Even ultracentrifugations copy, particularly of relatively immobile species, using the
are undergoing exciting developments concerning compo- techniques of cross polarization, high-power decoupling,
nent stoichiometry and interactions [30]. A major advance and magic angle spinning (CP-MAS) with 13C detection
in the eld of pulsed infrared (IR) spectroscopy has been [47,48]. Investigation of polysaccharidepolysaccharide
the development of two-dimensional IR-based spectrosco- interactions by high-resolution NMR is usually dicult,
py. Like 2-D NMR, spectra are characterized by diagonal because of signal broadening associated with reduced
and cross-peaks, which can be interpreted to obtain angu- mobility of the whole macromolecule or specic segments
lar and spatial information [31,32]. Other spectroscopies, of it. However, signal broadening is, in itself, a useful
such as pulsed electron paramagnetic resonance, have indication of stiening of polysaccharide chains, which
continued to develop for better resolution, even with may be due either to a more rigid conformation or to some
powder samples. The use of dipolar coupling technology kind of association. Also, loss of peak signal is expected to
greatly accelerates the speed at which structures can be be more pronounced for the less mobile polysaccharide
obtained from NMR methods. This also suggests that segments involved, for example, in junction zones [49,50].
studies require more than a single approach [30], for In contrast, more mobile systems still produce high-reso-
example NMR, IR spectroscopy, X-ray crystallography, lution spectra [51,52]. Multinuclear NMR (related to
or electron microscopy. Moreover, advances in electron nuclei other than proton and 13C) is another possible
cryo-microscopy has been a method of choice for obtaining approach to understanding the gel structure of various
structural information [30]. systems [53,54].
A direct measure of the thermodynamic parameter Restriction of macromolecular motion can be moni-
associated with a binding event can be obtained by using tored and quantied by measurement of the relaxation
isothermal titration calorimetry, including enthalpy and times T1 and T2 [5558]. The rst type of relaxation refers
entropy contributions. Atomic force microscopy has also to an energy transfer occurring between nuclear spins and
beneted from signicant advances in tip design, along neighboring molecules. The second involves the random-
with new methods for scanning molecular surfaces [33]. ization of nuclear spins. For two-phase materials, the
The method has been extended to include a range of relaxation process can be approximated by two exponen-
bacterial and plant polysaccharides, as well as network tial functions. For example, the free-induction decay sig-
structure formed in polysaccharide gels or even in the in nals (S) will generally be in the form
vivo biological systems [3437].
The X-ray method is still the royal road to determine S A expt=T2a B expt=T2b 1
the 3-D structures of polysaccharides. This feature was
shown for chitin, chitosane, and h-1,3-glucans [38], for the where A and B are the volume fractions of the two phases
molecular architecture of a galactoglucan from Rhizobium and T2a and T2b are the corresponding transverse relaxa-
meliloti [39], araban, and whelan [40], as well as for tion times. A third method of relaxation measurement
xanthangalactomannan interactions [41]. X-ray ber dif- (T1q) makes use of the rotating frame and is strongly
fraction is also considered to be a good method for dependent on the presence of nearby nuclei and on the
examining molecular models of gelation at atomic resolu- dynamics of the system.
tion. It may be used to test whether a binary gel composed The introduction of residual dipolar coupling meth-
of two components A and B, contains AB, AA, or BB odology has increased the scope of structural biological
junction zones. The preparation of bers requires that the problems addressed to NMR spectroscopists. Conforma-
gels be stretched and at least partially dehydrated. Such tional changes, the relative orientation of domains, inter-
methods have been applied to galactomannan- or konjac molecular complexes, and molecular dynamics can be
mannanalgal polysaccharide, galactomannan- or konjac accurately characterized [5961]. While this kind of ap-
mannanxanthan binary gels [42,43]. The results best proach has been followed mainly for oligosaccharides,
represent a hydrated solid material. Scattering techniques few studies concern polysaccharides [62,63]. Although
performed directly on gels are probably better, but, only this approach shows promise, the inuence of the inter-
recently, small-angle X-ray and neutron scattering have actions with the liquid crystalline medium on the molec-
been used to gain some insight into the molecular struc- ular structure and dynamics is still uncertain, in particular,
tures of agarose solutions and gels [44,45]. The structure for exible species or regions which should be treated
and intermolecular interactions between polysaccharide accordingly over contributing conformers requiring statis-
chains of guar galactomannan and hydroxypropyl guar tical weight of conformers and correct orientation tensors.
have been also studied by osmotic stress and X-ray scat- Nevertheless, pulsed-eld gradient techniques continue
tering. As osmotic pressure increases, the diraction peak to enhance the performances of all classes of NMR ex-
shifts to higher Q values, i.e., the intersheet spacing periments using a combination of 2-D homonuclear NMR,
decreases. Conclusions concerning phase transition, pack- 1-D selective excitation methods, and 2-D heteronuclear
ing, and crystallinity were drawn [46]. methods to determine microbial polysaccharide structure
An alternative means of assessing ordered structures [64].
at the molecular level in such systems (and therefore Advanced solid-state NMR methods were used to
complementing the information on long-range ordering characterize and quantify physicochemical properties of
284 Taravel et al.
gels [65], starch-based plastics [66], or functional constitu- surface attachment of galactomannans or glucomannans
ents of food [67]. Water binding was probed with the to aggregates or crystallites of the helix-forming polysac-
heteronuclear 2-D wideline separation experiment (wise- charide [8183]. For the type 2 gels, new X-ray patterns
NMR) [68]. suggest binding to stereochemically compatible polymers
In an initial study [69], it was shown by optical rotation such as denatured xanthan rather than to the xanthan
(OR) measurements that the helices of agarose and helix [42,84]. However, a loose gel is also formed with
n-carrageenan could bind in an ordered, cooperative fashion ordered xanthan involving interactions with disordered
to sequences of 1, 4-linked h-D-mannopyranose residues in fragments of xanthan or by a second mechanism in which
certain plant galactomannans, and that such mixed poly- side chains of ordered xanthan interact with galacto-
saccharide systems can lead to unexpected and useful rhe- mannan [41,8489].
ological properties. The form of the temperature-dependent It can be stressed that the conformational rearrange-
optical rotation for the agarosegalactomannan mixture ment of polymer to accommodate binding interactions
shows a complex buttery form instead of the usual loop, with other polysaccharides is an entirely reasonable inter-
and this was shown to originate from molecular eects [70]. pretation of current evidence [89]. Thermodynamic stabil-
This article reviews the extensive work on the interactions of ity and competition should be the key points. When an
agarose and h-1,4-glycans, performed by that group. attraction exists between the two species, whatever the
Other groups have used viscosity and dynamic visco- galactomannan or any general side chain composition,
elasticity measurements to study the synergistic interaction gelation properties will depend on the cooperative inter-
between xanthan or agarose and various galactomannans actions over long ranges or small and dynamic segments.
[7173]. These results support an interaction mechanism
between the polysaccharides, which was also proposed B. Theoretical Methods
from rheological studies [52,74]. It was shown that for very
low n-carrageenan concentrations (under 1%), the synergy There have been many dierent theoretical approaches in
phenomenon is very large because the blend modulus is the last 1015 years to model the three-dimensional struc-
much higher than the corresponding modulus of a n- ture of saccharides. The methods range from ab initio
carrageenan gel prepared at the same concentration. Also, quantum-chemical methods to simple distance criterion
the variation of blend modulus at low carrageenan con- maps for the evaluation of polysaccharide conforma-
centration is much sharper (by several decades) than that tions. The following sections contain a very brief descrip-
observed for the high carrageenan concentration range. It tion of dierent calculation methods. For a more detailed
was concluded that the blend gel structure must be based description of methods, the reader is referred to Refs. [90
on an interacting process between n-carrageenan helices 101]. Methods of theoretical conformational analysis can
and smooth zones of carob galactomannan [75]. In con- be classied in several ways. We have chosen, as most
trast, on the basis of rheological data, it was concluded that illustrative, the one in which they are divided into two
phase separation processes as a result of incompatibility groups, quantum-mechanical and classical mechanics pro-
between unlike polysaccharides occurred for various food cedures (the reader is referred to PolysaccharidesStruc-
mixtures [7678]. The eect of selected additives on the tural Diversity and Functional Versatility, rst edition,
ow parameters of 1:1 mixtures of carrageenanguar gum Ref. 102).
and carboxymethyl celluloselocust bean gum was also In the eld of molecular modeling of complex molec-
investigated by using a coaxial viscometer [79]. Those ular systems, very often a full treatment of many variables
techniques, rheological in nature, are concerned both with (degrees of freedom) is required to adequately describe the
small- or large-deformation studies and failure properties. properties. Therefore, to investigate such a system, one has
In general, they describe the physical and dynamic prop- to perform a numerical simulation of the behavior, which
erties of the systems in relation to some supramolecular produces statistical ensembles of the congurations repre-
model, but the conclusions are often at variance [80]. senting the system [98100]. The simulation of molecular
Recent studies using dierential scanning calorimetry systems requires the generation of a statistically represent-
and electron spin resonance spectroscopy were interpreted ative set of congurations, a so-called ensemble. The
in terms of the formation of mixed aggregates n-carra- properties of a system are dened as ensemble averages
geenan helices and konjac mannan, possibly involving or integrals over the conguration space. For many-parti-
bundles of self-aggregated n-carrageenan helices covered cle system, the averaging or integration will involve several
with surface-adsorbed konjac mannan chains [81]. degrees of freedom and, as a result, can only be carried out
Synergistic interactions between biopolymers can be over one part of the conguration space. The smaller the
divided into two types: type 1 gels, in which the neutral conguration space, the better the ensemble average or
polymer modies the gelation of the helical polysaccha- integral can be approximated. When choosing a model
ride; and type 2 gels, in which the mixing of the two non- from which a specic property is to be computed, one
gelling polysaccharide leads to gelation [82]. For the type 1 would like to explicitly include only those degrees of
gels, X-ray ber diraction studies failed to reveal new freedom on which the required property is dependent.
patterns as might be expected to occur for specic inter- Central to the use of molecular simulations is the
molecular binding between the two polysaccharides. The availability of a model with respect to atomic interactions.
binding should be random and dynamic and involves a Once the molecular model and force eld have been chosen,
a method to search the conguration space for low-energy the reader is referred to the rst version of Polysaccha-
congurations has to be selected. Various methods are ridesStructural Diversity and functional Versatility,
available, each with particular strength and weakness, Ref. 102).
depending on the size of the system and the force eld. If
the molecular system contains only a small number of A. Modeling of Polysaccharide Chains
degrees of freedom and if the potential energy surface does
not have too many relevant minima, it is possible to During the last few years, molecular modeling has become
systematically search the complete hypersurface of the a standard tool for the determination of the spatial molec-
system. If the latter contains too many degrees of freedom, ular structure of polysaccharides, both in solid phase [132
this search of the hypersurface is impossible. In that case, a 134] and solution [110113]. Starting from the primary
collection of congurations can be generated by random structure of a polysaccharide chain, the procedures de-
sampling using Monte Carlo methods or by molecular scribed in the previous sections are usually used to identify
dynamics. The commonly available and used molecular all the low-energy conformers, which are likely to occur for
simulation program packages are AMBER [103,104], the parent disaccharide in vacuum. This information is
CHARMM [105], DISCOVER [106], GROMOS [107], then used to model individual polysaccharide chains in
and MACROMODEL [108]. dierent environmental conditions. However, when mod-
At the present stage of molecular dynamics of bio- eling polysaccharide chain structure, the congurational
molecules, there is a general understanding of the motion space spanned by all atoms is still, by far, too large to be
that occurs on a subnanosecond time scale. For motions on searched for low-energy conformations. Therefore, the
a longer time scale, our understanding is more limited. For degrees of freedom characterizing internal motions in
motions that are slow because of their complexity and monosaccharide units are usually omitted and potential
because they involve large-scale structural changes, an function methods are used for an energy calculation. In an
extension of the available approaches is required. eort to facilitate the construction of complex carbohy-
Monte Carlo simulations, molecular dynamics as well drates, a carbohydrate-fragment library has been created
as the role of the environment, are described in Ref. 102. [135]. This data bank contains optimized geometry of many
monosaccharide residues and covers most of the units that
occur in polysaccharides. Because polysaccharides are
III. MOLECULAR MODELING OF SINGLE subjected to dierent constraints in the solid phase and in
SPECIES solution, dierent procedures have to be used for the
modeling of their structures [136143].
It is evident that the method applied for the modeling of a A new procedure for generating three-dimensional
molecular system depends on the complexity of the system structures of polysaccharides and complex carbohydrates
studied. Therefore, at the beginning, one has to decide how from their primary sequence has been described. The
small a system can be chosen without seriously aecting a POLYS computer program [144] combines a database of
proper representation of the property of interest. Then, the monosaccharide structures with a database containing
level of approximation with respect to computational information on populations of independent neighboring
methods has to be chosen. It is clear that for larger systems, glycosydic linkages in disaccharide fragments. The com-
more approximate methods of computation have to be puter program can cope with both the complexity and the
used. In practice, any choice involves a compromise be- diversity of carbohydrates and the unique topological
tween the type and number of structural variables and the features arising from multiple branching. The translation
extent of calculations on one hand, and the available of the primary structure is made through the use of a lexical
computing power on the other. analyzer and a command interpreter. However, it also
An attempt to understand polysaccharidepolysac- generates secondary and tertiary structures in the form of
charide interactions on the molecular level requires a Cartesian coordinates in formats used by most molecular
description of individual chains and, therefore, knowledge mechanics programs and packages. POLYS has been
of the detailed three-dimensional structure of monosac- tested with success on standard homopolysaccharide sys-
charide and oligosaccharide components. Several compre- tems such as cellulose, mannan [144], and pectic polysac-
hensive reviews have been written on dierent aspects of charides [140].
molecular modeling of monosaccharides, oligosaccharides, Various average properties of several pectic polysac-
and polysaccharides [109,110131]. Therefore, no attempt charide models were calculated by using metropolis Monte
will be made here to review the eorts to calculate confor- Carlo algorithm, based on the conformational energies for
mational energy surfaces. Instead, attention will be focused parent disaccharides [145]. Solvents eects were evaluated
only on a brief description of the main conformational by calculating the solvation energy for each conforma-
characteristics of oligosaccharides and polysaccharides. In tional state by estimating contributions from a cavity
what follows, we attempt to illustrate the approaches used formation, and from the electrostatic and dispersion inter-
to study the conformational properties of oligosaccharides actions between solvent and solute molecules. The behav-
and polysaccharides with a particular emphasis concerning ior of the mean characteristic ratio, the squared radius of
the inuence of exibility on the three-dimensional struc- gyration (Fig. 1), and the persistence length vs. chain
ture of these compounds (for modeling of oligosaccharides, length were discussed for various structural models, tem-
286 Taravel et al.
Figure 1 Comparison between experimental (,o) and theoretical (D) values of RG as a function of the molecular weight for
two samples of galactomannans. (From Ref. 146.)
perature and solvent for pectin substances [144], (1!4)- with n=4.52 and h=2.16 A, and n=2.67 and h=4.48
h-D-glucuronan and acetylated derivatives [138], acidic A, respectively. The third helix is right-handed with
polysaccharides [139], hyaluronan [141], alginates [142], or n=2.34 and h=5.18 A. The conformation of the latter is
galactomannans [146]. very close to the twofold helical structure of mannan I
inferred from electron diraction experiments [149]. The
1. Solid Phase small conformational dierences between both structures
In solid phase, crystalline polysaccharides usually adopt a are understandable and can be explained in terms of slight
regular helical shape. When subjected to the constraints changes imposed by crystal-packing forces with no large
imposed by a helical symmetry of the chain, the U and W energy cost.
angles are the same at every glycosidic linkage. Such Features concerning the deformations of the mannan
structures are described by a set of helical parameters, n helical conformations as a function of the number and
and h, where n is the number of residues per turn of the position of galactosyl residues were explored by adding
helix and h is the translation of one residue along the galactosyl residues to the mannan chain. In general, it was
helical axis. Discrimination between possible helical struc- found that mannan chains do not change their shape
tures is based on the potential energy. In the following, we signicantly as a result of these substitutions. However, it
illustrate an application of this procedure to describe a was observed that for equally substituted mannan back-
generation of helical and statistical structures of galacto- bones, the conformations of the rst helix were more often
mannan chains [147]. distorted than the other ones. It appeared that for the
Galactomannans are reserve carbohydrates found in structures generated from the third helix by branching with
the endosperm of various legume seeds. They consist of the galactosyl units, the mannan backbone chain maintained
(1!4)-h-D-mannan backbone to which are attached vari- the unsubstituted mannan helical structure. This observa-
ous amount of single (1!6)-a-D-galactosyl groups. The tion suggests that in the twofold helical structure of ga-
content and distribution of galactose in the chain depend lactomannan, interactions between consecutive galactosyl
on the source of galactomannans [148]. The investigation units are not severe, and that galactopyranosyl residues
of the possible structures of this polysaccharide started have a number of allowed conformational states. These
from two basic disaccharide models of galactomannan ndings are supported by experimental data. For example,
polymers, namely mannobiose and epimelibiose (Fig. 2). it was observed that both the ber repeat and the stacking
The systematic search of the minima for both disaccharides of the chains in the a direction remain nearly constant for
led to several dierent stable conformers, which were a galactomannan series diering in the extent and distri-
grouped into dierent families according to their torsion bution of galactose substitution [149].
angles across the glycosidic bonds. In agreement with
experimental evidences provided by NMR and linkage 2. Solution
rotation data, the conformational analysis of mannobiose In solution, helical constraints are removed and polysac-
in water solution established that three most stable con- charide chains tend to adopt a more or less coiled structure.
formations in equilibrium are available for the molecule. One of the most peculiar aspects of the physical chemistry
The geometries of these minima, characterized by the of polysaccharides is their ability to assume an enormous
glycosidic torsion angles (U=38j, W=122j), (41j, 16j), variety of spatial arrangements around the glycosidic link-
and (70j, 57j), were used to model helical structures. ages. This means that observable parameters describing the
Thus a propagation of the three lowest minima yielded solution behavior of polysaccharides are averages of the
three dierent helices. Their molecular drawings are pre- properties of the individual conformations. Theoretical
sented in Fig. 3. Two helices have a left-handed chirality polysaccharide models are based on studies of the relative
Figure 2 Schematic representations of mannobiose and epimelibiose with labeling of atoms.
abundance of the various conformations of a given poly- contributions of the cavity formation and of the interac-
saccharide in conjunction with the statistical mechanical tions between solvent and solute [156]. Signicant changes
theory of polymer-chain conguration [150]. Generally, in the proles of conformational surfaces were found in
calculations refer to the unperturbed state, that is, to the three solvents considered (water, 1,4 dioxane, and
conditions such that the long-range interactions are exactly dimethylsulphoxide). As a straightforward consequence,
compensated by intramolecular interactions. unperturbed chain dimensions are predicted to be solvent-
Representations of potential energy surfaces are usu- dependent.
ally performed by calculating two-dimensional maps of the
internal energy of appropriately chosen skeletal segment of
polysaccharide chain with respect to two glycosidic tor- IV. APPLICATIONS
sional angles, all the other internal parameters are kept
xed. The polysaccharide chains can be then generated and In this section, we attempt to describe the results of
used to calculate congurational properties, such as the molecular modeling of interactions between polysaccha-
mean persistence length, mean square radius of gyration, ride and small molecules and polysaccharidepolysaccha-
mean square end-to-end distance, dipole moment, and so ride interactions. We will not discuss carbohydrateprotein
forth [151,152]. Polysaccharide models of native polysac- interactions. Most of the papers appearing in the eld of
charides, rened to a various extent, have been presented carbohydrateprotein interactions deal with X-ray crystal-
[153]. Only recently, the Monte Carlo methods have been lography, NMR, microcalorimetry, circular dichroism,
applied to exploring the multiple conformations occurring and electron microscopy. Nevertheless, molecular model-
in a complex polysaccharide such as xyloglucan [154] or ing provides an alternative approach and can help to
investigating the solvent eects on the unperturbed dimen- understand how carbohydrate molecules, which are exi-
sions for two representative polysaccharides: cellulose and ble and experience continuous dynamic uctuations, can
amylose [155]. In this case, solvation energy terms were be recognized by protein receptors (including enzymes) in a
calculated for each conformational state by evaluating the highly specic manner. For carbohydrates, the species
288 Taravel et al.
Figure 3 Molecular drawings of the three helices obtained for unsubstituted oligomers starting from three lowest-energy minima
M1M3 (from left to right, respectively).
concerned range from monosaccharides to polysaccha- surfaces are identical. Native cellulose is a semicrystalline
rides, either free or conjugated, as in glycoproteins and material in which crystal phases coexist with amorphous
glycolipids. Results obtained in this very important eld of zones. To model the extreme complexity of the cellulose
glycobiology were reviewed and reported recently surfaces, not only the dominant crystal surfaces have to be
([3,157,158], and references cited therein). considered, but also crystallographic hydrophobic surfaces
The adsorption of polymers, such as polysaccharides, that protrude the CH groups. Amorphous surfaces show a
at surfaces of dierent natures is the subject of several complete disorder of chains.
experimental and theoretical investigations, because of its The benzophenone adsorption on cellulose has been
wide range of practical applications. The polymers may studied by following two distinct routes. First, crystalline
give to colloids some steric protection against aggregation. microbril models have been built. It is exclusively based
Thus, interactions between polysaccharides and phospho- on the monoclinic allomorph. It consists of 10 chains of 12
lipid bilayers have been demonstrated [159161]. However, residues each. In this model, a central chain is surrounded
little is known about the inuence of macromolecule by each of the (110), (110) surfaces as in the Congo red
conformation related to solvent characteristics (pH, ions, calculations and possesses a (200) hydrophobic surface. A
ionic strength). Interaction of gum arabic, maltodextrin, large number of adsorption sites have then been generated
and pullulan with lipids in emulsions could play a wall following a Monte Carlo procedure [166].
material role for encapsulation and protection [162164]. On the cellulose (200) hydrophobic surface, benzophe-
none molecules do interact by maximizing stacking inter-
A. Docking of Small Molecules actions between aromatic rings of the benzophenones and
the nonpolar CH groups of cellulose. Therefore, benzo-
The docking of small molecules on the surface of crystalline phenone molecules tend to be oriented in a parallel direc-
polymers is a problem that is suitable for investigation tion with the cellulose surface. A large number of
using molecular modeling. Computational experiments adsorption sites could be seen and, for each, adsorption
mostly relate to cellulose, for which many properties are takes place without a specic geometry. However, the
dependent on interactions occurring at the surface of the oxygen atom of the carbonyl group of benzophenone is
microbril. Results on molecular modeling of the interac- precisely located, being in an interaction with a surface
tion of Congo red [165] or benzophenone [166] with hydroxyl group (OH3 or OH2) of a glucose unit through a
cellulose provides a wealth of information about the hydrogen bond. The remaining part of the molecule is able
structure of the complex and helps to understand the to freely rotate to 360j without loss in the quality of the
features of these interactions. interaction. The electrostatic character of this interaction is
Direct dyes as Congo red have long been known to obvious and is a result of the creation of a hydrogen bond.
display specic and strong binding to h-(1!4)-glucans and However, the dominant component of the interaction is the
particularly to cellulose. Besides obvious textile applica- van der Waals term.
tions, they have been used for the histochemical observa- Despite minor structural dierences between the two
tions of plant cell walls and as beater additives in the pulp hydrophilic (110) and (110) surfaces, the benzophenone
and paper industry. They have also been shown to alter the adsorption process is the same for the two surfaces. The
biocrystallization of cellulose into microbrils during cel- calculated data show that electrostatic interactions are of
lulose biogenesis. greater importance. As a consequence of the topological
Starting from crystalline cellulose coordinates and characteristics of those two faces, adsorption sites are
Congo red models, the docking procedure was based on a specic. The probe molecules tend to orient their carbonyl
grid search exploring the surface repeat unit of the cellulose group toward the surface hydroxyl groups of cellulose that
crystals by using several orientations of Congo Red at each are located at the bottom of the grooves. Consequently,
grid point. The studied surfaces are the triclinic (100) and geometrical freedom of the interaction is restrained as
(010) and the monoclinic (110) and (110) ones. However, compared with the adsorption behavior of hydrophobic
results suggest that Congo red is able to adsorb onto all the surface. The carbonyl group of benzophenone is always
studied surfaces, with a preferential adsorption energy for hydrogen-bonded with the hydroxyl group of the surface of
the triclinic (010) and the monoclinic (110) surfaces. cellulose [166].
Results suggest also that the lower-energy conformers have The second method uses molecular dynamics proce-
a similar positioning and orientation with respect to the dures for traveling on cellulose surfaces subjected to peri-
cellulose chains at the surface repeat unit. In particular, odic boundary conditions. Comparisons are made between
two sulphonate groups of the dye molecule are ideally crystalline hydrophilic (110), crystalline hydrophobic
suited to form strong polar interactions with protruding (200), and amorphous surfaces (Figs. 46). Adsorption of
hydroxyl groups from cellulose. Furthermore, the amino the rst layer of benzophenone on each of the faces is
and the azo groups are also placed near the exposed oxygen studied following an iterative process to mimic the exper-
atoms [165]. imental conditions in which the probe molecule is primarily
The cellulose surfaces that have been considered show dissolved in solvent. To obtain a monomolecular layer,
only minor dierences that originate in ultrastructural between 16 and 18 benzophenone molecules are adsorbed
lateral organization of the surface chains. From the rough- onto each surface. The average covering level is about 93%
ness and hydroxyl accessibility point of view, all those of the total cellulose surface [166].
290 Taravel et al.
Figure 6 Molecular drawing of the adsorbed rst layer of

benzophenone on the amorphous face. The cellulose is dis-
played by its corresponding Connolly surface.
Figure 4 Molecular drawing of the adsorbed rst layer of
benzophenone on the (200) cellulosic face. The cellulose is
displayed by its corresponding Connolly surface. Adsorption geometry was quantied by using Euler
angles formalism. For ordered crystal surfaces, Euler
angles are grouped into dierent families, characterizing
the specic surfaces, which underlie the dierent geome-
tries of adsorption. For the amorphous surface, Euler
angles are randomly distributed, as benzophenone orien-
tation will depend on the local geometry of the surface.
For crystalline surfaces, interaction energy varies lin-
early with surface coverage. For the amorphous surface,
the rst molecules adsorb on the most favorable sites with a
better interaction energy. Then, when all these preferred
sites are occupied, benzophenone adsorbs on sites that are
energetically comparable to the crystalline sites. Therefore,
surface geometrical anisotropy creates two dierent ad-
sorption sites. With the exception of the rst few adsorbed
benzophenone molecules, the adsorption enthalpy is com-
parable for the three studied faces [166].
Conformational variations of the benzophenone are
observed. While interacting, this molecule does not stay
in the minimal energy conformation established in iso-
lated state. The relative orientation of both conjugated
benzene rings is adjusted to optimize intermolecular favor-
able contacts.
The dierence between the internal cohesion of the
monolayer and the interaction energy between benzophe-
none/cellulose layer surfaces suggests that crystalline cel-
lulose interface is stable, but not the amorphous interface.
Probe molecules diuse within the amorphous phase,
Figure 5 Molecular drawing of the adsorbed rst layer of promoting the large computed anity of the surface for
benzophenone on the (110) cellulosic face. The cellulose is the rst adsorbed molecules. To test this hypothesis,
displayed by its corresponding Connolly surface. complementary computations are carried out. The model
systems are interfaces composed by the cellulose surfaces, method with the cationic dye Azure A. The research was
the adsorbed benzophenone layer, and the empty space based on the fact that the absorption spectra of the free
above the benzophenone molecules lled by water mole- and bound dye are dierent. The solution equilibrium
cules. Then, 1 nsec of molecular dynamics is performed at of the reaction system was studied with the help of the
323 K. Normalized density proles of either cellulose or Scatchard model. Concentration of Azure A sodium chlo-
benzophenone along an axis perpendicular to the surface ride has a signicant eect on the interaction [167], as well
of cellulose suggests that a slight reorganization of the as characterization of the interaction between Methylene
whole crystalline system occurred during dynamics. On the Blue and glycosaminoglycans with the help of two math-
contrary, there is a real penetration of benzophenone ematical models [168]. Binding of calcouor white on
molecules within the amorphous cellulose phase. Simulta- the carbohydrate residues of acid-glycoprotein was also
neously, the cellulose phase is swollen as compared with studied [169].
the initial state [166].
Benzophenone adsorption on two dierent cellulose
samples of crystallinities of 73% and 40% has been exper- B. Modeling of PolysaccharidePolysaccharide
imentally studied by diuse reectance infrared (DRIFT) Interactions
spectroscopy. Through the observed modications of the Over the years, modeling of saccharides has been mainly
carbonyl-stretching band, it was possible to distinguish focused on intramolecular rather than intermolecular
three dierent environments for benzophenone: entrapped aspects. However, knowledge of polysaccharidepolysac-
between chains in crystalline domains, in amorphous charide interactions is crucial to understanding the func-
domains, and as crystallites adsorbed at the cellulose tions and properties of many systems. These interactions
surface. A straightforward comparison with the experi- are, e.g., responsible for chain packing in solid state and
mental investigations is dicult. The experimental data is govern the association of polysaccharides in gel networks.
averaged over many intermolecular arrangements, but The detailed information at atomic level obtained from
sampling is not currently accessible by molecular modeling molecular modeling can help to enlighten the ordered states
given the computational power needed. Another experi- of polysaccharides in solution and gel. In the rst part of
mental diculty concerns the technique that was used to this section, the application of molecular modeling meth-
prepare the samples. First, it involves swelling of cellulose ods to the resolution of three-dimensional models of poly-
induced by ethanol solvation. This treatment may change saccharides in solid phase is described. In the second part,
the original accessibility of the crystalline domains and the application of molecular modeling for a systematic
therefore could aect the conclusions of the study. It search of interaction potential energy surfaces is presented,
should nally be pointed out that the IR experimental using recent examples gathered from n-carrageenanman-
results are based on the carbonyl stretching band. Hydro- nan interactions in solution.
phobic interactions, arising from phenyl groups of benzo-
phenone and numerous CH groups of the cellulose, are not
seen in the experiments, whereas molecular modeling 1. Solid Phase
emphasizes their importance [166]. In spite of developments in experimental techniques, the
Despite the advance in the realism of the model secondary and tertiary structures of polysaccharides can-
surfaces of cellulose microbrils, dierent assumptions not be solved by direct experimental methods. Polysaccha-
restrict the impact of modeling and make comparison with ride crystals are not large enough for X-ray or neutron
experimental data dicult. For example, the situation in diraction analysis. Therefore, molecular modeling is re-
which benzophenone molecules are entrapped within the quired to supplement experimental data and to solve three-
cellulose (crystalline or amorphous) has not been directly dimensional structures [120,132134,170175]. The rst
considered in the modeling investigation, as modeled sys- step of such treatment involves molecular modeling of a
tems can deviate from the experimentally investigated one. parent disaccharide combined with ber diraction data.
Furthermore, it is impossible to evaluate to which extent This gives basic information on the helix type, and a
the real surfaces of the microbrils are correctly described number of molecular models can be constructed for a given
by those idealized model surfaces. These limitations con- polysaccharide. Providing that the data set is of sucient
cern surface accessibility, adsorption occurring at the quality and/or the unit cell dimensions and space-group
cellulose/vacuum interface, and the role of solvent for symmetry are well assigned, the nal stage of elucidation
Fourier-transform infrared spectroscopy (FTIR) experi- involves a complete structural determination of the unit cell
ence. Finally, thermodynamics of the adsorption process is content. Renement procedures have to match the ob-
assumed to be mainly governed by enthalpy components served and calculated X-ray amplitudes with simultaneous
[166]. optimization of polysaccharide chain structure, interchain
Dye binding heparin assays are commonly used in interactions, and preservation of the helical symmetry.
biochemical and clinical laboratories, primarily because of Models are rened until the t, or steric factors, allows
their high sensitivity and convenience. However, they are one model to be declared signicantly superior to the
not understood at the molecular level. Understanding the others by some standard statistical test. Two programs
interaction between heparin and small ions or molecules are the most widely used methods for analysis and rene-
is necessary, and has been studied by spectrophotometric ment of three-dimensional polysaccharide models: the
292 Taravel et al.
linked-atom least-squares (LALS) method [132,172] and

the variable virtual bond (PS87) method [133,173]. The
principles of these two methods are the same and they have
been shown to give similar results [174].
Recently, molecular modeling treatment, called CHA-
CHA [175], was used to predict the packing relationship of
several polysaccharide chains: that is, the dierent ways for
a polysaccharide chain of known conformation to interact
with other chain-like molecules. Given a rigid model of an
isolated (simple or double) helix, its interaction with a
second (simple or double) helix was studied while moving
the helices as close to each other as possible without
causing interpenetration of the van der Waals radii of
atoms of the two dierent helices. After the helices were
positioned to the shortest interhelical distance for a given
rotation and helixhelix translation, the energy was calcu-
lated using atomatom potentials that includes compensa-
tion for hydrogen bonding without violation of van der
Waals contacts (Fig. 7). This computational procedure
was used to study the polymorphism of starch [175] and
cellulose [176].
The results for starch [175] were in good agreement
with the experimental ones. Models were based on the ber
repeat distance extracted from ber diraction patterns
and correspond to double helices composed of left-handed
single strands related by twofold rotational symmetry. Two
stable relationships were found for both the parallel and
antiparallel models. The structure predicted to be the most
stable corresponds to a duplex of parallel double helices as
found in both the crystalline A and B allomorphs. This
duplex was maintained during transition from the B to the
A form [175].
Another study concerns native cellulose [176]. Exper-
imental diraction data for most of samples are extremely
dicult to analyze because of crystalline polymorphism
(two allomorphs, Ia and Ih, are present within the same
microbril) and the presence of amorphous regions. The
CHACHA algorithm has been applied. A large number
of favorable parallel interhelix settings were obtained.
Few of these arrangements are capable of generating an
eciently packed three-dimensional array. Two structures
were used for comparison with experimentally derived
data. Agreement between the predicted unit cell dimen- Figure 7 Interhelical parameters used to dene the geo-
sions and the published experimental ones has provided metric orientation of the two parallel cellulose chains (A and
some degree of validation of the methodology. The two B): chain rotations lA and lB, interchain contact distance Dx,
most favorable predicted crystalline arrangements corre- and longitudinal oset Dz.
spond to a triclinic P1 space group, and to a monoclinic
space group P21. These structures correspond closely to
those which have been reported for cellulose Ia and Ih,
respectively. The cellulose chains in the selected models ments of cellulose chains allows building realistic macro-
form layers, stabilized by interchain hydrogen bonding. molecular models of cellulose microbrils. All the com-
Stacking of the layers gives rise to the complete crystal puted stable arrangements are believed to be pertinent to
lattice. Layer stacking in the triclinic model is stabilized situations such as the amorphous state or at the surface of
only by van der Waals interactions. For the monoclinic cellulose crystalline domains.
model, the layers are linked through two interplane hydro- X-ray diraction patterns from stretched bers of
gen bonds per cellobiose unit, one to each neighboring xanthan, guaran, and the complex between the two have
layer. In addition, the study provides insights into transi- been studied [41]. They are indicative of good orientation
tions between the two allomorphs. Also, the exhaustive and reasonable crystallinity. Xanthan forms a vefold helix
exploration of the low-energy, three-dimensional arrange- of pitch 47.7 A [177], while guaran can form a twofold helix
of pitch 10.3 A similar to that of mannan itself [178]. The bodies, while during the model building analysis the main
diraction pattern of the complex is a hybrid of those of chain conformation angles were tethered to the
the individual components. Both xanthan and guaran in corresponding low energy domains or to values in a known
the complex may adopt cellulose-like helices having a related structure [41]. Several models have been proposed
slightly longer pitch of 10.5 A, and form a noncoaxial emphasizing the importance of the structural roles of
duplex. Alternately, the complex may also adopt a xan- pyruvyl and acetyl groups for association (Figs. 8 and 9).
than-like, coaxial, vefold double helix, in which one
strand is xanthan and the other is guaran. The morpholo-
gies of these arrangements have been visualized by com- 2. Amorphous Structures
puter modeling. The two starting molecular structures Besides the perfect ordered crystals, most of the polysac-
(mannobiose and cellobiose) have been treated as rigid charides can exist under amorphous solids. At tempera-
Figure 8 Stereo views of two turns of cellulose-like models showing the parallel (a) and antiparallel (b) association of xanthan
(open bonds) with guaran (lled bonds). The models are stabilized by main chainmain chain O6F . . . O6A hydrogen bonds and
main chainside chain O6F . . . O7C hydrogen bonds respectively. The helix axes are 7.9 A apart (a) and 12.5 A apart (b). (From
Ref. 41.)
294 Taravel et al.
Figure 9 Stereo view of one turn of double-helical model displaying the side chains on the periphery. Note the side chainside
chain O4H. . . O7C hydrogen bonds on the helix surface between the two polymers. (From Ref. 41.)
tures below Tg, polymeric glasses are solids for all practical This model [181] follows the widely accepted concept
purposes. Characteristic times for volume relaxation are of glasses being in a state of frozen-in liquid disorder. It
of the order of years, and molecular motion consists pre- rests on the following two assumptions: (1) the model does
dominantly of solid-like vibrations of atoms around their not incorporate thermal motion (i.e., it is static). Temper-
average equilibrium positions. Chain packing in the amor- ature enters only indirectly, through specication of the
phous bulk has been experimentally studied. Data from density; (2) the polymer is represented as an ensemble of
neutron scattering suggest that the chain macromolecules microscopic structures that are in mechanical equilibrium.
assume essentially unperturbed random coil conforma- Assumption (1) reects the fact that attention is focused on
tions, in the equilibrium melt and even in well-relaxed the atomic positions of static mechanical equilibrium, as
glasses. Flory [179] suggested this concept decades ago. A one would do in a crystal. By thus stripping the system of its
quantitative computer model of molecular structure in an thermal motion (and introducing only a mean eld
amorphous synthetic polymer below its glass formation temperature), a dramatic reduction in the degrees of free-
temperature (Tg) has emerged [180]. dom is achieved. Alternatively, a full simulation of the
system, in both conguration and momentum space, could mers. Only one application of this method to polysaccha-
be attempted by molecular dynamics. With the present ride structures has been reported so far [181,182]. The
computational capabilities, however, dynamic simulations model systems serve as a point of departure for the
could only cover an exceedingly short time span, and one prediction of structural (strongly networked by hydrogen
would not depart signicantly from the vicinity of the bonds) and thermodynamic properties of amorphous
initial guess structure. According to assumption (2), the starch.
requirement that the modeled microstates be in mechanical Starch is made of linear (amylose) and branched
equilibrium (at local minima of the total potential energy) (amylopectin) a-linked chains of D-glucose. It can be trans-
should not be interpreted as implying thermodynamic formed into a thermoplastically processable material
equilibrium (a minimum of the Helmholtz energy would through a thermomechanical treatment and the use of a
be required for this). Each structure generated in the suitable plasticizer. This thermoplastic starch has an amor-
original investigation is only one microstate, and the phous structure, whereas the native material occurs in
microstates do not comprise an equilibrium ensemble. partially crystalline form. The amorphous structure of
The model system is a cube of glassy polymer with three- dry starch at a molecular level has been investigated using
dimensional periodic boundaries, lled with chain seg- X-ray diraction and molecular modeling [181].
ments at a density corresponding to the experimental value The glassy starch structures were modeled following
for the polymer. The entire contents of the cube are formed the TheodorouSuter method [180]. Starch was modeled
from a single parent chain. The cube can thus be con- by linear amylose chains only. The simulation box
sidered as part of an innite medium, consisting of dis- contained one single chain of amylose having a degree of
placed images of the same chain [180]. polymerization of 80. Several initial guess structures were
A model structure satisfying the conditions of detailed prepared at dierent starting densities. Then the structures
mechanical equilibrium is obtained by an iterative process were compressed by using NPT molecular dynamics to
that starts with an appropriately chosen initial guess. reach the density of amorphous starch (1.5 g/cm3). A set of
Hence, two stages in the evolution of a realistic model three microstructures each was started at three dierent
structure can be identied: (1) the creation of an initial densities corresponding to a ctitious gaseous state (0.001
guess structure, and (2) the relaxation of this structure to g/cm3). The partial density of the starch component in
a state of minimal potential energy. amorphous starch with 18% of water was 1.0 g/cm3,
Accepting the view that glasses are in a state of frozen- whereas its experimental density was 1.5 g/cm3 (Fig. 10).
in liquid disorder also implies that the conformational Local and global conformational parameters, pictured
statistics of the chains are not too dierent from those of by the distribution of the conformation angles at the
unperturbed macromolecules. A satisfactory initial guess glycosidic linkages and the end-to-end distance, depend
could be obtained by the generation of an unperturbed on the choice of the density for the initial guess structure.
parent chain and subsequent use of this chain to ll the Using a large box (at low density), the chain grew into an
cube to the correct density. Monte Carlo generation of single extended helical conformation and the glycosidic bonds
unperturbed chains involves the generation of (1) a chain explored the lowest energy area of the dimer conformation
conguration, (i.e., a dyad tacticity sequence), and (2) a map. However, the chain became coiled during the com-
chain conformation, (i.e., a sequence of rotation angles). pression resulting in a decrease of the end-to-end distance.
The conformational statistics of unperturbed chains are well In smaller boxes (at higher densities), the long-range
described by the rotational isomeric state (RIS) theory [180]. interactions became more important and the glycosidic
The goal of this step is twofold. First, the structure bonds explored all the accessible area of the dimer confor-
should correspond to an energy minimum of the potential mation map. The helical character of the growing chain
energy, and second, it should be exempt from internal was less pronounced. The compression step did not change
tension or compression. Each generated structure is then their end-to-end distances. The structures that started at
relaxed by energy minimization. However, a straightfor- the higher densities give a better agreement with the
ward molecular mechanics scheme is likely to trap the experimental end-to-end distance measured by light scat-
simulated system in a metastable local high-energy mini- tering in a theta-solvent [181].
mum. Molecular dynamics simulation was used to prevent The hydrogen-bonding structure in these systems was
the system from such entrapments by providing thermal investigated in detail. A variation in the geometry of the
energies to cross energy barriers between local minima. A hydrogen bonds was found from the radial distribution
typical relaxation cycle consists of a short dynamics run function of the oxygen atoms. On average, every repeat
during constant volume (NVT) at dierent elevated tem- unit made 7.8 hydrogen bonds, of which 56% were inter-
peratures, each followed by energy minimization runs. molecular bonds. Most (6.9 per repeat unit or 88%) of the
Then a longer dynamics at the desired nal temperature hydrogen bonds contained one of the hydroxyl groups. The
(below Tg ) was performed at constant pressure ensemble hydroxyl group of the C6 carbon atom was the most
(NPT), in which the simulation box was allowed to vary in exible, with the maximum number of intermolecular
size and shape. This was again followed by an energy hydrogen bonds. The OH2 group was the one that was
minimization [180]. most often involved in intramolecular hydrogen bonds.
Originally developed on atactic polypropylene, this More than half of the hydroxyl groups were donors as well
method has been used to build numerous synthetic poly- as acceptors. Because of the decit of hydrogen bond
296 Taravel et al.
Dry starch was extremely hydrophilic and, under

normal atmospheric conditions, starch contained water.
The investigation [182] was therefore extended to amor-
phous structures containing 823% water (Fig. 11). The
possibility to form hydrogen bonds was increased by the
introduction of the small water molecules. With increasing
water content, the number of starch-to-water hydrogen
bonds increased. It was found to be eight hydrogen bonds
per repeat unit of starch. Manifestations of the plasticizing
eect of the water on the starch on a molecular level were
seen as suggested by the lowering of the starch-to-starch
interaction energy and the increase of the starch-to-starch
distance. On the macroscopic level, this was reected in the
increase of the cohesive energy density. Calculations of the
chemical potential of the water conrmed the high anity
of starch toward water. The chemical potential of the water
was lowest in dry structures and sharply increased at low
water contents.
3. Solution
As mentioned in Section I, the combination of aqueous
glycan solutions provides a very eective means of obtain-
ing systems with new and well-suited properties. The
phenomena involved are of interest in dierent areas
(composite materials, food industry, biological processes).
Among the most exploited mixed gels of food hydrocol-
loids leading to synergistic interactions are those involving
galactomannans, such as locust bean gum, (or other struc-
turally related plant polysaccharides), in combination with
n-carrageenan, furcellaran, agar, or xanthan, all of which
Figure 10 Selected chains of the three types of model

structures, which had been started at a density of (a) 0.001 g/
cm3, (b) 1.0 g/cm3, and (c) 1.5 g/cm3. Hydrogen atoms are
not displayed and a ribbon is drawn along the backbone for
clarity. (From Ref. 181.)
donors, three center hydrogen bonds occurred. The model

structures of amorphous starch contained 67% more oxy-
gen than hydroxyl hydrogen atoms, and approximately
40% of the donor atoms were bonded to two acceptor
atoms [181].
The dense network of hydrogen bonds indicates strong
interactions between the molecules. The cohesive energy
density and the solubility parameters have been evaluated
and compared to the experimental value. From the results,
it was concluded that the model structures starting at the
lowest density did not represent the starch structure cor-
rectly, whereas structures starting at larger densities agreed
well with the experimental value. Finally, the dierential
radial distribution functions derived from the model struc- Figure 11 Comparison of the radial distribution functions
ture and from the X-ray scattering intensity showed good calculated from the model structures with dierent water
agreement for distances up to 6 A [181]. contents. (From Ref. 182.)
adopt rigid, ordered structures [183]. It has been stressed in lengths, bond angles, and torsion angles) derived from the
Section II.A that the various investigations were performed crystal and molecular structure of h-D-galactopyranosyl
to obtain a complete description of the types of possible gel and 3,6-anhydro-a-D-galactopyranosyl residues [190], and
structures, which could explain the synergistic properties. the proposed torsion angles [191]. For sulfate groups and
Under conditions in which they do not exhibit rigid potassium counterions, coordinates were taken from pub-
or weak gel properties, galactomannans can cause non- lished crystal structures [192]. The mannan fragment was
gelling concentrations of carrageenan to form rm rigid generated from published mannobiose features [193] and
gels. The extent of these gelling interactions is controlled torsion angles proposed for the mannan chain [194].
and modied by chemical structure variations. Thus fur- The analysis of energy contributions showed that the
cellaran, which contains half the level of O-sulfation of complexes of two n-carrageenan chains are stabilized by
n-carrageenan gels better with galactomannan, while van der Waals and hydrogen-bonding energies, whereas
L-carrageenan, which contains twice the level of O-sulfa- the electrostatic contribution is very small. In the most
tion, exhibits no gelation interaction [184]. Structural var- stable complex (Fig. 12), CUH bonds are localized inside
iations in the galactomannan molecule also aect the the double helix, creating a kind of hydrophobic cavity,
gelling interactions. It was shown that the degree of whereas sulfate groups and hydroxyl groups are on the
interaction (and also the synergy) of galactomannan chains outside of the structure.
with other polysaccharides decreases as the D-galactose From the lowest energy minimum, this complex con-
content increases [185], and that the intermolecular binding tains two intertwining parallel chains, oset by a relative
involved occurs mainly via the unsubstituted D-mannose rotation of 49.7j and 3.3 A translation along the helix axis
units of the galactomannan chains [51,186]. Galacto- with a pitch of 25 A. The potential energy surface of
mannans with a larger proportion of longer regions of interaction between mannan and n-carrageenan double
unsubstituted blocks or sides along the mannan backbone helix is represented in Fig. 13. Four minima determined
interact best with agars, carrageenans, and xanthan. on the map were used for the nal renement of the
To explain all the preceding results, the structure- complex structures. The best complex structure is shown
dependent interaction specicity has to be acknowledged. in Fig. 14.
This interaction, although less sensitive here because of the It was found that the interaction between n-carra-
nonregular structure of galactomannan, is, in essence, geenan and mannan required the exibility of the mannan
identical to all other molecular recognition phenomena chain as well as structural adjustments of the n-carrageen-
implying specic interactions between pairs of molecules, an double helix. An analysis of the energy terms revealed
an example of which is found with proteincarbohydrate that the main contributions to the interaction energies
interactions [157,187]. These interactions are in general occurred from van der Waals and hydrogen-bonding en-
unique and reproducible. ergies, but contributions from intramolecular stabilization
To bring more information concerning this interaction of individual chains were also important. Several possibil-
specicity responsible for the synergistic properties of ities of intermolecular hydrogen bonds were found for the
mixed gels and to predict the structure of polysaccharide dierent complex structures determined (sulfate groups can
complexes, the computer program SAINT (SAccharide also be involved in intermolecular hydrogen bonds). The
INTeraction) has been developed. All its basic features most stable complex displays the larger number of hydro-
were described in a paper [188]. This procedure permits a gen bonds. In addition, the results showed that for the best
systematic search of the conformational space describing complex, hydrogen bonding involves mainly two residues
the interactions between chains, and at the same time, an of the mannan segment. This implies that a disaccharide
estimation of the inuence of these interactions on the sequence could be required for these interactions.
structure of the individual chains. The total energy includes As already observed [188], both polysaccharide chains
the internal energy of each chain and their interaction involved in the complex structures undergo conformation-
energy. The optimization procedure, based on the nonde- al modications of their individual structure. These con-
rivative method of conjugated directions, allows the simul- formational changes are characteristic properties of
taneous relaxation of all relevant geometrical parameters carbohydrates (not unique to carbohydrates but more
and the imposition of any constraint on the chain structure keenly felt for these molecules than for other biomole-
and/or on the mutual orientation of the chains. The force cules). Current eorts to deal with this concept of exibility
eld used is composed of four contributions: the Lennard are undertaken to fully understand all its functional or
Jones nonbonded atomatom interaction energy, the hy- informative implications concerning carbohydrates [195].
drogen bond energy, the torsion energy, and the electro- Results of these calculations show clearly the possibility
static energy. for the individual chains involved in the formation of
This method was used to investigate the interactions a complex to change their conformation of low energy
between mannan and n-carrageenan [189]. In the rst step, while maximizing van der Waals and hydrogen bonding
the double helical arrangement of n-carrageenan has been contributions. In the gel state, several mixed complexes
modeled. Then, modeling the interactions between n-car- are allowed, each with a weak occurrence preventing a
rageenan double helix and mannan was performed. The clear detection by X-ray diraction methods. The results
models were built by using geometrical parameters (bond corroborate also an associating gel network already
298 Taravel et al.
Figure 12 Molecular drawing for the best complex of two n-carrageenan chains: (a) perpendicular to the helical axis and
(b) along the helical axis.
mentioned [196] and are in agreement with some recent

experimental results [41,51,84,88,197].
Recently, the synergistic interaction between xanthan
and glucomannan in solution and in the gel phase has been
studied by circular dichroism and dierential scanning
calorimetry [88]. The structure for the complex was fol-
lowed by the potential energy as a function of distance and
orientation between the chains. The oligosaccharide struc-
tures were treated as rigid bodies and examined for asso-
ciation in parallel and antiparallel modes. After
minimization, two molecular models were examined, one
with a pseudo 21 helical conformation (cellulose-like xan-
than and glucomannan helices), the other with a xanthan-
like vefold helical conformation. The rst one can be
excluded because of marginal side chain involvement from
the circular dichroism ndings. The experimental and
calculation results clearly indicate the involvement of the
side chains of xanthan and suggest that the ordered por-
Figure 13 Two-dimensional interaction potential energy tions of the macromolecular complex in solution act in the
surface (ro,/o) between the double helical structure of n- gel phase as junction zones. The results are also formulated
carrageenan and mannan chains. The (ro,/o) location of in terms of 1:1 and 2:1 glucomannan/xanthan molecular
main minima is indicated by 1. assemblies [88].
Figure 14 Molecular drawing for the best complex of mannan and the double helix of n-carrageenan: (a) perpendicular to the
helical axis and (b) along the helical axis.
V. CONCLUSIONS AND PERSPECTIVES parison with experimental data is necessary to test the
accuracy of calculated results and to provide criteria for
In this review, we have emphasized the problem of molec- methodology improvement. When calculations are mean-
ular modeling of polysaccharidepolysaccharide inter- ingful, they can provide detailed results even if they are not
actions. Knowledge of the structure and stability of available by measurements.
polysaccharide complexes will contribute to our under- The expanding role of computational methods in the
standing of their biological functions and to our ability to eld of saccharides has been fueled by the steady and rapid
modify their properties for dierent applications. Molecu- increase in computing power and advances in conceptual
lar modeling of interaction potential energy surfaces is far and computational techniques over the last 20 years.
from routine. It is evident that knowledge of the structural Introduction of new technologies in computer architecture
features of single species is a prerequisite to quantitative will insure that the latter will continue to increase for some
studies of these interactions. years. This means that it will be possible to handle more
Current works on polysaccharidepolysaccharide complex molecular structures by using more reliable com-
interactions indicate the coupling of motion along inter- putational methods.
molecular coordinates with intramolecular motion. There-
fore, studies of these interactions require potential surfaces
which are not limited to intermolecular degrees of freedom,
that is, surfaces in all internal and external coordinates.
Methods of molecular modeling are a potent tool, REFERENCES
although versatile, in polysaccharide structural chemistry. 1. Sharon, N. Complex Carbohydrates, their Chemistry, Bio-
Obviously, the experiment plays an essential role in vali- synthesis, and Functions; Addison-Wesley Publishing
dating the molecular modeling methods; that is, a com- Company: Reading, MA, 1975.
300 Taravel et al.
2. Marchessault, R.H. Carbohydrate polymers: Natures Muller, M.; Netter, P.; Lapicque, F. Hyaluronatealginate
high-performance materials. Chemtech 1984, 14, 542. combination for the preparation of new biomaterials:
3. Imberty, A.; Bourne, Y.; Cambillau, C.; Rouge, P.; Perez, investigation of the behaviour in aqueous solutions.
S. Oligosaccharide conformation in protein/carbohydrate Biochim. Biophys. Acta 1999, 1426, 185.
complexes. Adv. Biophys. Chem. 1993, 3, 71. 23. Carver, J.P. Experimental structure determination of
4. Sakurai, K.; Mizu, M.; Shinkai, S. Polysaccharide oligosaccharides. Curr. Opin. Struct. Biol. 1991, 1, 716.
polynucleotide complexes. 2. Complementary polynu- 24. Peters, T.; Meyer, B.; Stuike-Prill, R.; Somorjai, R.;
cleotide mimic behavior of the natural polysaccharide Brisson, J.-R. A Monte Carlo method for conformational
schizophyllan in the macromolecular complex with analysis of saccharides. Carbohydr. Res. 1993, 238, 49.
single-stranded RNA and DNA. Biomacromolecules 25. Meyer, C.; Perez, S.; Herve du Penhoat, C.; Michon, V.
2001, 2, 641. Conformational analysis of 4,1V,6V-trichloro-4,1V,6V-tri-
5. Whitney, S.E.C.; Brigham, J.E.; Darke, A.H.; Grant Reid, deoxy-galacto-sucrose (sucralose) by a combined molec-
J.S.; Gidley, M.J. Structural aspects of the interaction of ular-modeling and NMR spectroscopy approach. J. Am.
mannan-based polysaccharides with bacterial cellulose. Chem. Soc. 1993, 115, 10300.
Carbohydr. Res. 1998, 307, 299. 26. Philippopoulos, M.; Lim, C. Internal motions in the molec-
6. Averous, L.; Fringant, C.; Moro, L. Plasticized starch ular tumbling regime. Eect on NMR dipolar cross-
cellulose interactions in polysaccharide composites. Poly- relaxation and interproton distance determination. J. Phys.
mer 2001, 42, 6565. Chem. 1994, 98, 8264.
7. Femenia, A.; Rigby, N.M.; Selvendran, R.R.; Waldron, 27. Akiyoshi, K.; Sunamoto, J. Supramolecular assembly of
K.W. Investigation of the occurrence of pecticxylan hydrophobized polysaccharides. Supramol. Sci. 1996, 3,
xyloglucan complexes in the cell walls of cauliower stem 157.
tissues. Carbohydr. Res. 1999, 39, 151. 28. Sivakama Sundari, C.; Balasubramanian, D. Hydrophobic
8. Weimer, P.J.; Hackney, J.M.; Jung, H.-J.G.; Hateld, surfaces in saccharide chains. Prog. Biophys. Mol. Biol.
R.D. Fermentation of a bacterial cellulose/xylan compos- 1997, 67, 183.
ite by mixed ruminal microora: implications for the role 29. Clark, A.H.; Ross-Murphy, S.B. Structural and mechanical
of polysaccharide matrix interactions in plant cell wall properties of biopolymer gels. Adv. Polym. Sci. 1987, 83, 57.
biodegradability. J. Agric. Food Chem. 2000, 48, 1727. 30. Kay, L.E.; Petsko, G.A. Biophysical methods. Curr. Opin.
9. Neville, A.C. Biology of Fibrous Composites; Cambridge Struct. Biol. 2001, 11, 513.
University Press: New York, 1993. 31. Zanni, M.T.; Hochstrasser, R.M. Two-dimensional infra-
10. Jardetsky, O.; Roberts, G.C.K. Time-dependent phenom- red spectroscopy: a promising new method for the time
ena and problems of averaging. NMR in Molecular resolution of structures. Curr. Opin. Struct. Biol. 2001, 11,
Biology; Academic Press: New York, 1981; 115 pp. 516.
11. Matveev, Y.I.; Grinberg, V.Y.; Tolstoguzov, V.B. The 32. Wilson, R.H.; Smith, A.C.; Kacurakova, M.; Saunders,
plasticizing eect of water on proteins, polysaccharides and P.K.; Wellner, N.; Waldron, K.W. The mechanical proper-
their mixtures. Glassy state of biopolymers, food and ties and molecular dynamics of plant cell wall polysac-
seeds. Food Hydrocoll. 2000, 14, 425. charides studied by Fourier-Transform Infrared
12. Kaliannan, P.; Michael Gromiha, M.; Elanthiraiyan, M. Spectroscopy. Plant Physiol. 2000, 124, 397.
Solvent accessibility studies on polysaccharides. Int. J. 33. Yip, C.M. Atomic force microscopy of macromolecular
Biol. Macromol. 2001, 28, 135. interactions. Curr. Opin. Struct. Biol. 2001, 11, 567.
13. Perez, S. Polysaccharideswater interactions. In Water and 34. Kirby, A.R.; Gunning, A.P.; Morris, V.J. Imaging Poly-
Biological Macromolecules: Topics and Structural Biology; saccharides by atomic force microscopy. Biopolymers
Westhof, E., Ed.; The Macmillan Press: London, 1993; 295 pp. 1995, 38, 355.
14. Morris, E.R. The eect of solvent partition on the mechani- 35. Gad, M.; Itoh, A.; Ikai, A. Mapping cell wall polysac-
cal properties of biphasic biopolymer gels: an approximate charides of living microbial cells using atomic force
theoretical treatment. Carbohydr. Polym. 1992, 17, 65. microscopy. Cell Biol. Int. 1997, 21, 697.
15. Irie, M. Stimuli-responsive poly(N-isopropylacrylamide). 36. Cowman, M.K.; Li, M.; Balazs, E.A. Tapping mode
Photo- and chemical-induced phase transitions. Adv. atomic force microscopy of hyaluronan: extended and
Polym. Sci. 1993, 110, 49. intramolecularly interacting chains. Biophys. J. 1998, 75,
16. Kaetsu, I. Stimuli-sensitive hydrogels. In Polysaccharides 2030.
in Medicinal Applications; Dumitriu, S., Ed.; Marcel 37. Camesano, T.A.; Logan, B.E. Probing bacterial electro-
Dekker, Inc.: New York, 1996; 243 pp. steric interactions using atomic force microscopy. Environ.
17. Pelton, R. Temperature-sensitive aqueous microgels. Adv. Sci. Technol. 2000, 34, 3354.
Colloid Interface Sci. 1999, 85, 1. 38. Ogawa, K. Three-D structures of polysaccharides. X-ray
18. Milas, M.; Rinaudo, M. On the electrostatic interactions of method is still the royal road. Kagaku to Seibutsu 1997, 35,
ionic polysaccharides in solution. Curr. Trends Polym. Sci. 714.
1997, 2, 47. 39. Chandrasekaran, R.; Lee, E.J.; Thailambal, V.G.; Zeven-
19. Amaike, M.; Senoo, Y.; Yamamoto, H. Sphere, honey- huizen, L.P.T.M. Molecular architecture of a galactoglu-
comb, regularly spaced droplet and ber structures of can from Rhizobium meliloti. Carbohydr. Res. 1994, 261,
polyion complexes of chitosan and gellan. Macromol. 279.
Rapid Commun. 1998, 19, 287. 40. Chandrasekaran, R.; Radha, A.; Lee, E.J.; Zhang, M.
20. Yamamoto, H.; Senoo, Y. Polyion complex ber and Molecular architecture of araban, galactoglucan and
capsule formed by self-assembly of chitosan and gellan at welan. Carbohydr. Polym. 1994, 25, 235.
solution interfaces. Macromol. Chem. Phys. 2000, 201, 84. 41. Chandrasekaran, R.; Radha, A. Molecular modelling of
21. Ohkawa, K.; Takahashi, Y.; Yamada, M.; Yamamoto, H. xanthan:galactomannan interactions. Carbohydr. Polym.
Polyion complex ber and capsule formed by self-assembly 1997, 32, 201.
of chitosan and poly(a,L-glutamic acid) at solution 42. Cairns, P.; Miles, M.J.; Morris, V.J.; Brownsey, G.J. X-ray
interfaces. Macromol. Mater. Eng. 2001, 286, 168. bre-diraction studies of synergistic, binary polysaccha-
22. Oerther, S.; Payan, E.; Lapicque, F.; Presle, N.; Hubert, P.; ride gels. Carbohydr. Res. 1987, 160, 411.
43. Cairns, P.; Atkins, E.D.T.; Miles, M.J.; Morris, V.J. eld-oriented proteins: information for structure determi-
Molecular tranforms of kappa carrageenan and furcel- nation in solution. Proc. Natl. Acad. Sci. U. S. A. 1995, 92,
laran from mixed gel systems. Int. J. Biol. Macromol. 1991, 9279.
13, 65. 61. Tjandra, N.; Bax, A. Direct measurement of distances and
44. Djabourov, M.; Clark, A.H.; Rowlands, D.W.; Ross- angles in biomolecules by NMR in a dilute liquid
Murphy, S.B. Small-angle X-ray scattering character- crystalline medium. Sciences 1997, 278, 1111.
ization of agarose sols and gels. Macromolecules 1989, 62. Martin-Pastor, M.; Bush, C.A. Rened structure of a
22, 180. exible heptasaccharide using 1H13C and 1H1H NMR
45. Rochas, C.; Brulet, A.; Guenet, J.M. Thermoreversible residual dipolar coupling in concert with NOE and long
gelation of agarose in water/dimethyl sulfoxide mixtures. range scalar coupling constants. J. Biomol. NMR 2001, 19,
Macromolecules 1994, 27, 3830. 125.
46. Cheng, Y.; Rau, D.C.; Chik, J.K.; Prudhomme, R.K. 63. Almond, A.; Duus, J.O. Quantitative conformational
Structure and intermolecular polysaccharides chains: an analysis of the core region of N-glycans using residual
osmotic stress and X-ray scattering study. Polym. Mater.: dipolar coupling, aqueous molecular dynamics, and steric
Sci. Eng. 2001, 85, 174. alignment. J. Biomol. NMR 2001, 20, 351.
47. Stipanovic, A.J.; Giammatteo, P.J.; Robie, S.B. Cross- 64. Uhrin, D.; Brisson, J.-R. Structure determination of
polarization/magic-angle spinning 13C NMR of (1,6)-h-D- microbial polysaccharides by high resolution NMR
glucan (pustulan): mechanism of gelation. Biopolymers spectroscopy. NMR in Microscopy; Horizon Scientic
1985, 24, 2333. Press: Wymondham, UK, 2000; 165 pp.
48. Gidley, M.J.; McArthur, A.J.; Underwood, D.R. 13C 65. van Duynhoven, J.P.M.; Kulik, A.S.; Jonker, H.R.A.;
NMR characterization of molecular structures in powders, Haverkamp, J. Solid-like components in carbohydrate gels
hydrates and gels of galactomannans and glucomannans. probed by NMR spectroscopy. Carbohydr. Polym. 1999,
Food Hydrocoll. 1991, 5, 129. 40, 211.
49. Saito, H.; Ohki, T.; Sasaki, T. A 13C nuclear magnetic 66. Smits, A.L.M.; Hulleman, S.H.D.; Van Soest, J.J.G.; Feil,
resonance study of gel-forming (1,3)-h-D-glucans. Evi- H.; Vliegenthart, J.F.G. The inuence of polyls on the
dence of the presence of single-helical conformation in a molecular organization in starch-based plastics. Polym.
resilient gel of a curdlan-type polysaccharide 13140 from Adv. Technol. 1999, 10, 570.
Alcaligenes faecalis var. myxogenes IFO 13140. Biochem- 67. Newman, R.H. Editing the information in solid-state
istry 1977, 16, 908. carbon-13 NMR spectra of food. Advances in Magnetic
50. Casu, B. Nuclear magnetic resonance studies of polysac- Resonance in Food Science; Royal Society of Chemistry:
charide structure and interactions. In Polysaccharides. Cambridge, UK, 1999; 144 pp.
Topics in Structure and Morphology; Atkins, E.D.T., Ed.; 68. Schmidt-Rohr, K.; Clauss, J.; Spiess, H.W. Correlation of
The Macmillan Press: London, 1985; 1 pp. structure, mobility and morphological information in
51. Rochas, C.; Taravel, F.R.; Turquois, T. NMR studies of heterogeneous polymer materials by two dimensional
synergistic kappa carrageenancarob galactomannan gels. wideline-separation NMR spectroscopy. Macromolecules
Int. J. Biol. Macromol. 1990, 12, 353. 1992, 25, 3272.
52. Turquois, T.; Taravel, F.R.; Rochas, C. Synergy of the 69. Dea, I.C.M.; McKinnon, A.A.; Rees, D.A. Tertiary and
agarosecarob galactomannan blend inferred from NMR quaternary structure in aqueous polysaccharide systems
and rheological studies. Carbohydr. Res. 1993, 238, 27. which model cell wall cohesion. Reversible changes in con-
53. Belton, P.S.; Morris, V.J.; Tanner, S.F. Interactions of formation and association of agarose, carrageenan and
group I cations with iota, kappa and lambda carrageenans galactomannan. J. Mol. Biol. 1972, 68, 153.
studied by multinuclear NMR. Int. J. Biol. Macromol. 70. Dea, I.C.M.; Rees, D.A. Anity interactions between
1985, 7, 53. agarose and h-1,4-glycans: a model for polysaccharide
54. Piculell, L.; Nilsson, S.; Strom, P. On the specicity of the associations in algal cell walls. Carbohydr. Polym. 1987, 7,
binding of cations to carrageenans: counterion NMR 183.
spectroscopy in mixed carrageenan systems. Carbohydr. 71. Tako, M.; Nakamura, S. Synergistic interaction between
Res. 1989, 188, 121. xanthan and guar gum. Carbohydr. Res. 1985, 138, 207.
55. Darke, A.; Morris, E.R.; Rees, D.A.; Welsh, E.J. Spectro- 72. Tako, M.; Nakamura, S. Synergistic interaction agarose
scopic characterisation of orderdisorder transitions for and D-galactoD-mannan in aqueous media. Agric. Biol.
extracellular polysaccharides of Arthrobacter species. Chem. 1988, 52, 1071.
Carbohydr. Res. 1978, 66, 133. 73. Tako, M. Synergistic interaction between xanthan and
56. Ablett, S.; Lillford, P.J.; Baghdadi, S.M.A.; Derbyshire, tara-bean gum. Carbohydr. Polym. 1991, 16, 239.
W. Nuclear magnetic resonance investigations of 74. Turquois, T.; Rochas, C.; Taravel, F.R. Rheological
polysaccharide lms, sols and gels. I. Agarose. J. Coll. studies of synergistic kappa carrageenancarob galacto-
Interface Sci. 1978, 67, 355. mannan gels. Carbohydr. Polym. 1992, 17, 263.
57. Smith, I.C.P.; Saito, H. Composition, conformation, se- 75. Turquois, T.; Doublier, J.L.; Taravel, F.R.; Rochas, C.
quence and dynamics of complex carbohydrates as Synergy of the n-carrageenancarob galactomannan blend
revealed by carbon-13 NMR spectroscopy. Pure Appl. inferred from rheological studies. Int. J. Biol. Macromol.
Chem. 1980, 27, 213. 1994, 16, 105.
58. Piculell, L.; Nilsson, S. Anion-specic salt eects in 76. Alloncle, M.; Lefebvre, J.; Llamas, G.; Doublier, J.L. A
aqueous agarose systems. 2. Nuclear spin relaxation of rheological characterization of cereal starchgalactoman-
ions in agarose gels and solutions. J. Phys. Chem. 1989, 93, nan mixtures. Cereal Chem. 1989, 66, 90.
5602. 77. Fernandes, P.B.; Goncalves, M.P.; Doublier, J.L. Inuence
59. Tolman, J.R. Dipolar couplings as a probe of molecular of locust bean gum on the rheological properties of kappa-
dynamics and structure in solution. Curr. Opin. Struct. carrageenan systems in the vicinity of the gel point. Car-
Biol. 2001, 11, 532. bohydr. Polym. 1993, 22, 99.
60. Tolman, J.R.; Flanagan, J.M.; Kennedy, M.A.; Preste- 78. Fernandes, P.B.; Gonc alves, M.P.; Doublier, J.L. Rheo-
gard, J.H. Nuclear magnetic dipole interactions in the logical behaviour of kappa-carrageenan/galactomannan
302 Taravel et al.
mixtures at a very low level of kappa-carrageenan. J. Text. 101. Davidson, E.R.; Feller, D. Basis set selection for molecular
Stud., 1994, 25, 267. calculations. Chem. Rev. 1986, 86, 681.
79. Gencer, G. Eect of selected additives on the ow param- 102. Tvaroska, I.; Taravel, F.R. In Polysaccharides, Structural
eters of 1:1 mixtures of carrageenanguar and CMClocust Diversity and Functional Versatility; Dumitriu, S., Ed.;
bean gum. J. Text. Stud. 1989, 20, 473. Marcel Dekker, Inc.: NY, 1998; 173 pp.
80. Stading, M.; Hermansson, A.-M. Rheological behaviour 103. Weiner, S.J.; Kolman, P.A.; Case, D.A.; Sing, U.C.; Ghio,
of mixed gels of n-carrageenanlocust bean gum. Carbo- C.; Alagona, G.; Profeta, S.P.; Weiner, D. A new force eld
hydr. Polym. 1993, 22, 49. for mechanical simulation of nucleic acids and proteins. J.
81. Williams, P.A.; Clegg, S.M.; Langdon, M.J.; Nishinari, K.; Am. Chem. Soc. 1984, 106, 765.
Piculell, L. Investigation of the gelation mechanism in 104. Weiner, S.J.; Kolman, P.A.; Nguyen, D.T.; Case, D.A. An
n-carrageenan/konjac mannan mixtures using dierential all atom force eld for simulation of proteins and nucleic
scanning calorimetry and electron spin resonance spectro- acids. J. Comput. Chem. 1986, 7, 230.
scopy. Macromolecules 1993, 26, 5441. 105. Brooks, B.R.; Bruccoleri, R.E.; Olafson, B.D.; States, D.J.;
82. Morris, V.J.; Wilde, P.J. Interactions of food biopolymers. Swaminathan, S.; Karplus, M. CHARMM: a program for
Curr. Opin. Colloid Interface Sci. 1997, 2, 567. macromolecular energy minimization, and dynamics cal-
83. Parker, A.; Lelimousin, D.; Miniou, C.; Boulenguer, P. culations. J. Comput. Chem. 1983, 4, 187.
Binding of galactomannans to kappa-carrageenan after 106. DISCOVER, version 2.8.0; Biosym Technologies, Inc.,
cold mixing. Carbohydr. Res. 1995, 272, 91. San Diego, CA, 1992.
84. Rinaudo, M.; Milas, M.; Bresolin, T.; Ganter, J. Physical 107. van Gunsteren, W.F. GROMOS, Groningen Molecular
properties of xanthan, galactomannan, and their mixtures Simulation Package; University of Groningen: The
in aqueous solutions. Macromol. Symp. 1999, 140, 115. Netherlands, 1987.
85. Lundin, L.; Hermansson, A.-M. Supermolecular aspects of 108. MACROMODEL, Schrodinger, Inc., Portland, U.S.A.,
the xanthanlocust bean gels based on rheology and 2001.
electron microscopy. Carbohydr. Polym. 1995, 26, 129. 109. Brady, J.W. Theoretical studies of oligosaccharide struc-
86. Schorsch, C.; Garnier, C.; Doublier, J.-L. Microscopy of ture and conformational dynamics. Curr. Opin. Struct.
xanthan/galactomannan mixtures. Carbohydr. Polym. Biol. 1991, 1, 711.
1995, 28, 319. 110. Brant, D.A. Conformational analysis of biopolymers:
87. Goycoolea, F.M.; Milas, M.; Rinaudo, M. Associative Conformational energy calculations. Annu. Rev. Biophys.
phenomena in galactomannandeacetylated xanthan sys- Bioeng. 1972, 1, 368.
tems. Int. J. Biol. Macromol. 2001, 29, 181. 111. Brant, D.A. Conformational theory applied to polysac-
88. Paradossi, G.; Chiessi, E.; Barbiroli, A.; Fessas, D. charide structure. Q. Rev. Biophys. 1976, 9, 527.
Xanthan and glucomannan mixtures: synergistic interac- 112. Brant, D.A. Conformational behavior of polysaccharides
tions and gelation. Biomacromolecules 2002, 3, 498. in solution. In Biochemistry of Plants; Preiss, J., Ed.;
89. Morris, E.R.; Foster, T.J. Role of conformation in syner- Academic Press: New York, 1980; Vol. 3, 425 pp.
gistic interactions of xanthan. Carbohydr. Polym. 1994, 23, 113. Tvaroska, I. The structure and conformational properties
133. of carbohydrates. In Theoretical Chemistry of Biological
90. Pople, J.A.; Beveridge, D.L. Approximate Molecular Systems; Naray-Szabo, G., Ed.; Elsevier: Amsterdam,
Orbital Theory; New York: McGraw-Hill, 1970. 1986; 283 pp.
91. Clarke, T.A. A Handbook of Computational Chemistry. 114. Tvaroska, I. Computational methods for studying oligo-
A Practical Guide to Chemical Structure and Energy Cal- and polysaccharide conformations. Pure Appl. Chem.
culations; Wiley: New York, 1985. 1989, 61, 1201.
92. Hehre, W.J.; Radom, L.; Schleyer, P.v.R.; Pople, J.A. Ab 115. French, A.D., Brady, J.W., Eds.; Computer Modeling
Initio Molecular Orbital Theory; Wiley: New York, 1986. of Carbohydrate Molecules; ACS Symposium Series;
93. Parr, R.G.; Yang, W. Density-Functional Theory of Atoms American Chemical Society: Washington, DC, 1990; Vol.
and Molecules; Oxford University Press: New York, 1989. 430.
94. Tvaroska, I.; Andre, I.; Carver, J.P. Ab initio molecular 116. Meyer, B. Conformational aspects of oligosaccharides.
orbital study of the catalytic mechanism of glycosyltrans- Top. Curr. Chem. 1990, 154, 141.
ferases: description of the reaction pathways and determi- 117. Brady, J.W. Molecular dynamics simulations of carbohy-
nation of transition-state structures for inverting N- drate molecules. Adv. Biophys. Chem. 1990, 1, 155.
acetylglucosaminyltransferases. J. Am. Chem. Soc. 2000, 118. Tvaroska, I. Theoretical aspects of structure and con-
122, 8762. formation of oligosaccharides. Curr. Opin. Struct. Biol.
95. Niketic, S.R.; Rasmussen, K. Lecture notes in chemistry. 1991, 2, 661.
The Consistent Force Field: A Documentation; Springer- 119. Koehler, J. Molecular dynamics simulations of carbohy-
Verlag: Berlin, 1977; Vol. 3. drates. Top. Mol. Struct. Biol. 1991, 16, 69.
96. Burkert, U.; Allinger, N.L. ACS Monograph. Molecular 120. Perez, S. Molecular modeling and electron diraction of
Mechanics; American Chemical Society: Washington, DC, polysaccharides. Methods Enzymol. 1991, 203, 510.
1982; Vol. 177. 121. Bush, C.A.; Cagas, P. Three-dimensional conformation of
97. Rasmussen, K. Lecture notes in chemistry. Potential complex carbohydrates. Adv. Biophys. Chem. 1992, 2, 149.
Functions in Conformational Analysis; Springer-Verlag: 122. French, A.D.; Dowd, M.K. Exploration of disaccharide
Heidelberg, 1985; Vol. 37. conformations by molecular mechanics. J. Mol. Struct.
98. McCammon, J.A.; Harvey, S.C. Dynamics of Proteins and (Theochem.) 1993, 286, 183.
Nucleic Acids; Cambridge University Press: London, 1987. 123. Perez, S.; Imberty, A.; Carver, J.P. Molecular modeling: an
99. van Gunsteren, W.F.; Berendsen, H.J.C. Computer essential component in the structure determination of
simulation of molecular dynamics: methodology, applica- oligosaccharides and polysaccharides. Adv. Comput. Biol.
tions, and perspectives in chemistry. Angew. Chem. Int. 1994, 1, 147.
Ed. Engl. 1990, 29, 992. 124. Engelsen, S.B.; Koca, J.; Braccini, I.; Herve du Penhoat,
100. Richard, W.G.; King, P.M.; Reynolds, C.A. Solvation C.; Perez, S. Travelling on the potential energy surfaces of
eects. Protein Eng. 1989, 2, 319. carbohydrates: comparative application of an exhaustive
systematic conformational search with an heuristic search. 143. Petkowicz, C.L.O.; Reicher, F.; Mazeau, K. Conforma-
Carbohydr. Res. 1995, 276, 1. tional analysis of galactomannans: from oligomeric seg-
125. Imberty, A.; Mikros, E.; Koca, J.; Mollicone, R.; Oriol, R.; ments to polymeric chains. Carbohydr. Polym. 1998, 37,
Perez, S. Computer simulation of histo-blood group 25.
oligosaccharides: energy maps of all constituting disac- 144. Engelsen, S.B.; Cros, S.; Mackie, W.; Perez, S. A molecular
charides and potential energy surfaces of 14 ABH and builder for carbohydrates: application to polysaccharides
Lewis carbohydrate antigens. Glyconj. J. 1995, 12, 331. and complex carbohydrates. Biopolymers 1996, 39, 417.
126. Woods, R.J. Three-dimensional structures of oligosac- 145. Boutherin, B.; Mazeau, K.; Tvaroska, I. Conformational
charides. Curr. Opin. Struct. Biol. 1995, 5, 591. statistics of pectin substances by Metropolis Monte Carlo
127. Bizik, F.; Tvaroska, I. Conformational analysis of di- study. Carbohydr. Polym. 1997, 32, 255.
saccharide fragments of blood group determinants in 146. Petkowicz, C.L.O.; Milas, M.; Mazeau, K.; Bresolin, T.;
solution by molecular modeling. Chem. Pap. 1995, 49, 202. Reicher, F.; Ganter, J.L.M.S.; Rinaudo, M. Conformation
128. Bizik, F.; Tvaroska, I. On the exibility of the Lewis x, of galactomannan: experimental and modeling ap-
Lewis a, Sialyl Lewis x, and Sialyl Lewis a oligosaccharides. proaches. Food Hydrocoll. 1999, 13, 263.
Conformational analysis in solution by molecular model- 147. Bergamini, J.-F.; Boisset, C.; Mazeau, K.; Heyraud, A.;
ling. Chem. Pap. 1996, 50, 84. Taravel, F.R. Conformational behavior of oligo-galacto-
129. Woods, R.J. The application of molecular modeling tech- mannan chains inferred from NMR spectroscopy and
niques to the determination of oligosaccharide solution molecular modeling. New J. Chem. 1995, 19, 115.
conformations. In Reviews in Computational Chemistry; 148. McCleary, B.V.; Clark, A.H.; Dea, I.C.M.; Rees, D.A. The
Lipkowitz, K.B., Boyd, D.B.,Eds.; VCH Publishers: New ne structures of carob and guar galactomannans.
York, 1996; 129 pp. Carbohydr. Res. 1985, 139, 237.
130. Peters, T.; Pinto, B.M. Structure and dynamics of 149. Winter, W.T. Structural consequences of h (1!4) glycan
oligosaccharides: NMR and modeling studies. Curr. Opin. modication. In Cellulose and Wood Chemistry and Tech-
Struct. Biol. 1996, 6, 710. nology; Schuerch, C., Ed.; Wiley: New York, 1989; 283 pp.
131. Qasba, P.K.; Balaji, P.V.; Rao, V.S.R. Conformational 150. Brant, D.A.; Goebel, K.D. A general treatment of the
analysis of Asn-linked oligosaccharides: implications in congurational statistic of polysaccharides. Macromole-
biological processes. J. Mol. Struct. (Theochem.) 1997, cules 1975, 8, 522.
395396, 333. 151. Jordan, R.C.; Brant, D.A.; Cesaro, A. A Monte Carlo
132. Arnott, S.; Scott, W.E. Accurate X-ray diraction analysis study of the amylosic chain conformations. Biopolymers
of brious polysaccharides containing pyranose rings. 1978, 17, 2617.
Part. I. The linkaged-atom approach. J. Chem. Soc. Perkin 152. Gagnaire, D.; Perez, S.; Tran, V. Congurational statistics
Trans. II. 1971, 324. of single chains of a-linked glucans. Carbohydr. Polym.
133. Zugenmeier, P.; Sarko, A. The variable virtual bond: 1982, 2, 171.
Modeling technique for solving polymer crystal structures. 153. Brant, D.A.; Christ, M.D. In Computer Modeling of Carbo-
In Fiber Diraction Methods; French, A.D., Gartner, K.H., hydrate Molecules; French, A.D., Brady, J.W., Eds.; ACS
Eds.; ACS Symposium Series Vol. 141; American Chemical Symposium Series Vol. 430; American Chemical Society:
Society: Washington, DC, 1980; 225 pp. Washington DC, 1990; 42 pp.
134. Perez, S. A priori crystal structure modeling of polymeric 154. Levy, S.; York, W.S.; Stuike-Prill, R.; Meyer, B.; Staehelin,
materials. In Electron Crystallography of Organic Mole- L.A. Simulation of the static and dynamic molecular
cules; Fryer, J.R., Dorset, D.L., Eds.; Kluwer Academic conformation of xyloglucan. The role of the fucosylated
Publ.: Amsterdam, 1990; 33 pp. side chain in surface-specic side chain folding. Plant J.
135. Perez, S.; Delage, M.M. A database of three-dimensional 1991, 1, 195.
structures of monosaccharides from molecular mechanics 155. Urbani, R.; Cesaro, A. Solvent eect on the unperturbed
calculations. Carbohydr. Res. 1991, 212, 253. chain conformation of polysaccahrides. Polymer 1991, 32,
136. Perez, S.; Kouwijzer, M.; Mazeau, K.; Engelsen, S.B. 3013.
Modeling polysaccharides: present status and challenges. J. 156. Tvaroska, I.; Kozar, T. Theoretical studies on the confor-
Mol. Graphics 1996, 14, 307. mation of saccharides. 3. Conformational properties of
137. Ferro, D.R.; Pumilia, P.; Ragazzi, M. An improved force the glycosidic linkage in solution and their relation to the
eld for conformational analysis of sulphated polysaccha- anomeric and exoanomeric eects. J. Am. Chem. Soc.
rides. J. Comput. Chem. 1997, 18, 351. 1980, 102, 6929.
138. Braccini, I.; Heyraud, A.; Perez, S. Three-dimensional 157. Imberty, A.; Hardman, K.D.; Carver, J.P.; Perez, S. Mo-
features of the bacterial polysaccharide (1!4)-h-D-glucur- lecular modeling of protein/carbohydrate interactions.
onan: a molecular modelling study. Biopolymers 1998, 45, Docking of monosaccharides in the binding site of con-
165. cavalin A. Glycobiology 1991, 1, 456.
139. Braccini, I.; Grasso, R.P.; Perez, S. Conformational and 158. Imberty, A.; Perez, S. Molecular modelling of protein
congurational features of acidic polysaccharides and their carbohydrate interactions. Understanding the specicities
interactions with calcium ions: a molecular modelling of two legume lectins towards oligosaccharides. Glycobi-
investigation. Carbohydr. Res. 1999, 317, 119. ology 1994, 4, 351.
140. Perez, S.; Mazeau, K.; Herve du Penhoat, Catherine The 159. Sunamoto, J.I.; Iwamoto, K.; Kondo, M.; Shinkai, S.
three-dimensional structures of the pectic polysaccharides. Liposomal membranes. VI. Polysaccharide-induced aggre-
Plant Physiol. Biochem. 2000, 38, 37. gation of multilamellar liposomes of egg lecithin. J.
141. Haxaire, K.; Braccini, I.; Milas, M.; Rinaudo, M.; Perez, S. Biochem. 1980, 88, 1219.
Conformational behaviour of hyaluronan in relation to its 160. Mobed, M.C.; Chang, T.M.S. Kinetic aspects of polyelec-
physical properties as probed by molecular modelling. trolyte adsorption: Adsorption of chitin derivatives onto
Glycobiology 2000, 10, 587. liposomes as a model system. Art. Cells Blood Subs.
142. Braccini, I.; Perez, S. Molecular basis of Ca2+-induced Immob. Biotech. 1997, 25, 367.
gelation in alginates and pectins: the egg-box model re- 161. Girod, S.; Cara, L.; Maillols, H.; Salles, J.P.; Devoisselle,
visited. Biomacromolecules 2001, 2, 1089. J.M. Relationship between conformation of polysaccha-
304 Taravel et al.
rides in the dilute regime and their interaction with a 180. Theodorou, D.N.; Suter, U.W. Detailed molecular struc-
phospholipid bilayer. Luminescence 2001, 16, 109. ture of a vinyl polymer glass. Macromolecules 1985, 18,
162. Imagi, J.; Kako, N.; Nakanishi, K.; Matsuno, R. Retarded 1467.
oxidation of liquid lipids entrapped in matrixes of 181. Trommsdor, U.; Tomka, I. Structure of amorphous
saccharides or proteins. J. Food Eng. 1990, 12, 207. starch. 1. An atomistic model and X-ray scattering study.
163. Minemoto, Y.; Adachi, S.; Matsuno, R. Autoxidation of Macromolecules 1995, 28, 6128.
linoleic acid encapsulated with polysaccharides of diering 182. Trommsdor, U.; Tomka, I. Structure of amorphous
weight ratio. Biosci. Biotechnol. Biochem. 1999, 63, 866. starch. 2. Molecular interactions with water. Macro-
164. Matsumura, Y.; Satake, C.; Egami, M.; Mori, T. Inter- molecules 1995, 28, 6138.
action of gum Arabic, maltodextrin and pullulan with 183. Morris, E.R. In Mixed Polymer Gels, Food Gels; Harris, P.,
lipids in emulsions. Biosci. Biotechnol. Biochem. 2000, 64, Ed.; Elsevier Applied Science Series: London, 1990; 291 pp.
1827. 184. Dea, I.C.M. Industrial polysaccharides. Pure Appl. Chem.
165. Woodcock, S.; Henrissat, B.; Sugiyama, J. Docking of 1989, 61, 1315.
Congo red to the surface of crystalline cellulose using mo- 185. Dea, I.C.M.; Morris, E.R.; Rees, D.A.; Welsh, E.J.;
lecular mechanics. Biopolymers 1995, 36, 201. Barnes, H.A.; Price, J. Associations of like and unlike
166. Mazeau, K.; Vergelati, C. Atomistic modeling of the ad- polysaccharides: mechanism and specicity in galactoman-
sorption of benzophenone onto cellulosic surfaces. Lang- nans, interacting bacterial polysaccharides, and related
muir 2002, 18, 1919. systems. Carbohydr. Res. 1977, 57, 249.
167. Jiao, Q.; Liu, Q. A mathematical model for interaction of 186. Dea, I.C.M.; Clark, A.H.; McCleary, B.V. Eect of the
spectroscopic probe with polysaccharides. Spectrosc. Lett. molecular ne structure of galactomannans on their inter-
1998, 31, 1353. action properties: The role of unsubstituted sides. Food
168. Jiao, Q.; Liu, Q. Characterization of the interaction be- Hydrocoll. 1986, 1, 129.
tween methylene blue and glycosaminoglycans. Spectro- 187. Quiocho, F.A. Proteincarbohydrate interactions: basic
chim. Acta Part A 1999, 55, 1667. molecular features. Pure Appl. Chem. 1989, 61, 1293.
169. Albani, J.R. Eect of binding of calcouor white on the 188. Tvaroska, I.; Rochas, C.; Taravel, F.R.; Turquois, T.
carbohydrate residues of a1-acid glycoprotein (orosomu- Computer modeling of polysaccharidepolysaccharide
coid) on the structure and dynamics of the protein moiety. interactions: an approach to the kappa-carrageenan
A uorescence study. Carbohydr. Res. 2001, 334, 141. mannan case. Biopolymers 1992, 32, 551.
170. Perez, S.; Chanzy, H. Electron crystallography of linear 189. Turquois, T.; Rochas, C.; Taravel, F.R.; Tvaroska, I.
polysaccharides. J. Electron Microsc. Tech. 1989, 11, 280. Computer modeling of kappa carrageenanmannan inter-
171. Perez, S.; Vergelati, C. Unied representation of helical actions. J. Mol. Recogn. 1994, 7, 243.
parameters: Application to polysaccharides. Biopolymers 190. Rees, D.A. Conformational analysis of polysaccharides.
1985, 24, 1809. Part II. Alternating copolymers of the agarcarrageenan
172. Winter, W.T. Program LALS; Syracuse, NY: SUNY-ESF, chondroitin type by model building in the computer with
1990. calculation of helical parameters. J. Chem. Soc. (B) 1969,
173. Sarko, A.; Zugenmaier, P. Program PS87; Syracuse, NY: 217.
SUNY-ESF, 1987. 191. Millane, R.P.; Chandrasekaran, R.; Arnott, S.; Dea,
174. Millane, R.P. Polysaccharide structures: X-ray ber dif- I.C.M. The molecular structure of kappa-carrageenan
fraction studies. In Computer Modeling of Carbohydrate and comparison with iota-carrageenan. Carbohydr. Res.
Molecules; French, A.D., Brady, J.W., Eds.; ACS 1988, 182, 1.
Symposium Series Vol. 430; American Chemical Society: 192. Watson, W.H.; Taira, Z. Potassium allylglucosinate
Washington, DC, 1990; 315 pp. monohydrate, K(C10H16O9NS2). H2O, Cryst. Struct.
175. Perez, S.; Imberty, A.; Scaringe, R.P. Modeling of inter- Commun. 1977, 6, 441.
actions of polysaccharide chains. In Computer Modeling 193. Sheldrick, B.; Mackie, W.; Akrigg, D. The crystal and
of Carbohydrate Molecules; French, A.D., Brady, J.W., molecular structure of O-h-D-mannopyranosyl-(104)-a-D-
Eds.; ACS Symposium Series Vol. 430; American Chemical mannopyranose (mannobiose). Carbohydr. Res. 1984,
Society: Washington, DC, 1990; 281 pp. 132, 1.
176. Vietor, R.J.; Mazeau, K.; Lakin, M.; Perez, S. A priori 194. Mackie, W.; Sheldrick, B.; Akrigg, D.; Perez, S. Crystal
crystal structure prediction of native celluloses. Biopoly- and molecular structure of mannotriose and its relation-
mers 2000, 54, 342. ship to the conformations and packing of mannan and
177. Millane, R.P.; Narasaiah, T.V.; Arnott, S. On the molec- glucomannan chains and mannobiose. Int. J. Biol. Macro-
ular structures of xanthan and genetically engineered mol. 1986, 8, 43.
xanthan variants with truncated side chains. In Biochemical 195. Drickamer, K.; Homans, S. Carbohydrates and glycocon-
and Biotechnological Advances in Industrial Polysaccha- jugates. Curr. Opin. Struct. Biol. 1993, 3, 667.
rides; Crescenzi, V., Dea, I.C.M., Paoletti, S., Stivala, S.S., 196. Dea, I.C.M.; Morrison, A. Chemistry and interactions of
Sutherland, I.W., Eds.; Gordon and Breach: New York, seed galactomannans. Adv. Carbohydr. Chem. Biochem.
1989; 469 pp. 1975, 31, 241.
178. Chien, Y.Y.; Winter, W.T. Accurate lattice constants for 197. Turquois, T.; Rochas, C.; Taravel, F.R.; Doublier, J.L.;
tara gum. Macromolecules 1985, 18, 1357. Axelos, M.A.V. Small-angle X-ray scattering of n-carra-
179. Flory, P.J. Principles of Polymer Chemistry; Cornell geenan based systems: sols, gels, and blends with carob
University Press: Ithaca, NY, 1953; 602 pp. galactomannan. Biopolymers 1995, 36, 559.
12
Interactions Between Polysaccharides and Polypeptides
Delphine Magnin and Severian Dumitriu
Sherbrooke University, Sherbrooke, Quebec, Canada
I. INTRODUCTION Since 1996, many studies have been carried out to

understand the structure and the role of heparin and
The interactions between polysaccharides and polypepti- heparan sulfate in broblast growth factor signaling [90].
des have been investigating since the end of the 19th century Fibroblast growth factors are proteins with a broad range
with the mixing behavior of starch and gelatin [1,2]. of biological activities. Heparan sulfate interacts with
Their signicance is notorious in many elds: biology broblast growth factor and broblast growth factor re-
[3], biotechnology [417], pharmaceutical sector [1824], ceptor by a ternary association on the surface of the cell.
medical applications [20,23,2537], food industry [22,38 Heparin sulfate regulates the transduction of broblast
51], cosmetics [22,5255], tissue engineering [5665], agri- growth factor signal and probably regulates the activity of
culture [66], and environment [6770]. several other signaling factors [8489]. Fig. 1 shows an
The key consideration is that under specic condi- example of the interaction between heparin and broblast
tions, polysaccharidepolypeptide hybrids enhance func- growth factor.
tional properties in comparison to polysaccharides and Hyaluronan is a polysaccharide with diversied bio-
polypeptides alone [52]. logical activities. It is a vital structural component of
In the nature, it is possible to nd many examples of connective tissues; it forms loose hydrated matrices that
interactions between polysaccharides and polypeptides. allow the division and the migration of cells, the regulation
Polysaccharides, most abundant in the natural peptido- of immune cell adhesion, and the activation of intracellular
glycans (or muccopolysaccharide), are heparin, heparan signaling. Moreover, there are large number of hyalu-
sulfate, keratan sulfate, chondroitin sulfate, and hyal- ronan-binding proteins (called hyaladherins) that exhibit
uronic acid [71]. Ongoing studies on peptidoglycans have signicant dierences in their tissue expression, cellular
highlighted the important functions in many biological localization, specicity, anity, and regulation [91,92]. In
mechanisms: starting with fertilization, extending to pa- some cases, the link between hyaluronan and proteins is
thologies, and mediating diverse cellular properties or/ covalent [77]. Kahmann et al. [93] have investigated the
and activities (apoptosis, growth control, and cell recog- hyaluronan binding site on the TSG-6 and have found that
nition) [7280]. Table 1 shows some examples of natural the interactions were possible by the formation of ionic
peptidoglycans. bonds.
In 1999, Berneld et al. [81] have presented a very
interesting review of interactions between heparin/heparan
sulfate and polypeptide. Table 2 shows some examples of II. INTERACTIONS AND PHYSICOCHEMICAL
these interactions [81]. Heparan sulfate proteoglycans are
PARAMETERS INVOLVED IN THE
very important in animal tissues and play an important role
FORMATION OF POLYSACCHARIDE
in many phenomena such as control of cellular growth,
dierentiation, organogenesis, bone formation, and pro- POLYPEPTIDE COMPLEXES
cess of malignancy [81,82]. A. Types of Interactions Between Polysaccharides
Faham et al. [83] were the rst to show the structure and Polypeptides
and interactions between heparin and broblast growth
factors. These interactions have been studied due to the When polysaccharides are mixed with polypeptides, many
importance of complex between heparin and broblast phenomena are possible. Fig. 2 shows these phenomena.
growth factors [8489]. Attractions and repulsions are the two most important
305
306 Magnin and Dumitriu
Table 1 Natural Peptidoglycans (Nonexhaustive List)
Polysaccharide Origin Peptide Reference
Heparan sulfate Bovine liver Heparin lyase I and II [75]

Heparan sulfate Crude porcine [76]
intestinal mucosal
Hyaluronan Extracellular matrix of SHAP protein [77]
dermal broblasts
of mouse
Polysaccharide K Mycelia culture [78]
from Coriolus versicolor
Alternating h-1-4 linked Vegetative cell of Tripeptide and [79]
N-acetyl glucosamine and Bacillus subtilis tripeptidetetrapeptide
N-acetyl muramic acid
Alternating h-1-4 linked Bacterial endospore Pentapeptide and [80]
N-acetyl glucosamine and tetrapeptidetetrapeptide
N-acetyl muramic acid
types of interactions between polysaccharides and poly- ical situation in view of the nature of polysaccharides and
peptides since they lead to immiscibility or complexation polypeptides and due to the presence of many functional
[57]. groups on the molecular structure of these biopolymers
If a polysaccharide and a polypeptide are soluble in the [95].
same aqueous solution, it is possible to have a homogenous The interactions can be incompatible (Fig. 2b). In this
mixing (Fig. 2a) and the more so at higher temperature [94]. case, there is a segregation between polysaccharides and
Such homogeneous noninteractive mixing is the least typ- polypeptides. In the past decade, the study of the phase
Table 2 Proteins of Cellular Microenvironment Bound by Heparin/Heparan Sulfate (Nonexhaustive List)
Morphogenesis and tissue repair Host defense
Morphogens Coagulation Anti-angiogenic factors

Activin Antithrombin III Angiostatin
BMP-2,4 Factor Xa Endostatin
Chordin Leuserpin Cell adhesion molecules
Frizzled-related peptides Tissue factor pathway inhibitor L-selectin
Sonic hedgehog Thrombin MAC-1
Sprouty peptides Growth factors N-CAM
Wnts (113) EGF family PECAM-1
Extracellular matrix components Amphiregulin Chemokines
Fibrin Betacellulin CC
Fibronectin Heparin-binding EGF CC
Interstitial collagens Neuregulin Cytokines
Laminins FGFs (115) IL-2,-3,-4,-5,-7,-12
Pleiotropin (HB-GAM) IGF-II GM-CSF
Tenascin PDGF-AA Interferon-g
Thrombospondin TGF-h1,2 TNF-a
Vibronectin VEGF-165,189 EC superoxide dismutase pro-
Tissue remodeling factors Growth factor binding proteins (BP) arg-rich antimicrobial peptides
Tissue plasminogen activator Follistatin Bac-5,-7
Plasminogen activator inhibitor IGF BP-3,-5 Pr-39
Protease nexin I TGF-h BP Energy metabolism
Underline proteinases Agouti-related protein
Neutrophil elastase ApoB, ApoE
Cathepsin Lipoprotein lipase
Triglyceride lipases
Source: Ref. 81.
Polysaccharides and Polypeptides 307
Figure 1 Interactions between heparin and polypeptide. (a) Stereo view of the linkage between heparin derivated hexasaccharide
to broblast growth factor-2. (From Ref. 83.) (b) Stereo view detailing the side chain-mediated interactions between heparin
derivated hexasaccharide to broblast growth factor-2. (From Ref. 85.)
behavior of aqueous polypeptidepolysaccharide solutions

has been given great attention. Mixtures of polysaccharides
and globular polypeptides have been found to segregate
into two liquid phases: one rich on polypeptides and the
other rich in polysaccharide [95,96]. In a recent paper,
Wang et al. [97] have studied combinations of polypeptides
and polysaccharides mixing (h-lactoglobulin/pullulan and
a-lactalbumin/pullulan). The main conclusion is that
phase separations between polypeptides and polysaccha-
rides are compatible with the theory of depletion interac-
tions [97].
Glycoproteins are heteroproteins resulting from the
covalent interaction (Fig. 2c) between a glucidic fraction
and a polypeptidic fraction. The glucidic fraction must be
equivalent to 5% to have a glycoprotein. In a few cases, this
percentage is near 40%. The glycoprotein class is divided Figure 2 Interactions between polysaccharides and poly-
into two subfamilies. The glycopeptides are macromole- peptides.
Table 3 Noncovalent Networks Involving Polysaccharides and Polypeptides

(Nonexhaustive List)
Polysaccharide Peptide Reference
Sodium alginate Gelatin [107]

Alginate Proteins from abattoir euent [108]
Alginate Proteins from whey [108]
Alginate Bovine serum albumin [108]
Pectate Bovine serum albumin [108]
Carboxymethylcellulose Bovine serum albumin [108]
Gellan gum Gelatin [109]
Chitosan Collagen [103]
cules with small glucidic fractions linked to polypeptidic entrapped networks involving polysaccharides and poly-
structure, while the peptidoglycans are macromolecules peptides [5,6,99,110119].
with peptidic fractions linked to a polysaccharide struc-
ture. Several conjugate vaccines are glycoproteins. The
group C meningococcal polysaccharide-tetanus toxoid is B. Links Between Polysaccharides
an example [98]. and Polypeptides
The formation of insoluble complexes (networks) is
1. Covalent Bonds
also possible between polysaccharides and polypeptides.
Three types of networks can be realized: entrapped net- Covalent bonds are stable links that confer an irreversible
work (Fig. 2d) [99], covalent network (Fig. 2e) [100102], behavior at polysaccharide and polypeptide complexes
and noncovalent network (Fig. 2f) [103,104]. For example, [120]. Fig. 3 shows the four types of interactions [121].
the interaction between propylene glycol alginate and The most common interaction is the chemical reaction
gelatin is a covalent network [105,106]. The linkages are between amino groups from polypeptides and carboxylic
amide bonds involving carboxyl groups of alginate and groups from the polysaccharides (Fig. 3d).
amino groups of gelatin.
Table 3 shows some examples of noncovalent net- 2. Hydrogen Bonds
works involving polysaccharides and polypeptides An example of this type of interaction is the formation of
[103,107109], whereas Table 4 shows some examples of coacervates between gelatin and pectin [102]. In this study,
Table 4 Entrapped Networks Involving Polysaccharides and Polypeptides

(Nonexhaustive List)
Chitosan and xanthan Lipase [6]

Chitosan and xanthan Protease [5]
Chitosan and alginate Interleukin-2 [99]
Alginate and xanthan Urease [110]
Alginate Immunoglobulin G [111,112]
Alginate Fibrinogen [111]
Alginate Insulin [113]
Alginate Endothelial cell growth factor [114]
Alginate Fibroblast growth factor [115]
Alginate Gamma globulin [111]
Alginate nerve growth factor [116]
Alginate Ovalbumin [117]
Alginate CD40L [118]
Alginate Tumor necrosis factor receptor [119]
Alginate Interleukin-1 receptor [119]
Alginate Interleukin-4 receptor [119]
Alginate Granulocyte macrophage colony [119]
stimulating factor
static interactions between an enzyme, the h-galactosidase,

and a polyelectrolyte is shown [131].
5. Repulsive Interactions
Contrary to other interactions, repulsive interactions can
exist. Repulsive interactions can be found in the mixtures
between nonionic polysaccharides and polypeptides for
example. The rst incompatibility between gelatin and
potato starch was reported in 1910 [2]. Polyakov et al.
[96] and Tolstoguvoz [95] report many other incompatibil-
ities.
C. Thermodynamic Considerations in the

Interactions Between Polysaccharides
and Polypeptides
1. Solubility of Macromolecules in a Solvent
Figure 3 Four types of interactions between polysaccha-
rides and amino acid. (From Ref. 121.) The solubility of polymers in solvent have been described
by a thermodynamic model. In 1942, Flory [132] and
Huggins [133] have developed in the same time a model
where the net interaction energy between the solvent and
the complex between gelatin and pectin can be obtained the macromolecule segment is expressed by the Flory
over a wide range of pH, including 4.8, the isoelectric point Huggins parameter v. v takes dierent values; it is equal
of gelatin. Moreover, when the degree of esterication of to zero for a polymer in its ideal solvent. The interactional
pectin increases, the compatibility of pectin and gelatin also parameter, v12, between the solvent and the polymer is
increases. More recent study conrms this phenomenon for dened by Eq. (1) as described by Flory [132] and Huggins
two systems: gelatinpectin and gelatinalginate [122]. [133].
Another example of hydrogen bonds between polysaccha-
ride and polypeptide is the interactions between chitosan v12 zDw12 r1 =kT 1
and collagen [123125]. These interactions are very strong where z is the lattice coordination number, Dw12 is the
and modify the conformational structure of collagen [125]. energy of interaction of the system linked with the creation
of a new contact of type (12) in the mixture between 1
3. Hydrophobic Interactions (solvent) and 2 (macromolecules), r1 is the number of
segments contained by the whole molecule of component
Hydrophobic interactions are a very important group of
1, k is the Boltzmann constant, and T is the temperature.
force that interact in the formation of complexes between
The energy of interaction of the system can be dened
polysaccharides and polypeptides. These forces are entro-
by Eq. (2)
py-driven and can be promoted by the increasing temper-
ature, by conformational and structural modications of Dw12 2w12 w11 w22 2
polymers. Such modications allow to make some con-
nections between polysaccharides and polypeptides, but where wij is the energy of interaction between i and j.
also, the solvent is an important parameter in the exposure
of hydrophobic polymer portions [107,120,126,127].
4. Electrostatic Interactions
In aqueous solutions, the interactions between polysac-
charides and polypeptides take place between opposite
charges of polymers. The rst cause of electrostatic inter-
actions is a decrease of electrostatic free energy. The loss of
entropy of rigid polymers on the formation of interaction
may be compensated by the enthalpy contribution arising
from the interactions between opposite polyions and the
liberation of counterions and water molecules [126,128].
The rst electrostatic interactions between gelatin and
acacia gum were reported in 1911 by Tiebackx [129]. More
recently, Ambjerb Pedersen and Jorgensen [130] have Figure 4 Precipitation resulting in a matrix formation for
described electrostatic interactions in a system between the polyelectrolyte complexation of multimeric enzyme by
casein and pectin. In Fig. 4, a schematic process of electro- electrostatic interactions. (From Ref. 131.)
Three important eects interact in the stability of tion, integrated in the FloryHuggins parameter. Scott has
mixing between polymers and solvent: the combinatorial dened the critical values of the Flory parameter [deriva-
entropy of mixing, the interactional contribution, and the tive of Eq. (6) at zero] as follows:
free volume eect. Schmitt et al. [134] have detailed well h i2
these factors. 1=2 1=2
v12 =V1 crit 1=2 1=V1 1=V2 7
The Combinatorial Entropy of Mixing
The rst eect is the combinatorial entropy of mixing The critical value of FloryHuggins parameter can be
[135], DSmix. It represents the number of possible permu- equal to 1/2 when there are no predominant interactions
tations of molecules in the mixing. Eq. (3) gives the relation between solvent/solvent, and solvent/polymer, and poly-
between DSmix, the gas constant R, the molar volume of the mer/polymer. In a good solvent, the critical value of Flory
component, and the volume fraction of component u (1 Huggins parameter is lesser than 1/2 and, inversely, in a
and 2 represent the solvent and the macromolecule, respec- poor solvent, it is greater than 1/2.
tively)
2. Mixing Between Polysaccharides and Polypeptides
DSmix =RV u1 lnu1 =V1 u2 lnu2 =V2 3
In a mixture containing polysaccharides, polypeptides, and
Interactional Contribution solvent, the FloryHuggins model may be valid to analyze
The second eect is due to the interactional contribu- the possible interactions. The FloryHuggins theory, using
tion (or heat mixing), DHmix. Repulsive and attractive the chemical potential equality, allows to make the illus-
forces allow the formation or nonformation of mixture. tration of interactions in a ternary mixture [138,139]. Fig. 5
Sperling [136] has dened the interactional contribution by shows these schematic interactions in a mixture containing
Eq. (4). two polymers and a solvent [139]. In this case, three
parameters of FloryHuggins should be used to determine
DHmix Vm d1 d2 2 u1 u2 a thermodynamic model: v12 dening the interactions
between one biopolymer and the solvent, v13 dening
and the interactions between the other biopolymer and the
solvent, and v23 dening the interactions between the two
di DEi =Vi 1=2 4 biopolymers.
v23 is a more important parameter to control the sta-
with Vm the total volume of the mixture, di the solubility bility of the ternary mixture because of the size of macro-
parameter, DEi the vaporization energy to a gas at zero molecules compared to that of the size of solvent. In a
pressure, and DEi/Vi represents the cohesive energy study, Dickinson [140] has shown that v23 determines the
density. conditions for the phase separation in a ternary mixture.
The interactional contribution can be written by using Table 5 resumes the interactions between polysaccha-
the FloryHuggins parameter [Eq. (5)]. rides and polypeptide [95,107,128,141145] and some ex-
amples of interactions [146158].
DHmix =RTV zDw12 =kTVs u1 u2
5 3. Phenomenon of Coacervation
v12 =V1 u1 u2
Bungenderg De Jong [144] was the rst to show the
where Vs is the molar volume of a segment. phenomenon of coacervation. A coacervate is a complex
By using Eqs. (3) and (5) in the second principle of that remained liquid rather than precipitated. This phe-
thermodynamics, Eq. (6) is obtained. nomenon can be reversible or irreversible. It occurs often in
the mixtures between two polymers with opposite charges
DG=RTV u1 lnu1 =V1 such as polysaccharides and polypeptides. There are two
6 types of coacervation: simple coacervation and complex
u2 lnu2 =V2 v12 =V1 u1 u2
coacervation. The simple coacervation is not developed in
this chapter because it results from a mixing of one polymer
In Eq. (6), the two rst terms can be negligible due to
with a poor solvent (v12 > 1/2) [159].
the high molecular weight of polymers. In fact, V1 and V2
In a complex coacervation, the mixture is composed of
tend to innity in the case of macromolecules. So the
a solvent and two or more polymers. Bungenderg De Jong
FloryHuggins parameter becomes the governing term
[144,160] has observed this phenomenon with a mixing
for the mixing solubility and stability. Moreover, v12/V1
between gelatin and acacia gum. In this case, the charges of
is a function of 1/T.
biopolymers are the main parameter that inuences the
The Free Volume Eect formation of this coacervate because the pH and the ionic
The last important eect for the stability of mixture is strength values inuence very well the properties of this
the free volume eect. The free volume eect is the dier- coacervate [161].
ence between the nal volume of mixture and the sum of Four big theoretical thermodynamic models exist to
volumes of solvent and macromolecules. It is, by conven- describe the phenomenon of complex coacervation. They
Figure 5 Schematic illustrations of interactions in a mixture containing two polymers and a solvent. The chemical potentials
equality based on the FloryHuggins theory allows to predict experimental tie lines. (From Ref. 139.)
allow to understand the fundamental theory of the phase of the VoornOverbeek theory and the VeisAranyi theory,
separation and to explain the stability of coacervates. respectively.
The VoornOverbeek theory [162167] and the Veis Table 6 resumes the two rst thermodynamic theories:
Aranyi theory [168] are two dierent views of theoretical the system that was studied, the process, the interaction,
thermodynamic modeling. Moreover, the NakajimaSato the thermodynamic mechanism, and the other details
theory [169] and Tainaka theory [170,171] are an extension [167,168,172,173].
Table 5 Resume of Interactions Between Polysaccharides and Polypeptides
FloryHuggins
parameter Interactions Reference Examples
v23 > 0 Thermodynamic incompatibility [107] BSA and dextran or

Segregative phase separation [95] g-globulin and dextran or
Net repulsion between the two [128] lysozyme and dextran [146,147]
polymers [141] Whey protein and neutral
Interactions solventpolymer are [142] polysaccharides (pH 5 at 7) [148]
higher than interactions [143] BSA and carboxymethylcellulose
polymerpolymer and at pH 5.3 [149]
solventsolvent Gelatin and dextran [150]
Two phases (polymer1solvent Locust bean gum and dextran [151]
and polymer2solvent) and
thermodynamic equilibrium
of two phases
v23<0 Thermodynamic compatibility [128] Ovalbumin and dextran sulfate [152]
Associative phase separation [144] BSA and j-carrageenan [153]
Coacervation phenomenon [145] Gelatin and chitosan [154]
Two phases (coacervate and solvent) h-Lactoglobulin and
and thermodynamic equilibrium gum arabic [155]
of two phases h-Lactoglobulin and pectin [156]
Whey proteins and pectin [157]
Milk protein and xanthan [158]
Table 6 Resume of the VoornOverbeek Theory and the VeisAranyi Theory
VoornOverbeek theory [167] VeisAranyi theory [168]
Year 1957 1960

System Gelatin and acacia gum Oppositely charged gelatins
[144] where r3r z 0.53 where r3r < 0.53
Process One step Two steps
Types of interactions Electrostatic interactions form a First, interaction between opposite
coacervate that entraps solvent charges and then rearranging and
molecules. electrostatic interactions and
formation of aggregates
Duration of process Spontaneous 1 hr to 1 day
Thermodynamic Solvent increases entropy of system, Increase of congurational
mechanism so a number of arrangement of entropy through the formation of a
macromolecules is possible. randomly mixed concentrated
coacervate phase and the dilution
of aggregate phase
Equations DebyeHuckel for electrical Function of polymer charge density
interactions [172,173] and polymer volume fraction
FloryHuggins theory of the Huggins interactions using v12
liquid quasilattice
where r is the charge density and r is the molecular weight.
Table 7 is an extension of Table 6 and it presents the 1. Chemical Parameters

two derivative models: the NakajimaSato theory and
Role of pH
Tainaka theory. In Table 7, the base and modications of
the NakajimaSato theory and Tainaka theory and some The inuence of pH is due to the eect on the
examples are presented [169171,174,175]. ionization degree of the functional groups carried by the
However, none of these four theories present a general biomacromolecules. For polypeptides, amino groups and
model of all biomacromolecule interactions and associative carboxylic groups can play a role in the formation of
phase (coacervate). complexes. For polysaccharides, the diversication of
functional groups are more important (amino group, car-
boxylic group, sulfur group, etc.). Due to the inuence of
D. Parameters that Influence the Formation pH, the maximum of yield for a complexation between
of Interactions Between Polysaccharides polypeptides and anionic polysaccharide is obtained for a
and Polypeptides pH that is below the isoelectric point of polypeptides and
more precisely for the electric equivalence pH. At the
Two types of parameters inuence the formation of inter- electric equivalence pH, polypeptides and polysaccharides
actions between polysaccharides and polypeptides: chem- are exactly oppositely charged, so there is a maximum of
ical parameters including pH, molecular weight, ionic electrostatic charges [161].
strength, polymer concentration, and ratio between bio- Burgess and Carless [176] have shown that the maxi-
polymers and other factors such as temperature and shear- mum yield for a coacervation phenomenon is obtained for
ing conditions. a pH near 4 for two systems: acacia gum and type A gelatin
Table 7 Resume of the NakajimaSato Theory and the Tainaka Theory
NakajimaSato theory [169] Tainaka theory [170,171]
Year 1972 1980

System Synthetic polyion oppositely Polyanionpolycation
charged pair of PVA Gelatin/acacia gum [174]
where r3r=0.025 Albumin/acacia gum [175]
Base of this model VoornOverbeek theory VeisAranyi theory
Modication Huggins interaction parameter v12 Without specic charge pairing
Uniform charges distribution Virial coecient expansion
in the two phases procedure for the two phases
and acacia gum and type B gelatin, even if the isoelectric

point is very dierent (8.3 for type A and 4.8 for type B). In
this case, it is the electric equivalence pH of acacia gum
(3.9) that plays an important role [176].
In a more recent study, Velings and Mestdagh [177]
have shown the inuence of pH on the complexation
between alginate gel beads and cytochrome c or myoglo-
bin. Fig. 6 shows the inuence of pH [177] (in this gure, N
represents the number of moles of proteins per bead). The
quantity (number of mole) of proteins absorbed on the
alginate beads is inuenced by pH. The quantity of myo-
globin was often lower than that of cytochrome c. In Fig. 6,
the ratio between the amount of protein adsorbed to the
amount of maximum adsorption vs. pH is presented. This
ratio increases when the pH increases from 2 to 6 and also
after it decreases. The maximum adsorption is near the
isoelectric point of the protein.
Role of Molecular Weight
Overbeek and Voorn [167] have proposed a theoretical
thermodynamic model, where the molecular weight is an
important factor. The increase of molecular weight of the
biopolymers expects to lower compatibility of the biopoly-
mer into the solution because the combinatorial entropy
mixing is lower. In this case, these authors have concluded
that the increase of molecular weight increases the forma-
tion of coacervate.
Only few studies are reported on the importance of
molecular weight of polysaccharide for the formation of
coacervate between polysaccharides and polypeptides. A
study involving 11S soy globulin and dextran shows the
eect of the molecular weight of dextran on the formation
of coacervate [178]. The light scattering has been used in
Figure 7 Yield in the precipitate of large (o) and small (.)
order to increase the molecular weight of dextran. So the subunits of RDPhC and pectin (E) upon acid titration of a
complexation between 11S soy globulin and dextran is 0.05% solution of the protein (ac) and its mixtures with
favored. Authors explain this phenomenon by an increase pectin (dh) vs. pH, charge density, and ionic strength of the
solution. (a, d, and h: 0.002 M NaCl; b: 0.1 M NaCl; c: 0.5 M
NaCl). Pectin concentration (d: 0.01%; e: 0.05%; f: 0.25%),
as well as degree of pectin esterication (df: DE=70%; h:
DE=90%; g: DE=0%). (From Ref. 95.)
of the space lled by dextran in solution; therefore it is

more accessible for 11S soy globulin.
Role of Ionic Strength
Burgess [174] has shown the role of ionic strength for
the system between acacia gum and gelatin. The coacerva-
tion has been optimized for a salt concentration of 10 mM.
At higher concentration, the yield of coacervation
decreases due to an interaction between acacia gum and
salts. Moreover, at lower concentration, the yield of coac-
ervation decreases also. This phenomenon cannot be
explained by the combinatorial entropy model. Authors
propose a hypothesis to explain this one: the macromole-
cules are in extended congurations due to the high charge
Figure 6 Relative amount of protein adsorbed per alginate of biopolymers, then only small aggregates are formed
gel bead as a function of pH: (.) cytochrome c and (n) [174].
myoglobin. N is number of mole of proteins adsorbed. (From In the study of Tolstoguvoz [95], the formation of
Ref. 177.) complexes between pectins (with esterication degree be-
tween 0% and 100%) and ribulose diphosphate carboxyl-

ase (RDPhC) has been investigated. Fig. 7 shows the
inuence of ionic strength [95]. In Fig. 7df, pectins allow
the precipitation of RDPhC. At high ionic strengths (0.5M
NaCl) in Fig. 7g, the complexes break down and the
protein undergoes precipitation at the isoelectric point
and contains no pectin [95].
In another study involving alginate, cytochrome c, and
myoglobin, the eect of NaCl concentration is investigated
[177]. In this study, the pH is xed at 7.5 for adsorption of
cytochrome c and at 6.2 for myoglobin (Fig. 8). Cyto-
chrome c adsorption is a reversible phenomenon because
the absorption of cytochrome c is inhibited by 0.2 M of
NaCl. For myoglobin, the adsorption is very reduced
(about 25%) for 0.2 M NaCl.
Role of Biopolymer Charge Density
The biopolymer charge density is the number of
charges on the chain of biopolymer per unit length. The
coacervation phenomenon is not possible under a critical
biopolymer charge density.
In the study of Tolstoguvoz [95], the formation of
complexes between pectins (with esterication degree
between 0% and 100%) and ribulose diphosphate car-
boxylase (RDPhC) has been investigated. Fig. 7 shows
the inuence of charge density [95]. At higher esterica-
tion degree of pectins (smaller net charge on the poly-
anion), its relative content in insoluble complexes does
not increase, as is the case with electrostatic complexing.
Inversely, the pectin content goes down monotonically
until it is completely absent from the precipitate at near Figure 9 The eect of biopolymer ratio on the formation of
100% (Fig. 7h). coacervates of 0.1% (w/w) h-lactoglobulin/acacia gum
Role of the Concentration of Biopolymer mixtures. (From Ref. 134.)
When the concentration of the two biopolymers are
high, the coacervation yields are low. The thermodynamic
incompatibility is important due to the competition be- In the study of Tolstoguvoz [95], the formation of
tween macromolecules for the solvent [107,126]. complexes between pectins (with esterication degree be-
tween 0% and 100%) and ribulose diphosphate carboxyl-
ase (RDPhC) has been tested for many factors. Fig. 7
shows the inuence of the concentration of pectin (Fig.
7df) [95].
Role of the Ratio Between Biopolymers
A maximum coacervation yield is obtained for a
specic ratio for each system. Schmitt et al. [134] have
shown that the specic ratio for the formation of complexes
between acacia gum and h-lactoglobulin is 1:4.
Fig. 9 shows the inuence of the ratio between bio-
polymers. The optical density is proportional at the quan-
tity of coacervate formed: in fact, the higher the quantity of
coacervate, the higher the optical density is. In Fig. 9, the
optimal ratio between biopolymers is 4. An increase of the
ratio polypeptide/polysaccharide decreases the coacerva-
tion [134].
2. Physical Parameters
Figure 8 Relative amount of protein adsorbed per alginate
gel bead as a function of NaCl concentration: (.) cyto- Role of Temperature
chrome c and (n) myoglobin. N is number of mole of The temperature plays a key role in the formation of
proteins adsorbed. (From Ref. 177.) complexes between polysaccharides and polypeptides.
at 750 bars in a microuidization chamber has allowed a

decrease of the size of brous complexes (0.2 mm compared
to 20 mm for a nontreated complex). He explained this
decrease by a disruption of the protein aggregates and by a
fragmentation of the xanthan molecules. This treatment
combines high pressure with turbulence, cavitation, and
shearing phenomena [182].
The second one is a high hydrostatic pressure into a
vacuum chamber. Dickinson and Pawlowsky [184] have
worked with a complex composed by BSA and dextran
sulfate. They have measured the shear viscosity of com-
plexes where the BSA is high-pressure-treated or not
treated. Dispersions containing BSA treated at 3000 MPa
exhibited no variation in the shear viscosity in comparison
with nontreated BSA. This phenomenon is due to the
partial denaturation of BSA by the high-pressure treat-
ment. This treatment increases the formation of stronger
Figure 10 Schematic representation of the eect of high-
electrostatic interactions between BSA and dextran sulfate.
pressure processing on the electrostatic polysaccharides
polypeptides complex. Application of pressure leads to the Moreover, the increase of the viscoelastic modulus is in
dissociation of the biopolymers; the globular protein unfolds accordance with the formation of electrostatic interactions
and exposes more changed groups during maintenance of [40,183185].
pressure-unfolded BSA with dextran sulfate reforms during Fig. 10 illustrates the schematic eect of high pressure
pressure release. (From Ref. 186.) processing on the electrostatic interactions between poly-
saccharides and polypeptides [186].
Galazka et al. [186] have shown the eect of high
pressure on the surface hydrophobicity So (Table 8), the
First, the temperature interacts on the FloryHuggins endothermic peak temperature Tm, and the total calori-
parameter as shown in Eq. (1); when the temperature metric enthalpy DH (Table 9) of BSA and BSA-carrageen-
increases, the v12 decreases and the associative phase an. High-pressure processing allows to decrease the protein
separation is easier [132,133]. Moreover, the temperature surface hydrophobicity and the presence of carrageenan
inuences the formation of bonds. Hydrophobic interac- allows a further decrease. The pressure and carrageenan
tions and covalent bondings are enhanced when the tem- allow to decrease also the endothermic peak temperature
perature increases. The increase of temperature allows and total calorimetric enthalpy [186].
conformational changes of polysaccharide structures and
thermal denaturation of globular protein (so a conforma- 3. Mechanical Parameters
tional change of polypeptide structures). On the contrary, Role of Shearing Rate
the decrease of temperature enhances the formation of
In the literature, few studies have reported the role of
hydrophobic and hydrogen bonds [179].
this parameter [126]. Tirkkonen et al. [187] have shown the
Harding et al. [180] have shown the eect of temper-
role of the shearing rate. Microcapsules of a complex,
ature on the formation of complexes between BSA and
involving acacia gum and gelatin, have been studied. The
alginate. The mixture of BSA and alginate has received a
shearing rate inuences the diameter of microcapsules: the
thermic treatment (from 25jC to 90jC). The apparent
molecular weight of the complex has been determined
according to the Svedberg equation [181]. At 25jC, com-
plexes have been found with a MWapp of 2 106 Da, while
between 35jC and 70jC, MWapp was equal to 0.2 106 Da Table 8 Inuence of High Pressure on the Surface
(this value corresponds at the MW of free alginate) so no Hydrophobicity
complex has been found. However, between 70jC and
90jC, complexes have been found. At 55jC, BSA is Treatment So (absorbance
Sample pressure units/mol)
thermally denaturated, resulting in the exposure of more
reactive sites that can interact with alginate [180]. BSA 0 275
Role of Pressure BSA 600 126
High pressures can play an important role on the BSA:n-carrageenan (2.5:1) 0 148
BSA:n-carrageenan (2.5:1) 600 85
formation of complexes between polysaccharides and poly-
BSA:L-carrageenan (5:1) 0 157
peptides. In the literature, two types of high-pressure
BSA:L-carrageenan (5:1) 600 98
treatments are found [40,134,182186]. BSA:L-carrageenan (2.5:1) 0 67
The rst one is a microuidization treatment into a BSA:L-carrageenan (2.5:1) 600 35
microuidization chamber. Le Hena has shown that a
treatment of a complex between xanthan and whey protein Source: Ref. 186.
Table 9 Inuence of High Pressure on the Endothermic Peak Temperature Tm and

Total Calorimetric Enthalpy DH
Treatment
Sample pressure Tm (jC) DH (kcal/mol)
BSA 0 70.5 17.5 104

BSA 600 70.5 9.7 104
BSA:n-carrageenan 0 69.0 14.1 104
BSA:n-carrageenan 600 69.9 7.3 104
BSA:L-carrageenan 0 68.5 13.3 104
BSA:L-carrageenan 600 69.5 3.1 104
n-Carrageenan or 600 0 0
L-carrageenan
Source: Ref. 186.
mean diameter was 2.2 Am at 420 rpm while it was 3.1 Am at coacervate could be controlled by the shearing time [134].
200 rpm. The relation between shearing rate and mean Fig. 11 shows the inuence of shearing time [134].
diameter is inversely proportional [187].
Role of Shearing Time
III. COMPLEXES INVOLVING
Schmitt et al. [134] have shown that the yield of POLYSACCHARIDES AND POLYPEPTIDES:
coacervation is a function of the time of shearing. The PROPERTIES AND STRUCTURAL
higher the duration of shearing, the higher the yield is.
CONFORMATION
Moreover, they have concluded that the size of particles of
A. Interactions Between Alginate
and Polypeptides
1. Alginate Beads: Inclusion of Polypeptide
or Adsorption of Polypeptide
Inclusion of Polypeptide
Two methods have been used in order to include
polypeptide into alginate gel: the rst one is based on
alginate beads that are prepared by an extruding meth-
odthe alginate solution, containing the polypeptides, is
added dropwise into a cationic divalent cross-linking solu-
tion [119]. The second method based on the preparation of
microbead involved polypeptide encapsulation with an
emulsion technique [116].
The release of polypeptides that are included into
alginate bead can be induced by two processes: the diu-
sion through the network pores or/and erosion (degrada-
tion) of network [119]. In a study of 1991, Martinsen et al.
[188] have shown that the molecular weight of alginate and
the alginate concentration can inuence the release rate of
large polypeptide, while it has no inuence for small
molecule [188]. Moreover, the composition of alginate
has an inuence on the release: when alginate with high
a-L-guluronic acid is used, the porosity of network is higher
and therefore the release rate is higher [188].
Adsorption of Polypeptide
Adsorption of cytochrome c and myoglobin on algi-
nate gel beads has been studied [177]. The gelation of
alginate has been inducted by calcium. The charge of
proteins is governed by the isoelectric point. Below this
Figure 11 The eect of shearing time on the coacervation of isoelectric point, they are positive; at the isoelectric point,
0.1% (w/w) h-lactoglobulin/acacia gum mixtures, pH 4.0. the charge is null, and above this isoelectric point, they are
(From Ref. 134.) negative. Alginate is always negative; therefore proteins
should be positive. At pH 2, alginate is uncharged due to a Ala-Ala), which is characterized by a charge density one-
complete protonation. When pH increases, the negative third that of poly(L-lysine). Alginates interacted with
charges increase and at pH 6.5, the alginate is completely poly(Lys-Ala-Ala) rather intensively. The dierence in
deprotonated. The best interaction between alginate and eciency of interaction of L-guluronan and D-galactu-
proteins is between pH 5 and below the isoelectric point. ronan with poly(L-lysine) results from the dierence in
For cytochrome c, the maximum of adsorption is at pH 7.5 the conformational exibility of their polyanionic chains
and for myoglobin, it is at pH 6.2. This result shows that in solution. L-Guluronan maintains the rigid twofold sym-
the interactions between alginate and proteins were elec- metry in solution, and D-galacturonan is conformationally
trostatic charges [177]. Figs. 6 and 8, presented in the adaptable in the course of interaction [196]. Bystricky and
second part of this chapter, show the inuence of pH and Malovikova [197] have presented circular dichroism anal-
ionic strength in the formation of interactions between ysis for complexes involving polyguluronate-rich alginate
alginate, cytochrome c, and myoglobin [177]. Moreover, poly-L-lysine, polyguluronate-rich alginatepoly-D-lysine,
the dierence of the two proteins is certainly due to the polymannuronate-rich alginatepoly-D-lysine, and poly-
dierence of molecular weight or/and isoelectric point mannuronate-rich alginatepoly-L-lysine The conclusion
[177]. is that the poly-L-lysine does not interact with polygulu-
ronate-rich alginate. The interactions between polymannu-
2. Alginate and Gelatin ronate-rich alginate and poly-D-lysine are more eective
Freshly prepared gels of gelatin with alginate or pectate (50%), while the interactions between polymannuronate-
below the isoelectric point of gelatin (e.g., at pH 3.9) melt rich alginate and poly-L-lysine are only 20% [197].
over the same temperature range as gelatin (3040jC), but
on aging, they become thermostable. The gels are also B. Interactions Between Carboxymethylcellulose
stable in 7 M urea, arguing against hydrogen bonding or and Polypeptides
hydrophobic interactions, but the enhanced thermal sta-
bility can be eliminated by high salt (e.g., 0.3 M NaCl) or by 1. Carboxymethylcellulose and Egg White
raising the pH to above the isoelectric point of gelatin, as Proteins and Ovalbumin
expected for an ionic network [105]. The authors who have modeled the various aspects of
precipitation of proteins with carboxymethylcellulose in-
3. Alginate and a-Galactosidase clude Clark and Glatz [198,199] and Fisher and Glatz [200].
The versions of the a-galactosidase (an enzyme) possessing In their work, involving the precipitation of egg white
fusion tails would contain multiple strong electrostatic proteins lysozyme and ovalbumin by carboxymethylcellu-
interaction sites. These sites would be capable of forming lose (CMC), Clark and Glatz [198] presented several con-
cross-links between enzymepolyelectrolyte complexes by clusions concerning the eects of pH, polymer dosage, and
the binding of a single enzyme to multiple polyelectrolytes ionic strength on protein recovery and fractionation [131]:
[131]. A schematic of this process is shown in Fig. 4. Only proteins possessing a charge opposite to that of
the polyelectrolyte are precipitated, and those of
4. Structural Conformation of Complexes higher charge density are precipitated preferen-
Involving Alginate and Polylysine tially.
Alginate, a polyanion, can interact with polylysine, a The eciency of precipitation increases with protein
polypeptide formed only by one basic amino acid: lysine. charge.
Many papers deal with complexes involving alginate and Up to an optimal polymer dosage, protein removal
poly-L-lysine [189197]. In these studies, this complex is increases with polyelectrolyte dosage.
used to make articial pancreas. An increase in ionic strength increases the required
The interactions involving alginates with various com- polymer dosage to eect the same protein
positions of basic polypeptides, such as poly(L-lysine) and removal, reduces the maximum possible precipi-
poly(Lys-Ala-Ala), have been studied by circular dichro- tation, and reduces the eect which precipitation
ism. The lower complexation eciency with alginate II pH has on protein recovery.
(MM, 33%, GG, 30%, MG, 37%) than with alginate I Fractional precipitation can be attained by the proper
(MM, 30%, GG, 20%, MG, 50%) and the near-zero adjustment of pH or polymer dosage.
eciency with alginate III (MM, 33%, GG, 47%, MG,
20%) are due to the presence of considerable amounts of Glatz et al. [131,201203] focused on the enhancement
noncomplexing L-guluronate sequences in the alginate of polyelectrolyte precipitation through the genetic fusion
structure. of charged polypeptides. A model is presented for the
On the basis of the results of complexation achieved in polyelectrolyte precipitation of proteins possessing
this study, it may be suggested that the L-guluronan chain is charged fusion tails [131]. A schema of the precipitation
more rigid, i.e., less adaptable to the changes in the of such fusion enzymes by polyelectrolytes is depicted in
surrounding medium than the D-galacturonan chain. The Fig. 12 [131]. The precipitation of ovalbumin and lysozyme
inuence of charge density of a polycation on complexa- by CMC is modeled as a multiple equilibrium binding
tion was studied with the sequentially regular poly(Lys- phenomenon [199]. As shown in Fig. 13 [199] for the
C. Interactions Between Pectate and Polypeptides

The interactions between pectate and polylysine have been
studied by circular dichroism [197]. Figs. 14 and 15 show
circular dichroism spectra of acidic polysaccharides and
polypeptides, poly-L-lysine and potassium pectate com-
plex, and poly-D-lysine and potassium pectate complex,
respectively.
Fig. 14, for interactions between pectate and poly-L-
lysine, shows a regular increase that corresponds to the
increase of amount of component added. In this spectrum,
the authors have demonstrated that the interactions be-
tween pectate and poly- L -lysine have a rapport 1:1
(COO:NH3+). Moreover, the spectrum indicates that
the structure of poly-L-lysine is an a-helix. The complexa-
tion eciency between pectate and poly-L-lysine is 100%
[197].
Fig. 15, for interactions between pectate and poly-D-
lysine, shows a regular arrangement. For poly-D-lysine,
there are small conformational modication caused by
very low complexation eciency. Poly-D-lysine shows
unchanged random coil arrangement. The complexation
eciency between pectate and poly-D-lysine is only 10%
[197].
Figure 12 Schematic of polyelectrolyte precipitation for

enzymes possessing charged fusion polypeptides. (From Ref.
131.)
precipitation of lysozyme by CMC, virtually all CMC is

precipitated for dosages less than the optimum. For pro-
teins with a high surface charge density, the iondipole
interaction dominates, but for the proteins with lower
charge density, hydrophobic considerations become more
important [199].
Figure 14 The CD spectrum of poly-L-lysine in complex

with potassium pectate: (-) pectate with addition of 20% (1),
Figure 13 Lysozyme and carboxymethylcellulose precipita- 40% (2), 60% (3), and 70% (4) poly-L-lysine; (- - -) poly-L-
tion at pH 7.5 and ionic strength 0.07N. D: Lysozyme; o: lysine with addition of 20% (1), 40% (2), 60% (3), and 70%
carboxymethylcellulose. (From Ref. 199.) (4) pectate. (From Ref. 197.)
Figure 16 Eect of pH and heparin/gelatin A ratio on coa-

cervate yield (I=10 mM). (From Ref. 205.)
Many factors indicated that this coacervation ts with

the VeisAranyi and Tainaka models [168,170,171]: coac-
ervation in two steps, aggregate formation, inuence of
temperature, and coacervation intensity increasing with
time [205].
Figure 15 The CD spectrum of poly-D-lysine, in the pres-
ence of potassium pectate: potassium pectate with addition of 2. Interactions Between Heparin and Antithrombin
20% (1), 40% (2), 60% (3), 80% (4), and 100% (5) poly-D-
In 1987, Gettins [206] has compared the structure of three
lysine. (From Ref. 197.)
antithrombin: human, bovine, and porcine antithrombin.
He has compared the structure of free human antithrombin
and its interaction with heparin [206] by 1H NMR spec-
troscopy. This study has shown that the amino composi-
D. Structural Conformation of Complexes tions of the three polypeptides are very similar. The binding
Involving Anionic Polysaccharides, of heparin on antithrombin III has an inuence on the
Myoglobin, and Bovine Serum Albumin structure and this latter can be visible in 1H NMR spec-
troscopy [206].
Imeson et al. [204] have investigated the conformational
structure of two polypeptides (myoglobin and bovine
3. Interactions Between Heparin and
serum albumin) in presence of alginate, pectin, or carboxy-
Fibroblast Growth Factors
methylcellulose. Dierential scanning calorimetry, adsorp-
tion spectrometry, and Sephadex chromatography have Interactions between heparin and broblast growth factors
been used in order to characterize these complexes. The have been studied due to the importance of complex
authors have shown that the dierential scanning calorim- between heparin and broblast growth factors [8490].
etry spectra of polypeptides in presence of polysaccharide
were modied compared to the dierential scanning calo-
rimetry spectra of polypeptides without polysaccharides.
This observation has allowed to conclude that there are
interactions between polypeptides and polysaccharides,
but the authors could not make hypothesis on the structure
of these complexes.
E. Interactions Between Heparin

and Polypeptides
1. Interactions Between Heparin and Gelatin
A complete study involving heparin and gelatin have been
investigated by Tsung and Burgess [205]. The eect of pH
(Fig. 16), ionic strength, time, temperature, and heparin/
gelatin ratio (Figs. 16 and 17) was studied. The optimized
conditions are the following: pH=2.6, ionic strength=10 Figure 17 Eect of heparin/gelatin A ratio on coacervate
mM, and a heparin/gelatin A ratio=1:2. yield (I=10 mM, pH=2.6). (From Ref. 205.)
Fibroblast growth factors are a large family of poly- and a dimer of human native and selenomethionyl acidic
peptides that interact in many biological phenomena due to broblast growth factor using x-ray crystallography. Fig.
their capacity to bind with transmembrane receptors (these 18 shows the structure of heparin-linked acidic broblast
transmembrane FGF receptors are tyrosine kinase) that growth factor (aFGF) dimers. Fig. 18a is electron density
allow to regulate many biological responses [207210]. map at 2.9-A resolution. Fig. 18b is a stereo view and Fig.
These transmembrane FGF receptors should be activated, 18c is the secondary structure.
and this activation requires heparin or heparin derivated As shown in Fig. 18, heparin has a 1C4 helical confor-
[211213]. mation and interacts with aFGF-dimer in two distinct
Many studies have presented crystal structure of com- orientations [89]. It is clear that heparin binds two poly-
plexes involving heparin-like sugar and broblast growth peptide chains: the aFGF is bridged by heparin. This case is
factors [83,89,90,214,215]. In 1998, DiGabriele et al. [89] a model of a polypeptide dimerization via a polysaccha-
have described the crystal structure at 2.9-A resolution of a ride. The linkage between heparin and aFGF is made by
complex formed with fully sulfated heparin decasaccharide sulfate groups of 56 monosaccharide units on opposite
side of helix axis [89] (this axis is not a true symmetric axis
due to the polarity of heparin). In fact, this complex is
assembled around a central heparin molecule linking with
two FGF molecules. This bridge (heparin molecule) is not
rigid and this complex has an appreciable exibility (Fig.
18c).
In another study, Pelligrini et al. [90] have shown the
structure of a ternary complex involving heparin decasac-
charide, human FGFR2: FGF receptor (splice variant IIIc)
and FGF1 with a ratio of 1:2:2. Size-exclusion chromatog-
raphy has allowed to determine the molecular weight of
this complex: 83 600. The study of the structure of this
ternary complex has been made by x-ray crystallography
[90]. Fig. 19a is the electron density map while Fig. 19b is
the summary of interactions, and Fig. 19c and d shows
some details of these interactions.
In Fig. 19a, the total length of heparin decasaccharide
is visualized by experimental electrodensity map. The
authors have shown that there is a distortion in the heparin
helix when it is complexed with FGFR2 and FGF1 com-
pared to the structure of heparin-FGF [83,89]. This distor-
tion makes a 34-A kink between disaccharide 2 and
Figure 18 Structures of heparin-linked aFGF dimers. (a)

This sigma-weighted electron-density map at 2.9-A resolu-
tion in the region of the bound heparin was generated
without the decasaccharide in the model. Blue contour is at
1r and pink contour is at 4r. (b) A stereo view of heparin-
linked dimer A (orthorhombic asymmetric unit) of human
aFGF. The amino and carboxy termini of the protomers, and
the O1 and O4 ends of the sugar, are labeled. The location of
the alpha carbon atom of every 10th residue is shown by a
numbered black circle. (c) The superposition of one aFGF
protomer (A1, B3, and C5) (bottom) from each aFGF dimer
in the orthorhombic asymmetric unit, using program O [320],
shows the variability in positions of the second protomers
(A2, blue, B4, cyan, C6, yellow worms; top) and heparin
chains (Asugar, blue, Bsugar, cyan, Csugar, yellow; center).
Protomer A1 is depicted as a blue ribbon (bottom) showing
labeled secondary structure [321]. The hexagonal asymmetric
unit (H7, H8, and Hsugar are not shown) is most similar to
aFGF dimer A. Rotations and translations that superimpose
the second protomers B4, H8, and C6 on A2 are 16.2j and
0.6 A, 7.5j and 0.4 A, and 16.8j and 1.5 A, respectively.
Prepared with program O (a) and SETOR [322] (b and c).
(From Ref. 89.)
Figure 19 Heparinprotein interactions in the FGF1FGFR2heparin complex. (a) Experimental 3.0-A electron density of the
heparin decasaccharide contoured at 1.2r. (b) Summary of heparinprotein interactions. The ve disaccharides of the heparin
decasaccharide are numbered from the reducing end, indicated by a hydroxyl group. Each disaccharide begins with glucosamine
(G1cN) followed by iduronic acid (IdoA). Solid lines represent interactions between amino acids and monosaccharide(s).
Heparin comes in close contact with the backbone atoms of amino acids marked by an asterisk. (c) Details of the FGF1heparin
interaction in binary complex B. Heparin and amino-acid side chains are shown as a ball-and-stick model and the protein
backbone as a coil. Dashed lines represent hydrogen bonds (yellow), van der Waals contacts (green), and electrostatic
interactions (purple). (d) FGFR2heparin interactions. Only heparin disaccharide 5 is depicted for clarity. Dashed lines colored
as in (c). (From Ref. 90.)
disaccharide 3. The interactions that are present in this ter- contribute substantially to these interactions. Moreover, in
nary complex, as shown in Fig. 19b, are the following [90]: Fig. 19b, it is possible to show that FGF1A interacts with
six monosaccharides while FGF1B interacts with 5 mono-
For the molecule FGF1FGFR2 A, there are three saccharides [90].
interactions: two between FGFR2A and heparin
and one between FGF1A and heparin.
For the molecule FGF1FGFR2 B, there is only one F. Interactions Between Sodium
interaction: between FGF1B and heparin. Chondroitin-6-Sulfate and Polypeptides
The total surface of interactions between FGF1 and Complex formation of bovine serum albumin (BSA) with
heparin is 2240 A with 617 A with domain D1 of FGF1A, sodium chondroitin-6-sulfate (Na2Chs) was studied by
631 A with domain D2 of FGF1A, and 992 A with FGF1B Nakagaki and Sano [217] by means of static light scatter-
[90]. Thompson et al. [216] have characterized the nature of ing. They concluded that the binding of BSA to the
interaction and have shown that the van der Waals forces polymer chain of Na2Chs follows the Langmuir-type bind-
ing isotherm, where the anity between them decreases Fig. 20 shows ternary phase diagram of BLG/AG/W
with increases in pH and ionic strength. A water-insoluble (a) and AF-BLG/AG/W (b) dispersions at pH 4.2. This
polyelectrolyte complex (PEC) was formed by mixing BSA gure shows both drop-shaped two-phase region in water-
with Na2Chs at a pH lower than an isoelectric point of BSA rich corner and tie lines on each sides of the water corner
[218]. The binding ratio (R) of BSA to Na2Chs in the PEC bisector, which is the hallmark of the coacervation or
was determined by means of chemical analysis. A PEC was associative phase separation [220]. Moreover, the shift of
also formed on the surface of kaolin via concurrent and/or the diphasic region can be explained by the enthalpic eect
cooperative adsorption of these two polymers on it. As a [220].
result, the aggregation of kaolin particles was induced and
accelerated by virtue of an interparticle bridging eect of
the PEC formed on the surface [218]. I. Interactions Between Chitosan
and Polypeptides
G. Interactions Between Gellan Gum 1. Chitosan and Bovine Serum Albumin
and Polypeptides In an interesting study, Yoshida et al. [221] have studied the
adsorption of BSA on strongly basic chitosan with or
1. Gellan Gum and Gelatin without phosphate buer. In this study, the eect of BSA
Synergistic interactions have been reported between gellan initial concentration, pH, phosphorus, and NaCl concen-
gum and gelatin [219]. High-strength gel is the result of tration have been studied [221].
these interactions [219]. The use of gellan gum in combi- Fig. 21 shows the eect of initial concentration on
nation with gelatin has been explored by other authors equilibrium isotherm for strongly basic chitosan (Chito-
[109,219]. Coacervation was observed only in the pH range pearl 2503 pH 6.9). The experimental equilibrium coe-
3.55.0, where the gelatin and gellan gum had net positive cients and the saturation capacities have been determined
and negative charge, respectively [109,219]. Microscopy by Eq. (8) that is an alternate Langmuir equation [Eq. (9)].
has been used to prove the coacervation and/or occula- With Eqs. (8) and (9), it is possible to correlate experimen-
tion [109]. tal data with Langmuir equation and Freundlich equation.

1 C
C q0 8
H. Interactions Between Acacia Gum K q
and Polypeptides Kq0 C
q 9
In a recent study, Sanchez and Renard [220] have shown 1 KC
the stability and the structure of polysaccharidepolypep- C represents the liquid phase equilibrium concentration of
tide coacervates in the presence of polypeptide aggregate BSA (kg/m3); C0 is the liquid phase initial concentration of
(acacia gum/h-lactoglobulin/water-containing protein ag- BSA (kg/m3); CE is the liquid phase concentration of NaCl
gregate: BLG/AG/W, or free protein aggregate: AF-BLG/ (mol/m3); K is the Langmuir coecient (m3/kg); q is the
AG/W). adsorbed phase equilibrium concentration of BSA (kg/m3
Figure 20 Ternary phase diagram of BLG/AG/W (a) and AF-BLG/AG/W (b) dispersions at pH 4.2. Numbers I and II refer to
monophasic and biphasic regions, respectively, according to the cloudy-point method. (From Ref. 220.)
the molecular weight of gelatin inuence the reaction of

complexation: a chitosan with a high molecular weight
increases the formation of the complex, while a gelatin with
a high molecular weight increases the solubilization of the
hydrogel [222].
3. Chitosan and Collagen

A complex involving chitosan and collagen has been
studied by Taravel and Domard [103,124]. In this work,
physicochemical tests are described. The charge of these
two polymers depends on the pH of the media. The
interactions between chitosan and collagen have been
Figure 21 Eect of initial concentration of BSA on carried out by pH-metry, conductometry, and infrared
equilibrium isotherm for strongly basic chitosan (pH 6.9). spectrometry. The results show that chitosan interacts with
() Eq. (9). (From Ref. 221.) collagen according to a purely electrostatic process of a
polyanionpolycation complex type. All the charges of
chitosan are consumed in this reaction. The reactional
wet resin); and q0 is the saturation capacity of BSA (kg/m3 mechanism depends largely on the initial state of collagen
wet resin). The equilibrium isotherms at pH 6.9 (Fig. 21) (A or B) and more especially of the extent of the gel which
are independent of the initial concentration of BSA; there- limits the polyanionpolycation formation to an encapsu-
fore the absorption follows the phosphate form resin by lation process [103,124].
Langmuir-type reaction [221]. Taravel and Domard [103,124,125] have synthesized a
The strong inuence of pH on the equilibrium iso- complex between chitosan and bovine atelocollagen. The
therm for BSA adsorption on chitosan (C0=1 kg/m3) has complex is formed by mixing a chitosan solution (in HCl)
been shown (Fig. 22). The isotherms are favorable when and a bovine atelocollagen solution at pH 7.8. During the
pH z pI of BSA (4.8); when pH increases, the amount of complexation, the protein is dispersed and is microencap-
BSA adsorbed increases also. The equilibrium constant sulated. During the microencapsulation, the three-helix
increases with pH. For saturation capacities, they are equal alpha of bovine atelocollagen is not destabilized. Two
for pH 4.8 and 6.9, while it is larger at pH 8.79 [221]. methods can be used to form this hydrogel with a ratio
1:1. The rst technique is the formation of this hydrogel at
2. Chitosan and Gelatin temperature higher than the temperature of bovine atelo-
Remunan-Lopez and Bodmeier [222] have studied the collagen denaturation. In this case, the bovine atelocolla-
formation of a hydrogel between chitosan and gelatin. gen is not coagulated. The second technique corresponds to
The optimization of pH, the ratio between the two poly-
mers, the polymer concentration, the temperature, and the
time of reaction are very important factors that have been
optimized. Moreover, the molecular weight of chitosan and
Figure 23 CD spectra of various lms of (a) chitosan

Figure 22 Eect of pH on equilibrium isotherm for ad- hydrochloride; (b) collagen; (c) the complex with 11.5% of
sorption of BSA on strongly basic chitosan (pH 6.9). () chitosan; (d) after subtraction of reduced b spectra to the c
Eq. (9). (From Ref. 221.) spectra. (From Ref. 103.)
Figure 24 Turbidimetric titration curves of legumin (.), chitosan (D), and complexes at ratio between leguminchitosan 0.3
(o). Ionic strength 0.25 M; 25jC; titration with 0.01N HCl or NaOH. (From Ref. 44.)
working with an excess of chitosan (>1200%). In the dichroism at 200 nm. For complex, there is a very large
second case, the excess of chitosan prevents from the red shift that is due to modications in intramolecular
protein precipitation. The infrared spectroscopy shows interactions, particularly linked at hydrogen bondings.
that interactions between chitosan and bovine atelocolla- Moreover, with the complex, two new bands appear: one
gen are hydrogen bondings. Bovine atelocollagen can be at 255 nm and another (after subtraction of the spectrum of
degraded easily by environmental enzyme; the interaction collagen) at 280 nm. This observation allows to conclude
between chitosan and bovine atelocollagen protects the that the spectrum of complex resulted to a combination of
protein against enzyme. This phenomenon is due to the two spectra: one of pure collagen and one of complex. The
stabilization of the three-helix alpha [103,124,125]. main conclusion of this study is that only a small part of
Native collagen and chitosan collagen IR spectra have collagen interacts with chitosan [103].
been compared and the spectra are quite similar [103].
Small modications exist: in the 8001200 cm1 range
(presence of pyranose ring) and in band at 3450 cm1 4. Chitosan and Faba Bean Legumin
(band of OH adsorption). These results show that only Plashchina et al. [44] have studied the complexation be-
electrostatic interactions between NH3+ of chitosan and tween chitosan and faba bean legumin. Faba bean legumin
COO of collagen occur in the complex formation [103]. is an oligomeric, with 6 subunits, protein having a molec-
Fig. 23 shows the circular dichroism spectra of chito- ular weight of 350 kDa. Interactions between chitosan
san, collagen, and complex. For chitosan, the amino group and legumin have been investigated by ultraviolet spec-
gives no signal in the range of 180440 nm. For collagen, troscopy.
the triple helix is shown via pp* O transition in positive Moreover, the complexation of legumin with chitosan
band at 189 nm and pp* ? transition with negative modies the solubility of this polypeptide. Fig. 24 shows
Figure 25 Applications of physicochemical methods to ensure the identity and integrity of polysaccharideprotein conjugate
vaccines. (From Ref. 37.)
that the complexation with chitosan increases respectively chitosan can interact with polypeptides via hydrophobic
the solubilization in the vicinity of the isoelectric point of interaction. To elucidate this capacity, ALCHEMIE 11
legumin and legumin-T [44]. The hysteresis phenomenon at (TRIPOS ASS) and PC MODEL PI (3.2) have been used to
pH 67 can be induced by peculiarities of chitosan solubil- calculate the conformation with consideration of x-ray
ity, and small hysteresis at pH 46 for legumin-T is not structural research. The results have shown that at pH
explained, but the same phenomenon is observed for 6.3 (pKb value of chitosan amino groups), the hydrophobic
globulin complexed with acidic polysaccharides [44]. surface of chitosan represents a fraction of 51.1%, while if
The structure of chitosan has been studied when it is the ionization degree is xed at 100%, the fraction of
complexed with polypeptides [44]. It is well known that hydrophobic surface is 52.4%. These results show that
Figure 26 Proteincarbohydrate interactions. (a) A schematic detailing hydrogen bonding interactions and distances.
(b) Proteinsugar stacking interactions along the substrate-binding cleft. (From Ref. 223.)
the single helix 2/1 conformation is unfolded and that the

major part of chitosan surface is accessible to react with
polypeptides [44].
J. Interactions Between Polysaccharide and

Polypeptide in Conjugate Vaccines
Polysaccharidepolypeptide conjugate vaccines have very
interesting properties for the prophylaxis against bacterial
infection [37]. The interaction between polysaccharide and
polypeptide in conjugate vaccines is due to a covalent
bond. This new link can inuence the structure and the
stability of the new conjugate; therefore the standard
control of this new product is very important [37]. Jones
et al. [37] have developed spectroscopic methods (NMR,
optical spectroscopy, circular dichroism, and uorescence)
in order to analyze the structure and the stability of these Figure 27 Schematic diagram of the interactions of CBM15
components. They summarized analytical methods used to with xylopentaose. (From Ref. 224.)
analyze the structure (Fig. 25).
K. Interactions Between Polysaccharides other hand, between carbohydrate residue and water (hy-
and Enzymes During the Hydrolysis drogen bonding), and Fig. 26b shows the enzyme-sugar
of Polysaccharide stacking interactions along the substrate-binding cleft (ex-
cept for the sugar residue 1) [223].
1. Interactions Between Pectin and Ribulose Water molecules play an important role in the com-
Diphosphate Carboxylase plexation via hydrogen bonding. Hydrogen bonding dis-
In 1993, Braudo and Antonov [102] have studied the plays highly anisotropic temperature factors or split
structure of a complex involving pectin and ribulose di- positions in alternate sugar conformations. Moreover,
phosphate carboxylase. The concentration of pectin in water molecules facilitate solvation and release of the
complexes modies the denaturation temperature and product during the hydrolysis [223].
denaturation enthalpy of ribulose diphosphate carboxyl-
ase. The circular dichroism spectra have shown that this 3. Interactions Between Xylan
modication is due to the conformational stability of the (or Xylooligosaccharides) and Family 15
protein in presence of pectin. The denaturation tempera- Carbohydrate-Binding Module
ture and denaturation enthalpy of ribulose diphosphate In 2001, Szabo et al. [224] have shown the structure of
carboxylase decrease when pectin concentration in the family 15 carbohydrate-binding module (CBM) in complex
complexes increases caused by the decrease of conforma- with xylopentaose. CBMs are a key role in the catalytic
tional stability. In the case of increase of pectin concentra- activity of plant cell wall hydrolases because CBMs can
tion, the content of a-helix for ribulose diphosphate interact and bind with structural polysaccharides that are
carboxylase decreases. The authors have suggested that present in plant [224].
the decrease of a-helix for ribulose diphosphate carboxyl- In Fig. 27, the interactions between CBM15 and
ase is linked at the formation of hydrogen bonding between xylapentaose are schematized [224]. Two tryptophan resi-
OH groups of tyrosine and NH groups of tryptophan of dues of CBM15 interact by hydrophobic interactions with
polypeptide with ester groups of pectin [102]. Xy12 and Xy14. The chair ring plane of Xy12 and Xy14
forms angle of 117j and 99j, respectively. Moreover,
2. Interactions Between Cellopentaose and Glycosidase hydrogen bonds allow these interactions: Xy13 via O-5
In a recent paper, Guerin et al. [223] have studied the interacts with amide of Gln171, O-3 of Xy12 with amide of
atomic resolution structure of a glycosidase and its sub- Gln171, and O-2 of Xy12 with amide of Asn106 and Gln217.
strate: cellopentaose. The crystal structure of Clostridium
thermocellum endoglucanase CelA complexed with this
substrate has been determined at 0.94 A with a nal IV. APPLICATIONS
crystallographic R factor of 9.4%. The nal model includes A. Applications in the Biotechnological Area:
363 amino acids and 6 enzyme-bound D-glucosyl residues.
Enzymes Immobilization
Fig. 26 shows the interactions between enzyme and
oligosaccharide and the role of water molecules in these The immobilization of enzymes is a technique extensively
interactions [223]. Fig. 26a shows detailing hydrogen studied since the late 1960s [225]. The knowledge base
bonding interactions and distances, on one hand, between accumulated on enzyme immobilization studies has grown
carbohydrate residue and amino acid residue, and on the steadily [226228]. In general, ve types of immobilization
enzyme immobilization [11]. Roig et al. [11] have entrapped

high alkaline protease (HAP) in calciumalginate bead.
They have tested two types of substrates with dierent sizes
(neutral casein and a synthetic chromogenic tripeptide).
Fig. 28[11] shows the eect of CaCl2 concentration and
storage on the relative activity. The increase of CaCl2
concentration, used to form the capsule, decreased the
relative activity of HAP [11]. The pH of calcium chloride
solution has an eect on the relative activity of HAP (Fig.
29) [11].
In a study of Dashevsky [10], the problem of enzyme
loss by the microencapsulation in alginate beads is
reported. Microencapsulation of lactase into alginate
beads resulted in a protein loss of about 36%. A similar
protein loss (34.4%) is described by Pommersheim et al.
[230]. Moreover, the pH inuences the percent of protein
adsorption.
Arruda and Vitolo [12] have investigated the invertase
immobilization into calciumalginate beads. Table 10
shows the eect of the manuronic/glucoronic ratio (M/G)
Figure 28 Eect of making and storing capsule in various

concentrations of calcium chloride on the activity of gel-
entrapped HAP with (A) casein at pH 11 and 37jC and (B)
tripeptide substrate at pH 10 and 25jC. Activity at 1% w/v
CaCl2 is taken as 100%. (D, 5) Storage in water for 1 hr and
for a further 2 hr. (E, n) Storage in CaCl2 for 1 hr and water
for a further 2 hr. (From Ref. 11.)
method are known [225]: (1) binding to carriers by covalent

bonds; (2) binding to carriers by adsorptive interactions;
(3) entrapment in gels, beads, or lms; (4) cross-linking or
co-cross-linking with bifunctional reagents; (5) encapsula-
tion in microcapsules or membranes.
1. Microencapsulation in Alginate
Alginate/Glutaraldehyde
Oh et al. [9] have studied the immobilization of L-
arabinose isomerase for the tagatose production. Alginate
beads treated with glutaraldehyde gave the most stable and
economic method [9]. L-Arabinose isomerase immobilized
in a packed-bed reactor produced an average of 30 g
tagatose per liter and per day from 100 g galactose per Figure 29 Eect of making and storing encapsulated HAP
liter for 8 days. at various pHs of calcium chloride. Assay (A) casein
substrate at pH 11 and 37jC and (B) tripeptide substrate at
Alginate/CaCl2
pH 10 and 25jC. The activity from 2% CaCl2 at pH 5.0 is
Many papers present the immobilization into chito- taken as 100%. (D, 5) Storage in water for 1 hr and for a
sancalcium complexes [1012,229]. Calcium alginate gels further 2 hr. (E, n) Storage in CaCl2 for 1 hr and water for a
have proven to be versatile, reliable, and useful matrix for further 2 hr. (From Ref. 11.)
Table 10 The Variation of Lost Protein Index

(%) Against the pH of the 0.1 M CaCl2
Solution Using Two Alginate as Support for the
Entrapment
pH M/G=0.5 M/G=1.2
4 50 47
7 48 45
8 34 34
of alginate on the lost protein index (LPI); the LPI depends

on alginate solution pH and M/N ratio.
The kinetic constants for free and immobilized invert-
ase have been determined. For immobilized enzymes in Figure 31 Comparison of immobilized (.) and free (n)
general, a decrease in Vmax is a common result, but a large LOX at room temperature. (From Ref. 14.)
diminution of KM is seldom observed [12]. Moreover, the
activation energies, determined in this study, are 28 and 24
kJ/mol for free and immobilized invertase, respectively
[12]. They have tested dierent drying treatments of immobi-
Alginate/Silicate lized LOX. The drying treatment aected generally the
catalytic activity of enzyme [14]. After drying, the relative
The bilirubin oxidase has been eciently entrapped in
activity of immobilized LOX varies between 15% and
alginatesilicate solgel matrix [13]. The enzyme was not
30%. The immobilized LOX maintains same activity dur-
released from the beads after 7 days at ambient tempera-
ing 25 days, while free LOX lost all activity in 24 hr (Fig.
ture. Moreover, the bilirubin oxidase entrapped present a
31). In another study, the same conclusion has been shown
very good resistance at high temperature. Fig. 30 shows the
for the stability of h-glucosidase immobilized in alginate
relative activity vs. temperature. The free bilirubin oxidase
silicate gel [231] during several months.
is stable below 20jC, while immobilized bilirubin oxidase is
stable up to 50jC [13]. The kinetics of this free enzyme and
immobilized enzyme followed Michaelis and Menten ki-
netics.
Hsu et al. [14] have shown interest at the lipoxygenase
(LOX) immobilization in alginatesilicate solgel matrix.
Figure 32 Batch operational stability test for immobilized

Figure 30 Eect of temperature on the stability of free and lipase. Residual activity is plotted against operational time
immobilized bilirubin oxidase. The enzyme was incubated in baking the esterication activity of 109 Amol/(g m) as 100%.
pH 8.4 Tris buer at temperature indicated for 1 hr before Esterication assay was carried out with substrate containing
measuring its residual activity under the standard assay 250 mM butyric acid in heptane. Initial hydrolytic activity
conditions. (From Ref. 13.) was 32.8 U/mg. (From Ref. 15.)
In this case, the formation of salt bridges between

porcine pancreatic a-amylase and sodium alginate has
been conrmed by the presence of multiple combination
bands from infrared spectra in the region of 28002000
cm1, which correspond at the primary amine groups (link
between enzyme and sodium alginate).
Enzyme Covalently Linked with Chitosan
Cellulase has been stabilized by covalent conjugation
with chitosan [16]. The conjugation between chitosan and
cellulase has been made under reducing conditions to
periodate-oxidized sugar moieties. Fig. 33 shows the ther-
mal stability of native and modied cellulase [16]. The
thermostability has been evaluated after heating for 10
min. The T50 for native and modied cellulase has been
Figure 33 Thermal stability prole of native (o) and
calculated at 68.4jC and 77.3jC, respectively. Half-life
chitosan-modied (.) cellulase. (From Ref. 16.)
times of native and modied cellulase have been investi-
gated for four temperatures as shown in Table 11 [16].
2. Chitosan
4. Entrapment in a Matrix Based on the Complexation
Pereira et al. [15] have investigated the immobilization of
Between Chitosan and Xanthan
lipase from Candida rugosa into chitosan beads. The
concentration of lipase has been the most important pa- In seven papers [57,233236], a polyionic hydrogel
rameter and its inuence has been positive [15]. Maximum obtained by complexation between chitosan and xanthan
yield of catalytic reaction has been 83% and has been has been used as support for enzyme immobilization
found at 37jC, with 2.5% of lipase (w/v) and with a molar (proteases, xylanase, and lipase). The hydrogel has been
ratio acid/alcohol of 1.5. Moreover, Pereira et al. [15] have made by mixing a 0.65% xanthan solution and enzyme,
tested the stability of the immobilized lipase in time. Fig. 32 and after this, the solution is added dropwise in a 0.65%
shows that the half-life time of immobilized lipase is 86 hr chitosan solution [5]. The hydrophilic microenvironment
for repeated use in batch esterication of butanol with of this hydrogel bead (high water content: swelling degree
butyric acid [15]. between 300% and 4000%) makes this matrix a favorable
The molecular weight and degree of acetylation inu- system for enzyme immobilization [57,233236].
ence the lipase-loaded and lipase activity [232]. High Dumitriu et al. [5] have demonstrated that the percent
molecular weight and high degree of acetylation allow a of immobilization is function of the concentration of
higher loading of lipase into chitosan beads, but a lower enzyme in the xanthan solution. Table 12 [5] shows the
activity is observed with high degree of acetylation. Mo- properties of immobilized xylanase and protease in the
lecular weight seems to not aect the activity of lipase. chitosanxanthan hydrogels. For endo-1,4-h xylanase
Moreover, the release rate of lipase is higher with chitosan from Trichoderma viride and for protease type XIX fungal
having a low molecular weight [232]. from Aspergillus sojae, the enzymatic activity of immobi-
lized enzyme increases with an increase of enzyme concen-
3. Enzyme Covalently Linked with Polysaccharides tration in the xanthan solution [5]. Moreover, the
immobilization increases the thermal stability (Table 12)
Enzyme Covalently Linked with Alginate [5]; therefore the hydrogel confers a favorable structure on
In a recent study of Gomez et al. [8], porcine pancreatic enzymes.
a-amylase has been covalently linked to sodium alginate, In a more recent study, Magnin et al. [7,236] have
activated by periodate oxidation, via a reductive alkylation shown the inuence of the concentration of protease type
with NaBH4. The stability of porcine pancreatic a-amylase XIX fungal from A. sojae in xanthan, the storage tem-
has been tested for temperature, pH, and time. perature, and the molarity of phosphate buer storage
Table 11 Half-Life Times of Native and Chitosan-Modied Cellulase
t1/2 (hr1) t1/2 (hr1)

Temperature (jC) native cellulase modied cellulase Dt1/2 (hr1)
65 4.28 6.42 2.14

70 0.95 4.28 3.33
75 0.25 0.87 0.62
80 0.11 0.29 0.18
Source: Ref. 16.
Table 12 Properties of Immobilized Xylanase and Protease in the ChitosanXanthan Hydrogels
Concentration of
enzyme in the xanthan Measured activitya Relative activityb Limit of thermal
Enzyme solution (wt.%) (mU/g) (%) activity (%)
Xylanase 0.21 8.889 +69 95c

0.29 11.737 +61 95
0.36 16.000 +77 95
1.00 26.000 +2 98
1.20 28.000 7 98
Protease 0.29 52.595 0 77d
0.69 67.900 0 77
0.96 72.187 2 77
1.88 104.294 8 77
a
Enzyme activity refers to the unit weight of dry hydrogel.
b
Calculated with respect to the initial activity of the xylanase (2.500.000 mU/g) and protease (0.4U/mg).
c
Thermal stability of free xylanase=45jC.
d
Thermal stability of free protease=42jC.
Source: Ref. 5.
(Fig. 34). Moreover, this study has demonstrated that the immobilize lipase [6]. Lipase immobilization inuences the
molecular weight of chitosan and time of reaction of com- structures of the matrix (Fig. 37). Fig. 37b (matrix with
plexation are important factors that inuence the enzy- lipase) shows globular structures in scanning electronic
matic activity (Fig. 35). microscopy, while these structures do not appear in Fig.
For lipase immobilization [6,7,233,236], the eciency 37a (matrix without lipase). To conrm that lipase has
varies with the initial enzyme concentration [233]. Magnin formed globular structures into the matrix, the authors
et al. [6,7] have shown that the immobilization of lipase into have used a cytochemical method (Gomori method) to
chitosanxanthan hydrogel has allowed to double the stain the lipase [6]. With this method, lipase is stained by
enzymatic activity (Fig. 36a) [6]. Fig. 36b shows the lipasic lead nitrate; and, in backscattering scanning electronic
activity in organic three-organic media (toluene, isooctane, microscopy, lead appears in light gray with other structure,
and cyclohexane) [6]. containing carbon, and azote and oxygen appear in dark
An electronic microscopic study has been presented gray. In Fig. 38b, in backscattering scanning electronic
for the complex between chitosan and xanthan, used to microscopy, globular structures appear lighter than other
Figure 35 Inuence of molecular weight of chitosan and

Figure 34 Inuence of the concentration of protease XIX reaction time between the solution of xanthan containing
(0.2% at 1% in xanthan solution), the temperature of the protease and the chitosan solution. In this experiment,
storage, and the molarity of phosphate buer use to storage. the percent of protease in xanthan solution is 0.8% and the
(From Ref. 7.) buer is 0.2 M phosphate buer. (From Ref. 7.)
Figure 38 (a) Transmission electron microscope image of

the hydrogel with lipase. This hydrogel have been treated
with Gomori method. 16.300. (b) Backscattering electron
microscope image of immobilized lipase in chitosan. This
hydrogel has been treated via the Gomori method. 20.000.
(From Ref. 6.)
B. Applications in the Pharmaceutical Field

1. Controlled Release of Polypeptides
The degradation of polypeptides in the gastric tract is the
most important problem for the oral formulation of poly-
peptides. If therapeutic polypeptides can be protected by
this degradation, the oral route could become very inter-
esting for new formulations [237].
Figure 36 (a) Lipasic activities of free enzyme and Controlled release systems are often used to extend the
immobilized lipase in aqueous medium. (b) Lipasic activities time of the therapeutic dose from a single administration
of free enzyme and immobilized lipase in three organic and to prevent or minimize the drug concentration in the
solvents. (From Ref. 6.) body. These systems oer many advantages [238]: decrease
the variability in systemic drug concentrations; easier,
more accurate, and less frequent dosing; ability to deliver
a drug more selectively in a specic site; absorption that is
structures; therefore the globular structure contains lipases more consistent with the site and mechanism of action; and
[6]. Transmission electronic microscopy observation has reduction of toxic metabolites.
been investigated also in this study [6]. The matrix with Hydrogels Based on Complexation of Polysaccharides
lipase, treated with Gomori method, has been observed in and Polypeptides
TEM. In this case, the lipase stained by lead nitrate appears
in black dot in the micrograph (Fig. 38a). Lipase is Alginate and Poly-L-Lysine. Wheatly et al. [239]
concentrated in two zones: in the interface between a have shown that a hydrogel, involving alginate and poly-
L-lysine HBr, can be used to control the release of myo-
pseudomembrane and the inner part of the bead, and in
the inner part of the bead, lipase is located in agglomer- globin. In another study, a biocompatible capsule mem-
ations (clusters) [6]. brane forming with alginate and poly-L-lysine is used to
release insulin [192].
Complexes Based on Polysaccharides Used to Release
Polypeptides
Ecient delivery of biologically active polypeptides
has received great attention in modern chemotherapy. The
protection of polypeptides against enzymatic degradation
is an important factor in the case of oral administration
[240]. Macromolecular system has attracted large attention
for making vehicle for drug delivery system [241]. In more
recent years, bioadhesive polymers have considerable in-
terest as auxiliary agents for oral administration of poly-
peptides [242,243].
Alginate. Microspheres of alginate have been used to
Figure 37 Scanning electron microscope images of the hy- the delivery of antigens [244]. The gelation is due to the
drogels without (a) or with lipase (b) 20.000. (From Ref. 6.) adding of Ca2+. The alginate microspheres are interesting
because they are easily prepared in aqueous solutions at

room temperature. In physiological conditions, the micro-
spheres swell and gradually disintegrate with time [244].
In a study involving alginate bead and tumor growth
factor-h1 (TGF-h1), Mumper et al. [245] have shown the
release of this inhibitor of the growth of many epithelial
cells. To avoid doing irreversible binding between alginate
and TGF-h1, polyacrylic acid is added in the alginate bead
as an excipient. In vitro study has shown that no release is
possible in acid media (0.1N HCl, pH 1), while the release
exists in phosphate-buered saline (PBS) at pH 7.4. Fig. 39
shows the release of TGF-h1 [245]. For the release in PBS
after an incubation of 1 day in HCl, the release rate is
higher because it is possible that the incubation in HCl has
began to hydrolyze alginate [245]. This example is very
interesting in the case of oral administration because the
alginate beads protect polypeptides of acid stomach media
and after a rapid release in the small intestine (where the pH
is not acid).
In vivo tests have been investigated with alginate beads
loading TGF-h1 [246]. Fig. 40 shows the proliferating and
mitotic indices in three rats organs. Rats have been treated
with alginate bead loading with TGF-h1 or controls via
oral or intraperitoneal administration. For rats treated
with alginate bead loading with TGF-h1, a signicantly
reduced of proliferating index and mitotic index have been
observed compared to the controls [246].
Basic broblast growth factor (bFGF), polypeptide
that has a short half-time, has been binding to heparin-
sepharose [247] and then encapsulated in alginate micro-
beads with an eciency of 77%. In vitro, the release is very
slow, more than 14 days. The release rate can be enhanced
by adding heparinase in the release media [247]. In another
study involving leukemia inhibiting factor, the release
takes more than 80 days [248]. Figure 40 Proliferating and mitotic indices of intestinal cells
Gombotz and Wee [119] have studied the in vitro in rats after administration of (5) control vehicle (square
release of some recombinant proteins encapsulated in with points inside), TGF-h1 intraperitoneally (square with
lines inside) and TGF-h1 in phosphate-buered saline
perorally, and (n) TGF-h1 in alginate beads perorally.
(From Ref. 246.)
alginate beads. Fig. 41 shows the cumulative release percent

vs. time of tumor necrosis factor receptor (TNFR), inter-
leukin 1 receptor (IL1R), interleukin 4 receptor (IL4R), and
granulocyte macrophage colony stimulating factor
(GMCSF). These four polypeptides are acidic; therefore
they are likely to interact with anionic alginate compared to
basic polypeptides such as TGF-h1 and bFGF that can
interact with alginate. For the basic polypeptide, the release
rates are smaller than for acidic polypeptides [119]. In the
case of basic polypeptides, the release is primarily due to the
erosion, while in the case of acidic polypeptides, the release
can be due to diusion through the pores of network [119].
In Fig. 41, GMCSF, the one with the lowest polypeptide
molecular weight (16 kDa), is released the fastest. Next are
Figure 39 Cumulative percent in vitro release of 125I-TGF- IL4R and IL1R that have the same molecular weight of
h1 at 37jC in (n) PBS, pH 7.4 and (.) 0.1N HCl, pH 1.0 about 50 kDa, and the last is TNFR with a molecular
transferred to PBS after 24 hr. (From Ref. 245.) weight of 180 kDa. The main conclusion of this study is that
the release rate for acidic polypeptide is linked to the

molecular weight of polypeptides [119]. This conclusion is
in accord with another study [249]. Gombotz and Wee [119]
have investigated an in vivo test for interleukin 17 receptor
(IL17R) encapsulated in alginate matrix. Alginate bead
containing IL17R having a diameter of 1 mm has been
implanted subcutaneously in inamed B6 mice having
lymph nodes higher (due to an irradiation) than normal
B6 mice. Mice having received this implant have a reduction
of lymph node weights similar to inamed B6 mice having
received an injection of IL17R. But the dierence is the
dose: in alginate matrix, the dose was only one-third
compared to direct injection [119].
Maysinger et al. [116] have shown the advantage of
included nerve growth factor (NGF) in alginate. This
system can prevent central cholinergic degeneration in rats.
The inclusion of NGRF in alginate has created a protection
against hydrolytic cleavages. This system gives same results
than NGF implanted via Alza mini-osmotic pumps [116].
Chitosan. Burgamelli et al. [250] have investigated
the production of chitosan microparticles containing in-
Figure 41 The cumulative percent in vitro release proles of sulin by interfacial cross-linkage of chitosan solution in the
TNFR (n), IL1R (.), IL4R (E), and GMCSF (x) from 70% aqueous phase of water/oil dispersion in presence of
G content alginate beads in PBS at 37jC. (From Ref. 119.) ascorbyl palmitate that permitted covalent bond forma-
Figure 42 Diusion of BSA during 24 hr in gastric simulation media from microcapsules produced via external gelation (x: 2%
alginate w/w), internal gelation (.: 2% alginate w/w and E: 4% alginate w/w), and in presence of 0.25 M NaCl in the gelling
media (n). (From Ref. 18.)
Table 13 Time of Release of FITC-BSA taken more than 5 days. In vitro activity has been investi-
from Three Systems gated by an incubation of this alginate bead coated with
chitosan hydrochloride and loaded with IL2 with tumor
System Time cells and lymphocytes. The induction of cytotoxicity has
Alginatechitosan 4 days been measured and the main conclusion is that IL2 incor-
AlginateCaCl2 6 hr porated in the alginate system remained active and it is
AlginatepolyLlysine 24 hr more ecient in triggering the induction of cytotoxicity
than free IL2 [99].
In the study of Polk et al. [251], the release of BSA and
vibrio bacterin has been tested. For the release of BSA, the
tion with the amino group of chitosan when its oxidation inuence of molecular weight of chitosan and pH has been
to dehydroascorbyl palmitate takes place during the investigated. Tables 14 and 15 show the inuence of
formation of microparticles. The charac terization of mi- molecular weight of chitosan and pH, respectively [251].
croparticle has shown that this method produced micro- Okhamafe et al. [252] have shown the modulation of
particle having high loading levels of insulin. Insulin is BSA release from chitosanalginate microcapsules (300
completely released in about 80 hr at an almost constant 500 Am) using the pH-sensitive polymer hydroxypropyl
release rate [250]. methylcellulose acetate succinate (HPMCAS). Fig. 43
Chitosan and Alginate Complexes. Vandenberg and shows the release of BSA from chitosanalginate micro-
De La Noue [18] have evaluated a complex involving capsule (Fig. 43a), the release of BSA from chitosan
chitosan and alginate for polypeptide release. The authors alginate microcapsule coated with HPMCAS solution
have tested the loss of BSA during the manufacture and (Fig. 43b), and the release of BSA from alginateHPMCAS
during an acid incubation for two types of microcapsules: microcapsules (Fig. 43c).
one produced via external gelation and the second via In another study [253], hemoglobin has been encapsu-
internal gelation [18]. The type of gelation inuences sig- lated in chitosancalcium alginate beads. The molecular
nicantly the loss of BSA. Fig. 42 shows the diusion of weight of chitosan (Mv=245.000 or 390.000) and the pH
BSA during 24 hr into a gastric simulation medium. The (2.4 or 5.4) of this solution had only a slight eect on the
loss of BSA was higher for microcapsules produced by release of hemoglobin. With pH at 5.4, the best retention is
internal gelation than microcapsules produced by external obtained [253].
gelation. Moreover, the loss is higher when NaCl is present
in the gelling media during the formation of microcapsules 2. Controlled Release of Polypeptides
(A=external gelation and B=internal gelation) [18]. Via Oral-Colon Specic Delivery
In another study, uorescein isothiocyanate-labeled
bovine serum albumin (FITC-BSA) has been included in Chitosan
alginatechitosan beads [99]. The release rate of this system Tozaki et al. [254] have studied the colon-specic
has been compared with two other systems: alginateCaCl2 insulin delivery with chitosan capsules. A marked adsorp-
and alginatepoly-L-lysine [99]. Table 13 shows the time of tion of insulin and a corresponding decrease in plasma
total release of these systems. glucose levels have been observed following the oral ad-
The short release rate of FITC-BSA from alginate ministration of these capsules that contain 20 IU of insulin
CaCl2 is due to the low stability of the chelation, while the and sodium glycocholate (PA=3.49%), as compared with
high release rate from alginatechitosan is due to a strong the capsules containing only lactose or only 20 IU of insulin
interaction between the two biopolymers [99]. (PA=1.62%). The hypoglycemic eect started from 8 hr
In the same study, Liu et al. [99] have shown the release after the administration of chitosan capsules when the
of interleukin 2 (IL2) from alginate bead coated with capsules entered the colon, as evaluated by the transit time
chitosan hydrochloride. In vitro, the complete release has experiments with chitosan capsules [254].
Table 14 Eect of Chitosan Molecular Weight on Elution of BSA in Eluent pH 6.2
Fraction entrapped
albumin released
Chitosan molecular
System weight (106) At 4 hr At 24 hr
Capsules 0.25 0.78 0.82

1.25 0.36 0.75
0.25+1.25 0.20 0.45
Control None 0.72 0.75
(alginate bead)
Source: Ref. 251.
Table 15 Eect of pH on Elution of BSA from Capsules Prepared with

Mixing of High and Low Molecular Weight Chitosan (0.25106+1.25106)
Fraction entrapped
albumin released
System Eluent pH At 4 hr At 24 hr
Capsules 3.0 0.15 0.16

chitosanalginate 6.2 0.20 0.48
8.0 0.40 0.70
Control 6.2 0.72 0.75
(alginate bead)
Source: Ref. 251.
3. Controlled Release of Polypeptides To develop nasal power preparations with high bio-
via Mucosal Delivery availability for polypeptide delivery, Suzuki and Makino
Alginate microspheres containing a conjugate of polysac- [258] have tested the eect of a combination of hydroxy-
charide antigen and cholera toxin B subunit (CTB) have propylcellulose (HPC) and microcrystalline cellulose
been prepared for a novel mucosal immunization [255]. An (MCC). The addition of a small amount of HPC to MCC
alginate solution (1.05.0% w/v) has been added dropwise allow a signicant enhanced adsorption of leuprolide and
into n-octanol containing one emulsier (HCO-10 or HCO- calcitronin. It is suggested that MCC works as an absorp-
60 or Span80). Afterwards, n-octanol containing calcium tion enhancer by causing a locally high concentration of
chloride (1.08.0% w/v) was added. After 10 min, CaCl2 at drugs in the vicinity of the mucus surface. Moreover, HPC
8% is added followed by addition of isopropyl alcohol. A works to increase retention of drugs on the nasal mucus due
pneumococcal capsular polysaccharide type 19 (PS19) has to its gel-forming property [258].
been conjugated to the CTB, and this conjugate has been Alginate
encapsulated into the microspheres with a loading percent Alginate beads can be used as a vaccine delivery system
of 60%. The alginate microspheres have a diameter smaller [119,259]. Alginate microbeads coated with poly-L-lysine
than 5 Am, and also they can be used to mucosal immuni- and loaded with ovalbumin have been intranasally admin-
zation because the transport through the eerent lymphatic istrated in mice. The level of antigen specic antibody in the
system is possible with particles of this size. After 1 day, serum of these mice is high compared to two controls (mice
80% of CTB-PS19 has been released. In vivo, a study with received free ovalbumin and mice received empty alginate).
mice has been investigated and has shown an antibody Moreover, IgA have been detected in the bronchial alveo-
response after oral administration of CTB-PS19 entrapped lar lavage uids in mice that received primary and second-
in alginate microspheres [255]. ary immunizations via nasal administration [119,259]. In
other studies [117,260], alginate microbeads containing
4. Controlled Release of Polypeptides ovalbumin have been administrated orally and an immu-
via Nasal Delivery nity is detected at mucosal sites.
Chitosan
In 1994, Illum et al. [256] have demonstrated that 5. Controlled Release of Polypeptides
chitosan was able to enhance the transnasal adsorption Via Subcutaneous Injection
of polypeptides such as insulin. A double mechanism has Murine CD40Ligand (CD40L) has been encapsulated in
been proposed to explain this enhancement: the bioadhe- alginate and implanted subcutaneously in CD40L-decient
sion of chitosan and the transient widening of the tight mice (having hypogammaglobulinemia and opportunistic
junction in the membrane [256,257]. infections due to a lack in the immune system: Hyper IgM
In 1996, Aspen et al. [257] have studied the inuence of syndrome). The mice that received the implant mounted a
many chitosan salts as promoters for insulin adsorption via strong antigen-specic delayed-type hypersensitivity re-
nasal delivery [257]. sponse, while it is not the case for the mice that received
When insulin is administrated with chitosan as pro- a control (ovalbumin encapsulated in alginate) [118,119].
moter, a decrease of less than 20% is observed in the blood
glucose levels (Fig. 44) [257]. This decrease is very impor- 6. Controlled Release of Polypeptides
tant compared to the baseline measurements (saline insu- via Intravenous Injection
lin). The one-way analysis of variance test of these values
shows that the dierence between chitosan is not signicant Pullulan
( p<0.05), and no correlation between the degree of deace- In a study of 1998, Akiyoshi et al. [261] have investi-
tylation or molecular weight has been found [257]. gated the complexation of insulin on nanoparticle of
Figure 44 Eect on blood glucose levels of nasally dosing

rats with various 0.25% chitosan solutions all containing
insulin 40 IU/ml: D: 113Cl; .: 211Cl; 5: 210G; E: 313Cl; n:
411Cl; : saline (n=57). (From Ref. 257.)
complexes decrease the enzymatic degradation of insulin

compared to free insulin and dodecyl maltoside (DDM)
and insulin complexes [261].
The release of insulin is shown in Fig. 47 after adding
BSA. The BSA is important because there is an exchange
between BSA and insulin (due to binding strength to the
CHP) that allows to release insulin. The binding constant
of BSA (k = 8 106 M) is higher than the binding constant
of insulin (k = 2 106 M). The concentration of BSA
inuences the release of insulin because it is probably due
to an exchange between insulin and BSA [261].
The biological activity of CHP binding insulin has
shown that this complex decreases the blood glucose level
down to 50% at 60% of the initial value after 30 min of
intravenous injection. Compared with free insulin, the
Figure 43 (A) BSA release from unmodied chitosan

alginate microcapsules at pH 1.2 and pH 3.0; (B) BSA release
from chitosanalginate microcapsule coated with HPMCAS
solution [0.5% (., o), 1% (5, n), 2% (D, E)]; (C) BSA
release from microcapsules with a core blend of alginate/
HPMCAS in a ratio of 1:1 (D=microcapsules with an
additional HPMCAS membrane coating, 5=microcapsules
without an additional HPMCAS coating, .=unmodied
microcapsules). (From Ref. 252.)
cholesterol-bearing pullulan (CHP). The complexation

between CHP and insulin has been followed by high-
performance size exclusion (HPSEC). Fig. 45 shows chro-
matogram of HPSEC of the aggregation.
The stability of complexes CHP and insulin has been
tested and no dissociation has been observed after 1 week at Figure 45 HPSEC chromatograms of 35 AM insulin (-), 1.8
room temperature [261]. Moreover, enzymatic degradation AM CHP self-aggregate (- -), and 35 AM insulin after the
has been also investigated with a-chymotrypsin [261]. Fig. incubation with 1.8 AM CHP self-aggregate (- - -) for 1 hr in
46 shows this enzymatic degradation. The CHP and insulin 0.1 M PBS at pH 8.0 and 25jC. (From Ref. 261.)
Figure 48 Eect of equimolar doses of native free insulin

(5) and insulin complexed with CHP (o) on in vivo blood
glucose levels. (From Ref. 261.)
Figure 46 Enzymatic degradation of 7 AM insulin com-
plexed with 0.9 AM CHP self-aggregate (5), 7 AM insulin
complexed with 2.5 mM DDM (.), and 7 AM free insulin (o)
by a-chemotrypsin (30 Ag/ml) as a function of time at 37jC.
(From Ref. 261.) Interferon (IFN) has been conjugated with pullulan in
order to target the liver with ecient exertion of its anti-
viral activity [264]. The cyanuric chloride method enabled
dierence is not signicant (Fig. 48). The authors of this the preparation of this conjugation and retained about 7
study have concluded that the complexation of insulin with 9% of biological activity of IFN. Pullulan conjugation
CHP has preserved the bioactivity of insulin. enhanced the liver accumulation and the retention period
Pullulan is a polysaccharide, having a high anity of IFN. In vivo tests have shown that the pullulanIFN
with liver; consequently, Gu et al. [262] and Wang et al. injected intravenously to mice enhanced the activity of 25
[263] have developed a novel hydrophobized polysaccha- A synthetase in the liver and the retention time was 3 days
ride/oncoprotein complex vaccine inducing in vitro and in (compared to free IFN where the retention time is 1 day)
vivo cellular and humoral immune responses against [264].
HER2-expressing murine sarcomas [262,263]. This protein Diethylenetriamine pentaacetic acid (DTPA), a chela-
has been complexed with cholesteryl group bearing pullu- tion residue, is coupled with pullulan. DTPApullulan is
lan (CHP) and cholesteryl group bearing mannan (CHM). conjugated with IFN via Zn2+ coordination. Intravenous
Mice immunized with CHM-HER2 and CHP-HER2 have injection of IFNDTPApullulan conjugate induced an
rejected HER2-transfected tumors. Moreover, a complete antiviral enzyme in the liver [265].
rejection of tumors has been observed after 3 days with
CHM-HER2 [262,263].
C. Applications in the Medical Field
1. Prophylaxis
First, many authors have been interested to the polysac-
charidepolypeptide conjugates during the last 20 years.
Polysaccharidepolypeptide conjugate vaccines have very
interesting properties for the prophylaxis against bacterial
infection [37].
In a study, Devi et al. [31,32] have tested a group B
Neisseria meningitidis and Escherichia coli K92 capsular
polysaccharidepolypeptide conjugate in animal model: in
juvenile rhesus monkeys. The aim of this study is the
evaluation of conjugate to induce active antibody in a
nonhuman primate model [31]. Meningitis and septicemia
cause high morbidity and mortality worldwide; N. menin-
Figure 47 Release of insulin from the CHP (0.22 AM)- gitidis is a pathogen that can cause this pathology [31,32].
insulin (1.8 AM) complex after adding (.) 50 mg/ml, (o) 6.7 Fig. 49 shows the levels of IgG and IgM in monkeys that
mg/ml, and (5) 1.3 mg/ml of BSA in PBS at pH 7.4 and have been immunized with a conjugate (the N-propiony-
37jC. (From Ref. 261.) lated B polysaccharide, N-pr.B, coupled with group B
meningococcal recombinant outer membrane protein 3,

OMP3) [31].
In another study, Konadu et al. [36] have tested a
polysaccharideproteins conjugate vaccine in human
(adults, teenagers, and 2- to 4-year-old children). They
are made phases 1 and 2 studies of Salmonella enterica
serovar paratyphi A O-specic polysaccharidetetanus
toxoid conjugate [36]. S. enterica can cause enteric fever
with septicemia.
After the vaccination with S. enterica serovar para-
typhi A O-specic polysaccharidetetanus toxoid conju-
gate, no signicant secondary eect has been reported [36].
Table 16 shows the level of IgG in serum of human that has
been vaccinated with the conjugate [36]. Vaccines with
SPA-TT2 have a level of IgG higher than vaccines with
SPA-TT1, but this dierence is not signicant [36]. The
authors of this study have concluded that S. enterica
serovar paratyphi A O-specic polysaccharidetetanus
toxoid conjugate is safe and allows the synthesis of IgG
antibodies with bacterial activity in the three groups [36].
2. Transplantation: Articial Pancreas

An alternative at islet transplantation is to enclose islet
tissue in a semipermeable membrane to protect the islets
against the cell-mediated immunity and humoral immunity
(that causes the rejection of articial pancreas). The mem-
brane should be permeable at nutrients, glucose, and
insulin that modulate the islet activities [189].
Alginate and Poly-L-Lysine
Figure 49 IgG and IgM titers of individual monkeys (F2V,
GIB, GID, E4D, and GON) immunized with N-pr.B-OMP3 In a study, a biocompatible capsule membrane form-
conjugate in Al(OH)3 as measured by ELISA with N-pr.B- ing with alginate and poly-L-lysine is used to protect trans-
HSA as the coating antigen. Arrows indicate the weeks at planted islets [190192].
which monkeys were immunized. Data correspond to A405S The determination of the fate of transplanted micro-
normalized to 100 min times the dilution factor. (From capsulated islets is linked at the permeability of the capsule
Ref. 31.) of alginate and poly-L-lysine. Many papers deal with this
Table 16 Age-Related IgG Levels of Anti-Lipopolysaccharide Antibody in Serum Before and 180 Days after Vaccination with
SPA-TT Conjugate
GM lipopolysaccharide antibody titer (EU)

on days after vaccination:
No. of Fold rise at
Age (year) Conjugate injections 0 day 42 days 68 days 180 days 180 days
1844 SPA-TT1a 1 1.26 9.82 NAb 3.73 3.0

SPA-TT2c 1 1.69 27.3 NAb 8.28 4.9
1317 SPA-TT1a 1 1.69 12.8 NAb 6.72 4.0
SPA-TT2c 1 1.70 17.4 NAb 7.37 4.3
24 SPA-TT2c 1 0.91 19.3 11.7 3.47 3.8
SPA-TT2c 2 0.77 16.7 11.9 4.08 5.3
a
Salmonella enterica serovar paratyphi A O-specic polysaccharide was activated with 1-cyano-4-dimethylaminopyrinium tetrauoroborate and
bound to tetanus toxoid with adipic acid dihydrazide as a linker.
b
Not applicable.
c
Salmonella enterica serovar paratyphi A O-specic polysaccharide was activated with 1-cyano-4-dimethylaminopyrinium tetrauoroborate and
bound to tetanus toxoid directly.
Source: Ref. 36.
permeability, but the results have been very dierent authors have presented the synthetic and natural polymers
[190,193195]. In one study, the microcapsules are imper- that can be used to form wound [270].
meable (with a poly-L-lysine of 14 kDa) [193] to immuno- Yasutomi et al. [271] have developed a biosynthetic
globulin, while it is not the case in other studies wound composed of a spongy mixture of chitosan and
[190,194,195]. derivated collagen.
In vivo evaluation of microcapsule involving alginic Yannas et al. [272] have synthesized a dermal substi-
acid and poly-L-lysine, and that entrapped islets, has been tute involving glycosaminoglycan chondroitin-6-sulfate
investigated [266269]. Lim and Sum [266] have trans- and collagen. This material is a layer of 2 mm thick and
planted microcapsules that have been coated with poly- the size of pores is between 70 and 200 Am. The growth of
ethyleneimine into rats; the normoglycemia in diabetic rats brovascular system are possible with this structure [272].
has been maintained only during 2 and 3 weeks. Sum et al. Similar system can be used as composite substitute, in this
have used microcapsules coated with alginic acid in order case human broblasts have been seed in collagenglyco-
to increase the biocompatibility of these systems. In this aminoglycan membrane and the growth of this cell have
case, these systems can maintain a normoglycemia in been observed [273,274]. Cooper and Hansbrough have
diabetic animal models for long time [267]. In another tested this membrane in athymic mouse model [275].
study, Wu et al. [269] also reported a clinical study of
allotransplantation of fetal human islets in the insulin- 4. Matrix or Surface Used for Hemocompatibility
dependent diabetes recipients.
Matrix for Hemoperfusion: Chitosan Beads
3. Wound for Immobilization of Poly-L-Lysine
In a recent paper, Sheridan et al. [270] have presented the Hemoperfusion is an alternative for the treatment of
role of biomaterial for wound dressing. In this review, the hyperbilirubinemia. Poly-L-lysine immobilized into chito-
Figure 50 SEM micrographs of bovine endothelial cells cultured on various photopolymerized and covalently xed surfaces:
(A) polystyrene; (BC) nontreated surface; (D) surface with albumin; (E) surface with heparin and albumin; (F) surface with
albumin and bronectin; (G) surface with heparin, albumin, and basic broblast growth factor; and (H) surface with heparin,
albumin, bronectin, and basic broblast growth factor. (From Ref. 284.)
san bead is a superabsorbent for bilirubin [276]. Moreover, poly(acrylic acid)-grafted surface. Styrenated heparin and
the capacity to bind bilirubin on these systems is directly styrenated albumin have been adsorbed on a photoirradi-
proportional to the number of lysine residues. In this study, ated poly(acrylic acid) surface, and a chemical reaction
the main conclusion is that the chitosan bead coated with took place after photoirradiation and condensation of this
poly-L-lysine and albumin show an very good adsorbent surface [284].
power for hemoperfusion. Moreover, the coating with The double treatment with heparin and albumin
poly-L-lysine and albumin give an excellent blood compat- decreases the platelet adhesion, while this surface increases
ibility of this system [277]. the adhesion and proliferation of bovine endothelial cells
Matrix Involving Heparin and Albumin as Sealants (Figs. 50 and 51). Moreover, the co-immobilization of
for Prosthetic Vascular Grafts bronectin and basic broblast growth factor enhances
the bovine endothelial cell adhesion and proliferation. The
Many papers [278283] deals with the used of albu-
main conclusion of this study is that this surface treated
min-heparin conjugate as sealants for prosthetic vascular
with heparin and albumin can be used for articial and
grafts. Albumin plays an important role because it reduce
tissue-engineered devices due to the adhesion and prolifer-
platelet adhesion while heparin interact with antithrombin
ation of endothelial cell and the nonadhesion of platelets
III to prevent thrombus formation [278282]. Cross-linked
[284].
gels of albumin as well as heparinized albumin gels as
potential sealant of prosthetic vascular grafts, have been Surface Layer for Hemodialysis
studied with regard to in vitro stability, binding of basic
Hemodialysis is a therapeutic treatment of chronic
broblast growth factor, and cellular interactions [283].
renal disease to remove an excess of water and uremic
The authors have been concluded that the hydrogel involv-
toxins from the patient blood by dialysis under extracor-
ing albumin and heparin is a best candidate for sealant of
poreal blood circulation. A membrane is used in the
prosthetic vascular grafts and suitable substrates for endo-
dialyzer to remove blood toxins. Synthetic membranes
thelial cell seeding [283].
[20,285], natural membrane [20,285], and polysaccha-
Surface Layer for Cellular Adhesion Responses: ridesprotein membrane can be used [20,286]. Ohno et al.
Polymerized Mixed Heparin/Albumin Surface [286] have shown that cellulose acetate membrane has an
Magoshi and Matsuda [284] have studied the forma- excellent blood compatibility if this monolayer of cellulose
tion of dense albuminated and heparinized layer on a acetate coverage of the surface with serum albumin.
Figure 51 Time-dependent number of bovine endothelial cells adhered on various photopolymerized and covalently xed
surfaces: (A) polystyrene; (BC) nontreated surface; (D) surface with albumin; (E) surface with heparin and albumin; (F) surface
with albumin and bronectin; (G) surface with heparin, albumin, and basic broblast growth factor; and (H) surface with
heparin, albumin, bronectin, and basic broblast growth factor. (From Ref. 284.)
Covalent AntithrombinHeparin Complexes better if this implant allows the colonization by functional
Used as Anticoagulant cellules [58].
In a recent review, Berry et al. [23] presented the ad- Three-dimensional scaold systems are generally po-
vantages, the limitations, the potential uses, and the con- rous microstructure which allows cellular growth and
cept to make the antithrombinheparin complexes. scaold colonization in 3-D [58]. Some important proper-
Antithrombin is a protein that plays an important role ties of three-dimensional scaold system are biocompati-
in the anticoagulation cascade due to its inhibitive proper- bility, bioadhesion, biodegradability, interaction with cells,
ties against many enzymes that have been secreted during tissue mechanical properties, macromolecular permeabili-
the coagulation cascade in vivo [23]. Heparin is a polysac- ty, protein binding or repulsion, and tissue adhesion or
charide well known for its anticoagulant properties [23]. lubricity. Moreover, three-dimensional scaold system can
These complexes can be used to inhibit rapidly the release drugs or important factors or biologically active
thrombin in the case of anticoagulant prophylaxis, in the agents to enhance the cell migration and proliferation into
antithrombic treatment, and in selective organ treatment the matrix or to target tissue [58].
(for respiratory distress syndrome, for example, due to the
big size of the complex) [23]. In another paper, Chan et al. 1. Hydroxyapatite/ChitosanGelatin Network
[287] have shown that antithrombinheparin complex can Hydroxyapatite/chitosangelatin (HA/CSGel) network
be a possible alternative to heparin for arterial thrombosis composite has been developed as a three-dimensional
prevention [287]. biomimetic network by phase separation method. This 3-
Fig. 52 shows the eciency of standard heparin (SH) D network can be used to bone tissue engineering [56]. Fig.
and antithrombinheparin complex (ATH) on the weight 53 shows the porous structure of this network. The sizes of
of intravascular clot in rabbit model [287]. The antithrom- the pores vary between several microns and 500 Am.
bic eect is greater with ATH (the clot weight is smaller) Fig. 54 shows the adhesion, proliferation, and expres-
than with SH. There are no clots with ATH 33 and 65 IU/ sion of rat calvaria osteoblasts on the hydroxyapatite/
kg. These results have been conrmed with blood ow and chitosangelatin network with a porosity of 90.6% and
the cumulative blood ow is more rapid for rabbit that has with pores of 300500 Am. In Fig. 54, the culture of
been treated with ATH compared to those who have been osteoblast on hydroxyapatite/chitosangelatin network
treated with SH [287]. has been stained by hematoxylin and eosin for visualization
of the osteoblasts.
D. Application in Tissue Engineering and Moreover, with an immunohistologic observation,
Three-Dimensional Scaffold System Zhao et al. [56] have shown that the osteoblasts proliferat-
ed along the pore wall structures of the matrix and the cell/
The aim of tissue engineering is to develop new materials scaold constructs had good biomineralization eect after
that facilitate the reparation, the regeneration, or the 3 weeks in culture.
replacement of damaged tissues [58]. In the case of repara-
tion or regeneration, tissue engineering interacts in order to 2. Hydroxyapatite/Hyaluronic
improve natural reparation process with new functional AcidCollagen Network
tissue in place of damaged tissue. In case of replacement,
implanted materials should replace the tissue, and it is A composite material involving a network composed of
hyaluronic acid and collagen and inorganic component:
hydroxyapatite has been studied by Bakos et al. [59]. The
structure evaluation of the composite showed more dense
arrangement due to the formation of collagenhyaluronic
acid conjugate, and particles of hydroxyapatite are closely
anchored in the structure. The test of contact cytotoxicity
showed a very good biocompatibility of this biomaterial
and confers at this matrix a potential for scaold system
[59].
3. Calcium Alginate Network and Bone

Morphogenetic Proteins
Bone morphogenetic proteins (BMPs) are unique mole-
cules with a specic biological activity for inducing ectopic
bone formation when implanted with a suitable carrier
matri x [ 288]. A n ovel BMP-2-der ived pepti de,
NSVNSKIPKACCVPTELSAI was covalently coupled to
alginate. NSVNSKIPKACCVPTELSAI-linked alginate
Figure 52 Inhibition of intravascular clot formation by hydrogel composites were implanted into the calf muscle
ATH and SH. Clot weight in treated groups was expressed as of rats and harvested 3 or 8 weeks after surgery. Ectopic
a fraction (%) of control saline. (From Ref. 287.) bone formation was observed in alginate hydrogel linked
collagen-blended chitosan substrate integrated well with

the hydrogel matrix and survived for at least 2 weeks under
in vivo conditions. Collagen-blended chitosan substrates
produce signicantly improved bovine adrenal medullary
chroman cell attachment characteristics, which can be
considered for neural tissue engineering studies. Morpho-
logical examinations revealed that the chroman cells
survived for at least 2 weeks with collagenchitosan scaf-
folds [60].
The eectiveness of chitosan as a scaold of hepato-
cyte attachment was examined [61]. Since chitosan gel was
too fragile to use for cell culture, its free amino groups were
cross-linked by glutaraldehyde to increase its strength. Rat
hepatocytes seeded onto glutaraldehyde-cross-linked chi-
tosan (GA-chitosan) gel could attach with stability to the
surface, retaining its spherical form, the same as in vivo,
and then release a very small amount of lactate dehydro-
genase during the 5-day culture period. By contrast, hepa-
tocytes on a collagen-coated surface spread at, and they
release much more lactate dehydrogenase than those on the
GA-chitosan gel. Hepatocytes on GA-chitosan also
retained higher urea synthesis activity, a liver-specic
function, than those on the collagen-coated surface. These
results indicate that chitosan is a promising biopolymer as
a scaold of hepatocyte attachment which can be applied
to an eective bioarticial liver support system [61].
With an aim of improving bone regeneration, chitosan
sponge containing platelet-derived growth factor-BB
(PDGF-BB) was developed. PDGF-BB was incorporated
into the chitosan sponge by soaking chitosan sponge into
the PDGF-BB solution. Release kinetics of PDGF-BB, cell
attachment, proliferation capacity, and bony regenerative
potentials of PDGF-BB-loaded chitosan sponge were in-
Figure 53 SEM micrographs of HA/CSGel scaolds vestigated [292]. Release rate of PDGF-BB could be con-
prepared from HA/CSGel/acetic acid mixtures with dier- trolled by varying initial loading content of PDGF-BB to
ent CSGel concentrations: (a) CSGel %: 1.0%, HA/CS obtain optimal therapeutic ecacy. PDGF-BB-loaded chi-
Gel: 50/50 and (b) CSGel %: 2.5%, HA/CSGel: 30/70. tosan sponge induced signicantly high cell attachment
(From Ref. 56.) and proliferation levels, which indicated good cellular
with BMP-2-derivated peptide. Moreover, the authors

suggested that alginate hydrogel linked with an oligopep-
tide derivated from BMP-2 might provide an alternative
system for topical delivery of the morphogenic signal of
BMP-2 [288].
4. Chitosan Network
It is known that glycosaminoglycans are involved in cell
cell and cellmatrix interactions and probably act as
modulators of cell morphology, dierentiation, movement,
synthesis, and function [289291]. Chitosan, having struc-
tural similarity to glucosaminoglycans, was modied using
several proteins (collagen, albumin, and gelation) to in-
crease surface area and improve biocompatibility. In vitro,
collagen-blended chitosan matrices were found to attach Figure 54 SEM micrographs of osteoblasts/scaolds con-
more readily to chroman cells than gelatin-blended ma- struct. The osteoblasts were well attached to the pore wall
trices or albumin-blended matrices [60]. Morphological and surrounded by an excreted interlaced brous network.
evidence showed that the chroman cells attached to (From Ref. 56.)
adaptability. PDGF-BB-loaded chitosan sponge demon- calcium, sodium, magnesium, iron, or potassium. For
strated marked increase in new bone formation and rapid example, if the complexation is made above isoelectric
calcication. Degradation of the chitosan sponge was point of polypeptide, the metal ions allow to facilitate the
proceeded at defect site and subsequently replaced with reaction.
new bone. Histomorphometric analysis conrmed that
PDGF-BB-loaded chitosan sponge signicantly induced 2. Protease Coupled to a Polysaccharide
new bone formation. These results suggested that chitosan In two patents, cosmetic products involving a modied
sponge and PDGF-BB-loaded chitosan sponge may be protease by coupling to polysaccharide have been pre-
benecial to enhance periodontal bone regeneration sented [54,55].
[292]. The ability of cultured human foreskin broblasts Modied protease is produced in three steps: (1) the
to bind and to contract lattices of collagen, collagen synthesis of triazine ring-bound polysaccharide by the
chitosan, and collagenchitosan sulfate was determined reaction between polysaccharide and cyanuric trichloride;
[62]. Scanning electron microscopy of the cellular lattices (2) the reaction between triazine ring-bound polysaccha-
showed bers of the collagenchitosan mixture to be the ride and protease where the triazine ring varies between 0.4
thickest and with altered organization. These results show and 1.2 mmol/g and the molar ratio between triazine ring
that chitosan sulfation markedly enhances broblast ad- and amino group of protease is superior at 2:1; and (3) heat
hesion and promotes contraction of a collagen lattice treating [54].
compared to the unsulfated material. By analogy to the The aim of this modication is to decrease the antige-
in vivo sequence of hyaluronan followed by sulfated gly- nicity, decrease the skin sensibilization, and improve the
cosaminoglycans in wounds, the results suggest that gly- activity and stability of this enzyme [54].
cosaminoglycan sulfation may be a contributing signal for The protease used are trypsin and chymotrypsin of
phenotypic transformation during wound healing [62]. animal origin, microbial proteases. The polysaccharides
used are agarose, guar gum, inulin, starch, dextran, pul-
5. Collagen and Glycosaminoglycans Network lulan, xanthan, carrageenan, pectin, alginate, and some
The association of collagen and glycosaminoglycans via cellulose derivated. Polysaccharides can decrease the anti-
electrostatic and covalent linkages can be used as three- genicity [54].
dimensional scaold system [58]. After controlled freeze- This modied protease can be used for skin cream,
dried method, the complex involving collagen and glycos- lotion, or milk; cleansing cream, lotion, or milk; cold
aminoglycan (GAG) forms a porous 3-D microstructure cream; cream soap; makeup; calamine lotion; T zone
that can be used to skin repair/regeneration, cartilage essence; hand cream; essence, whitening, or soap powder;
repair, and nerve regeneration [58,6365]. transparent soap; lipstick or lip cream; nourishing essence;
face or eye-shadow powder; nail remover; hair tonic,
liquid, cream, or treatment; shampoo, sun oil; etc. [54,55].
E. Applications in the Cosmetical Industry
Polysaccharides and polypeptides are used in the cosmetic F. Applications in the Food Industry
composition. Polysaccharides have some important prop-
erties as humectants, lm formers, gelling ability, high 1. Emulsion Properties
viscosity, and skin moisturizers. Polypeptides are known Interpolymer complexes involving polysaccharide and
to be excellent lm formers, conditioning agents, and polypeptides are known to have the capacity to form and
moisturizers, but their uses are dicult due to their stabilize emulsions [39,44,185]. This type of interaction can
unstability and they are easy hydrolyzable [97]. modify the rheology of the systems [185].
The stabilization of interpolymer complexes involving
1. Complexes of Polysaccharides and Polypeptides polysaccharide and polypeptides is very interesting for
In a recent patent, Wang et al. [97] have presented the many applications [293], particularly for food application
invention of protein/polysaccharide complex (PPC) in- because these macromolecules are biodegradable [134].
volving anionic polysaccharide, and this PPC is a com- Table 17 shows some examples of complexes involving
pound of emulsion cosmetic for skin or hair. The polysaccharide and polypeptide and used as emulsier.
polysaccharide used contains a large number of hydro- The interactions between BSA and L-carrageenan have
philic groups to have a net negative charge density, and the been studied by rheology measurement. Fig. 55 shows that
polypeptide contains a large number of carboxyl groups. the complex shear modulus G* is maximum for 0.04 wt.%
Polysaccharides and polypeptides can interact via ionic L-carrageenan. This gure allows to conrm that the high
bond or electrostatic interaction. Many PPCs have been pressure changes the interactions between polypeptides
studied with the following polysaccharides: galactan, ga- and polysaccharide [185].
lactomannan, glucomannan, agarose, agar, carrageenan,
and mixture thereof, and following polypeptides: casein, 2. Gelling Properties
milk protein, hydrolyzed vegetable protein, and mixture Vaslin et al. [22] have patented a complex involving at least
thereof [97]. one polysaccharide and at least one polypeptide. Alone,
The complexation of polypeptides and polysaccharide these compounds have no gelling properties. The ratio
can be made alone or in presence of metal ions such as between polypeptide and polysaccharide can be between
Table 17 Some Examples of Complexes Involving Polysaccharide and

Polypeptide and Used as Emulsier (Nonexhaustive List)
Alginate Gelatin [311]

Alginate Soy protein [312]
Alginatedimethyl cellulose Gelatin [311]
Carboxymethylcellulose Whey protein [313]
Dextran BSA [314]
L-Carrageenan BSA [184,185]
n-Carrageenan BSA [185]
Acid polysaccharides Casein [315]
Carboxymethyl dextran h-Lactoglobulin [316]
Pectin Whey proteins [317]
Xanthan Whey proteins [182]
Xanthan Casein [318]
Carboxymethylcellulose Casein [318]
Acacia gum Gelatin [319]
2 and 15, preferably 5 and 10. Polysaccharides can be protein (SWP). Fig. 57 shows the gelling time vs. the
galactomannanes, glucomannanes, or xanthan. percent of SWP; an increase of gelling time is observed
Complex forming with xanthan and h-lactoglobulin when the percent of polypeptides into the mixing increases
has been used in order to make preparation that has the [39]. Moreover, the mixture (creamy gel) has a lower GV
texture of butter [22]. Cogranules composed of lactoserum after 2 hr, when SWP concentration increases (Fig. 57).
proteins and carrageenan can be used as gelling agents in SWP can interfere in the formation of gel due to the
cream and ice cream [45]. presence of aggregates [39]. The SWP concentration can
In a recent study, Plashchina et al. [44] have shown the modulate the gelling force.
emulsion properties and stability of legumin and legumin-
T complexed with chitosan. Fig. 56 shows the fraction of 3. Preserving Properties
emulsied decane vs. the protein concentration. First, the Dorsman et al. [50] have developed a complex involving
emulsion stability of legumin-T is dierent that the emul- polysaccharide and polypeptide as preserving agent in the
sion stability of legumin and this dierence is retained food product. Globular protein as albumin, egg, whey, or
when these polypeptides are complexed with chitosan. At
low concentration, there are no signicant dierences
between emulsion stability of free protein compared to
proteinchitosan complexes, but at high concentration, the
complexes are more stable.
Howell et al. [39] have shown the gelling properties of
sodium alginate mixing with deaminated soluble wheat
Figure 56 Dependence of emulsion stability of legumin and

Figure 55 Eect of added L-carrageenan concentration c on legumin-T at pH 7.3 on protein concentration (.: free
complex shear modulus G* at 1 Hz and 30j for BSA- legumin and o: free legumin-T) and complexes with chitosan
stabilized emulsions (40 vol.% oil, 2.7 wt.% protein, 5 mM, at pH 6.3 (E: leguminchitosan and D: legumin-Tchitosan)
pH 6). High-pressure treatment: n: 0 MPa; D: 200 MPa; and (chitosan concentration 0.125% w/w and ionic strength 0.25
o: 500 MPa. (From Ref. 185.) M and T=25jC). (From Ref. 44.)
ylcellulose, pectin, alginate, gellan, xanthan, carrageenan,

chitin, or chitosan are some examples of polysaccharide
that can be used to make this complex [4648]. Casein, soy
protein, egg protein, peanut protein, cottonseed protein,
sunower protein, pea protein, whey protein, sh protein,
albumin, globulin, prolamine, gliadin, etc. are some exam-
ples of polypeptide used in the complexation with polysac-
charide [4648].
Jacobson et al. [49] have studied the fabrication of
nonfat natural cheese. This product is made with skim milk
and an important additive is a complex forming with poly-
saccharide and polypeptide. This complex is a microgel and
the size of particle varies between 0.05 and 30 Am [49].
Complexes involving acacia gum and gelatin as fat
Figure 57 Time taken for mixture of 1% alginate solution substitutes have been patented in 1994 by Bakker et
with varying amounts of SWP, to read gelling point as al.[298]. The ratio between acacia gum and gelatin are
indicated by GV/GW crossover point from a time sweep 1:55:1. The coacervates resulting from this complexation
experiment at 1-Pa stress, strain 3%, and frequency of 1 Hz
are spherical particles having a mean diameter of 11 Am
at 25jC. (From Ref. 39.)
[298].
7. Gustatory Product
soya protein, casein, and carrageenan or alginate can be In a Japan patent, Omura et al. [299] have conjugated zinc
used for the formation of this complex. The advantages of with polysaccharide and polypeptide in order to make a
this complex are preservation and emulsication [50]. safety product with excellent gustatory behavior and avor
for a zinc supplement.
4. Textural Properties: Fiber
Tolstoguzov et al. [294] have patented some complexes G. Applications in the Environmental Field
involving alginate, pectate, or pectins with caseins, soy
protein, or yeast protein, in which their texture is compa- 1. Complexes Involving Chitosan and Polyanion
rable to ber meat. Therefore polysaccharidepolypeptide Used to the Protein Flocculation
complexes can be used as meat analog [294]. In a recent paper, Savant and Teorres [68] have studied the
Soucie and Chen [295] have also patented a complex eect of chitosanpolyanion complexes on the occulation
involving xanthan and soy protein or albumin, and this of protein in suspended solid wastes in cheese whey. Three
brous structure can be used in low-fat meat foods [295]. polyanion have been tested: carrageenan, alginate, and
pectin. Fig. 58 shows the coagulation eciency for three
5. Nutritional Aspect complexes. The coagulation eciency is based on the
Chango et al. [296] used calcium alginate to precipitate decrease of turbidity. In this gure, the eect of time and
proteins from Lipnus luteus L. The process consists of monomeric mixing ratio between chitosan and polyanion
mixing alginate, CaCl2, and bitter yellow lupin at alkaline has been investigated [68]. Measurements beyond 39 hr did
pH. This process allows to separate low alkaloid protein not decrease the turbidity. The mixing ratio is not a
concentrate, alkaloids, and a solid residue rich in protein signicant eect on the decrease of turbidity [68]. The
[296]. The low alkaloid protein concentrate can be used for analysis of coagulated solids has shown an adsorption of
human foodstus [296]. 71.3% of protein with chitosanalginate complex, having a
mixing ratio of 0.2 [68].
6. Low-Fat Products
In the food product, fat is a very important function due to 2. Complexes Involving Polysaccharide and
its avor, aroma, texture, and emulsion properties [134], Polypeptide Used as an Adsorbent and Filtering Aid
but more and more, the consumers want low-fat product In recent patents, McArdle [69,70] has presented a poly-
with the same organoleptic properties [297]. The food saccharidepolypeptide complex (PPC) in removal of con-
industries have developed many fat replacer, and the taminants. This complex can be used as an adsorbent and
complexes involving polysaccharides and polypeptides ltering aid to absorb or decompose some compound
are an alternative for fat replacer [297]. including PCB and dioxins, an odor suppressant or clean-
Chen et al. [4648] have patented an ionic polysaccha- ing, coagulant, or clarifying agent [69].
ridepolypeptide complex. It is an aqueous dispersion Polypeptides used in this PPC are vegetable protein
containing insoluble microfragmented polysaccharide such as corn prolamine or zein, barley or hordein, or wheat
polypeptide complex. This complex can be used as fat prolamine or gliadin. Polysaccharides that can be used are
substitute in many food products, e.g., ice cream, salad alginate, carrageenan cellulose derivated, arabic gum, guar
dressing, dips, spreads, sauces, etc. [4648]. Carboxymeth- gum, pectin, xanthan gum, or mixtures [69].
Figure 58 Eect of polyanion, mixing ratio, and treatment time on turbidity reduction of Cheddar cheese whey at two chitosan
polyanion concentration: (a) 10 mg polymer/L whey and (b) 30 mg polymer/L whey. (From Ref. 68.)
H. Applications in Technological Field anity between polysaccharide and polypeptide as a li-

gand/substrate and the anity ligand conjugated to a
1. Purication of Proteins reversible solubleinsoluble polymer [300305].
Many technologies and processes, involving polysacchar-
ides, have been developed to make the purication of Carboxymethylcellulose
polypeptides [300306]. The bioseparation method is based In 1971, Hidalgo and Hansen [307] have shown that
on the anity precipitation and this technique is highly carboxymethylcellulose can be used to make a selective
selective [300]. This bioseparation is based on the macro- precipitation of whey polypeptides. At pH 4.0, carboxy-
alginate chains, and these later give better displacement.

Fig. 59 shows the chromatographic spectra of separation of
albumin on DEAE-cellulose by displacement chromatog-
raphy using unmodied commercial alginate and sonicated
alginate as displacers [309].
Heparin
In a recent paper, Heger et al. [310] have used heparin
anity chromatography to separate active and inactive
forms of human antithrombin. Antithrombin is a polypep-
tide having a molecular weight of 58.000 Da and can be
used as pharmaceutical product (inhibitor activity against
factor Xa), but before the commercialization, antithrom-
bin is treated by heating (60j) to inactive infective virus.
Figure 59 Separation of albumin on DEAEcellulose by This process can alter the conformation of the polypeptide,
displacement chromatography using unmodied commercial and this latter becomes inactive; therefore the high anity
alginate as displacer. Chromatographic conditions were as between heparin and antithrombin has been used in anity
follows: column 10010 mm was packed with 2.5 ml of
chromatography [310].
DEAEcellulose A-52; carrier buer: 20 mM sodium
Fig. 60 shows an illustration of the dierent steps to
phosphate, pH 7.5; sample: 30 mg albumin in 1.0-mL carrier
buer; displacer: 5 mg/mL native alginate in carrier buer;
separate dierent forms of antithrombin by heparin anity
ow rate: 0.2 mL/min; fraction size: 0.4 mL/fraction. (From chromatography [310].
Ref. 309.) The rst step of purication is the separation of a- and
h-forms by heparin anity chromatography. The elution
of a- and h-forms are realized with 1.3 and 2.0 M NaCl,
respectively, and the human antithrombin used contains
methylcellulose interacts with h-lactoglobulin and bovine
serum albumin after cooling at 2jC. Moreover, the yield of
protein recovery is 5.1 g/L for a ratio of carboxymethyl-
cellulose/protein equal to 0.28.
With the same ratio of 0.28, the precipitation of h-
lactalbumin by carboxymethylcellulose is possible if the pH
is adjusted at 3.2. In this case, the yield of protein recovery
is 0.7 g/L.
Alginate
In a recent study, Teotia et al. [300] have shown that it
is possible to purify in one step a sweet potato h-amylase by
anity precipitation using sodium alginate and CaCl2 with
a yield of 80%.
2. Chromatographic matrix
Alginate
A matrix formed with calcium alginate and epichlo-
rohydrin has been used for anity and ion exchange
chromatography of proteins [308]. In this study, the
authors have compared the separations of proteins with
alginate beads and alginate cross-linking with epichloro-
hydrin. Beads formed with alginate cross-linking with
epichlorohydrin are stable in eluant having pH between 1
and 13 in a temperature range between 0jC and 100jC, and
they resist at high ionic strength and polar solvents [308].
Due to its characteristics, alginate cross-linking with epi-
chlorohydrin is more suitable as chromatographic matrix
(high mechanical resistance).
In another study, alginate can be used as displacer for
protein displacement chromatography. Chen and Scouten
[309] have tested unmodied alginate and alginate treated
by ultrasound as displacers for protein displacement chro- Figure 60 Schema of the production of dierent active and
matography. Ultrasound treatment allows to form smaller inactive forms of antithrombin. (From Ref. 310.)
approximately 7% of h-antithrombin [310]. The second The authors think that this new method is a very good
step of purication allows to separate active and inactive way to study the interactions between polysaccharide and
forms of antithrombin. The last step is separation of polypeptides.
dierent inactive forms by heparin anity chromatogra-
phy [310].
REFERENCES
3. ELISA Test
1. Beijerinck, M.W. Uber eine eigentumlichkeit der loslichen
Fibrinogen is a protein that increases in the blood in the starke. Zent.bl. Bakteriol. Parasitenkd. Infekt.krankh.
case of cardiovascular disease. The level of brinogen in the 1896, 2, 697.
blood could be quantied by a new method: SPC-ELISA: 2. Beijerinck, M.W. Uber emulsionsbilding bei der vermi-
sulfated polysaccharide-coating enzyme-liked immunosor- schung wasseriger losungen gewisser gelaninierender
bent assay [3]. This new method is based on classic ELISA kolloide. Z. Chem. Ind. Kolloid. 1910, 7, 16.
3. Alban, S.; Gastpar, R. Development of SPC-ELISA: A
technology, which allows high throughput screening. This new assay principle for the study of sulfated polysaccha-
new technique uses the binding properties of sulfated rideprotein interactions. J. Biomol. Screen. 2001, 6, 393.
polysaccharides to polystyrene and allows to coat the 4. Dumitriu, S.; Bulacovschi, V. Bioactive polymers vii.
microplates with sulfated polysaccharides. Moreover, Oxidoreductases and hydrolases immobilized on cellulose
these sulfated polysaccharides can interact with protein. derivatives. Rev. Roum. Chim. 1980, 25, 989.
Heparin, dermatan sulfate, fucoidans, and semisynthetic 5. Dumitriu, S.; Magny, P.; Montagne, D.; Vidal, P.F.;
Chornet, E. Polyionic hydrogel obtained by complexation
glucan sulfates have been investigated. These compounds
between xanthan and chitosan: Their properties as sup-
have dierent anity with brinogen, and polysaccharides ports for enzyme immobilization. J. Bioact. Compat.
with high anity to this polypeptide might be used to lower Polym. 1994, 9, 184.
brinogen concentration. The quantity colorimetric dos- 6. Magnin, D.; Dumitriu, S.; Magny, P.; Chornet, E. Lipase
age is made with antihuman brinogen antibody and immobilization into porous chitosan beads: Activities in
secondary antibody [3]. aqueous and organic media and lipase localization. Bio-
The binding of brinogen to sulfated polysaccharides technol. Prog. 2001, 17, 734.
7. Magnin, D.; Dumitriu, S.; Chornet, E. Two applications
is time-dependent, and the concentration of primary anti- of a polyionic hydrogel: enzymes immobilization and poly-
body and secondary antibody inuence is an important peptides-drug delivery systems. In Advanced materials for
parameter and should be xed. biomedical applications. Symposium on Advanced Materi-
Fig. 61 shows the inuence of polysaccharides used to als for Biomedical Application; COM2002: The Confer-
coat the microplate. The binding prole is dependent on ence of Metallurgists, 2002.
brinogen concentration and varies with polysaccharides 8. Gomez, L.; Ramirez, H.L.; Villalonga, R. Modication of
alpha-amylase by sodium alginate. Acta Biotechnol. 2001,
used. Fibrinogen anity with heparin was too weak to 3, 265.
observe a complete sigmoid curve, while this prole is ob- 9. Oh, D.K.; Kim, H.J.; Ryu, S.A.; Rho, H.J.; Kim, P. De-
served for sulfated glucunogalactan and xanthan sulfate velopment of an immobilization method of L-arabinose
[3]. isomerase for industrial production of tagatose. Biotech-
nol. Lett. 2001, 23, 1859.
10. Dashevsky, A. Protein loss by the microencapsulation of
an enzyme (lactase) in alginate beads. Int. J. Pharm. 1998,
161, 1.
11. Roig, M.G.; Rashid, D.H.; Kennedy, J.F. High-alkaline
protease from Bacillus pb92 entrapped in calcium alginate
gel. Physicochemical and microscopic studies. Appl.
Biochem. Biotechnol. 1995, 55, 95.
12. Arruda, L.M.O.; Vitolo, M. Characterization of invertase
entrapped into calcium alginate beads. Appl. Biochem.
Biotechnol. 1999, 81, 23.
13. Chen, J.P.; Wang, H.Y. Improved properties of bilirubin
oxidase by entrapment in alginatesilicate solgel matrix.
Biotechnol. Tech. 1998, 12, 851.
14. Hsu, A.F.; Foglia, T.A.; Piazza, G.J. Immobilization of
lipoxygenase in an alginatesilicate solgel matrix: For-
mation of fatty acid hydroperoxides. Biotechnol. Lett.
1997, 19, 71.
15. Pereira, E.B.; De Castro, H.F.; De Moraes, F.F.; Zanin,
G.M. Esterication activity and stability of Candida
rugosa lipase immobilized into chitosan. Appl. Biochem.
Figure 61 Fibrinogen binding proles of sulfated polysac- Biotechnol. 2002, 98100, 977.
charides: unfractionated heparin (x), a sulfated glucurono- 16. Darias, R.; Villalonga, R. Functional stabilization of
galactan from red algae (.), and a semisynthetic xanthan cellulase by covalent modication with chitosan. J. Chem.
sulfate (n). The amount of immobilized polysaccharide was Technol. Biotechnol. 2001, 76, 489.
10 Ag/well. (From Ref. 3.) 17. Chivers, M.L. Clarication of protein precipitate suspen-
sions using anionic polymeric occulants. U.S. Patent 34. Passwell, J.H.; Harlev, E.; Ashkenazi, S.; Chu, C.; Miron,
0058786, 2002. D.; Ramon, R.; Farzan, N.; Shiloach, J.; Bryla, D.A.;
18. Vandenberg, G.W.; De La Noue, J. Evaluation of protein Majadly, F.; Roberson, R.; Robbins, J.B.; Schneerson, R.
release from chitosanalginate microcapsules produced Safety and immunogenicity of improved shigella o-specic
using external or internal gelation. J. Microencapsul. 2001, polysaccharideprotein conjugate vaccines in adults in
18, 433. Israel. Infect. Immun. 2001, 69, 1351.
19. Chang, K.L.B.; Lin, J. Swelling behavior and the release of 35. Konadu, E.Y.; Donohue-Rolfe, A.; Calderwood, S.B.;
protein from chitosanpectin composite particles. Carbo- Pozsgay, V.; Shiloach, J.; Robbins, J.B.; Szu, S.C.
hydr. Polym. 2000, 43, 163. Syntheses and immunologic properties of Escherichia coli
20. Dumitriu, S. Polysaccharides as biomaterials. In Polymeric o157 o-specic polysaccharide and shiga toxin 1 b subunit
Biomaterials, Second Edition, Revised and Extended; conjugates in mice. Infect. Immun. 1999, 67, 6191.
Dumitriu, S., Ed.; Marcel Dekker, Inc.: New York, 36. Konadu, E.Y.; Lin, F.Y.; Ho, V.A.; Thuy, N.T.; Van Bay,
2002; 1 pp. P.; Thanh, T.C.; Khiem, H.B.; Trach, D.D.; Karpas, A.B.;
21. Hejazi, R.; Amiji, M. Chitosan-based delivery systems: Li, J.; Bryla, D.A.; Robbins, J.B.; Szu, S.C. Phases 1 and
Physicochemical properties and pharmaceutical applica- 2 studies of Salmonella enterica serovar paratyphi a o-
tions. In Polymeric Biomaterials, Second Edition, Revised specic polysaccharidetetanus toxoid conjugate in adults,
and Extended; Dumitriu, S., Ed.; Marcel Dekker, Inc.: teenagers, and 2- to 4-years-old children in Vietnam. In-
New York, 2002; 213 pp. fect. Immun. 2000, 68, 1529.
22. Vaslin, S.; Simonet, F.; Doublier, J.L.; Garnier, C. Com- 37. Jones, C.; Crane, D.T.; Lemercinier, X.; Bolgiano, B.;
position contenant un melange de polysaccharides et de Yost, S.E. Physicochemical studies of the structure and
proteine(s) globulaire(s), son procede de preparation et stability of polysaccharideprotein conjugate vaccines.
leurs utilisations. Fr. Patent 2821244, 2002. Dev. Biol. Stand. 1996, 87, 143.
23. Berry, L.R.; Andrew, M.; Chan, A.K.C. Antithrombin 38. Turgeon, HS.L.; Beaulieu, M. Improvement and modi-
heparin complexes. In Polymeric Biomaterials, Second cation of whey protein gel texture using polysaccharides.
Edition, Revised and Extended; Dumitriu, S., Ed.; Marcel Food Hydrocoll. 2001, 15, 583.
Dekker, Inc.: New York, 2002; 661 pp. 39. Howell, N.; Bristow, E.; Copeland, E.; Friedli, G.L.
24. Villa, A.; Sanchez, A.; Tobio, M.; Calvo, P.; Alonso, M.J. Interaction of deamidated soluble wheat protein with
Design of biodegradable particles for protein delivery. sodium alginate. Food Hydrocoll. 1998, 12, 317.
J. Control. Release 2002, 78, 15. 40. Galazka, V.B.; Dickinson, E.; Ledward, D.A. Inuence of
25. Ito, H.; Shimura, K.; Itoh, H.; Kawade, M. Antitumor high pressure processing on proteinpolysaccharide inter-
eects (ATOM) prepared from agaricus blazei (iwade actions in emulsions. Spec. Publ. R. Soc. Chem. 2001, 258,
strain 101) himematsutake and its mechanisms in 315.
tumor-bearing mice. Anticancer Res. 1997, 17, 277. 41. Ibanoglu, E. High pressure eect on foaming properties of
26. Shafer, D.E.; Schuman, R.F.; Lees, A. Rapid and complete h-lactoglobulin and dextran sulfate mixture. Nahrung
adsorption of unconjugated protein from protein 2001, 45, 342.
polysaccharide conjugate vaccines. Vaccine 2001, 19, 1547. 42. He, M.; Horikawa, Y. Partial deocculation of mutual
27. Lees, A. Coupling of unmodied proteins to haloacyl or ocs of allophane and halloysite by xanthan and chitosan
dihaloacyl derivatized polysaccharides for the preparation and relevance to particle arrangement in the ocs. Soil Sci.
of proteinpolysaccharide vaccines. U.S. Patent 6087328, Plant Nutr. 2000, 46, 81.
2000. 43. McArdle, B. Proteinpolysaccharide complex composition
28. Lees, A. Proteinpolysaccharide conjugate vaccines and and method of use. U.S. Patent 5645880, 1997.
other immunological reagents prepared using homobi- 44. Plashchina, I.G.; Mrachkovskaya, T.A.; Danilenko, A.N.;
functional and heterobifunctional vinylsulfones, and pro- Kozhevnikov, G.O.; Starodubrovskaya, N.Yu; Braudo,
cesses for preparing the conjugates. U.S. Patent 6309646, E.E.; Schwenke, K.D. Complex formation of faba bean
2001. legumin with chitosan: Surface activity and emulsion
29. Jenning, H.J.; Kasper, D.L. Group b streptococcus type v properties of complexes. Spec. Publ. R. Soc. Chem. 2001,
polysaccharideprotein conjugate vaccines, U.S. Patent 258, 293.
5795580, 1998. 45. Vaslin, S.; Bourriot, S.; Kiefer, J.C.; Bost, P.E.; Barachon,
30. Jenning, H.J.; Kasper, D.L. Group b streptococcus type ii N.; Lambert, D. Cogranules de proteines et depolysac-
and type v polysaccharideprotein conjugate vaccines. charides, leur procede dobtention et leurs utilisations.
U.S. Patent 5993825, 1999. Fr. Patent 2817261, 2002.
31. Devi, S.J.N.; Zollinger, W.D.; Snoy, P.J.; Tai, J.Y.; 46. Chen, W.S.; Henry, G.A.; Gaud, S.M.; Miller, M.S.;
Costantini, P.; Norelli, F.; Rappuoli, R.; Frasch, C.E. Kaiser, J.M.; Balmadeca, E.A.; Morgan, R.G.; Baer,
Preclinical evaluation of group b Neisseria meningitidis C.C.; Borwankar, R.P.; Hellgeth, L.C.; Strandholm, J.J.;
and Escherichia coli k92 capsular polysaccharideprotein Hasenheutti, G.L.; Kerwin, P.J.; Chen, C.C.; Kratochvil,
conjugate vaccines in juvenile rhesus monkeys. Infect. J.F.; Lloyd, W.L. Microfragmented ionic polysaccharide/
Immun. 1997, 65, 1045. protein complex dispersions. E.P. Patent 0340035, 1989.
32. Zollinger, W.D.; Moran, PE.E.; Devi, S.J.N.; Frasch, C.E. 47. Chen, W.S.; Henry, G.A.; Gaud, S.M.; Miller, M.S.;
Bacterial antibody responses of juvenile rhesus monkeys Kaiser, J.M.; Balmadeca, E.A.; Morgan, R.G.; Baer,
immunized with group b Neisseria meningitidis capsular C.C.; Borwankar, R.P.; Hellgeth, L.C.; Strandholm, J.J.;
polysaccharideprotein conjugate vaccines. Infect. Im- Hasenheutti, G.L.; Kerwin, P.J.; Chen, C.C.; Kratochvil,
mun. 1997, 65, 1053. J.F.; Lloyd, W.L. Microfragmented ionic polysaccharide/
33. Devi, S.J.; Hayat, U.; Frasch, C.E.; Kreger, A.S.; Morris, protein complex dispersions. U.S. Patent 5104674, 1989.
J.G. Jr. Capsular polysaccharideprotein conjugate vac- 48. Chen, W.S.; Henry, G.A.; Gaud, S.M.; Miller, M.S.; Kai-
cines of carbotype 1 vibrio vulnicus: Construction, im- ser, J.M.; Balmadeca, E.A.; Morgan, R.G.; Baer, C.C.;
munogenicity, and protective ecacy in a murine model. Borwankar, R.P.; Hellgeth, L.C.; Strandholm, J.J.; Hasen-
Infect. Immun. 1995, 63, 2906. heutti, G.L.; Kerwin, P.J.; Chen, C.C.; Kratochvil, J.F.;
Lloyd, W.L. Microfragmented ionic polysaccharide/pro- 68. Savant, V.D.; Teorres, J.A. Chitosan-based coagulating
tein complex dispersions. W.O. Patent 10068, 1989. agents for treatment of cheddar cheese whey. Biotechnol.
49. Jacobson, K.A.; Hamann, A.C.; Alm, W.L.; Kerrigan, Prog. 2000, 16, 1091.
G.L.; Borwankar, R.P.; Hynes, J.T.; Carrell, N.A.; Nath, 69. McArdle, B. Use of proteinpolysaccharide complex in
K.R.; Reddy, D.S. Non-fat natural cheese. W.O. Patent removal of contaminants. U.S. Patent 6197199, 2001.
06598, 1992. 70. McArdle, B. Method of using a lter aid proteinpolysac-
50. Dorsman, C.; Van Roon, J.H.; Twaalhoven, L.C. Food charide complex composition, U.S. Patent 5942123, 1999.
compositions. U.K. Patent 1440181, 1972. 71. Bernkop-Schnurch, A. Mucoadhesive polymers. In Poly-
51. Braudo, E.E.; Plashchina, I.G.; Schwenke, K.D. Plant meric Biomaterials, Second Edition, Revised and Extended;
protein interactions with polysaccharides and their inu- Dumitriu, S., Ed.; Marcel Dekker, Inc.: New York, 2002;
ence on legume protein functionality. A review. Nahrung 147 pp.
2001, 45, 382. 72. Jimenez-Barbero, J.; Asensio, J.L.; Javier-Canada, F.;
52. Benichou, A.; Aserin, A.; Garti, N. Proteinpolysacchar- Poveda, A. Free and protein-bound carbohydrate struc-
ide interactions for stabilization of food emulsions. J. tures. Curr. Opin. Struct. Biol. 1999, 9, 549.
Dispers. Sci. Technol. 2002, 23, 93. 73. Taipale, J.; Keski-oja, J. Growth factors in the extra-
53. Wang, T.X.; DiGirolamo, D.M.V.; Russ, J.G. Cosmetic cellular matrix. FASEB J. 1997, 11, 51.
compositions containing polysaccharide/protein com- 74. Conrad, H.E. Heparin-Binding Proteins; Academic Press:
plexes. U.S. Patent 6197319, 2001. San Diego, 1998.
54. Nakayama, H.; Fukunage, S.; Fujino, Y.; Mori, K. Modi- 75. Hernaiz, M.J.; Yang, H.O.; Gunay, N.S.; Toida, T.;
ed protease, method of producing the same and cosmetic Linhardt, R.J. Purication and characterization of hepar-
products containing the modied protease, U.S. Patent an sulfate peptidoglycan from bovine liver. Carbohydr.
5133968, 1992. Polym. 2002, 48, 153.
55. Nakayama, H.; Fukunage, S.; Fujino, Y.; Mori, K. Modi- 76. Van Gorp, C.L.; Toida, T.; Hileman, R.E.; Schubert, R.L.;
ed protease, method of producing the same and cosmetic Grin, C.C.; Lindhart, R.J.; Brown, S.E. Isolation and
products containing the modied protease, U.S. Patent characterization of heparan sulfate from crude porcine
5230891, 1993. intestinal mucosal peptidoglycan heparin. Carbohydr.
56. Zhao, F.; Yin, Y.; Lu, W.W.; Leong, J.C.; Zhang, W.; Res. 1995, 276, 183.
Zhang, J.; Zhang, M.; Yao, K. Preparation and histological 77. Kimata, K.; Zhuo, L. SHAP, a protein covalently bound
evaluation of biomimetic three-dimensional hydroxyapa- to hyaluronan. On the World Wide Web http://www.gly-
tite/chitosan-gelatin network composite scaolds. Bioma- coforum.gr.jp/science/hyaluronan/HA22/HA22E.html.
terials 2002, 23, 3227. 78. Garcia-Lora, A.; Pedrinaci, S.; Garrido, F. Protein-bound
57. Tanabe, T.; Okitsu, N.; Tachibana, A.; Yamauchi, K. polysaccharide k and interleukin-2 regulate dierent
Preparation and characterization of keratinchitosan nuclear transcription factors in the nkl human natural
composite lm. Biomaterials 2002, 23, 817. killer cell line. Cancer Immunol. Immunother. 2001, 50,
58. Matthew, H.W.T. Polymers for tissue engineering scaf- 191.
folds. In Polymeric Biomaterials, Second Edition, Revised 79. Warth, A.D.; Strominger, J.L. Structure of the peptido-
and Extended; Dumitriu, S., Ed.; Marcel Dekker, Inc.: glycan from vegetative cell wall of bacillus subtilis.
New York, 2002; 167 pp. Biochemistry 1971, 10, 4349.
59. Bakos, D.; Soldan, M.; Hernandez-Fuentes, I. Hydroxy- 80. Popham, D.L. Specialized peptidoglycan of bacterial
apatitecollagenhyaluronic acid composite. Biomaterials endospore: the inner wall of the lockbox. Cell. Mol. Life
1999, 20, 191. Sci. 2002, 59, 426.
60. Elcin, A.E.; Elcin, Y.M.; Peppas, G.D. Neural tissue 81. Berneld, M.; Gotte, M.; Park, P.W.; Reizes, O.;
engineering: adrenal chroman cell attachment and via- Fitzgerald, M.L.; Lincecum, J.; Zako, M. Functions of
bility on chitosan scaold. Neurol. Res. 1998, 20, 648. cell surface. heparan sulfate proteoglycans. Ann. Rev.
61. Kawase, M.; Michibayashi, N.; Nakashima, Y.; Kuri- Biochem. 1999, 68, 729.
kawa, N.; Yagi, K.; Mizoguchi, T. Application of glutar- 82. Blackhall, F.H.; Merry, C.L.R.; Davies, E.J.; Jayson, G.C.
aldehyde-crosslinked chitosan as a scaold for hepatocyte Heparan sulfate proteoglycans and cancer. Ann. Rev.
attachment. Biol. Pharm. Bull. 1997, 20, 708. Biochem. 2001, 85, 1094.
62. Mariappan, M.R.; Alas, E.A.; Williams, J.G.; Prager, 83. Faham, S.; Hileman, R.E.; Fromm, J.R.; Linhardt, R.J.;
M.D. Chitosan and chitosan sulfate have opposing eects Rees, D.C. Heparin structure and interactions with basic
on collagenbroblast interactions. Wound Repair Regen. broblast growth factor. Science 1996, 271, 1116.
1999, 7, 400. 84. Ornitz, D.M. FGFs, heparan sulfate and FGFRs: complex
63. Ellis, D.L.; Yannas, I.V. Recent advances in tissue interactions essential for development. BioEssays 2000, 22,
synthesis in vivo by use of collagenglycosaminoglycan 108.
copolymers. Biomaterials 1996, 17, 291. 85. Faham, S.; Lindardt, R.J.; Rees, D.C. Diversity does make
64. Yannas, I.V. Applications of ecm analogs in surgery. J. a dierence: Fibroblast growth factorheparin interac-
Cell. Biochem. 1994, 56, 188. tions. Curr. Opin. Struct. Biol. 1998, 8, 578.
65. Nehrer, S.; Breinan, H.A.; Ramappa, A.; Shortkro, S.; 86. Pellegrini, L. Role of heparan sulfate in broblast growth
Young, G.; Minas, T.; Sledge, C.B.; Yannas, I.V.; Spector, factor signalling: A structural view. Curr. Opin. Struct.
M. Canine chondrocytes seeded in type i and type ii Biol. 2001, 11, 629.
collagen implants investigated in vitro. J. Biomed. Mater. 87. Powell, A.; Turnbull, J.; Guimond, S. Heparan sulfate:
Res. 1997, 38, 95. Decoding a dynamic multifunctional cell regulator. Trends
66. McArdle, B. Herbicidal and insecticidal proteinpolysac- Cell Biol. 2001, 11, 75.
charide delivery compositions and methods for controlling 88. Mulloy, B.; Linhardt, R.J. Order out of complexity-
plant and insect populations. U.S. Patent 5747416, 1998. protein structures that interact with heparin. Curr. Opin.
67. Hansen, P.M.T.; Hidalgo, J.; Gould, I.A. Reclamation of Struct. Biol. 2001, 11, 623.
whey protein with carboxymethylcellulose. J. Dairy Sci. 89. DiGabriele, A.D.; Lax, I.; Chen, D.I.; Svahn, C.M.; Jaye,
1971, 54, 830. M.; Schlessinger, J.; Hendrickson, W.A. Structure of a
heparin-linked biologically active dimer of broblast D.J., Williams, P.A., Eds.; Oxford: Pergamon Press, 1984;
growth factor. Nature 1998, 393, 812. 189 pp.
90. Pelligrini, L.; Burke, D.F.; Von Delft, F.; Murroy, B.; 109. Chilvers, G.R.; Morris, V.J. Coacervation of gelatin
Blunder, T.L. Crystal structure of broblast growth factor gellan gum mixtures and their use in microencapsulation.
receptor ectodomain bound to ligand and heparin. Nature Carbohydr. Polym. 1987, 7, 111.
2000, 407, 1029. 110. Elqin, Y.M. Encapsulation of urease enzyme in xanthan
91. Day, A.J. Understanding hyaluronanproteins interac- alginate spheres. Biomaterials 1995, 18, 1157.
tions. On the World Wide Web http://www.glycoforum. 111. Tanaka, H.; Matsumara, M.; Veliki, I.A. Diusion char-
gr.jp/science/hyaluronan/HA16/HA16E.html. acteristics of substrates in caalginate gel beads. Biotech-
92. Day, A.J.; Sheehan, J.K. Hyaluronan: polysaccharide nol. Bioeng. 1984, 26, 53.
chaos to protein organisation. Curr. Opin. Struct. Biol. 112. Chevalier, P.; Consentino, G.P.; de la Noue, J.; Rakhit, S.
2001, 11, 617. Comparative study on the diusion of an IgG from various
93. Kahmann, J.D.; OBrien, R.; Werner, J.M.; Heinegard, hydrogel beads. Biotechnol. Tech. 1987, 1, 201.
D.; Ladbury, J.E.; Campbell, I.D.; Day, A.J. Localization 113. Gray, C.J.; Dowsett, J. Retention of insulin in alginate gel
and characterization of the hyaluron-binding site on the beads. Biotechnol. Bioeng. 1988, 31, 607.
link module from human tsg-6. Structure 2000, 8, 763. 114. Ko, C.; Dixit, V.; Shaw, W.; Gitnick, G. In vitro slow
94. De Kruif, C.G.; Tuinier, R. Polysaccharide protein release prole of endothelial cell growth factor immobi-
interactions. Food Hydrocoll. 2001, 15, 555. lized within calcium alginate beads. Artif. Cells Blood
95. Tolstoguvoz, V.B. Functional properties of food proteins Substit. Immobil. Biotechnol. 1995, 23, 143.
and role of proteinpolysaccharide interaction. Food 115. Downs, E.C.; Robertson, N.E.; Riss, T.L.; Plunkett, M.L.
Hydrocoll. 1991, 4, 429. Calcium alginate beads as a slow-release system for de-
96. Polyakov, V.I.; Grinberg, V.Y.; Tolstoguzov, V.B. Ther- livering angiogenic molecules in vivo and in vitro. J. Cell.
modynamic incompatibility of proteins. Food Hydrocoll. Physiol. 1992, 152, 422.
1997, 11, 171. 116. Maysinger, D.; Jalsenjak, I.; Cuello, A.C. Microencapsu-
97. Wang, S.; Van Dijk, J.A.P.P.; Odijk, T.; Smit, J.A.M. lated nerve growth factor: eects on the forebrain neurons
Depletion-induced demixing in aqueous proteinpolysac- following devascularizing cortical lesion. Neurosci. Lett.
charide solutions. Biomacromolecules 2001, 2, 1080. 1992, 140, 71.
98. Michon, F.; Huang, C.H.; Farley, E.K.; Hronowski, L.; 117. Bowersock, T.L.; HogenEsch, H.; Suckow, M.; Davis-
Di, J.; Fusco, P.C. Structure activity studies on group c synder, E.; Borie, D.; Park, H.; Park, K. Oral admin-
meningococcal polysaccharideprotein conjugate vac- istration of mice with ovalbumin encapsulated in alginate
cines: Eect of o-acetylation on the nature of the protective microspheres. Polym. Preprints 1994, 35, 405.
epitope. In Physico-Chemical Procedures for the Character- 118. Fanslow, W.C.; Campbell, K.A.; Wee, S.F.; Cliord,
ization of Vaccines; Brown, F., Corbel, M., Griths, E., K.N.; Maliszewski, C.; Hall, R.; Peterson, M.; Whitney,
Eds.; Dev. Biol. Karger: Basel, Switzerland, 2000; 151 pp. L.; Kiinke, R.; Armitage, R. Administration of puried
99. Liu, L.S.; Liu, S.Q.; Ng, S.Y.; Froix, M.; Ohno, T. recombinant trimeric cd401 augments humoral and cell-
Controlled release of interleukin-2 for tumour immuno- mediated immune responsiveness in cd401-decient mice.
therapy using alginate/chitosan porous microsphere. J. Presentation at the 37th American Society of Hematology
Control. Release 1997, 43, 65. Meeting, Seattle, WA, 1995.
100. Braun, J.; Le Chanu, P.; Le Goc, F. The immobilization 119. Gombotz, W.R.; Wee, S.F. Protein release from alginate
of penicillin g acyclase on chitosan. Biotechnol. Bioeng. matrices. Adv. Drug Deliv. Rev. 1998, 31, 267.
1989, 33, 242. 120. Stainsly, G. Proteinaceous gelling systems and their
101. Gonzalez Siso, M.I.; Lang, E.; Carreno-Gomez, B.; complexes with polysaccharides. Food Chem. 1980, 6, 3.
Beccera, M.; Otero Espinar, F.; Blanco Mendez, J. 121. Louisot, P. Biochimie Generale et Medicale; SIMEP: Paris,
Enzyme encapsulation on chitosan microbeads. Process 1989.
Biochem. 1997, 32, 211. 122. Antonov, Y.A.; Lashko, N.P.; Glotova, Y.K.; Maloviko-
102. Braudo, E.E.; Antonov, Y.A. Non-coulombic complex va, A.; Markovich, O. Eect of the structural features of
formation of proteins as a structure forming factor in food pectins and alginates on their thermodynamic compati-
systems. In Proteins, Structure and Functionality; bility with gelatin in aqueous media. Food Hydrocoll.
Schwenke, K.D., Mothes, R., Eds.; VCH: Weinheim, 1996, 10, 1.
1993; 210 pp. 123. Domard, A.; Domard, M. Chitosan: Structureproperties
103. Taravel, M.N; Domard, A. Relation between the phys- relationship and biomedical applications. In Polymeric
icochemical characteristics of collagen and its interactions Biomaterials, Second Edition, Revised and Extended;
with chitosan: I. Biomaterials 1994, 14, 930. Dumitriu, S., Ed.; Marcel Dekker, Inc.: New York, 2002;
104. Leer, C.C.; Muller, B.W. Inuence of the acid type on 187 pp.
the physical and drug liberation properties of chitosan- 124. Taravel, M.N.; Domard, A. Competition between gel and
gelatin sponges. Int. J. Pharm. 2000, 194, 229. polyanionpolycation complex formation in a collagen
105. Morris, E.R. Mixed polymer gels. In Food Gels; Harris, P., chitosan binary system. Macromol. Rep. 1994, A31, 1237.
Ed.; Elsevier Applied Science: London, 1990; 291 pp. 125. Taravel, M.N.; Domard, A. Collagen and its interactions
106. McKay, J.E.; Stainsby, G.; Wilson, E.L. A comparison of with chitosan: Ii. Inuence of the physicochemical char-
the reactivity of alginate and pectate esters with gelatin. acteristics of collagen. Biomaterials 1995, 16, 865.
Carbohydr. Polym. 1985, 5, 223. 126. Tolstoguzov, V.B. Proteinpolysaccharide interactions. In
107. Tolstoguzov, V.B. Functional properties of proteinpoly- Food Proteins and Their Applications; Damodaran, S. Paraf,
saccharide mixtures. In Functional Properties of Macro- A., Eds.; Marcel Dekker, Inc.: New York, 1997; 171 pp.
molecules; Mitchell, J.R., Ledward, D.A., Eds.; Elsevier 127. Samant, S.K.; Singhal, R.S.; Kulkarni, P.R.; Rege, D.V.
Applied Science Publishers: London, 1986; 385 pp. Proteinpolysaccharide interactions: a new approach in
108. Imeson, A.P. Recovery and utilisation of proteins using food formulations. Review. Int. J. Food Sci. Technol.
alginate. In Gums and Stabilisers for the Food Industry 2 1993, 28, 547.
Applications of Hydrocolloids; Phillips, G.O., Wedlock, 128. Piculell, L.; Lindman, B. Association and segregation in
aqueous polymer/polymer, polymer/surfactant and sur- 151. Garnier, C.; Chorsch, C.; Doublier, J.L. Phase separation
factant/surfactant mixtures: Similarities and dierences. in dextran/locust bean gum mixtures. Carbohydr. Polym.
Adv. Colloid Interface Sci. 1992, 41, 149. 1995, 28, 313.
129. Tiebackx, F.W. Gleichzeitige ausockung zweier kolloide. 152. Matsudomi, N. Characteristics of heat-induced trans-
Z. Chem. Ind. Kolloide 1911, 8. parent gels from egg white by the addition of dextran
130. Ambjerb Pedersen, H.C.; Jorgensen, B.B. Inuence of sulfate and the proteinpolysaccharide interactions. Nah-
pectin on the stability of casein solutions studied in rung 1998, 42, 238.
dependence of varying pH and salt concentration. Food 153. Neiser, S.; Draget, K.I.; Smidsrd, O. Gel formation in
Hydrocoll. 1991, 5, 323. heat-treated bovine serum albuminn-carrageenan sys-
131. Niederauer, M.Q.; Glatz, C. Model of the polyelectrolyte tems. Food Hydrocoll. 2000, 14, 95.
precipitation of genetically engineered enzymes possessing 154. Schliwa, M. The Cytoskeleton. An Introduction Survey;
charged polypeptide tails. J. Macromol. Sci. Part A, Pure Springer: Wien, 1986.
Appl. Chem. 1994, A31, 127. 155. Schmitt, C.; Sanchez, C.; Thomas, F.; Hardy, J. Complex
132. Flory, P.J. Thermodynamics of high polymer solutions. J. coacervation between h-lactoglobulin and acacia gum in
Chem. Phys. 1942, 10, 51. aqueous medium. Food Hydrocoll. 1999, 13, 483.
133. Huggins, M.L. Theory of solutions of high polymers. J. 156. Dickinson, E.; James, J.D. Inuence of high-pressure
Am. Chem. Soc. 1942, 64, 1712. treatment on h-lactoglobulinpectin associations in emul-
134. Schmitt, C.; Sanchez, C.; Desorbry-Banon, S.; Hardy, J. sions and gels. Food Hydrocoll. 2000, 14, 365.
Structure and technofunctional properties of protein 157. Zaleska, H.; Ring, S.G.; Tomasik, P. Apple pectin com-
polysaccharide complexes: A review. Crit. Rev. Food Sci. plexes with whey protein isolate. Food Hydrocoll. 2000,
Nutr. 1998, 38, 689. 14, 37.
135. Olabisi, O.; Robeson, L.M.; Shaw, M.T. Thermodynamics 158. Sanchez, C.; Zuniga-Lopez, R.; Schmitt, C.; Despond, S.;
of polymerpolymer miscibility. PolymerPolymer Misci- Hardy, J. Microstructure of acid-induced skim milklocust
bility; Academic Press: New York, 1979; 19 pp. bean gumxanthan gum gels. Int. Dairy J. 2000, 10, 199.
136. Sperling, J.H. Introduction to Polymer Science, 2nd Ed. 159. Burgess, D.J. Complex coacervation: Microcapsule for-
John Wiley and Sons, Inc.: New York, 1992. mation. In Macromolecular Complexes in Chemistry and
137. Scott, R.L. The thermodynamics of high polymer so- Biology; Dubin, P.L., Bock, J., Davis, R., Schulz, D.N.,
lutions. v. phase equilibria in the ternary system: Poly- Thies, C., Eds.; Springer Verlag: Berlin, 1994; 281 pp.
mer1polymer2solvent. J. Chem. Phys. 1949, 17, 279. 160. Bungenderg De Jong, H.G. Chapter 8 and 10. In Colloid
138. Clark, A.H. Direct analysis of experimental tie line data Sciences, Reversible Systems; Kruyt, H.R., Ed., Elsevier:
(two polymerone solvent systems) using FloryHuggins Amsterdam, 1949; Vol. 2.
theory. Carbohydr. Polym. 2000, 42, 337. 161. Xia, J.; Dublin, P.L. Cproteinpolyelectrolyte complexes.
139. Doublier, J.L.; Garnier, C.; Renard, D.; Sanchez, C. In Macromolecular Complexes in Chemistry and Biology;
Proteinpolysaccharide interactions. Curr. Opin. Colloid Dubin, P.L., Bock, J., Davis, R., Schulz, D.N., Thies, C.,
Interface Sci. 2000, 5, 202. Eds.; Springer Verlag: Berlin, 1994; 247 pp.
140. Dickinson, E. Colloidal aspects of beverages. Food Chem. 162. Voorn, M.J. I. General theoretical consideration: Complex
1994, 51, 343. coacervation. Recl. Trav. Chim. 1956, 75, 313.
141. Albertsson, P.A. Partition of Cell Particles and Macro- 163. Voorn, M.J. II. Thermodynamic calculations of a specic
molecules, 2nd Ed.; Wiley Interscience: New York, 1971. model, with application to two component systems:
142. Dobry, A.; Boyer-Kawenoki, F. Sur lincompatibilite des Complex coacervation. Recl. Trav. Chim. 1956, 75, 405.
macromolecules en solution aqueuse. Bull. Soc. Chim. 164. Voorn, M.J. III. Thermodynamic calculations on three-
Belge 1948, 57, 280. component systems: Complex coacervation. Recl. Trav.
143. Zeman, L.; Patterson, D. Eects of the solvent on polymer Chim. 1956, 75, 427.
incompatibility in solution. Macromolecules 1972, 5, 513. 165. Voorn, M.J. IV. Thermodynamic calculations on four
144. Bungenderg De Jong, H.G. In La coacervation complexe et component systems: Complex coacervation. Recl. Trav.
son importance en Biologie; Faure-Fremiet, E., Ed.; Chim. 1956, 75, 925.
Hermann et Cie: Paris, 1936. 166. Voorn, M.J. Experiment on the distribution of salts.
145. Hsu, C.C.; Prausnitz, J.M. Thermodynamics of polymer comparison of theory and experiment. Recl. Trav. Chim.
compatibility in ternary systems. Macromolecules 1974, 7, 1956, 75, 925.
320. 167. Overbeek, J.T.J.; Voorn, M.J. Phase separation in poly-
146. Hoskins, R.; Robb, I.D.; Williams, P.A; Warren, P. Phase electrolyte solutions. theory of complex coacervation. J.
separation in mixtures of polysaccharides and proteins. Cell. Comp. Physiol. 1957, 49, 7.
J. Chem. Soc. Faraday Trans. 1996, 92, 4515. 168. Veis, A.; Aranyi, C. Phase separation in polyelectrolyte
147. Hoskins, R.; Robb, I.D.; Williams, P.A. Selective separa- systems. I. complex coacervates of gelatin. J. Phys. Chem.
tion of proteins from mixtures using polysaccharides. 1960, 64, 1203.
Biopolymers 1998, 45, 97. 169. Nakajima, A.; Sato, H. Phase relationships of a equivalent
148. Syrbe, A.; Bauer, W.J.; Klostermeyer, H. Polymer science mixture of sulfated polyvinyl alcohol and aminoacetalyzed
concepts in dairy systemsAn overview of milk protein polyvinyl alcohol in microsalt aqueous solution. Biopoly-
and food hydrocolloid interaction. Int. Dairy J. 1998, 8, mers 1972, 10, 1345.
179. 170. Tainaka, K.I. Study of complex coacervation in low
149. Renard, D.; Boue, F.; Lefebvre, J. Solution and gelation concentration by virial expansion method. I. Salt free
properties of protein polysaccharide mixture: Signature by systems. J. Phys. Soc. Jpn. 1979, 46, 1899.
small-angle neutron scattering and rheology. In Gums and 171. Tainaka, K.I. Eect of counterions on complex coacerva-
Stabilisers for the Food Industry 9; William, P.A., Phillips, tion. Biopolymers 1980, 19, 1289.
G.O., Eds.; Royal Society of Chemistry: Cambridge, 1998; 172. Debye, P.; Huckel, E. Zur theorie der elektrolyte. Phys. Z.
189 pp. 1923, 24, 185.
150. Owen, A.J.; Jones, R.A.L. Rheology of simultaneously 173. Debye, P.; Huckel, E. Bemerkungen zu einem satze uber
phase separating and gelling biopolymer mixtures. Macro- die kataphoretische wanderungs-geschwindigkeit suspen-
molecules 1998, 31, 7336. dierte teilchen. Phys. Z. 1924, 25, 49.
174. Burgess, D.J. Practical analysis of complex coacervate Langerhans: An insulin diusion study. Artif. Organs
systems. J. Colloid Interface Sci. 1990, 140, 227. 1983, 7, 208.
175. Burgess, D.J.; Kwok, K.K.; Megremis, P.T. Character- 193. Goosen, M.F.A.; OShea, G.M.; Gharapetian, H.M.;
ization of albuminacacia gum complex coacervation. J. Chou, S.; Sun, A.M. Optimization of microencapsulation
Pharm. Pharmacol. 1991, 43, 232. parameters: Semipermeable microcapsules as a bioarti-
176. Burgess, D.J.; Carless, J.E. Microelectrophoretic studies of cial pancreas. Biotechnol. Bioeng. 1985, 27, 146.
gelatin and acacia for prediction of complex coacervation. 194. Tze, W.J.; Tai, J. Biocompatibility and immunological
J. Colloid Interface Sci. 1984, 88, 1. studies of microencapsulation with cross-linked alginic
177. Velings, N.M.; Mestdagh, M.M. Protein adsorption in acid capsule. Transplantation 1982, 33, 563.
calcium alginate bead. J. Bioact. Biocompat. Polym. 1994, 195. Weber, C.J.; Koschitsky, T.; Constanzo, M.K.; DAgati,
9, 133. V.; Zabinski, S.; Rajotte, R.V.; Krekun, S.; Reemtsma, K.
178. Semenova, M.G. Factor determining the character of Xenografts of microencapsulated rat, canine, porcine and
biopolymerbiopolymer interactions in multicomponent human islets. In Pancreatic Islet Cell Transplantation;
aqueous solutions modeling food systems. In Macro- Ricordi, C., Ed.; R.G. Landes: Austin, TX, 1992; 177 pp.
molecular Interactions in Food Technology; Parris, N., 196. Bystricky, S.; Malovikova, A.; Sticzay, T. Interaction of
Kato, A., Creamer, L.K., Pearce, J., Eds.; ACCS alginates and pectins with cationic polypeptides. Carbo-
Symposium Series 650: Washington, DC, 1996; 37 pp. hydr. Polym. 1990, 13, 283.
179. Kelly, R.; Gudo, E.S.; Mitchell, J.R.; Harding, S.E. Some 197. Bystricky, S.; Malovikova, A. Conformation presumption
observations on the nature of heated mixture of bovine for polysaccharidespolylysine complexation. In Macro-
serum albumin with alginate and a pectin. Carbohydr. molecular Complexes in Chemistry and Biology; Dublin, P.,
Polym. 1994, 23, 115. Bock, J., Davis, R., Schulz, D.N., Thies, C., Eds.; Springer-
180. Harding, S.E.; Jumel, K.; Kelly, R.; Gudo, E.S.; Horton, Verlag: Berlin, 1994; 175 pp.
J.C.; Mitchell, J.R. The structure and nature of protein 198. Clark, K.M.; Glatz, C.E. Protein fractionation by precipi-
polysaccharide complexes. In Food Proteins, Structure and tation with carboxymethyl cellulose. In ACS Symposium
Functionality; Schwenke, K.D., Mothes, R., Eds.; VCH: Series 419: Downstream Processing and Bioseparation;
Weinheim, 1993; 216 pp. Hamel, J.F.P., Hunter, J.B., Sikdar, S.S., Eds.; ACS Press:
181. Park, J.M.; Muhoberac, B.B.; Dubin, P.L. Eects of Washington, DC, 1990; 170 pp.
protein charge heterogeneity in proteinpolyelectrolyte 199. Clark, K.M.; Glatz, C.E. A binding model for the
complexation. Macromolecules 1992, 25, 290. precipitation of proteins by carboxymethyl-cellulose.
182. Le Hena. S. Microparticules de complexes de proteines de Chem. Eng. Sci. 1992, 47, 215.
lactoserum et xanthan comme substitut de matie`re grasse, 200. Fisher, R.R.; Glatz, C.E. Polyelectrolyte precipitation of
mise en place dun protocole de fabrication et de fonctio- proteins. ii. Models of the particle size distributions.
nalite dans une Emulsion mode`le allegee. Masters thesis, Biotechnol. Bioeng. 1988, 32, 789.
University of Laval, Canada, 1996. 201. Zhao, J.; Ford, C.F.; Glatz, C.E.; Rougvie, M.A.; Gendel,
183. Dickinson, E.; Pawlowsky, K. Eect of high-pressure S.M. Polyelectrolyte precipitation of h-galactosidase
treatment of protein on the rheology of occulated fusions containing poly-aspartic acid tails. J. Biotechnol.
emulsions containing protein and polysaccharide. J. Agric. 1990, 14, 273.
Food Chem. 1996, 44, 2992. 202. Parkerr, D.E.; Glatz, C.E.; Ford, C.F.; Gendel, S.M.;
184. Dickinson, E.; Pawlowsky, K. Proteinpolysaccharide Souminen, I.; Rougvie, M.A. Recovery of a charge-fusion
interactions in emulsions containing high pressure treated protein from cell extracts by polyelectrolyte precipitation.
protein. Spec. Publ. R. Soc. Chem. 1998, 218, 314. Biotechnol. Bioeng. 1990, 36, 467.
185. Pawlowsky, K.; Dickinson, E. Proteinpolysaccharide 203. Souminen, I.; Ford, C.F.; Stachon, D.; Heimo, H.;
interactions in emulsions containing high pressure treated Niederauer, M.; Nurmela, H.; Glatz, C.E. Enhanced
protein. Spec. Publ. R. Soc. Chem. 1998, 222, 214. recovery and purication of Aspergillus glucoamylase
186. Galazka, V.B.; Smith, D.; Ledward, D.A.; Dickinson, E. from Saccharomyces cerevisiae by the addition of poly
Complexes of bovine serum albumin with sulphated (aspartic acid) tails. Enzyme Microb. Technol. 1993, 15,
polysaccharides: Eect of pH, ionic strength and high 593.
pressure treatment. Food Chem. 1999, 64, 303. 204. Imeson, A.P.; Ledward, D.A.; Mitchell, J.R. On the nature
187. Tirkkonen, S.; Turakka, L.; Paronen, P. Microencapsula- of the interactions between some anionic polysaccharides
tion of indomethacin by gelatinacacia complex coacerva- and proteins. J. Sci. Food Agric. 1977, 28, 669.
tion in presence of surfactants. J. Microencapsul. 1994, 11, 205. Tsung, M.; Burgess, D.J. Preparation and stabilization of
615. heparin/gelatin complex coacervate microcapsules. J.
188. Martinsen, A.; Skjak-Braek, G.; Smirod, O.; Zanetti, F.; Pharm. Sci. 1996, 86, 603.
Paoletti, S. Comparison of dierent methods for determi- 206. Gettins, P.G.W. Antithrombin iii and its interactions with
nation of molecular weight and molecular weight distri- heparin. Comparison of the human, bovine and porcine
bution of alginates. Carbohydr. Polym. 1991, 15, 171. proteins by 1h nmr spectroscopy. Biochemistry 1987, 26,
189. Iwata, H. Bioarticial pancreas. In Polysaccharides in 1391.
Medical Applications; Dumitriu, S., Ed.; Marcel Dekker, 207. Basilico, C.; Moscatelli, D. The fgf family of growth
Inc.: New York, 1996; 603 pp. factors and oncogenes. Adv. Cancer Res. 1992, 59, 115.
190. Halle, J.P.; Bourassa, S.; Leblond, F.A.; Chevalier, S.S.; 208. Galzie, Z.; Kinsella, A.R.; Smith, J.A. Fibroblast growth
Beaudry, M.; Chapdelaine, A.; Cousineau, S.; Saintonge, factors and their receptors. Biochem. Cell. Biol. 1997, 75,
J.; Yale, J.F. Protection of islets of Langerhans from 669.
antibodies by microencapsulation with alginic acid-poly-L- 209. Jaye, M.; Schlessinger, J.; Dionne, C.A. Fibroblast growth
lysine membrane. Transplantation 1993, 55, 350. factor receptor tyrosine kinases: molecular analysis and
191. Sun, A.M.; OShea, G.M.; Van Rooy, H.; Goosen, M.F. signal transduction. Biochem. Biophys. Acta 1988, 1135,
Microencapsulation of the islands of Langerhans and the 185.
articial pancreas. J. Ann. Diabetolog. 1982, 20, 161. 210. Johnson, D.E.; Williams, L.T. Structural and functional
192. Leung, Y.F.; OShea, G.M.; Goosen, M.F.; Sun, A.M. diversity in the fgf receptor multigene family. Adv. Cancer
Microencapsulation of crystalline insulin or islets of Res. 1993, 60, 1.
211. Rapraeger, A.C.; Krufka, A.; Olwin, B.B. Requirement of 230. Pommersheim, R.; Schrezenmeir, J.; Vogt, W. Immobili-
heparan sulfate for bfgf-mediated broblast growth and zation of enzymes by multilayer micro-capsules. Macro-
myoblast dierentiation. Science 1993, 252, 1705. mol. Chem. Phys. 1994, 195, 1557.
212. Spivak-Kroizman, T. Heparin-induced oligomerization of 231. Heichal-Segal, O.; Rappoport, S.; Braun, S. Immobiliza-
fgf molecules is responsible for fgf receptor dimerization, tion in alginatesilicate solgel matrix protects h-glucosi-
activation and cell proliferation. Cell 1994, 79, 1015. dase against thermal and chemical denaturation. Biotech
213. Lin, X.; Bu, E.M.; Perrimon, N.; Michelson, A.M. 1995, 13, 798.
Heparan sulfate proteoglycans are essential for fgf 232. Alsaara, I.A.; Betigeri, S.S.; Zhang, H.; Evans, B.A.;
receptor signaling during drosophila embryonic develop- Neau, S.H. Molecular weight and degree of deacetylation
ment. Development 1999, 126, 3715. eects on lipase-loaded chitosan bead characteristics.
214. Zhu, X.; Hsu, B.; Rees, D.C. Structural studies of the Biomaterials 2002, 23, 3637.
binding of the anti-ulcer drug sucrose octasulfate to acidic 233. Dumitriu, S.; Chornet, E.; Vidal, P.F.; Moresoli, C.
broblast growth factor. Structure 1993, 1, 27. Polyionic hydrogel as supports for immobilization of
215. Ornitz, D.M., et al. Fgf binding and fgf receptor activation lipase. Biotechnol. Tech. 1995, 9, 883.
by synthetic heparan-derived di- and trisaccharides. Science 234. Dumitriu, S.; Chornet, E. Inclusion and release of proteins
1995, 268, 432. from polysaccharide-based polyion complexes. Adv. Drug
216. Thompson, L.D.; Pantoliano, M.W.; Springer, B.A. Deliv. Rev. 1998, 31, 223.
Energetic characterization of the basic broblast growth 235. Dumitriu, S.; Chornet, E. Polysaccharides as support for
factor-heparin interaction: Identication of the heparin enzyme and cell immobilization. In Polysaccharides;
binding domain. Biochemistry 1994, 33, 3831. Dumitriu, S., Ed.; Marcel Dekker, Inc.: New York, 1998;
217. Nakagaki, M.; Sano, Y. Light-scattering studies of the 629 pp.
interaction of sodium chondroitin sulfate c with bovine 236. Magnin, D. Master thesis: Immobilisation denzymes dans
serum albumin. Bull. Chem. Soc. Jpn. 1972, 45, 1011. un hydrogel polyionique a` base de chitosane: le chitoxan,
218. Shimabayashi, S.; Hirao, K.; Bando, J.; Shinohara, C. Universite de Sherbrooke, Sherbrooke, Canada, 2001.
Formation of a polymer complex between bovine serum 237. Morgan, D. Firms via for lead in oral drug delivery. New
albumin and chondroitin-6-sulfate in an aqueous phase Sci. 1991, 1, 9.
and a solid/water interface of kaolin. J. Macromol. Sci. 238. Robbins, D.H.; Mauger, J.W. Drug deliver systems. Am.
Part A, Pure Appl. Chem. 1994, A31, 65. J. Hosp. Pharm. 1991, 48, 514.
219. Shim, J.L. Gellan gum/gelatin blends. U.S. Patent 239. Wheatly, M.A.; Chang, M.; Park, E.; Langer, R. Coated
4517216, 1985. alginate microsphere: factors inuencing the controlled
220. Sanchez, C.; Renard, D. Stability and structure of protein delivery of macromolecules. J. Appl. Polym. Sci. 1991, 43,
polysaccharide coacervates in the presence of protein 2123.
aggregates. Int. J. Pharm. 2002, 242, 319. 240. Haga, M.; Saito, K.; Shimaya, T.; Maezawa, Y.; Kato, Y.;
221. Yoshida, H.; Nishihara, N.; Kataoka, T. Adsorption of Kim, S.W. Hypoglycemic eect of intestinally adminis-
bsa on strongly basic chitosan: Equilibria. Biotechnol. trated monosaccharide-modied insulin derivated in rat.
Bioeng. 1993, 43, 1087. Chem. Pharm. Bull. 1990, 38, 1983.
222. Remunan-Lopez, C.; Bodmeier, R. Eect of formulation 241. Tsuruta, T., Reisfeld, R., Ed.; In Advances in Polymer
and process variables on the formation of chitosan-gelatin Science; Springer Verlag: Berlin, 1996; 1 pp.
coacervates. Int. J. Pharm. 1996, 135, 63. 242. Bernkop Schnurch, A. Strategien zur peroralen applika-
223. Guerin, D.M.A.; Lascombe, M.B.; Costabel, M.; tion von peptid- und proteinwirkstoen. Sci. Pharm. 1997,
Souchon, H.; Lamzin, V.; Beguin, P.; Alzari, P.M. 65, 61.
Atomic (0.94 A) resolution structure of an inverting 243. Lehr, C.M. Bioadhesion technologies for the delivery tract.
glycosidase in complex with substrate. J. Mol. Biol. 2002, Crit. Rev. Ther. Drug 1994, 11, 119.
316, 1061. 244. Kwok, K.K.; Groves, M.J.; Burgess, D.J. Production of 5
224. Szabo, L.; Jamal, S.; Xie, H.; Charnock, S.J.; Bolam, D.N.; 15 microns diameter alginatepolylysine microcapsules by
Gilbert, H.J.; Davies, G.J. Structure of a family 15 an air-atomization technique. Pharm. Res. 1991, 8, 341.
carbohydrate-binding module in complex with xylopen- 245. Mumper, R.J.; Homan, A.S.; Puolakkainen, P.; Bou-
taose. evidence that xylan binds in an approximate 3-fold chard, L.S.; Gombotz, W.R. Calcium alginate beads for
helical conformation. J. Biol. Chem. 2001, 276 (52), 49061. the oral delivery of transforming growth factor-h1:
225. Silman, H.; Katchalski, E. Water insoluble derivated of Stabilization of tgf-h1 by the addition of polyacrylic acid
enzymes, antigens, and antibodies. Ann. Rev. Biochem. within acid treated beads. J. Control. Release 1994, 30, 241.
1966, 35, 873. 246. Pualakkainen, P.A.; Ranchalis, J.E.; Gombotz, W.R.;
226. Crumbliss, A.L.; Stonehuerner, J.G.; Henkens, R.W. Homan, A.S.; Mumper, R.J.; Twardzik, D.R. Novel
Carrageenan hydrogel stabilized colloidal gold multi- delivery system for inducing quiescence in intestinal stem
enzyme biosensor electrode utilizing immobilized horse- cell in rats by transforming growth factor h1. Gastro-
radish peroxidase and cholesterol oxidase/cholesterol enterology 1994, 107, 1319.
esterase to detect cholesterol in serum and whole blood. 247. Edelman, E.R.; Mathiowitz, E.; Langer, R.; Klagsbrun,
Biosens. Bioelectron. 1993, 8, 331. M. Controlled and modulated release of basic broblast
227. Klibanov, A.M. Immobilized enzymes and cells as growth factor. Biomaterials 1991, 12, 619.
practical catalysts. Science 1983, 219, 722. 248. Austin, L.; Bower, J.J.; Muldoon, C. The controlled
228. Ariga, O.; Kato, M.; Sano, T.; Nakazawa, Y.; Sano, Y. release of leukaemia inhibitory factor (lif) from alginate
Mechanical and kinetic properties of pva hydrogel gels. Proc. Int. Symp. Control. Release Bioact. Mater.
immobilizing beta-galactosidase. J. Ferment. Bioeng. 1996, 23, 739.
1993, 76, 203. 249. Martinsen, A.; Skjak-Braek, G.; Smirod, O. Alginate as
229. Das, N.; Kayastha, A.M.; Malhotra, O.P. Immobilization immobilization material: I. Correlation between chemical
of urease from pigeon pea (Cajanus cajan L.) in poly- and physical properties of alginate gel beads. Biotech.
acrylamide gels and calcium alginate beads. Biotechnol. Bioeng. 1989, 33, 79.
Appl. Biochem. 1998, 27, 25. 250. Burgamelli, F.; Raggi, M.A.; Orienti, I.; Zecchi, V.
Controlled insulin release from chitosan microparticles. 267. Sum, A.M.; OShea, G.M.; Goosen, M.F.A. Injectable,
Arch. Pharm. Med. Chem. 1998, 331, 133. biocompatible islets microencapsules as a bioarticial
251. Polk, A.E.; Amsden, B.; Scarratt, D.J.; Gonzal, A.; pancreas. Prog. Artif. Organs 1980, 1983, 769.
Okhamafe, A.O.; Goosen, M.F.A. Oral delivery in 268. Krestow, M.; Lum, Z.P.; Tai, I.T.; Sum, A.M. Xeno-
aquaculture: controlled release of proteins from chito- transplantation of microencapsulated fetal rat islets.
sanalginate microcapsules. Aquac. Eng. 1994, 13, 311. Transplantation 1991, 51, 51.
252. Okhamafe, A.O.; Amsden, B.; Chu, W.; Goosen, M.F.A. 269. Wu, Z.G.; Shi, Z.Q.; Lu, Z.N.; Yang, H.; Shi, F.Y.; Zheng,
Modulation of protein release from chitosanalginate X.R.; Sum, A.M. In vitro culture and transplantation of en-
microcapsules using the pH-sensitive polymer hydroxy- capsulated human fetal islets as an articial endocrine pan-
propyl methylcellulose acetate succinate. J. Microencap- creas.Trans. Am. Soc. Artif. Intern. Organs 1989, 35, 736.
sul. 1996, 13, 497. 270. Sheridan, R.L.; Morgan, J.R.; Mohammad, R. Biomate-
253. Huguet, M.L.; Groboillot, A.; Neufeld, R.J.; Poncelet, D.; rials in burn and wound dressing. In Polymeric Biomate-
Dellacherie, E. Hemoglobin encapsulation in chitosan/ rials, Second Edition, Revised and Extended; Dumitriu, S.,
calcium alginate beads. J. Appl. Polym. Sci. 1994, 51, 1427. Ed.; Marcel Dekker, Inc.: New York, 2002; 451 pp.
254. Tozaki, H.; Komoide, J.; Tada, C.; Maruyama, T.; Terabe, 271. Yasutomi, Y.; Nakakita, N.; Shioya, S.; Kuroyanagi, Y.
A.; Suzuki, T.; Yamamoto, A.; Muranishi, S. Chitosan Burn Inj. 1993, 19, 102.
capsules for colon-specic drug delivery: Improvement of 272. Yannas, I.V.; Burke, J.F.; Orgill, D.P.; Skrabut, E.M.
insulin absorption from the rat colon. J. Pharm. Sci. 1997, Wound tissue can utilize a polymeric template to
86, 1016. synthesize a functional extension of skin. Science 1982,
255. Cho, N.H.; Seong, S.Y.; Chun, K.H.; Kim, Y.H.; Know, 215, 174.
I.C.; Ahn, B.Y.; Jeong, S.Y. Novel mucosal immunization 273. Boyce, S.T.; Christianson, D.J.; Hansbrough, J.F. Struc-
with polysaccharideprotein conjugate entrapped in algi- ture of collagen-gag dermal skin substitute optimized for
nate microspheres. J. Control. Release 1998, 53, 215. cultured human epidermal keratinocytes. J. Biomed.
256. Illum, L.; Farraj, N.F.; Davis, S.S. Chitosan as a novel Mater. Res. 1988, 22, 939.
nasal delivery system for peptide drugs. Pharm. Res. 1994, 274. Boyce, S.T.; Hansbrough, J.F. Biologic attachment,
11, 1186. growth, and dierentiation of cultured human epidermal
257. Aspden, T.J.; Illum, L.; Skaugrud, . Chitosan as a nasal keratinocytes on a graftable collagen and chondroitin-6-
delivery system: evaluation of insulin absorption enhance- sulfate substrate. Surgery 1988, 103, 421.
ment and eect on nasal membrane integrity using rat 275. Copper, M.L.; Hansbrough, J.F. Use of a composite skin
models. Eur. J. Pharm. Sci. 1996, 4, 23. graft composed of cultured human keratinocytes and
258. Suzuki, Y.; Makino, Y. Mucosal drug delivery using broblasts and a collagen-gag matrix to cover full-thick-
cellulose derivates as a functional polymer. J. Control. Rel. ness wounds on athymic mice. Surgery 1991, 109, 198.
1999, 62, 101. 276. Zhu, X.X.; Brown, G.R.; Saint-Pierre, L.E. Adsorption of
259. Wee, S.F.; Gombotz, W.R.; Fanslow, W. Evaluation of bilirubin with polypeptide coated resins. Biomater. Artif.
alginate microbeads for intranasal delivery of ovalbumin. Cells Artif. Organs 1990, 18, 75.
Proc. Int. Symp. Control. Release Bioact. Mater. 1995, 22, 277. Beena, M.S.; Paul, W.; Chandy, T.; Sharma, C.P.
566. Chitosan: A novel matrix for hemoperfusion. In Poly-
260. Bowersock, T.L.; HogenEsch, H.; Suckow, M.; Porter, saccharides in Medical Applications; Dumitriu, S., Ed.;
R.E.; Jackson, R.; Park, K. Oral administration with Marcel Dekker, Inc.: New York, 1996; 651 pp.
alginate microspheres systems. J. Control. Release 1996, 278. Hennink, W.E.; Feijen, J.; Ebert, C.D.; Kim, S.W.
39, 209. Covalently bound conjugates of albumin and heparin:
261. Akiyoshi, K.; Kobayashi, S.; Shichibe, S.; Mix, D.; synthesis, fractionation and characterization. Thromb.
Baudys, M.; Kim, S.W.; Sunamoto, J. Self-assembled Res. 1983, 29, 1.
hydrogel nanoparticle of cholesterol-bearing pullulan as a 279. Hennink, W.E.; Dost, L.; Feijen, J.; Kim, S.W. Inter-
carrier of protein drugs; complexation and stabilization of actions of albuminheparin conjugates preadsorbed sur-
insulin. J. Control. Release 1998, 54, 313. faces with blood. Trans. Am. Soc. Artif. Intern. Organs
262. Gu, X.G.; Schmitt, M.; Hiasa, A.; Nagata, Y.; Ikeda, H.; 1983, 29, 200.
Sasaki, Y.; Akiyoshi, K.; Sunamoto, J.; Nakamura, H.; 280. Hennink, W.E.; Ebert, C.D.; Kim, S.W.; Breemhaar, W.;
Kuribayashi, K.; Shiku, H. A novel hydrophobized Bantjes, A.; Feijen, J. Interactions of antithrombin iii with
polysaccharide/oncoprotein complex vaccine induces in preadsorbed albuminheparin conjugates. Biomaterials
vitro and in vivo cellular and humoral immune responses 1984, 5, 264.
against her2-expressing murine sarcomas. Cancer Res. 281. Hennink, W.E.; Kim, S.W.; Feijen, J. Inhibition of surface
1998, 58, 3385. induced coagulation by preadsorption of albuminheparin
263. Wang, L.; Ikeda, H.; Schmitt, M.; Myahara, Y.; Takaha- conjugates. J. Biomed. Mater. Res. 1984, 18, 911.
shi, Y.; Gu, X.G.; Nagata, Y.; Sasaki, Y.; Akiyoshi, K.; 282. Engbers, G.H.M.; Dost, L.; Hennink, W.E.; Aarts, P.A.;
Sunamoto, J.; Nakamura, H.; Kuribayashi, K.; Shiku, H. Sixma, J.J.; Feijen, J. An in vitro study of the adhesion of
Bone marrow-derivated dendritic cells incorporate and blood platelets onto vascular catheters. part I. J. Biomed.
process hydrophobized polysaccharide/oncoprotein com- Mater. Res. 1987, 21, 613.
plex as antigen presenting cells. Int. J. Oncol. 1999, 14, 695. 283. Bos, G.W.; Scharenborg, N.M.; Poot, A.A.; Engbers,
264. Xi, K.; Tabata, Y.; Uno, K.; Yoshimoto, M.; Kishida, T.; G.H.M; Beugeling, W.G.; Van Aken, T.; Feijen, J.
Sokawa, Y.; Ikada, Y. Liver targeting of interferon Endothelialization of crosslinked albuminheparin gels.
through pullulan conjugation. Pharm. Res. 1996, 13, 1846. Thromb. Haemost. 1999, 82, 1757.
265. Tabata, Y.; Matsui, Y.; Uno, K.; Sokawa, Y.; Ikada, Y. 284. Magoshi, T.; Matsuda, T. Formation of polymerized
Simple mixing of ifn with a polysaccharide having high mixed heparin/albumin surface layer and cellular adhe-
liver anity enables ifn to target to the liver. J. Interferon sional responses. Biomaterials 2002, 3, 976.
Cytokine Res. 1999, 19, 287. 285. Ikada, Y. Membrane as biomaterials. In Polysaccharides in
266. Lim, F.; Sum, A.M. Microencapsulated islets as bioarti- Medical Applications; Dumitriu, S., Ed.; Marcel Dekker,
cial endocrine pancreas. Science 1980, 210, 908. Inc.: New York, 1996; 663 pp.
286. Ohno, M.; Suzuki, M.; Miyagi, M.; Yagi, T.; Sakurai, H.; 303. Gupta, M.N.; Guoqiang, D.; Mattiason, B. Purication of
Kai, T.U. Cta hemodialysis membrane design for h2-mi- endo-polygalactouronase by anity precipitation using
croglobulin removal. In High-Performance Cellulosic Ma- alginate. Biotechnol. Appl. Biochem. 1993, 18, 321.
terials: Cellulosic: Chemical, Biochemical and Material 304. Sharma, S.; Sharma, A.; Gupta, M.N. One step purica-
Aspects; Kennedy, J.F., Phillips, G.O., Williams, P.A., tion of peanut phospholipase d by precipitation with
Eds.; Ellis Horwood, edition, 1993; 663 pp. alginate. Bioseparation 2000, 9, 93.
287. Chan, A.K.C.; Rak, J.; Berry, L.; Liao, P.; Vlasin, M.; 305. Sharma, A.; Sharma, S.; Gupta, M.N. Purication of
Weitz, J.; Klement, P. Antithrombinheparin complex. a wheat germ amylase by precipitation. Protein Expr. Purif.
possible alternative to heparin for arterial thrombosis 2000, 18, 111.
prevention. Circulation 2001, 106, 261. 306. Sardar, M.; Gupta, M.N. Alginate beads as an anity
288. Suzuki, Y.; Tanihara, M.; Suzuki, K.; Saitou, A.; Sufan, material for alpha-amylases. Bioseparation 1998, 7, 159.
W.; Nishimura, Y. Alginate hydrogel linked with synthetic 307. Hidalgo, J.; Hansen, P.M.T. Selective precipitation of
oligopeptide derivated from bmp-2 allows ectopic osteoin- whey proteins with carboxy methylcellulose. J. Dairy Sci.
duction in vivo. J. Biomed. Mater. Res. 2000, 50, 405. 1971, 54, 1270.
289. Muzzarelli, R.; Baldassare, V.; Conti, F.; Ferrara, P.; 308. Grasselli, M.; Diaz, L.E.; Cascone, O. Beaded matrices
Biagini, G.; Gazzanelli, G.; Vasi, V. Biological activity of from cross-linked alginate for anity and ion exchange
chitosan: ultrastructural study. Biomaterials 1988, 9, 247. chromatography of proteins. Biotechnol. Tech. 1993, 7, 707.
290. Hirano, S.; Tsuchida, H.; Nagao, N. N-acetylation in chi- 309. Chen, G.; Scouten, W.H. Alginate as a displacer for
tosan and the rate of its enzymatic hydrolysis. Biomaterials protein displacement chromatography. J. Mol. Recognit.
1989, 10, 574. 1996, 9, 415.
291. Bartone, F.F.; Adickes, E.D. Chitosan: Eects on wound 310. Heger, A.; Grunert, T.; Schulz, P.; Josic, D.; Buschacher,
healing in urogenital tissue: Preliminary report. J. Urol. A. Separation of active and inactive forms of human anti-
1988, 140, 1134. thrombin by heparin anity chromatography. Thromb.
292. Park, Y.J.; Lee, Y.M.; Park, S.N.; Sheen, S.Y.; Chung, Res. 2002, 106, 157.
C.P.; Lee, S.J. Platelet derived growth factor releasing 311. Le Gloahec, V.C.H. Proteinous compound and the manu-
chitosan sponge for periodontal bone regeneration. Bio- facture thereof. U.S. Patent 2430180, 1947.
materials 2000, 21, 153. 312. Lippi, M.S.; Taranto, M.V. Soy proteinacidic polysac-
293. Chappat, M. Some applications of emulsions. Colloids charide interaction: Modication of the emulsion proper-
Surf. A Physicochem. Eng. Asp. 1994, 91, 57. ties of soy protein isolate. Lebensm.-Wiss. Technol. 1981,
294. Tolstoguzov, V.B.; Izjumov, D.B.; Gringer, V.Y.; Maru- 14, 55.
sova, A.N.; Chekhovskaya, V.T. Method of making pro- 313. Ganz, A.J. Proteinpolymer complexes and process. U.S.
tein-containing foodstus resembling minced-meads. U.S. Patent 3407076, 1968.
Patent 3829587, 1974. 314. Larichev, N.A.; Gurov, A.N.; Tolstoguzov, V.B. Protein
295. Soucie, W.G.; Chen, W.S. Edible xanthan gumprotein polysaccharide complexes at the interface I. Character-
brous complexes. U.S. Patent 4563360, 1986. istics of decane/water emulsions stabilized by complexes of
296. Chango, A.; Villaume, C.; Bau, H.M.; Nicolas, J.P.; bovine serum albumins with dextran sulfate. Colloids Surf.
Mejean, L. Debittering of lupin (Lipnus luteus L.) protein 1983, 6, 27.
by calcium alginate and nutritional evaluation. J. Sci. 315. Tokaev, E.S.; Gurov, A.N.; Rogov, I.A.; Tolstoguzov, V.B.
Food Agric. 1993, 63, 195. Properties of oil/water emulsions stabilized by caseinacid
297. Armbruster, B.L.; Desai, N. Identication of proteins and polysaccharide mixtures. Nahrung 1987, 31, 825.
complex carbohydrates in some commercial low-fat 316. Nagasawa, K.; Takahashi, K.; Hattori, M. Improved
products by means of immunolocalization techniques. emulsifying properties of h-lactoglobulin by conjugating
Food Struct. 1993, 12, 289. with carbomethyl dextran. Food Hydrocoll. 1996, 10, 63.
298. Bakker, M.A.E.; Konig, M.M.G.; Visser, J. Fatty ingre- 317. Einhorn-Stoll, U.; Glasenapp, N.; Kunzek, H. Modied
dient. W.O. Patent 14334, 1994. pectins in whey protein emulsions. Nahrung 1996, 40, 60.
299. Omura, T.; Suganuma, O.; Maeda, H. Food material for 318. Cao, Y.; Dickinson, E.; Wedlock, D.J. Inuence of
zinc supplement and zinc-conjugated protein and/or zinc- polysaccharides on the creaming of casein-stabilized
conjugated polysaccharide protein, and their production. emulsions. Food Hydrocoll. 1991, 5, 443.
J.P. Patent 078715, 2001. 319. Suzuki, S.; Kondo, T. Interaction of gelatinacacia micro-
300. Teotia, S.; Khare, S.K.; Gupta, M.N. An ecient capsules with surfactants. Colloids Surf. 1982, 4, 163.
purication process for sweet potato beta-amylase by af- 320. Jones, T.A.; Zou, J.Y.; Cowan, S.W.; Kjeldgaard, M.
nity precipitation with alginate. Enzyme Microb. Tech- Improved methods for binding protein models in electron
nol. 2001, 28, 792. density maps and the location of errors in these models.
301. Senstad, C.; Mattiason, B. Purication of wheat germ Acta Crystallogr. A 1991, 47, 110.
agglutin using occulation with chitosan, and a subsequent 321. Laskowski, R.A.; MacArthur, M.W.; Moss, D.S.; Thorn-
centrifugation or otation. Biotechnol. Bioeng. 1989, 34, ton, J.M. Procheck: a program to check stereochemical
387. quality of protein structures. J. Appl. Crystallogr. 1993, 26,
302. Tyagi, R.; Kumar, A.; Sardar, A.; Kumar, M.; Gupta, 283.
M.N. Chitosan as an anity macroligand for precipitation 322. Evans, S.V. Setor: Hardware lighted three-dimensional
of n-acetyl glucosamine binding proteins/enzymes. Isol. solid model representations of macromolecules. J. Mol.
Purif. 1996, 2, 217. Graph. 1993, 11, 134.
13
Rheological Behavior of Polysaccharides Aqueous Systems
Jacques Lefebvre and Jean-Louis Doublier
INRA-Laboratoire de Physico-Chimie des Macromolecules, Nantes, France
I. INTRODUCTION A remarkable variety of physical chemical properties

reect the structural diversity of polysaccharides. Even
The relevance of rheology to polysaccharide studies con- chemically related polysaccharides can behave quite dier-
cerns both their practical and their fundamental aspects. ently in aqueous media, and, furthermore, in a way which
The capacity of polysaccharides to extensively modify strongly depends on solvent conditions. The same applies
the rheology of aqueous media into which they are intro- by way of consequence to their rheological properties.
duced, even at fairly low concentrations, is the basis of their Polysaccharides are seldom homopolymers; in most
functional properties as thickening and gelling agents. It cases, their backbones comprise several types of sugar
is also involved in many other types of applications, such as monomers linked in sequences which, when indeed not
encapsulation, controlled release, etc. Some degree of totally unknown, are not characterized in detail. The
rheological characterization is essential in particular to heteropolymeric character is responsible for the capacity
evaluate the potential uses of a polysaccharide as extracted of many polysaccharides to form gels in certain conditions.
from a natural source or subsequently modied. Moreover, polysaccharides are often branched polymers;
On the other hand, rheology provides precious tools to the degree and pattern of branching, the lengths of the side
explore and understand the properties of polysaccharides in chains, their composition itself in many cases, are generally
aqueous systems. The rheological behavior of polymer ill dened. Among the polysaccharides that behave on the
systems manifests the underlying structure of the systems. whole as linear homopolymers from a rheological point of
In the simplest case, that of polymer solutions, viscosity is view, many can be nevertheless grafted with short side
directly related to fundamental molecular properties (mo- chains, in which the number, length, and distribution along
lecular conformation, molecular weight and molecular the backbone do play an important role in their properties.
weight distribution, intramolecular and intermolecular inter- On the other hand, the presence of a few of sugar hetero-
actions). In the case of more structured polymer systems, monomers inserted along an otherwise uniform linear
gels, for example, their viscoelastic properties are related to sequence has a strong eect on the conformation of the
supramolecular organization. Although the relation between chain and its physical chemical behavior. Such limited
structure and rheological properties subsequently becomes structural dierences often result in a large variety of
more complex and less direct, rheology allows us to probe rheological properties, which can be observed among
the structure of the systems at dierent scales, in conditions polysaccharides as belonging to the same chemical class
where other physical methods are impossible or dicult to but arising from dierent biological origins.
use. Rheological techniques are especially useful to monitor Extraction, purication, and fractionation processes
and to probe structural changes in the systems, such as are another source of structural diversity. First, some
gelation or phase separation processes. polysaccharide or protein impurities, the nature and quan-
Polymer rheology, which has been thoroughly studied tity of which depend on the purication procedure, are
and set up on rm theoretical foundations, provides a likely to remain in the sample. Their presence has been
general frame for the investigation and the interpretation found to deeply aect the properties of the polysaccharide
of the rheological behavior of polysaccharide systems. under consideration, or can be suspected to do so. Sup-
However, usually the parallel cannot be drawn very far posing this problem is solved, because in the initial material
into the details, particularly on quantitative grounds, as the the polysaccharide of interest certainly presents some
following short discussion will make clear. degree of structural heterogeneity of natural origin, the
357
358 Lefebvre and Doublier
exact composition and structure of the puried sample will cations can be distributed schematically in three classes:
depend on the way it has been prepared. This is the very solutions, gels, and polysaccharide/polysaccharide (or
consequence of the selectivity of the extraction and frac- polysaccharide/protein) mixtures in aqueous media. The
tionation operations themselves. Finally, chemical modi- last class comprises an extremely broad spectrum of sys-
cations of the polysaccharide are likely to occur during the tems ranging from mixed solutions to complex structured
preparation of the sample, giving rise to an artifactual multiphase systems. Biopolymers, even diering little in
heterogeneity; an example is backbone hydrolysis and composition or structure, are in eect generally incompat-
partial demethylation of pectins during their extraction ible in aqueous media. As a consequence, the simultaneous
and purication. Indeed, many dierent samples may be presence of two polysaccharides (or of a polysaccharide
obtained with as many preparation procedures! As a result, and a protein) in the system results in a variety of micro-
puried polysaccharide samples generally show at the same structures through phase separation processes. These pro-
time very large polydispersity, i.e., they contain macro- cesses can interfere or combine with gelation processes in a
molecules with widely dierent molecular weights and way that is highly dependent on the details of the structure
polymolecularitythey contain chains with qualitatively of the biopolymers and on the experimental conditions.
similar compositions, but diering in their quantitative This gives rise to a fascinating variety of morphologies at
compositions and in their structures. With much work, dierent spatial scales, and, among other applications,
polysaccharide fractions with relatively narrow polydis- allows tuning the rheological behavior of the systems
persity can be obtained (to be used as molecular weight to suit specic requirements. In spite of the theoretical and
standards, for example), but in most cases, it is practically practical interest of polysaccharide mixtures, we shall
impossible to avoid some degree of polymolecularity. restrict this chapter to simple solutions and gels, the
One more source of complexity in the behavior of rheology of which marks out the eld of the behaviors of
polysaccharide systems is the special nature of the sol- polysaccharide aqueous systems. Only shear deformation
ventwaterand the delicate balance between chain will be considered. Finally, the discussion of viscoelasticity
chain and chainsolvent interactions (hydrogen bonding, will be limited to the linear domain.
hydrophobic interaction, ionic interactions). Small
changes in ionic strength or ionic composition, and limited
shifts of temperature can induce a change in the physical II. POLYSACCHARIDE SOLUTIONS:
nature of the system, e.g., a transition from the state of a GENERAL REMARKS
macromolecular solution to that of a gel, causing a drastic
modication of its rheological behavior. True polymer solutions are dened by the conjunction of
The polydisperse and polymolecular character of poly- the two following characteristics: (1) they are thermody-
saccharides, as they are available in practice, and the fact namically stable systems; (2) only physical interactions
that their macromolecular structure is generally only quite exist between the coilshydrodynamic interactions and
vaguely known put severe limitations to the quantitative topological constrains which develop above a critical con-
application of polymer rheology theories, whereas their centration (coil overlap concentration).
structural diversity and complexity give rise to an extremely Rheology of polymer solutions has been extensively
rich phenomenology, which largely gives rise to their studied and thorough theoretical treatments are available,
functional versatility. However, within a dened physical at least for linear neutral chains. This provides a frame to
state, the rheological behaviors of polysaccharide/(aque- understand the rheology of polysaccharide solutions.
ous) solvent systems are amenable to a general pattern, and However, because of polydispersity, polymolecularity,
they essentially dier on quantitative grounds. Structural and molecular interactions, departures from polymer laws
dierences mainly show in the conditions controlling the are often observed.
shift from one physical state to the other (from the solution In particular, obtaining true polysaccharide solutions
to the gel and conversely, or from the solution to the is often not trivial. Polysaccharides are well-known to
dispersion or to the precipitate in phase separating sys- manifest a strong propensity to associate via hydrogen
tems). Accordingly, on one hand, theoretical considera- bonds, because of the abundance of hydroxyl groups. This
tions will be kept to a minimum in this chapter. On the is indeed the basis of the gelling properties of polysaccha-
other hand, the rheology-relevant specic features perti- rides such as amylose and amylopectin, agarose, etc. but
nent to the dierent classes of polysaccharides will not be reversible and/or irreversible association occurs also in
surveyed. Our intention is neither to scan the dierent many other cases, although it does not lead to gel forma-
classes of polysaccharides, nor to draw a panorama of the tion in usual observation conditions. In any event, solubi-
application of dierent rheological techniques in this eld; lization of polysaccharides is always dicult when the
several valuable books and extensive reviews, written along concentration is not low. Aggregation and incomplete
these lines, are available and will be quoted in due place. We solubilization result in the presence of microgels of more
shall instead consider the main types of polysaccharide or less swollen particles in the system. The system is then
aqueous systems, and show schematically how rheological actually no longer a solution, but a suspension in a polymer
studies can provide an insight into their organization, solution. These problems increase with polysaccharide
focusing more on the common features than on the specic concentration, with the consequence that polysaccharide
properties of each polysaccharide. The main types of solutions can be prepared in practice only up to rather
polysaccharide systems that are encountered in the appli- limited concentrations. In many cases, the range of con-
Rheological Behavior of Polysaccharides 359
centrations over which the rheological behavior can be distant along the chain cannot occupy the same volume
studied is moreover limited on the lower side because of element at the same time when approaching each other, as a
relatively low MW, hence low viscosity and low viscoelas- consequence of their nite volume. This is the so-called
ticity that would require high sensitivity instruments to be excluded volume interaction. On the other hand, there
measured. Few systematic rheological studies have been are always attractive and repulsive monomer/monomer
carried out on polysaccharide solutions (with the exception and monomer/solvent interactions. The balance between
of cellulose derivatives) for the above reasons, and also these interactions will result in coil contraction or coil
because of the tedious extraction, purication, and frac- expansion. Whereas backbone rigidity and molecular
tionation operations necessary to obtain polysaccharide weight are intrinsic characteristics of the polymer chain
samples suitable for quantitative characterization. considered, this balance depends on solvent quality (the
chemical nature of the solvent and the temperature). The
eective excluded volume will therefore depend on
III. SOLUTIONS OF NONCHARGED CHAINS solvent and temperature. In good solvent conditions,
A. The Isolated Polymer Coil the chain conguration is more expanded, because solva-
tion of chain segments increases excluded volume; then, its
A typical polymer molecule is a long, exible or semiex- average square end-to-end distance will beL 2>Lo2 and can
ible chain that can adopt in solution any conguration be written as:
compatible with its xed bond lengths and angles and other
possible steric restrictions. The set of these spatially and 2
L b2 N2m ; with m z 0:5 1
temporally uctuating congurations denes the equilibri-
um statistical conformation of the polymer coil in the Parameter m is the exponent of the radius of gyration
solvent. The polymer coil is a swollen structure; the volume molecular weight relationship of the coil (Rg~Mm), its
occupied by the monomers accounts only for a small value depending on chain exibility: obviously, m=1 for
fraction of the total volume pervaded by the coil; monomer fully extended rigid chains, and m=1/3 for compact
density decreases from the center of gravity of the coil to its spheres. For polymer coils, m lies of course in between these
periphery. If the volume of the chain itself is neglected, and two limiting values. However, it depends not only on the
if sterical and physical chemical interactions between more or less exible character of the backbone, but also on
monomers or between monomers and solvent molecules polymersolvent interactions, which govern coil expan-
are absent, the spatial distribution of the monomers (or of sion. Exponent m has the value of 0.5 for Gaussian chains,
the chain segments) within the coil would be Gaussian as we have seen; for typical exible polymers in good
(random coil). The volume of the Gaussian coil depends solvents, it is close to 0.6. In bad solvent conditions,
only on the length (or the molecular weight M) of the chain, attraction between chain segments dominates and causes
and on the size bo of the monomer. In real polymer coil collapse: the coil contracts, forming a dense particle
molecules, bond angles are xed and there are steric and (m!1/3), which eventually precipitates from the solution.
energetic restrictions to rotations, resulting in a less-exible Somewhere in between these two situations, repulsion
chain (nonfreely jointed chain). Nevertheless, polymer between chain segments can be exactly compensated for
molecules can still be treated as an equivalent Gaussian by attraction; such solvent conditions are called Q con-
chain by replacing as statistical units the monomers by ditions or Q solvent. In Q conditions, the polymer coil
segments comprising n monomers; the rigidity of the behaves indeed as if it were actually Gaussian and has its
backbone will be reected in the length b=nbo of the unperturbed dimensionLo2=b2N (m=0.5).
statistical elements the articulation of which forms the It is classical to express coil dimension as:
chain. This length b (twice the persistence length Lp)
can be directly determined from small angle neutron or 2 2 2 2
small-angle X-ray scattering experiments. An appropriate L a2LLo ; or Rg a2g Rg o 1b
chain stiness parameter is the ratio of the contour length L
of the chain to the length b of the statistical element: for a aL2 or ag2, called the expansion factor of the polymer, is equal
given polymer, chain exibility increases with the molecu- to 1 in Q conditions and >1 in good solvents. Because the
lar weight. A ratio L/b > 10 would be required for the probability that one segment comes to take the place already
polymer conformation to be regarded as a coil; this corre- occupied by another increases with N, the expansion factor
sponds to M higher than some limiting value Mc. Most increases with the molecular weight of the polymer. It is a
polysaccharides are relatively sti chains, with statistical complicated function of L, b, m, and of the second virial
element length of f10 nm and Mcf3 104. coecient A2 of the polymer in the solution [1].
N, being the number of statistical elements of the
chain, the average square end-to-end distance of the equiv- B. Solutions of Noncharged Chains at Finite
2
alent Gaussian chain is Lp b2 N kM: The diameter of
o Concentrations: The Three Concentration
the coil will be equal to b N , its radius of gyration (R g2)1/2
o Regimes
such as (R g2)o=L o2/6; the average monomer (or segment)
density within the coil will decrease as N1/2. Three concentration domains can be distinguished in so-
The coil volume is actually somewhat larger than that lutions of polymers with molecular weights above the
of the equivalent Gaussian coil, because two segments critical value Mc.
1. Dilute Regime (c < c*) zones exhibit lifetimes much larger than that of entangle-
In a very dilute solution, the volume available to each ments. The system then has shifted from the state of an
polymer molecule is much higher than that of the individ- entangled solution to that of a physical gel (cf. Section
ual coil. The coils remain statistically far from each other, VII). The dierence between the characteristics of the two
and encounters are infrequent. The coils maintain the types of systems is primarily a dierence in degree, and not
dimensions of the isolated chain. This situation prevails in nature. However, the establishment of such junction
up to the critical overlap concentration c*, at which the zones generally involves a change in the conformation of
coils ll the volume of the solution. The volume of each coil the chains, which lose their character of random coils.
2 2 (cf. Section VII).
being proportional to Rg 3=2 1=63=2 L 3=2 ,
2 3. Concentrated Regime (c > c**)
c ~M=L 3=2 ~N13m 2
At certain concentrations c**> c *, the coils reach their Q
dimension; the excluded volume interaction is completely
2. Semidilute Regime (c* < c < c**) screened o. Above c**, coils will shrink no more; the
When polymer concentration is increased above c*, there is polymer solution becomes an entanglement network where
a progressive interpenetration of the coils, concomitant the chains have completely lost their individual character.
with a contraction of their individual volume. Coil con- The only characteristic length in the system is now the mesh
traction is a result of the progressive screening o of the size f of the network, which continues to decrease as
excluded volume interaction: as a result of interpenetra- concentration increases, tending toward its limit value b
tion, segments of foreign chains interpose themselves in the melt.
between segments belonging to the same chain. The solu-
tion becomes a transient network of entangled chains, an IV. THE CASE OF POLYELECTROLYTES
entanglement being the topological constraint correspond-
ing to a point of contact between two chains, and being due A polyelectrolyte is a exible polymer electrically charged
to the fact that the chains cannot cross each other. A given because its structure includes monomers bearing ionizable
polymer with M>Mc statistically contracts a determined groups with charges of the same sign. Many polysaccha-
number of entanglements at a given concentration c>c*, rides, such as alginates, low-methoxyl pectins, carra-
but, because of chain conformation uctuations, these geenans, etc., are anionic polyelectrolytes, negatively
entanglements continuously unfasten to be reformed on charged at pH values above the pK of ionization of their
other points along the chain contour; their lifetime is very acid groups. The only commonly found cationic polysac-
short. If M<Mc, the length of the chain is shorter than the charide is chitosan.
minimum distance required between entanglement points. The distinctive feature of polyelectrolytes is that the
An alternative view [2] is to consider the system as equiv- conformation of the macromolecule depends sharply on
alent to a cage between the rails of which one given chain the ionic strength of the solvent, because the range of the
has to reptate along its own contour in order to move away; electrostatic interaction decreases as ionic concentration
the chain is conned within a tube, the diameter of which is increases. The Debye screening length j1~I 1/2, where I
the mesh size of the temporary network. is the ionic strength, classically measures the range of the
In the semidilute domain, the mesh size f of the network electrostatic interaction in simple electrolyte solutions. In
(average distance between entanglement coupling) and the the case of polyelectrolyte solutions, the polymer itself,
size of the coil decrease as concentration increases. f2 because of its proper charge and of the counterions sur-
measures the mean square end-to-end distance of the chain rounding its charged groups, as well as the salt dissolved in
segment comprised between two successive entanglement the solvent, both contribute to electrostatic screening. The
points along one chain. The chain segment thus dened, Debye length is now j2 j2p j2s, where the indices p and s
made up of g statistical elements, constitutes a blob, and a refer to the polymer and to the small ions contributions
chain of N/g blobs forms the polymer molecule. The (including the counterions of the polyion), respectively.
excluded volume interaction between two adjacent blobs Thus, the conformation depends on both the polymer and
along the chain being screened o by an interposed foreign the salt concentrations; j2p ~fcp, where cp is the concentra-
chain, the chain of blobs is Gaussian, so that the mean tion of the polyelectrolyte and f is the fraction of charged
square end-to-end distance a polymer chain can be written: monomers in its chain. However, the eective contribution
2
L f2 N=g. Within the blob, on the contrary, the excluded of the polymer is generally lower than expected from its
volume interaction is eective and f2=b2g2m. Scaling laws theoretical charge density, as a result of the binding of
2
give [3]: f~cm/13m andL ~c2m1=13m . counterions on the macroion. Ion binding (condensa-
In the semidilute regime, there are two characteristic tion) results from strong attraction of counterions by
lengths in the system: the size of the coilas the coils still the polyelectrolyte when its charge density is high; the ionic
retain some degree of individualityand the entanglement atmosphere surrounding the xed charges can then dier
spacing (or size of the blob). widely from that of the DebyeHuckel approximation.
Looking at the system as a network can be readily Obviously, electrostatic repulsion between the charged
extended to the case where low-energy physical chemical segments of the polyelectrolyte will favor more expanded
interactions develop between chains in the regions of conformations of the chain than if excluded volume eect
entanglements, giving rise to junction zones. Junction were the only long-range interaction existing between chain
segments. Thus, the ionized polyelectrolyte will exhibit tion, the chain becomes exible at all polyelectrolyte
larger mean-square end-to-end length and radius of gyra- concentrations (DF regime). The conformation is then
tion values than the uncharged chain would have in good analogous to that of the neutral chain in good solvent, but
solvent. This can be accounted for by introducing an the expansion factor is controlled by the electrostatic
electrostatic contribution to the persistence length of the screening length, which depends on both c and cs. Above
chain (see, for example, Ref. [4]). In very dilute salt-free c* (SF regime), the chain is likewise exible, but now the
solutions, the macromolecule tends to adopt an extended classical excluded volume interaction screening eect of
rodlike conformation in order to minimize the electrostatic neutral polymers combines with the electrostatic screening
2
contribution to the free energy of the chain; then,L cL~ eect in governing the expansion factor. Whereas both
N. As I increases, electrostatic interaction between charged eects depend on c, the contribution of the latter
segments is progressively screened o. At moderate values decreases as cs increases, and at high enough salt concen-
of I the chain conformation resumes a spherical symmetry, trations the situation becomes similar to that for the
but with a larger radius of gyration than the equivalent neutral chain. Because at low and intermediate ionic
uncharged chain in the same solvent, and at higher values strength values, polyelectrolyte dimension is strongly
of I, the dimension of the coil approaches that of the dependent on its concentration, the semidilute regime is
uncharged chain. very wide; it can extend over three or four polymer
Even a very schematic analysis of the eect of cou- concentration decades.
lombic repulsion of ionized groups on polymer conforma- The extremely low values of c* and the existence of
tion would be beyond the scope of this chapter. The reader strong intermolecular interactions for c < c* make the
can refer to Refs. [5,6] for a thorough theoretical treatment. experimental characterization of the isolated macromole-
Many points about the behavior of polyelectrolytes remain cule dicult at low and intermediate cs values. At large salt
indeed unclear. Therefore, we shall just point out here a few concentrations, experimental problems also arise, because
remarks of practical importance. the added salts aect solvent quality, independently of their
Since its conformation strongly depends on its own electrostatic screening eect; actually, many exible neu-
concentration c, as well as on salt concentration cs, the tral water-soluble polymers approach unperturbed (Q)
phase diagram of a polyelectrolyte is more complex than dimensions as salt concentration increases; they eventually
that of neutral polymers. An example of such a diagram is even precipitate (salting out). The density of charges
shown on Fig. 1. Because the macromolecule is highly along the chain, i.e., the relative number of monomers
expanded, the coil overlap concentration c* is extremely bearing ionizable groups and the degree of ionization are
low at low and intermediate salt concentrations. Below c*, the primary intrinsic characteristics of the chain aecting
the situation is in fact complex. At low salt concentra- polyelectrolyte expansion. Theories take into account an
tions, the polyelectrolyte is in its extended conformation average value for charge density; they introduce, e.g., an
(DR regime of Fig. 1), far dierent from that of a neutral average number of monomers between charges. But the
polymer, and furthermore, there are strong intermolecular distribution of the charged groups along the chain also
interactions. However, for large enough salt concentra- plays a role: the repulsion between a pair of charges is likely
to have a larger eect on the overall conformation when the
charges are distant along the chain than when they are
adjacent. In the case of polysaccharides, charge distribu-
tion is neither even nor random along the chain, and is
generally completely unknown; it is susceptible to consid-
erable variation for a given polysaccharide with a given
average charge density.
V. FLOW BEHAVIOR OF POLYSACCHARIDE

SOLUTIONS
A. Origin of Rheological Properties of
Polymer Solutions
In the dilute regime, Newtonian ow behavior and absence
of viscoelasticity are generally observable in practical
conditions, at least for noncharged polymers,* because
statistically, macromolecules are spatially and temporally
Figure 1 Theoretical phase diagram for an aqueous poly- noncorrelated. Nevertheless, in principle, polymer coils are
electrolyte solution (molar concentration c) in presence of
able to deform when submitted to velocity gradients, and to
added salt (molar concentration cs). DR: dilute rodlike regime.
recover their equilibrium conformation after cessation of
DF: dilute regime of exible conformation. SF: semi-dilute
regime. The diagram has been calculated for a chain with
N=3350 monomers, an average number of monomers between * As we shall see later, this is not the case for dilute polyelectrolyte
charges A=5, and Nb/A=3, where A is the actual extended solutions in low added salt conditions, because of long-range
length of the chain. Reproduced from Dobrynin et al. [5]. electrostatic interactions.
the mechanical excitation, hence intrinsic non-Newtonian great theoretical interest, it has little practical importance
and viscoelastic properties. However, these properties are and the topic will not be considered here. In this section,
very faint and are exhibited in practice only at fairly high viscosity and intrinsic viscosity will refer implicitly to
deformation rates. They are, in consequence, of little direct measurements performed within the Newtonian domain,
concern for applications, as far as polysaccharide dilute so- i.e., at shear rates low enough for the dilute solutions to
lutions are considered. However, internal coil deformation display shear rate-independent viscosity. The condition of
modes are responsible for the short-time or high-frequency Newtonian behavior, in most cases, easy to achieve for di-
viscoelastic response of polymer nondilute solutions and luted polymer solutions, can be in some others (when
gels. On the other hand, the characteristics of the polymer working on polymers with very high molecular weight, or
that are reected in the viscosity of its dilute solutions with rather sti chains, and on polyelectrolytes at low salt con-
govern, to a large extent, its viscous behavior in nondilute centrations) more dicult to accomplish experimentally.
solutions. In consequence, no characterization of the semi-
dilute and concentrated regimes of polysaccharides, which 1. The Intrinsic Viscosity of Noncharged Polymers
are the relevant issues regarding applications, is possible The perturbation to the ow of a liquid resulting from the
without the knowledge of the behavior of the polysacchar- dispersion in this liquid of rigid, monodisperse, noninter-
ides in dilute solutions. This is why dilute solution proper- acting, and randomly distributed spheres has been given by
ties have to be given some development. Einstein as a series expansion of the viscosity g of the sus-
In practice, non-Newtonian ow behavior and visco- pension as a function of the volume fraction u of the particles:
elasticity of polymer solutions develop for c > c*. In the
g gs 1 k1 u k2 u2 : : : 3
case of exible or semiexible chains, these properties
originate from the disentanglement/re-entanglement pro- where gs is the viscosity of the dispersing liquid, and kis are
cesses resulting from the opposite actions of ow and of positive coecients. Einstein calculated the coecient of the
thermal agitation. They are manifestations of the transient term of the rst degree in u to be k1=2.5. Later, Eq. (3) was
network structure. Dispersions of rigid macromolecules or generalized to ellipsoidal particles; in the case of Brownian
particles exhibit similar properties above some critical suspensions of ellipsoids of revolution, k1>2.5 and increases
concentration; but they are mainly due, in this case, to with the axial ratio a/b; the relation k1=f(a/b) was estab-
the spontaneous establishment of a local order at rest, as a lished and can be solved numerically (Simha, 1940).
consequence of crowding; strain perturbs this order and Eq. (1) can be written as:
thermal agitation tends to restore it. When macromolecules
gsp 1 g
or particles are asymmetrical, orientation in the direction u 1 k1 k2 u : : : 4
of ow plays, in addition, an important role (liquid crystal u u gs
structures). For solutions of exible chains as well as for The quantity gsp u ([g/gs]1) is called the specic viscosity
dispersions of rigid particles, non-Newtonian ow and of the suspension. Extending Eq. (4) to the case of the
viscoelastic properties are therefore of entropic origin. solutions of rigid macromolecules, we can replace the
volume fraction u by the usual weight/volume concentra-
B. Viscosity of Dilute Solutions tion c of the macromolecule; because u = c Nat/M, where t
is the molecular eective volume in solution and Na is
Only a very schematic and short account will be provided Avogadros number, it comes for spherical rigid macro-
here on viscosity of dilute solutions of polymers in general, molecules:
and of polysaccharides in particular. The matter, which has
gsp 1 g t t
direct bearing on macromolecular conformation studies, u 1 Na 2:5 k2 c : : : 5
has been the subject of an enormous amount of theoretical c c gs M M
and of experimental work, and books or extensive reviews and
are available on it (for example, Refs. [79]). The main g
sp t
information that can be extracted from viscosity measure- lim u g 2:5Na 6
c!0 c M
ments on dilute macromolecular solutions is contained in
the intrinsic viscosity of the macromolecule. Intrinsic vis- Eq. (6) denes the intrinsic viscosity [g] of the macromol-
cosity is not a viscosity at all, but actually a measure of the ecule, which is generally expressed in mL/g. The intrinsic
hydrodynamic volume of the coil in the case of noncharged viscosity of rigid spherical macromolecules is actually
polymeric chains, or of the asymmetry of the particle in the independent of their size, but depends on their specic
case of rigid macromolecules. The case of polyelectrolytes hydrodynamic volume t/M.
is specic, and remains poorly understood. The last step is to generalize Eqs. (5) and (6) and the
As already alluded to, for most polymers, non-New- concept of intrinsic viscosity to the case of solutions of
tonian behavior is exhibited from the lowest concentra- polymer coil macromolecules. The underlying assumption,
tions, as a result of the deformation and of the orientation approximately veried for exible polymer chains with
of the polymer coil in ow. The intrinsic viscosity itself is molecular weight>Mc, is that polymer coils in dilute
therefore shear rate-dependent, in a way that is determined solutions behave as equivalent solid spheres with a hydro-
by the structure of the macromolecule, its expansion due to dynamic radius Rh = nRg. This is called the nondraining
polymersolvent interactions and its deformation in ow. coil approximation, meaning that the solvent inside the
Although the shear dependence of intrinsic viscosity has a coil is considered to move as if trapped in it. The factor n has
a theoretical value of 0.875 for Gaussian chains; experi- linear chains. However, such low values of the exponent
mental values decrease from 0.860.83 in Q conditions to could reect as well some degree of branching: obviously, a
0.775 in good solvents [9], as a consequence of lowered branched polymer molecule pervades a smaller volume
segment density within the coil in good solvents. Because than the linear chain with the same molecular weight.
2
t 4=3pR3h ~L 3=2 , combining Eqs. (1) and (6) gives: The bilogarithmic plot of [g] against M for polymers in
good solvents often displays a break at a molecular weight
g~b3 N3m =M~M3m1 7 M*fMc; for M < M*, the exponent is f0.5; the MHS
exponent a refers to M>M*.
Eq. (7) shows that the intrinsic viscosity of exible or
semirigid macromolecules depends on the rigidity and on
2. Viscosity of Dilute Polyelectrolyte Solutions
the molecular weight of the chain. It is generally cast in the
form of the FloryFox equation: As a consequence of Section IV, it can be expected that the
reduced viscosity gsp/c of polyelectrolyte solutions, for low
g 63=2 UR3g =M 8 values of c and for low and intermediate salt concentra-
tions, will show a complex variation with c and with cs,
where the proportionality factor U = 2.5(4/3)pNan3 is a because the conformation of the macromolecule itself
universal constant for Gaussian coils (U = 2.86 1023 depends on these parameters. Indeed, in contrast to neutral
kg1), but decreases from f2.8 1023 in Q conditions to polymers, for which it is a monotonously increasing func-
f2.11023 kg1 in good solvents [9]; in Eq. (8), the number tion of concentration (cf. Eq. (5)), one observes that the
averages of the molecular weight and of the radius of gyra- reduced viscosity gred = gsp/c of polyelectrolyte solutions
tion are to be used when polydisperse samples are considered. at low ionic strength is a strongly decreasing function of c
Yamakawas general theory of the hydrodynamics of within a certain concentration domain (Fig. 2). This be-
wormlike chains [10] gives an expression of the intrinsic havior has been accounted for by the Fuoss equation:
viscosity as a function of polymer chain parameters,
encompassing the complete range of polymer conforma- c 1 B
c1=2 10
tions, from the rigid rod to the completely exible coil [11]. gsp A A
The Yamakawa expression for the intrinsic viscosity can be
approximated by the equation of Bohdanecky [12]: Fuoss equation (as well as other relations of the same
general form in 1/c1/2 that can be found in Ref. [13]) is
13 empirical and fails to describe the molecular weight and
M2 1
P QM 2 8b the charge eects on the viscosity. Nevertheless, it has
g
found support, to some extent, in a general theory for
where P = 1.516 108 AoML and Q = 1.516 108 Bo electrolytes, which predicts a gred~c1/2 dependence at the
(ML/2Lp)1/2 in CGS units, with ML = M/L. The factors P limit c H cs [6]. But the usual interpretations of Fuoss
and Q are functions of d/2Lp, d being the diameter of the equation as reecting a coil to rod transition due to the
wormlike cylinder, which are tabulated in Bohdanecky decrease of the ionic strength upon dilution of the poly-
paper. It has to be pointed out that the excluded volume electrolyte, and of A as the intrinsic viscosity of the fully
interaction is not taken into account in Eq. (8b). stretched chain, are grossly in error, as it will be briey
The relation between intrinsic viscosity and molecular discussed presently.
weight of polymer-like macromolecules is usually For low salt and intermediate concentrations, the plot
expressed in the form of the empirical Mark-Houwink of the reduced viscosity against polyelectrolyte concentra-
Sakurada (MHS) equation: tion actually exhibits a maximum located at a very low
polymer concentration value cmax, as it can be seen on the
g KM a 9
examples of Fig. 2. Therefore, the parameters of Eq. 10
where K and a are empirical parameters depending on the have no physical meaning. The peak vanishes above a
polymersolvent pair and on the temperature, and are both certain salt concentration and the solution resumes the
related to chain stiness. The value of the exponent, since usual polymer solution behavior (Fig. 2). The existence of
theoretically a = 3m1 (compare Eqs. (7) and (9)), gives an this reduced viscosity maximum has been observed long
indication about the general conformation of the polymer. ago, for sodium pectinate solutions containing dierent
For exible linear chains, 0.5 V a V 1; it typically assumes concentrations of NaCl in the range 00.05 M [14]. It was
values between 0.5 and 0.8, according the excluded volume later experimentally conrmed by many studies on poly-
eect is more or less signicant (cf. Section III.A and Eqs. electrolytes diering in structure and charge density.
(7) and (8)). Sti chains display larger values of a (values as Precise measurements of extremely small specic viscosity
high as 1.8 are reported for rodlike chain conformations), increments on dilute solutions of well-dened fractions of
and these values are less dependent on solvent quality. synthetic polyelctrolytes, as well as accurate control of
However, sti chains with negligible volume exclusion can minute concentrations of polymer and very low ionic
also display a values in the same interval as typical exible strengths, made possible the study of the eect of dierent
chains, as a result of partial draining, with nevertheless the factors on the reduced viscosity peak (see, for example,
dierence that in the former case, a is not signicantly Refs. [1519]). These studies have been carried out on
aected by solvent quality. On the other end of the scale, polyelectrolytes with high charge density. As salt concen-
a < 0.5 indicates some degree of coil collapse in the case of tration was increased in the solution, the peak shifted to
the behavior of dilute solutions of polyelectrolytes at low

and intermediate salt concentrations is governed by long-
range electrostatic intermolecular interactions or by the
formation of relatively large clusters [17,19]. The latter
interpretation is corroborated by that of the characteristic
intensity proles of low salt polyelectrolyte solutions in
small angle light scattering experiments, by Ermi and Amis
[20]. They attribute the steep intensity upturn at low
scattering vector values to multichain domains existing
even at low polyelectrolyte concentration; regrading the
characteristic intensity peak at nite scattering vector
value, it would be attributable to a network structure with
short-range order within the domains.
The marked shear dependence of viscosity in the peak
region and its further increases for c<cmax, which have
been frequently observed since long ago (rst, for dilute
pectin and carboxymethyl cellulose solutions containing
low NaCl concentrations [21]), pointed to the same direc-
tion. Finally, dilute dispersions of colloidal ionic polymer
latices in low-salt conditions have been shown to exhibit
Figure 2 Reduced viscosities of dilute pectin solutions (25jC,
reduced viscosity dependence on colloid concentration, on
pH 7) in the presence of dierent added salt concentrations,
salt concentration, and on shear rate, which are similar to
plotted against pectin concentration.
. Pals and Hermans data [21]: squares (in water), circles those of polyelectrolytes [22,23]. Polymer latices are rigid
(in 2 104 M NaCl), triangles (in 4 104 M NaCl), spheres. Therefore, the similitude is a compelling indica-
lozenges (in 0.05 M NaCl); the continuous line tion that conformational changes of polyelectrolytes relat-
represents the linear t of the data for 0.05 M NaCl. ed to intramolecular repulsion between charged groups
The intrinsic viscosity of the sample in 0.05 M NaCl might well not play an essential role as regards the specic
was 295 mL/g. features of the viscous behavior of dilute polyelectrolytes.
. M. A. V. Axelos data in water at 25jC (unpublished A model for high charge density polyelectrolyte so-
results): crosses and dotted line. The intrinsic vis- lutions has been derived [15] from a general theory for
cosity of the sample in 0.1 M NaCl was 282 mL/g, chargecharge interactions in Brownian spheres hydrody-
its content in galacturonic acid was 80% and its namics. It successfully accounts for the existence of the
degree of methylation, 30%. reduced viscosity peak and its variations with salt concen-
tration, polyelectrolyte charge, and molecular weight, as
well as for the Fuoss law behavior at higher polyelectrolyte
higher polymer concentrations and its height decreased. concentration [1517]. Borsali et al. [24], on the same
Besides, the reduced viscosity and the polyelectrolyte theoretical basis, have given a more rigorous treatment
concentration at the peak maximum were found to infor weakly charged polyelectrolytes.
crease linearly with the molecular weight of the polyelec- The theory of the wormlike chain model of Yama
trolyte; the position of the peak proved to be extremely kawa has been extended to the intrinsic viscosity of poly-
sensitive to temperature and to shift to higher polymer electrolytes (electrostatic wormlike chain theory) by
concentration values as temperature increases [17]. taking into account the electrostatic contributions to the
The reduced viscosity peak has been for long consid- persistence length and to the excluded volume [4,25,26].
ered as a consequence of polylectrolyte conformation In the study of polysaccharidic polyelectrolyte dilute
changes with concentration, and therefore to be related solutions in low-salt conditions, the diculties of polyelec-
to intramolecular interactions (see, for example, Refs. trolyte behavior combine with those inherent to biomacro-
[18,19]). However, several arguments have progressively molecules. Besides, from a utilitarian point of view,
emerged in favor of a dominant implication of intermolec- polysaccharide polyelectrolytes are always used in practice
ular electrostatic interactions in the distinctive behavior of at concentrations larger than c* and in solutions containing
very dilute polyelectrolyte solutions at low salt concentra- relatively large ionic concentrations. So that the approach
tion [6]. remains empirical in essence, and is generally based on
It has been often pointed out that the height of the determination of the intrinsic viscosity at dierent added
reduced viscosity peak exceeds by far the theoretical in- salt concentrations; the isoionic dilution method of Pals
trinsic viscosity of an equivalent rodlike molecule. Cohen and Hermans [21] is generally followed, as it will be briey
and Priel [17] observed that reduced viscosity data obtained described in Sections V.B.3 and V.B.4.
at extreme dilution (c b cmax) of their polyelectrolytes in
salt-free conditions could be linearly extrapolated to c=0; 3. Determination of Intrinsic Viscosity
they found that the apparent intrinsic viscosities thus Let us rst examine the case of rigid macromolecules and of
obtained were several orders larger than those calculated noncharged polymer coils, or of polyelectrolytes at salt con-
for the fully stretched polymers. These facts suggested that centration high enough for the electrostatic inter- and intra-
molecular interactions to be screened o. Eq. (4) can be

written in the form of a virial expansion of the concentration:
gsp
g 1 kH gc Oc2 11
c
Limited to its rst two terms, it is called the Huggins
equation; the dimensionless Huggins coecient kH accounts
for hydrodynamic pair interactions between the macromol-
ecules. Huggins equation is linear in c, and thus it is used to
obtain by extrapolation to c=0 the intrinsic viscosity of
macromolecules. Many other linearized forms of Eq. (4) in
the low concentration range have been proposed for this
purpose; one the use of which is the most widely spread is
Kramers equation:
lngsp 1
g kVg2 c 12
c
It is easy to show that Eqs. (11) and (12) are equivalent if
kH+kV=0.5. Coecients kH and kV are normally positive.
Linear extrapolation using Eq. (11) or Eq. (12)
requires the specic viscosity data to be obtained in a range Figure 3 An example of determination of the intrinsic
of low enough polymer concentrations, with c being kept viscosity of a polysaccharide by the double extrapolation
below c*. As a practical rule, it is often considered that only method. Xanthan solutions at 25jC. The reduced viscosities
data such as gsp<0.7 should be used for extrapolation, but were obtained from the low shear rate Newtonian plateau
curvature of the reduced viscosity plot can be observed in viscosity values measured with a LS40 Contraves rotational
viscometer. Huggins extrapolation [Eq. (11), continuous line]
some cases even for gsp<0.7. On the other hand, the
gives [g]=6388 mL/g and Kramer extrapolation [Eq. (12),
experimental errors on viscosity and on concentration
dotted line] [g]=6495 mL/g.
measurements increase as concentration decreases, making
extrapolation somewhat tricky. It is therefore advisable to
simultaneously use Eqs. (11) and (12); they should extrap- values are consistent with those predicted by some theories
olate to a common intercept [g]. An example of this double [7]. Concentration-dependent aggregation causes kH to
extrapolation procedure is given in Fig. 3. increase, and explains why sometimes very large values of
The viscosity of the solvent and of the dilute solutions the Huggins coecient are found. From a practical point of
is generally measured with capillary viscometers. Such view and especially in the eld of polysaccharides, one
instruments give very sensitive and precise measurements, should refrain from giving to an experimental value of kH
but do not allow proper control of the shear rate; the shear too much a signicance. Usually, kH is aected by a large
rate changes continuously during the measurement as the error, because it is obtained by dividing the slope of the
liquid ows down the capillary, and its average values are Huggins plot (gsp/c vs. c) by the square of the extrapolated
generally quite largeof the order of 100 s1. This can be a intercept. Nevertheless, the value of kH can be used as an
real problem with large molecular weight polymers or with alarm signal; kH>f0.8 would indicate either that mea-
very asymmetric rigid macromolecules, which display non- surements at too high concentrations have been taken into
Newtonian ow behavior even below c*, because the account, or that the polysaccharide is aggregating. How-
viscosity data to be used in the determination of [g] have ever, if the content in aggregates of the solution does not
to be the low-shear Newtonian ones. In addition, viscosity depend on concentration, kH is not aected, but [g] is.
measurements with capillary viscometers require the den- Determining the intrinsic viscosity of polyelectrolytes
sity of the solutions to be accurately known. Rotational is by no means a simple question. At salt concentrations
viscometers allow absolute measurement of viscosity and cs z f0.1 M, the contribution of the polyion to the ionic
to investigate the eect of shear rate. Therefore, they are in strength is usually negligible, and the solution can be
principle preferable, if available instruments could be diluted with the salt solution and treated as that of an
sensitive enough; unfortunately, this is not the case for uncharged polymer. At lower salt concentrations, this is no
most commercial instruments. longer the case (Section V.B.2). The method usually fol-
Much eort has been spent to interpret the Huggins lowed is then that of isoionic dilution [21]. It consists of
coecient. The theoretical value for rigid spheres itself, diluting the polyelectrolyte solution with solutions of a uni-
kH=0.69, was not rigorously established before the 1960s. valent electrolyte of such a molarity as to keep constant the
For polymer coils, the problem is more complex, because total eective concentration of small ions (salt ions+poly-
they can interpenetrate and shrink as concentration ion mobile counterions). This amounts to keep constant
increases. Then, kH is related to the expansion coecient the eective ionic strength I=Is+kc (where Is is the
[7]. Experimentally, values of kHf0.5 are found in Q contribution of the added salt and k is a coecient depend-
conditions, and k H decreases as solvent quality ing on the eective charge of the polyion). In this way, the
improves, approaching f0.3 in very good solvents; these Huggins plots for the polysaccharides become linear and
dissolved material in polysaccharide solutions is often the

cause of such discrepancies. This has been recently exem-
plied and discussed in the case of guar by Picout et al. [27].
The other main problem is the identity of the structure of
the calibration samples, and their similarity in this respect
with the unknown samples to be characterized, because
fractionated samples dier often not only as regards their
molecular weights, but also with respect to their structure.
Polymolecularity is generally associated with polydispersi-
ty in the case of polysaccharides, as we have already
stressed in the introduction to this chapter. Dextran pro-
vides a good illustration of this point. Figure 5 shows the
intrinsic viscosityweight average molecular weight plots
on bilogarithmic scales of two sets of data (from Refs.
[28,29], respectively), obtained in water on dextran frac-
tions with reduced polydispersity; the fractions were sepa-
rated from acid-hydrolyzed puried native dextran
preparations. The plots show clearly a breakpoint: their
slope decreases steadily as MW increases above MWf105,
Figure 4 An illustration of the isoionic dilution method. whereas it is constant and close to 0.5 below this limit.
Reduced viscosity of pectin solutions at pH 7 and 25jC is Native dextran is a very high molecular weight microbial
plotted against the concentration of the pectin at dierent glucan exopolysaccharide with a very large MW distribu-
constant eective ionic strengths in presence of NaCl. tion. It is a branched polymer, with alpha-1,6 glucosidic
Replotted from the original data of Pals and Hermans [21]. backbone bonds and alpha-1,3 branching. Partial acid
Total eective ionic strength: a: 3.75 104 M; b: 7.5 104 M; hydrolysis breaks both types of linkages, so that the lower
c: 1.5 103 M; d: 3 103; e: 0.012 M; f: 0.096 M. the molecular weight, the lower the degree of branching:
this explains the downward curvature of the MHS plot for
high MW fractions. To interpret Fig. 5, we could imagine
can be treated the same way as noncharged polymers. The
isoionic dilution procedure, illustrated in Fig. 4, has found
a basis in the theory of Hess and Klein, which yields gred~c
when ks=constant [6]. However, in view of the discussion
of Section V.B.2, it is clear that the intrinsic viscosity
values of polyelectrolytes obtained with the method of
isoionic dilution are to be considered rather as empirical
quantities.
4. Practical Relevance of Intrinsic
Viscosity Determinations
Molecular Weight Determinations.
Intrinsic viscosity determination allows handy and
cheap evaluation of the average molecular weight of a
polymer sample, provided that the parameters K and a of
Eq. (9) relative to this polymer have been determined
beforehand on a series of samples with low polydispersity
index and known molecular weight values, in the same
conditions of solvent and temperature. Weight average
values of the molecular weights (such as those determined
by light scattering) should be used to establish the [g]M
correlation, preferably to number-average values. The
calibration samples should cover a large enough molecular
weight range, and all of them should have molecular Figure 5 Intrinsic viscositymolecular weight relationship
weights higher than Mc. Comprehensive (K,a) data can for dextran fractions. Empty circles and dotted line: data of
be found in the literature [8,13]. Soeteman and Peeters [29]; 30jC; molecular weights deter-
However, because of many practical problems, the mined by GPC; polydispersity ratios MW/Mn=1.33. Filled
method should be applied with caution and it is wise not to circles and continuous line: data of Senti et al. [28]; 25jC;
expect more than a rough estimate of molecular weights molecular weights determined by light scattering; polydisper-
from [g]MW correlations found in the literature, which are sity ratios MW/Mn=1.2. The lines represent the power law
often diverging even for relatively simple polysaccharides. ts of the two sets of data for MW V 105; the exponent values
The presence of a small amount of aggregated or non- are 0.53 and 0.49, respectively.
that above a certain degree of hydrolysis, nearly linear uous than the exponent a does. In Q conditions, the exible
dextran fractions are obtained; consequently, the MHS or semiexible polymer chain is Gaussian; then, its mean
2
plot becomes linear and the value of the slope (f0.5) would square end-to-end distance is Lo b2 N and its intrinsic
2 3=2 2
indicate that water at 2530jC is a Q solvent for dextran. viscosity gQ ULo =M KQ M1=2. The ratio C Lo =
2 2
However, even comparatively low MW dextran fractions Lf , Lf , being the mean square end-to-end distance of the
do exhibit a small but denite degree of branching [30]. equivalent freely jointed chain, reects the eect of steric or
Indeed, intrinsic viscosity determinations on 18 dextran energetic hindrances on coil dimension. Therefore, the
fractions with quite narrow MW distributions (MW / characteristic ratio C measures the intrinsic exibility
Mnf1.045) in the MW range of 12,000150,000 have of the chain. If the freely jointed chain contains Nf segments
2
shown that the slope of the MHS plot actually decreases of length bf,Lf b2f Nf. Since L=bN=bfNf, it comes readily
steadily, from 0.483 to 0.396 as the molecular weight that C b=bf L2o =b2f m=M, where m is the molecular
increases [31]. Therefore, the low apparent value (af0.5) weight of the segment (i.e., the monomer) of the freely
of the MHS exponent in this MW range is certainly to be jointed chain, and so KQ UCb2f =m3=2. However, nding
ascribed to the branched character of the chains rather than Q conditions is never an easy task, and in the case of
to the quality of the solvent. The MHS exponent of polysaccharide, it is generally an impossible one. On the
hypothetical linear dextran in water has been calculated other hand, in solvent conditions better than Q conditions,
to be f0.68 [32]. the problem of relating the intrinsic viscosity to the char-
For most polysaccharides, values found for exponent acteristic ratio becomes extremely dicult because now the
a lie within the range expected for typical exible polymers. excluded volume eect has to be taken into account: for
This is the case even for chains recognized to have a rather example, via approximate expressions of the viscosity ex-
sti character, such as cellulose derivatives or xanthan, and pansion factor, ag3=[g]/[g]Q. Several procedures have been
results probably from the partially draining character of proposed [7]. The most convenient one is that of Stock-
rather sti chains (cf. Section V.B.1). mayerFixmanBurchard (SFB), which plots [g]/M1/2
In the presence of salt concentration cs high enough to against M1/2; extrapolation to M=0 gives KQ; in not too
screen o the electrostatic interactions, polyelectrolyte good solvents, the plot is linear and extrapolation is easy.
behave similarly to neutral polymers. Scaling theory pre- In this way, Robinson et al. [36] found KQ=0.42 mL/g and
dicts in such conditions [g]~M4/5 in good solvent, the value C=12.6 (and therefore Lp=3.4 nm) for guar gum. Picout
of the exponent being independent of cs in the high salt limit et al. [27,37] have recently reported persistence lengths of
[5]. At low and intermediate salt concentrations, the intrin- 3<Lp<5 nm for all galactomannans; these values were
sic viscosity is expected to depend on cs. But in practice, the obtained via the SFB procedure on solutions submitted to a
true intrinsic viscosity cannot be experimentally deter- heat/pressure treatment in order to insure complete solu-
mined, as it has been discussed in Sections V.B.2 and V.B.3. bilization. They found that the persistence length was
Taking for [g] the linear extrapolation of the reduced insensitive to the degree of substitution of the chain (the
viscosity measured at extremely low polymer concentra- ratio mannose/galactose is typically f4 for locust bean
tions (c b cmax), Cohen and Priel [17] found [g]~M for gum, f3 for tara gum, and f2 for guar gum).
sodium polystyrene sulfonate in salt-free conditions. Noda The SFB method requires a series of samples with
et al. [33] studied solutions of sodium polyacrylate at known molecular weights, spanning a large enough range,
dierent concentrations of NaBr and for dierent degrees and with reasonably narrow molecular weight distribu-
of ionization of the polyelectrolyte; they determined ap- tions. On the other hand, it is limited to rather exible
parent intrinsic viscosity values by the usual Huggins and chains and to high molecular weights. Application of the
Kramer extrapolations for polymer concentrations higher Yamakawa wormlike chain model circumvents these lim-
than f103 g/mL. As the salt concentration was increased itations, at the expense of some computation; the model
from 0.01 to 0.5 M, the MHS exponent decreased from allows the length of the statistical segment b=2Lp to be
f0.9 to 0.384 at low charge density, and from f0.9 to calculated from the intrinsic viscosity if the molecular
f0.66 for the fully ionized polymer. Vreeman et al. [34] weight of the polymer is known and an estimation of the
found similar results for n-carrageenan in the disordered diameter of the chain is available [11]. As already discussed,
form; the MHS exponent decreased from 0.90 in 0.028 M the model spans the full range of conformations, from the
NaCl to an extrapolated value of 0.67 at innite salt exible coil to the rigid rod. Cros et al. [38] applied this
concentration; intrinsic viscosities were determined by model to citrus pectins with degrees of methylation (DM)
Huggins extrapolation of data obtained at carrageenan ranging from 28% to 73%, in 0.1 M NaCl pH7 solutions at
concentrations c V 103 g/mL. The behavior of xanthan 25jC; the molecular weights, determined from light scat-
seems dierent; over the NaCl range of 0.010.3 M, Tin- tering experiments, fell between 70,000 and 200,000,
land and Rinaudo [35] found a to decrease from 1.36 to 1. depending on the DM of the sample. The results were
The authors related the high value of the exponent to the compared to Lp values obtained from small-angle neutron
high intrinsic stiness of xanthan, which is in a helical scattering (SANS) experiments; both sets are reported in
conformation in this salt concentration range. Table 1. The fact that Lp values from [g] are systematically
in excess over those from SANS was ascribed to the
Intrinsic Viscosity and Chain Stiness. presence of aggregates in the solutions, to which SANS
In principle, factor K in the MHS equation contains data are not sensitive, but which does aect viscosity.
information on macromolecular conformation less ambig- SANS results show that Lp represents 1017 monomers,
Table 1 Comparison of the Persistence Lengths Determined the macromolecule. The results agreed with those of Sho et
from Intrinsic Viscosity with Those Obtained By Small Angle al. [39] as to the little sensitivity of [g] to the ionic strength,
Neutron Scattering Experiments (SANS) for Pectin Samples and to the correlative smallness of the contribution of Lpe
with Dierent Degrees of Methylation (DM) to Lp. But Sho et al. found a much larger value for the
persistence length at innite ionic strength (Lp=106 nm).
[g] (mL/g), 0.1
103 M NaCl, pH 7, Lp Lp The discrepancy is probably attributable to the fact that
DM Mwa 25jC (nm)b (nm)c Sho et al. used Eq. (8b) and therefore neglected the eect of
the excluded volume interaction, which was taken into
73d 196 562 10.1 5.4 account by Tinland and Rinaudo.
48d 160 536 11.5 4.5 Extensive data on polysaccharides persistence lengths
44d 114 362 10.2 5.3 are tabulated in Lapasin and Pricl [13]. They show that
40d 127 329 8.5 6.0 polysaccharides display a very large range of chain sti-
28d 70 271 12.6 7.5 ness. However, as most of these data were obtained from
28e 176 282 5.9 5.1 intrinsic viscosity, they can be biased as a result of aggre-
a
Average molecular weight measured by HPSEC/MALLS.
gation. This could explain, in addition to the large vari-
b
Persistence length from intrinsic viscosity [g] (Yamakawa model). ability of the detailed structure of polysaccharides, the
c
Persistence length from SANS measurements. large scatter of the data for the same type of polysaccha-
d
Citrus pectin. ride. The scatter is particularly large for xanthan. The
e
Apple pectin. conformation and the state of aggregation of this polyelec-
Source: Ref. 38. trolyte depend not only on the fermentation process, the
molecular weight, the solvent conditions, but also on the
preparation steps following fermentation [40].
In the intermediate salt concentration range, the in-
meaning that pectins are semiexible coils in the solvent trinsic viscosity of polyelectrolytes, determined by the
conditions of the study. Table 1 indicates that pectin isoionic dilution method, decreases as ionic strength
exibility is maximum at a degree of methylation f50%; increases and reaches a constant value [g]l at high salt
this maximum level probably results from the interplay of concentration. This variation is well represented [21] by:
hydrophobic and hydrophilic interactions, favored at high
and at low DM, respectively, on the short range confor- g gl SI1=2 13
mation. Table 1 includes for comparison the results for one
sample of LM pectin from apple, showing that its chain is The parameter S shows a low power (exponent<1) depen-
appreciably more exible than the citrus pectin with the dence on molecular weight. Smidsrd suggested to write
same degree of methylation. This illustrates the importance S=B([g]0.1)n, as [g]0.1 itself is related to M by a power law
of the botanical source as regards the structure and the (the MHS equation), and found n to vary in a narrow range
properties of plant polysaccharides. 1.21.4 [41]. Smidsrd parameter B is often used as an
The persistence length Lp of a polyelectrolyte at a empirical measure of the stiness of polyelectrolyte chains
given ionic strength is the sum of two terms, Lpe and Lpo, [41]. The lower B is, the stier the chain becomes. This is
corresponding to the contributions of electrostatic interac- illustrated in Table 2; for citrus pectins with dierent
tion and of nonionic eects (including the excluded volume degrees of methylation, B indicates that exibility shows
interaction), respectively; Lpe is a decreasing function of a maximum around DMf40%, in a similar way to the
ionic strength and tends toward a small limiting value at persistence length measured by SANS in NaCl 0.1 M
high salt concentration (it can be assumed that this limiting (Table 1).
value is reached in the above-mentioned study on pectins in
0.1 M NaCl). Davis [4] applied the electrostatic wormlike
chain theory (cf. Section V.B.2) to carboxymethyl cellulose Table 2 The Polyelectrolyte Chain Stiness Parameter B of
(CMC); he obtained Lpo=5.4 nm for the uncharged back- Smidsrd for a Few Selected Polysaccharides
bone of sodium carboxymethyl cellulose, a value showing B
that the intrinsic exibility of CMC is comparable to that of
pectin. In 0.01 M NaCl, the ratio Lpe/Lp was f0.45 for Citrus LM-pectins DM = 0.72 0.017 [107]
CMC (degree of substitution 1.06); it dropped to f0.13 in DM = 0.53 0.018 id
0.2 M NaCl, a solvent in which Lp=6.2 nm. The behavior DM = 0.45 0.023 id
of CMC, a strong polyelectrolyte with a rather intrinsically DM = 0.38 0.027 id
exible chain, contrasts with that of xanthan: the results of DM = 0.30 0.026 id
Tinland and Rinaudo [35] yielded Lpe/Lp=0.094 in 0.01 M DM = 0.044 0.017 id
NaCl and f3.5 103 in 0.3 M NaCl, with Lp=31 nm in n-Carrageenan 0.11 [34]
the latter condition. Xanthan is indeed a weak polyelec- E-Carrageenan 0.05 In [13]
Carboxymethyl DS = 1.06 0.065 [41]
trolyte with a sti helical conformation in presence of salt;
cellulose
the electrostatic contribution to the persistence length is
Xanthan 5.25 103 [35]
modest and soon becomes negligible as salt concentration Dextran sulphate 0.23 [41]
increases, when compared to the large intrinsic rigidity of
Intrinsic Viscosity as a Probe of Conformational solution statistically becomes a uniform distribution of

Transitions or of Intermolecular Phenomena. chain segments.
Actually, above c*, the rheological behavior of the
Because of its simplicity, determination of the intrinsic
solution is gradually dominated by the development of en-
viscosityor often just the measurement of the specic or
tanglement coupling between chains, provided their molec-
the reduced viscosity at some low concentration of the
ular weight is >Mc (Section III.B.2). Entanglement coupling
polymeras a function of temperature, pH, salt concen-
is a consequence of coil overlap, but represents a dierent
tration, etc., is commonly used to monitor conformational
type of physical interaction, imposing constraints on large-
changes of macromolecules. A change in conformation is
scale local motions of the chain and so prolonging them.
usually indicated by a more or less abrupt change of the
Because all these phenomena occurring in polymer
viscometric quantity. The method is sometimes used alone,
solutions at nite concentrations are primarily governed by
but frequently in conjunction with polarimetric, spectro-
the fraction of the total volume of the solution occupied by
metric, or potentiometric measurements, for instance,
the polymer coils, they will be reected in the viscosity* vs.
which yield information on conformation at a more local
concentration curves of polymer solutions, and such curves
scale. Let us just cite a couple of examples extracted from
can be expected to share common features. This is illus-
an extremely large literature: the monitoring of conforma-
trated on Fig. 6, where the data relative to a few poly-
tional transition of gellan induced by increasing concen-
saccharides diering widely in structure and/or in
trations of some cations [42], and the study of the
molecular weight are plotted as specic viscosity vs. re-
interaction of hydroxypropyl cellulose with ionic and
duced concentration c[g]. The reduced concentration c[g] is
nonionic surfactants [43]. Viscosity can be used to follow
a measure of the volume the coils would pervade in the
reactions such as enzymatic hydrolysis of polysaccharide
solution if they maintain their innite dilution dimension,
backbone, etc.; the activity of an endo-hydrolase will result
volume which depends on the polymer, its molecular
in a much larger eect on viscosity than that of an exo- or a
weight and on polymersolvent interactions, as reected
debranching enzyme.
in [g]. Accordingly, c[g] should be f1 at c=c*. So, c/c* or
Specic viscosity reects changes not only in confor-
c[g] can be used as the reduced concentration. The curves
mation and size, but also in intermolecular interactions
on Fig. 6 can be divided into three regions, limited by the
(association, aggregation), and in the physical state of the
critical concentrations c* and c** (Section III.B2).
system. This can be a cause of ambiguity when one is
Below c[g]f1, i.e., for c<c*, the curve is linear on
interested in molecular characteristics; however, in return,
bilogarithmic scales, with a slope adf1.2. This is the dilute
it provides a simple means to probe changes at supramo-
regime, where the increase in gsp is attributable to the
lecular scales in dilute polymer solutions. For instance,
hydrodynamic interaction between the polymer coils be-
viscosity measurements have been used to study spinodal
having as independent impermeable spheres. The increase
phase separation in agarose solutions at concentrations
in viscosity remains modest; at c*f1/[g], the viscosity of
well below the coil overlap critical concentration [44].
the solution is no more than about three times that of the
solvent (Fig. 6).
C. Flow Behavior of Semidilute and Concentrated Above c=c*, the semidilute regime is entered. The
Polysaccharide Solutions curve shows an upward concavity; its shape results from
the opposite eects of progressive coil contraction and of
1. ViscosityConcentration Curves increasing entanglement density on viscosity, the former
The size and the conformation of individual polymer eect dominating in the lower region of the semidilute
molecules determine intrinsic viscosity. As the concentra- regime, and the latter in its upper region. Although it is
tion of the solutions increases, intermolecular interactions actually concave, this part of the curve is often approxi-
of increasing order contribute to viscosity; pair hydrody- mated by a linear segment with a slope asdf2.5. At the
namic interactions are reected in the Huggins coecient. upper limit of the semidilute domain, the solution is about
Above the critical overlap concentration c* of the polymer, 100 times more viscous than the solvent, because of the
coil contraction and entanglements govern the rheological increase of entanglement density.
behavior at low shear rates for exible and semiexible Finally, in the concentrated regime (for c>c**), log gsp
polymers (Sections III.B and V.A). vs. log(c[g]) curves resume linearity with a slope acf45.
Coil overlap gives rise to intermolecular hydrodynam- In fact, whereas c[g] eectively acts as a reduced
ic interaction because the ow incident on a chain is variable in the dilute regime, this no longer the case above
perturbed by the segments of the neighboring chains which c* (as can be expected), because of coil contraction and
overlap with the considered molecule. At the same time, the entanglements. One observes some dierences in the values
hydrodynamic interaction between segments belonging to of c*[g] and c**[g] and shifts on the reduced viscosity scale,
the same chain (intramolecular hydrodynamic interaction) as well as limited dierences in the values of the slope in the
is screened by the segments of the overlapping chains, in a
parallel manner to the screening of the excluded volume * For c>c*, polymer solutions become non-Newtonian from
interaction. Polymer coils, nondraining at high dilution, moderate and even low shear rates; the viscosity values to be
become gradually free-draining as their concentration considered are those corresponding to the low shear Newtonian
increases. At concentration c**, coils have reached their plateau, which are related to the equilibrium state of the solution at
unperturbed dimension (Q-condition dimension); the rest (see Section V.C). They will be written as go from now on.
Figure 6 Specic viscosity-reduced concentration curves, showing the three concentration regimes. (a): Guar and hydroxyethyl
cellulose solutions at 25jC. Guar (empty circles): data from Robinson et al. [36] for ve samples diering in molecular weight
(450 V [g] V 1250 mL/g). Hydroxyethyl cellulose (lled triangles): data from Castelain et al. [46] ([g]=807 mL/g). The lines show
the slopes 1.2 and f5, relative to the dilute and to the concentrated regimes, respectively. (b): Xanthan samples with dierent
molecular weights in 0.1 M NaCl at 25jC. Symbols: data from Takada et al. [51]. Results shown for the samples with the
following intrinsic viscosities: empty circles: [g]=68.5 mL/g; lled circles: [g]=577 mL/g; triangles: [g]=2520 mL/g; squares:
[g]=7100 mL/g. Continuous line: data from Cuvelier and Launay [74] ([g]=4930 mL/g). (c): Narrowly distributed polystyrene
sample (MW=7.01 105) in toluene at 25jC. Data from Clasen and Kulicke [62].
semidilute regime, because of dierences in structure (ex- in aqueous solutions than for typical polymers in good
ibility) and in interactions with the solvent. The broadness (organic) solvents, as suggested by the comparison of
c**/c* of the semidilute domain depends on the compres- polysaccharide curves with that of the polystyrene solution
sion possibilities of the coil, and consequently the better the in toluene (Fig. 6). Close to Q conditions, the semidilute
solvent for the polymer, the broader the semidilute domain regime becomes very narrow and may not be observable in
[45,46]. This could explain, besides the greater intrinsic all systems.
stiness of polysaccharide chains, why the semidilute The failure of the universal behavior above c* is in
domain seems, in general, narrower for polysaccharides fact observed even in the case of typical exible polymers in
good solvent conditions. It is related to the existence of two In the case of rather sti chains, such as xanthan, at
characteristic lengths in the semidilute domain (Section high concentration, orientational eects can also be im-
III.B.2). As long as entanglements do not contribute to plied in the high value of x. For rigid rodlike polymers, the
viscosity, scaling arguments give: static correlation of the rods becomes important at high
c 1 concentration, and they tend to orient in the same direction
3m1
go cgs 14 as their neighbors. This results in an alignment at the
c macroscopic scale in the solution at rest when concentra-
Universal behavior is therefore expected for exible chains tion exceeds c**~ML/dL (rod length L, diameter d, mo-
in good solvents; but, when entanglements do contribute to lecular weight M=L ML) [2]. Below c**, the solution is still
viscosity, dynamical scaling shows that: isotropic, but it becomes anisotropic for c>c** and shows
liquid crystalline solution properties. In the isotropic semi-
go cgs c=c 3=3m1 =N2e 15 dilute regime (c*~ML/L2<<c<<c**~ML/dL), the New-
tonian low shear viscosity of the rigid rodlike polymer
where Ne is the number of blobs per entanglement, which
solution becomes [2]:
depends on the molecular weight and which causes univer-
sality to fail [47]; in the initial form of the scaling theory, the M6
Ne2 term does not appear in Eq. (15) (e.g., Ref. [2]). The mo- go ~c3 16
lnL=d
lecular weight dependence of Ne could account for the
small discrepancies that can be observed on Fig. 6 in the Thus, viscosity shows very strong molecular weight
semidilute regime for guar or for xanthan fractions with dependence. Therefore, rather sti polysaccharides such
dierent molecular weights. A smooth transition between as xanthan can be expected to show exponent x values
the two limiting behaviors represented by Eqs. (14) and intermediate between 3.5 (the value for exible chains)
(15) will be observed over the c* c** concentration range. and f6 (the value for rigid rods). Takada et al. [51] have
The slope of the plots of Fig. 6 increases from adf1.3, shown that the viscosity behavior of xanthan fractions
which is indeed close to the value given by Eq. (14) with solutions in 0.1 M NaCl shifted progressively from that
m = 0.6 (good solvent), to acf45 above c**. From Eq. of the rigid rod model to that of the typical polymer
(15), one expects acf3.75 in good solvent, but acf6 in Q chain as the molecular weight of the fraction increased.
conditions, which would be more appropriate as the coils This is attributable to the fact that the exibility of the
reach their Q dimensions in the concentrated regime. chain, which can be measured by the ratio p=L/2Lp (cf.
However, in this regime, the segmental friction coecient Section III.A), increases as L, and therefore M, increases.
depends on the concentration in a nontrivial way, making For samples with M>f30 104 ( p>f0.8), chain
the exact concentration dependence of viscosity impossible exibility became an important factor as regards viscos-
to establish [2]. Experimentally, ac values close to 4.5 are ity of xanthan fractions solutions in the isotropic semi-
generally found. Kulicke and Kniewske [48] suggested the dilute regime.
empirical approximation acf3.4/a. This stems from the The transition from the isotropic to the anisotropic
experimentally well established fact that, in the concentrat- regimes causes a spectacular reversal of the viscosity
ed regime, as in the melt, g~Mx, with x close to 3.43.5; as concentration relation for rigid rodlike macromolecules.
1/c*~[g]~Ma, Eq. (15) gives (aac)=3.43.5, instead of the Whereas viscosity was an increasing function of concen-
value (aac)=3 expected since a=3m1. tration [theoretically, a c3 dependence, cf. Eq. (16)] in the
The reptation theory, modied to take into account isotropic semidilute regime, it becomes a decreasing func-
chain contour length uctuations [49,50], actually predicts tion of c in the concentrated, anisotropic one [2]. This has
x=3.4, whereas the original reptation theory gave x=3. been observed, e.g., for hydroxypropyl cellulose solutions
However, exponent x is often found to be larger for (MWf60,000 and f100,000) in acetic acid [53]. The tran-
polysaccharides than for typical polymers; for example, sition occurred in the 2735% concentration interval;
in the data of Takada et al. [51] for xanthan at xed below c=27%, viscosity varied as c5.2; it increased enor-
concentration c >c** in 0.1 M NaCl, a value x=5.45.5 mously in the 27<c<30% interval; a maximum was
can be estimated. observed at c=30%, the concentration at which the rst
The eect of chainchain association at high concen- liquid crystal phase was detected by birefringence measure-
trations can be suspected in many cases of abnormally ments. Then viscosity showed a sudden and sharp drop,
high x values. Molecular associations are obviously sus- before it increased again above 35% concentration. The
ceptible to aect solution viscosity in dierent ways but, range 30<c<35% corresponded to a biphasic domain
among other eects, they generally interfere by increasing (coexistence of isotropic and cholesteric phases). Allain et
the eective molecular weight, and the eective radius of al. [54] have reported qualitatively similar results for
gyration, in a concentration-dependent manner above c*. xanthan aqueous solutions; the viscosity maximum was
Concomitantly, there is a shift from more or less linear and observed at much lower concentrations (of the order of 10
exible chains to branched and less interpenetrable objects. g/L); this was attributed to the high molecular weight of the
A recent paper [52] outlined a phenomenological treatment xanthan sample (MWf1.8 106). Indeed, Inatomi et al.
of this question. Furthermore, it should be remembered [55] have shown that the phase boundary concentrations of
that association generally leads to phase separation or to aqueous xanthan in NaCl solutions increased sharply with
gelation, i.e., it drives the system out of thermodynamical decreasing molecular weight below fMW f4 105. They
equilibrium, inducing time-dependent behavior. also found that these concentrations decreased sharply
with the added salt concentration, a fact which they high shear rates, the viscosity tends toward a second
interpreted as an illustration of the importance of electro- plateau gl; this high-shear Newtonian plateau is in fact
static interaction in the isotropic-liquid crystalline phase seldom experimentally observed because of instrumental
equilibrium. There is no doubt about its importance; limitations and because of practical problems (temperature
however, the results showed that the eect of salt concen- rise, mechanical degradation, ow instability) at high shear
tration remained about as important between 0.1 and 1 M rates. In the central part of the shear-thinning region, a
NaCl as it was between 0.01 and 0.1 M NaCl; this creates power law relation g~c-n is accurately followed, with
diculty. The study of the relations between rheological usually nf0.60.8.
behavior and sti chain phase transitions is certainly This type of behavior is called shear-thinning (or
rendered more dicult in the case of polyelectrolytes by pseudoplastic). It must be stressed that ow curves such
the typical polyelectrolyte eects (Section V.B.2) at low salt as Fig. 7 are steady-state ow curves. Concentrated poly-
concentrations, and by aggregation (due to the generally mer solutions being viscoelastic, they do not instanta-
low solubility of noncharged polysaccharidic polyelectro- neously respond to changes in shear rate or stress values.
lyte backbones) at higher salt concentrations. Upon imposition of the given shear rate or stress, their
viscosity varies with the time of shearing, and these changes
2. Shear Rate Dependence of the Viscosity
are reversible; the constant viscosity value, which is the one
We shall restrict the discussion of the shear rate depen- to be considered when plotting ow curves, is reached only
dence of the viscosity to the case of semidilute and concen- after some time. Viscoelastic time eects are to be distin-
trated polysaccharide solutions (c>c*). The reasons have guished from thixotropic time dependence, or from aging
been already stated (Section V.A.2). phenomena. Thixotropy corresponds to a modication of
Polysaccharide solutions at concentrations higher the structure of the ow units induced by shear and
than c* display the non-Newtonian shear behavior typical depending on the time the system is sheared; in the case
of polymer melts and polymer semidilute or concentrated of aging, the structure changes simply with time. Thixo-
solutions (provided that M>Mc). This type of behavior is tropic and aging time dependence eects are not observed
illustrated by the ow curve of Fig. 7, where the (steady- in the case of true polymer solutions; they manifest the
state) viscosity of a 0.4% aqueous solution of polyacryl- departure of the system from the solution state, generally
amide is plotted against the shear rate c on bilogarithmic because of aggregation/disaggregation or phase separation
scales (data from Ref. [56]). Below a critical shear rate value processes, and they often occur in concomitance. However,
cc, the ow curve shows an initial region, the low-shear chemical changes at the molecular level, such as chain
Newtonian plateau, where the viscosity keeps a constant hydrolysis, can cause apparent time-dependent behavior,
value go. A region (c>cc) where the viscosity decreases as without causing changes in the physical state of the system,
the shear rate increases (shear-thinning region) follows. At which remains a molecular dispersion.
The ow curve of a concentrated polymer solution
reects the eect of shear rate on entanglement density.
The shear eld tends to disentangle the chains, whereas
Brownian motion tends to restore the entangled congu-
ration corresponding to the equilibrium state at rest. In the
classical interpretation of the shear rate eect [57], it is
considered that a given chain can entangle only with chains
the centers of gravity of which lie within a sphere of a
certain radius R and at the condition that their centers of
gravity remain within this sphere at a time long enough for
entanglements to form. The formation of an entanglement
requires in eect thermal random motion of sizable sec-
tions of the chains following complex paths in the space,
and is therefore a kinetic process with an associated
characteristic time se. Two chains will form an entangle-
ment when their centers of gravity remain within a distance
R during a time k>se. The transit time varies as 1/c,
because the greater the shear rate, the faster the two
molecules are moved apart by ow. If c is low enough,
the system remains in its equilibrium fully entangled state,
and g=go. As c increases, the entanglement density
decreases, and so does the viscosity; at each shear rate
value corresponds a given entanglement density that results
from the balance between the ow-induced disentangle-
Figure 7 Flow curve of an aqueous solution of polyacry- ment process and the re-entanglement process driven by
lamide (c=0.4%) illustrating the three regions of the typical Brownian motion. In the reptation theory [2], the charac-
polymer solutions shear thinning behaviour. Replotted from teristic time sd, which is the time required for the chain to
Boger [56]. diuse one chain length L along its own contour (to
escape out of the tube), plays the role of se. Both charac- discrepancy are briey discussed in Ref. [2]. The most
teristic times are logically expected to be of the order useful expression is probably the semiempirical equation
of magnitude of the longest Rouse relaxation time of Cross:
sR=6(gogs)M/p2cRT of the system, which is the time n 1
necessary for the complete reorganization of the chain g gl c
conguration (cf. Section VI.A). The expression of the 1 18
go gl co
Rouse terminal relaxation time is valid at all polymer con-
centrations; at innite dilution, it becomes (sR)o=6gs[g]M/ Fitting Eq. (18) to the data of Fig. 7 gives go=1.82 Pa
p2RT. On the other hand, se (or sd) should be comparable sec, gl=2.6 103 Pa sec, co=0.67 sec1, and n=0.60.
to 1/cc. The parameter co is related to the critical shear rate, but
The ow curves of nondilute polymer solutions span co>cc, as can be seen on Fig. 7. The data used for this
several logarithmic decades of shear rates and of viscosity. gure spanned a very large shear rate range and reached
On the other hand, go and cc depend on the nature of the the second Newtonian plateau. However, since normally,
polymer, its molecular weight, and its concentration, on go H gl and considering that, in general, the experimen-
the solvent, and of course on the temperature. They may tal data do not extend to shear rate values high enough
change by several orders of magnitude from one system to for gl to contribute appreciably, gl can be, in most
another. Nevertheless, the above discussion suggests that cases, neglected.
a universal ow curve can be recovered by using g/go The considerations developed above constitute the
and c/cc as reduced variables, provided the exponent n fundamental background of the dierent reduction
maintains the same value in all cases. In fact, n is found to schemes, which are used to built master curves combin-
be an increasing function of the coil overlap parameter ing the data for dierent molecular weights, concentra-
c[g], but this function levels o for c[g]>20 [57]. The tions, temperatures, or solvents. A master curve simplies
theory of Graessley [57,58] predicts an asymptotic value the treatment of data for dierent systems and conditions;
n=9/11f0.82 for narrow distribution polymers. Increas- discrepancies from the master curve allow for pointing out
ing the distribution breadth results in an earlier onset of dierences in polymer chains structures. Master curves are
the non-Newtonian behavior, a less sharp transition from built either by graphically superimposing the individual
the Newtonian initial plateau to the power-law region, ow curves g(c) by shifting them along the viscosity and the
and in a lower asymptotic value (n=0.74) of the power- shear rate axes, or by using the reduced variables g/go and
law exponent [58]. Despite these limitations, usually the c/cc. However, because cc is dicult to determine in prac-
reduction scheme works remarkably well for c[g] zf30, tice, it is generally replaced either by cz, the shear rate at
i.e., in the concentrated regime where entanglements
govern the behavior of the systems. As concentration
decreases, deformation of the coils in the ow direction
and its consequences on the interaction potential of the
molecules makes an increasing contribution to the shear-
rate dependence of viscosity relative to that of entangle-
ments. The reptation theory predicts in the semidilute
regime
33m
1=c c csd ~M3 c 3m1 T1 17
This gives sd~M3 c 3/2 in good solvent (m=0.6) and

sd~M3c3 in Q conditions. The latter concentration de-
pendence would be expected in the concentrated regime;
however, the theoretical concentration dependence of sd
becomes actually tricky to derive for c>c**, as in the case
for the concentration dependence of go and for the same
reason (cf. Section V.C.1). Experimentally, the absolute
value of the concentration exponent of cc is often found
>3 in the concentrated regime.
Many equations have been proposed to describe the
shear rate dependence of viscosity of polymer solutions
(or melts). Most of them are empirical or semiempirical. A
few were derived from theoretical considerations; but they Figure 8 Flow curves of hydroxyethyl cellulose solutions in
are not the most successful as a rule. A striking example is 0.1 M NaCl at 25jC for dierent polysaccharide concen-
that of the reptation theory; it predicts [2] that at high trations. High-viscosity hydroxyethyl cellulose (HV HEC)
shear rates (csd H 1), gcgo(csd)3/2. A value n>1 for the sample ([g]=1557 mL/g in water). The gure shows also the
shear rate exponent is obviously unrealistic (the stress master curve obtained by shifting the curves parallel to the
would decrease as the shear rate increases) and in com- axes in order to collapse them on that relative to c=22.18 g/L.
plete contradiction with experience; the reasons for the Data kindly provided by C. Castelain.
which g=zgo, with z=0.8 [57] or z=0.1 [59,60] for exam-

ple, or by the parameter co of the Cross equation. A
universal ow curve for polysaccharides has been thus
published, which combined the data for solutions of a large
number of dierent polysaccharides at dierent concen-
trations, and this curve followed the simplied Cross
equation (gl omitted) with n=0.76 [5961].
The eect of concentration on the steady ow behav-
ior of polysaccharide solutions is shown in Fig. 8 in the case
of a high viscosity hydroxyethyl cellulose sample (HEC
HV; [g]=1557 mL/g; MWf9.57105) with concentrations
ranging over a large interval in the semidilute and concen-
trated regimes (3.5<c[g]<35). The master curve on Fig.
8 was drawn by collapsing the dierent ow curves on
that relative to the most concentrated solution, using the
graphical shift procedure. This master curve, which repre-
sents the ow curve of the 22.18 g/L solution over nearly six
logarithmic decades of shear rate, is accurately tted with
the simplied (three parameters) Cross equation. The t
gives Do =112.5 F 0.5 Pa sec, co =0.19F 0.004 sec 1 ,
n=0.642 F 0.002.
Figure 9 illustrates the procedure by using the reduced Figure 10 Comparison of master ow curves of dierent
variables g/go and c/co for a few concentrations of a locust polysaccharides at 25jC. The reduced variables used to plot
bean gum (LBG) solution ([g]=1490 mL/g; MWf2.2 the data are as in Figure 9. See text. Continuous line:
106) such as 7<c[g]<23. The master curves for HEC HV, replotted from the universal master curve for polysac-
LBG, and for concentrated solutions (c ranging from f40 charides [59]. Segmented line: data relative to the HV HEC
to f100 g/L, 9<c[g]<25) of a highly methylated sample of Figure 8. Squares: data relative to the LBG sample
(DM=71%) pectin sample* with a relatively low molecu- of Figure 9. Triangles: data relative to a very concentrated
solution of a high methoxyl pectin sample (DM=71%,
[g]=238 mL/g) in water at pH 3 (HM pectin) (unpublished
results from M. Axelos).
lar weight ([g]=238 mL/g; MWf9 104), are compared on

Fig. 10. On this gure, the data of the master curve of
Morris et al. [59] have been also plotted by using the
reduced shear rate c/co, instead of c/c0.1, which was used
by the authors. Whereas the LBG and Morris curves are
close to each other, with nf0.77, as expected, that of HEC
is less steep (n=0.64). The relatively low power law expo-
nent value for HEC is perhaps related to the stier char-
acter of the chain. From the relationships given in Ref. [62],
it follows that n=0.66 for an aqueous solution at 25jC of a
narrow molecular weight distribution hydroxypropyl cel-
lulose sample with the same molecular weight as HEC HV.
The data given in Ref. [62] show that, for aqueous solutions
of narrow molecular weight distribution fractions of cellu-
lose derivatives at 25jC, the asymptotic value of n (f0.81)
is reached at values of the overlap parameter c[g], which
appreciably depend on the nature of the substituent:
c[g]f20, c[g]f35, and c[g]f90 for hydroxypropyl cellu-
lose, for hydroxypropylmethyl cellulose, and for carbox-
ymethyl cellulose (in 0.01 M NaCl), respectively. The
Figure 9 Master ow curve of a locust bean gum sample authors also show that dierences in the positions of the
(LBG) solution in water at 25jC. The data are collapsed by OH groups substituted on the glucose ring are likely to
using g/go and c/co as reduced variables, go and co being
obtained by tting Cross Eq. (18) to the viscosity-shear rate
data recorded on 0.5%, 0.75%, 1% and 1.5% LBG * The solvent was water and the pH of the solutions was adjusted to
solutions. The continuous line represents the t of Eq. (18) the value pH=3; the carboxylic groups are practically not ionized
on the reduced data. The intrinsic viscosity of the LBG at this pH, so that the pectin behaves as a neutral polymer. High
sample was 1490 mL/g, and its mannose/galactose ratio was concentrations were reached by dialysis against dextran T40
3.8. Data kindly provided by S. Durand. solutions (osmotic concentration).
approximately as cMW, the data for pectin show a steep

decrease in spite of the fact that they are located in the
concentrated range (c[g] zf10), because of a lesser prev-
alence of entanglement eects in relation with the rather
low molecular weight of the pectin sample. Although
discussion of the non-Newtonian behavior of polymer
solutions focus on chain entanglements, it must be kept
in mind that viscosity depends on shear rate even in the
absence of entanglement eects. Therefore, nonlinear ow
behavior is not exclusively governed by entanglements.
The non-Newtonian ow behavior of semidilute and
concentrated solutions of sti chains in the isotropic do-
main is similar to that of exible chains. But when the
concentration at which the solution becomes anisotropic is
approached, the critical shear rate cc for the onset of non-
Newtonian behavior decreases steeply as concentration
increases, and becomes very low for anisotropic solutions.
This is why the initial Newtonian plateau is often dicult to
observe experimentally in the case of concentrated solutions
of sti polymers. Xanthan solutions have been long con-
Figure 11 Hydroxyethyl cellulose in water at 25jC (HV sidered (and sometimes are still considered) to display yield
HEC sample). Variation of the terminal Rouse relaxation stress behavior. This erroneous idea arose from ow curves
time sR (empty triangles) and of the critical shear rate co of relatively concentrated xanthan solutions displaying a
(lled circles) with the coil overlap parameter c[g]. The power law shape with nf0.8 all over the shear rate range
terminal relaxation time sR=6(go gs)M/k2cRT was usually explored (f0.01f100 sec1). In fact, one does
calculated using the molecular weight M=957000 obtained observe an initial Newtonian plateau provided the measure-
from intrinsic viscosity by application of the MHS relation ments are carried out at suciently low shear rates, and the
given by Kamide and Miyazaki [108].
ow curve follows Cross equation [64,65] (cf. Section
V.C.3). The time required to reach a constant viscosity
cause large dierences in the ow behavior of cellulose value at low shear rates becomes relatively long, which was
derivatives having similar average degrees of substitution related by Lim et al. [66] to the formation of liquid
and similar molecular weights, through their eect on the crystalline domains they observed, using optical birefrin-
aggregation behavior. In contrast, the eect of the average gence measurements, in xanthan solutions with concentra-
degree of substitution itself is small at low shear rates and tions zf1% (unfortunately, the intrinsic viscosity or the
becomes negligible at high shear rates. molecular weight of their samples were not specied).
Figure 11 shows that the variation of the critical shear
rate with HEC HV concentration parallels that of 1/sR, as
expected, and that the two quantities do not dier much in
magnitude. The bilogarithmic plots are shifted relatively to
each other by a constant factor because we have used the
Cross parameter co as critical shear rate value. A break on
the bilogarithmic plots of co and 1/sR is clearly visible
around c[g]f10, corresponding to the transition from the
semidilute to the concentrated regimes. The slope of the
plots in the semidilute range is close to 1.5 in agreement
with the reptation theory; but in the concentrated regime
the slope is f4.5, much higher than the value of 3 predicted
by Eq. (17). Lapasin et al. [63] reported co~c3.5 for
concentrated solutions (go~c4.2) of hydroxyethyl guar.
The product boV=cosR is equivalent to the reduced shear
rate bo=(gogs)Mc0.8/cRT of Graessley. Graessley [57]
observed that bo, instead of being a constant, shows in
the concentrated regime a residual linear dependence in
cM. In the semidilute regime, bo generally decreases as cM
increases, but this has been considered as an artifact, which
is met except with very high molecular weight polymer
samples [57]. In Fig. 12, boV has been plotted against cMW
for the HEC HV sample in the concentrated regime, and Figure 12 The dimensionless critical shear rate boV=co sR
compared to the data for the LBG and the pectin samples. for the solutions of HV HEC (circles), LBG (triangles) and
Whereas in the two rst cases, boV increases, indeed HM pectin (squares) samples in the concentrated regime. See
3. Practical Problems curves should superimpose. On the other hand, in the case
For concentrated solutions of high MW polysaccharides, it of relatively low-viscosity solutions, inertia eects, espe-
can prove dicult to approach go because it is shifted to cially when constant stress rheometers are used, can inter-
the left of the experimental shear rate range. This applies fere with, or can be mistaken for, time dependency.
even at moderate concentrations to high molecular weight
xanthan solutions, as we have earlier pointed out. Com- VI. VISCOELASTICITY OF POLYSACCHARIDE
pleting viscometric measurements with creep experiments SOLUTIONS
performed on a controlled stress rheometer (cf. Section
A. General Features of the Linear Viscoelastic
V.3.1) can circumvent the diculty; creep test allows
measuring very high viscosities at very low shear rates. Behavior of Liquid Polymer Systems
Figure 13 shows an example. The viscoelastic properties of polymeric liquids (solutions
Another important point we have already stressed is and melts), to a large extent, comply with a universal
that viscosity characterizes the steady ow regime. There- pattern, although dierences can be observed in the details
fore, ow curves are to be recorded in equilibrium con- of the behavior.
ditions, i.e., when a constant stress value (or a constant In consequence of the multiple spatial scales of the
shear rate value in a creep test) is reached in response to the polymer molecule, the relaxation of polymeric liquids
shear rate or to the shear stress applied. Because semidilute (solutions and melts) spans many orders of magnitude in
and concentrated polymer solutions are viscoelastic, it can time. Whereas rearrangements at the scale of the monomer
take an appreciable time before steady ow is reached upon or of the chain segments are relatively rapid, those which
imposition of a constant shear rate (or a constant shear govern the ow properties (terminal relaxation process-
stress). For example, in the case of the 0.8% xanthane es), implying the rearrangement of the conformation of
solution of Fig. 13, the creep test had to be carried out for at the entire chains, are much longer and depend strongly on
least 5 min to determine reliable steadystate shear rate and molecular weight and possible long chain branching. Ter-
viscosity values [65]. This is why it can prove risky to record minal relaxation processes are still slower in the case of
ow curves by applying a shear rate or a shear stress ramp, entangled systems, because the chains are now coupled.
which is the usual practice. It is preferable to proceed by
changing the applied shear rate (or the shear stress) step- 1. Dilute Polymer Solutions
wise, waiting at each step for a constant response to be Let us rst consider the case of a linear exible long-chain
reached. In both cases, it is advisable to check for the polymer in dilute solution, where the eect of entangle-
absence of appreciable time eects by recording the data in ments is not to be considered. The Rouse model represents
both the up and down modes (increasing and decreas- the polymer chain in the solution as a necklace of N+1
ing the shear rate or stress); the up and down ow identical beads connected by N identical linear springs. It is
in a way the mechanical equivalent of the Gaussian chain.
The chain segments are viewed as entropic springs of length
b and stiness 3kT/b2, whereas uid friction is considered
to be concentrated on the equidistant small spherical
beads, and to obey Stokes equation with the assumption
that there is no hydrodynamic interaction between the
motions of the beads. The dynamics of the polymer chain
is modeled by the Brownian motion of the coupled oscil-
lators constituting the necklace. This leads to the following
equation expressing the relaxation modulus of the dilute
solution containing n=cNa/M molecules of polymer per
unit volume, neglecting the contribution of the solvent [67]:
X
N
t=sp
Gt nkT e 19
p1
with:
s1 fo N2 b2 6go gs M
sp and s1 20
p2 6p2 kT p2 cRT
where fo is the bead (segmental) translational friction
coecient. The longest relaxation time, s1, was noted sR
in Section V.C.
Figure 13 Flow curve of a xanthan solution obtained by Equation (19) is that of a generalized Maxwell model
combining the data of creep experiments (lled symbols) with with equal relaxation strengths and with the relaxation
those of usual viscometric measurements (empty symbols). time distribution specied by Eq. (20). The expressions for
Results for a 0.8% xanthan solution at 25jC (c[g]=48), total the storage modulus ( GV(x)) and for the loss modulus
ionic strength 0.045 M. Replotted from [65]. ( GU(x)), or for the real (gV(x)) and the imaginary
(gU(x)) components of the complex viscosity, follow read-

ily [67]; the angular frequency x in dynamic measurements
is equivalent to 1/t in transient tests. The mechanical
spectrum (i.e., the plot of GV(x) and GU(x) on bilo-
garithmic scales) of the Rouse model exhibits two charac-
teristic regions with a rather sharp transition between
them. For xs1 b 1, GV and GU are proportional to x2
and to x, respectively, with GU>GV. This is the terminal
region, where the behavior is dominated by viscous ow.
Slopes 2 and 1 in the low-frequency region are those
expected indeed for any linear viscoelastic liquid [67].
For xs1 H 1, GV=GU~x1/2; in this region, GV and GU do
not depend on the molecular weight.
The neglect of the hydrodynamic interaction between
the beads corresponds to the free-draining assumption, and
so it restricts a priori the applicability of Rouse model to Q
conditions as far as dilute solutions of high molecular
weight polymers are concerned (Section V.C.1). But this
model predicts that [g]~M, which is in contradiction with
the well-established dependence of the intrinsic viscosity on
M1/2 in Q conditions (Section V.C.1). However, the free-
draining assumption is also relevant in the case of concen-
trated solutions, and Rouse model will turn out to be useful
in this context.
The bead-spring model has been extended by Zimm Figure 14 Typical mechanical spectrum of an entangled
so as to take into account the hydrodynamic interaction. polymer system over a very wide frequency range. Poly-
This results in a spacing of the relaxation times dierent styrene melt of narrow molecular weight distribution,
from that of the Rouse theory; the Zimm model gives M=600, 000. Filled symbols: GV; empty symbols: GW; 2 and
sp=s1/pl, with l=3m, so that l=3/2 in Q conditions 1: characteristic slopes of GV and GW in the terminal region.
(m=0.5), and l=9/5 for the typical value of m (m=0.6) in See text for te and td. Creep data of Plazek and ORourke,
good solvent [2]. The shape of the mechanical spectrum reduced to 100jC, reported in [67]. These data have been
remains the same as that of the Rouse model in the converted from the time to the frequency domain.
terminal region, but at higher frequencies, GV and GU vary
now as x1/l with GU>GV. Experimental results on dilute
solutions of polymers with narrow molecular weight dis- the GU curves, respectively)and the frequency region
tributions show generally good agreement with the theory where GV and GU follow approximately the Rouse depen-
of Zimm [67]; moreover, this theory predicts the right dence on x1/2. The latter region is called the transition
molecular weight dependence for the intrinsic viscosity zone, meaning the transition from the plateau to the
([g]~M3m1, cf. Eq. (7)). glassy behavior observed at very high frequencies. Glassy
behavior corresponds to frequencies so high that no
2. Entangled Polymer Liquids congurational rearrangements of the chain backbones
As we move away from dilute solutions to more concen- have time enough to take place; energy dissipation occurs
trated polymer systems, we move from the nondraining only through limited local motions, such as of lateral
case to the free-draining one. This is a consequence of the chemical groups. In the transition zone, the viscoelastic
screening of the hydrodynamic interaction because of coil response is a result of the congurational changes of those
overlap, so that we can expect a progressive shift from regions of the chain which are shorter than the distance
Zimm-like to Rouse-like behavior in the semidilute regime between entanglements (or, in the tube model, shorter than
c*<c<c**. On the other hand, entanglements will play an the tube diameter). These short-scale motions are not
increasing role in the rheological behavior, as already aected by the topological constraints of the network,
discussed. In the melt (concentration u specic weight q and thus the system follows Rouse dynamics down to the
of the polymer), the system will be fully entangled and frequency 1/se, i.e., up to the time se at which the segmen-
free draining. tal displacement becomes comparable to the entangle-
A typical mechanical spectrum of a polymer melt with ments spacing (tube diameter). For t>se, Rouse be-
narrow molecular weight distribution is shown on Fig. 14. havior is stopped by the topological constraints and the
The eect of entanglement coupling shows up as a plateau plateau region begins. In the terminal region (x<1/sd or
region, where GV is nearly at and where GU becomes t>sd), reptation dominates the behavior.
smaller than GV, so that the curves cross in two points. Leaving aside the glassy region, which is not accounted
This characteristic viscoelastic or rubbery plateau for by bead-spring or tube models, the relaxation behavior
develops between the terminal zonein the low-frequency of a monodisperse polymer melt can be considered as the
range (characterized by the slopes 2 and 1 of the GV and of sum of the contributions of three processes reecting
uncorrelated diusion mechanisms which involve the same lower molecular weight (M<Me) ones. Jeo is extremely
statistical unit of length b [68]: sensitive to molecular weight distribution.
1. The Rouse-like relaxation between entanglement Mechanical spectra of entangled polymer solutions are
points. The characteristic time sA of this process is qualitatively similar to Fig. 14. We have already given the
the longest relaxation time of the Rouse chain the expression for sd in the semidilute regime as a function of
molecular weight of which is Me, the molecular the concentration and the molecular weight (Section V.C.2,
weight between entanglements: sA =f o N e2 b 2 / Eq. (17)). Reptation theory gives in the semidilute regime:
6k2kT, where Ne is the number of chain segments 1 3m
~GNo ~M0 c 3m1 23
between entanglement points. The chain statistical o
Je
segment is commonly identied with the mono-
mer. Accordingly, fo is replaced by the monomeric The exponent of c in Eq. (23) would be typically f2.25 in
friction coecient fo. good solvent conditions (m=0.6); together with the non-
2. The recovery of the equilibrium monomer density dependence of GNo or Jeo on M, this is in good agreement
along the chain contour by the retraction of the with experimental results [69,70]. In Q conditions and in
chain within the tube (process B). The character- the concentrated regime, on the contrary, one observes GNo
istic time of this process is H B = ~ oNe2 N2b2/3k2kT ~1=Joe ~M0 cy with yf22.3 [69,70], instead of the value of
= 2sA(N2/Ne2); se<sB<sd. 3 expected from Eq. (23). This is due to the complex
3. The reptation process, with the characteristic time dependence of Me and fo on c in the entangled regime [2].
sd=6sA(N3/Ne3). Actually, no crossover at c** was observed by Takahashi et
al. [69] on log( Jeo) vs. log(c) of solutions of high molecular
Whereas sA does not depend on the molecular weight of the weight polystyrene fractions with narrow molecular weight
polymer, sB and sd vary as M2 and M3, respectively. The distributions, and they practically obtained the same curve
tube model gives se=6p2sA, and for the longest
prelaxation in a good solvent and in a Q solvent.
time in the system H maxuDoJeo=sd(11= N=Ne )2csd [2]. So far, we have considered polymers with rather
Therefore, the width of the viscoelastic plateau (smax z t z exible backbones. Solutions of elongated rigid or semi-
se) decreases sharply as the molecular weight decreases rigid macromolecules above c* in the isotropic regime
and disappears as N!Ne. Because both sd and go vary behave qualitatively in the same way as entangled classical
as M 3 , the steady state recoverable compliance J eo polymers. Their viscoelastic plateau zone is very wide. The
is independent of the molecular weight. The main contri- tube model predicts for rigid rod solutions in this regime [2]:
bution to the moduli in the plateau region comes from the
3cRT s c2 M7
reptation process: g*x with s~ 24
5M 1 ixs lnM
8 X 1 xsd =p2 2 Figure 14 shows that the viscoelastic response of an
GVxd GNo 21
p2 p:odd p2 1 xsd =p2 2 entangled polymer system encompasses many orders of
magnitude of frequency (or time). No single experiment
GNo 1=JNo qRT=Me is the plateau modulus ( JNo, the can span the entire range. The curves of Fig. 14 are in fact
plateau compliance), independent of the molecular weight master curves built from results recorded on the melt at
but dependent on the molecular weight between entangle- dierent temperatures and reassembled by shifting. The
ments. Eq. (21) shows that, for the modulus of a monodis- so-called timetemperature superposition principle states
perse polymer, the transition from the terminal region to that changing the temperature of the measurements simply
the plateau is very sharp. Because of the contribution of changes the time scale of the experiment and shifts the
process B, although it is comparatively small, the relaxa- moduli by a constant factor, without changing the shape
tion modulus or the storage modulus is not completely at of the viscoelastic functions [67]. In fact, the modulus shift
in the plateau region; this contribution is given by [68]: is attributable only to the eect of temperature on the
density q of the melt, and it is small. The main eect of an
Xe
N=N
Ne xsB =p2 2 increase (a decrease) in temperature is to shift the visco-
GVxB GNo 22 elastic response to higher (lower) frequencies. The assump-
p1
N 1 xsB =p2 2
tion underlying the principle is that temperature
Experimentally, one generally observes that smax~M3.4, changes the rate of Brownian motion without changing
with the same molecular weight dependence as go (cf. the conformation of the polymer and aects all the
Sections V.C.1 and V.C.2); on the other hand, GNoJeo c2, relaxation processes in the same way and at the same
instead of the value 6/5 predicted by reptation theory. extent. The principle is generally (but not always) veried
The presence of molecular weight distribution makes in the case of polymer melts. Obviously, its application to
the transition between the plateau and the terminal region polymer solutions is more problematic: changing the
less steep; the plateau of GV becomes less at temperature changes the thermodynamical state of the
and GU becomes smoother. The characteristic slopes are polymersolvent system (polymersolvent interactions);
reached at much lower frequencies than for a narrow this is especially critical when the solvent is aqueous. In
distribution polymer with the same molecular weight av- any case, the temperature window for timetemperature
erage, and the plateau modulus tends to be lower because superposition is limited to f45jC because of the freezing
the higher molecular weight fractions are diluted by the and boiling points of water. Generally speaking, the
[72] over a range of much higher frequencies (10350 103

rad/sec); the intrinsic storage and loss moduli followed the
Zimm frequency dependence in the upper part of the
frequency window. The authors tted the data with a
modied Zimm model proposed for semirigid macromole-
cules; however, the persistence length of native xanthan in
NaCl solutions they obtained from this model (500800
nm) seems unrealistically large and exceeds by far most of
the values available in the literature (cf. the section In-
trinsic Viscosity and Chain Stiness). Zirnsack et al. [73]
found quantitative agreement with Zimm theory for the
storage and loss moduli of f0.0140.055 g/L xanthan
solutions in high viscosity solvents. Although the authors
qualied these solutions as semidilute, the rheological
results showed that they were rather in the dilute range,
and they were indeed treated as such. However, the normal
stress measurements in steady shear were consistent with
theories of suspensions of rigid particles.
2. Concentrated Polysaccharide Solutions

Figure 15 shows some examples of the mechanical spectra
Figure 15 Mechanical spectra of hydroxyethyl cellulose [ GV(x) and GU(x) plots] of the solutions of HEC HV, the
(HV HEC sample) solutions in 0.1 M NaCl at 25jC (c z ow behavior of which are illustrated in Fig. 8. It was not
c**). See text. Filled symbols: GV; empty symbols: GW; con- possible, due to instrumental issues, to record reliable
tinuous and dotted lines: master curves. Circles: c=22.18 g/L; spectra over a large enough frequency range when the
squares: c=16.62 g/L; triangles: c=11.11 g/L. Data kindly concentration was below f11 g/L. Therefore, all spectra
provided by C. Castelain. were obtained for HEC HV solutions in the concentrated
regime (c**f1011 g/L, cf. Fig. 6). On the other hand,
true homogeneous solutions of HEC HV, and of high
temperature window is often further limited by the occur- molecular weight polysaccharides, in general, cannot be
rence of phase separation phenomena. Master curves of directly prepared at concentrations larger than about
polymer solutions are more often obtained by shifting f2.5%. The master curves of GV(x) and GU(x) were built
curves recorded for dierent concentrations. by the graphical shift procedure as it was carried out for
the master ow curve. The experimental frequency win-
B. Viscoelastic Behavior of Polysaccharide dow, extending over three logarithmic decades, encom-
Solutions passes the transition from the terminal region to the very
1. Dilute Polysaccharide Solutions
Let us reiterate once more that, although the viscoelastic
properties of dilute polymer solutions are of great theoret-
ical importance, only those of entangled systems matter in
practice, as far as the usual applications of polysaccharides
are concerned. The study of the viscoelastic behavior of
dilute polymer solutions is dicult and requires special
instrumentation. Such studies have been seldom attempted
in the case of polysaccharides. An exception is xanthan; the
very high molecular weight and intrinsic viscoelasticity of
this polysaccharide make this type of investigation more
tractable than for other polysaccharides. Tam and Tiu [71]
have studied the viscoelasticity of solutions of xanthan in
water in the frequency range 1041 rad/sec, at concen-
trations as low as 50 ppm. The results were generally in
qualitative agreement with the theory of Zimm, as far as it
can be concluded because the frequency range did not
extend actually much beyond the terminal region. Howev-
er, the longest relaxation time was about 10 times that Figure 16 The components of the complex viscosity in the
predicted by theory, perhaps because of the rigidity of the terminal region plotted in the complex plane for hydroxy-
backbone or because of the eect of electrostatic interac- ethyl cellulose HV HEC sample solutions (c z c**) in 0.1 M
tion. The mechanical spectrum of dilute solutions of xan- NaCl at 25jC. See text. Symbols: dynamic data; lines: Cole
than (0.1<c<0.4 g/L) was determined by Carriere et al. Cole ts; a: 19.42 g/L; b: 16.62 g/L; c: 13.88 g/L.
Figure 17 Concentration dependence of the viscoelastic constants for hydroxyethyl cellulose HV HEC solutions (0.1 M NaCl,
25jC) in the concentrated regime. The lines represent the power law ts of the results. (a) Zero-shear Newtonian viscosity values
determined by steady shear (empty symbols) and by dynamic measurements (lled symbols). Steady shear values of go were
obtained by tting Cross equation to the ow curves. The values of go were obtained from dynamic measurements by tting the
ColeCole model to the complex viscosity data in the complex plane. (b) Maximum relaxation times smax as determined from
steady shear measurements (empty symbols); smax=1/co, co being the critical shear rate in the Cross equation; and from dynamic
measurements (lled symbols); smax=1/xc, xc being the characteristic frequency in the ColeCole equation of the complex
viscosity. (c) Coordinates of the low frequency GV, GW crossover point of HV HEC solutions. See text.
beginning of the plateau region, stopping just beyond the expected for a polydisperse sample. Yet, the polydispersity
rst G,VGU intersection. The master curve results in an of the HEC HV sample is probably not as large as that
enlargement of the window in the low-frequency direction; commonly found in the case of unfractionated polysac-
actually however, this enlargement does not exceed one charide samples. This illustrates some of the limitations
decade, and it does not even allow the slope of 2 to be encountered in the study of the viscoelasticity of typical
reached for GV(x). We can observe as a corollary that the polysaccharides.
transition from the terminal region to the plateau is much Complex viscosity is particularly adapted to the rep-
less steep than that featured by Eq. (21), as can be resentation of the terminal region of the mechanical spec-
viscoelastic plateau GNo . The values of the moduli at the

intersections of GV(x) and GU(x) curves, directly obtained
from the experimental data, can be taken as approximate
values for GNo , whereas the location xoV of these intersec-
tions on the frequency scale provides an alternative esti-
mation of smaxf1/xoV. The coordinates of the intersections
are plotted against concentration on Fig. 17c. As far as the
restricted concentration range allows us to t these data
with power laws, we observe that the concentration expo-
nent for GNo is f2, as expected theoretically and determined
experimentally for polymer solutions; but smax~c4, which
is again a much steeper dependence than is usually found
for polymer solutions. According to the CoxMerz rule,
the curve of the steady state viscosity g against the shear
rate c and that of the modulus of the complex viscosity
Ag*A against the angular frequency x should superimpose.
This empirical rule is well veried for polymer melts and
homogeneous solutions within the limits of experimental
accuracy, whereas deviations are observed when the system
contains some degree of shear-sensitive supramolecular
structure. The CoxMerz rule provides a sensitive practical
Figure 18 Comparison of steady shear and dynamic vis- test to detect the presence of aggregates in the solution.
cosities of HV HEC solutions (c z c**), showing progressive Figure 18 shows that our HEC solutions follow indeed very
deviation from the CoxMerz rule as hydroxyethyl cellulose accurately the rule for the lowest concentrations of the
concentration increases. Symbols: modulus of the complex range. But a slight departure, increasing with c, is observed
viscosity; lines: steady-shear viscosity. for the highest ones, Ag*A remaining slightly higher than
the steady shear viscosity over the entire experimental
tra. Results such as those of Fig. 15 lend themselves more window. The discrepancies are thus probably the conse-
readily to quantitative analysis when they are set in terms of quence of slight aggregation in concentrated solutions of
the components of the complex viscosity.* On Fig. 16, gU HEC; HEC chains tend to associate in water via hydro-
has been plotted against gV on linear scales for some of the phobic interactions [62].
HEC HV solutions. The viscoelastic parameters of the In contrast, with HEC HV, the viscoelastic behavior of
terminal relaxation domain can be easily derived from this puried guar gum solutions in water closely follows that of
plot (ColeCole plot) by tting the data with the ColeCole
function for complex viscosity:
go
g*x 25
1 ix=xc n
go is the low shear Newtonian steady-state viscosity; 1/xc
and the exponent n are measures of the average relaxation
time and of the broadness of the relaxation time distribu-
tion, respectively, in the terminal region. Eq. (25) is the
parametric equation of an arc of circle passing through
the origin in the complex plane representation (Fig. 16).
The values of go obtained in this way agree very well with
those obtained from steady shear measurements (Fig. 17a).
As indeed expected from the denition of co, the critical
shear rate in Eq. (18), 1/co<1/xc; but the variations with
concentration of both measures of smax are parallel (Fig.
17b). The dependence of go, 1/xc, and co on concentration
seem to follow much steeper power laws than those usually
observed for polymer solutions in good solvents; however,
the very narrow concentration range probably precludes
calculation of reliable exponent values. The complex vis-
cosity representation does not give the height of the
* Very simple relations link gV and gU (real and imaginary component Figure 19 Mechanical spectra of concentrated solutions of
of the complex viscosity, respectively), or JV and JU (components of guar gum ([g]=1130 mL/g) in water at 20jC. Circles: 18.7
the complex compliance) with GV and GU (components of the g/L; triangles: 11.6 g/L; squares: 4.7 g/L. Filled symbols: GV;
complex modulus); see Ref. [67]. empty symbols: GW.
normal polymer solutions. The plot of GV(x) and GU(x) for

guar gum solutions in the concentrated regime are dis-
played in Fig. 19, and the components of g*(N) are
replotted on Fig. 20 in the complex plane. Treated as in
the same way as it was done for HEC, the dynamic data
gave go, xo, GNo, xoV, the variations of which with concen-
tration are shown on Fig. 21. The power law exponent for
go(c) is 4.5, which is the value found for most polysaccha-
rides in the concentrated regime and which is actually
predicted by the theory with go~M3.5. That for GNo(c) is
1.9, close to the expected value of 2. The exponent for
smaxc1/xoc1/xoV is 3.1, a value slightly higher than the
one we could expect (2.5) if we consider that smax go Joe
~go =GNo ; however, the plateau modulus was not directly
measured, so that some systematic error is possible. Cox
Merz rule was satised (Fig. 22), suggesting that guar gum
solutions are indeed molecular solutions.
While for the concentrated solutions of most polysac-
charides, the plateau region begins only toward the upper
end of the experimental frequency range usually accessible,
it extends over a large frequency window in the case of
Figure 20 Complex plane plots of the components of the xanthan solutions. This is illustrated on Fig. 23, where the
complex viscosity of guar gum solutions (c z c**) in water at mechanical spectrum of a solution of a native xanthan
20jC (same guar gum sample as in Fig. 19). The continuous sample is compared to that of a guar gum solution at the
lines are the ts with ColeCole model. same concentration; whereas the low-frequency intersec-
tion of GV and GU is located at xoV=94.6 rad/sec in the case
of the guar solution, xoV is visibly well below 0.01 rad/sec
for the xanthan solution. And yet the two solutions have
obviously rather similar plateau moduli values GNo. Be-
cause of the large extension of the plateau modulus,
concentrated solutions of xanthan are frequently described
Figure 21 Concentration dependence of the viscoelastic constants for guar gum solutions (water, 20jC; same sample as in Figs.
19 and 20) in the concentrated regime. The lines represent the power-law ts of the results. (a): Zero shear Newtonian viscosity
and characteristic frequency of the terminal regime determined by tting the ColeCole model to the complex viscosity data.
Filled symbols: zero shear viscosity go; empty symbols: characteristic frequency xo. (b): Coordinates of the low frequency
o
crossover of GV and GW, giving approximations to GN and smax (see text). Filled and empty symbols: values of GV and of x,
respectively, at the crossover.
as weak gels and their viscoelastic properties have been

related to side-by-side association of xanthan short
branches (for example, Ref. [74]). Indeed, deviation from
the CoxMerz rule is generally observed for xanthan
solutions (Fig. 24). However, the xanthan sample
([g]=10,300 mL/g) studied by Milas et al. [64] complied
with CoxMerz rule at the concentration of 5 g/L; this
sample was prepared from an unpasteurized broth, at the
dierence of most commercial samples which are heated
during the manufacturing process. The main reason for the
existence of a broad plateau is certainly that, as already
discussed (Section V.C.2), the terminal relaxation times of
concentrated solutions of sti chains are shifted to much
longer values as compared to similar but more exible ones.
C. Departure from Solution Behavior

We have insisted on several occasions on the fact that
obtaining true homogeneous solutions of polysaccharides
is not actually easy and commonly achieved as it would
seem. In the preceding section, we have seen how departure
from the solution state can be spotted out by comparing
Figure 22 Comparison of steady shear and dynamic
go(c) and Ag*(x)A curves. The violation of the CoxMerz
viscosities of guar gum solutions in water at 20jC (same
rule by HEC HV solutions was probably a result of the
sample as in Figs. 19, 20 and 21), showing they follow Cox
Merz rule. Symbols: modulus of the complex viscosity; lines: propensity of HEC to aspontaneously aggregate in aque-
steady shear viscosity. ous solutions, at least in the concentrated regime. In the
case of xanthan, we had an illustration of the importance of
the conditions in which the samples are prepared as to the
properties of their aqueous dispersions. However, the
deviation of the mechanical spectra of xanthan solutions
from the typical polymer solution behavior is certainly
largely due to the stiness of this polysaccharide. In the
case of HEC, the discrepancies from the typical solution
behavior that we have observed remained limited and were
Figure 23 Mechanical spectrum of a 0.5% xanthan solution

(in 0.13 M KCl, 20jC) compared to that of a guar gum
solution (in water, 20jC) with a similar concentration Figure 24 Comparison of steady shear and dynamic vis-
(0.48%). The intrinsic viscosities of the two polysaccharides cosities of xanthan solutions (0.13 M KCl, 25jC), showing
were 6500 and 1130 mL/g, respectively. Triangles: xanthan; departure from the CoxMerz rule. Symbols: modulus of the
circles: guar gum. Filled symbols: GV; empty symbols: GW. complex viscosity; lines: steady shear viscosity.
not conspicuous when considering the shape of the me- any change of the chemical or physical conditions in the
chanical spectra. But with nonpuried or ill-dissolved medium. This is indicative that a spontaneous aggregation
polysaccharide samples, the shape of the GV(x) curve process takes place. Rheological methods are generally
frequently shows at low frequencies a second plateau due very eective in detecting such phenomena at an early
to the presence of swollen, microgel-like particles. Figure stage, e.g., before a signicant turbidity develops.
25 compares the mechanical spectrum of a solution of a Locust bean gum (LBG) galactomannan diers from
crude commercial guar gum sample (dierent from the one guar gum only by a lower content in galactose and the fact
studied above) to that of the same solution after ltration that its galactose side groups are not uniformly distributed
on sintered glass; the solutions were prepared by dispersing on the mannan backbone, but form hairy zones, sepa-
the material in water under strong agitation at 20jC rated by smooth zones devoided of galactose. Its lower
followed by heating the dispersion at 80jC. The unltered content in galactose makes LBG still more dicult to
solution clearly displays a low-frequency plateau; after solubilize properly than guar gum. Fresh solutions of
ltration, the plateau disappears, although the GV(x) curve locust bean gum have the rheological properties expected
still shows an inection. The two spectra are practically for solutions of exible chains, very close in all respects to
identical at higher frequencies, indicating that ltration has those of guar gum. But if the solutions are left to age,
removed only a very small amount of guar gum material. rheological changes become apparent after a few days (the
Incomplete solubilization is very likely the reason for higher the concentration, the earlier it appears), whereas
the abnormal rheological behavior observed by Wientjes et guar gum solutions remain stable.
al. [75] for guar gum solutions; GV(x) displayed a plateau A 10 g/L locust bean gum solution in water has been
and Ag*(x)A an upright turn at low frequencies, whereas left to age at 20jC during 15 days within the measuring
the steady shear viscosity reached the expected Newtonian device of the rheometer; every 24 hr it was examined by
plateau at low shear rates. These authors used puried guar dynamic measurements. Because the evolution of the sys-
samples, but solutions were prepared either by adding the tem was very slow, it was possible to characterize the
dry guar to water and letting it to hydrate at 4jC without viscoelastic behavior at each time over a reasonable fre-
stirring, or by using a food blender at room temperature. quency range [77]. Some of the mechanical spectra
Such solubilization conditions are known to be inadequate recorded are shown on Fig. 26. As already stressed, the
for galactomannans [27]. Every time that appropriate fresh sample behaves as expected for a true solution of
procedures have been followed for their preparation, guar galactomannan in this range of molecular weight; we see
gum solutions have been found to display normal mechan- that at the lower end of the frequency range, GV and GU
ical spectra of the type shown on Fig. 19 (see, for example, curves comply with the theoretical slopes of 2 and 1,
Ref. [76]). respectively. The spectrum of the 2-day aged system shows,
Solutions of some macromolecules can display rheo- as compared to the fresh solution, noticeably higher GV
logical properties that depart progressively, while aging, values in the low-frequency range, and the slope of the GV
from the typical behaviors we have overviewed, without curve in this region is no longer close to 2, but smaller;
however, GV values remain practically unchanged at higher
frequencies. On the other hand, the GU curve is nearly
unaected all over the frequency window. On more aged
samples, the development of a plateau at low frequencies
becomes more and more conspicuous on the GV curve.
After 15 days, this phenomenon results in the appearance
of an elastic plateau at low frequency, with a at GV curve
and GV higher than GU, and the GU curve now looks very
dierent in the low-frequency region. This low-frequency
viscoelastic plateau is dierent from the viscoelastic pla-
teau of the solution, which begins at the far right end of the
frequency window and does not seem, as far as it is possible
to appreciate this on the experimental frequency range, to
change actually during aging.
As we shall see in the next section, the evolution of the
mechanical spectra of the LBG solution with time shown
on Fig. 26 is qualitatively similar to that occurring during a
typical solgel transition in a polymer system. One inter-
pretation could be that we are monitoring spontaneous
gelation of LBG due to the association of chains involving
their naked sections, i.e., the regions unsubstitued by
galactose along the backbone. The junction zones formed
Figure 25 Mechanical spectrum of a 0.5% solution (water, in this way, stabilized by physicalchemical interactions,
25jC) of a nonpuried guar gum sample before (triangles) can be expected to have a much longer lifetime than the
and after (squares) it has been ltered on sintered glass. entanglements; after some time, a three-dimensional net-
Filled symbols: GV; empty symbols: GW. work is formed, with a mesh size larger than that of the
Figure 26 Changes in the mechanical spectrum of a locust bean gum ([g]=1450 mL/g) solution (10 g/L in water, 20jC) during
ageing, showing the progressive development of a low frequency viscoelastic plateau. (a): Circles: fresh solution; squares: same
solution 2 days old; triangles: 4 days old. Filled symbols: GV; empty symbols: GW. The two straight-line segments indicate the
characteristic slopes 2 and 1 of GV and GW, respectively, in the terminal region of the mechanical spectrum of a viscoelastic liquid.
(b): Circles: fresh solution; squares: 6 days old; triangles: 15 days old. Filled symbols: GV; empty symbols: GW.
entanglement network. Hence the development of a sec- A. Chemical and Physical Gels
ondary GV plateau lower than the entanglement plateau
and extending in the lower frequency range. An alternative What is a gel is not easy to dene. The best denition, topo-
explanation is that the evolution of the mechanical spec- logical and quite abstract, would be that a gel is a biphasic
trum reects a phase separation process resulting from system of two continuous phases. A less elegant but more
aggregation of LBG through the same mechanism. Draw- operative one would describe a gel as a three-dimensional
ing the distinction between these two processes would network with a more or less permanent character, formed
require quantitative analysis of the spectra (see below). by the association of molecules (or of particles) and entrap-
Confocal laser scanning microscopy observations, carried ping a solvent. It has the merit to implicitly indicate that gel
out in parallel on LBG covalently labeled with a uorescent state derives from solution state (solgel transition), but
probe, suggest the formation of a cloudy three-dimensional adding that the network must invade the whole volume has
network in the 15-day aged system [77]. to complete it, introducing the idea of macroscopic con-
Although it is generally far from being as spectacular nectivity. In this respect, it is easy to understand that a
as in the case of the LBG solution described above, time- structural element must be linked to statistically more than
dependent aggregation actually occurs in polysaccharide two others for the network to form. The latter denition
solutions rather frequently; it is an often overlooked cause amounts to confer to the gel something of the character of a
of inconsistency in the rheological studies. solid, although it can contain more than 99% of solvent;
this brings us to rheology. In practice, will be qualied as
gels materials containing a large proportion of solvent but
VII. POLYSACCHARIDE GELS showing a wide viscoelastic plateau extending to long
enough times range, i.e., displaying a solidlike character
Whereas the conditions in which polysaccharides form at the time scale of current observation.
gels, the mechanisms of gelation and the structure of the Typical gels, the formation (solgel transition) and the
gels vary from one polysaccharide to another and are very properties of which are quantitatively accounted for by
dependent on the structure details of the biopolymer, the well-developed theories, are those resulting from the es-
viscoelastic behaviors of the gels are all qualitatively sim- tablishment of pointlike covalent cross-links between ex-
ilar, in spite of large quantitative variations. The mechan- ible polymer chains. Such a type of gel, sometimes called
ical spectra show little variation of GV and GU over the usual chemical, cannot be more than of marginal occurrence
frequency range, so that they have been described some- in our eld of interest. However, as chemical gelation of
times as rather uninteresting [80]. However, we shall see polymers is the best understood among gelation processes,
that a quantitative analysis of the viscoelastic response can it provides a rm stand from which to look to the other
be often attempted and is susceptible to yield more infor- types of gels where connectivity is realized by noncovalent
mation than when using rheological methods to monitor interactions between the structural elements, which are
gelation and compare gel strengths. called physical gels. In the case of chemical gels, the
solgel transition is a percolation transition. The percola- Conversely, most biological macromolecules form
tion theory [78] describes at thermodynamical equilibrium physical gels, the connectivity of which rests on low-
the changes in the behavior of a system where the connec- energy interactions (hydrogen bonds, ionic bridges, hydro-
tivity changes. Beyond the point where a certain fraction -c phobic interactions, van der Waals interactions) between
of the total possible bonds has been eectively realized the chains. The category of macromolecular physical
between the structural elements of the system, an innite gels is a very heterogeneous one and receives variable
aggregate forms which establishes a continuous path degrees of extension. The rheological properties of such
through the whole system; -c is the percolation threshold. gels cover the whole range from the quasi purely elastic
Close to the percolation threshold, which marks a discon- solid behavior to that of a typical viscoelastic liquid resem-
tinuity in those properties of the system that relate to its bling a polymer solution. From the structural point of view,
connectivity, many physical quantities diverge, that is, one can distinguish between two broad classes: catenary
they tend asymptotically to zero or to innity. These gels formed from chain-like macromolecules and colloidal
quantitiessuch as the mass of the largest aggregate, the gels formed from corpuscular macromolecules (globular
zero-shear viscosity go, the equilibrium relaxation modulus proteins, for instance), or from corpuscular aggregates of
Gein the vicinity of the transition follow scaling laws of macromolecules (e.g., micellar casein gels). Polysaccharide
the form c=A[(--c)/-c]q, where q is a critical exponent gels normally belong to the rst class. These gels form via
the value of which the theory predicts to be independent side-by-side association of polysaccharide chains resulting
of the system considered. In the case of the solgel transi- in the formation of more or less extended junction zones,
tion, the percolation threshold is the gelation point, and the which are maintained by low-energy interactions and
rheological behavior in its vicinity will be described in the which are often highly ordered. The presence along the
frame of the theory by a set of universal exponents that chains of residues that cannot be incorporated in junction
will depend only on the distance to the gelation point. In zones and remain highly solvated prevents the formation of
practice, p will be represented by the value of the control insoluble precipitates. The solgel transition is triggered by
parameter R, i.e., the factor the variation of which induces decreasing the temperature, or by changing polysaccha-
gelation; for chemical gelation, R is the concentration of ridesolvent interactions through the pH, the addition of
the cross-linking agent. At the gel point ( p=pc), R=Rc. specic ions or that of other solutes. Physical gels of
Then, in the vicinity of the gelation point, the rheological polysaccharides thus result from a kind of compromise
behavior should be described by go ~es , G e ~et , between solubilization and precipitation. They are meta-
GVcGU~xD, with e=A(RRc)/RcA. The scalar percola- stable, and they are generally thermoreversible, an increase
tion theory states the following values for the exponents: (a decrease) in temperature resulting in a decrease (an
s=0.75, t=1.94, D=0.72. When gelation point is increase) in the strength of the hydrogen bonds or the ionic
approached from below (R<Rc), viscosity grows to interactions stabilizing the junction zones.
innity, GVcGU~xD holds over the entire frequency do- Because ultimately the criterion for the gel state is
main, and the equilibrium modulus is zero. Just above Rc, rheological, it is no wonder that the overall viscoelastic
Ge takes nite values and the mechanical spectra develop behavior of polysaccharide physical gels qualitatively
into the viscoelastic solid type. The viscoelasticity of poly- exhibits a large degree of uniformity and contrasts with
mer chemical gels is entropic, indeed of the same nature as the extreme diversity of gelation mechanisms and of gel
that of entangled polymer systems, the dierence being that structures. However, this is only true as long as the eect of
the crosslinks in the covalent network have a permanent the dierent factors on solgel transition and on gel
character, entailing the suppression of the terminal relax- characteristics are not considered.
ation region. The viscoelasticity of covalently crosslinked The formation, the structure, and the properties of
polymer networks can be described by using modications physical gels of the dierent gelling polysaccharides have
of the Rouse model [67,79]. Eqs. (19) and (20) are now been and are still extensively studied. Besides a very large
replaced by the following expressions for the relaxation number of papers dealing with dierent specic systems,
modulus and the relaxation times [79]: there are several reviews on the subject to which one can
! refer [13,8082]. Several books can help to replace the topic
XN
in the general frame of physical networks and contain
t=sp
Gt mkT 1 1 e 26
p2
sections of variable importance on polysaccharide gels
[8388]. Our purpose is just to illustrate a few particular
1 fo R2g o M aspects of polysaccharide gelation and gels viewed from the
sp 27 rheologists standpoint.
p 12 6p2 kTMo
which are written here for the case where the isotropic state B. Solgel Transition in Polysaccharide Systems
of the network, when it is submitted to stress, is the same as
that during its formation. Eq. (26), where m represents the In the classical case of chemical gelation of polymers,
number of elastically active network chains per unit vol- the process is directly controlled by the stoichiometry of
ume, is the same as Eq. (19), except for the rst term which the cross-linking reaction. Of course, a small fraction of the
represents the equilibrium modulus Ge of the covalent cross-links formed will not perhaps contribute to the
network. Eq. (27) is a modied Rouse distribution [cf. Eq. connectivity (intrachain cross-links), and, on the other
(20) and Lo2=b2N] with innite longest relaxation time s1. hand, entanglements entrapped between cross-linking
points are likely to contribute to viscoelasticity. But as a

rst approximation, p is proportional to the concentration
of the cross-linking agent. Moreover, the reaction is quick
and is not reversible, so that the system can be considered
to be in a state of equilibrium at any stage, and changing
the temperature or the concentration of the cross-linked
network does not modify the number of cross-links per
chain. The only case of chemical gelation to have attracted
some attention in the eld of polysaccharides is the cova-
lent cross-linking of water extractable arabinoxylans
through the chemically or enzymatically mediated oxida-
tion of ferulic acid [8992]. However, this complex cross-
linking reaction is very dicult to control. It involves
specic (phenolic) groups along the chains; determining
the number and positions of those actually reacting in the
conditions of the experiments would be a formidable task. Figure 27 Master curve of the mechanical spectra of fully
In any case, this type of polysaccharide gels has been much cured LM pectincalcium gels close to the gel point. Here, Go
less studied than physical gels. stands for the equilibrium modulus Ge, and xo for the
The situation is less comfortable when dealing with frequency of the GV, GW low frequency crossover point.
Ca
physical gels. The cross-links are now junction zones Gelation was controlled by the ratio R 2 COO , which was
with nite energy and lifetime. Their number, size, and varied by adjusting the calcium chloride concentration. The
position uctuate with time and with temperature. Their gel point of the system (R=Rc) was found at R=0.086.
formation is almost always linked to that of ordered Experimental conditions:
- apple pectin, galacturonic acid content: 28%, degree
secondary structure (such as intertwined or stacked helices,
of methyllation: 28%; [g]=298 mL/g at pH 7 in 0.1 M
egg-boxes caging divalent cations, etc.) involving sizable
NaNO3, 25 C;
sections of the chains. Moreover, in concentrated gels,
- gelation at pH 7 and 20 C in the presence of NaCl;
junction zones often subsequently associate in bundles. total ionic strength: 0.1 M; pectin concentration: 14.9
Consequently, gelation is a kinetic process, the properties g/L (c[g]=4.5); gels cured during f24 h.
of the system change with time sometimes in a period Reproduced from [94].
lasting several hours. Generally, equilibrium is never
reached; with aging, concentrated gels often show synere-
sis, which reects a trend toward solid/liquid phase sepa- so that the system, when R>Rc, crosses the gel point before
ration because of secondary extension, rearrangement, or stabilizing at some distance from it. Although the time
stacking of the already formed junction zones. necessary to eventually reach this (quasi-)equilibrium state
Nevertheless, low-methoxyl (LM) pectinscalcium can be fairly long, the solgel transition occurs within a
ions systems have been observed to lend themselves to a restricted span of time and cannot be followed by per-
thorough investigation of their solgel transition [9395]. forming frequency sweeps to record mechanical spectra.
Interchain association of LM pectins proceeds by dimer- Complex waveform mechanical spectroscopy can then be
ization of homogalacturonic acid chain sequences via Ca2+ used. This type of method has been used to monitor alginate
complexation. The degree of crosslinking of LM pectins gelation induced by calcium ions [96]; the mechanism of this
with moderate degrees of esterication is directly con- gelation process is basically the same as for LM pectins.
trolled by the ratio R=2[Ca2+]/[COO]; for suciently Figure 28 shows the evolution with time of the mechanical
low values of R above the gel point, the low-frequency spectrum recorded on the same sample of alginatecalcium
complex modulus of the fully cured gels is proportional to system; to delay the solgel transition, calcium ions are
R [93]. Axelos and Kolb [94,95] determined the scaling released progressively in the sample from its complex with
exponents for the rheological functions on both sides of the ethylenediaminetetraacetic acid (EDTA) by action of y-
gel point. They found values in good agreement with the gluconolactone. The total time needed to obtain one spec-
scalar percolation theory. Above the gel point, the dynamic trum on the 0.1100 rad/sec range is about 2 min, including
data collapsed on a master curve when GV/Ge, GU/Ge, and data logging and le processing. The solgel transition of
x/xc were used as reduced variables, xc being the GV and alginate can be nicely approached in this way. Two hours
GU the crossover frequency (Fig. 27). Above xc, GU is and forty minutes after the addition of y-gluconolactone,
slightly above GV, and both moduli increase as xD, with the system has probably just passed the transition point; the
D=0.71. Below xc, GV=Ge and GU~x. curves of GV and GU are parallel on bilogarithmic plot and
In the study of LM pectin gelation by Axelos and Kolb, their slope (0.71) is very close to the theoretical one for
the systems were characterized in quasi-equilibrium con- scalar percolation transition. Fifteen minutes earlier, the
ditions (for R>Rc, the mechanical spectra were recorded slopes were dierentindicating that the system was still
on fully cured gels) and playing on the concentration of below the transition. After 2 h 52 min, the elastic plateau
calcium, which acted as a cross-linking agent, allowed a extends up to the higher limit of the frequency window.
step-by-step approach of the percolation threshold from Determining the gel point according to either of the
both sides. However, physical gelation is a kinetic process, above procedures is never easy, and it is actually often
>
by Michon et al. [97,98] in the case of gelatin and iota-

carrageenan systems with time and temperature, respec-
tively, as the controlling parameter of gelation. They found
that D was a decreasing function of iota-carrageenan
concentration in 0.2 M NaCl, with values much less than
the scalar percolation one. The authors concluded that the
value of the exponent D was not universal. However,
departure from the theoretical behavior can indeed be
expected as concentration increases, because secondary
aggregation processes take place. Moreover, iota-carra-
geenan gels are brous networks, the elasticity of which
will be determined by the bending and stretching of the
brous elements; their setting requires double helix forma-
tion [82]. Therefore, as it is indeed the case for many
polysaccharide gels, they are very dierent from classical
polymer networks and also from LM pectin calcium gels,
the formation of which does not entail important structur-
al changes at the macromolecular level [93].
The changes in the shape of the mechanical spectra
across the temperature-controlled solgel transition of a
iota-carrageenanNaCl system is illustrated on Fig. 30.
Figure 29 Application of the method of the tangent The transition visibly occurs very close to 42jC; at this tem-
(Winter and Chambon criterion) to the determination of perature, the curves of GV(x) and GU(x) are practically
the gel point and of the critical exponent D of the alginate superposed in the frequency range of the measurements,
calcium system of Figure 28. with GV~x0.53 and GU~x0.50. For temperatures lower
than the transition temperature but approaching it, the
viscoelastic plateau is no longer visible within the experi-
impossible in many cases. Very commonly, gelation is mental window, so that the spectra cannot be quantitative-
monitored at some xed frequency and the gel point is ly analyzed. This becomes possible when the data are
dened as the time, the temperature, or the concentration plotted in terms of the components of the complex com-
at which, starting from the sol state, the storage modulus of pliance. Figure 31 shows that in the experimental frequency
the system takes o, or at which GV crosses GU. This is range the complex compliance corresponds to a ColeCole
obviously empirical, as the apparent threshold value of the distribution of retardation times (cf. Section VI.B.2). The
controlling variable will depend on the sensitivity of the plateau compliance JNo=1/GNo, as well as the central fre-
instrument and the choice of the frequency. In some cases, quency xc and the broadness parameter of the high-fre-
the transition could even be undetected if monitored at an quency loss compliance peak, are obtained by tting the
inappropriate frequency, such as when LBG aging is ColeCole functions to the JV,JU data. We cannot enter here
observed at 10 rad/sec (Fig. 26). But the fact is that, at into technical details of this classical question; for an
the transition, GVcGU~xD provides a theoretically based example of practical application to a complex system, see
and practically applicable method to determine the gel for instance Ref. [99]. Figure 32 gives the results of the
point, often called the Winter and Chambon criterion or treatment, illustrating the divergence of JNo and of xc when
the method of the tangent: GV and GU are measured at a the transition is approached from the viewpoint of low
few frequencies for dierent values of the parameter temperatures. One of the points of this exercise in classical
controlling the transition, and tan(y)=GU/GV is plotted phenomenological rheology is to demonstrate that the
against this parameter; the curves of tan(D) corresponding usual handling of dynamic data in terms of the complex
to the dierent frequencies intersect at the critical value of modulus components is not necessarily the most appropri-
the parameter. At the transition, tan(y)=tan(kD/2), giving ate. Representing the dynamic data in terms of JV(x), JU(x)
in principle the value of the critical exponent; modied gives more weight to slow dissipative processes compared
Rouse (Zimm) models (Section VI.A.26) predict D=0.5 to GV(x), GU(x). This leads to shift the observation window
(2/3) (the frequency exponent value in the transition zone toward lower frequencies, allowing, in the case of the
of entangled networks), whereas scalar percolation theory experiment considered, to frame an utilizable section of
gives D=0.72 [94,95]. The method is applied on Fig. 29 to the loss peak that marks the end of the viscoelastic plateau
the results of the alginate gelation experiment of Fig. 28; it and the beginning of the transition zone. On the other
gives Df0.70, which is very close to that of the LM pectin hand, an example of the usefulness of the complex viscosity
calcium system of Axelos and Kolb. The method was used representation to analyze the terminal region of the spec-
Figure 28 Monitoring the gelation of an alginate-calcium system at 20jC by Fourier transform mechanical spectroscopy (see
text). Calcium ions are progressively released in the system from EDTAcalcium complex by the action of y-gluconolactone. (a):
immediately after addition of y-gluconolactone. (b): after 2h 25 min. (c): after 2h 40 min. (d): after 2h 52 min. (e): after 4h.
Figure 30 Cold setting of a 1% iota-carrageenan gel in 0.5% NaCl. Mechanical spectra of the system recorded at dierent
temperatures: (a): 45j, 42j, and 40jC (b): 39j, 30j, and 20jC. The gel point temperature is close to 42jC.
trum of viscoelastic liquids has been given above (Section The distinction reects dierences in the linearity limit, the
VI.B.2). viscoelastic behavior remaining linear up to much higher
strain values for strong gels than for weak gels.
C. Weak Gels vs. Strong Gels, or the However, it remains arbitrary because it does not take into
Lifetime of Junction Zones account the time-scale of observation; the same gel, behav-
ing as a brittle material when submitted to a rapidly
A distinction is sometimes made among physical gels increasing strain, could ow when deformed slowly, the
between strong and weak gels, based on the behavior nal strain being the same. In eect, physical gels, in
at large deformations: the former still exhibit a solidlike contrast to chemical gels, are not necessarily viscoelastic
character at large deformations, while the latter ow [100]. solids. Most of them, even when they display very high
rigidity, seem susceptible to ow, or at least to show
irreversible deformation, when they are observed over a
period of time long enough. A remarkable case is that of
gelatin, a protein of course, but the thermoreversible
gelation properties of which are similar in many respects
to those of several polysaccharides. Gelatin gels would be
classied as strong at any rate without hesitation. And
yet, ten Nijenhuis [85] published the mechanical spectrum
of a f2% aqueous gelatin gel after 2 days of aging at
2.6jC, which clearly shows a loss peak in the low-frequency
region. This peak is centered around x=105 rad/sec and
it is separated from the high-frequency loss peak (located
ca. 10 rad/sec) by a deep valley corresponding to the
region of the at viscoelastic plateau. At frequencies below
104 rad/sec, GV decreases with x. Although the low
frequency crossover of GV and GU remains on the left of
the frequency window, the terminal region of the spectrum
is visibly approached. Mechanical spectra of polysaccha-
ride gels are generally recorded over a limited frequency
range, rarely extending below 0.01 rad/sec; thus the possi-
ble liquidlike character of these gels is not usually
evidenced. However, a minimum of GU(x) can often be
Figure 31 Complex plane plot of the components of the observed within the frequency window, giving an indica-
complex compliance for the iota-carrageenan system of Fig. 30, tion of the presence of a low-frequency loss peak. For
at two temperatures below the gel point. The continuous lines example, the LM pectincalcium gel of Fig. 33 practically
are the ts of the ColeCole model to the experimental data. maintains a constant GV value (f150 Pa) all over the
Figure 32 Divergence of the compliance plateau (a) and of the characteristic frequency of the low frequency loss compliance
peak (b) of the iota-carrageenan gel when approaching gel point temperature from below (see text).
frequency window, whereas the GU(x) curve is V-shaped dierent polysaccharides can form gels with qualitatively
with a minimum located at f0.4 rad/sec. This mechanical similar mechanical spectra through very dierent mecha-
spectrum has the same shape as that of the 1% iota- nisms, while the same polysaccharide is susceptible to yield
carrageenan gel at 20jC (Fig. 30b), except that the latter gels with very dierent viscoelastic behaviors, depending
displays a lower viscoelastic plateau value (f25 Pa). On on the experimental conditions.
the contrary, it diers from those of the LM pectin gels of The existence of a low-frequency loss peak does not
Fig. 27. The characteristics of the two pectin samples are necessarily imply that this peak is related to steady-state
rather similar, but the conditions of gelation dier in some ow. However, the liquid character of several polysaccha-
points (c[g], R; cf. the legends of Figs. 27 and 33). Thus, two ride gels has been demonstrated by the creep experiments
of Plashchina et al. [101103] and of Braudo et al. [104];
they found that the steady-state viscosity of these gels was a
power law function of the polysaccharide concentration,
with an exponent close to 4.5. Among the gels studied by
these authors, kappa-carrageenan (12.5% concentration),
at least, cannot be denied the quality of being strong. We
can therefore hypothesize that this type of highly visco-
elastic liquid behavior has some character of generality
among polysaccharide physical gels. The slow relaxation
modes of such materials would certainly deserve deeper
investigation.
Xanthan dispersions are usually given as an example
of weak gels [100]. However, we have seen that these
systems can be described as concentrated and slightly
ordered and aggregated solutions of a high molecular
weight semirigid polymer. Iota-carrageenans gels would
be better candidates; in eect, they are known to ow when
submitted to shear rate ramps, showing just increasing
partial thixotropy behavior as the distance from the gel
point increases [105]. At any rate, if the distinction between
weak and strong gels can be maintained, the dier-
Figure 33 Mechanical spectrum of a fully cured low ence in their viscoelastic behaviors is a dierence in degree,
methoxyl citrus pectincalcium gel. Pectin characteristics: not in nature. It is probably mainly related to the magni-
70% galacturonic acid, DM=28%, [g]=234 ml/g. Gelation tude of the longest relaxation times and to the broadness of
conditions: 20jC in 0.086 M NaCl; c=1% (c[g]=2.34); their distribution, which certainly depend on the distance
R=1.19. Filled symbols: GV; empty symbols: GW. from the gel point among other factors.
It should be stressed that the low-frequency plateau, REFERENCES

the development of which is the practical signature of the
1. Chen, Z.Y.; Noolandi, J. Extended two-parameter theory
formation of a physical gel, is distinct from the entangle-
for exible polymer chains. Macromolecules 1992, 25,
ment viscoelastic plateau existing in the semidilute or 4978.
concentrated sol state of high molecular weight polysac- 2. Doi, M.; Edwards, S.F. The Theory of Polymer Dynamics;
charides. The latter is normally located at higher frequen- Oxford Science Publications, Clarendon Press: Oxford,
cies, and very often does not appear in the experimental 1986.
window (cf. Figs. 28 and 30). However, in the case of the 3. de Gennes, P.G. Scaling Concepts in Polymer Physics;
spontaneous gelation of LBG upon aging (Fig. 26), the Cornell University Press: Ithaca, 1979.
4. Davis, R.M. Analysis of dilute solutions of (carboxy-
window encompassed the beginning of the entanglement methyl)cellulose with the electrostatic wormlike chain
plateau because of the very high molecular weight of the theory. Macromolecules 1991, 24, 1149.
polysaccharide. As the number of junction zones increases 5. Dobrynin, A.V.; Colby, R.H.; Rubinstein, M. Scaling
in the system, the gel plateau extends in the direction of theory of polyelectrolyte solutions. Macromolecules 1995,
higher frequencies and the frequency gap separating it from 28, 1859.
the entanglement plateau shrinks progressively. The pro- 6. Forster, S.; Schmidt, M. Polyelectrolytes in solution. Adv.
Polym. Sci. 1995, 120, 51.
gression of the high-frequency limit of the gel plateau as 7. Bohdanecky, M.; Kovar, J. Viscosity of Polymer Solutions;
temperature decreases below the gel point is illustrated in Elsevier: Amsterdam, 1982.
Fig. 32b for the iota-carrageenanNaCl system. 8. Harding, S.E. Dilute solution viscometry of food bio-
polymers. In Functional Properties of Food Macromole-
D. Practical Problems cules; Hill, S.E., Ledward, D.A. Mitchell, J.R., Eds.;
Aspen Publishers: Gaithersburg, 1998; 1 pp.
A system undergoing a solgel transition generally spans 9. Tanford, C. Physical Chemistry of Macromolecules; John
an enormous range of rheological characteristics. It can Wiley and Sons: New York, 1961.
prove dicult or impossible to measure them with a good 10. Yamakawa, H. Polymer chain dynamics: the helical
accuracy all over the process in the relevant frequency or wormlike chain. In Molecular Conformation and Dynamics
of Macromolecules in Condensed Systems; Nagasawa, M.,
time windows, because of the limited range of operation of Ed.; Elsevier: Amsterdam, 1988; 21 pp.
the rheometers. Moreover, close to the gelation point and 11. Yamakawa, H.; Fuji, M. Intrinsic viscosity of wormlike
on both sides of it, the linearity range is likely to be chains. Determination of the shift factor. Macromolecules
restricted, because the system becomes structured, but its 1974, 7, 128.
structure is weak. In such conditions, the course of the 12. Bohdanecky, M. New method for estimating the parame-
gelation process or the gel structure can be even modied if ters of the wormlike chain model from the intrinsic viscos-
ity of sti-chain polymers. Macromolecules 1983, 16, 1483.
the system is submitted to considerably large strain or
13. Lapasin, R.; Pricl, S. Rheology of Industrial Polysaccha-
stress values. On the other hand, far beyond the gel point, rides; Blackie Academic & Professional: London, 1995.
or during aging, syneresis could occur. Then there will no 14. Pals, D.T.F.; Hermans, J.J. Concentration dependence of
longer be adhesion of the surface of the gel on the surfaces the viscosity of sodium pectinate in solutions of NaCl. J.
of the measuring device, because of the formation of the Polym. Sci. 1948, 3, 897.
liquid lm; the measurements will be dominated by slip- 15. Cohen, J.; Priel, Z.; Rabin, Y. Viscosity of dilute
page, thus losing any signicance. In such cases, the use of polyelectrolyte solutions. J. Chem. Phys. 1988, 88, 7111.
16. Cohen, J.; Priel, Z. Viscosity of dilute polyelectrolyte
measuring devices with sanded or grooved surfaces can be a solutions: concentration dependence on sodium chloride,
solution. magnesium sulfate, and lanthanum nitrate. Macromole-
Very often, temperature-controlled gelation is moni- cules 1989, 22, 2356.
tored at one or a few frequencies by performing tempera- 17. Cohen, J.; Priel, Z. Viscosity of dilute polyelectrolyte solu-
ture ramps. The choice of the frequency can be critical for tions: temperature dependence. J. Chem. Phys. 1990, 93, 9062.
the interpretation. Besides, it is generally observed that the 18. Hodgson, D.F.; Amis, E.J. Viscometric titration of a
linear polyelectrolyte. J. Chem. Phys. 1989, 91, 2635.
results depend on the cooling or on the heating rate; as an 19. Kurucsev, T. Viscosity of dilute solutions of linear poly-
example among many, Labropoulos et al. [106] recently electrolytes. Rev. Pure Appl. Chem. 1964, 14, 147.
reported that the onset of gelation of agar occurs at lower 20. Ermi, B.D.; Amis, E.J. Domain structures in low ionic
temperatures with increasing cooling rate. This is not strength polyelectrolyte solutions. Macromolecules 1998,
surprising because in such conditions, the system, the 31, 7378.
gelation of which is a kinetic process, is in general con- 21. Pals, D.T.F.; Hermans, J.J. Sodium salts of pectin and of
carboxymethyl cellulose in aqueous sodium chloride. Rec.
stantly out of equilibrium. This type of experiments is
Trav. Chim. Pays-Bas 1952, 71, 433.
certainly not advisable for thorough work. 22. Yamanaka, J.; Matsuoka, H.; Kitano, H.; Ise, N. Revisit
to the intrinsic viscositymolecular weight relationship of
ionic polymers. J. Colloid Interface Sci. 1990, 134, 92.
ACKNOWLEDGMENTS 23. Yamanaka, J.; Araie, H.; Matsuoka, H.; Kitano, H.; Ise,
We are indebted to our colleagues Monique Axelos, for N.; Yamaguchi, T.; Saeki, S.; Tsubokawa, M. Revisit to
the intrinsic viscositymolecular weight relationship of
useful comments and communication of unpublished ionic polymers. 4. Viscosity behavior of ethylene glycol/
results, Chantal Castelain for original data, and Sylvie water solutions of sodium poly(styrenesulfonate). Macro-
Durand, for some experiments carried out for the purpose molecules 1991, 24, 3206.
of this article. 24. Borsali, R.; Vilgis, R.T.A.; Benamouna, M. Viscosity of
weakly charged polyelectrolyte solutions: the modemode 45. Graessley, W.W. Polymer chain dimensions and the de-
coupling approach. Macromolecules 1992, 25, 5313. pendence of viscoelastic properties on concentration, mo-
25. Davis, R.M.; Russel, W.B. On the theory of dilute poly- lecular weight and solvent power. Polymer 1980, 21, 258.
electrolyte solutions: extensions, renements and exper- 46. Castelain, C.; Doublier, J.-L.; Lefebvre, J. A study of the
imental tests. J. Polym. Sci. B Polym. Phys. 1986, 24, 511. viscosity of cellulose derivatives in aqueous solution.
26. Davis, R.M.; Russel, W.B. Intrinsic viscosity and Huggins Carbohydr. Polym. 1987, 7, 1.
coecients for potassium poly(styrene sulfonate) solu- 47. Adam, M.; Lairez, D.; Raspaud, E. Non universality of
tions. Macromolecules 1987, 20, 518. scaling laws in semi-dilute and good solvent conditions. J.
27. Picout, D.R.; Ross-Murphy, S.B.; Errington, N.; Harding, Phys. II 1992, 2, 2067.
S.E. Pressure cell assisted solution characterization of 48. Kulicke, W.-M.; Kniewske, R. The shear viscosity depen-
polysaccharides. 1. Guar gum. Biomacromolecules 2001, dence on concentration, molecular weight, and shear rate
2, 1301. of polystyrene solutions. Rheol. Acta 1984, 23, 75.
28. Senti, F.R.; Hellman, N.N.; Ludwig, N.H.; Babcock, G.E.; 49. Lin, Y.-H. A general linear-viscoelastic theory for nearly
Tobin, R.; Glass, C.A.; Lamberts, B.I. Viscosity, sedi- monodisperse exible linear polymer melts and concen-
mentation, and light-scattering properties of fractions of trated solutions and comparison of theory and experi-
an acid-hydrolyzed dextran. J. Polym. Sci. 1955, 17, 527. ment. Macromolecules 1984, 17, 2846.
29. Soeteman, A.A.; Peeters, F.A.H. Diusie-, visceus en 50. Lin, Y.-H. Comparison of experiment and the proposed
GPC-gedrag van dextranen in water als functie van hun general linear viscoelastic theory. 3. Zero-shear viscosity
molecuulgewicht. Bull. Soc. Chim. Belg. 1982, 91, 875. and steady-state compliance. Macromolecules 1986, 19, 168.
30. Wales, M.; Marshall, P.A.; Weissberg, S.G. Intrinsic 51. Takada, Y.; Sato, T.; Teramoto, A. Dynamics of sti-
viscositymolecular weight relationships for dextran. J. chain polymers in isotropic solution. 2. Viscosity of
Polym. Sci. 1953, 10, 229. aqueous solutions of xanthan, a rigid double-helical
31. Kiciak, M.W. Oznaczanie sredniej masy czasteczkowej polysaccharide. Macromolecules 1991, 24, 6215.
dekstranu. II: Bledy powodowane niestaloscia wspolczyn- 52. Burchard, W. Structure formation by polysaccharides in
nikow Kg i a ze wzoru Marka-KuhnaHouwinka. Chem. concentrated solution. Biomacromolecules 2001, 2, 342.
Anal. 1976, 21, 1105. 53. Navard, P.; Naudin, J.M. Rheology of hydroxypropyl cel-
32. Granath, K. Solution properties of branched dextrans. J. lulose solutions. J. Polym. Sci.: Polym. Phys. Ed. 1986, 24, 189.
Colloid Sci. 1958, 13, 308. 54. Allain, C.; Lecourtier, J.; Chauveteau, G. Mesophase
33. Noda, I.; Tsuge, T.; Nagasawa, M. The intrinsic viscosity formation in high molecular-weight xanthan solutions.
of polyelectrolytes. J. Phys. Chem. 1970, 74, 710. Rheol. Acta 1988, 27, 255.
34. Vreeman, H.J.; Snoeren, T.H.M.; Payens, T.A.J. Phys- 55. Inatomi, S.-I.; Jinbo, Y.; Sato, T.; Teramoto, A. Isotropic-
icochemical investigation of n-carrageenan in the random liquid crystal phase equilibrium in semiexible polymer
state. Biopolymers 1980, 19, 1357. solutions: eects of molecular weight and ionic strength in
35. Tinland, B.; Rinaudo, M. Dependence of the stiness of polyelectrolyte solutions. Macromolecules 1992, 25, 5013.
the xanthan chain on the external salt concentration. 56. Boger, D.V. Demonstration of upper and lower New-
Macromolecules 1989, 22, 1863. tonian uid behaviour in a pseudoplastic uid. Nature
36. Robinson, G.; Ross-Murphy, S.B.; Morris, E.R. Viscos- 1977, 265, 126.
itymolecular weight relationships, intrinsic chain exi- 57. Graessley, W.W. The entanglement concept in polymer
bility, and dynamic solution properties of guar rheology. Adv. Polym. Sci. 1974, 16, 1.
galactomannan. Carbohydr. Res. 1982, 107, 17. 58. Graessley, W.W. Viscosity of entangling polydisperse
37. Picout, D.R.; Ross-Murphy, S.B.; Jumel, K.; Harding, polymers. J. Chem. Phys. 1967, 47, 1942.
S.E. Pressure cell assisted solution characterization of 59. Morris, E.R.; Cutler, A.N.; Ross-Murphy, S.B.; Rees, D.A.
polysaccharides. 2. Locust bean gum and tara gum. Concentration and shear rate dependence of viscosity in
Biomacromolecules 2002, 3, 761. random coil polysaccharide solutions. Carbohydr. Polym.
38. Cros, S.; Garnier, C.; Axelos, M.A.V.; Imberty, A.; Perez, 1981, 1, 5.
S. Solution conformations of pectin polysaccharides: 60. Morris, E.R. Rheology of hydrocolloids. In Gums and
determination of chain characteristics by small angle Stabilisers for the Food Industry2; Phillips, G.O.,
neutron scattering, viscometry, and molecular modeling. Wedlock, D.J., Williams, P.A., Eds.; Pergamon Press:
Biopolymers 1996, 39, 339. Oxford, 1984; 57 pp.
39. Sho, T.; Sato, T.; Norisuye, T. Viscosity behaviour and 61. Morris, E.R. Shear thinning of random coil poly-
persistence length of sodium xanthan in aqueous sodium saccharides: characterisation by two parameters from a
chloride. Biophys. Chem. 1986, 25, 307. simple linear plot. Carbohydr. Res. 1990, 13, 85.
40. Muller, G.; Anrhourrache, M.; Lecourtier, J.; Chauve- 62. Clasen, C.; Kulicke, W.-M. Determination of viscoelastic
teau, G. Salt dependence of the conformation of a single- and rheo-optical material functions of water-soluble
stranded xanthan. Int. J. Biol. Macromol. 1986, 8, 167. cellulose derivatives. Prog. Polym. Sci. 2001, 26, 1839.
41. Smidsrd, O.; Haug, A. Estimation of the relative stiness 63. Lapasin, R.; Pricl, S.; Tracanelli, P. Rheology of hydroxyethyl
of the molecular chain in electrolytes from measurements guar gum derivatives. Carbohydr. Polym. 1991, 14, 411.
of viscosity at dierent ionic strengths. Biopolymers 1971, 64. Milas, M.; Rinaudo, M.; Knipper, M.; Schuppiser, J.L.
10, 1213. Flow and viscoelastic properties of xanthan gum solu-
42. Cesa`ro, A.; Gamini, A.; Navarini, L. Supramolecular tions. Macromolecules 1990, 23, 2506.
structure of microbial polysaccharides in solution: from 65. Giboreau, A.; Cuvelier, G.; Launay, B. Rheological
chain conformation to rheological properties. Polymer behaviour of three biopolymer/water systems, with
1992, 33, 4001. emphasis on yield stress and viscoelastic properties. J.
43. Drummond, C.J.; Albers, S.; Furlong, D.N. Polymer Texture Stud. 1994, 25, 119.
surfactant interactions: (hydroxypropyl)cellulose with ion- 66. Lim, T.; Uhl, J.; Prudhomme, R.K. Rheology of self-
ic and non-ionic surfactants. Colloids Surf. 1992, 62, 75. associating concentrated xanthan solutions. J. Rheol. 1984,
44. Emanuele, A.; Palma-Vittorelli, M.B. Time-resolved 28, 367.
experimental study of shear viscosity in the course of 67. Ferry, J.D. Viscoelastic Properties of Polymers, 3rd Ed.;
spinodal demixing. Phys. Rev. Lett. 1992, 69, 81. John Wiley & Sons: New York, 1980.
68. Benallal, A.; Marin, G.; Montfort, J.P.; Derail, C. Linear linking of pentosans by a fungal laccase and horseradish
viscoelasticity revisited: the relaxation function of mono- peroxidase: mechanism of linkage between feruloylated
disperse polymer melts. Macromolecules 1993, 26, 7229. arabinoxylans. Cereal Chem. 1998, 75, 259.
69. Takahashi, Y.; Noda, I.; Nagasawa, M. Steady-state 92. Pruska-Kedzior, A.; Kedzior, Z.; Michniewicz, J.; Lefeb-
compliance of linear polymer solutions over a wide range vre, J.; Kolodziejczyk, P. Rheological properties of water-
of concentration. Macromolecules 1985, 18, 2220. soluble wheat pentosans solutions and gels obtained from
70. Colby, R.H.; Fetters, L.J.; Funk, W.G.; Graessley, W.W. various cultivars of common wheat. Proceedings of
Eects of concentration and thermodynamic interaction ISFRS 20033rd International Symposium on Food
on the viscoelastic properties of polymer solutions. Rheology and Structure; Zurich: ETH, 2003, 175179.
Macromolecules 1991, 24, 3873. 93. Durand, D.; Bertrand, C.; Busnel, J.-P.; Emery, J.;
71. Tam, K.C.; Tiu, C. Steady and dynamic shear properties Axelos, M.A.V.; Thibault, J.-F.; Lefebvre, J.; Doublier,
of aqueous polymer solutions. J. Rheol. 1989, 33, 257. J.-L.; Clark, A.H.; Lips, A. Physical gelation induced by
72. Carriere, C.J.; Amis, E.J.; Schrag, J.L.; Ferry, J.D. Dilute- ionic complexation: pectincalcium systems. In Physical
solution dynamic viscoelastic properties of xanthan Networks. Polymers and Gels; Burchard, W., Ross-
polysaccharide. J. Rheol. 1993, 37, 469. Murphy, S.B., Eds.; Elsevier: London, 1990; 283 pp.
73. Zirnsack, M.A.; Boger, D.V.; Tirtaatmadja, V. Steady 94. Axelos, M.A.V.; Kolb, M. Crosslinked biopolymers:
shear and dynamic rheological properties of xanthan gum experimental evidence for scalar percolation theory. Phys.
solutions in viscous solvents. J. Rheol. 1999, 43, 627. Rev. Lett. 1990, 64, 1457.
74. Cuvelier, G.; Launay, B. Concentration regimes in xan- 95. Axelos, M.A.V.; Kolb, M. Solgel transition in biopoly-
than gum solutions deduced from ow and viscoelastic mers. Makromol. Chem., Makromol. Symp. 1991, 45, 23.
properties. Carbohydr. Polym. 1986, 6, 321. 96. Doublier, J.-L.; Durand, S.; Lefebvre, J. Application of
75. Wientjes, R.H.W.; Duits, M.H.G.; Jongschaap, R.J.J.; Fourier transform mechanical spectroscopy to polysac-
Mellema, J. Linear rheology of guar gum solutions. Mac- charide systems undergoing structural changes. Proceed-
romolecules 2000, 33, 9594. ings of the XIIth International Congress on Rheology,
76. Dressler, M.; Fischer, P.; Windhab, E.J. Rheological Quebec 1996; 843 pp.
characterization and modeling of aqueous guar gum 97. Michon, C.; Cuvelier, G.; Launay, B. Concentration
solutions. Proceedings of ISFRS 20033rd International dependence of the critical properties of gelatin at the gel
Symposium on Food Rheology and Structure; ETH: point. Rheol. Acta 1993, 32, 94.
Zurich, 2003; 249253. 98. Michon, C.; Cuvelier, G.; Launay, B.; Parker, A.
77. Garnier, G.; Bouchet, B.; Gallant, D.; Doublier, J.-L. Concentration dependence of the properties of gelatin
Ultrastructure et comportement rheologique de melanges and iota-carrageenan systems at the gel point. J. Chim.
de biopolyme`res a` base de galactomannanes. Sci. Ali- Phys. 1996, 93, 819.
ments 1999, 19, 459. 99. Lefebvre, J.; Popineau, Y.; Deshayes, G.; Lavenant, L.
78. Stauer, D. Introduction to Percolation Theory; Taylor Temperature-induced changes in the dynamic rheological
and Francis: London, 1985. behavior and size distribution of polymeric proteins for
79. Ilavsky, M. Viscoelastic behaviour of network materials. glutens from wheat near-isogenic lines diering in HMW
In Polymer Networks. Principles of their Formation, glutenin subunit composition. Cereal Chem. 2000, 77, 193.
Structure and Properties; Stepto, R.F.T., Ed.; Blackie 100. Ross-Murphy, S.B. Structureproperty relationships in
Academic & Professional: London, 1998. food biopolymer gels and solutions. J. Rheol. 1995, 39, 1451.
80. Clark, A.H.; Ross-Murphy, S.B. Structural and mechan- 101. Plashchina, I.G.; Fomina, O.A.; Braudo, E.E.; Tolsto-
ical properties of biopolymer gels. Adv. Polym. Sci. 1987, guzov, V.B. Creep study of high-esteried pectin gels. 1.
83, 57. The creep of saccharose-containing gels. Colloid Polym.
81. Clark, A.H. Biopolymer gels. Curr. Opin. Colloid Inter- Sci. 1979, 257, 1180.
face Sci. 1996, 1, 712. 102. Plashchina, I.G.; Grinberg, N.V.; Braudo, E.E.; Tolsto-
82. Morris, V.J. Gelation of polysaccharides. In Functional guzov, V.B. Viscoelastic properties of kappa-carrageenan
Properties of Food Macromolecules; Hill, S.E., Ledward, gels. Colloid Polym. Sci. 1980, 258, 939.
D.A., Mitchell, J.R., Eds.; Aspen Publishers: Gaithers- 103. Plashchina, I.G.; Gotlieb, A.M.; Braudo, E.E.; Tolstogu-
burg, MD, 1998; 143 pp. zov, V.B. Creep study of high-esteried pectin gels. 2. The
83. Burchard, W., Ross-Murphy, S.B., Eds. Physical Networks, creep of glycerol-containing gels. Colloid Polym. Sci.
Polymers and Gels; Elsevier: London, 1990. 1983, 261, 672.
84. Guenet, J.-M. Thermoreversible Gelation of Polymers and 104. Braudo, E.E.; Plashchina, I.G.; Tolstoguzov, V.B. Struc-
Biopolymers; Academic Press: London, 1992. tural characterisation of thermoreversible anionic poly-
85. ten Nijenhuis, K. Thermoreversible Networks; Springer- saccharide gels by their elastoviscous properties. Carbo-
Verlag: Berlin, 1997. hydr. Polym. 1984, 4, 23.
86. ten Nijenhuis, K. , Mijs, W.J., Eds. Chemical and Physical 105. Parker, A.; Tilly, G. Thixotropic carrageenan gels and
Networks. Volume 1: Formation and Control of Properties; dairy desserts. In Gums and Stabilisers for the Food
John Wiley & Sons: Chichester, UK, 1998. Industry7; Phillips, G.O., Williams, P.A. Wedlock,
87. Stokke, B.T.; Elgsaeter, A., Eds. Chemical and Physical D.J., Eds.; IRL Press: Oxford, 1994.
Networks. Volume 2: Synthetic versus Biological Networks; 106. Labropoulos, K.C.; Niesz, D.E.; Danforth, S.C.; Kevre-
John Wiley & Sons: Chichester, UK, 1998. kidis, P.G. Dynamic rheology of agar gels: theory and
88. Stepto, R.F.T., Ed. Polymer Networks. Principles of their experiments. Part II. Gelation behavior of agar sols and
Formation, Structure and Properties; Blackie Academic & tting a theoretical rheological model. Carbohydr. Polym.
Professional: London, 1998. 2002, 50, 407.
89. Hoseney, R.C.; Faubion, J.M. A mechanism for the 107. Axelos, M.A.V.; Thibault, J.-F. Inuence of the sub-
oxidative gelation of wheat our water-soluble pentosans. stituents of the carboxyl groups and of the rhamnose
Cereal Chem. 1981, 58, 421. content on the solution properties and exibility of
90. Izydorczyk, M.S.; Biliaderis, C.G.; Bushuk, W. Physical pectins. Int. J. Biol. Macromol. 1991, 13, 77.
properties of water-soluble pentosans from dierent 108. Kamide, K.; Miyazaki, Y. The partially free draining
wheat varieties. Cereal Chem. 1991, 68, 145. eect and unperturbed chain dimensions of cellulose,
91. Figueroa-Espinoza, M.-C.; Rouau, X. Oxidative cross- amylose and their derivatives. Polym. J. 1978, 10, 409.
14
Stability and Degradation of Polysaccharides
Valdir Soldi
Federal University of Santa Catarina, Florianopolis, SC, Brazil
I. INTRODUCTION The degradation of polysaccharides has been analyzed

by chemical, enzymatic, thermal, mechanical (ultrasonic),
The interest in degradation of polysaccharides is strongly and radiative processes, which are generally dependent on
associated with a variety of applications in the food, paper, the structure, conformation, and applicability of the poly-
pharmaceutical, cosmetic, textile, and oil industries. More saccharides, reactive agents, etc. For example, chemical
specically, polysaccharides have been used in drug deliv- degradation reactions depend on the nature of initiation
ery, medical devices, oil exploration, water-based paints, (thermal, high energy, UV radiation, etc.) and reactive
emulsions, dispersions, wastewater treatment, etc. Besides agents such as oxygen and hydrogen ions. Depending on
the dependence on their chemical structure, these functions the structure, conformation, and reactive agents, dierent
are also dependent on a controlled molecular size. For chemical bonds of the polymer can be attacked. In dextran,
example, the potential biomedical and food applications for example, the bonds near the ends of a polymer chain are
for lower molecular weight chitosan encouraged the de- more reactive than those at the center [9]. In cellulose, the
velopment of viable processes for controlling the degrada- cleavage of the glycosidic bond is catalyzed either by H3O+
tion of this polysaccharide [1,2]. Through decreasing the (acid hydrolysis) or by enzyme (cellulase) [10]. At the same
molecular weight of chitosan (and other polysaccharides), time, the acid hydrolysis in chitosan breaks preferentially
it is possible to control properties like viscosity, solubility, the ether linkages in the macromolecule increasing the
and biological activity. In a similar manner, the water- polymer reactivity [11]. For chitosan, nitrous acid attacks
soluble polysaccharide, guar, needs to be depolymerized the amine groups but not the N-acetyl moieties, and
for use in applications such as food and oil production subsequently cleaves the h-glycosidic linkages [12]. Xan-
[3,4]. The primary degradation products from the thermal than is able to retain a high molecular weight (106 g mol1)
degradation of polysaccharides are also of great interest. when submitted to depolymerizing conditions such as acids
For example, monomeric anhydrosugars have been or free radicals [13,14]. This is because the double-helical
obtained from the pyrolysis of polysaccharides. By this structure of xanthan is stabilized by noncovalent bonds
process, levoglucosan was obtained from cellulose [5,6] between adjacent chains. In contrast to other polysaccha-
and starch [7], and 2-acetamido-1,6-anhydro-2-deoxy-h-D- rides, in xanthan, the cellulosic regions are the primary sites
glucopyranose from chitin [5,6]. Most of the applications that suer the attack by cellulases (enzymatic degradation)
for cellulose depend on enzymatic modication or other [15,16].
structural changes. This behavior is in general associated The application of ultrasonic degradation in the lab-
with a total absence of solubility either in water or in oratory and the food industry (not discussed in detail in this
aqueous alkaline solutions due to the existence of intra- chapter) has received more attention in recent decades
and intermolecular hydrogen bonds in solid-phase cellu- mainly to depolymerize macromolecules, prepare emul-
lose. To solve this problem, it is necessary to activate sions, and disrupt biological cells. For polysaccharides,
cellulose by swelling and degradation processes (mechan- the preliminary studies were related to cellulose derivatives
ical or thermal treatment). In another example, cellulose is [17], starches [18,19], dextrans [20], chitosans [21], and
easily dissolved in aqueous solution (9% NaOH) after xanthans [22]. The main eect observed in these macro-
modication with cellulase (enzyme) and treatment with molecules was a reduction in solution viscosity or gel
sodium hydroxide [8]. strength due to a decrease in molecular weight [20,21,23].
395
396 Soldi
Recently, Lii et al. [24] studied the degradation kinetics of

agarose and carrageenans by ultrasound, observing that
for solutions with 0.5 wt.% polysaccharide, the degrada-
tion rate decreases linearly with the reduced viscosity. This
result indicated that the ultrasonic degradation of agarose
and carrageenans follows a rst-order kinetics, and is
dependent on molecular size (decrease in viscosity). The
degradation mechanism has been attributed to cavitation
(mechanical eect); that is, the formation and collapse of
microscopic vapor bubbles generated by the strong sound
waves [25].
The aim of this chapter is to analyze the stability and
degradation of polysaccharides considering three main
general topics: (1) chemical (acid and alkaline) degrada-
tion in aqueous solution, (2) processes of biodegradation,
and (3) thermal stability (thermal degradation) of poly-
saccharides. The rst topic was chosen because alkaline
degradation was the rst process studied concerning the
stability of mono- and polysaccharides. Although most
work related to chemical degradation deals with cellulose
and cellulose derivatives, it is important to point out the
eects of temperature, concentration, and mechanism of
degradation (reaction products). In biodegradation, dif-
ferent aspects are considered such as the hydrolysis pro-
cess, eect on the molecular weight distribution, and eect
of pH. The thermal stability was analyzed in terms of the
kinetic parameters of degradation, viscosity (intrinsic vis-
cosity), temperature, depolymerization, and mechanism of
degradation. To better understand the mechanism of Figure 1 Reduced viscosity (gred) vs. concentration (A) and
degradation, a section with the main products associated log intrinsic viscosity (log [g]) vs. log molecular weight (log
with the degradation of polysaccharides was included in MW) (B). In A, 5 represents undegraded N-succinyl-chitosan
this chapter. and o, 4 represent two degraded samples of N-succinyl-
chitosan. (From Ref. 26.)
II. CHEMICAL DEGRADATION

lower and a slight reduction was observed; that is, practi-
In this section, dierent polysaccharides were analyzed in cally independent of the concentration. At the same time,
terms of acid and alkaline degradation processes. For the intrinsic viscosity, obtained from the intercept of the
example, the depolymerization of N-succinyl-chitosan so- straight lines of Fig. 1A, increased linearly with the molec-
dium salt was studied in an aqueous solution of hydro- ular weight (Fig. 1B). The behavior in terms of viscosity
chloric acid by Kato et al. [26] in terms of the molecular and molecular weight, associated with the analysis of the
weight and viscosity behavior. The acid treatment was composition of the degraded products indicated that the
performed by stirring a solution of N-succinyl-chitosan in chain scission of the N-succinyl-chitosan probably oc-
7.5 M aq HCl at room temperature or 3.3 M aq HCl at curred through the breakdown of the ether linkages in
40jC. In the depolymerization process, two degraded the macromolecule, as was observed for chitosan [11].
products were characterized in terms of the molecular Basedow et al. [9] studied the depolymerization reac-
weight by the authors, using size-exclusion chromatogra- tion of dextran at 80jC in 0.12 N sulfuric acid. During the
phy-multiangle light scattering (SEC-MALS). Studies uti- reaction, the molecular weight distribution was determined
lizing 1H NMR and elemental analysis indicated that the by the gel permeation chromatography (GPC) method.
degraded products retained the fundamental structure of Dierent samples with initial concentrations of dextran in
the N-succinyl-chitosan. The molecular weights (Mw) for the range of 0.252% were used. Considering the results, a
the two degraded products were 70,000 and 28,000 g cm3, rst-order reaction with respect to dextran concentration
very low values in comparison to the N-succinyl-chitosan was suggested by the authors. The average molecular
for which Mw = 310,000 g cm3. The viscosity behavior weight (Mw) changed from 117,000 to 3650 Da in the
[reduced viscosity (gred)] was analyzed as a function of the reaction time range of 0450 min and the rate constant
concentration in salt solution at 37jC (Fig. 1A). As can be (k) was 6.30 106 sec1.
observed, the viscosity of N-succinyl-chitosan, despite its Changes in the molecular weight were also analyzed by
higher values, increased with the concentration. However, the size exclusion chromatography-refractive index meth-
for the depolymerized products, the viscosity values were od (SEC-RI) considering the acid hydrolysis of (n-, L-, E-)
Stability and Degradation of Polysaccharides 397
carrageenans, dextran sulfate, and heparin [27]. The desul- is the gas constant, A is the pre-exponential factor, and T is
fation and depolymerization processes were studied at pH the temperature. The activation energy can be obtained
2 (0.008 M LiCl + 0.012 M HCl buer) at 35jC and 55jC. through the slope of the ln k vs. 1/RT plot. The activation
After hydrolysis, the results showed that for carrageenans energy values were in the range of 100120 kJ mol1 except
and heparin, the sulfation was unaected by the hydrolysis for L-carrageenan, which was 162 kJ mol1. The higher
conditions. However, for dextran sulfate, the desulfation stability for L-carrageenan was attributed to the steric eect
increased linearly with the hydrolysis time especially at caused by the sulfate group in the polymer structure. Under
55jC. The dierence in terms of the observed behavior was the above hydrolysis conditions, only the dextran sulfate
attributed to the conformation exibility of the polymer was sensitive to the process of desulfation. However, a
chain. The average molecular mass (hMmi) measured as a depolymerization process was characterized by the authors
function of hydrolysis time is shown in Fig. 2. A signicant for the carrageenans and dextran sulfate.
decrease in hMmi was observed for the carrageenans and The cleavage of the glycosidic bond of cellulose is
dextran sulfate (Fig. 2ad) in comparison with the theo- catalyzed by H3O+ (acid hydrolysis), producing glucose as
retical values determined assuming that desulfation was the the main reaction product (yield > 95%) and, in general,
only mechanism for molecular mass loss. However, a very the mechanism of degradation included the formation of
slight eect was observed by the authors for heparin (Fig. cationic intermediates that are responsible for the rate
2e). The comparative analysis with the theoretical values constant. The degradation of cellulose bers was analyzed
for dextran sulfate (Fig. 2d) indicated that besides the by Phillip [10] in HCl (100jC) and 1 N H2SO4 (80jC). In
sulfate loss, a chain scission occurred, signicantly decreas- both mediums, a signicant decrease in the degree of
ing the average molecular mass. From Fig. 2 and using Eq. polymerization was observed. The mechanism of degrada-
(1) [28,29] and the Arrhenius equation [Eq. (2)], the authors tion includes a selective attack on the more unstable
determined the kinetic parameters (activation energy) for glycosidic bonds (less-ordered regions) by the H3O+ ions.
the carrageenans and dextran sulfate. In Eq. (1), hMm(t)i As described by the author, the acid hydrolysis aects also
and hMm(0)i are the molecular mass at time t and time zero, the brillar structure of the cellulose bers.
respectively In the degradation of xanthan in dilute acid (pH 14)
at 80jC and at high (500 mM) and low (10 mM) ionic
1 1 kt strengths, the viscosity was described by a single exponen-
D ED E 1
t
Mm
0
Mm m tial-decay constant (the viscosity decreased with the hy-
drolysis time). At the same time, the stability increased with
the ionic strength [30].
ln k ln A Ea =RT 2
The thermochemical degradation of starch and cellu-
where k is the rst-order rate constant and m is the lose to organic acids was studied by Krochta et al. [3133] in
molecular weight of the repeating unit. In the Arrhenius alkaline solution. Experimentally, the reactions were per-
equation [(Eq. (2)], Ea is the apparent activation energy, R formed at dierent temperatures (range of 180240jC) and
Figure 2 Average molecular mass as a function of hydrolysis time at (.) 35jC and (o)55jC, for (a) n-carrageenan, (b) L-
carrageenan, (c) E-carrageenan, (d) dextran sulfate, and (e) heparin. Theoretical molecular mass as a function of hydrolysis time
based on the desulfation eect is plotted as () at 35jC and (- - -) at 55jC. (From Ref. 27.)
398 Soldi
products (organic acids) were determined by high-perform- experimental conditions, polysaccharide structure, confor-
ance liquid (HPLC) and gas (GC) chromatography. For mation, and other characteristics. The predominant mech-
both polysaccharides, the degradation was described as a anism for the chain scission was the breakdown of the
second-order kinetics by Eq. (3) glycosidic bonds in the macromolecule, a well-known mech-
anism, which occurs in a wide range of polysaccharides.
Ca
ln lnM Cp0 M Nka t 3
Cp
III. BIODEGRADATION PROCESSES
where Ca and Cp are the alkali and polysaccharide con-
centrations, respectively; M is the ratio between the initial Biodegradation was dened as an event that take place in
concentrations (Ca/Cp); N is the relation ((Ca0)/(Ca) / (Cp0)/ the natural environment and in living organisms [38,39].
(Cp)); ka is the rate constant, and t is the reaction time. The Biodegradation, as well as other processes of degradation,
rate constant (ka) determined by Eq. (3) increased from is dependent on the chemical structure of the polymers (or
0.0026 L mol1 min1 (180jC) to 0.3733 L mol1 min1 biopolymers) because it is fundamental how easily they can
(240jC) for starch and from 0.0034 L mol1 min1 be attacked by microorganisms. The hydrolysis process is
(180jC) to 0.6000 L mol1 min1 (240jC) for cellulose. also dependent on the local order of the macromolecule.
A signicant and similar eect due to the temperature, was Unordered macromolecules are more susceptible to hydrol-
observed for both systems. The activation energy deter- ysis (acid hydrolysis). Complex macromolecules, such as
mined using the Arrhenius equation [Eq. (2)] was 165.10 kJ lignin, which is a natural polymer, are less degradable than
mol1 for the degradation of starch and cellulose. A synthetic polymers like aliphatic polyesters, conrming
similar value (162 kJ mol1) was determined by Karlsson that the molecular composition and architecture determine
and Singh [27] for the degradation of L-carrageenan under the accessibility of a polymer to degradation by micro-
acidic conditions. organisms. As described by Ratajska and Boryniec [40], the
The stability of starch, cellulose, and rice straw in mechanism of biodegradation by microorganisms primar-
various NaOH solutions was compared by Krochta et al. ily occurs through an attack on the surface of the biode-
[33]. The conversion of starch to water-soluble products gradable fragments. By removing the fragments, the
(degradation) in 4% NaOH occurred just by heating to porosity of the material increases, allowing the permeation
240jC. For cellulose and rice straw, the degradation of water with the microorganisms that may cause mechan-
occurred in 40 min at 280jC, suggesting a higher stability ical stress facilitating the process of degradation by the
in comparison to the starch. Less time was required for the enzymes produced.
degradation when 6% NaOH was used. By HPLC, the au- The processes of biodegradation described in the
thors identied seven organic acids resulting from the literature are, in general, associated with the hydrolysis
alkaline degradation of 10% (w/w) starch, cellulose, rice caused by added enzymes or those produced by the micro-
straw, and glucose in 6% (w/w) NaOH at 280jC for 10 min. organisms, and can be followed by changes in molecular
For both systems, the yields of lactic and formic acids were weight, viscosity, and morphology. For example, in Fig.
more signicant. For example, the starch degradation 3A,B, the molecular weight distribution for the biodegra-
mainly produced 19.8% lactic acid, 13.0% formic acid, dation process of pure viscose (cellulosic) bers and mix-
and 5.7% glycolic acid. Other organic acids such as acetic ture with 30% of cellulose carbamate, respectively, are
acid, 2-hydroxybutyric acid, 2-hydroxyisobutyric acid, and shown under conditions of controlled temperature and
2-hydroxyvaleric acid were formed with ca. 2% content. humidity of the air and soil [40]. For both systems, a
Similar percentages were determined for cellulose; signicant decrease in the molecular weight was observed
however, the percentages determined for the glucose were after the enzymatic degradation. The biodegradation pro-
totally dierent. For example, for glucose the amount of cess was also analyzed by the authors in terms of the degree
lactic acid produced was 36.1%, for formic acid 5.4%, and of polymerization (DP) observing that for pure viscose, it
for 2-hydroxybutyric and 2-hydroxyisobutyric acids ca. decreased from 268 (untreated) to 53 after 6 months in
1%. In general, for both systems, the amount of organic contact with the microorganisms. For the viscose with 5%
acids produced corresponds to the NaOH equivalents. (gure not shown) and 30% of cellulose carbamate, the
Recently, Knill and Kennedy [34] discussed the cellulose ranges of DP variation under the same conditions were
degradation under alkaline conditions in terms of the 27848 and 15955, respectively.
mechanism and physical aspects, which aect the degrada- The treatment of pectin extracted by autoclave from
tion reaction and products. The predominant mechanism sugar beet pulp (ABP), with enzymes to modify their
suggested by the authors under alkaline conditions and at physicochemical properties, was studied by Ooterweld et
temperatures <170jC, based on the literature [3537], is al. [41]. The experimental procedure included the dissolu-
the formation of D-glucoisosaccharinic acids, as in the tion of the samples in 0.04 sodium acetate buer containing
alkaline degradation of 4-O-methyl-D-glucose. 0.01% of NaN3 (pH 5.0) to a nal concentration of 5 mg/
The dierent processes of polysaccharides depolymer- mL. The enzymes arabinofuranosidase B (AF), endo-
ization by acid hydrolysis discussed above, were in general, arabinanase plus arabinofuranosidase (EA + AF), rham-
described in terms of eects on the viscosity and the average nogalacturonase plus rhamnogalacturonan acetyl esterase
molecular weight. The extent of the eect depends on the (Rgase + RGAE), polygalactorunase plus pectin methyl
radation was observed by the authors when polygalactor-

unase plus pectin methyl esterase (PG + PE) was used,
suggesting that changes in the physicochemical properties
depend on the chemical structure of the ABP. These
conclusions seem consistent if we consider that the struc-
ture of ABP consists mainly of homogalacturonans and the
enzyme used was polygalactorunase.
The eect on the molecular weight and structure of N-
acetylated chitosan by hemicellulase was studied by Qin et
al. [42]. For a sample with a degree of deacetylation of 73.2,
the Mw decreased from 659,000 to 4200 kDa when the
chitosan was submitted to degradation by hemicellulase for
4 hr at 50jC and pH 5.5. The eect observed in the Mw was
accompanied by an increase in the water solubility and
decrease in the thermal stability. For the authors, the
results suggested that the enzymatic hydrolysis was endo-
action and mainly occurred in a random fashion.
Figure 3 Molecular weight distribution of (A) 100% viscose

bers and (B) viscose bers containing 30% of cellulose
carbamate: (a) untreated, (b) treated for 3 months, (c) treated
for 6 months. (From Ref. 40.)
esterase (PG + PE), were added to a nal concentration of

1 Ag protein/mL. Incubations were carried out at 20jC for
42 hr. The digests were analyzed in terms of the molecular
weight (Mw) at several stages of the incubation by high-
performance size-exclusion chromatography (HPSEC).
During the enzyme treatment, other parameters such as
the intrinsic viscosity ([g]w) and radius of gyration (Rgw) of
the polysaccharides were monitored. The behavior ob-
served for the above parameters is shown in Fig. 4AC.
The Mw decreased from 271 to 112 kDa when ABP was
treated with RGase + RGAE. This eect was associated
with the hydrolysis of the rhamnogalacturonan backbone
present in the ABP. For the same enzymes, no signicant
changes were observed in the intrinsic viscosity ([g]w) and
radius of gyration (Rgw). By adding EA + AF to ABP, the
Mw decreased to 207 kDa but a small change occurred in
the ([g]w) and (Rgw). However, by adding PG + PE, the
intrinsic viscosity ([g]w) and radius of gyration (Rgw) Figure 4 Eect of enzymatic modication of sugar beet
showed a signicant decrease (Fig. 4B,C) and the Mw pulp, ABP, on (A) Mw, (B) [g]w, and (C) Rgw: () EA + AF;
decreased to 5 kDa. A greater eect on the enzyme deg- (z) Rgase + RGAE; (.) PG + PE. (From Ref. 41.)
400 Soldi
The biodegradation of the ethyl(hydroxyethyl cellu- degradation was observed for the sample with DSethyl =
lose) (EHEC) by endoglucanases was studied by Richard- 0.9 (E481-02506) (Fig. 5B). The Mw decreased from
son et al. [43]. Ethyl(hydroxyethyl cellulose) is a nonionic, 772,000 (intact sample) to 57,000 and 67,000 g mol1, for
water-soluble cellulose derivative produced by introduc- products obtained by hydrolysis with the same enzymes,
tion of ethyl and ethylene oxide groups to the hydroxyl respectively. The action of endoglucanase on caboxy-
groups of the cellulose backbone. In general, the endoglu- methylcellulose (CMC) also decreased the molecular
canases, which are enzymes for the cellulose hydrolysis, weight when samples with DS 0.6 and 1.2 were submitted
cleave the internal (14)-h-D-glucosidic linkages in the to enzyme degradation (0.2% solution) for 92 hr at 45jC
cellulose chain, producing smaller fragments of mono- [46]. For the sample with DS 0.6, the molecular weight
and oligosaccharides [44]. From the literature, is known decreased from 249,000 to 10,700 g mol1. The signicant
that endoglucanases hydrolyze unmodied cellulose to decrease observed by the authors in the above value, in
low-molar-mass products such as glucose, cellobiose, and comparison to the sample with DS 1.2 in which the Mw
cellotriose [45]. For EHEC, the authors used two types of changed from 248,000 to 72,000 g mol1, indicated that the
endoglucanase from Trichoderma reesei (Cel7B, Mw f endoglucanase accessed more easily the sample with low
50,000 Da and Cel12A, Mw f 24,500 Da). The hydrolysis DS. However, for EHEC, the greater eect was observed
products were analyzed by size-exclusion chromatography for the sample with DSethyl 0.9, suggesting that the access of
with multiangle light scattering and refractive index detec- the enzyme to the internal (14)-h-D-glucosidic linkages
tion (SEC-MALS-RI). The molar mass distribution for was more favorable. These opposite eects indicated that a
two samples of EHEC with degrees of substitution (DS; more heterogeneous distribution of substituents occurred
average number of hydroxyl groups in the monomer unit in the sample with DSethyl 0.9.
substituted with ethyl) 0.9 (E481-02506) and 0.7 (EHMO), The eects on the viscosity were also described as
are shown in Fig. 5A and B, respectively. For the EHEC being associated with enzymatic degradation processes.
with DSethyl = 0.7 (EHM0), the molar weight of the For example, the enzymatic degradation of caboxymethyl-
hydrolysis products after being treated with Cel7B and cellulose (CMC) by endoglucanase signicantly decreased
Cel12A were 83,000 and 102,000 g mol1, respectively, the intrinsic viscosity [46]. For samples with DS 0.6 and 1.2,
which are values signicantly lower than those for the the viscosity decreased from 750 to 8.4 mL g1 and from
intact EHM0 (Mw = 359,000 g mol1). However, a higher 690 to 108 mL g1, respectively. As was pointed out above,
Figure 5 Molar mass distribution of (A) intact E481-02506, E481-02506 hydrolyzed by Cel7B, and E481-02506 hydrolyzed by
Cel12A; (B) intact EHMO, EHMO hydrolyzed by Cel7B, and EHMO hydrolyzed by Cel12A. (From Ref. 43.)
the viscosity values indicate that the endoglucanase concentration, and experimental conditions. For structur-
accessed more easily the sample with a low degree of ally related series of polysaccharides, the chemical reac-
substitution. Chitosan and substituted chitosans (methyl tions of degradation are associated with changes in
pyrrolidinone chitosan, N-carboxymethyl chitosan) also enthalpy and entropy, which can be described by kinetic
showed a rapid decrease in viscosity when samples were parameters such as Ea (activation energy) and A (pre-
submitted to degradation in solutions with wheat germ exponential factor) derived from the Arrhenius equation
lipase [47]. [(Eq. 2)]. Depolymerization processes are commonly de-
scribed by equations that consider that the chain scission of
the glycosidic linkage of polysaccharides follows a pseudo
IV. THERMAL DEPOLYMERIZATION rst-order reaction [48,49]. The rate constant will be related
to the molecular weight through Eq. (1), which indicates
As was pointed out in the above sections, the depolymer- that the inverse of the molecular weight increased linearly
ization processes of polysaccharides are normally studied with the depolymerization time and, in consequence, the
in terms of hydrodynamic properties such as intrinsic rate constant (k) can be obtained from the slope of the
viscosity ([g]) and average molecular weight (Mw). In corresponding plot.
general, both properties decreased during thermal, hydro- The degradation rate (k) can also be described in terms
lytic, and oxidative processes and are aected by pH, of the intrinsic viscosity by Eq. (4), which was derived
heating temperature range (reaction temperature), process- combining Eq. (1) and the MarkHouwinkSakurada
ing time, mechanical shear, solvent quality, polysaccharide equation ([g] = KM a), where K and a are constants for a
Figure 6 Apparent viscosity of 1% solutions of thermally degraded chitosan chlorides with degree of acetylation (*) 0.02, (5)
0.16, and (.) 0.35 vs. degradation time at (A) 60jC, (B) 80jC, (C) 105jC, and (D) 120jC. The degradation time for A and B is
given in days, while for C and D it is given in hours. (From Ref. 48.)
402 Soldi
given system. In Eq. (4), [g]t and [g]0 are the viscosities at independently of the temperature or acetylation degree.
time t and 0, respectively With the extended period of time, the viscosity decreases at
a lower rate as a consequence of the chain scission. At the
1 1 k same time, a more accentuated decreased was observed
t 4
g1=a g
1=a Mm K1=a with the increase in temperature and degree of acetylation.
t 0
The degradation rates were determined by the authors
For processes such as thermal- or acid-catalyzed degrada- from the slopes of the D1/[g](1/a) vs. t plots (Fig. 7),
tion, the rate constant can be obtained by plotting the considering Eq. (4). The values increased with both acety-
dierences between the inverses of the viscosities vs. the lation degree and temperature. For example, at 120jC, k
degradation time. For a given system, the degradation rates increased from 120 106 to 1450 106 hr1 when the
at dierent temperatures can be used to determine the acetylation degree increased from 0.02 to 0.35, respec-
kinetic parameters through the Arrhenius equation [Eq. tively. At the same time, for an acetylation degree of 0.35,
(2)]. k increased from 3.5 106 to 1450 106 hr1 when
Polysaccharides such as dextran [50], chitosan [11], the temperature increased from 60jC to 120jC. There-
chitosan chloride [48], N-succinyl chitosan [26], agarose fore the authors observed that regardless of the temper-
[24,49], and n-carrageenan [24,49], are examples in which ature, the degradation rates of the chitosan chloride with
the thermal depolymerization was followed by measuring an acetylation degree of 0.35 was about ten times greater
the apparent and intrinsic viscosity. For example, the than those determined for a chitosan with an acetylation
viscosity behavior of 1% solutions of thermally degraded degree of 0.02, indicated a dependence on the chemical
chitosan chloride [48] at four temperatures and three composition. On the other hand, the results also indicated
acetylation degrees is shown in Fig. 6. An exponential that the eect on the viscosity was more signicant as the
decrease was initially observed for all the systems studied temperature increased.
Figure 7 Time course of thermal degradation of chitosan chlorides with degree of acetylation (*) 0.02, (5) 0.16, and (.) 0.35 at
(A) 60jC, (B) 80jC, (C) 105jC, and (D) 120jC. The degradation time for A and B is given in days, while for C and D it is given in
hours. (From Ref. 48.)
Food polysaccharides such as agarose and n-carra-

geenan were studied by Lai et al. [49] in terms of the kinetics
of the depolymerization process. As observed in Fig. 8, the
intrinsic viscosity decreased linearly with time, showing a
certain dependence on temperature within a range of 75
95jC. From the slope of the plots using Eq. (4) (Fig. 9), the
authors determined the rate constants for the depolymer-
ization processes, observing that the values increased by
two- to threefold for a temperature increment of 10jC. The
rate constants were in the range of 0.21.6 104 and 0.2
1.3 106 sec1 for agarose and n-carrageenan, respec-
tively, within the temperature range of 7595jC. Appar-
ently, the access to the glycosidic linkage was more
favorable for agarose.
The thermal degradation of cellulose was studied by
Phillip [10] within the temperature range 100200jC con-
sidering the eect on the zero-order rate constant. For the
degradation reactions of cellulose in N2 + O2 and N2 +
Figure 9 Time dependencies on viscosity considering Eq. (4)

for agarose (A) and n-carrageenan (B) depolymerized at 75
95jC. (From Ref. 49.)
H2O, the rate constant values were in the range of 0.640

105 DP1 hr1 when the temperature changed from
100jC to 200jC. However, in comparison to the degrada-
tion reaction in N2 atmosphere, the above rate constants
were up two to eight orders of magnitude higher. Under
these conditions, the chain scission produced dierent
amounts and species of volatile low-molecular compounds
and of char. When only air was used, the mechanism of
thermal degradation of cellulose was described as having
three stages, which included dehydroxylation (preheating
and preliminary drying for removing absorbed water),
carbonization, and oxidative degradation [51]. Depending
Figure 8 Plots of intrinsic viscosity, [g], against heating time on the heating rate, conjugate stages occurred. In the
for agarose (A) and n-carrageenan (B) depolymerized at 75 process of the thermal degradation of cellulose and chitin
95jC. (From Ref. 49.) in supercritical acetone, a very slight evolution of gases was
404 Soldi
observed [6]. The authors assumed that approximately In the processes of thermal degradation, the kinetic
98% of the cellulose is in fact liqueed. parameters (activation energy and pre-exponential factor)
The intrinsic viscosity and average molecular weight of are important to analyze the reaction mechanism and
high methoxy pectin was evaluated at various temperatures thermal stability of dierent macromolecules. In Table 1,
(2060jC) [52]. The results presented in Fig. 10A,B, for the activation energy and pre-exponential factor associated
viscosity and Mw, respectively, indicated a similar behav- with dierent processes of degradation for a series of
ior; that is, a decrease with temperature. For viscosity, the polysaccharides were compared. In general, and regardless
observed eect must be associated with the molecular of the degradation conditions, the activation energy change
breakdown of the chain and a conformational change to with the mass loss fraction in polysaccharides, also indi-
a more compact structure. The decrease in molecular cating changes in the reaction mechanism. For example, in
weight was apparently due to the a h-elimination mecha- the thermal degradation of sodium hyaluronate, xanthan,
nism suggested by the authors. and methylcellulose in nitrogen atmosphere, the activation
The thermal stability of natural polymers, such as, energy determined by the Ozawa method [55,56] changed
sodium hyaluronate, xanthan, and methylcellulose in ni- from ca. 100 to 170 kJ mol1 [53]. For the above-mentioned
trogen atmosphere were compared considering the kinetic polysaccharides, the activation energy in Table 1 represents
and thermogravimetric parameters [53]. The results indi- average values. Variation in the activation energy with the
cated a higher thermal stability for methylcellulose, which mass loss fraction was also observed for chitosan and a
is a neutral polysaccharide. This conclusion was supported mercaptan derivative of chitosan in both nitrogen and air
by both the maximum degradation temperature and acti- atmospheres [57]. For these systems, the activation energy
vation energy. Similar behavior was described by Khomu- varied from ca. 100 to 250 kJ mol1. Another interesting
tov at al. [54] in studies of thermooxidative degradation of aspect relating to the values in Table 1 concerns the
anionic polysaccharides. They concluded that the thermal comparison between processes of thermal degradation that
stability increased with a decrease in the charge of the occurred in air (oxidative) and nitrogen atmosphere. Cel-
macromolecule. lulose and starch showed similar activation energy values
(58.5 and 50.0 kJ mol1, respectively) when the thermal
degradation process occurred in air [58]. In nitrogen atmo-
sphere, the activation energies were 242 and 474 kJ mol1
for cellulose and starch, respectively. The extremely high
value observed for starch in nitrogen was not expected if we
consider the similarity in the chemical structures of both
polysaccharides. However, the authors explained it by the
small mass loss (carbonaceous residue) observed for cellu-
lose at temperatures above 400jC.
The thermogravimetric curves for sodium alginate in
air showed two main stages of degradation with Tmax
(temperature of maximum degradation rate) at 240jC
and 380jC [59]. The activation energy determined by the
Broido method [60] were 171.4 and 174.4 kJ mol1 for the
above two stages of degradation, respectively. These values
were about three times higher than those observed for
starch and cellulose under the same conditions (air), indi-
cating a high dependence on the structure, and probably on
the conformation, of the polysaccharides. For copolymers
of sodium alginate with acrylonitrile, methyl acrylate, ethyl
acrylate, and methyl methacrylate, the activation energies
decreased to values in the range 6090 kJ mol1 in the rst
stage (Tmax = 240jC) and increased up to ca. 240 kJ mol1
in the second stage.
The activation energy values presented in Table 1
clearly show some dierences in magnitude when dierent
degradation processes are used. For example, in the chem-
ical degradation of chitosan, values in the range 80100 kJ
mol1 were determined. When chitosan was degraded by a
thermal process, values of 181 kJ mol1 (in nitrogen) and
160 kJ mol1 (in air) were obtained. The signicant dier-
Figure 10 Eect of increased temperature on the intrinsic ence was apparently associated to the easier access of the
viscosity, [g] (A) and average molecular weight, Mw (B) for a chemical agents (acid) to the glycosidic bonds. Another
high-methoxy pectin in standard phosphate chloride buer interesting point is the similarity in the activation energy
(pH = 6.8, I = 0.1 M). (From Ref. 52.) observed for most of the polysaccharides in Table 1,
Table 1 Activation Energy and Pre-Exponential Factor for Polysaccharide Depolymerization

Polysaccharides E (kJ mol1) A (min1) Degradation process Reference
Cellulose 58.5 (air) 3.4 1016 Thermal 58
83.7 (air) 3.4 109 Thermal 61
242.(N2) 6.3 1020 Thermal 58
189199.(N2) Thermal 62
D-glucose 174. (N2) Thermal 62
Cornstarch 50. (air) Thermal 58
474.(N2) 2.0 1042 Thermal 58
Sodium alginate 171.4 (air) Thermal 59
Xanthan 130 Thermal 53
Sodium hyaluronate 135 Thermal 53
Methycellulose 140 Thermal 53
Chitosan 87.1 Nitrous acid 12
130 Acid hydrolysis 63
92 Alkaline hydrolysis 64
181.(N2) 1.6 1014 Thermal 57
160.(Air) 4.8 1012 Thermal 57
Chitosan chloride 109 Thermal 48
Chitin 124 Acid hydrolysis 65
n-Carrageenan 105 5.3 1012 Hydrolyze, pH 2 27
113 2.5 1013 Hydrolyze, pH 2 28
126 4.6 1015 Hydrolyze, pH 2 29
120 3.2 1016 Hydrolyze, pH 2 14
97.2 7.0 107 Water, pH 7 49
L-Carrageenan 162 1.3 1021 Hydrolyze, pH 2 27
133 7.4 1017 Hydrolyze, pH 2 14
E-Carrageenan 116 1.9 1013 Hydrolyze, pH 2 27
Dextran sulfate 103 9.7 1010 Hydrolyze, pH 2 27
Agarose 103 7.8 1010 Water, pH 7 49
submitted to thermal degradation in nitrogen atmosphere. heated isothermally at 250jC was determined through
The values varied from ca. 100 to 150 kJ mol1, suggesting thermogravimetry-Fourier transform infrared (TG-FTIR)
that the mechanism of degradation was, generally, by techniques (Fig. 11A) [51]. The water is produced in a great
random scission of the chain. This conclusion was consis- amount in comparison with other products as shown in
tent with the large number of dierent products (residue Fig. 11A. Another signicant eect was observed when
and volatile products) detected in the degradation of poly- cellulose was heated to 170jC in air (oxygen) (Fig. 11B)
saccharides which is partially discussed in the next section. [51]. The water evolution was observed to be much higher
The activation energy values of cellulose (242 kJ mol1) in air than in nitrogen, because under these conditions,
and starch (474 kJ mol1) determined by thermal degra- hydroperoxide groups are formed, which can dissociate to
dation in nitrogen atmosphere were in apparent discrep- give hydroxyl radicals. The water is formed from hydroxyl
ancy with most of the polysaccharides in Table 1. radicals by hydrogen abstraction. A detailed mechanism of
cellulose degradation was described by Scheirs et al. [51]
considering the water evolution from cellulose after heat-
V. PRODUCTS OF DEGRADATION ing to dierent temperatures. Up to ca. 300jC, a dehydra-
tion process occurred that corresponded to the evolution of
Products of degradation in polysaccharides have been physical and chemical water. As suggested by the authors,
determined mainly in systems submitted to thermal and the evolution of chemical water occurs by intramolecular
pyrolytic degradation or chemical degradation at high elimination (from carbons 2 and 3 of the monomeric unit)
temperatures [33,51,53,6675]. In this section, some exam- with the formation of anhydrocellulose (enol or keto
ples concerning these processes are analyzed. forms). At the same temperature, cross-linking by ether
Cellulose has been one of the most extensively studied linkages due to the intermolecular elimination between the
polysaccharides not only in terms of the processes of hydroxyl groups may occur. The degradation at temper-
degradation, but also in terms of the reaction products of atures higher than 300jC induced elimination of water
degradation and pyrolysis under dierent conditions. For from the carbon 6 forming a vinylene group. Other reac-
example, the amount of water evolved from cellulose tions such as ring rearrangement, which apparently oc-
406 Soldi
was preferentially formed. These results were supported by

previous studies related to the pyrolysis of curdlan a
(1!3)-h-D-glucan in which the presence of sodium chlo-
ride favored lactone formation [68]. The degradation of
glucose in alkaline media [aqueous solution of Ca(OH)2] at
100jC produced a complex mixture with more than 50
compounds (saccharinic acids) [69].
Recently, Chen et al. [70] analyzed the volatile com-
pounds generated by the thermal degradation of gluco-
samine and N-acetylglucosamine, which are the monomer
forms of chitosan and chitin, respectively. Gas chromatog-
raphy and gas chromatography/mass spectrometry tech-
niques were used to identify the main degradation products
after maintaining the samples at 200jC for 30 min. The
degradation of N-acetylglucosamine produced mainly 3-
acetoamido-5-acetylfuran and 2-acetylfuran. According to
the authors, the formation mechanism for the above com-
pounds basically included successive dehydration steps
that occurred when the monomeric unit was maintained
at 200jC. Another 13 compounds were identied in the
same degradation reaction, which to include furans, pyr-
idines, pyrroles, and pyrazine derivatives. Studies related to
the thermal degradation of glucosamine were previously
reported by Chen and Ho [71]. They identied a series of
furyl-substituted pyrazines.
The glucan pyrolysis produced one-, two-, and three-
carbon compounds: glycolaldehyde, acetol (hydroxipropa-
none), acetic and formic acids [72]. The pyrolytic behavior
for all the glucans studied was the same [73]. This behavior
suggests that the nature of the glycosidic linkages in a given
glucan has little eect on major reaction pathways.
The FTIR analysis of the residual products of the
thermal degradation of sodium hyaluronate was described
by Villetti et al. [53]. The analysis at 280jC (the tempera-
ture of maximum degradation rate) indicated that the
Figure 11 Water evolution from (A) cellulose as measured bands associated with exocyclic groups (1150, 1079, and
by TG-FTIR technique. (B) Heating cellulose at 170jC in 1042 cm1) and NH stretching (1320 cm1) disappeared
oxygen and nitrogen. (From Ref. 51.) characterizing the cleavage (scission) of the glycosidic
linkage in the main chain. With a temperature increase
(>400jC), the scission of strong links in the backbone
curred after the initial water elimination, lead to further occurred.
water loss producing furanic species. Radlein et al. [74] considered the existence of two
Richards [66] used gas chromatography and 1H NMR decomposition pathways in the pyrolysis of cellulose. At
techniques to identify the pyrolysis products of lter paper temperatures lower than 300jC (slow decomposition), the
(cellulose) under nitrogen at 350jC. Glycolaldehyde, for- pyrolysis reaction produced mainly char and gases, con-
mic acid, 1-hydroxypropan-2-one, acetic acid, and ethylene rming previous results described by Shazadeh [75], which
glycol were identied as the main reaction products. Gly- suggested that in the range 150190jC, a reduction in the
colaldehyde was the major product of the pyrolysis with degree of polymerization, elimination of water, formation
yields up to 9.2%. As described in the literature [67], the of carbonyl, carboxyl and hydroperoxide groups, and
formation of glycolaldehyde includes two initial steps: (1) evolution of carbon mono- and dioxide occurred. Practi-
the formation of levoglucosan from cellulose and (2) cally the same products were identied by TG-FTIR (Fig.
glucose from levoglucosan. Reaction products, such as 11A) from cellulose heated isothermally to 250jC [51]. A
formic, acetic, glycolic, and lactic acids (in a total yield of second pathway was related to the pyrolysis at temper-
30% based on cellulose), were formed by alkaline degra- atures higher than 300jC (fast decomposition). At temper-
dation of cellulose [33]. In the presence of 10% sodium atures close to 500jC, for example, the main products of
chloride, the yield of glycolaldehyde decreased to 4.8%, the degradation were hydroxyacetaldehyde and levoglucosan
yield of 1-hydroxypropan-2-one (4.2%) increased at the with yields up to 15.3% and 38.4%, respectively, depending
same time, suggesting that in the presence of salt, lactone on the cellulose source and temperature. Other prod-
ucts such as acetic acid, formic acid, and acetol were thermal-degradation products of chitin. Z. Lebensm.
identied by the authors with yields in the range of 510%. Unters. Forsch. 1979, 169, 111.
6. Koll, P.; Metzger, J.O. Thermal-degradation of cellulose
and chitin in super-critical acetone. Angew. Chem. Int. Ed.
1978, 17, 754.
VI. CONCLUSIONS 7. Cerny, M.; Trnka, T.; Redlich, H. Praktische darstellung
von 1,6-anhydro-h- D -glucopyranose durch vakuum-
In this chapter, three dierent polysaccharide degradation pyrolyse von Starke. Kriterien fur den bau eines stahl-
processes were analyzed in terms of eects on viscosity and reaktors. Carbohydr. Res. 1988, 174, 349.
the average molecular weight. The processes clearly indi- 8. Cao, Y.; Tan, H. The properties of enzyme-hydrolyzed
cate a dependence on experimental conditions (pH, tem- cellulose in aqueous sodium hydroxyde. Carbohydr. Res.
2002, 337, 1453.
perature, reactive agents) and, on the structure and
9. Basedow, A.M.; Ebert, K.H.; Ederer, H.J. Kinetic studies
conformation of the polysaccharides. The eects on vis- on the acid hydrolysis of dextran. Macromolecules 1978, 11
cosity and average molecular weight were quite similar if (4), 774.
we consider that both decreased with the extent of degra- 10. Phillip, B. Degradation of cellulosemechanisms and
dation (treated time). In chemical and thermal degradation applications. Pure Appl. Chem. 1984, 56, 391.
processes, the predominant mechanism for the chain scis- 11. Rege, P.R.; Block, L.H. Chitosan processing: inuence of
sion is the breakdown of the glycosidic bonds in the process parameters during acid and alkaline hydrolysis and
eect of the processing sequence on the resultant chitosans
macromolecule, which occurs in a wide range of polysac- properties. Carbohydr. Res. 1999, 321, 235.
charides. In biodegradation, the local order of the macro- 12. Allan, G.G.; Peyron, M. Molecular weight manipulation of
molecule seems to be important. As initially only one bond chitosan. I: Kinetics of depolymerization by nitrous acid.
is hydrolyzed by enzymatic attack, unordered macromole- Carbohydr. Res. 1995, 277, 257.
cules are more susceptible to hydrolysis. Secondary cleav- 13. Christensen, B.E.; Myhr, M.; Smidsrd, O. Degradation of
ages occur on one of the fragments produced by the initial double-stranded xanthan by hydrogen peroxide in the
presence of ferrous ions: comparison to acid hydrolysis.
scission. Enzymatic degradation does not follow any of the Carbohydr. Res. 1996, 280, 85.
typical modes (random, gaussian) of chain scission. Dif- 14. Hjerde, T.; Kristiansen, T.S.; Stokke, B.T.; Smidsrd, O.;
ferently to the biodegradation, the thermal degradation of Christensen, B.E. Conformation dependent depolymeriza-
polysaccharides occurs by random scission of the chain as tion kinetics of polysaccharides studied by viscosity
indicated by the activation energy values that are higher measurements. Carbohydr. Polym. 1994, 24, 265.
than 100 kJ mol1 for most of the polysaccharides. Simi- 15. Sutherland, W. Hydrolysis of unordered xanthan in so-
lution by fungal cellulases. Carbohydr. Res. 1984, 131, 93.
larly, the degradation of guar, agarose, and carrageenans
16. Cheetham, N.W.H.; Mashimba, E.N.M. Characterization
by ultrasound occurs also by random scission of glycosidic of some enzymic hydrolysis products of xanthan. Carbo-
linkages [2325]. In the degradation of cellulose and starch hydr. Polym. 1991, 15, 195.
in the presence of air, the activation energy has been found 17. Mason, T.J.; Lorimer, J.P. Sonochemistry: Theory, Appli-
to be lower than 100 kJ mol1 (58.5 and 50.0 kJ mol1, cations and Uses of Ultrasound in Chemistry; Ellis Hor-
respectively), indicating a lower thermal stability for both wood: Chichester, UK, 1988; 99 pp.
polysaccharides. In terms of the products of degradation 18. Gallant, D.; Degrois, M.; Sterling, C.; Guilbot, A. Micro-
scopic eects of ultrasound on the structure of potato
resulting from thermal processes, polysaccharides produce starch. Die Starke 1972, 24, 116.
mainly acids and other specic compounds depending on 19. Seguchi, M.; Higasa, T.; Mori, T. Study of wheat starch
the structure and conformation of the macromolecules and structures by sonication treatment. Cereal Chem. 1994, 71,
experimental conditions. For example, glycolaldehyde is 636.
formed in great yields from cellulose degradation. As 20. Lorimer, J.P.; Mason, T.J.; Cuthbert, T.C.; Brookeld,
glycolaldehyde is formed from the glucose monomeric unit, E.A. Eect of ultrasound on the degradation of aqueous
two initial steps occur: the formation of levoglucosan from native dextran. Ultrason. Sonochem. 1995, 2, 55.
21. Chen, R.H.; Chang, J.R.; Shyur, J.S. Eects of ultrasonic
cellulose and of glucose from levoglucosan. conditions and storage in acidic solutions on changes in
molecular weight and polydispersity of treated chitosan.
Carbohydr. Res. 1997, 299, 287.
REFERENCES 22. Milas, M.; Rinaudo, M.; Tinland, B. Comparative depo-
lymerization of xanthan gum by ultrasonic and enzymic
1. Kumar, M.N.V.R. A review of chitin and chitosan treatments: Rheological and structural properties. Carbo-
applications. React. Funct. Polym. 2000, 46, 1. hydr. Polym. 1986, 6, 95.
2. Shahidi, F.; Arachchi, J.K.V.; Jeon, Y.J. Food applications 23. Lai, M.F.; Kuo, M.L.; Li, C.F.; Lii, C.Y. Ultrasonic and
of chitin and chitosans. Trends Food Sci. Technol. 1999, heat eects on the chemical and gelling properties of red al-
10, 37. gal polysaccharides. Food Sci. 1996, 23, in Chinese. 717.
3. Tayal, A.; Kelly, R.M.; Khan, S.A. Rheology and 24. Lii, C.-Y.; Chen, C.-H.; Yeh, A.-I.; Lai, V.M.F. Prelimi-
molecular weight changes during enzymatic degradation nary study on the degradation kinetics of agarose and
of a water-soluble polymer. Macromolecules 1999, 32, 294. carrageenans by ultrasound. Food Hydrocoll. 1999, 13,
4. Tayal, A.; Pay, V.B.; Khan, S.A. Rheology and micro- 477.
structural changes during enzymatic degradation of a guar- 25. Tayal, A.; Khan, S.A. Degradation of a water-soluble
borax hydrogel. Macromolecules 1999, 32, 5567. polymer: Molecular weight changes and chain scission
5. Koll, P.; Metzger, J.O. Detection of acetamide in the characteristics. Macromolecules 2000, 33, 9488.
408 Soldi
26. Kato, Y.; Onishi, H.; Machida, Y. Depolymerization of N- 47. Muzzarelli, R.A.A.; Xia, W.; Tomasetti, M.; Ilari, P.
succinyl-chitosan by hydrochloric acid. Carbohydr. Res. Depolymerization of chitosan and substituted chitosans
2002, 337, 561. with the aid of a wheat germ lipase preparation. Enzyme
27. Karlsson, A.; Singh, S.K. Acid hydrolysis of sulfated Microb. Technol. 1995, 17, 541.
polysaccharides. Desulfation and the eect on molecular 48. Holme, H.K.; Foros, H.; Pettersen, H.; Dornish, M.;
mass. Carbohydr. Polym. 1999, 38, 7. Smidsrd, O. Thermal depolymerization of chitosan
28. Myslabodski, D.; Stancio, D.; Heckert, R. Eect of acid chloride. Carbohydr. Polym. 2001, 46, 287.
hydrolysis on the molecular mass of kappa carrageenan by 49. Lai, V.M.-F.; Lii, C.-y.; Hung, W.-L.; Lu, T.-J. Kinetic
GPC-LS. Carbohydr. Polym. 1996, 31, 83. compensation in depolymerisation of food polysaccharides.
29. Singh, S.K.; Jacobsson, S.P. Kinetics of acid hydrolysis of Food Chem. 2000, 68, 319.
n-carrageenan as determined by molecular weight (SEC- 50. Cote, G.L.; Willet, J.L. Thermomechanical depolymeriza-
MALLS-RI), gel breaking strength, and viscosity measure- tion of dextran. Carbohydr. Polym. 1999, 39, 119.
ments. Carbohydr. Polym. 1994, 23, 89. 51. Scheirs, J.; Camino, G.; Tumiatti, W. Overview of water
30. Christensen, B.E.; Smidsrd, O. Hydrolysis of xanthan in evolution during thermal degradation of cellulose. Eur.
dilute acid: Eects on chemical composition, conformation, Polym. J. 2001, 37, 933.
and intrinsic viscosity. Carbohydr. Res. 1991, 214, 55. 52. Morris, G.A.; Foster, T.J.; Harding, S.E. A hydrodynamic
31. Krochta, J.M.; Hudson, J.S.; Drake, C.W. Alkaline thermo- study of the depolymerisation of a high methoxy pectin at
mechanical degradation of cellulose to organic acids. Bio- elevated temperatures. Carbohydr. Res. 2002, 48, 361.
technol. Bioeng. 1984, S14, 37. 53. Villetti, M.A.; Crespo, J.S.; Soldi, M.S.; Pires, A.T.N.;
32. Krochta, J.M.; Hudson, J.S.; Tillin, S.J. Kinetics of alka- Borsali, R.; Soldi, V. Thermal degradation of natural
line thermochemical degradation of polysaccharides to or- polymers. J. Therm. Anal. 2002, 67, 295.
ganic acids. Am. Chem. Soc. Div. Fuel Chem. 1987, 32, 54. Khomutov, L.I.; Ptichkina, N.M.; Sheenson, V.A.; Lashek,
148. N.A.; Panina, N.I. Thermal degradation of polysaccha-
33. Krochta, J.M.; Tillin, S.J.; Hudson, J.S. Degradation of rides. Russ. J. Appl. Chem. 1994, 67, 574.
polysaccharides in alkaline solution to organic acids: Prod- 55. Ozawa, T. A new method of analyzing thermogravimetric
uct characterization and identication. J. Appl. Polym. Sci. data. Bull. Chem. Soc. Jpn. 1965, 38, 1881.
1987, 33, 1413. 56. Ozawa, T. Critical investigation of methods for kinetic-anal-
34. Knill, C.J.; Kennedy, J.F. Degradation of cellulose under ysis of thermoanalytical data. J. Therm. Anal. 1975, 7, 601.
alkaline conditions. Carbohydr. Polym. 2003, 51, 281. 57. Covas, C.P.; Monal, W.A.; Roman, J.S. A kinetic study of
35. Colbran, R.L.; Davidson, G.F. The degradative action of the thermal degradation of chitosan and a mercaptan
hot dilute alkalis on hydrocellulose. J. Text. Inst. 1961, 52, derivative of cellulose. Polym. Degrad. Stab. 1993, 39, 21.
T73. 58. Aggarwal, P.; Dollimore, D.; Heon, K. Comparative
36. Richards, G.N. In Alkaline Degradation; Whistler, R.L., thermal analysis study of two biopolymers, starch and
Ed.; Academic Press: New York, 1963; 154 pp. cellulose. J. Therm. Anal. 1997, 50, 7.
37. Nevell, T.P. Degradation of Cellulose by Acids, Alkalis, and 59. Shah, S.B.; Patel, C.P.; Trivedi, H.C. Thermal behaviour of
Mechanical Means; Nevell, T.P., Zeronian, S.H., Eds.; Ellis graft copolymers of sodium alginate. Angew. Makromol.
Horwood: Chichester, 1985; 223 pp. Chem. 1996, 235, 1.
38. Albertsson, A.-C. Degradable polymers. JMS-Pure Appl. 60. Broido, A. A simple, sensitive graphical method of treating
Chem. 1993, A30, 757. thermogravimetric analysis data. J. Polym. Sci. A-2 Polym.
39. Albertsson, A.C. Biodegradation of polymers in historical Phys. 1969, 7, 1761.
perspective versus modern polymer chemistry. In Hand- 61. Rychly, J.; Strlic, M.; Rychla, L.M.; Kolar, J. Chemilumi-
book of Polymer Degradation; Hamid, S.H., Ed.; Marcel nescence from paper I. Kinetic analysis of thermal
Dekker: New York, 2000; 421 pp. oxidation of cellulose. Polym. Degrad. Stab. 2002, 78, 357.
40. Ratajska, M.; Boryniec, S. Physical and chemical aspects of 62. Jain, R.K.; Lal, K.; Bhatnagar, H.L. A kinetic study of the
biodegradation of natural polymers. React. Funct. Polym. thermal degradation of cellulose and some related model
1998, 38, 35. compounds. Indian J. Chem. Sect. A 1984, 23, 828.
41. Ooterweld, A.; Beldman, G.; Voragen, A.G.J. Enzymatic 63. Varum, K.M.; Otty, M.H.; Smidsrd, O. Acid hydrolysis
modication of pectic polysaccharides obtained from sugar of chitosans. Carbohydr. Polym. 2001, 46, 89.
beet pulp. Carbohydr. Polym. 2002, 48, 73. 64. Sannan, T.; Kurita, K.; Iwakura, Y. Studies on chitin. 5.
42. Qin, C.; Du, Y.; Zong, L.; Zeng, F.; Liu, Y.; Zhou, B. Kinetics of deacetylation reaction. Polym. J. 1977, 9, 649.
Eect of hemicellulase on the molecular weight and 65. Allan, G.G.; Altman, L.C.; Bonsigner, R.E.; Ghost, D.K.;
structure of chitosan. Polym. Degrad. Stab. 2003, 80, 435. Hirabayashi, Y.; Neogi, A.A.; Neogi, S. In Chitin, Chitosan
43. Richardson, S.; Lundqvist, J.; Wittgren, B.; Tjerneld, F.; and Related Enzymes; Zikakis, J.P., Ed.; Academic Press:
Gordon, L. Initial characterization of ethyl(hydroxyethyl) Orlando, FL, 1984; 119134.
cellulose using enzymic degradation and chromatographic 66. Richards, G.N. Glycolaldehyde from pyrolysis of cellulose.
methods. Biomacromolecules 2002, 3, 1359. J. Anal. Appl. Pyrolysis 1987, 10, 251.
44. Gohdes, M.; Mischnick, P. Determination of the substitu- 67. Piskorz, J.; Radlein, D.; Scott, D.S. On the mechanism of
tion pattern in the polymer chain of cellulose sulfates. the rapid pyrolysis of cellulose. J. Anal. Appl. Pyrolysis
Carbohydr. Res. 1998, 309, 109. 1986, 9, 121.
45. Karlsson, J.; Momcilovic, D.; Wittgren, B.; Schulein, M.; 68. Richards, G.N.; Shazadeh, F. Formation of glucometa-
Tjerneld, F.; Brinkmalm, G. Enzymatic degradation of saccharinolactones in the pyrolysis of curdlan, a (1!3)-h-
carboxymethyl cellulose hydrolyzed by the endoglucanases D-glucan. Carbohydr. Res. 1982, 106, 83.
Cel5A, Cel7B, and Cel45A from Humicola insolens and 69. Yang, B.Y.; Montgomery, R. Alkaline degradation of glu-
Cel7B, Cel12A and Cel45A core from Trichoderma reesei. cose: eect of initial concentration of reactants. Carbohydr.
Biopolymers 2002, 63, 32. Res. 1996, 280, 27.
46. Horner, S.; Plus, J.; Saake, B.; Klohr, E.-A.; Thielking, H. 70. Chen, J.; Wang, M.; Ho, C.-T. Volatile compounds
Enzyme-aided characterization of carboxymethylcellulose. generated from thermal degradation of N-acetylgluco-
Carbohydr. Polym. 1999, 40, 1. samine. J. Agric. Food Chem. 1998, 46, 3207.
71. Chen, J.; Ho, C.-T. Volatile compounds generated from glu- charides: A study of several glucans. J. Anal. Appl.
cosamine in dry system. J. Agric. Food Chem. 1998, 46, 1971. Pyrolysis 1992, 22, 217.
72. Ponder, G.R.; Richards, G.N. Pyrolysis of some 13C- 74. Radlein, D.; Piskorz, J.; Scott, D.S. Fast pyrolysis of
labeled glucans: A mechanistic study. Carbohydr. Res. natural polysaccharides as a potential industrial process. J.
1993, 244, 27. Anal. Appl. Pyrolysis 1991, 19, 41.
73. Ponder, G.R.; Richards, G.N.; Stevenson, T.T. Inuence of 75. Shazadeh, F. Introduction pyrolysis of biomass. J. Anal.
linkage position and orientation in pyrolysis of polysac- Appl. Pyrolysis 1982, 3, 283.
15
Biosynthesis, Structure, and Physical Properties
of Some Bacterial Polysaccharides
Roberto Geremia
Laboratoire dAdaptation et de Pathogenie des Microorganismes, Joseph Fourier University, Grenoble, France
Marguerite Rinaudo
Grenoble, France
I. INTRODUCTION II. BIOSYNTHESIS OF BACTERIAL

POLYSACCHARIDES
Polysaccharides consist of a large variety of natural poly-
mers biosynthesized in wood, plants, and algae, but are also A. Introduction and General View
produced by bacteria and fungi. Their chemical structures of the Biosynthetic Pathway
and physical properties in solution or in the solid state may
vary in a large extent. Nevertheless, now, for industrial The physicochemical properties of polysaccharides are
purposes, many new polysaccharides are developed from determined mainly by their sugar sequence derived
bacteria; exocellular polysaccharides are produced in a through biosynthetic enzymes. Thus, understanding of
large scale by the usual techniques of microbiology and the biosynthetic process is necessary to eventually engineer
fermentationthis procedure allows a good control of the polysaccharide sequence and, hence, physicochemical pro-
characteristics of polymers and allows the purication perties. In this chapter, we are going to deal with the bio-
of polysaccharides more easily than from other natural synthesis of succinoglycan, xanthan, and gellan. Besides
sources [14]. Extension of such production also allows their importance in the industry, succinoglycan and xan-
reducing the price and extending the range of applications than play important biological roles; Sinorhizobium meli-
[5]. A good example is hyaluronan (HA), previously pro- loti strains failing to produce succinoglycans are unable to
duced by extraction from animal source but in which some invade alfalfa nodules [6], whereas Xanthomonas campest-
fractions of proteins remain when the bacterial hyaluronan ris lacking xanthan are less aggressive toward plants
is prepared in a very pure form. [7]. No biological role was reported so far for gellan-
This chapter will introduce the mechanisms of biorelated polysaccharides.
synthesis of bacterial polysaccharides and some informa- It can be considered that the biosynthetic pathway
tion on the engineering of polysaccharides, allowing, in the starts with the biosynthesis of activated glycosidic and
near future, the production of a polysaccharide with a nonglycosidic donors, many of these molecules being
choice chemical structure having a set of predictable phys- involved in other metabolic pathways. We are not going
ical properties. to refer to them here. However, it is worth mentioning that
411
412 Geremia and Rinaudo
the sugar donors are nucleotide diphospho-sugars, and availability of X. campestris and S. meliloti mutant strains,
their biosynthetic genes are often located in polysaccharide unable to synthesize xanthan and succinoglycan, respec-
clusters. The biosynthesis of bacterial polysaccharides tively, allowed the cloning and sequencing of the biosyn-
proceeds by three pathways: (1) by a processive enzyme thetic gene cluster [6,7,1219]. The genes present in these
(polysaccharide synthase); (2) by using a wzy-dependent clusters comprise the enzymes involved in ORU assembly
pathway; and (3) by using a wzy-independent pathway [8]. (transfer of glycosidic and nonglycosidic residues), trans-
Although the polysaccharide synthase is able to synthesize location, polymerization, and regulatory genes. In the
the complete polysaccharide, both wzy-dependent and particular case of succinoglycan, it is interesting to note
wzy-independent pathways involve the use of several gly- the presence of glycosidases in the gene cluster [15,20,21]. It
cosyltransferases as well as a lipid anchor (polyprenol- is also worth noting that the regulatory protein ExoP
phosphate). In the case of the wzy-independent pathway, contains an autotyrosine kinase cytoplasmic domain [22].
the whole polysaccharide is synthesized on the lipid anchor The removal or mutation of a proline-rich domain results
and the growth of the polysaccharide is coupled to secre- in enhanced production of low-molecular-weight (LMW)
tion [8]. For the wzy-dependent pathway, the oligosaccha- succinoglycan (mainly the ORU) [23]. This eect is prob-
ridic repeat unit (ORU) is synthesized on the lipid anchor, ably due to the alteration of the tyrosine phosphorylation
translocated, and nally polymerized in a wzy-dependent status of the ExoP because either the inactivation of the
way (Fig. 1). The assembly of the oligosaccharidic repeat ATP binding motif or the amino acid replacement of
unit is largely the best-studied step, whereas very little is phosphorylated tyrosine residues results in an increase of
known about translocation and polymerization. The rst LMW succinoglycan production. The presence of secreted
step of ORU assembly is the transfer of a sugar-1-phos- ORU in that mutants indicates that neither ORU assembly
phate to polyprenol-phosphate. The remaining sugars are nor translocation is aected by the phosphorylation status
transferred sequentially by specic glycosyltransferases. of ExoP. Therefore, the regulatory target seems to be
Actually, the glycosyltransferases are responsible for the ORUs polymerization.
ORU sugar sequence. The biosynthesis of succinoglycan
and xanthan ORU was accomplished in vitro [9,10] by C. Glycosyltransferases
using permeabilized cells. These experiments established
that the biosynthesis of succinoglycan ORU starts by For xanthan and succinoglycan, biochemical characteriza-
addition of a Gal-1-P [9] to the polyprenol-phosphate, tion of glycolipids accumulated by mutant strains allowed
whereas for xanthan and gellan, the rst sugar of the to propose the function of several glycosyltransferases
biosynthetic repeat unit is a-D-glucose-1-phosphate [10,11]. [7,1618,24]. In the case of gellan gum, the characterization
of glycolipids formed in Escherichia coli expressing the cor-
B. Molecular Genetics of Polysaccharide responding genes allowed the identication of three genes
Biosynthesis involved in the assembly of gellan [11]. The proposed func-
tion of the glycosyltransferases is summarized in Fig. 2.
Molecular microbiology has been of big help in the under- Although the biochemical phenotype of the mutants is very
standing of polysaccharide biosynthesis. Actually, the useful, the precise function can only be biochemically
Figure 1 Schema of succinoglycan biosynthetic machinery, as an example of wzy-dependent pathway.

Biosynthesis, Structure and Physical Properties 413
Figure 2 Glycosyltransferases involved in polysaccharide biosynthesis biochemically characterized. The inset corresponds to a
uorography of TLC in which the in vitro-formed oligosaccharides were separated. The genes proposed to add the corresponding
sugars are indicated on the structure.
attributedwhat implies the use of isolated enzymes and These studies revealed several features shared by these
well-characterized substrates (nucleotide sugars as well as proteins. First, even in the absence of predicted transmem-
glycolipids). At rst glance, the complexity of the ORU-P2- brane helix, these proteins are associated with the mem-
polyprenol intermediates complicates these studies. This brane. Moreover, when it is possible to produce soluble
obstacle can be partially overcome by isolating the authen- proteins, they have a strong propensity to aggregation (R.
tic glycolipid from the relevant bacterial strains. The A. Geremia, unpublished results). Thus, biochemical and
incubation of permeabilized cells with radiolabeled sugar structural studies are limited. Second, the free oligosac-
donors provides a reliable acceptor. The modication of charides are not used as acceptors, indicating that features
the oligosaccharide can be assessed by thin-layer chroma- of the polyprenol-pyrophosphate moiety are needed. It is
tography (TLC) and uorography. Using this strategy, we tempting to speculate that the pyrophosphate linkage of the
have characterized the glycosyltransferases ExoM, ExoO, acceptor serves to anchor correctly the oligosaccharide in
and ExoU from the succinoglycan pathway [25] (R. A. the glycosyltransferase. Third, only one sugar moiety was
Geremia and S. Drouillard, personal communication), transferred to the acceptor. Finally, the exquisite acceptor
GelK from the gellan pathway [26], and the GumH-ho- specicity of these proteins was conrmed in vitro. On the
mologous AceA from the xanthan pathway [27] (Fig. 2). basis of these results, it is tempting to predict that the
recognition of the acceptor molecule is driven by the EX7E motif, secondary structure alignment suggests that
pyrophosphate moiety of Und-P2, and that specic inter- this motif is involved in sugar donor recognition.
actions of the saccharidic moiety, including the length of The structural questions concerning substrate recog-
the oligosaccharide, are also important for anchoring the nition are important for eventually engineering the speci-
correct sugar in the reaction center. Structural and bio- city of glycosytransferases. However, there is still so much
chemical studies of several glycosyltransferases with similar to understand from classical biochemistry. Actually, there
substrate specicity are necessary to better understand are no other biochemical constants available for any of
these phenomena. these proteins. This point is central to understanding the
It is important to point out here that in the absence of kinetics of polysaccharide biosynthesis, and eventually
structural data for glycosyltransferase involved in ORU engineering these enzymes.
biosynthesis, the bioinformatic analysis became an im-
portant tool for prediction. Analysis of glycosyl- D. Major Issues in Polysaccharide Engineering
transferases amino acid sequence resulted in important
contributions for glycobiology. Actually, two main motifs It is conceivable that understanding the molecular basis of
were predicted by analysis of these glycosyltransferases polysaccharide biosynthesis may be of important help for
[18,2830]. These studies implied the use of hydrophobic the production of more and better polysaccharides. Also, it
cluster analysis, and allowed the identication of conserved is worth reminding that already notable eects can be
secondary structure features. The two identied motifs are achieved by just changing the culture conditions; for
the so-called DXD or DD and EX7E [29], which instance, it is known that the culture media composition
are characterized by a set of conserved amino acids. Based aects gellan gum nonglycosidic substitution [36]. On the
on the scarce structural data available from other glycosyl- other hand, much more knowledge is necessary to actually
transferases, these two motifs most probably represent two proceed to rational engineered polysaccharides. We are
dierent structural superfamilies of glycosyltransferases. going to discuss a few points, whose understanding is
The role of the conserved amino acids was studied in the necessary for that purpose.
glycosyltransferases ExoM (DXD) and AceA (EX7E). In the rst instance, the factors aecting the amount of
In the case of ExoM, the DXD motif is present in the NH2 polysaccharide produced are not known. It is possible to
terminal domain [28]. Replacement of either Asp-44 or Asp- predict that the ORU assembly pathway is not the bottle-
96 by Ala resulted in the inactivation of the enzyme, whereas neck because mutant ExoM or AceA that exhibits less than
the conservative replacement by Glu rendered an enzyme 10% of wild-type activity in vitro can restore polysaccha-
with residual activity [31]. These data are compatible with ride production almost at the same level as wild-type
such a domain being involved in the binding of the sugar enzymes [31,33]. These experiments indicate that the gly-
donor, as in the glycosyltransferase SpsA [32]. On the other cosyltransferase machinery would produce more ORU if
hand, COOH seems to be much less conserved. However, more precursors are available. If this turns out to be the
based on the structure of SpsA, Asp-191 was identied as case, it is probably the availability of glycosidic donors or
putative catalytic residue. Either the conservative (by Glu) polyprenol-phosphate that limits polysaccharide produc-
or nonconservative (by Ala) replacement of Asp-191 led to tion. Kinetic data of ORU assembly (glycosyltransferases)
the inactivation of the activity, which is consistent with a as well as estimation of the number of biosynthetic com-
direct role in catalysis. In the case of AceA, the EX7E plexes per cell are needed to assess this point.
motif is located in the COOH terminal domain (Glu-286 Concerning sugar substitution in the polysaccharide,
and Glu-295) [29]; some conserved residues are present in we need to consider that it is necessary to change the
the NH2 domain. Replacement of Asp-109, His-127, and specicity of one or several glycosyltransferases, and prob-
Ser-162 by Ala resulted in an important decrease in the ably the polymerase. Actual knowledge on the atomic basis
enzymatic activity. Replacement of the second Glu of the of sugar recognition is scarce, and more structures of
EX7E motif, Glu-295 by Ala, resulted in a protein with glycosyltransferases in complex with their substrates are
barely detectable activity. Finally, replacement of either needed. Regarding polymerization, to date, the specicity
Lys-211 or the rst Glu of the motif, Glu-286, resulted in of this reaction has not been biochemically assessed.
a complete abolition of the mannosyltransferase activity Finally, controlling the size of the polysaccharide
[33]. Interestingly, it has been found that EX7E motif is seems to be the most attractive and dicult task, mainly
also present in an unrelated glycosyltransferase: human when trying to reduce the size of the physiologically
glycogen synthase (HMGS). Amino acid replacement produced polysaccharide. This issue is directly related to
experiments showed the same biochemical phenotype as the polymerization reaction that, as was pointed out
AceA, namely, replacement of the rst Glu completely before, needs to be biochemically assessed.
inactivates the enzyme, whereas replacement of the second
Glu dramatically decreases the glycogen synthase activity
[34]. By both comparison of the predicted secondary struc- III. STRUCTURE AND PHYSICAL PROPERTIES
ture and fold recognition, it was found that both AceA and OF SOME BACTERIAL POLYSACCHARIDES
the HMGS are related to the very distant glycosyltrans-
ferases MurG and T4-h-glucosyltransferase [33,35]. Al- In the following, the characteristics of some important
though the latter enzymes do not possess a typical industrial polysaccharides will be examined and summa-
rized. The example of hyaluronan is not described because

a separate chapter is included in this book and it has been
described abundantly recently in the literature [37].
On a general point of view, polysaccharides are espe-
cially important in the domain of water-soluble polymers;
they play an important role as thickening, gelling, emulsi-
fying, hydrating, and suspending polymers. Especially
important is the fact that some polysaccharides give phys-
ical gels in well-dened thermodynamic conditions. It
constitutes a very important class of materials in food,
cosmetics, or pharmaceutical applications.
In our studies, we have developed specic methods for
the purication of polysaccharides that are usually isolated
under their sodium salt forms by precipitation from aque-
ous solution with ethanol or isopropanol. This step is the
most important one to get reproducible experimental
characteristics. In fact, due to the presence of many OH Figure 4 Proton NMR spectrum of a succinoglycan from R.
groups in the molecule, the polysaccharides have a tend- meliloti, strain M5N1 (C = 5 g/L in D20 at 85jC) in the
ency to form cooperative intrachain and interchain H- presence of an internal standard (5 mM sodium acetate).
bonds, causing some insolubility or at least the presence d ppm is the chemical shift from TMS. (From Ref. 43. n
of aggregates when solutions are prepared. Due to their Elsevier Science, 2003.)
stereoregularity, they are often able to form helical con-
formations in solution; their ordered conformation has a
semirigid character and its stability depends on tempera- The content in the substituent is easily determined by
1
ture and ionic concentration if the polysaccharide structure H nuclear magnetic resonance (NMR) in the presence of
contains uronic acid unit or ionic substituents [38]. an internal standard to calibrate the signals and to get a
Details on the chemical structure of bacterial polysac- quantitative determination of the substituent contents
charides were given previously by Kenne and Lindberg [39], (Fig. 4).
Dutton [40], and Kennedy et al. [41]. In the following, the The molecular weight distribution is obtained by steric
main industrial bacterial polysaccharides will be described. exclusion chromatography (SEC) using a multidetector
equipment. The average molecular weight was found
A. Succinoglycans around 106 g/mol or more. The intrinsic viscosity in 0.1
M NaCl is high due to the stiness of the molecule in the
Succinoglycans are produced by many soil bacteria of the ordered (helical) conformation; an example is [g] = 13,600
genus Pseudomonas, Rhizobium, Agrobacterium, and Alca- mL/g when Mw = 4.2 106 g/mol [46]. Nevertheless, an
ligenes. These polysaccharides have a general chemical original behavior is observed: from native state, heating
structure based on a height sugar repeat unit (D-glucose/ causes a conformational transition to a exible coil (with a
D-galactode ratio, 7:1); this structure is given in Fig. 3. much lower viscosity), but on decreasing temperature, it
Dierent substituents are also present in the molecule such goes to a viscosity lower than the initial one [4648]. This
as acetyl, succinyl, and pyruvyl groups, depending on the point will be discussed later in relation with chain associ-
strain and conditions of fermentation or isolation [4245]. ation [49,50].
It was rst produced on a large scale by Shell Cy (UK)
from the strain Pseudomonas sp. NCIB 11592 and later by
Rhodia (France) from Agrobacterium tumefaciens I-736
under the trade name RheozanR.
As with many bacterial polysaccharides, succinogly-
can, whatever its origin, is water-soluble and, being stereo-
regular, adopts a single-chain helical conformation at least
in dilute solution; this ordered conformation was demon-
strated from the existence of a conformational transition
induced by temperature change using optical rotation,
NMR, calorimetry, viscosity, and conductivity [47,51].
As shown by the dierent techniques, the conformational
transition is reversible and very cooperative at least in the
presence of some salt excess (Fig. 5). The stability of the
ordered conformation depends on the salt concentration in
solution as is usual for charged polysaccharides. On both
sides of the transition, the stiness is completely dierent:
Figure 3 Chemical structure of a succinoglycan. in the helical conformation, the chain behaves as a semi-
Figure 5 Inuence of the temperature on the specic optical rotation [a] of succinoglycan at 1 g/L. Curve 1, no added salt; curve
2, 0.1 M NaCl. (From Ref. 47. n Elsevier Science, 2003.)
rigid chain having an intrinsic persistence length (Lp) Succinoglycan is produced by Rhodia and commer-
estimated as 35 nm, becoming more exible at higher cialized under the trade name Rheozan. It has a thickening
temperatures in the coiled conformation, with Lpf5 nm. behavior as that of xanthan, with some specic uses in
The persistence length in the helical conformation was relation with its larger stability in acidic conditions. It is a
conrmed by atomic force microscopy (AFM) from the thickener used in detergents, toilet bowl cleaners, etc.
analysis of chain curvature [52].
The rheological behavior in dilute and semidilute so- B. Xanthan
lution is quite usual and typical of viscoelastic systems [48].
In dilute aqueous solutions, in the absence of external Xanthan was the rst bacterial polysaccharide produced by
salt, a polyelectrolytic specic behavior was observed in the strain X. campestris and developed on a large scale. It
light and/or neutron scattering experiments: a maximum was rst commercialized by Kelco Cy (USA) and it is now
( qmax) in the scattered intensity is observed as a function of available under the trade names KeltrolR for food appli-
the wave vector (q). This involves a correlation between cations, KeloR for feed-grade xanthan, or KelzanR for
charged chain neighbors due to electrostatic repulsion industrial uses; Rhodia also developed production under
[53,54]; qmax was shown to vary with the polymer concen- the name RhodopolR.
tration Cp as Cp1/2 in agreement with a hexagonal packing The chemical structure of xanthan is recalled in Fig. 8;
of the ionic chains (Fig. 6). In semidilute solution, after a it was conrmed by NMR: the 13C spectrum allows to
rst conformational transition, succinoglycan restores the identify the dierent sugar units as well as the substituents
ordered conformation in a helical single chain up to a [56]. 1H allows to quantify easily the yield in substituents
critical polymer concentration around 5 g/L. Then, for (Fig. 9). Xanthan is an ionic polysaccharide for which the
higher polymer concentration, and favored by the chain polyelectrolytic character was investigated in detail [57
stiness, a double helical structure was suggested from the 59]. In addition, an ordered conformation is stabilized in
distance of packing of the chains [54] (Fig. 7) but also from solution but its stability depends on the temperature and
apparent molecular weight determination [50]. This was ionic concentration. The xanthan conformation was abun-
recently conrmed by Kido et al. [55]. dantly discussed in the literature: the debate was between a
Figure 6 Eect of polymer concentration (Cp) on light scattering intensity I plotted as I( q)/kCp vs. q in salt-free solution.
Succinoglycan concentration: (E) 1.59 105 g/cm3; (n) 2.38 105 g/cm3; (.) 3.03 105 g/cm3; (5) 3.84 105 g/cm3; (o)
4.53 105 g/cm3. q is the scattering wave vector. qm represents the position of the maximum in elastic intensity [A1]. (From
Ref. 54. n American Chemical Society, 2003.)
Figure 7 Variation of log( qm) as a function of log[Cp/ML] for all the polymer concentrations tested by light scattering as well as
by small-angle neutron scattering (SANS) (o). The two lines correspond to hexagonal packing for single-chain (low
concentration) and double chain (double linear mass density ML; higher concentration). For comparison, NaPSS results (.) are
also represented. (From Ref. 54. n American Chemical Society, 2003.)
Figure 8 Chemical structure of xanthan.
single or a double helix in aqueous solution. The data In the native state, as it is produced by bacteria, and in
obtained in our group will be exposed later in this chapter, the absence of drying or thermal treatment, xanthan is a
but it must be mentioned that after thermal treatment, single extended chain under helical conformation as shown
many commercial xanthans provide much aggregation in by transmission electron microscopy (TEM) [60]. In the
dilute or semidilute aqueous solutions, causing diculties solid state, it was demonstrated that the conformation is a
in the interpretation of physical behavior (especially from 51 helix [61]. Depending on the conditions of fermentation
rheology and light scattering experiments). posttreatment, dierent yields in acetyl and pyruvyl sub-
stituents exist on the side chain; it was demonstrated that
the substituents have no fundamental role on the main
properties of xanthan in solution [62,63]. For that purpose,
a series of samples having dierent yields in acetyl and
pyruvate substituents and dierent molecular weights was
prepared; it was demonstrated that all the samples behave
equally in a plot relating intrinsic viscosity/molar mass in
salt excess (Fig. 10). In Table 1, we give the temperature of
conformational change Tm for native xanthan and modi-
ed xanthan to evidence the role of the substituents on the
Figure 9 Proton NMR spectra of (A) partially depolymer- Figure 10 Representation of the intrinsic viscosity depend-
ized xanthan and (B) a modied xanthan in D2O (C = 5 mg/ ence as a function of the weight average molecular weight of
mL, T = 90 jC). (From Ref. 56. n American Chemical dierent modied xanthans. (From Ref. 62. n Elsevier
Society, 2003.) Science, 2003.)
Table 1 Temperatures for Conformational Change tion of microgels necessary for application in tertiary oil
as a Function of the Chemical Structure of Xanthan recovery was developed [81,82].
The thermal stability was also investigated in relation
Samplesa Tm (jC)
with the oil recovery process [8386]. At 90jC, a decrease
Initial xanthan 76 of the viscosity and molar masses associated with the
Acetyl free xanthan 65 production of oligomers and a decrease in the substituent
Pyruvyl free xanthan 82 contents were observed. The ordered conformation is also
Pyruvyl and acetyl free xanthan 72.5 more stable in this thermal degradation mechanism; some
pieces of evidence of free radical mechanism of degradation
a
Polymer concentration, 1 g/L; solvent, 4 102 M NaCl. were given.
Source: Ref. 62.
The mechanism of conformational transition is much
less cooperative for xanthan than for succinoglycan, but
the exact origin of this dierence is not yet established [87].
conformation [62]. Taking o the pyruvate-charged group As for succinoglycan, neutron scattering also demons-
stabilizes the helical conformation whereas taking o the trated that at higher concentration, a double-stranded
acetyl group destabilizes the helix. chain, at least locally, is formed [88,89].
But the substituents have a role in interactions such as Nevertheless, the rheological behavior of xanthan is
galactomannan/xanthan-specic interactions, as we have recognized as remarkable in relation with its local rigidity
demonstrated previously [6469]; the interaction with ga- [9093]. Even with a xanthan having a moderate molecular
lactomannan is stronger at low temperature (20jC) when weight (Mw around 106 g/mol), a non-Newtonian behavior
xanthan is under coiled conformation and deacetylated. of viscosity is observed in a wide range of polymer concen-
Xanthan is a semirigid polymer for which, in the native tration at 25jC in 0.1 M NaCl to stabilize the helical
conformation, a persistence length of around 40 nm is conformation. The rheological behavior of xanthan was
found [7072]; it was demonstrated that when the native investigated by many authors. In our work, we studied the
form is heated to coil conformation (with a much lower role of polymer concentration, shear rate in ow experi-
stiness) and renatured by cooling, the conformation is ments (Fig. 12), and frequency in dynamic experiments
changed [7376]. This can be represented as follows: (Fig. 13) [91,93]. It was shown that at zero shear rate, the
specic viscosity is given by the relation:
Native xanthan ! Denatured xanthan X Renaturated xanthan
gsp c!0 Cg kH Cg2 BCgn 2
Single helix Irreversible Coil Reversible Double helix
This renaturated conformation obtained in dilute where kH = 0.44, B = 1.73 102, and n = 4.24.
solution is stier (Lp = 160 nm) but shorter, the viscosity
being usually higher than that of the native form. At a given
ionic concentration, the helical conformation is stabilized
at low temperature; when temperature increases, a confor-
mational change occurs at a characteristic temperature Tm
(determined for 50% of transition). The value of Tm can be
predicted as a function of the total ionic concentration CT,
taking into account the external salt and the polyelectrolyte
concentrations, from the following relation [67]:
T m1 2:16 103 0:5 103 logCT 1

Due to the intrinsic stiness of the xanthan chain, we
have shown that it gives liquid crystalline phase in solution;
it was identied as a right-handed cholesteric [77,78]. This
ordering in solution appears in native xanthan solution at
relatively low concentration (i.e., birefringence appears
over 3 g/L). At the same time, a characteristic evolution
of viscosity was observed with a drop in viscosity when
entering the biphasic domain (Fig. 11). It was clearly
demonstrated that the helical conformation is needed to
observe the cholesteric organization. The conformation
was also shown to be important in the enzymic degradation
of xanthan; it was shown that the rate of viscosity decreases
under helical conformation compared with the coiled
conformation [79,80]. These results were obtained with a Figure 11 Reduced viscosity and volume fraction of the
commercial cellulase able to hydrolyze the h(1!4)D-glu- cholesteric phase as a function of xanthan concentration (Mw
can chains. Then, combining a glycanase with a protease, a = 235,000; T = 25jC; solvent, 0.1 M NaCl; shear rate, 1
process for the clarication of xanthan broth and elimina- sec1). (From Ref. 78. n Springer, 2003.)
Figure 12 Dependence of the relative viscosity as a function of the shear rate and polymer concentration for xanthan in the
helical conformation (Mw = 107; T = 25jC; solvent, 0.1 M NaCl). (From Ref. 91. n Springer, 2003.)
Figure 14 Dependence of the intrinsic viscosity as a

Figure 13 GV(x) and GW(x)(x) as a function of the function of the molar mass for xanthan () and sodium
frequency x for dierent polymer concentrations in 0.1 M polystyrene sulfonate () at 25jC (a) in 102 M NaCl (b)
NaCl at 25jC. (From Ref. 93. n American Chemical Society, values extrapolated to innite salt concentration (l). (From
2003.) Ref. 90.)
It was also established the MarkHouwink relation at other dierent biopolymers such as carrageenans, algi-
zero shear rate is: nates, pectins, gelatins, galactomannans, etc.
g mL=g 1:7 104 M1:14 3

C. Gellan and Gellan Family
in 0.1 M NaCl at 25jC [91]. A series of bacterial polysaccharides having a similar main
The role of the stiness of the xanthan chain is clearly chain based on a four-sugar repeat unit was developed
demonstrated in Fig. 14: The intrinsic viscosity is nearly (Fig. 15). Their respective names are gellan [95,96], rham-
independent of the ionic concentration in the opposite of san [97], welan [98,99], S-88, S-198, and S-657 [100],
the exible polyelectrolyte Na-polystyrene sulfonate [90]; introduced by Kelco Cy [101]; we have compared the
the stiness was also analyzed in free diusion process [94]. conformation and thermal stability of four of them in the
Xanthan has now many industrial applications due to native state or after deacetylation [102]. The conclusions
its suspending character (stabilizer of solid suspensions or are given in Table 2.
foams); pseudoplastic and thickening properties; good In fact, we identied a new polysaccharide, called
stability of rheological properties as a function of pH, RMDP17, produced by the bacteria Sphingomonas pauci-
temperature, and salt concentration; and its ability to be bilis strain I-886, having a structure very similar to that of
crosslinked and gelled. It is used in paints and coatings, rhamsan. Their structures were determined and compared
cosmetics, ceramics, etc. [103,104] as well as their properties in solution [105].
Xanthan is accepted as a food additive by the United Welan is developed by Kelco Cy under the trademark
States Food and Drug Administration (U.S. FDA) regu- Kelco-CreteR, designed for use in cementitious systems. Its
lation and European authorities under reference E 415. In polyelectrolyte properties in solution were investigated,
this domain of applications, it is mainly used as stabilizer of particularly its suspending character [106109].
emulsion, and thickener and stabilizer of suspension. It has Gellan is the most important polymer in this series; it is
good stability during sterilization. It can be associated with produced by the bacteria Sphingomonas elodea [110,111].
Figure 15 Chemical structure of the repeat unit of gellan family polysaccharides.

Table 2 Possible Conformationa in Solution for the Dierent Polysaccharides
Gellan Welan Rhamsan S 657

b,c
Native (DH, SC) DH DH DH
Deacetylated DH, SCb DH DH, SCb DH
a
SC, single coil; DH, double helix.
b
Gel formation under certain conditions.
c
Aggregation prevents determination of the structure.
Source: Ref. 70.
The native gellan, as produced by the bacteria, contains temperature for conformational change Tm increases when
two additional substituents (acetyl and L-glyceryl) com- the salt concentration increases as usually for stereoregular
pared to the commercial polymer (Fig. 16). The native polyelectrolytes; the helical conformation is more stable in
gellan gives highly viscous solutions with a loose gel-like the presence of divalent counterions but no ionic selectivity
behavior. After deacylation, gellan gives a strong and rigid appears among monovalent counterions (Li, Na, K, and
gel; recently, a comparison between the native and deacyl- TMA) on one side and among divalent counterions (Ca,
ated gellan was developed [112,113]. The chemical struc- Mg) on the other side (Fig. 19) [111,114]. When the salt and
tures of the repeat units of the native and deacylated gellans polysaccharide concentrations increase, the double helices
are easily compared by 1H NMR, as shown in Fig. 17; in interact to give a physical gel with a strong ionic selectivity.
the native gellan, the signal at 2 ppm is attributed to acetyl The mechanism of gelation proposed is the same as that
substituents when the complex spectrum between 4 and 4.8 proposed previously for kappa carrageenans [114,115]. The
ppm occurs in the presence of L-glyceryl substituents. In the sequence of selectivity is:
deacylated gellan, the four H-1 signals are identied as well
as the CH3 of the rhamnose unit around 1.2 ppm. By K > Na > Li
microcalorimetry (Fig. 18), on decreasing temperature, the K+ promotes gel formation and gives the higher elas-
role of the localization and the presence of the substitu- tic modulus [114], as shown in Table 3 and Fig. 20. The role
ents on the thermal stability of the double helix of gellan of purication is clearly shown, with the commercial sam-
are demonstrated: ple of gellan containing few calcium counterions increasing
Taking o the acetyl groups does not modify the the stability.
stability of the double helix (but favors double Gellan in the deacylated form is the mainly used form;
helix interaction with gel formation; the methyl it was rst commercialized in Japan in 1988 and in the
groups are outside the double helices). United States in 1993; GelriteR produced by Kelco Cy rst
Hydrolysis of the glyceryl groups destabilizes the competed with agarose for microbiological applications
double helix, decreasing the temperature of but gives a clear gel that sets faster than agarose. KelcogelR
conformational change at the same time as the is the gellan grade proposed for industrial or food uses. A
enthalpy of conformational change; the glyceryl new product commercialized by Kelco Cy in 1998 is
groups interact inside the double helix. Kelcogel LT100, a high-acyl gellan giving soft and elastic
thermoreversible gels used for food applications: dressings,
Deacylated gellan adopts an ordered conformation in jellies, milk puddings, dairy and food beverages, etc. Gellan
double helix in salt excess as mentioned in Table 2. The also presents synergism with other polymers such as gelatin
Figure 16 Structure of native gellan (a) and deacylated gellan (b).

Figure 17 1H NMR spectra of (a) native gellan and (b) deacetylated gellan. Solution at 30 g/L in D2O at 85jC (a) and 75jC (b),
respectively. (From Ref. 113. n Elsevier Science, 2003.)
or gum arabic. A series of papers on gellan was published A very specic polysaccharide was obtained from
recently [116]. Alteromonas sp. strain 1644 bacteria originating from
deep-sea hydrothermal vents; these bacteria produce two
dierent polysaccharides (one is an exopolysaccharide
D. Other Polysaccharides excreted in the medium and the other remains self-associ-
ated with the bacteria), which are isolated depending on
Many bacterial polysaccharides are described in the liter- experimental conditions. They were named polysaccharide
ature but only a few are industrially developed. In our 1644 and polysaccharide 1644 bis [117121]. The rst one
laboratory, new polysaccharides under development have has a chemical structure based on eighth sugars, ve being
been identied with valuable properties. in the main chain and three on a side chain as described
Figure 18 Dierential scanning calorimetry (DSC) scans on cooling at 0.3jC/min for (a) native gellan (b), deacetylated gellan,
and (c) deacylated gellan in 0.01 M NaCl. Polymer concentration, 10 g/L. (From Ref. 113. n Elsevier Science, 2003.)
before [118]; it is very rich in uronic acid (three uronic units) ment, it turns to a thermoreversible gelling polymer. The
and contains also a dicarboxylic sugar described by polysaccharide adopts an ordered conformation assumed
Dubreucq [122] and Dubreucq et al. [123]. It strongly xes to be a double helix in the native state; the gel forms after
Ca2+ counterions and gives very elastic gel. The physical partial denaturation and the solgel transition is located in
properties of this gel are completely dierent from alginate the range 6070jC, depending on the thermal treatment.
or pectins rigid gels. These characteristics have been de- The polysaccharide contains some acetyl groups as sub-
scribed previously. The 1644 bis polysaccharide has a com- stituents, which are needed for gelation. Completely deacet-
pletely dierent structure and no dicarboxylic unit [120]. ylated YAS 34 exists only as a random coil and never forms
A new polymer produced by a strain from sunower a gel. A mutation was realized on a R. meliloti strain; this
rhizosphere is under development by Soliance Cy (France) strain is named R. meliloti NCIMB 40472. It produces a
under the tradename Soligel. It is produced by a Rhizobi- new polysaccharide by reference to the normal succinogly-
um-type strain and named YAS 34 [124128]. Its specic can; its structure and properties were investigated. It is a
property is that it is a thickening polysaccharide under high-molecular-weight polyglucuronic acid forming strong
native conformation but, after controlled thermal treat- gels in the presence of Ca2+ [129135].
Figure 19 Dependence of the inverse of the temperature of

conformational change (Tm1) as a function of the activity of
monovalent and divalent counterions (in log scale). (+) Na-
gellan in NaCl; (o) K-gellan in KCl; (D) TMA-gellan in
TMACl; (E) Li-gellan in LiCl; (5) Ca-gellan in CaCl2; (n)
Mg-gellan in MgCl2. (From Ref. 113. n Elsevier Science,
2003.) Figure 20 Elastic modulus of deacylated gellan as a
function of polymer concentration at constant salt concen-
tration (0.1M) under dierent ionic forms. (.) Commercial
K-gellan in KCl; () K-gellan puried in KCl; (o) Na-gellan
The physical properties of two commercial polysac- in NaCl. (From Ref. 114. n Elsevier Science, 2003.)
charides known as FucogelR [136] and RhamnosoftR [137]
and developed for cosmetic applications by Solabia Cy
(France) were recently investigated in our group. Fucogel is this condition, a gel is obtained when the polymer concen-
produced by a Klebsiella pneumoniae-type strain and has a tration is larger than 1 g/L.
chemical structure very near that from Klebsiella-type
bacteria I-156, K63, and K42, described previously in the
literature [138140]. The structures are given in Fig. 21. No IV. CONCLUSION
ordered conformation can be demonstrated in solution and
Fucogel is only a moderate thickening polymer as well as As was described in this chapter, many polysaccharides
Rhamnosoft [141,142]. Another polymer, BEC1615, is from bacterial sources can be identied as having original
produced by the same Klebsiella-type strain as that pro- chemical structures, produced with dierent yields in sub-
ducing K54 described by Dutton and Merrield [143] and stituents and dierent molecular weights. Then, a large
the polysaccharide XM6 [144]. This polysaccharide is variety of physical properties are found, allowing extended
partially acetylated; after deacetylation, it turns to a ther- applications of polysaccharides in many industrial
moreversible gelling system associated with the existence of domains. They are recognized not only for their functional
an ordered conformation shown by optical rotation, calo- properties but also for their biological activities. All these
rimetry, or rheology. The temperature for thermal confor-
mational transition becomes independent of the ionic
concentration (Tm c 35jC) when the salt concentration
is larger than 0.1 M without any ionic selectivity [141]; in
Table 3 Physical Properties of Gellan Gel Under Dierent

Ionic Formsa
K+ Na+ Li+
E 104 (Pa) 31.5 11.9 0.44
Fm (N) 23.1 13.3
Figure 21 Chemical structure of the repeat unit of (a)
a
E is the modulus, Fm is the breaking force; the gels are formed at a Fucogel, (b) Rhamnosoft, and (c) BEC1615. (From Ref.
polymer concentration Cp = 13g/L in the presence of 0.1 M XCl. 141.)
polymers are water-soluble, usually biocompatible; they 13. Vanderslice, R.W.; Doherty, D.H.; Capage, M.A.; Bet-
are often used to increase the viscosity of the solvent and, in lach, M.R.; Hassler, R.A.; Henderson, N.M.; Ryan-
Graniero, J.; Tecklenburg, M. Genetic engineering of
some cases, to form physical gels stabilized by divalent
polysaccharide structure, Xanthomonas campestris. In
counterions or H-bonds. Biomedical and Biotechnological Advances in Industrial
Bacterial engineering will allow, in the future, to better Polysaccharides; Crescenzi, V., Dea, I.C.M., Paoletti, S.,
control the chemical structure and physical properties. Sutherland, I.W., Eds.; Gordon and Breach Science
Publ.: New York, 1988; 145157.
14. Becker, A.; Ruberg, S.; Baumgarth, B.; Bertram-Drogatz,
P.A.; Quester, L.; Puhler, A.; Niemeyer, D.; Sharypova,
ACKNOWLEDGMENT L.A.; Yurgel, S.N.; Keller, M.; Simarov, B.V.; Quester, I.;
The cooperation of M. Milas in our experimental work on Kuster, H.; Niehaus, K. Regulation of succinoglycan and
galactoglucan biosynthesis in Sinorhizobium meliloti. J.
bacterial polysaccharides is sincerely recognized. Mol. Microbiol. Biotechnol. 2002, 4, 187190.
15. Becker, A.; Kleickmann, A.; Arnold, W.; Puhler, A.
Analysis of the Rhizobium meliloti exoH/exoK/exoL
REFERENCES fragment: ExoK shows homology to excreted endo-beta-
1,3-1,4-glucanases and ExoH resembles membrane pro-
1. Sutherland, I.W. Microbial polysaccharides. Biotechno- teins. Mol. Gen. Genet. 1993, 238, 145154.
logical products of current and future potential. 16. Becker, A.; Kleickmann, A.; Kuster, H.; Keller, M.;
In Biomedical and Biotechnological Advances in Industrial Arnold, W.; Puhler, A. Analysis of the Rhizobium meliloti
Polysaccharides; Crescenzi, V., Dea, I.C.M., Paoletti, S., genes exoU, exoV, exoW, exoT, and exoI involved in
Stivala, S.S., Sutherland, I.W., Eds.; Gordon and Breach exopolysaccharide biosynthesis and nodule invasion:
Science Publ.: New York, USA, 1989; 123132. exoU and exoW probably encode glucosyltransferases.
2. Yalpani, M.; Sanford, P.A. Commercial polysaccharides: Mol. PlantMicrob. Interact. 1993, 6, 735744.
recent trends and developments. In Industrial Polysaccha- 17. Becker, A.; Kleickmann, A.; Keller, M.W.A.; Puhler, A.
rides: Genetic Engineering, Structure/Property Relations Identication and analysis of the Rhizobium meliloti
and Applications; Yalpani, M., Ed.; Elsevier: Amsterdam, exoAMONP genes involved in exopolysaccharide biosyn-
1987; 311335. thesis and mapping of promoters located on the exoH-
3. Jann, K.; Jann, B. Bacterial polysaccharides related to KLAMONP fragment. Mol. Gen. Genet. 1993, 241, 367
mammalian structures. In Polysaccharides. Structural 379.
Diversity and Functional Versatility; Dimitriu, S., Ed.; 18. Glucksmann, M.A.; Reuber, T.L.; Walker, G.C. Family
Marcel Dekker, Inc.: New York, 1998; 211235. of glycosyl transferases needed for the synthesis of
4. Morin, A. Screening of polysaccharide-producing micro- succinoglycan by Rhizobium meliloti. J. Bacteriol. 1993,
organisms, factors inuencing the production, and recov- 175, 70337044.
ery of microbial polysaccharides. In Polysaccharides. 19. Glucksmann, M.A.; Reuber, T.L.; Walker, G.C. Genes
Structural Diversity and Functional Versatility; Dimitriu, needed for the modication, polymerisation, export, and
S., Ed.; Marcel Dekker, Inc.: New York, 1998; 275296. processing of succinoglycan by Rhizobium meliloti: a
5. Morris, V.J. Bacterial polysaccharides. In Food Polysac- model for succinoglycan biosynthesis. J. Bacteriol. 1993,
charides and Their Applications; Stephen, A.M., Ed.; 175, 70457055.
Marcel Dekker, Inc.: New York, USA, 1995; 341375. 20. Becker, A.; Kuster, H.; Niehaus, K.; Puhler, A. Extension
6. Leigh, J.A.; Lee, C.C. Characterisation of polysaccharides of the Rhizobium meliloti succinoglycan biosynthesis gene
of Rhizobium meliloti exo mutants that form ineective cluster: identication of the exsA gene encoding an ABC
nodules. J. Bacteriol. 1988, 170, 33273332. transporter protein, and the exsB gene which probably
7. Katzen, F.; Ferreiro, D.U.; Oddo, C.G.; Ielmini, M.V.; codes for a regulator of succinoglycan biosynthesis. Mol.
Becker, A.; Puhler, A.; Ielpi, L. Xanthomonas campestris pv. Gen. Genet. 1995, 249, 487497.
campestris gum mutants: eects on xanthan biosynthesis 21. York, G.M.; Walker, G.C. The Rhizobium meliloti ExoK
and plant virulence. J. Bacteriol. 1998, 180, 16071617. and ExsH glycanases specically depolymerize nascent
8. Whiteld, C. Biosynthesis of lipopolysacchairde O anti- succinoglycan chains. Proc. Natl. Acad. Sci. USA. 1998,
gens. Trends Microbiol. 1995, 3, 178185. 95, 49124917.
9. Tolmasky, M.E.; Staneloni, R.J.; Leloir, L.F. Lipid- 22. Niemeyer, D.; Becker, A. The molecular weight distribu-
bound saccharides in Rhizobium meliloti. J. Biol. Chem. tion of succinoglycan produced by Sinorhizobium meliloti
1982, 257, 67516757. is inuenced by specic tyrosine phosphorylation and
10. Ielpi, L.; Couso, R.O.; Dankert, M.A. Sequential ATPase activity of the cytoplasmic domain of the ExoP
assembly and polymerisation of the polyprenol-linked protein. J. Bacteriol., 2001; 51635170.
pentasaccharide repeating unit of the xanthan polysac- 23. Becker, A.; Puhler, A. Specic amino acid substitutions in
charide in Xanthomonas campestris. J. Bacteriol. 1993, the proline-rich motif of the Rhizobium meliloti ExoP
175, 24902500. protein result in enhanced production of low-molecular-
11. Pollock, T.J.; van Workum, W.A.; Thorne, L.; Mikolajc- weight succinoglycan at the expense of high-molecular-
zak, M.J.; Yamazaki, M.; Kijne, J.W.; Armentrout, R.W. weight succinoglycan. J. Bacteriol. 1998, 180, 395399.
Assignment of biochemical functions to glycosyl trans- 24. Reuber, T.L.; Walker, G.C. Biosynthesis of succinogly-
ferase genes which are essential for biosynthesis of can, a symbiotically important exopolysaccharide of
exopolysaccharides in Sphingomonas strain S88 and Rhizobium meliloti. Cell 1993, 74, 269280.
Rhizobium leguminosarum. J. Bacteriol. 1998, 180, 586593. 25. Lellouch, A.C.; Geremia, R.A. Expression and study of
12. Yamazaki, M.; Thorne, L.; Mikolajczak, M.; Armentrout, recombinant ExoM, a beta 14 glucosyltransferase
R.W.; Pollock, T.J. Linkage of genes essential for involved in succinoglycan biosynthesis in Sinorhizobium
synthesis of a polysaccharide capsule in Sphingomonas meliloti. J. Bacteriol. 1999, 181, 11411148.
strain S88. J. Bacteriol. 1996, 178, 26762687. 26. Videira,P.;Fialho,A.;Geremia,R.A.;Breton,C.;Sa-Correia,
I. Biochemical characterisation of the beta-1,4-glucuronosyl- 43. Heyraud, A.; Rinaudo, M.; Courtois, B. Comparative
transferase GelK in the gellan gum-producing strain Sphingo- studies of extracellular polysaccharide elaborated by
monas paucimobilis ATCC 31461. Biochem. J. 2001, 358, 457 Rhizobium meliloti strain M5N1 in dened medium and
464. in non-growing cell suspensions. Int. J. Biol. Macromol.
27. Geremia, R.A.; Roux, M.; Ferreiro, D.U.; Dauphin- 1986, 8, 8588.
Dubois, R.; Lellouch, A.C.; Ielpi, L. Expression and 44. Hebbar, K.P.; Gueniot, B.; Heyraud, A.; Colin-Morel, P.;
biochemical characterisation of recombinant AceA, a Heulin, T.; Balandreau, J.; Rinaudo, M. Characterization
bacterial alpha-mannosyltransferase. Mol. Gen. Genet. of exopolysaccharides produced by rhizobacteria. Appl.
1999, 261, 933940. Microbiol. Biotechnol. 1992, 38, 248253.
28. Saxena, I.M.; Brown, R.M. Jr.; Fevre, M.; Geremia, R.A.; 45. Courtois, B.; Courtois, J.; Heyraud, A.; Rinaudo, M.
Henrissat, B. Multidomain architecture of beta-glycosyl Eect of biosynthesis conditions on the chemical compo-
transferases: implications for mechanism of action. J. sition of the water-soluble polysaccharides of fast-growing
Bacteriol. 1995, 177, 14191424. rhizobium. J. Gen. Appl. Microbiol. 1986, 32, 519526.
29. Geremia, R.A.; Petroni, E.A.; Ielpi, L.; Henrissat, B. 46. Gravanis, G. Relation entre la structure moleculaire et les
Towards a classication of glycosyltransferases based on proprietes en solution dun polysaccharide hydrosoluble.
amino acid sequence similarities: prokaryotic alpha- Ph.D. Thesis; France: University of Grenoble, 1985.
mannosyltransferases. Biochem. J. 1996, 318, 133138. 47. Gravanis, G.; Milas, M.; Rinaudo, M.; Clarke-Sturman,
30. Breton, C.; Bettler, E.; Joziasse, D.H.; Geremia, R.A.; A.J. Conformational transition and polyelectrolyte be-
Imberty, A. Sequencefunction relationships of prokary- haviour of a succinoglycan polysaccharide. Int. J. Biol.
otic and eukaryotic galactosyltransferases. J. Biochem. Macromol. 1990, 12, 195200.
(Tokyo) 1998, 123, 9991000. 48. Gravanis, G.; Milas, M.; Rinaudo, M.; Clarke-Sturman,
31. Garinot-Schneider, C.; Lellouch, A.C.; Geremia, R.A. A.J. Rheological behaviour of a succinoglycan polysac-
Identication of essential amino acid residues in the charide in dilute and semi-dilute solutions. Int. J. Biol.
Sinorhizobium meliloti glucosyltransferase ExoM. J. Biol. Macromol. 1990, 12, 201206.
Chem. 2000, 275, 3140731413. 49. Boutebba, A. Proprietes du succinoglycane: transition
32. Charnock, S.J.; Davies, G.J. Structure of the nucleotide- conformationnelle et gelication en milieu aqueux. Ph.D.
diphospho-sugar transferase, SpsA from Bacillus subtilis, Thesis; University of Grenoble: France, 1998.
in native and nucleotide-complexed forms. Biochemistry 50. Boutebba, A.; Milas, M.; Rinaudo, M. On the interchain
1999, 38, 63806385. associations in aqueous solutions of a succinoglycan
33. Abdian, P.L.; Lellouch, A.C.; Gautier, C.; Ielpi, L.; polysaccharide. Int. J. Biol. Macromol. 1999, 24, 319327.
Geremia, R.A. Identication of essential amino acids in 51. Boutebba, A.; Milas, M.; Rinaudo, M. Orderdisorder
the bacterial alpha-mannosyltransferase aceA. J. Biol. conformational transition in succinoglycan: calorimetric
Chem. 2000, 275, 4056840575. measurements. Biopolymers 1997, 42, 811819.
34. Cid, E.; Gomis, R.R.; Geremia, R.A.; Guinovart, J.J.; 52. Balnois, E.; Stoll, S.; Wilkinson, K.J.; Bue, J.; Rinaudo,
Ferrer, J.C. Identication of two essential glutamic acid M.; Milas, M. Conformations of succinoglycan as
residues in glycogen synthase. J. Biol. Chem. 2000, 275, observed by atomic force microscopy. Macromolecules
3361433621. 2000, 33, 74407447.
35. Cid, E.; Geremia, R.A.; Guinovart, J.J.; Ferrer, J.C. 53. Morn, I.; Reed, W.F.; Rinaudo, M.; Borsali, R. Further
Glycogen synthase: towards a minimum catalytic unit? evidence of liquid-like correlations in polyelectrolyte
FEBS Lett. 2002, 528, 511. solutions. J. Phys. II 1994, 4, 10011019.
36. Fialho, A.M.; Martins, L.O.; Donval, M.L.; Leitao, J.H.; 54. Borsali, R.; Rinaudo, M.; Noirez, L. Light scattering and
Ridout, M.J.; Jay, A.J.; Morris, V.J.; Sa-Correia, I. small-angle neutron scattering from polyelectrolyte solutions:
Structures and properties of gellan polymers produced by the succinoglycan. Macromolecules 1995, 28, 10851088.
Sphingomonas paucimobilis ATCC 31461 from lactose 55. Kido, S.; Nakanishi, T.; Norisuye, T. Ordered conforma-
compared with those produced from glucose and from tion of succinoglycan in aqueous sodium chloride.
cheese whey. Appl. Environ. Microbiol. 1999, 65, 24852491. Biomacromolecules 2001, 2, 952957.
37. Milas, M.; Rinaudo, M. On the electrostatic interactions 56. Rinaudo, M.; Milas, M.; Lambert, F.; Vincendon, M. 1H
of ionic polysaccharides in solution. Curr. Trends Polym. and 13C NMR investigation of xanthan gum. Macro-
Sci. 1997, 2, 4767. molecules 1983, 16, 816819.
38. Lindberg, B. Bacterial polysaccharides: components. In 57. Rinaudo, M.; Milas, M. Polyelectrolyte behaviour of
Polysaccharides. Structural Diversity and Functional Ver- bacterial polysaccharide from Xanthomonas campestris.
satility; Dimitriu, S., Ed.; Marcel Dekker, Inc.: New Comparison with carboxymethylcellulose. Biopolymers
York, 1998; 237273. 1978, 17, 26632678.
39. Kenne, L.; Lindberg, B. Bacterial polysaccharides. In The 58. Rinaudo, M.; Milas, M. Xanthan properties in aqueous
Polysaccharides; Aspinall, G.O., Ed.; Academic Press: solution. Carbohydr. Polym. 1982, 2, 264269.
New York, 1983; Vol. 2, 287363. 59. Lambert, F.; Milas, M.; Rinaudo, M. Sodium and
40. Dutton, G.G.S. Polysaccharides Encyclopedia of Polymer calcium counterion activity in presence of xanthan
Science and Technology, 2nd Ed.; Mark, H.F., Bikales, polysaccharide. Int. J. Biol. Macromol. 1985, 7, 4952.
N.M., Overberger, C.G., Menges, G., Eds.; Wiley 60. Milas, M.; Rinaudo, M.; Tinland, B.; de Murcia, G.
Interscience: New York, 1988; Vol. 13, 87147. Evidence for a single stranded xanthan chain by electron
41. Kennedy, J.F., Phillips, G.O., Williams, P.A., Eds,; microscopy. Polym. Bull. 1988, 19, 567572.
Hyaluronan, Chemical, Biomedical and Biological As- 61. Moorhouse, R.; Walkinshaw, M.D.; Arnott, S. Xanthan
pects; Vol. 1; Hyaluronan, Medical and Clinical Aspects; gummolecular conformation and interaction. Extrac-
Vol. 2; Woodhead Publishing, Ltd., 2002. ellular Microbial Polysaccharides; Sandford, P.A., Laskin,
42. Courtois, B.; Courtois, J.; Heyraud, A.; Rinaudo, M. A., Eds.; ACS Symposium Series, American Chemical
Eect of biosynthesis conditions on the chemical compo- Society (USA), 1997; Vol. 45, 90102.
sition of the water-soluble polysaccharides of fast-growing 62. Callet, F.; Milas, M.; Rinaudo, M. Inuence of acetyl
Rhizobium. J. Gen. Appl. Microbiol. 1986, 32, 519526. and pyruvate content on rheological properties xanthan
in dilute solution. Int. J. Biol. Macromol. 1987, 9, 291 83. Lambert, F. Caracterisation du xanthane en solution.
293. Application a` letude de la stabilite thermique. Ph.D.
63. Callet, F. Inuence de la structure, de la conformation et Thesis; University of Grenoble: France, 1983.
des traitements popst fermentation sur les proprietes du 84. Lambert, F.; Rinaudo, M. On the thermal stability of the
xanthane en solution. Ph.D. Thesis; University of xanthan gum. Polymer 1985, 26, 15491553.
Grenoble: France, 1987. 85. Callet, F.; Milas, M.; Rinaudo, M. On the role of thermal
64. Lopes, L.; Andrade, C.T.; Milas, M.; Rinaudo, M. Role treatments on the properties of xanthan solutions.
of conformation and acetylation of xanthan on xanthan Carbohydr. Polym. 1989, 11, 127137.
guar interaction. Carbohydr. Polym. 1992, 17, 121126. 86. Milas, M.; Linossier, J.L.; Contat, F. The eect of thermal
65. Bresolin, T.; Milas, M.; Rinaudo, M.; Ganter, M. Xanthan aging on xanthan solutions. J. Appl. Polym. Sci. 1988, 35,
galactomannan interactions as related to xanthan con- 11151122.
formations. Int. J. Biol. Macromol. 1998, 23, 263275. 87. Gravanis, G.; Milas, M.; Rinaudo, M.; Tinland, B. Com-
66. Bresolin, T.; Milas, M.; Rinaudo, M.; Reicher, F.; Ganter, parative behaviour of the bacterial polysaccharides xanthan
J. Role of galactomannan composition on the binary gel and succinoglycan. Carbohydr. Res. 1987, 160, 259265.
formation with xanthan. Int. J. Biol. Macromol. 1999, 26, 88. Milas, M.; Rinaudo, M.; Duplessix, R.; Borsali, R.;
225231. Lindner, P. Small angle neutron scattering from polyelec-
67. Rinaudo, M.; Milas, M.; Bresolin, T.; Ganter, J. Physical trolyte solutions: from disordered to ordered xanthan
properties of xanthan, galactomannan and their mixtures chain conformation. Macromolecules 1995, 28, 31193124.
in aqueous solution. Macromol. Symp. 1999, 140, 115124. 89. Milas, M.; Lindner, P.; Rinaudo, M.; Borsali, R. Inuence
68. Goycoolea, F.M.; Milas, M.; Rinaudo, M. Heterotypic of the shear rate on the small-angle neutron scattering
interactions of deacetylated xanthan with a galactomannan pattern of polyelectrolyte solutions: the xanthan example.
of high galactose substitution during synergistic gelation. Macromolecules 1996, 29, 473474.
In Gums and Stabilizers for the Food Industry; Phillips, G.O. 90. Tinland, B. Etude dun polysaccharide microbien: le xan-
Williams, P.A., Eds., RSC: Cambridge, 2000; 229240. thane, mode`le de molecules semi-rigides. Ph.D. Thesis,
69. Goycoolea, F.M.; Milas, M.; Rinaudo, M. Associative France, 1988.
phenomena in galactomannandeacetylated xanthan sys- 91. Milas, M.; Rinaudo, M.; Tinland, B. The viscosity
tem. Int. J. Biol. Macromol. 2001, 29, 181192. dependence on concentration, molecular weight and shear
70. Chazeau, L.; Milas, M.; Rinaudo, M. Conformations of rate of xanthan solutions. Polym. Bull. 1985, 14, 157164.
xanthan in solution: analysis by steric exclusion chroma- 92. Tinland, B.; Rinaudo, M. Dependence of the stiness of
tography. Int. J. Polym. Anal. Charact. 1995, 2, 2129. the xanthan chain on the external salt concentration.
71. Milas, M.; Reed, W.F.; Prinz, S. Conformations and Macromolecules 1989, 22, 18631865.
exibility of native and renatured xanthan in aqueous 93. Milas, M.; Rinaudo, M.; Knipper, M.; Schuppiser, J.L.
solutions. Int. J. Biol. Macromol. 1996, 18, 211221. Flow and viscoelastic properties of xanthan gum solu-
72. Rinaudo, M. Wormlike chain behaviour of some bacterial tions. Macromolecules 1990, 23, 25062511.
polysaccharides. In Macromolecules; Kahovec, J., Ed.; 94. Tinland, B.; Maret, G.; Rinaudo, M. Reptation in
VSP: Utrecht, The Netherlands, 1992; 207219. semidilute solutions of wormlike polymers. Macromole-
73. Milas, M.; Rinaudo, M. Conformational investigation on cules 1990, 23, 596602.
the bacterial polysaccharide xanthan. Carbohydr. Res. 95. Jansson, P.E.; Lindberg, B.; Sandford, P.A. Structural
1979, 76, 186196. studies of gellan gum, an extracellular polysaccharide
74. Milas, M.; Rinaudo, M. Investigation on conformational pro- elaborated by Pseudomonas elodea. Carbohydr. Res. 1983,
perties of xanthan in aqueous solutions. In Solution Properties 124, 135139.
of Polysaccharides; Brant, D., Ed.; ACS Symposium Series, 96. ONeill, M.A.; Selvendran, R.R.; Morris, J.V. Structure of
American Chemical Society (USA), 1981; Vol. 150, 2530. the acidic extracellular gelling polysaccharide produced by
75. Milas, M.; Rinaudo, M. On the existence of two dierent Pseudomonas elodea. Carbohydr. Res. 1983, 124, 123133.
secondary structures for the xanthan in aqueous solutions. 97. Jansson, P.E.; Lindberg, B.; Lindberg, J.; Maekawa, E.;
Polym. Bull. 1984, 12, 507514. Sandford, P.A. Structural studies of a polysaccharide (S-
76. Milas, M.; Rinaudo, M. Properties of xanthan gum in 194) elaborated by Alcaligenes ATCC 31961. Carbohydr.
aqueous solutions: role of the conformational transition. Res. 1986, 156, 157163.
Carbohydr. Res. 1986, 158, 191204. 98. Jansson, P.E.; Lindberg, B.; Widmalm, G.; Sandford,
77. Maret, G.; Milas, M.; Rinaudo, M. Cholesteric order in P.A. Structural studies of an extracellular polysaccharide
aqueous solutions of the polysaccharide xanthan. Polym. (S-130) elaborated by Alcaligenes ATCC 31555. Carbo-
Bull. 1981, 4, 291297. hydr. Res. 1985, 139, 217223.
78. Milas, M.; Rinaudo, M. Properties of the concentrated 99. ONeill, M.A.; Selvendran, R.R.; Morris, J.V.; Eagles, J.
xanthan gum solutions. Polym. Bull. 1983, 10, 271273. Structure of the extracellular polysaccharide produced by
79. Rinaudo, M.; Milas, M. Enzymic hydrolysis of the the bacterium Alcaligenes (ATCC 31555) species. Carbo-
bacterial polysaccharide xanthan by cellulase. Int. J. Biol. hydr. Res. 1986, 147 (5), 295313.
Macromol. 1980, 2, 4548. 100. Chowdhury, T.A.; Lindberg, B.; Lindquist, U.; Baird, J.
80. Milas, M.; Rinaudo, M.; Tinland, B. Comparative Structural studies of an extracellular polysaccharide, S-
depolymerization of xanthan gum by ultrasonic and 657, elaborated by Xanthomonas ATCC 53159. Carbo-
enzymic treatments. Rheological and structural proper- hydr. Res. 1987, 164, 117122.
ties. Carbohydr. Polym. 1986, 6, 95107. 101. Moorhouse, R. Structure/property relationships of a
81. Kohler, N.; Milas, M.; Rinaudo, M. Elimination of family of microbial polysaccharides. In Industrial Poly-
xanthan microgels by new enzymatic treatments. Soc. Pet. saccharides: Genetic Engineering, Structure/Property Re-
Eng. AIME 1983, 8191. lations and Applications; Yalpani, M., Ed.; Elsevier:
82. Rinaudo, M.; Milas, M.; Kohler, N. Enzymic process for Amsterdam, 1987; 187206.
the treatment of xanthan gums to improve the ltrability 102. Campana, S.; Ganter, J.; Milas, M.; Rinaudo, M. On the
of their aqueous solutions. U.S. Patents 4,416,990 and solution properties of the bacterial polysaccharides of
4,431,734. gellan family. Carbohydr. Res. 1992, 231, 3138.
103. Falk, C.; Jansson, P.E.; Rinaudo, M.; Heyraud, A.; saccharide excrete par la bacterie Alteromonas sp. 1644
Widmalm, G.; Hebbar, P. Structural studies of the isolee du milieu hydrothermal profond. Ph.D. Thesis;
exocellular polysaccharide from Sphingomonas paucimo- Lille: France, 1996.
bilis strain I-886. Carbohydr. Res. 1996, 285, 6979. 123. Dubreucq, G.; Domon, B.; Fournet, B. Structure deter-
104. Bian, W.; Chandrasekaran, R.; Rinaudo, M. Molecular mination of a novel uronic acid residue isolated from the
structure of the rhamsan-like exocellular polysaccharide exopolysaccharide produced by a bacterium originating
RMPDP17 from Sphingomonas paucimobilis. Carbohydr. from deep sea hydrothermal vents. Carbohydr. Res. 1996,
Res. 2002, 337, 4556. 290, 175181.
105. Villain-Simonnet, A.; Milas, M.; Rinaudo, M. Compar- 124. Alami, Y.; Heulin, T.; Milas, M.; de Baynast, R.; Hey-
ison between the physicochemical behavior of two micro- raud, A.; Villain, A. Polysaccharide, microorganisme et
bial polysaccharides: RMDP17 and rhamsan. Int. J. Biol. procede pour son obtention, composition le contenant et
Macromol. 1999, 26, 5562. application. Patent FR 97 01624.
106. Lopes, L.; Andrade, C.; Milas, M.; Rinaudo, M. Welan 125. Villain-Simonnet, A.; Milas, M.; Rinaudo, M. A new
microbial polysaccharide. In Polymeric Materials Ency- bacterial exopolysaccharide (YAS34): I. Characterization
clopedia; Salamone, J.C., Ed.; CRC Press, Inc.: Boca of the conformations and conformation transition. Inter.
Raton, USA, 1996; Vol. 11, 86988706. J. Biol. Macromol. 2000, 27, 6575.
107. Campana, S.; Andrade, C.; Milas, M.; Rinaudo, M. 126. Villain-Simonnet, A.; Milas, M.; Rinaudo, M. A new
Polyelectrolyte and rheological studies on the polysac- bacterial exopolysaccharide (YAS34): II. Inuence of
charide welan. Int. J. Biol. Macromol. 1990, 12, 379384. thermal treatments on the conformation and structure.
108. Lopes, L.; Milas, M.; Rinaudo, M. Inuence of the Relation with gelation ability. Int. J. Biol. Macromol.
method of purication on some solution properties of 2000, 27, 7787.
welan gum. Int. J. Biol. Macromol. 1994, 16, 253258. 127. Villain-Simonnet, A. Nouveaux poplysaccharides dori-
109. Lopes, L.; Andrade, C.T.; Milas, M.; Rinaudo, M. gine bacterienne. Structures et proprietes. Ph.D. Thesis;
Inuence of aggregates on the sedimentation properties University of Grenoble: France, 1999.
of welan gum. Polym. Bull. 1995, 34, 655662. 128. Dargelas, F. Structures et proprietes dun nouveau
110. Milas, M.; Shi, X.; Rinaudo, M. On the physicochemical polysaccharide bacterien. Nouvelles applications. Ph.D.
properties of gellan gum. Biopolymers 1990, 30, 451464. Thesis; University of Grenoble: France, 2002.
111. Shi, X. Relation entre la conformation et les proprietes 129. Courtois, J.; Courtois, B.; Heyraud, A.; Colin-Morel, P.;
dun polysaccharide bacterien, le gellane. Ph.D. Thesis; Rinaudo, M. Composes polyme`res de lacide glucuron-
University of Grenoble: France, 1990. ique, procede de preparation et utilisation notamment en
112. Mazen, F.; Milas, M.; Rinaudo, M. Conformational tant que moyens geliants, epaississant, hydratant, stabi-
transition of native and modied gellan. Int. J. Biol. lisant, chelatant ou oculants. Patent FR 92 02510.
Macromol. 1999, 26, 109118. 130. Courtois, J.; Seguin, J.P.; Declomesnil, S.; Heyraud, A.;
113. Rinaudo, M.; Milas, M. Gellan gum, a bacterial gelling Colin-Morel, P.; Dantas, L.; Barbotin, J.C.; Courtois, B.
polymer. In Novel Macromolecules in Food Systems; A (14)-h-D-glucuronan excreted by a mutant of the
Doxastakis, G. Kiosseoglou, V., Eds.; Elsevier: Amster- Rhizobium meliloti M5N1 strain. J. Carbohydr. Chem.
dam, The Netherlands, 2000; 239263. 1993, 12, 441448.
114. Milas, M.; Rinaudo, M. The gellan solgel transition. 131. Dantas, L. Etudes structurales et proprietes en solution
Carbohydr. Polym. 1996, 30, 177184. dun exopolysaccharide secrete par une souche mutee de
115. Rochas, C.; Rinaudo, M. Mechanism of gel formation in Rhizobium meliloti. Ph.D. Thesis; University of Grenoble:
kappa-carrageenan. Biopolymers 1984, 23, 735745. France, 1992.
116. Nishinari, K., Kremer, F., Lagaly, G., Eds.; Physical 132. Dantas, L.; Heyraud, A.; Courtois, B.; Courtois, J.; Milas,
Chemistry and Industrial Application of Gellan Gum. M. Physicochemical properties of Exogel exocellular h-
Progress in Colloid and Polymer Science; Springer-Verlag: (14)-D-glucuronan from Rhizobium meliloti strain M5N1
Berlin-Heidelberg, Germany, 1999; Vol. 114, 1131. CS (NCIMB 40 472). Carbohydr. Polym. 1994, 24, 185191.
117. Bozzi, L.; Milas, M.; Rinaudo, M. Characterization and 133. Braccini, I.; Heyraud, A.; Perez, S. Three-dimensional
solution properties of a new exopolysaccharide excreted features of the bacterial polysaccharide (14)-h-D-glucu-
by the bacterium Alteromonas sp. strain 1644. Int. J. Biol. nonan: a molecular modeling study. Biopolymers 1998,
Macromol. 1996, 18, 917. 45, 165175.
118. Barbier, G.; Guezennec, J.; Pignet, P.; Bozzi, L.; Rinaudo, 134. Heyraud, A.; Dantas, L.; Courtois, J.; Courtois, B.; Helbert,
M.; Milas, M.; Fournet, B.; Leroy, Y.; Dubreucq, G.; W.; Chanzy, H. Crystallographic data on bacterial h-
Prieur, D.; Jeanthon, C.; Vincent, P. Bacteries du type (14)-D-glucuronan. Carbohydr. Res. 1994, 258, 276279.
Alteromonas, polysaccharides produits par ces bacteries, 135. Dantas, L.; Courtois, J.; Courtois, B.; Seguin, J.P.; Gey,
oses contenus dans ces polysaccharides et applications. C.; Heyraud, A. NMR spectroscopic investigation of
Patent FR 93 01687. oligoglucuronates prepared by enzymic hydrolysis of a
119. Bozzi, L.; Milas, M.; Rinaudo, M. Solution and gel (14)-h-D-glucuronan. Carbohydr. Res. 1994, 265, 303
rheology of a new polysaccharide excreted by the 310.
bacterium Alteromonas sp. strain 1644. Int. J. Biol. 136. Paul, F.; Perry, D.; Monsan, P. New strain of Klebsiella
Macromol. 1996, 18, 8391. pneumoniae subsp. pneumoniae and process for producing
120. Samain, E.; Milas, M.; Bozzi, L.; Dubreucq, G.; Rinaudo, a polysaccharide containing L -fucose. U.S. Patent
M. Simultaneous production of two dierent gel-forming 9623057, 1996.
exopolysaccharides by an Alteromonas strain originating 137. Adam, O.; Rivie`re, M.; Vercellone, A.; Monsan, P.; Puzo,
from deep sea hydrothermal vents. Carbohydr. Polym. G. Complete NMR structural elucidation of the capsular
1997, 34, 235241. polysaccharide adjuvant from Klebsiella I-714. Eur. J.
121. Bozzi, L. Production et etude physico-chimique de nou- Biochem. 1996, 241, 602610.
veaux polysaccharides synthetises par des bacteries ma- 138. Johansson, A.; Jansson, P.E.; Wildmalm, G. Structure of the
rines. Ph.D. Thesis; Universty of Grenoble: France, 1994. polysaccharide S-156 elaborated by Klebsiella pneumoniae
122. Dubreucq, G. Contribution a` letude structurale dun poly- ATCC 316 46. Carbohydr. Res. 1994, 253, 317322.
139. Joseleau, J.P.; Marais, M.F. Structure of the capsular M. Structure and properties of a bacterial polysaccharide
polysaccharide of Klebsiella K-type 63. Carbohydr. Res. named FucogelR. Biomacromolecules, 2003, 4, 1362
1979, 77, 183190. 1371.
140. Nieman, H.; Chakraborty, A.K.; Friebolin, H.; Stirm, S. 143. Dutton, G.G.S.; Merrield, E.H. The capsular polysac-
Primary structure of Escherichia coli serotype K42 charide from Klebsiella serotype K54; location of the O-
capsular polysaccharide and its serological identity with acetyl groups and a revised structure. Carbohydr. Res.
the Klebsiella K63 polysaccharide. J. Bacteriol. 1978, 133, 1982, 105, 189203.
390391. 144. ONeil, M.A.; Morris, V.J.; Slvendran, R.R.; Lindberg,
141. Guetta, O. Analyse structurale et etude du comportement B.; Lindquist, U.; Sutherland, I.W.W. Structure of the
en solution de dierents polysaccharides bacteriens. Ph.D. extracellular gelling polysaccharide produced by Entero-
Thesis; University of Grenoble, 2002. bacter (NCIB 11870) species. Carbohydr. Res. 1986, 148,
142. Guetta, O.; Mazeau, K.; Auzely, R.; Milas, M.; Rinaudo, 6369.
16
Microbial Exopolysaccharides
I. W. Sutherland
University of Edinburgh, Edinburgh, United Kingdom
I. INTRODUCTION are exceptions, which include the levans and dextrans

discussed below. The mass, polydispersity, and degree of
The ability to synthesize and secrete exopolysaccharides acylation may also be dependent on the physiological
(EPS)polysaccharides with rigid cell wall and mem- conditions employed.
braneis widespread among microorganisms. In some Many polysaccharides from aerobic bacterial species,
cases, it is a feature of all isolates of a genus and they are yeasts, and microalgae have now been analyzed and, in a
synthesized under virtually all physiological conditions. In large number of examples, have been fully characterized
other strains, production of such polymers results from structurally. Only strictly anaerobic bacteria have received
specic nutritional conditions, from the phase of growth, little attention, although even some of the EPS among these
or as a component of a stress response. In the latter case, in have been fully studied. In some bacterial genera, a wide
some prokaryotes, it may also result from developmental range of chemotypes and serotypes is found and this is
or morphogenetic changes such as microcyst or myxospore typied by genera of the Enterobacteriaceae such as
formation of the type seen in the gram-negative Azotobac- Escherichia or Klebsiella/Enterobacter species. It is also a
ter spp. and myxobacteria, respectively, or sporulation as characteristic of the gram-positive species Streptococcus
seen in gram-positive rods of the genera Bacillus and pneumoniae. The result for these three groups of bacteria is
Clostridium. EPS may be rmly attached to the microbial that each produces a range of almost 100 chemotypes and
cell surface, in which case some brillar structure may even serotypes. In other types of bacteria, a much more re-
be apparent in electron micrographs. Alternatively, the stricted range of chemotypes or even one polysaccharide
EPS may be excreted as an amorphous mass of slime, structure only may be found. Thus, almost all Xanthomo-
which can be readily separated from the microbial cells nas isolates produce xanthan (albeit varying in its acyla-
by centrifugation of the latter. In some bacterial species tion, vide infra), whereas all Azotobacter vinelandii strains
such as the prosthecate bacterium Hyphomonas, polysac- synthesize a form of bacterial alginate. Similarly, colanic
charide may be associated with the external surface of the acid production is widespread among certain members of
stalk, where it functions as an eective adhesive. EPS are the Enterobacteriaceae [1], although it may only be
also major components of microbial biolms in which expressed under conditions such as reduced incubation
mixed cultures attach to inert surfaces. It should also be temperature, high salt concentrations, or other forms of
remembered that a number of bacterial strains may have nutritional stress. It should also be remembered that some
the capacity to form more than one type of polysaccharide, bacterial genera seldom yield polysaccharide-producing
although it is unusual for two or more EPS to be secreted species. Thus, few gram-positive Bacillus spp. produce
simultaneously (Table 1). Some strains yield dierent EPS EPS. An exception is Bacillus circulans, which has yielded
as a result of phenotypic variation, a factor that may be a number of EPS-synthesizing isolates. One of these proved
signicant under biolm conditions. A very small number to have hexasaccharide repeat unit (Fig. 1) [2].
of bacteria have the capacity to synthesize three dierent
EPS. Examples have also been found of cryptic EPS-
synthesizing capacity, only expressed following deletion II. EPS PRODUCTION AND SYNTHESIS
of genes essential for another EPS. Generally, the mono-
saccharide composition and structure of the EPS are Levan and dextran syntheses are dependent on the presence
independent of the carbon substrate available, but there of sucrose as a substrate for the extracellular or surface-
431
432 Sutherland
Table 1 Examples of Multiple EPS Production

Bacterium Polysaccharides produced
Azotobacter chroococcum Alginate and Type-specic EPS
E. amylovora Type-specic EPS or Levana
Escherichia coli Type-specic EPSb or Colanic acidb
E. agglomerans 1.15 Colanic acid typeb and/or Glucomannan,b cellulose
Pseudomonas spp. Alginate or Levana
R. meliloti Succinoglycanb or Galactoglucanb
Rhizobium/Agrobacterium spp. Succinoglycanb or Curdlanb
a
Only synthesized from sucrose.
b
One or more sugar nucleotide precursors shared.
bound enzymes that are responsible for the formation of common intermediates. In one study on Klebsiella aero-
these EPS. However, all other EPS are synthesized using a genes, a comparison of EPS and non-EPS-forming strains
wide range of potential carbon substrates depending on the revealed that glycogen synthesis was consistently lower in
species involved. The substrates are metabolized intracel- the EPS+ than in the EPS strain, indicating that the
lularly to intermediate compounds that serve as the pre- preferred use of the substrate was in EPS production [5].
cursors for activated intermediatessugar nucleotides The limiting nutrient is frequently nitrogen, but potassium,
(nucleoside diphosphate sugars or rarely nucleoside monophosphate, magnesium, or sulphur may also have the same
phosphate sugars). These compounds are synthesized in eect. In a recent study of the yeast Rhodotorula glutinis,
the microbial cytoplasm and, in turn, both form energy- the highest EPS yield was obtained with a C:N ratio of 15:1
rich monosaccharide donors for polysaccharide synthesis [6]. In contrast to bacterial EPS synthesis, which normally
and provide a means for interconversion or synthesis of occurs in the pH range 78.5, production was favored by
many sugars. Bacterial alginates are unique in that the the pH range 35. Incubation at suboptimal growth tem-
polymer formed initially is poly-D-mannuronic acid, which peratures may also increase EPS production, as may
is then modied at the polymer level by epimerases. The alternative stresses including increased osmotic pressure.
epimerase enzymes convert some of the D-mannuronosyl Although bacteria isolated from deep marine environ-
residues to L-guluronic acid. In A. vinelandii, a family of ments have, in several cases, proved to synthesize EPS
seven Ca2+-dependent polymer level epimerases has been (e.g., Ref. 7), the eects of hyperbaric conditions on these
identied [3]. The epimerase in Pseudomonas aeruginosa is a bacteria do not appear to have been examined.
55-kDa periplasmic protein, which only acts on nonacety- The complete gene clusters associated with synthesis of
lated mannuronosyl residues [4]. Unlike the corresponding several EPS have now been determined for both gram-
enzymes from A. vinelandii, only single guluronosyl resi- negative and gram-positive bacteria. In some bacteria, the
dues are introduced and no guluronic acid blocks result. genes are chromosomal, whereas in others, they are carried
The extent of EPS production may depend on the on plasmids. In the case of a simple structure such as a
availability of the sugar nucleotide precursors and also on galactoglucan, a 32-kb gene cluster was identied as a se-
the physiological conditions used. High carbon-to-limiting quence on a megaplasmid [8]. The corresponding proteins
nutrient ratios favor production, although such conditions included several involved in precursor synthesis, together
in bacteria also favor synthesis of internal storage polymers with six glycosyltransferases and an ATP-binding protein
such as glycogen and poly-h-hydroxybutyric acid (PHB). (ABC) transporter complex. The chromosomal genes and
This possibly reduces the amount of substrate available for the corresponding enzymes for succinoglycan synthesis in
EPS, although few comparisons have been made between Sinorhizobium meliloti were identied by Glucksman et al.
the demands of EPS and storage polymer synthesis for [9] (Table 2). Interestingly, whereas similar gene sequences
have been discovered in gram-positive bacteria, those for
EPS synthesis in Streptococcus thermophilus (strain Sf6) are
chromosomal [10] and those for Lactobacillus lactis species
cremoris (NIZO B40) are carried on a plasmid [11]. Despite
its relatively simple composition, the P. aeruginosa alginate
requires an 18-kb operon of 12 genes. Three of these (algI,
AlgJ, and AlgF ) are involved in the acetylation of the D-
mannuronosyl residues [12].
Attempts to alter EPS production through mutagene-
sis may have varying results. Two approaches have been
targeted: mutagenesis of enzymes directly involved in EPS
synthesis and mutation of enzymes suspected to be respon-
Figure 1 The structure of B. circulans EPS. sible for the formation of key metabolic intermediates. In
Microbial Exopolysaccharides 433
Table 2 Genes Involved in Rhizobium EPS Synthesis

Location EPS Function
R. meliloti
ndvA C* Glucan and succinoglycan transport
ndvB C* + Export
exoA P* Membrane attached Glucose 2 transferase (1,3 h)
exoB P* UDP-gal epimerase
exoC C* Phosphoglucomutase
exoD P* Variable
exoF P* Peripheral face Galactosyl transferase
exoG P* No polymerization
exoH P* Membrane spanning + (succ) Succinyl transferase
exoI P* +
exoJ P* + (Trace)
exoL P* Cytoplasm Glucose 3 transferase (1,4 h)
exoM P* Membrane attached Glucose 4 transferase (1,4 h)
exoN P* (Trace) UDP-glucose pyrophosphorylase
exoO P* Cytoplasmic + Glucose 5 transferase
exoP P* Peripheral face Chain length determination
exoQ P* Membrane spanning Polymerization
exoR C* Regulation
exoS C* Regulation
exoT P* Membrane spanning Export
exoU P* Cytoplasmic Trace Glucose 6 transferase
exoV P* Cytoplasmic Pyruvate ketalase
exoW P* Membrane attached Glucose 7 transferase
exoX P* Membrane bound Regulation
exoY P* Galactosyl transferase
exoZ P* Membrane spanning Acetylase
exsA P* ATP transporter
exsB P* Regulation (negative)
exo, form no infection threads; eps, synthesis essential for nodule invasion but not infection; C*, chromosomal, controlled; P*, megaplasmid 2,
controlled.
ExoL, ExoN, ExoO, ExoU, and ExoV proteins are cytoplasmic; ExoA protein anchored to membrane; ExoP has N-terminus in periplasm and C-
terminus in cytoplasm.
ExoH does not form any low-molecular weight succinoglycan.
The 24-kb region is bounded by exoP and exoU and contains 19 exo genes extending to 27 kb with exs genes.
many mutants, EPS synthesis is lost through elimination of surprising results from mutagenesis have come from exten-
a key enzyme in the biosynthetic process. If more than one sive studies on xanthan and acetan mutants in which it has
polysaccharide is formed simultaneously, elimination of been shown that side-chain sugars (and attached acyl
the second is necessary, as it is usually impossible to groups) can be successively eliminated. As a result, a whole
separate the product mixtures. This was applied to strains series of new polymers with increasingly shortened side-
of Alcaligenes faecalis var. myxogenes, which originally chain oligosaccharides and altered physical properties was
produced both curdlan and succinoglycan. Spontaneous produced [16,17]. It was therefore clear that the polymerase
mutants incapable of succinoglycan synthesis were eventu- needed to form the macromolecule was much less specic in
ally obtained for curdlan production [13]. As has already its action than had originally been thought, although yields
been mentioned, suppression of the original EPS synthesis of the mutant products might be lower than those of the
may also lead to production of a new, previously cryptic original polysaccharide. Attempts have also been made to
polymer. This is exemplied by the glucuronan formed by alter EPS production through mutagenesis of enzymes
S. meliloti [14]. Roblot et al. [15] observed that the acetyl involved in precursor synthesis. Recently, Levander et al.
content of this acetylated poly-D-glucuronic acid of S. [18] attempted to increase EPS production in S. thermophi-
meliloti M5N1CS strain was inuenced by the salt content lus through metabolic engineering. Tenfold overexpression
of the medium. The polymer synthesized by mutants may of the galU gene coding for uridine 5c-diphosphate (UDP)-
also be of either increased or decreased mass, with resultant glucose pyrophosphorylase failed to aect EPS produc-
alterations in the viscosity of aqueous solutions. The most tion. However, overexpression of both galU and pgmA (the
434 Sutherland
gene coding for phosphoglucomutase) enhanced EPS yield Enterobacter aerogenes [33], and from an isolate that has
almost twofold when lactose was used as carbon source. been named Alteromonas macleodii, from a deep-sea vent
[7]. The tetrasaccharide repeat unit of the EPS from the
Archaeon H. denitricans (strain 35960) contained a
III. COMPOSITION AND STRUCTURE trisaccharide sequence of three residues of 2,3-diaceta-
mido-2,3-dideoxy- D -glucopyranosiduronic acid [30].
Microbial EPS are composed of a very wide range of Apart from bacterial alginates in which only uronic acid
sugars, including some that, as yet, have either not been residues are present, most EPS contain a single uronic
found in other types of polysaccharides, or are only rarely acid residue in each repeat unit. There are inevitable
found in eukaryotic products. Among the less common exceptions, one of which is S. pneumoniae type 1 [34] in
neutral sugars, L-altrose has, as yet, only been found as which each trisaccharide contains 2-D-galacturonosyl res-
part of a pentasaccharide repeat unit in the EPS of the idues in addition to 2-acetamido-4-amino-2,4,6-trideoxy-
strictly aerobic bacterium Butyrivibrio brisolvens [19]. D-galactose. Pentoses are absent from the majority of
However, probably the great majority of EPS are com- bacterial EPS but are found in eukaryotic products and
posed of a fairly restricted range of common sugars. These in some of those from Cyanobacteria [35]. In various
are mainly neutral hexoses or methylpentoses, but may preliminary reports, pentoses have initially been reported
also include the corresponding uronic acids and N-acetyl- to be present in very small, nonstoichimetric amounts.
aminosugars. A range of unusual aminosugars has also After improved purication of the polymers, further study
been found in bacterial EPS. In only a single example so and detailed structural analysis have usually eliminated
far, a nonacetylated amino sugar has been found to form these from the genuine polysaccharide components. How-
part of an EPS structure. Many EPS are polyanionic due ever, in the EPS of the green microalga Botyrococcus
to the presence of uronic acids. The commonest uronic braunii, in which D -galactose is the major mono-
acid is D-glucuronic acid, but D-galacturonic acid is also saccharide, signicant amounts of 3-O-methyl fucose
frequently found. In bacterial alginates, both D-mannu- and 3-O-methyl rhamnose have been detected [36]. The
ronic acid and L-guluronic acid are present, whereas monosaccharide components of many EPS are listed in
mannuronic acid is also found in a number of other Table 3.
EPS. Very rarely, L-iduronic acid has been detected In addition to monosaccharides, bacterial EPS fre-
[20,21] as has D-riburonic acid (in the furanose form) quently carry other substituents, which may either be
[22]. Another acid sugar of rare occurrence in bacterial organic or inorganic in nature (Table 4). The commonest
EPS is D-methylglucuronic acid found in some Bradyrhi- organic substituents are either O-acetyl groups or pyruvate
zobium polymers [23]. A 2-deoxy-D-arabino-hexuronic ketals, whereas a number of monosaccharides esteried
acid was found in the hexasaccharide repeat unit of with lactic acid or succinic acid have also been reported.
Sphingomonas paucimobilis strain I-886 (Fig. 2) [24]. An The pyruvate ketals are most commonly found attached to
apparently identical monosaccharide was also part of the D-galactose, D-glucose, or D-mannose residues. However,
structure of the EPS of a Rhizobium spp. [25] and was in Rhodococcus equi type 3, the pyruvate is attached to the
present in the hexasaccharide repeat unit of an EPS from O2 and O3 positions of D-glucuronic acid [38]. Frequently,
A. indicus var. myxogenes [26]. Bian et al. [27] have now both acetyl and pyruvate substituents are present on the
reported the presence of 2-deoxy-D-glucuronic acid in a same polymer, but examples can also be found in which
rhamsanlike polysaccharide from another strain of S. only acetate D or only pyruvate is present. In the linear
paucimobilis, whereas 3-O-[(R)-1-carboxyethyl]-D-glucu- tetrasaccharide repeat unit of E. coli K50, the mannosyl
ronic acid was identied in the EPS from Nostoc commune residue is pyruvylated in the sequence:
DRH-1 [28]. Several aminouronic acids have been identi-
ed as components of bacterial EPS, including mannosa-
! 3-h-L-Rhap-1 ! 4-a-D-GlcpNAc-1 ! 2
minuronic acid from a P. uorescens strain [29] and
glucosaminuronic acid from the EPS of a Haloferax -h-D-Manp-1 ! 3-a-D-ManpNAc-1 !
denitricans isolate [30] and galactosaminuronic acid from
P. solanacearum [31]. Although most polyanionic EPS The only acetyl groups are on the amino groups of the
contain only a single type of uronic acid, several examples acetylamino sugars [39].
of structures have been described in which two uronic It is fairly unusual to nd both pyruvate and acetate
acids, usually D-glucuronic acid and D-galacturonic acid, attached to the same monosaccharide residue, but in the
are present. These include EPS from Cyanobacteria [32], EPS of Pseudomonas gingeri, the mannose residue of the
Figure 2 The EPS of S. paucimobilis I-886.

Table 3 Monosaccharide Components of Microbial EPS

Neutral sugars
D-glucose, D-galactose, D-mannose, D-fructose
L-fucose, L-rhamnose, L-altrose
D-xylose, D-ribose, D-arabinose
Colitosea,b
Acetylaminosugars
N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-mannosamine, quinovosamine, N-acetyl-L-fucosamine,
N-acetyl-L-talosamine
Uronic acids
a
D-glucuronic acid, D-galacturonic acid, D-mannuronic acid, L-guluronic acid, L-iduronic acid , D-riburonic acid, 2-deoxy-
D-arabino-hexuronic acid
Aminouronic acids
N-acetyl-D-mannosaminuronic acid
a
Rarer sugars are italicized.
b
This sugar is normally found in lipopolysaccharides in gram-negative bacteria, but has been found in the EPS from Vibrio cholerae O139
[37].
trisaccharide repeat unit is acetylated on the 2-position and acetate per repeat unit [34]. Inorganic substituents such as
also carries a pyruvate ketal on the 4- and 6-positions [40]. phosphate and sulphate are also found. Phosphate groups
Bacterial alginates are acetylated but carry no pyruvate, occur widely, especially among EPS from gram-positive
whereas other polysaccharides such as that from an Erwi- species including some of the pathogenic streptococci and a
nia sp. are pyruvylated but not acetylated [41]. Most of the number of the industrially important species from the same
reports of pyruvylated EPS come from gram-negative genus, which are employed in the fermentation of milk,
bacterial EPS, but as more structures from gram-positive yoghurt, and cheeses. A few phosphate-containing EPS
species are studied, a number of these have also been found have also been reported from E. coli serotypes (Table 5)
to contain pyruvate. One such is EPS from L. rhamnosus [4447]. These include a small number of structures that
RW-959M, in which a galactose residue carried a 4,6- closely resemble some of the teichoic acids found as wall
linked ketal [42]. The linear 1,3-a-linked GalMan polymers in many gram-positive bacteria. Other phos-
Rha trisaccharide repeat unit of R. equi serotype 7 carries phate-containing EPS from gram-negative bacteria con-
a pyruvate on the mannosyl residues [43]. In a number of tain the unusual octasaccharide ketodeoxyoctonic acid and
EPS, the acyl groups are present in nonstoichimetric more closely resemble the lipopolysaccharides found in the
amounts. Thus, some xanthans contain 0.3 mol of pyruvate outer membrane of the walls of these bacteria. Examples of
per repeat unit, whereas the trisaccharide repeat unit of these are emulsan [48] and the E. coli serotype K24 polymer
S. pneumoniae type 1 carries approximately 0.66 mol of [45]. Some of these substituents are relatively common in
Table 4 Noncarbohydrate Substituents Found in Exopolysaccharides

Substituent Linkage Source of EPS
Organic acids
Acetate Ester Very common (e.g., A. vinelandii, Klebsiella, etc.)
Glycerate Ester S. paucimobilis (gellan)
Hydroxybutanoate Ester R. trifolii, R. leguminosarum
Malonate Ester E. coli K10
Propionate Ester E. coli K52
Pyruvate Ketal Very common [e.g., Klebsiella, E. coli (colanic acid), etc.]
Succinate (Half ) Ester Rhizobium spp., Agrobacterium spp. (succinoglycan)
Taurine Ester Staphylococcus aureus
Amino acids
L-glutamate K. aerogenes K82
L-serine E. coli K40, E. coli K54
L-threonine E. coli K54
Inorganic acids
Phosphate Common (especially gram-positives)
Sulphate Cyanobacteria, Halobacterium spp.a, Sulfolobus spp.a
a
Archaea.
436 Sutherland
Table 5 Phosphate-Containing E. coli EPS possible linkages have been found, although some are
much commoner than others, especially among the back-
Serotype Major components Reference bone sequences of monosaccharides. Additionally, some
K2 Galactofuranose, phosphate 44 monosaccharides such as mannose and galactose have been
K24 Glycerol, phosphate, KDO 45 found both in the common D-conguration and much rarer
K51 Phosphate 46 L-conguration. Among the homopolysaccharides, several
K52 Fructose, propionic acid, 47 are formed from a single linkage type. Thus, curdlan from
phosphate, acetate Agrobacterium radiobacter is an a-(1!3)-linked D-glucan;
similarly, bacterial cellulose is h-(1!4)-linked. Alternan is
an unusual a-D-glucan in which 1!3 and 1!6 linkages
alternate on a regular basis and is a product of some
Leuconostoc mesenteroides strains [54]. Mixed linkage h-
certain bacterial species, whereas others such as sulphate D-glucans of the type found in barley or oat walls are
occur more rarely. Phosphate is also a common constituent apparently not formed by microorganisms, although more
of EPS from yeasts. The EPS from Pichia (Hansenula) than one linkage type may certainly be present. This is seen
hostii comprised a phosphomannan core attached to which in pullulan from Aureobasidium pullulans or in dextrans
were oligosaccharide diester phosphate side chains [49]. from L. mesenteroides. Indeed, the latter provided one of
Within the core, all mannosyl residues were a-linked, with the few examples of a highly branched bacterial EPS
a phosphate:mannose ratio of 1:6. in which the glucosyl residues may be (1!2)-, (1!3)-,
Sulphate, although it is a common constituent of (1!4)-, and (1!6)-linked. As well as dextrans, scleroglu-
marine algal polysaccharides, is much more limited in can and some other homopolysaccharides in the group of
prokaryotic EPS. The main groups in which sulphate has closely related fungal polymers contain more than one
been found in EPS include Cyanobacteria and some linkage type.
osmophilic Archae of the genera Haloferax [e.g., H. Although fructose is found as a substituent of a
mediterranea (Fig. 3) [50] and Sulfolobus [51]], together number of bacterial polysaccharides including several from
with the polymers from some isolates such as Pseudoalter- E. coli serotypes (e.g., Ref. 55), it is also the sole monosac-
omonas spp. [7] obtained from the proximity of deep charide component of fructans. These homopolysaccha-
hydrothermal vents. Sulphate, along with glucose, man- rides are synthesized by a number of bacterial species
nose, galactose, and glucosamine, was also identied in the including many oral isolates. As is the case for dextrans,
EPS from thermophilic Archaea of the genus Sulfolobus fructans or levans are only produced from sucrose as
[51]. The EPS from two strains of Synechocystis contained substrate. Fructans represent a group of high-mass homo-
ca. 8% sulphate, in addition to uronic acids and neutral polysaccharides and, in the case of Leuconostoc spp. and
sugars [52]. In addition to those listed in the table, a few related bacteria, are synthesized by extracellular enzyme
unique sugar derivatives have, as yet, been found only in systems utilizing sucrose as the source of the fructosyl
one single EPS. residues (glycansucrases). Many of the lactic acid bacteria
A large number of extracellular microbial polysac- also form fructans [56]. Similar polysaccharides are formed
charides are neutral polymers. However, as a result of the by many oral and plant pathogenic bacteria as well as by
presence of uronic acids, sulphate or phosphate groups, symbiotic species such as Acetobacter diazotrophicus. A.
pyruvate ketals, or succinyl half-esters, many of the other chroococcum is unusual in that it synthesizes alginate when
macromolecules in this group are negatively charged. A grown on glucose but forms a levan in the presence of
single polymer from Staphylococcus epidermidis provides sucrose [57]. Mannans are a common feature of fungal
an EPS, which is known to be positively charged [53]. The walls but are not normally found among bacterial EPS.
nature of the composition and resultant structure, and the However, a recent report suggested that the EPS from P.
presence or absence of certain sugars or acyl groups can syringae pv. ciccaronei is an a-linked D-mannan closely
also greatly aect the hydrophilicity (or otherwise) of EPS. resembling fungal types in being highly branched and
Large numbers of 6-deoxyhexose residues such as L-fucose phosphorylated [58]. It diered from the fungal polysac-
or L-rhamnose can thus increase the lipophilicity of the charides in that it carried a small number of D-glucosyl
molecule. Other amphipathic molecules include emulsan residues. Although most bacterial alginates are heteropoly-
and similar EPS in which the lipophilic portion of the mers, algG mutant strains of P. aeruginosa have been
molecule derives from acyl groups or esters. developed, which were unable to form guluronic acid by
Structurally, microbial EPS are either homopolysac-
charides, which are composed of a single type of monosac-
charide residue, or heteropolysaccharides, in which two or
more types of monosaccharides are present. Many of the
microbial homopolysaccharide EPS are D-glucans, either
a-linked or h-linked. The enormous range of possible
structures resulting from the dierent linkage types is
reected in the rapidly increasing numbers of reported
structures of bacterial EPS. Representatives of almost all Figure 3 H. mediterranei EPS.
epimerization of mannuronosyl residues and thus yielded from neutral sugars with the charge being contributed
an acetylated poly-D-mannuronic acid [59]. A similarly solely by the acyl substituents. Furthermore, the highly
acetylated poly-D-glucuronic acid has been obtained by conserved backbone tetrasaccharide carries a tetrasaccha-
mutagenesis of a S. meliloti strain [60]. In this EPS, about ride side chain, which is conserved in some species but
40% of the monosaccharide residues were acetylated on shows some variation in others, including, in some struc-
either the C3 or C2 positions. tures, the presence of uronic acids in place of neutral sugars
Probably, heteropolysaccharide EPS are commoner (e.g., Figs. 6 and 7) [63,64]. Thus, in isolates of Rhizobium,
than homopolysaccharide EPS, at least among prokaryotic either succinoglycan or related octasaccharide structures
species. These are formed from two or more monosaccha- may be present, although some very dierent structures
rides and may also carry a range of noncarbohydrate have also been reported from this genus.
substituents. Several molecules of some of the component One of the simplest bacterial polysaccharide struc-
monosaccharides such as glucose, galactose, and mannose tures, that of the gram-positive S. pneumoniae type 3, was
may be present in each repeat unit, whereas other sugars, also one of the rst to be elucidated. This polymer is
including the less common types, are represented by a composed of cellobiouronic acid repeat units, each of
single molecule. Thus, many EPS contain only a single which is linked by 1,3-h-linkages to the next. A polysac-
methylpentose molecule in each repeat unit. Inevitably, charide of mass 2 mDa with the same structure has also
exceptions may be found. Some heteropolysaccharide EPS been reported from the gram-negative Rhizobium sp. T1
may be relatively rich in rhamnose (or fucose). An example strain [14]. Thus, the same EPS structure is occasionally
can be seen in a recently studied polymer from an Erwinia found in totally unrelated gram-positive and gram-nega-
sp. in which the hexasaccharide repeat unit contains three tive bacteria. Another simple disaccharide repeat unit was
L-rhamnose residues, in addition to a single molecule each demonstrated for the neutral EPS produced by A. latus
of D-galactose, D-glucose, and D-glucuronic acid [41]. [65]. The structure of this polysaccharide is:
Another EPS from Erwinia chrysanthemi also contained
three molecules of rhamnose, together with mannose, ! 2-a-D-Manp-1 ! 3-a-l-Fucp-1 !
glucose, and glucuronic acid (Fig. 4) [61]. Similarly, the
tetrasaccharide repeat unit of a Bradyrhizobium sp. is Among the most widely and systematically studied
composed of three rhamnose molecules and one 4-O- bacterial polysaccharide structures are those found in the
methyl-D-glucuronic acid [62], and the heptasaccharide of genus Klebsiella/Enterobacter. This group of bacteria has
L. rhamnosus RW-9595 contains four rhamnose molecules yielded over 80 chemotypes with a fairly limited range of
[42]. However, even among polysaccharides composed monosaccharide components. Many of this group of poly-
solely of disaccharides of D -glucose and D -galactose mers are also acetylated and pyruvylated. These acyl
(galactoglucans), a wide range of structures synthesized adornments added to the serological complexity of these
by various gram-negative bacterial species can be found, as EPS. An example of this can be seen in the pentasaccharide
will be seen in Table 6. The potential complexity of this structure of K. pneumoniae ATCC 31314 (Fig. 8), in which
group of products is extended through the widely varying approximately 0.7 mol of acetate was present per repeat
acyl substituents that they carry. It is also clear that some of unit, distributed over at least three positions [66]. However,
these polymers are synthesized by closely related bacterial the basic carbohydrate structure was identical with that in
species, whereas others are obtained from very dierent the Klebsiella K30 and K33 serotypes.
bacterial types. Another group of polysaccharides normal- Most of the polysaccharides in which three or four
ly containing only glucose and galactose is the succinoglu- monosaccharides are present are formed from a uniform
cans formed by several Rhizobium species isolated from a backbone to which side chains are attached at regular
wide range of leguminous plants. These EPS are unusual in intervals. Thus, colanic acid and related structures pro-
that they are normally formed from octasaccharide repeat duced by E. coli, E. gergoviae, and other enteric bacteria as
units, which carry O-acetyl, pyruvate ketal, and succinyl well as E. chrysanthemi Ech6 [67] represent another family
half-ester substituents (Fig. 5). They are also unusual in of closely related structures. They are formed from four
that the polysaccharides, although polyanionic, are formed sugarsL-fucose, D-glucuronic acid, D-glucose, and D-
galactose in the molar ratio 2:1:1:2together with pyru-
vate ketal and O-acetyl groups. Although very similar in
their gross structure, some dierences in the individual
linkages may be seen (Fig. 9) [6870]. Several other EPS
share partial structural similarities to colanic acid and
other reports lacking detailed structural information may
indeed also be of colanic acid (e.g., Ref. 71). A common
type of structure found among bacterial EPS is a trisac-
charide or tetrasaccharide repeat unit on the main chain,
with one of the residues in it carrying a single sugar or a
disaccharide side chain. Occasionally, more complex struc-
tures with more branches are found. Thus, one strain of
Figure 4 E. chrysanthemi (strain SR260) EPS. Burkholderia cepacia produced an EPS in which a trisac-
438 Sutherland
Table 6 Galactoglucans
R. meliloti
!-4-a-D-G1cp-1 ! 3-h-D-Ga1p-1- !
6
z m
CH3 CO:O: Pyr
R. meliloti Strains YE-2(S1), Rm 1021

!-3-h-D-G1cp-1 ! 3-a-D-Ga1p-1- !
6
z m
CH3 CO:O: Pyr
A. radiobacter Strain II; B. cepacia

!-3-h-D-G1cp-1 ! 3-a-D-Ga1p-1- !
m
Pyr
Pseudomas marginalis HTO41B

!-3-h-D-G1cp-1 ! 3-a-D-Ga1p-1- !
z m
Succ Pyr
E. coli K37
!-3-h-D-G1cp-1 ! 3-a-D-Ga1p-1- !
4
z
Pyr Z a D-Ga1p
Klebsiella pnemoniae SK1

!-3-h-D-Ga1p-1 ! 3-h-D-Ga1p-1 ! 3-a-D-G1cp1 !
4 2
1z 1z
h-D-G1cAp a-D-Galp
charide repeat unit has three distinct side chainsone a repeat units formed from trisaccharide side chains attached
disaccharide and the others being single D-galactosyl resi- to every alternate glucose residue on a cellulose backbone
dues [72]. A mannosyl residue in the main chain is D- (Fig 10) [73], but varying in the degree and nature of their
galactosylated, whereas a D-glucuronic acid molecule car- acetyl and ketal substituents depending on the source of the
ries two side chains, yielding a heptasaccharide repeat unit. polysaccharides (Table 7). The range of this group of
Another bacterial polysaccharide family can be seen in polymers has also been further extended through the
the xanthans formed by most pathovars of Xanthomonas development of mutants altered in side chain and acyl
campestris. These are all composed of pentasaccharide group composition. Some strains of Gluconacetobacter
Figure 5 The structure of the EPS succinoglycan from R. meliloti, etc.

Figure 6 The structure of the octasaccharide repeat unit of the EPS of R. leguminosarum strains.
xylinum form an EPS (acetan) in which there is again a acylation and in the mass of the molecules. All were
cellulosic backbone but in which the attached side chain is a composed of three types of structures within the macro-
pentasaccharide rather than a trisaccharide and is of molecule: contiguous blocks of D-mannuronic acid, similar
dierent composition from that found in xanthan. In place blocks of L-guluronic acid, and mixed sequences of the two
of the trisaccharide with a side-chain terminal mannosyl monosaccharides (Fig. 13). In the Pseudomonas alginates,
residue in xanthan, a pentasaccharide composed of man- there was apparently no evidence for blocks of guluronosyl
nose, rhamnose, glucose, and glucuronic acid is present residues. Also, the polysaccharides from plant pathogenic
(Fig. 11) [74]. Acetan, unlike xanthan, lacks pyruvate Pseudomonas spp. [80] were normally of relatively low
groups but is O-acetylated with slightly less than two mass, although this may result from degradation by en-
residues per tetrasaccharide repeat unit in the polymer dogenous alginate lyase enzymes. Typical analyses for
produced by a mutant with truncated side chains [75]. some of these polymers can be seen in Table 8 and struc-
A trio of polysaccharides with considerable sharing tures are presented in Fig. 14. Mutant products without
of structure is also seen in K. aerogenes type 54 [76)], acetylation have also been obtained from P. aeruginosa [59]
Enterobacter XM6 [77], and E. coli K27 [78]. All are and a recent report from Schurks et al. [81] has indicated
formed from tetrasaccharide repeat units and, in the rst that such mutants are similar in their monosaccharide
two strains, the carbohydrate structures of these were composition to the products from the original wild-type
identical, but the former carried either one or two O- strain. A further interesting nding from this study was the
acetyl groups on each octasaccharide (i.e., only alternate lower-than-expected occurrence of triads of mannuronic
repeat units were acetylated in the former). In the EPS of acid (MMM sequences) and higher-than-expected triads
E. coli K27 EPS, the side-chain D-glucose residue was re- of MMG/GMM and also GMG in the P. aerugi-
placed by D-galactose (Fig. 12). nosa alginates studied. As a result of the availability of such
As has already been mentioned, various gram-negative a wide range of bacterial alginates, their physical properties
bacteria, either Pseudomonas spp. or species closely related can also now be studied.
to them such as Azotobacter spp., can produce EPS, which Another very signicant group of polysaccharide with
are eectively alginates. These dier from most other closely related structures is to be found in the gellan family.
bacterial EPS in lacking a regular structure but most are Most of these polymers have been obtained from bacterial
macromolecules of high molecular weight. Instead, there isolates now regarded as strains of S. paucimobilis, but
are completely random sequences of the two component earlier designated Alcaligenes or P. elodea. In all but two of
uronic acids. The gene sequences responsible for alginate the nine polysaccharides of this group, a common tetra-
formation are also widely distributed among related saccharide backbone contained two molecules of D-glucose
groups of gram-negative bacteria, although not all these and one each of D-glucuronic acid and either L-rhamnose
produce alginate (e.g., if one of the genes is missing) [79]. or L-mannose. In the two exceptions, the glucuronic acid
There were, however, very marked structural dierences was replaced by 2-deoxy-D-glucuronic acid; three EPS in
between the bacterial polymers and also between them and the group lack side chains. Attached to the repeat unit
the analogous commercial products obtained from marine structure in the remaining polymers were either single
algae, although all were composed of the same two uronic monosaccharides or disaccharides [82]. This series of struc-
acids. Unlike the algal material, the bacterial polymers tures is shown in Fig. 15 [83,84]. Several of the polysac-
were almost all heavily acetylated on the D-mannuronosyl charides have been the subject of intensive physical studies
residues, which may carry one or two acetyl groups. The and molecular modelling. This is in part due to the com-
EPS from Azotobacter spp. (vinelandii and chroococcum) mercialization of gellan and the potential applications of
most closely resembled algal alginates except in their some of the other members of this group.
Figure 7 Uronic acid-containing EPS of R. leguminosarum.

440 Sutherland
Figure 8 EPS of K. pneumoniae ATCC 31314.
Among species of Cyanobacteria, the EPS tend to be

much more complex in structure than those found in most Figure 10 The structure of the EPS from X. campestris
other prokaryotes. Some are highly branched and possess (xanthan) structures.
side chains of diering length as has been demonstrated by
Wolk [85]. Another feature dierentiating these EPS from
other prokaryotic products is the common appearance of
pentoses including xylose and arabinose. Garozzo et al. xylose, fucose, rhamnose, glucose, mannose, and galactose,
[86] have provided an example of such a complex structure and both glucuronic and galacturonic acids was proposed
in the polysaccharide from Cyanospira capsulata. It was for the EPS from a thermophilic cyanobacterium (Mastigo-
composed of octasaccharide repeat units and three cladus laminosus) [87]. A viscous EPS obtained from N.
branches were attached to the main chain. Seven mono- commune was composed of a 1,4-linked xylogalactan back-
saccharides in total were present, including L-arabinose bone and attached to it were D-ribofuranose and 3-O-[(R)-
and 4-O-(1-carboxyethyl)-mannose. An even more com- 1-carboxyethyl]-D-glucuronic acid residues [28]. Several
plex pentadecasaccharide repeat unit structure containing EPS from Cyanobacteria also carry sulphate groups.
Figure 9 Structures of colanic acid and closely related types of EPS.

Table 7 The Structure of the EPS From Mutant Strains of et al. [88]. Relatively minor alterations to the chemical
X. campestris structure may cause considerable changes to the physical
properties of these polymers. Thus, the side chains present
!-4-h-D-G1cp-1 ! 4-h-D-G1cp-1- ! on the cellulosic backbones of xanthan and acetan render
3
1z these polymers water-soluble and provide highly viscous
a-D-Manp-O:CO:CH3 aqueous solutions with excellent salt stability. Acetan,
1z
2 despite its longer side chains and lack of pyruvylation,
h-D-G1cpA formed double-stranded chains resembling those observed
for xanthan [89]. The polysaccharide also resembled xan-
than in that it underwent thermoreversible helixcoil tran-
Polytetramer sition in aqueous solution [90]. As is the case for xanthan,
ionic strength played a signicant role in determining the
!-4-h-D-G1cp-1 ! 4-h-D-G1cp-1- ! transition temperature for the ordered form, and acetyla-
3 tion did not inhibit helix formation.
1z
a-D-Man The inuence of noncarbohydrate substituents has
2 been widely studied for some polysaccharide families and
1z
h-D-G1cpA the eects are described below. Indeed, the availability of
many of the natural families of bacterial EPS and the more
recent selection of mutants with truncated side chains have
Polytetramer (Nonacetylated) provided a wealth of information about the role of poly-
saccharide components. The commercial development of
!-4-h-D-G1cp-1 ! 4-h-D-G1cp-1- ! such polymers as xanthan and gellan and the encourage-
1z
3 ment and support given by commercial manufacturers have
a-D-Manp-O:CO:CH3 led to a rapid increase in the number of physical studies on
microbial polysaccharides. As a result, considerable infor-
mation is now available on the physical properties, tertiary
Polytrimer structures, crystallinity, etc. of these polymers. In many
cases, associations between helices were promoted by cat-
!-4-h-D-G1cp-1 ! 4-h-D-G1cp-1- ! ions and water molecules. The tendency to adopt helical
1z
3 conformations was found both in the solid and liquid
a-D-Manp states. The physical properties observed were determined
by the shape of the helix, interactions between helices, and
interactions with ordered water molecules and cations [91].
Polytrimer (Nonacetylated) A number of the water soluble EPS can undergo
Polytrimer can also be prepared by h-D-glucuronidase treatment of physical gelation, which, as pointed out by Picullel [92],
Polytetramer. may involve coil-to-helix transition of the type observed
on cooling a heated random coil solution. The resultant gel
may be either strong and brittle, or weak. In the case of
gellan, the system can be modied to yield either strong
IV. PHYSICAL PROPERTIES or weak gels. The initial association of helical gellan
A. General Physical Properties molecules into rigid rods developed into a network struc-
Following extensive physical studies on many dierent

EPS, the existence of many of these polysaccharide families
has assisted in the determination of structureproperty
relationships. It has also provided evidence for the role
that acyl and some carbohydrate groups or linkages may
play in the development of certain physical properties. It
has also assisted in the determination of the eect of these
groups and linkages on both serological and enzymic
specicity, together with other biological properties. Be-
cause of the very large numbers of monosaccharides,
diverse linkage types, and noncarbohydrate substituents,
microbial EPS reveal a very wide range of physical prop-
erties. As numerous EPS are eectively linear macromole-
cules of high mass, many yield highly viscous aqueous
solutions; others with relatively rigid backbones are almost
insoluble as is the case for bacterial cellulose, curdlan, or Figure 11 The structure of the EPS from strains of
two E. agglomerans polysaccharides described by Hughes Acetobacter xylinum (acetan).
442 Sutherland
Figure 12 The structure of Enterobacter XM6 and K. aerogenes type 54 EPS.
ture. Succinoglycan and some other polysaccharides adop- 106 were reported. Use of pure material has now shown
ted a single-chain conformation with an intramolecular that for puried xanthan with diering acylation from
helixcoil transition [93]. In the case of xanthan, questions several dierent X. campestris strains, the mass was
have been asked over the initial state of the helices. Milas consistently in the range 1.11.7 106 [110].
et al. [94] suggested that native xanthan, which had not X-ray ber diraction analysis has proved useful for a
been exposed to either high temperature or low osmotic number of EPS in determining the three-dimensional
strength, remained in a single-stranded form, whereas structures. In the case of polymers such as either K.
material that had been heated above the transition tem- aerogenes type 54 or beijeran, the native polymer must
perature adopted the double-helical form. Gelation of rst be deacetylated before crystalline oriented lms can be
xanthan or acetan by trivalent cations was considered to prepared. Bian et al. [82] found that beijeran, a polymer
be due to interactions with the glucuronosyl residues of the with a trisaccharide repeat unit, [!3)-a-D-GalpA-(1!3)-
side chains, with the longer and bulkier side chains in h-L-Rhap-(1!3)-a-D -Glcp(1!], adopted an extended
acetan suppressing gelation through steric hindrance [95]. twofold helical structure in which the two chains were
The mass of many EPS may depend on the physi- tightly packed by hydrogen bonding and water molecules
ological conditions under which they were produced, but in an antiparallel manner, forming thick sheets. The sheets
other factors can also come into play. Thus, we now associated in a network with sodium ions and water
know that a number of bacterial species yield degrada- molecules. Using atomic force microscopy, Gunning et
tive enzymes (polysaccharases or polysaccharide lyases) al. [111] were able to visualize such networks and gels in
(Table 9), which, at least in gram-negative species, are gellan preparations. Lee and Chandrasekaran [112] and
normally found associated with the periplasm [9,96108]. Chandrasekaran et al. [113] demonstrated that gellan
They, thus normally do not come into direct contact adopted a double helical conformation in which each chain
with the EPS. However, as cells age and lyse, some was translated half a pitch ( p = 564 nm) with respect to
enzyme may be released and may then partially degrade the other, and in which interchain hydrogen bonds stabi-
the polymer, yielding material of lower mass. This was lized the adopted structure. A similar double-helical mor-
seen in alginate production by several Pseudomonas spp. phology was also present in the closely related polysaccharides
[105]. It has also proved possible to obtain stable of the gellan family that have been examined so far [114].
mutants synthesizing EPS of increased mass and, hence, Thus, the extensive collection of side-chain substituents
increased viscosity. This could be achieved with colanic failed to aect this ordered conformation, although deace-
acid-producing strains of E. coli K12, Enterobacter/Kleb- tylation has been shown to destabilize the helices formed by
siella strains, and a P. chlororaphis strain [109]. Some rhamsan and gellan [115]. Within aqueous samples of
estimates of EPS have proved misleading, as the material gellan, microcrystalline regions may be detected and these
used has represented associations of polysaccharide are thought to result from aggregates of double helices
chains rather than single molecules. This was noticeable [116]. A range of counterions promoted gelation of gellan
for xanthan for which initial values ranging up to 20 solutions. As with gelation of XM6, monovalent ions
Figure 13 Bacterial alginate from A. vinelandii.

Table 8 Composition and Diad Frequencies of Some Bacterial and Algal Alginates
Source FG FM FGG FMM FGM,MG Acetyl (%)
L. hyperborea 0.665 0.335 0.558 0.228 0.107 0

Macrocystis pyrifera 0.41 0.59 0.24 0.42 0.17 0
A. vinelandii 73 0.561 0.439 0.372 0.25 0.189 11
A. vinelandii 206 0.08 0.92 0.03 0.87 0.05 24
P. aeruginosa B 0.16 0.84 0 0.68 0.16 37
P. uorescens 0.31 0.69 0 0.38 16.9
Pseudomonas putida 0.22 0.78 0 0.56 21
G = L-guluronic acid; M = D-mannuronic acid.
within this series were required in higher concentrations acteristics as the extent of hydration as has been shown for
than divalent cations. High-strength gels were also formed hyaluronic acid [124].
in the proton form of gellan. Larwood et al. [117] proposed A number of EPS are eective emulsifying agents.
that gellan gels were formed when cations mediated the Emulsan and alasan are two of a group of biosurfac-
aggregation of double helices. For divalent ions such as tants from Acinetobacter lwoi (formerly calcoaceticus)
Ca2+, direct bridging between carboxyl oxygen atoms strain RAG-1 and related bacteria [125]. Other polymers
helped to promote the aggregation. Welan, another poly- with this type of activity have been obtained from Cyano-
mer in the gellan family, existed as a half-staggered, parallel bacteria including Phormidium spp. [126], whereas some
double helix similar to gellan [118]. The side chains of the food-grade yeasts have also been reported to produce
calcium salt folded back onto the main chain and hydrogen emulsifying agents [127]. Recently, genetic engineering
bonds were formed with the carboxylate groups, thus has been applied to the A. lwoi in attempts to enhance
enhancing the stability of the double helix. In dilute both emulsication and metal-binding activities [128,129].
solution, both gellan and welan chains tended to aggregate
and, after deacetylation, gelation of rhamsan occurred in B. The Effect of Acylation or Ketalation
the presence of Ca2+. Not all EPS adopted an ordered on Physical Properties
conformation. The EPS capsular material from K. pneumo-
niae K40 is relatively exible and, in solution, it adopted a Acetylation frequently has a very considerable eect on
random coil conformation [119]. Another polysaccharide the physical properties of a bacterial polysaccharide.
showing similar lack of ordered conformation is also from Several examples of this have now been demonstrated.
a K. pneumoniae strain (SK1) [120]. E. aerogenes serotype K54 and Enterobacter XM6 pro-
Due to the uronic acids or pyruvate ketals present in vided one such comparison, as both polysaccharides
many polysaccharides, they have an ability to bind cations; possessed the same carbohydrate structure. Each is
in a few cases, there may be some degree of specicity. composed of a tetrasaccharide repeat unit composed of
Thus, bacterial alginates, after deacetylation, showed spec- D-glucose, L-fucose, and D-glucuronic acid in the molar
icity for Ca2+ and, to a lesser extent, Mg2+ in manner ratio 2:1:1 (Fig. 12) [7678]. Only the K54 polymer was
similar to algal alginates [121]. Recently, Quintero et al. O-acetylated and carried either one acetyl group on each
[122] suggested that a polymer from Hyphomonas adhae- repeat unit, or one on every alternate unit. Each poly-
rens had the much rarer ability to sequester gold. This saccharide yielded highly viscous aqueous solutions.
undoubtedly resulted from the tertiary structure adopted However, very marked dierences could be seen when
by such polymers. Although many ions may have little they were examined by x-ray crystallography. The K54
eect on the structures adopted by microbial EPS, some polysaccharide was amorphous and failed to produce a
multivalent cations may disrupt the helical conformations clear pattern, whereas the XM6 material yielded well-
[123]. The counterions present may also aect such char- dened x-ray patterns signifying a well-developed crys-
Figure 14 The alginate from P. aeruginosa.

444 Sutherland
Figure 15 The structures of gellan and related polysaccharides.
talline microstructure [130]. The XM6 polysaccharide that the acetyl group had a critical role on the solution
also formed gels with a number of monovalent and conformation. They suggested that intramolecular hydro-
divalent cations, with the gels showing very sharp melting gen bonding took place between the acetyl substituents on
and setting points at about 38jC with virtually no hysteresis a mannose residue and the C(2)-hydroxyl group of the
(Fig. 16) [131]. The major dierences in properties between adjacent monosaccharide (glucose). Removal of the acetyl
the two polymers were ascribed to the inhibitory eect of groups greatly reduced the intrinsic viscosity. Studies on
acylation on intramolecular association. A similar eect was the selective deacylation of succinoglucan by Ridout et al.
observed in studies on beijeran, a galacturonic acid-contain- [134] demonstrated the role of these substituents in the
ing polymer from a strain of A. beijerinckia with a repeating functional behavior of this polysaccharide. Deacetylation
trisaccharide structure [132]. Only after deacetylation were caused a decrease in the orderdisorder transition temper-
gelation and higher crystallinity observed. However, unlike ature, whereas removal of the succinyl residues increased it.
XM6, the acetyl groups failed to disturb the regular array in The material lacking succinate also showed much im-
the crystal; the ordered conformation derived from extended proved pseudoplasticity and increased cooperativity in
twofold helices. the transition.
In studies on the solution conformation of the EPS Gellan double helices exist as crystal structures stabi-
from Pseudomonas gingeri, Gianni et al. [133] indicated lized by interactions with K+ and water. In the presence of
Table 9 Polysaccharases and Polysaccharide Lyases Associated with EPS Biosynthesis

Bacterial species Enzyme Reference
A. xylinum Endoglucanase (CM-cellulase) 96

A. tumefaciens Endoglucanase (CM-cellulase) 97
A. chroococcum Alginate lyase (polymannuronate lyase) 98
A. vinelandii Alginate lyase (polymannuronate lyase) 99
Cellulomonas avigena 1,3-h-D-glucanase 100
E. coli K5 N-acetyl-heparosan lyase 101
P. aeruginosa Alginate lyase (polymannuronate lyase) 102
P. uorescens Alginate lyase (polymannuronate lyase) K. Hughes, unpublished
P. uorescens (marginalis) Galactoglucanase 103
P. mendocina Alginate lyase (polymannuronate lyase) 104
P. putida Alginate lyase (polymannuronate lyase) 105
R. meliloti Succinoglycan depolymerase 9
R. meliloti Poly-D-glucuronic acid lyase 106
Sphingomonas spp. Gellan lyase 107,108
Streptococcus equi Hyaluronidase
divalent ions such as Ca2+, direct crosslinks between the ylic acid groups of uronic acid residues, lead to increased
chains permit gel formation at very low ion concentrations. adsorption of cations and to precipitation with quaternary
The L-glycerate groups carried by the native polymer tend ammonium compounds such as cetavlon.
to inhibit gelation and thus accounted for the weaker, A series of studies on xanthan preparations carrying
elastic type of gelation observed prior to removal of acyl varying degrees of O-acetylation and ketalation also
groups [135]. In those EPS carrying pyruvate groups, they indicated how these substituents aected their physical
provide increased polyanionic features as well as aect properties.
transition temperature. They, thus, along with the carbox- Table 7 shows the various acylation types of xanthan
available. These include multiply acetylated material, xan-
than carrying pyruvate without acetate, and acetate with
no pyruvate. It was thus possible to examine the eects of
such variants on the transition temperature, the eect of
salts, and also the eects on the synergistic interactions
with plant glucomannan and galactomannan. Most xan-
than preparations tested formed strong thermoreversible
gel networks with locust bean gum (LBG) and the strength
of the system depended greatly on the degree of acyl
substitution [136]. Mutants lacking the terminal mannose
of the side chain formed much weaker networks, indicating
the role of the xanthan side chains in establishing the
interactions with LBG. Xanthan also gelled with konjac
mannan solutions but much higher concentrations were
needed and the melting and setting temperatures were
higher than those observed with LBG [137]. Gel strength
again depended on the level of acetylation. Xanthan prep-
arations interacted much more weakly with guar gum, with
the exception of low acetyl xanthans [138]. The midpoints
of the helixcoil transition behavior of xanthans correlated
with the acetyl and pyruvyl contents [110]. Addition of
NaCl increased the melting temperatures and, in the case of
high pyruvate, low-acetate xanthan revealed an unusual
two-phase helixcoil transition. In further studies on the
LBGxanthan mixtures, Copetti et al. [139] suggested the
coexistence within mixed gels of both LBGLBG and
LBGxanthan junction zones. In the gels, the xanthan
retained its ordered helical structure. Studies have also
been made on xanthan in which the side chains had been
Figure 16 Gelation of XM6 EPS. partially degraded by mild acid hydrolysis [140]. The
446 Sutherland
transition temperature was not aected by removal of the unit; the second enzyme then split that into two tetrasac-
terminal h-mannosyl residuethe modied polymer charides (Fig. 17) [145]. Some EPS including gellan (but not
revealed orderdisorder properties very similar to the other structures in that series) are degraded by lyases from
native polymer and adopted double-stranded state. Con- homologous, gellan-forming bacteria [107,108] as well as
formational dierences have also been postulated to ex- by enzymes from heterologous bacteria [146]. A study by
plain the increased viscosity of the acetylated polymer, Craston et al. [147] indicated that the gellan lyase from
which had lost the terminal mannose (and pyruvate) when heterologous bacteria (a small, pink-pigmented pseudomo-
compared with the corresponding nonacetylated mutant nad) might prove useful in specic determination of gellan
material [141]. Light scattering measurements and other in processed foods.
procedures revealed that the pattern of acylation of the Several polysaccharides, including both bacterial and
xanthan molecule did not greatly inuence its inherent algal alginates, are only degraded by lyases. The latter are
stiness [110,142]. degraded by numerous alginate lyases from various sour-
ces, but bacterial alginates are often more resistant due to
the presence of the O-acetyl groups on D-mannuronosyl
V. DEGRADATIONPOLYSACCHARASES residues. This group of alginate lyases can be dierentiated
AND POLYSACCHARIDE LYASES into poly-D-mannuronic acid-specic and poly-L-guluronic
acid-specic lyases, but there may also be further specic
Very few microbial EPS are degraded by commercial activity depending on the monosaccharide sequences adja-
enzyme preparations. Almost all require highly specic cent to the linkages split. A similar polysaccharide that is
enzymes for their degradation. These are either polysac- degraded by a lyase is poly-D-glucuronic acid. This is one of
charide hydrolases (polysaccharases) or polysaccharide the groups of degradative enzymes associated with the
lyases [143]. This latter group cleaves bonds between bacteria that also synthesize the polymer substrate, in this
neutral sugars or uronic acids and adjacent uronosyl case S. meliloti. Da Costa et al. [148] identied a 20-kDa
residues, with abstraction of water and creation of an protein with glucuronan lyase activity, yielding oligosac-
unsaturated uronic acid at the nonreducing terminus of charides with four to ve uronic acid residues. As with
the oligosaccharides. Several of these enzymes are found in alginate lyases, this enzyme showed greater activity on the
the biosynthetic complex of EPS-forming bacteria. Others deacetylated substrate. In particular, 2,3-di-O-acetyl-D-
can be obtained from either heterologous microorganisms glucuronic acid residues in close proximity to the site of
or from bacteriophage. These viral particles have proved to cleavage of the lyase reduced depolymerization of the
be an excellent source of highly specic bacterial EPS- polysaccharide [149].
degrading enzymes and have been widely used to generate Xanthan can be degraded by mixtures of enzymes,
fragments for structural analyses [144]. They generally which usually include a xanthan lyase that cleaves the
release oligosaccharides that are equivalent to the repeat bond between the terminal mannosyl residue and glucu-
unit, or to dimers or trimers of the repeat unit, and have the ronic acid on the side chains. Such enzymes have been
advantage for structural studies that there is no loss of acyl isolated from mixed bacterial cultures as well as from pure
or other labile substituents [165]. In families of EPS with cultures of Bacillus sp. [150]. One of the latter was specic
closely related structures, the phage enzymes degrade both for pyruvylated mannose residues [151]. Despite the cellu-
acylated and nonacylated polysaccharides. In the case of losic main chain, xanthan is not normally degraded by
succinoglycans, two distinct enzymes have been obtained commercial cellulase preparations. However, when com-
from a bacterial source (Cytophaga arvensicola). One pletely deionized and in the disordered form, limited
enzyme degraded the polymer to its octasaccharide repeat degradation with Trichoderma cellulase has been observed
Figure 17 Enzymes acting on the succinoglycan group of EPS.

[152,153]. This limited hydrolysis was postulated to be due [159]. Another E. coli strain (K4) produced a polymer with
to sections of the xanthan chain lacking side chains. This some structural features in common with chondroitin.
hypothesis was later conrmed by Christensen and The backbone of h-D-glucuronosyl-(1!3)-h-N-acetyl-D-
Smidsrd [154]. Maximal hydrolysis required sequences galactosamine was not sulphated as would be true of
of 10 or more glucose residues lacking side chains. Similar chondroitin and each uronic acid residue carried a
results were obtained with acetan. (2!3)-linked h-D-fructosyl residue. P. multocida yields a
number of dierent EPS capsular macromolecules. In
addition to the hyaluronic acid of type A, it has now been
VI. POLYSACCHARIDES COMMON TO established that types D and F synthesize unmodied
PROKARYOTES AND EUKARYOTES heparin and chondroitin, respectively [160]. Sialic acids
form components of various glycoconjugates in eukar-
A number of bacterial species are able to produce EPS yotes. They are usually linked to hexoses or aminosugars
either identical to, or closely related to, polysaccharides in nonterminal positions. A number of bacterial species,
found in eukaryotic species. Thus, cellulose is an EPS including several dangerous pathogens such as E. coli K1
with no structural role, formed in several bacterial and Neisseria meningitidis capable of inducing meningitis,
cultures including G. xylinum (formerly designated A. produce capsular material in which sialic acid, varying in
xylinum) and Agrobacterium spp. This is in contrast to its linkages and in the presence or absence of O-acetylation
the polymer of identical structure found as a component and other sugars, is present [161]. The group C meningo-
of plant cell walls and closely associated with auxiliary cocci produced a sialic acid homopolysaccharide in which
wall polysaccharides. The number of bacterial species the residues were (2!9)-linked, whereas the polysaccha-
with the potential to form cellulose has recently been rides from E. coli K1 and N. meningitidis group B were
extended by the studies of Zogaj et al. [155], who (2!8)-a-linked. In the former, typically 200 sialyl residues
observed it as a cryptic product formed by various were present [162]. Nonacetylated bacterial sialic acids
species on Enterobacteriaceae under dened physiologi- composed of alternate (2!8) and (2!9) linkages are also
cal conditions. Bacterial cellulose thus represents a very found, as are EPS in which both sialic acid and other sugars
pure form of the polymer in which the microbrils are present (Table 10). However, sialic acid has also been
exhibit high crystallinity and purity. identied as a component of the capsular material of the
Another polysaccharide common to prokaryotes and fungal pathogen Cryptococcus neoformans [163]. In addi-
eukaryotes is hyaluronic acid. This was produced by tion to EPS composed solely of sialic acid, several patho-
several strains of groups A and C Streptococcus including genic bacterial species secreted EPS in which sialic acid
S. epizooticus [156], and by type A Pasteurella multocida played a dominant role in infection. These included S.
[157]. Reports of hyaluronic acid production by other agalactiae in which a common feature of all serotypes was
bacterial species and genera have also appeared in the N-acetylneuraminic acid a-2,3-linked to D-galactose [164].
literature, but many of these are poorly authenticated. Although various bacterial species produce alginates
Bacterial hyaluronic acid is identical in composition and as EPS, none of these conforms exactly to the structure of
structure to the eukaryotic product but may be variable in the corresponding algal products. The EPS from A. vine-
mass due to the action of a hyaluronidase present in the landii most closely resembles the commercial products
bacterial cultures. Until recently, it was thought that no from marine brown algae including Laminaria digitata
bacteria synthesized heparin or chondroitin, although a and M. pyrifera. All these polymers are composed of
few strains did yield polymers with close similarity to these block structures, irregular sequences of poly-D-mannur-
polysaccharides. The EPS of E. coli serotype K5 is eec- onic acid, and poly-L-guluronic acid. The bacterial poly-
tively a desulphatoheparin (Fig. 18) [158]. This structure saccharide was, however, heavily O-acetylated on many of
was identical with that of N-acetylheparosan, which is a the mannuronosyl residues, a feature that rendered its
precursor in heparin biosynthesis. It was degradable by physical properties rather dierent from those found in
heparin lyases and EPS-specic lyases from coliphage K5 the seaweed products. Despite this, many literature reports
incorrectly attribute properties to bacterial alginates that
are only present in the commercial materials. The other
main bacterial alginate producers are Pseudomonas spp.
(Table 11), including the very mucoid isolates of P. aeru-
ginosa from cystic brosis patients. The Pseudomonas EPS
vary in their mass. Those from plant pathogenic species
were of relatively low mass, whereas those from P. aerugi-
nosa, P. uorescens, and P. putida were 500 kDa or more.
Acetylation is again a major feature of these polysacchar-
ides, but unlike all the other alginates, no polyguluronic
acid blocks are present. Rather, polymannuronosyl
sequences are interspersed with single L-guluronosyl resi-
Figure 18 The structure of the EPS from E. coli strains K4 dues and the total content of guluronic acid tends to be
and K5 and of bacterial hyaluronic acid. much lower than on the Azotobacter alginates.
448 Sutherland
Table 10 Sialic Acid-Containing Microbial EPS

N. meningitidis Serotype B N-acetyl-neuraminic acid a-2,8
N. meningitidis Serotype C N-acetyl-neuraminic acid, acetate a-2,9
N. meningitidis Serotype W135 N-acetyl-neuraminic acid, galactose
N. meningitidis Serotype Y N-acetyl-neuraminic acid, glucose, acetate
E. coli K1 N-acetyl-neuraminic acid, acetate a-2,8
E. coli K92 N-acetyl-neuraminic acid a-2,8; a-2,9
Salmonella toucra N-acetyl-neuraminic acid
VII. BIOLOGICAL PROPERTIES genic bacteria was the ability of some species to mimic the
polysaccharides found in the human and animal body (vide
From the large number of bacterial polysaccharides for infra) and fail to generate antibodies.
which the structures have now been elucidated, it is clear Other polysaccharides possessed interesting biological
that many share at least some part of their structure, properties including tumor suppression and immune stim-
thus providing some immunological cross-reactions. ulation eects. Thus, lentinan and other fungal 1,3-h-D-
Such cross-reactions may be with other EPS or with glucans such as sceleroglucan have proved to possess such
lipopolysaccharides and other carbohydrate-containing properties (e.g., Ref. 168). They appeared to act as host
macromolecules. One example of common structures defense, potentiating agents rather than biological re-
within EPS can be seen in Enterobacter type 54 and E. sponse modiers. Both these polymers were capable of
coli serotype K27, which share a trisaccharide backbone adopting a triple-helical conformation [169], a feature that
but carry glucosyl and galactosyl side-chain residues, appears to be required for expression of biological activity.
respectively [76,78]. Preparations of alginate from Sargassum fulvellum have
Pathogenic bacterial frequently owe their pathogenic- been shown to possess antitumor activity against various
ity to their ability to form EPS. Consequently, antisera to tumors in mice [170], the highest antitumor activity being
some of these polymers may be protective agents and form apparently correlated to increasing content of poly-D-
essential components of vaccines. A problem with such mannuronic acid sequences. Bacterial alginates do not yet
vaccines is often the multiplicity of chemotypes found appear to have been tested for similar activity. Fungal h-D-
among the EPS and the resultant wide range of serotypes. glucans also stimulated coagulation of factor G from
Vaccines have, therefore, to be prepared from selections of horseshoe crab amoebocytes, but only polysaccharides of
the most widely encountered types, as is the case for S. relatively high mass, in the single helical conformation,
pneumoniae. This may result in up to 23 dierent EPS being were active [171]. The unbranched bacterial glucan also
included [34] for this species. However, the majority of EPS yielded gels with the limulus preparation [172]. Fungal h-D-
are poor antigens and production of a protective vaccine glucans lacking (1!3) linkages failed to induce gelation.
may require the presence of suitable adjuvants or the Within certain limits, molecular mass had much less eect.
conjugation of the EPS to a protein carrier. One example Another eect observed with an EPS from B. polymyxa
of this is Haemophilus type b polysaccharide conjugated to S-4, which contained both D-glucuronic and D-mannur-
N. meningitidis outer membrane protein [166]. Conjugate onic acids, was the ability to reduce cholesterol levels in
vaccines have also been prepared by coupling oligosaccha- experimental animals [173].
ride fragments from the EPS to suitable carriers [167]. As already mentioned, the EPS from E. coli serotype
Another problem encountered with EPS-forming patho- K5 resembles desulfatoheparin. As a result, it has been
used to prepare analogues of heparin [174], which have
been tested for their biological properties. An acid hetero-
polysaccharide from A. infernus, which contained glucose,
Table 11 Alginate-Producing Bacteria galactose, and both glucuronic and galacturonic acids, was
without observed activity [175]. However, sulphation and
Species D-mannuronic:L-guluronic acid ratio depolymerization yielded fragments from this macromol-
ecule with anticoagulant activity. Thus, although these two
A. vinelandii Wide range
A. chroococcum Wide range EPS do not, in themselves, possess signicant biological
P. aeruginosa from 1:0 to 0.6:0.4 activities, they can be readily converted into derivatives
P. cepacia that do.
P. uorescens ca. 0.6:0.4
P. maltophila
P. mendocina VIII. APPLICATIONS AND COMMERCIAL
P. phaseolicola 0.95:0.05 PRODUCTS
P. pisa 0.83:0.017
Microbial polysaccharides nd a very wide range of appli-
P. putida ca. 0.6:0.4
cations in industries as diverse as oil extraction, printing,
Table 12 Commercial Development of Microbial Polysaccharides

Intended Usage Development Possible limitations Successful products
Food Empirical GRAS acceptability Xanthan, gellan

Food Molecular genetics User acceptability LAB polymers
Nonfood Empirical Costs, enzymes Xanthan
Molecular genetics Cost, yields Xanthan and acetan derivatives
Ethical Eukaryotic substitutes Mass, purity Hyaluronic acid
Industrial Empirical, etc. Cost, physical properties Pullulan, etc.
food, cosmetics, and health care. There are, however, Microbial polysaccharides represent renewable
potential problems or limitations in the development of resources that oer a wide range of potentially useful
these polymers (Table 12). Even within these industries, products. They do, however, have to compete against
various physical and biological properties are utilized. established plant and algal products as well as synthetic
Most of the applications for EPS use bacterial, rather than materials. Despite extensive research and searches for new
eukaryotic, products. Although De Baets and Vandamme EPS and the many polysaccharide structures that have now
[176] suggested that yeasts might provide EPS with the been elucidated, only a relatively small number have
potential for novel applications, these appear to remain proved to be commercially successful and are now well-
mainly as future goals rather than currently available established products. Xanthan has proved to be extremely
commercial products. The most promising applications successful because of the very wide range of potential
for yeast and other fungal EPS and their derivatives appear applications in food and other industries (Table 13). It is
to be as potential anticancer or immune-modulating unlikely that this number will increase signicantly, as the
agents. This was exemplied by Ferro et al. [177], who cost of development of new polysaccharides, especially if
have developed a large-scale procedure for the preparation they are intended for food use, is likely to be very expensive.
of the phosphorylated manno-oligosaccharide used in the This limitation is also mainly due to the cheapness of
synthesis of the anticancer agent PI-88. competing plant or algal polysaccharides, which are avail-
Table 13 Some Functional Properties of Xanthan Used in Processed Foods and Other
Commercial Applications
Function Application
Adhesive Icings and glazes
Binding agent Pet foods
Coating Confectionary
Emulsifying agent Salad dressings
Encapsulation Powdered avors
Film formation Protective coatings, sausage casings
Foam stabilizer Beer
Stabilizer Ice cream, salad dressings
Swelling agent Processed meat products
Syneresis inhibitor Cheeses, frozen foods
Thickening agent Jams, sauces, syrups, and pie llings
Nonfood applications
Suspending agent Laundry chemicals
Agrichemical spraysherbicides, owable pesticides
Liquid feed supplements
Paper coatings
Flocculant Water clarication
Ore extraction
Shear thinning/viscosity control Oil-drilling muds
Viscosity/crosslinking Hydraulic fracturing
Viscosity control Abrasives
Jet printing
Stabilization Thixotrophic paints
Foam stabilization Fire-ghting uids
Gelling agent Explosives, blockage of permeable zones in oil reservoirs
450 Sutherland
able in large quantities even though their properties may mixtures of xanthan and galactomannans depended on the
not necessarily be quite as good as those of the microbial acylation of the xanthan molecules [136138]. Despite its
polymers. It should also be remembered that several mi- structural similarity to xanthan, native (acetylated) acetan
crobial EPS are natural ingredients of some traditional does not form synergistic gels with LBG or konjac mannan.
fermented foods. In these, they play an important role in However, after removal of the acetyl groups, thermorever-
developing texture, gelation, and viscosity of the nal sible gels were formed with the glucomannan [181].
product. Those microbial EPS that have been commercial- Most industrial applications of xanthan derive from its
ized are clearly acceptable to the user in their chemical and ability to dissolve in hot or cold water, yielding high-
physical properties and in their cost when compared to viscosity, pseudoplastic solutions, even at low polysaccha-
competing polymers. ride concentrations. These solutions are compatible with
Dextran from L. mesenteroides has been in commercial acids, bases, and salts, and are stable for long periods at
use for many years. Although it was originally approved ambient temperatures. Xanthan is used in the oil industry
for food applications, this role has lapsed. It is currently the as a lubricant in oil drilling muds where the capacity for
base for the Sephadex range of ion exchange and gel suspending barytes and viscosity regain after shear are
ltration agents used in the separation and purication of particularly useful. The pseudoplastic xanthan solution
biological macromolecules. Because of their low antigenic- provides low viscosity at the high-shear region of the drill
ity and viscosity in saline solutions, dextran fractions bit together with high viscosity in the annulus where the
are also suitable for use as a plasma substitute. They are shear is low [182]. Xanthan can also be used in the process
also convenient standards for molecular mass in size of selective pore blockage. Xanthan solutions can be
exclusion chromatography, whereas blue dextran (dex- injected into the reservoir rocks, in zones of high perme-
tran coupled to a blue dye) is used to determine column ability, and then cross-linked with Cr3+. Once in place, the
void volumes. gels have a long stability. There are many other industries
Xanthan has proved to be one of the most successful that make use of the physicochemical properties of xan-
commercial products with a wide range of applications in than. It has been incorporated as a stabilizer into emulsion
food, oil recovery, and other industries. Many industrial paints and laundry starch, as well as used as a carrier for
applications of xanthan originated in the food industry. It agrochemicals such as herbicides and fertilizers. Xanthan
received approval for food use from the United States can be incorporated into clay coatings to provide high-
Food and Drug Administration (U.S. FDA) in 1969. The quality nish on papers; it is also a potential thickening
polysaccharide is incorporated into foods to alter the agent for textile dyes in which it is incorporated to control
rheological properties of the water present and thus change rheological properties during textile printing. Although
the texture of the product. Applications of xanthan thus succinoglucan was also produced for a short while as an
utilize many of its physical properties (Table 13). In many industrial viscosifying and suspending agent with oil eld
foodstus, xanthan possesses further useful attributes, applications, it is apparently no longer readily available.
including rapid avor release, good mouthfeel, and com- Gellan from S. paucimobilis was approved for food use
patibility with other food ingredients including proteins, [generally regarded as safe (GRAS) listed] but has also
lipids, and polysaccharides. As most foodstus already found applications in plant biotechnology where it is
contain polysaccharides such as starch or pectin, in addi- marketed as PhytogelR, a gelling agent suitable for plant
tion to proteins and lipids, any added polymer such as tissue culture. Indeed, Doner and Douds [183] demonstrat-
xanthan must be totally compatible with them. Some of the ed that after purication of the commercial product to
foodstus to which polysaccharides are added are of low remove multivalent cations, solutions of gellan in mono-
pH. These include salad dressings, relishes, and yoghurts. valent cation form (Li+, Na+, NH4+, etc.) could be used
Clearly, any polysaccharide that is incorporated into foods to prepare gel beads with calcium ions in a manner similar
of these types must be acid-stable. Xanthan, which is stable to alginate beads. After obtaining the EPS in a pure K+
over a wide range of pH values, is thus very well suited to form, Watase and Nishinari [184] also noted that the
such applications. Some of the food applications of xan- critical polymer concentration required for gelation was
than result from its interaction with plant glucomannans or lowered. When the native polymer was deacylated, it
galactomannans. Bresolin et al. [178] have suggested that yielded excellent, optically clear, strong gel with the dier-
the galactomannan promotes an ordering of xanthan ence between melting and setting temperatures being 45
chains under conditions where the bacterial polymer alone 60jC [185]. A range of gel textures could be obtained
would remain in the disordered form. The temperature of through control of the extent of deacylation. It was also a
gelation and the gel strength for the synergistic mixtures suitable gelling agent for microbial culture media, provid-
did depend on the extent of side chains present on the plant ing an exceptionally clear gel without observable syneresis.
polysaccharide [179]. Annable et al. [180] demonstrated Perhaps surprisingly, bacterial cellulose from G. xy-
that the xanthan/konjac mannan (glucomannan) interac- linum has proved to be a high value product with specialist
tion occurred at temperatures that were considerably lower applications. It is a very pure, highly crystalline polysac-
than the transition temperature; the presence of electro- charide in the so-called type I form and is free from lignin.
lytes had the eect of promoting xanthan self-association It is also characterized by its high capacity for water
at the expense of xanthan/glucomannan interaction. We absorption and high strength in the wet state [186,187].
have also noted that the extent of formation of gels with Bacterial cellulose has been incorporated in the manufac-
ture of high-quality acoustic membranes, but has also gellan. Specialized new applications may be found such
found use in wound dressings (marketed as Biol) in which as the suggested usage of gellan for pharmaceutical
it apparently promotes wound healing as well as minimizes formulations [194]. Perhaps more biological activities
plasma loss and aids in keeping wounds uninfected. It was will be discovered. It is also possible that chemical
considered to be a valuable temporary skin substitute in modication of EPS will introduce novel properties that
the treatment of burns, ulcers, grafts, and dermal abra- can be used in microfabrication or microelectronics.
sions [188]. It may also provide useful products after
chemical derivatization.
Bacterial hyaluronic acid can replace materials of REFERENCES
animal origin provided that it is of satisfactory purity
and mass. Strains lacking hyaluronidase have been devel- 1. Grant, W.D.; Sutherland, I.W.; Wilkinson, J.F. Exopo-
lysaccharide colanic acid and its occurrence in the Entero-
oped to ensure minimal degradation of the product. As bacteriaceae. J. Bacteriol. 1969, 100, 11871193.
well as its use in wound management as a replacement for 2. Fontaine, T.; Wieruszeski, J.-M.; Talmont, F.; Saniez, M.;
hyaluronic acid lost during eye or joint operations, the Duot, P.; Leeu, J.; Fournet, B. Exopolysaccharide
polysaccharide is incorporated into cosmetics where it is a structure from Bacillus circulans. Eur. J. Biochem. 1991,
valuable hydrating agent. Water uptake of 1 kg/g has 196, 107113.
been claimed. 3. Svanem, B.I.G.; Strand, W.; Ertesvag, H.; Skjak-Braek,
G.; Hartmann, M.; Barbeyron, T.; Valla, S. The catalytic
Claims have been made for commercial applications activities of the bifunctional Azotobacter vinelandii man-
of emulsan as a biodegradable surfactant suitable for nuronan C-5-epimerase and alginate lyase AlgE7 prob-
cleaning oil from tanks or vessels or in emulsion stabiliza- ably originate from the same active site in the enzyme. J.
tion. As it is clearly a much more expensive product than Biol. Chem. 2001, 276, 3154231550.
chemically synthesized detergents, it is thus likely to have 4. Franklin, M.J.; Chitnis, C.E.; Gacesa, P.; Sonesson, A.;
only niche applications [189]. However, in natural environ- White, D.C.; Ohman, D.E. Pseudomonas aeruginosa algG
is a polymer level alginate C5 mannuron epimerase. J.
ments, biosurfactant production may prove to be a useful
Bacteriol. 1994, 176, 18211830.
and ecologically friendly means of cleaning up oil spills. A 5. de Souza, A.M.; Sutherland, I.W. Exopolysaccharide and
number of other similar EPS biosurfactants such as dis- storage polymer production in Enterobacter aerogenes
persan [190] have now been described and it is possible that type 8 strains. J. Appl. Bacteriol. 1994, 76, 463468.
some of these may eventually be commercialized, although 6. Cho, D.H.; Chae, H.J.; Kim, E.Y. Synthesis and
they are likely to prove considerably more expensive than characterization of a novel extracellular polysaccharide
synthetic products. by Rhodotorula glutinis. Appl. Biochem. Biotechnol. 2001,
95, 183193.
7. Rougeaux, H.; Guezennec, J.G.; Carlson, R.W.; Kerva-
rec, N.; Pichon, R.; Talaga, P. Structural determination of
IX. FUTURE SCENARIOS the exopolysaccharide of Pseudoalteromonas strain HYD
721 isolated from a deep-sea hydrothermal vent. Carbo-
As investigations into new microbial isolates from ex- hydr. Res. 1999, 315, 273285.
treme environments continue, it is highly likely that 8. Becker, A.; Rueberg, S.; Kuester, H.; Roxlau, A.; Keller,
M.; Ivashina, T.; Cheng, H.-P.; Walker, G.C.; Puhler, A.
some EPS with novel monosaccharides will be found. The 32-kilobase exp gene cluster of Rhizobium meliloti
Maybe more pentoses will be detected in cyanobacterial directing biosynthesis of galactoglucan: genetic organiza-
EPS. The existence of further acyls, amino acids, or tion and properties of the encoded gene product T. J.
other substituents is also possible. On the other hand, Bacteriol. 1997, 179, 13751384.
it is just possible that the extensive range of constituents 9. Glucksman, M.A.; Reuber, T.L.; Walker, G.C. Genes
of microbial EPS that have already been discovered is needed for the modication, polymerization, export and
processing of succinoglycan by Rhizobium meliloti: a
truly representative. Extensive recent studies on the EPS
model for succinoglycan biosynthesis. J. Bacteriol. 1993,
from lactic acid bacteria are beginning to reveal the 175, 70457055.
structures of many of these polymers (e.g., Refs. 191 10. van Kranenburg, R.; Marugg, J.D.; Van Swam, I.I.;
and 192) and the fact that they are produced by bacteria Willem, N.J.; de Vos, W.M. Molecular characterization of
already utilized by the food industry may lead to their the plasmid-encoded eps gene cluster essential for
further development for food usage. Currently, however, exopolysaccharide biosynthesis in Lactococcus lactis.
the relatively low yields and the diculties of production Mol. Microbiol. 1997, 24, 387397.
11. Stingele, F.; Vincent, S.J.F.; Faber, E.J.; Newell, J.W.;
in semisynthetic media present considerable problems, Kamerling, J.P.; Neeser, J.-R. Introduction of the eps
although some EPS including that from S. thermophilus gene cluster from Streptococcus thermophilus S6 into
SY show pseudoplastic behavior with viscosity possibly Lactococcus lactis MG1363: production and character-
higher than xanthan [193]. Proposals have been made for ization of an altered polysaccharide. Mol. Microbiol.
metabolic engineering to improve yields or to tailor 1999, 32, 12871295.
the EPS to alter or improve their functions [18,192]. 12. Franklin, M.J.; Ohman, D.E. Mutant analysis and
cellular localization of the AlgI, AlgJ and AlgF proteins
Almost certainly, the future development of microbial required for O acetylation of alginate in Pseudomonas
EPS will lie in the direction of high-value products with aeruginosa. J. Bacteriol. 2002, 184, 30003007.
specialized applications, rather than as bulk chemicals of 13. Amemura, A.; Hisamatsu, M.; Harada, T. Spontaneous
medium costa market already lled by xanthan and mutation of polysaccharide production in Alcaligenes
452 Sutherland
faecalis var. myxogenes. Appl. Environ. Microbiol. 1977, exopolysaccharide of Pseudomonas uorescens strain
34, 617620. H13. Carbohydr. Res. 1997, 300, 323327.
14. Guentas, L.; Pheulpin, P.; Heyraud, A.; Gey, C.; 30. Parolis, L.A.S.; Parolis, H.; Paramonov, N.A.; Boan, I.F.;
Courtois, B.; Courtois, J. Production of a glucoglucur- Anton, J.; Rodriguez-Valera, F. Structural studies on the
onan by a rhizobia strain infecting alfalfa. Structure of the acidic exopolysaccharide from Haloferax denitricans
repeating unit. Int. J. Biol. Macromol. 2000, 27, 269277. ATCC 35960. Carbohydr. Res. 1999, 319, 133140.
15. Roblot, C.; Seguin, J.-P.; Barbotin, J.N.; Michaud, P.; 31. Orgambide, G.; Montrozier, H.; Servin, P.; Roussel, J.;
Courtois, J.; Courtois, B.; Heyraud, A. Eect of salts on Trigalet-Demery, D.; Trigalet, A. High heterogeneity of
production and on O-acetylation of glucuronan excreted the exopolysaccharides of Pseudomonas solanacearum
by the Rhizobium meliloti M5N1CS strain. Int. J. Biol. strain GMI 1000 and the complete structure of the major
Macromol. 1995, 17, 365368. polysaccharide. J. Biol. Chem. 1993, 266, 83128321.
16. Vanderslice, R.W.; Doherty, D.H.; Capage, M.; Betlach, 32. De Phillipis, R.; Vincenzi, M. Exocellular polysaccharides
M.R.; Hassler, R.A.; Henderson, N.M.; Ryan-Graniero, from Cyanobacteria and their possible applications.
J.; Tecklenberg, M. Genetic engineering of polysaccharide FEMS Microbiol. Rev. 1998, 22, 151175.
structure in Xanthomonas campestris. In Biomedical and 33. Takeda, M.; Ishigami, M.; Shimada, A.; Matsuoka, H.;
Biotechnological Advances in Industrial Polysaccharides; Nakamura, I. Separation and preliminary characteriza-
Crescenzi, V., Dea, I.C.M., Paoletti, S., Stivala, S.S., tion of acidic polysaccharides produced by Enterobacter
Sutherland, I.W., Eds.; Gordon and Breach: New York, spp. J. Ferment. Bioeng. 1994, 78, 140144.
1989; 145156. 34. Stroop, C.J.M.; Xu, Q.; Retzla, M.; Abeygunawardana,
17. Edwards, K.J.; Jay, A.J.; Colquhoun, I.J.; Morris, V.J.; C.; Bush, C.A. Structural analysis and chemical depolyme-
Gasson, M.J.; Grin, A.M. Generation of a novel rization of the capsular polysaccharide of Streptococcus
polysaccharide by inactivation of the aceP gene from the pneumoniae type 1. Carbohydr. Res. 2002, 337, 335344.
acetan biosynthetic pathway in Acetobacter xylinum. 35. Bertocchi, C.; Navarini, L.; Cesaro, A.; Anastasio, M.
Microbiology 1999, 145, 14991506. Polysaccharides from Cyanobacteria. Carbohydr. Polym.
18. Levander, F.; Svensson, M.; Radstrom, P. Enhanced 1990, 12, 127154.
exopolysaccharide production by metabolic engineering 36. Allard, B.; Casedevall, E. Carbohydrate composition and
of Streptococcus thermophilus. Appl. Environ. Microbiol. characterization of sugars from green microalga Botyr-
2002, 68, 784790. ococcus brauni. Phytochemistry 1990, 29, 18751878.
19. Ferreira, F.; Kenne, L.; Cotta, M.A.; Stack, R.J. Structural 37. Linnerborg, M.; Weintraub, A.; Albert, M.J.; Widmalm,
studies of the extracellular polysaccharide from Buytrivi- G. Depolymerization of the capsular polysaccharide from
brio brisolvens. Carbohydr. Res. 1997, 301, 193203. Vibrio cholerae O139 by a lyase associated with the
20. Stack, R.J.; Plattner, R.D.; Cote, G.L. Identication of L- bacteriophage JA1. Carbohydr. Res. 2001, 333, 263269.
iduronic acid as a constituent of the major extracellular 38. Severn, W.B.; Richards, J.C. The acidic capsular poly-
polysaccharide produced by Butyrivibrio brisolvens strain saccharide of Rhodococcus equi serotype 3. Structural
X6C61. FEMS Microbiol. Lett. 1988, 51, 16. elucidation and stereochemical analysis of the lactate
21. Sheng, S.; Cherniak, R. Structure of the capsular ether and pyruvate acetal substituents. Can. J. Chem.
polysaccharide of Clostridium perfringens Hobbs 10 1992, 70, 26642675.
determined by NMR spectroscopy. Carbohydr. Res. 39. Grue, M.R.; Parolis, H.; Parolis, L.A.S. Structure of the
1998, 305, 6572. capsular antigen of Escherichia coli O8:K50:H. Carbo-
22. Amemura, A.; Hisamatsu, M.; Ghai, S.K.; Harada, T. hydr. Res. 1994, 258, 233241.
Structural studies on a new polysaccharide containing 40. Cescutti, P.; Osman, S.F.; Fett, W.F.; Weisleder, D. The
D-riburonic acid. Carbohydr. Res. 1981, 91, 5965. structure of the acidic exopolysaccharide produced by
23. Streeter, J.G.; Salminen, S.O.; Whitmoyer, R.E.; Carlson, Pseudomonas gingeri strain Pf9. Carbohydr. Res. 1995,
R.W. Formation of novel polysaccharides by Bradyrhi- 275, 371379.
zobium. Appl. Environ. Microbiol. 1992, 58, 607613. 41. Yang, B.Y.; Ding, Q.; Montgomery, R. Extracellular
24. Falk, C.; Jansson, P.-E.; Rinaudo, M.; Heyraud, A.; polysaccharides of a bacterium associated with a fungal
Widmalm, G.; Hebbar, P. Structural studies of the canker disease of Eucalyptus sp. Carbohydr. Res. 2002,
exocellular polysaccharide from Sphingomonas paucomo- 337, 731742.
bilis strain I-886. Carbohydr. Res. 1996, 285, 6979. 42. Van Calsteren, M.R.; Pau-Roblot, C.; Begin, A.; Roy, D.
25. Guentas, L.; Pheulpin, P.; Michaud, P.; Heyraud, A.; Structure determination of the exopolysaccharide pro-
Gey, C.; Courtois, B.; Courtois, J. Structure of a duced by Lactobacillus rhamnosus strains RW-9595M and
polysaccharide from a Rhizobium species containing 2- R. Biochem. J. 2002, 363, 217.
deoxy-h-D-arabino-hexuronic acid. Carbohydr. Res. 43. Masoud, H.; Richards, J.C. Structural elucidation of the
2001, 332, 167173. specic capsular polysaccharide of Rhodococcus equi
26. Gulin, S.; Kussak, A.; Jansson, P.-E.; Widmalm, G. serotype 7. Carbohydr. Res. 1994, 252, 223233.
Structural studies of S-7 another exocellular polysaccha- 44. Jann, K.; Jann, B.; Schmidt, M.A.; Vann, W.F. Structure
ride containing 2-deoxy-arabino-hexuronic acid. Carbo- of the E. coli K2 capsular antigen, a teichoic acid like
hydr. Res. 2001, 331, 285290. polymer. J. Bacteriol. 1980, 143, 11081115.
27. Bian, W.; Chandrasekaran, R.; Rinaudo, M. Molecular 45. Lenter, M.C.; Jann, B.; Jann, K. Structure of the K24
structure of the rhamnsan-like exocellular polysaccharide antigen of Escherichia coli O83:K24:H, a polymer that
RMDP17 from Sphingomonas paucimobilis. Carbohydr. consists of a-KDOp and glycerol phosphate. Carbohydr.
Res. 2002b, 337, 4556. Res. 1990, 208, 139144.
28. Helm, R.F.; Huang, Z.; Edwards, D.; Leeson, H.; Peery, 46. Jann, B.; Dengler, T.; Jann, K. The capsular (K51)
W.; Potts, M. Structural characterization of the released antigen of E. coli O1:K51:H. FEMS Microbiol. Lett.
polysaccharide of desiccation tolerant Nostoc commune 1985, 29, 257261.
DRH-1. J. Bacteriol. 2000, 182, 974982. 47. Hofmann, P.; Jann, B.; Jann, K. Structure of the fructose-
29. Osman, S.F.; Fett, W.F.; Irwin, P.; Cescutti, P.; Brouil- containing K52 capsular polysaccharide. Eur. J. Biochem.
lete, J.N.; OConnor, J.V. The structure of the 1985, 147, 601609.
48. Zuckerberg, A.; Diver, A.; Peeri, Z.; Gutnick, D.L.; M.; Murooka, Y. Structural characterization of a new
Rosenberg, E. Emulsier of Arthrobacter RAG-1: chem- acidic exopolysaccharide and cyclic (1!2) h-glucan
ical and physical properties. Appl. Environ. Microbiol. produced by Rhizobium huakuii forming nodules on
1979, 37, 414420. Astragalus sinicus. J. Ferment. Bioeng. 1997, 83, 315320.
49. Parolis, L.A.S.; Parolis, H.; Kenne, L.; Meldal, M.; Bock, 65. Nohata, Y.; Azuma, J.; Kurane, R. Structural studies of a
K. The extracellular polysaccharide of Picia (Hansenula) neutral polysaccharide produced by Alcaligenes latus.
holstii NRRL Y-2448: the phosphorylated side chains. Carbohydr. Res. 1996, 293, 213222.
Carbohydr. Res. 1998, 309, 7787. 66. Edebrink, P.; Jansson, P.-E.; Widmalm, G. Structural
50. Parolis, H.; Parolis, L.A.S.; Boan, I.F.; Rodriguez-Valera, studies of the capsular polysaccharide S21 from Klebsiella
F.; Widmalm, G.; Manca, M.C.; Jansson, P.-E.; Suther- pneumoniae ATCC 31314. Carbohydr. Res. 1993, 245,
land, I.W. The structure of the exopolysaccharide 311321.
produced by the halophilic Archaeon Haloferax medi- 67. Yang, B.Y.; Brand, J.; Montgomery, R. Pyruvated
terranea strain R4 (ATCC 33500). Carbohydr. Res. 1996, galactose and oligosaccharides from Erwinia chrysanthemi
295, 147156. Ech6 extracellular polysaccharide. Carbohydr. Res. 2001,
51. Nicolaus, B.; Manca, M.C.; Romana, I.; Lama, L. 331, 5967.
Production of an exopolysaccharide from two thermo- 68. Lawson, C.J.; McCleary, C.W.; Nakada, H.I.; Rees, D.A.;
philic Archaea belonging to the genus Sulfolobus. FEMS Sutherland, I.W.; Wilkinson, J.F. Structural studies of
Microbiol. Lett. 1993, 109, 203206. colanic acid from Escherichia coli by using methylation
52. Pano, J.; Priem, B.; Morvan, H.; Joset, F. Sulphated and base-catalysed fragmentation. Biochem. J. 1969, 115,
exopolysaccharides produced by two unicellular strains of 947958.
Cyanobacteria, Synechocystis PCC 6803 and 6714. Arch. 69. Cescutti, P.; Toanin, R.; Pollesello, P.; Sutherland, I.W.
Microbiol. 1988, 150, 558563. Structural determination of the acidic exopolysaccharide
53. Mack, D.; Fischer, W.; Krokotsch, A.; Leopold, K.; produced by a Pseudomonas sp strain 1.15. Carbohydr.
Hartmann, R.; Egge, H.; Laufs, R. The intercellular Res. 1999, 315, 159168.
adhesin involved in biolm accumulation of Staphylococ- 70. Yang, B.Y.; Gray, J.S.S.; Montgomery, R. Extracellular
cus epidermidis is a linear h-1,6-linked glucosaminoglycan: polysaccharide of Erwinia chrysanthemi Ech6. Int. J. Biol.
purication and structural analysis. J. Bacteriol. 1996, Macromol. 1994, 16, 306312.
178, 175183. 71. Isobe, Y.; Matsumoto, Y.; Yokoigawa, K.; Kawai, H.
54. Leathers, T.D.; Ahlgren, J.A.; Cote, G.L. Alternansucrase Properties of an extracellular polysaccharide produced by
mutants of Leuconostoc mesenteroides strain NRRL B- a strain of Enterobacter isolated from pond water. Biosci.
21138. J. Ind. Microbiol. Biotechnol. 1997, 18, 278283. Biotechnol. Biochem. 2001, 65, 13991401.
55. Rodriguez, M.L.; Jann, B.; Jann, K. Structure and 72. Cescutti, P.; Bosco, M.; Picotti, F.; Impallomeni, G.;
serological properties of the capsular K11 antigen of Richau, J.A.; SaCorreia, I. Structural study of the
Escherichia coli O13:K11:H11. Carbohydr. Polym. 1990, exopolysaccharide produced by a clinical isolate of
196, 101109. Burkholderia cepacia. Biochem. Biophys. Res. Commun.
56. Monsan, P.; Bozonnet, S.; Albenne, C.; Joucla, G.; 2000, 273, 10881094.
Willemot, R.-M.; Remaud-Simeon, M. Homopolysaccha- 73. Jansson, P.-E.; Kenne, L.; Lindberg, B. Structure of the
rides from lactic acid bacteria. Int. Dairy J. 2001, 11, 675 extracellular polysaccharide from Xanthomonas campest-
685. ris. Carbohydr. Res. 1975, 45, 275282.
57. Cote, G.L.; Krull, L.H. Characterization of the exocel- 74. Jansson, P.-E.; Lindberg, J.; Wimalsiri, K.M.; Dankert,
lular polysaccharides from Azotobacter chrococcum. Car- M.A. Structural studies of acetan, an exopolysaccharide
bohydr. Res. 1988, 181, 143152. elaborated by Acetobacter xylinum. Carbohydr. Res.
58. Corsaro, M.M.; Evidente, A.; Lanzetta, R.; Lavermicocca, 1993, 245, 303310.
P.; Molinaro, A. Structural determination of the phyto- 75. Colquhoun, I.J.; Defernez, M.; Morris, V.J. NMR studies
toxic mannan exopolysaccharide from Pseudomonas syrin- of acetan and the related bacterial polysaccharide CR1/4
gae pv. ciccaronei. Carbohydr. Res. 2001, 330, 271277. produced by a mutant strain of Acetobacter xylinum.
59. Franklin, M.J.; Ohman, D.E. Identication of algF in the Carbohydr. Res. 1995, 269, 319331.
alginate biosynthetic gene cluster of Pseudomonas aerugi- 76. Sutherland, I.W.; Wilkinson, J.F. The exopolysaccharide
nosa which is required for alginate acetylation. J. of Klebsiella aerogenes A3(Sl) (Type 54). Biochem. J.
Bacteriol. 1993, 175, 50575065. 1968, 110, 749754.
60. Courtois, J.; Seguin, J.-P.; Roblot, C.; Heyraud, A.; Gey, 77. ONeill, M.A.; Morris, V.J.; Selvendran, R.; Sutherland,
C.; Dantas, L.; Barbotin, J.-N.; Courtois, B. Exopolysac- I.W.; Taylor, I.T. Structure of the extracellular gelling
charide production by the Rhizobium meliloti M5N1 CS polysaccharide produced by Enterobacter (NCIB 11870)
strain. Location and quantitation of the sites of O- species. Carbohydr. Res. 1986, 148, 6369.
acetylation. Carbohydr. Polym. 1994, 25, 712. 78. Sutherland, I.W.; Jann, B.; Jann, K. The isolation of
61. Gray, J.S.S.; Brand, J.M.; Koerner, T.A.W.; Montgomery, O-acetylated fragments from the K antigen of Escherichia
R. Structure of an exopolysaccharide produced by Erwinia coli O8:K27(A)H by the action of phage-induced enzymes
chrysanthemi. Carbohydr. Res. 1993, 245, 271287. from Klebsiella aerogenes. Eur. J. Biochem. 1970, 12, 285
62. An, J.H.; Carlson, R.W.; Glushka, J.; Streeter, J.G. The 288.
structure of a novel polysaccharide produced by Bradyr- 79. Fialho, A.M.; Zielinski, N.A.; Fett, W.F.; Chakrabarty,
hizobium species within soyabean nodules. Carbohydr. A.M.; Berry, A. Distribution of alginate gene sequences in
Res. 1995, 269, 303317. the Pseudomonas rRNA homology group IAzomonas
63. Kuo, M.; Mort, A.J. Location and identity of the acyl Azotobacter lineage of superfamily B prokaryotes. Appl.
substituents on the extracellular polysaccharides of Environ. Microbiol. 1990, 56, 436443.
Rhizobium trifolii and Rhizobium leguminosarum. Carbo- 80. Fett, W.F.; Osman, S.F.; Fishman, M.L.; Siebles, T.S.
hydr. Res. 1986, 145, 247265. Alginate production by plant pathogenic pseudomonads.
64. Hisamatsu, M.; Nomura, S.; Shutsrirung, A.; Obata, H.; Appl. Environ. Microbiol. 1986, 52, 466473.
Teranishi, K.; Yamada, T.; Nuswantara, S.; Yamashita, 81. Schurks, N.; Wingender, J.; Flemming, H.C.; Mayer, C.
454 Sutherland
Monomer composition and sequence of alginates from 100. Voepel, K.C.; Buller, C.S. Formation of an extracellular
Pseudomonas aeruginosa. Int. J. Biol. Macromol. 2002, 30, energy reserve by Cellulomonas avigena strain KU. J.
105111. Ind. Microbiol. 1990, 5, 131138.
82. Bian, W.; Chandrasekaran, R.; Ogawa, K. X-ray struc- 101. Legoux, R.; Lelong, P.; Jourde, C.; Feuillerat, C.;
ture analysis of the sodium salt of beijeran. Carbohydr. Capdeviellle, J.; Sure, V.; Ferran, E.; Kaghad, M.;
Res. 2002a, 337, 305314. Delpech, B.; Shire, D.; Ferrara, P.; Loison, G.; Salome,
83. Lobas, D.; Nimtz, M.; Wray, V.; Schumpe, A.; Proppe, M. N-acetyl-heparosan lyase of Escherichia coli K5 gene:
C.; Deckwer, W.-D. Structure and physical properties of gene cloning and expression. J. Bacteriol. 1997, 178, 7260
the extracellular polysaccharide PS-P4 produced by 7264.
Sphingomonas paucimobilis P4 (DSM6418). Carbohydr. 102. Schiller, N.L.; Monday, S.R.; Boyd, C.M.; Keen, N.T.;
Res. 1994, 251, 303313. Ohman, D.E. Characterization of the Pseudomonas
84. ONeill, M.A.; Darvill, A.G.; Albersheim, P.; Chou, K.J. alginate lyase gene (algL): cloning, sequencing and
Structural analysis of an acidic polysaccharide secreted by expression in Escherichia coli. J. Bacteriol. 1993, 175,
Xanthobacter sp. (ATCC53272). Carbohydr. Res. 1990, 47804789.
206, 289296. 103. Osman, S.F.; Fett, W.F.; Irwin, P.L.; Bailey, D.G.; Parris,
85. Wolk, C.P., Brun, Y.V., Shimkets, L.J., Eds.; Prokaryotic N.; OConnor, J.V. Isolation and characterization of an
Development; ASM: Washington, 2000; 83104. exopolysaccharide depolymerase from Pseudomonas mar-
86. Garozza, D.; Impallomeni, G.; Spina, E.; Sturiale, L. The ginalis HTO41B. Curr. Microbiol. 1993, 26, 299304.
structure of the exocellular polysaccharide from the 104. Sengha, S.S.; Anderson, A.J.; Hacking, A.J.; Dawes, E.A.
cyanobacterium Cyanospira capsulata. Carbohydr. Res. The production of alginate by Pseudomonas mendocina in
1998, 307, 113124. batch and continuous culture. J. Gen. Microbiol. 1989,
87. Gloagen, V.; Morvan, H.; Homann, L.; Plancke, Y.; 135, 795804.
Wieruszeski, J.-M.; Lippens, G.; Strecker, G. Capsular 105. Conti, E.; Flaibani, A.; ORegan, M.; Sutherland, I.W.
polysaccharide produced by the thermophilic cyanobacte- Alginate from Pseudomonas uorescens and Pseudomonas
rium Mastigocladus laminosus. Eur. J. Biochem. 1999, putida: production and properties. Microbiology 1994,
266, 762770. 140, 11281132.
88. Hughes, K.A.; Sutherland, I.W.; Clark, J.; Jones, M.V. 106. Michaud, P.; Pheulpin, P.; Petit, E.; Barbotin, J.N.;
Bacteriophage and associated polysaccharide depoly- Heyraud, A.; Courtois, B.; Courtois, J. Identication of a
merasesnovel tools for study of bacterial biolms. J. glucuronan lyase from a mutant strain of Rhizobium
Appl. Microbiol. 1998, 85, 583590. meliloti. Int. J. Biol. Macromol. 1997, 21, 39.
89. Berth, G.; Dautzenberg, H.; Christensen, B.E.; Smidsrd, 107. Sutherland, I.W.; Kennedy, L. Polysaccharide lyases from
O. Physicochemical studies on xylinan (acetan): 2. gellan-producing Sphingomonas spp. Microbiology 1996,
Characterization by static light scattering. Biopolymers 142, 867872.
1996, 39, 721728. 108. Yamazaki, M.; Thorne, L.; Mikolajczak, M.J.; Armentr-
90. Ojinnaka, C.; Brownsey, G.J.; Morris, E.R.; Morris, V.J. out, R.W.; Pollock, T.J. Linkage of genes essential for
Eect of deacetylation on the synergetic interaction of synthesis of a polysaccharide capsule in Sphingomonas
acetan with locust bean gum or konjac mannan. strain S88. J. Bacteriol. 1996, 178, 26762687.
Carbohydr. Res. 1998, 305, 101108. 109. Sutherland, I.W. Enhancement of polysaccharide viscosity
91. Chandrasekaran, R. X-ray and molecular modeling by mutagenesis. J. Appl. Biochem. 1979, 1, 6070.
studies on the structurefunction correlations of poly- 110. Shatwell, K.P.; Sutherland, I.W.; Ross-Murphy, S.B.
saccharides. Macromol. Symp. 1999, 140, 1729. Inuence of acetyl and pyruvate substituents on the
92. Piculell, L. Gelling polysaccharides. Curr. Opin. Colloid solution properties of xanthan polysaccharide. In Physical
Interface Sci. 1998, 3, 643650. Networks; Burchard, W., Ross-Murphy, S.B., Eds.;
93. Gravanis, G.; Milas, M.; Rinaudo, M.; Clarke-Sturman, Elsevier: London, 1990; 315333.
A.J. Conformational transition and polyelectrolyte be- 111. Gunning, A.P.; Kirby, A.R.; Ridout, M.J.; Brownsey,
haviours of a succinoglycan polysaccharide. Int. J. Biol. G.J.; Morris, V.J. Investigation of gellan networks and
Macromol. 1990, 12, 195200. gels by atomic force microscopy. Macromolecules 1996,
94. Milas, M.; Reed, W.F.; Printz, S. Conformations and 29, 67916796.
exibility of native and re-natured xanthan in aqueous 112. Lee, E.J.; Chandrasekaran, R. X-ray and computer
solutions. Int. J. Biol. Macromol. 1996, 18, 211221. modelling studies on gellan related polymers: molecular
95. Christensen, B.E.; Smidsrd, O.; Stokke, B.T. The role of structures of welan, S657 and rhamsan. Carbohydr. Res.
side-chains in the Cr3+-induced gelation of xanthan and 1991, 214, 1124.
xylinan (acetan) variants. Carbohydr. Polym. 1994, 25, 113. Chandrasekaran, R.; Millane, R.P.; Arnott, S.; Atkins,
2529. E.D.T. The crystal structure of gellan. Carbohydr. Res.
96. Standal, R.; Iversen, T.; Coucheron, D.H.; Fjaervik, E.; 1988, 175, 115.
Blatny, J.M.; Valla, S. A new gene required for cellulose 114. Chandrasekaran, R.; Radha, A. Molecular architectures
production and a gene encoding cellulolytic activity in and functional-properties of gellan gum and related poly-
Acetobacter xylinum are colocalized with the bcs operon. saccharides. Trends Food Sci. Technol. 1995, 6, 143148.
J. Bacteriol. 1994, 176, 665672. 115. Campana, S.; Ganter, J.; Milas, M.; Rinaudo, M. On the
97. Matthysse, A.G.; White, S.; Lightfoot, R. Genes required solution properties of bacterial polysaccharides of the
for cellulose synthesis in Agrobacterium tumefaciens. J. gellan family. Carbohydr. Res. 1992, 231, 3138.
Bacteriol. 1995, 177, 10691075. 116. Chandrasekaran, R.; Puigjaner, L.C.; Joyce, K.L.; Arnott,
98. Pecina, A.; Paneque, A. Detection of alginate lyase by S. Cation interactions in gellan: an X-ray study of the
activity staining after SDS PAGE and subsequent potassium salt. Carbohydr. Res. 1988, 181, 2340.
renaturation. Anal. Biochem. 1994, 217, 124127. 117. Larwood, V.L.; Howlin, B.J.; Webb, G.A. Solvation eects
99. Kennedy, L.; McDowell, K.; Sutherland, I.W. Alginases on the conformational behaviour of gellan and calcium ion
from Azotobacter species. J. Gen. Microbiol. 1992, 138, binding to gellan double helices. J. Mol. Model. 1996, 2,
24652471. 175182.
118. Chandrasekaran, R.; Radha, A.; Lee, E.J. Structural roles Dea, I.C.M. Inuence of the acetyl substituent on the
of calcium ions and side-chains in welan: an X-ray study. interaction of xanthan with plant polysaccharides: I.
Carbohydr. Res. 1994, 252, 183207. XanthanLocust Bean gum systems. Carbohydr. Polym.
119. Flaibani, A.; Leonhartsberger, S.; Navarini, L.; Cescutti, 1991, 14, 2951.
P.; Paoletti, S. Solution properties of the capsular 137. Shatwell, K.P.; Sutherland, I.W.; Ross-Murphy, S.B.;
polysaccharide produced by Klebsiella pneumoniae K40. Dea, I.C.M. Inuence of the acetyl substituent on the
Int. J. Biol. Macromol. 1994, 16, 6570. interaction of xanthan with plant polysaccharides: III.
120. Cescutti, P.; Paoletti, S.; Navarini, L.; Flaibani, A. XanthanKonjac Mannan systems. Carbohydr. Polym.
Solution properties of the capsular polysaccharide pro- 1991, 14, 131147.
duced by Klebsiella pneumoniae SK1. Int. J. Biol. Macro- 138. Shatwell, K.P.; Sutherland, I.W.; Ross-Murphy, S.B.;
mol. 1993, 15, 201207. Dea, I.C.M. Inuence of the acetyl substituent on the
121. Geddie, J.L.; Sutherland, I.W. The eect of acetylation on interaction of xanthan with plant polysaccharides: II.
cation binding by algal and bacterial alginates. Biotech- XanthanGuar Gum systems. Carbohydr. Polym. 1991,
nol. Appl. Biochem. 1994, 20, 117129. 14, 115130.
122. Quintero, E.J.; Langille, S.; Weiner, R.M. The polar 139. Copetti, G.; Grassi, M.; Lapasin, R.; Pricl, S. Synergistic
polysaccharide capsule of Hyphomonas adhaerens MHS-3 gelation of xanthan gum with locust bean gum: a
has a strong anity for gold. J. Ind. Microbiol. Bio- rheological investigation. Glycoconj. J. 1997, 14, 951961.
technol. 2001, 27, 14. 140. Christensen, B.E.; Smidsrd, O.; Stokke, B.T. Xanthans
123. Koenig, M.K.; McLean, R.J.C. Inuence of metal ions with partially hydrolysed side chains: Conformation and
and temperature on the conformation of Escherichia coli transitions. In Carbohydrates and Carbohydrate Polymers;
K1 capsular polysaccharide. Biometals 1999, 12, 4752. ATL Press: Mt. Pleasant, 1993; 166173.
124. Jouon, N.; Rinaudo, M.; Milas, M.; Desbrieres, J. 141. Levy, S.; Schuyler, S.C.; Maglothin, R.K.; Staehelin, L.A.
Hydration of hyaluronic acid as a function of the coun- Dynamic simulations of the molecular conformations of
terion type and relative humidity. Carbohydr. Polym. wild type and mutant xanthan polymers suggest that
1995, 26, 6973. conformational dierences may contribute to observed
125. Rosenberg, E.; Ron, E.Z. Bioemulsansmicrobial poly- dierences in viscosity. Biopolymers 1996, 38, 251272.
meric emulsiers. Curr. Opin. Biotechnol. 1997, 8, 313316. 142. Shatwell, K.P.; Sutherland, I.W.; Ross-Murphy, S.B.;
126. Bar-Or, Y.; Shilo, M. Characterization of macromolecular Dea, I.C.M. Inuence of acetyl and pyruvate substituents
occulants produced by Phormidium sp. strain J1 and by on the solution properties of xanthan polysaccharide. Int.
Anabaenopsis circularis Pcc 6720. Appl. Environ. Micro- J. Biol. Macromol. 1990, 12, 7178.
biol. 1987, 53, 22262230. 143. Sutherland, I.W. Polysaccharide lyases. FEMS Microbiol.
127. Shepherd, R.; Rockey, J.; Sutherland, I.W.; Roller, S. Rev. 1995, 16, 323347.
Novel bioemusliers from microorganisms for use in 144. Sutherland, I.W. Polysaccharases for microbial polysac-
foods. J. Biotechnol. 1995, 40, 207217. charides. Carbohydr. Polym. 1999, 38, 319328.
128. Gutnick, D.L.; Bach, H. Engineering bacterial polymers 145. Oyaizu, H.; Komagata, K.; Amemura, A.; Harada, T. A
for the biosorption of heavy metals: new products and succinoglycan-decomposing bacterium, Cytophaga arven-
novel formulations. Appl. Microbiol. Biotechnol. 2000, sicola sp. nov. J. Gen. Appl. Microbiol. 1982, 28, 369388.
54, 451460. 146. Kennedy, L.; Sutherland, I.W. Gellan lyasesnovel poly-
129. Johri, A.K.; Blank, W.; Kaplan, D.L. Bioengineered saccharide lyases. Microbiology 1994, 140, 30073013.
emulsans from Acinetobacter calcoaceticus RAG-1 trans- 147. Craston, D.H.; Farnell, P.; Francis, J.M.; Gabriac, S.;
poson mutants. Appl. Microbiol. Biotechnol. 2002, 59, Matthews, W.; Saeed, M.; Sutherland, I.W. Determina-
217223. tion of gellan gum by capillary electrophoresis and CE-
130. Atkins, E.D.T.; Attwool, P.T.; Miles, M.J.; Morris, V.J.; Ms. Food Chem. 2001, 73, 103110.
ONeill, M.A.; Sutherland, I.W. Eect of acetylation on 148. Da Costa, A.; Michaud, P.; Heyraud, A.; Colin-Morel, P.;
the molecular interactions and gelling properties of a Courtois, B.; Courtois, J. Acetyl substitution of glucu-
bacterial polysaccharide. Int. J. Biol. Macromol. 1987, 9, ronan inuences glucuronan cleavage by GlyA from
115117. Sinorhizobium meliloti M5N1CS (NCIMB 40472). Carbo-
131. Nisbet, B.A.; Sutherland, I.W.; Bradshaw, I.J.; Kerr, M.; hydr. Polym. 2003, 51, 223228.
Morris, E.R.; Shepperson, W.A. XM6 a new gel-forming 149. Da Costa, A.; Michaud, P.; Petit, E.; Heyraud, A.; Colin-
bacterial polysaccharide. Carbohydr. Polym. 1984, 4, 377 Morel, P.; Courtois, B.; Courtois, J. Purication and
394. properties of a glucuronan lyase from Sinorhizobium
132. Ogawa, K.; Yui, T.; Nakata, K.; Kakuta, M.; Misaki, A. meliloti M5N1CS (NCIMB 40472). Appl. Environ.
X-ray study of beijeran sodium salts, a new galacturonic Microbiol. 2001, 67, 51975203.
acid-containing exopolysaccharide. Carbohydr. Res. 1997, 150. Hashimoto, W.; Miki, H.; Tsuchiya, N.; Nankai, H.;
300, 4145. Murata, K. Polysaccharide lyase: molecular cloning,
133. Gianni, R.; Cescutti, P.; Bosco, M.; Fett, W.F.; Rizzo, R. sequencing and overexpression of the xanthan lyase gene
Inuence of substituents on the solution conformation of of Bacillus sp. strain GL1. Appl. Environ. Microbiol.
the exopolysaccharide produced by Pseudomonas gin- 2001, 67, 713720.
geri strain Pf9. Int. J. Biol. Macromol. 1999, 26, 249 151. Ruijssenaars, H.J.; Debont, J.A.M.; Hartmans, S. A
253. pyruvated mannose-specic xanthan lyase involved in
134. Ridout, M.J.; Brownsey, G.J.; York, G.M.; Walker, G.C.; xanthan degradation by Paenibacillus alginolyticus xl-1.
Morris, V.J. Eect of O-acyl substituents on the func- Appl. Environ. Microbiol. 1999, 65, 24462452.
tional behaviour of Rhizobium meliloti succinoglycan. Int. 152. Rinaudo, M.; Milas, M. Enzymic hydrolysis of the
J. Biol. Macromol. 1997, 20, 17. bacterial polysaccharide xanthan by cellulase. Int. J. Biol.
135. Chandrasekaran, R.; Thailambal, V.G. The inuence of Macromol. 1980, 2, 4548.
calcium ions, acetate and L-glycerate groups on the gellan 153. Sutherland, I.W. Hydrolysis of unordered xanthan in
double helix. Carbohydr. Polym. 1990, 12, 431442. solution by fungal cellulases. Carbohydr. Res. 1984, 131,
136. Shatwell, K.P.; Sutherland, I.W.; Ross-Murphy, S.B.; 93104.
456 Sutherland
154. Christensen, B.E.; Smidsrd, O. Dependence of the 0.5 M NaCl aqueous solution characterized by light
content of unsubstituted (cellulosic) regions in prehydro- scattering. Polym. J. 2001, 33, 317321.
lysed xanthans on the rate of hydrolysis by Trichoderma 170. Fujihara, M.; Nagumo, T. The eect of the content of D-
reesei endoglucanase. Int. J. Biol. Macromol. 1996, 18, mannuronic acid and L-guluronic acid blocks in alginates
9399. on antitumour activity. Carbohydr. Res. 1992, 224, 343
155. Zogaj, X.; Nimtz, M.; Rohde, M.; Bokranz, W.; Romling, 347.
U. The multicellular morphotype of Salmonella typhimu- 171. Aketagawa, J.; Tanaka, S.; Tamura, H.; Shibata, Y.;
rium and E. coli produce cellulose as the second com- Saito, H. Activation of limulus coagulation factor G by
ponent of the extracellular matrix. Mol. Microbiol. 2001, several (1!3) h-D-glucans: comparison of the potency
39, 14521463. of glucans with identical degree of polymerization but
156. Stoolmiller, A.C.; Dorfman, A. The biosynthesis of hyal- dierent conformations. J. Biochem. 1993, 113, 683
uronic acid by Streptococcus. J. Biol. Chem. 1969, 244, 686.
236246. 172. Ohno, N.; Emori, Y.; Yadomae, T.; Saito, K.; Masuda,
157. Rosner, H.; Grimmeke, H.-D.; Knirel, Y.A.; Shashkov, A.; Oikawa, S. Reactivity of limulus amoebocyte lysate
A.S. Hyaluronic acid and a (1!4)-h-D-xylan, extracellu- towards (1!3)-h-D-glucans. Carbohydr. Res. 1990, 207,
lar polysaccharides of Pasteurella. Carbohydr. Res. 1992, 311318.
223, 329333. 173. Fukui, H.; Tanaka, M.; Misaki, A. Structure of a
158. Vann, W.F.; Schmidt, M.A.; Jann, B.; Jann, K. The physiologically active polysaccharide produced by Bacil-
structure of the capsular polysaccharide (K5 antigen) of lus polymyxa S-4. Agric. Biol. Chem. 1985, 49, 2343
urinary tract infective Escherichia coli O10:K5:H4. Eur. J. 2349.
Biochem. 1981, 116, 359364. 174. Casu, B.; Grazioli, G.; Razi, N.; Guerrini, M.; Naggi, A.;
159. Haening, P.; Shashkov, A.S.; Jann, B.; Jann, K. Analysis Torri, G.; Oreste, P.; Tursi, F.; Zoppetti, G.; Lindahl, U.
of the enzymatic cleavage (h elimination) of the capsular Heparin-like compounds prepared by chemical modica-
K5 polysaccharide of Escherichia coli by the K5-specic tion of capsular polysaccharide from Escherichia coli K5.
coliphage: a re-examination. J. Bacteriol. 1966, 178, 4747 Carbohydr. Res. 1994, 263, 271284.
4750. 175. Jouault, S.C.; Chevolot, L.; Helley, D.; Ratiskol, J.; Bros,
160. Deangelis, P.L.; Gunay, N.S.; Toida, T.; Mao, W.-J.; A.; Sinquin, C.; Roger, O.; Fischer, A.-M. Characterization,
Linhardt, R.J. Identication of the capsular polysaccha- chemical modications and in vitro anticoagulant proper-
rides of Type D and F Pasteurella multocida as ties of an exopolysaccharide produced by Alteromonas
unmodied heparin and chondroitin, respectively. Carbo- infernus. Biochim. Biophys. Acta 2001, 1528, 141151.
hydr. Res. 2002, 337, 15471552. 176. De Baets, S.; Vandamme, E.J. Yeasts as producers of
161. Lifely, M.R.; Nowicka, U.; Moreno, C. Analysis of the polysaccharides with novel application potential. Micro-
chain length of oligomers and polymers of sialic acid biology 1999, 49, 321328.
isolated from Neisseria meningitidis group B and C and 177. Ferro, V.; Fewings, K.; Palermo, M.C.; Li, C.P. Large-
Escherichia coli K1 and K92. Carbohydr. Res. 1986, 156, scale preparation of the oligosaccharide phosphate
123135. fraction of Pichia holstii NRRL Y-2448 phosphomannan
162. Pelkonen, S. Capsular sialyl chains of Escherichia coli K1 for use in the manufacture of PI-88. Carbohydr. Res.
mutants resistant to K1 phage. Curr. Microbiol. 1990, 21, 2001, 332, 183189.
2328. 178. Bresolin, T.M.B.; Milas, M.; Rinaudo, M.; Ganter,
163. Rodrigues, M.L.; Rozental, S.; Couceiro, J.N.S.S.; J.L.M.S. Xanthangalactomannan interactions as related
Angluster, J.; Alviano, C.S.; Travassos, L.R. Identica- to xanthan conformations. Int. J. Biol. Macromol. 1998,
tion of N-acetylneuraminic acid and its 9-O-acetylated 23, 263275.
derivative on the cell surface of Cryptococcus neofor- 179. Bresolin, T.M.B.; Milas, M.; Rinaudo, M.; Reicher, F.;
mansinuence on fungal phagocytosis. Infect. Immun. Ganter, J.L.M.S. Role of galactomannan composition on
1997, 65, 49374942. the binary gel form with xanthan. Int. J. Biol. Macromol.
164. Chan, D.O.; McKinnon, K.; Rubens, C.E. CpsK of 1999, 26, 225231.
Streptococcus agalactiae exhibits a2,3-sialyltransferase 180. Annable, P.; Williams, P.A.; Nishinari, K. Interaction in
activity in Haemophilus ducreyi. Mol. Microbiol. 2002, xanthan glucomannan mixtures and the inuence of
45, 109122. electrolyte. Macromolecules 1994, 27, 42044211.
165. Sutherland, I.W.; Jann, B.; Jann, K. The isolation of O- 181. Ojinnaka, C.; Jay, A.J.; Colquhoun, I.J.; Brownsey, G.J.;
acetylated fragments from the K antigen of Escherichia Morris, E.R.; Morris, V.J. Structure and conformation of
coli O8:K27(A)H by the action of phage-induced enzymes acetan polysaccharide. Int. J. Biol. Macromol. 1996, 19,
from Klebsiella aerogenes. Eur. J. Biochem. 1970, 12, 149156.
285288. 182. Kang, K.S.; Cottrell, I.W. Polysaccharides. In Microbial
166. Lenoir, A.A.; Pandey, J.P.; Grano, D.M. Antibody re- Technology. Peppler, H.J. Perlman, D., Eds,; Academic
sponses of black children to Haemophilus type b poly- Press: New York, 1979; 417481.
saccharideNeisseria meningitidis outer membrane protein 183. Doner, L.W.; Douds, D.D. Purication of commercial
conjugate vaccine in relation to the Km(1) allotype. J. gellan to monovalent cation salts results in acute mod-
Infect. Dis. 1988, 157, 12421245. ication of solution and gel properties. Carbohydr.
167. Pawlowski, A.; Svenson, S.B. Electron beam fragmenta- Polym. 1995, 273, 225233.
tion of bacterial polysaccharides as a method of producing 184. Watase, M.; Nishinari, K. Eect of potassium ions on the
oligosaccharides for the preparation of conjugate vac- rheological and thermal properties of gellan gum gels.
cines. FEMS Microbiol. Lett. 1999, 174, 255263. Food Hydrocoll. 1993, 7, 449456.
168. Chihara, G.; Maeda, Y.Y.; Suga, T.; Hamuro, J. Lentinan 185. Kang, K.S.; Veeder, G.T.; Mirrasoul, P.J.; Kaneko, T.;
as a host defence potentiator. Int. J. Immunother. 1989, 5, Cottrell, I.W. Agar-like polysaccharide produced by a
145154. Pseudomonas species: production and basic properties.
169. Zhang, L.N.; Zhang, X.F.; Zhou, P.Y.; Zhang, M.; Li, Appl. Environ. Microbiol. 1982, 43, 10861091.
X.L. Triple helix of beta-D-glucan from Lentinus edodes in 186. Geyer, U.; Heinze, T.; Stein, A.; Klemm, D.; Marsch, S.;
Schumann, D.; Schmauder, H.-P. Formation, derivatiza- calcoaceticus A2. Appl. Environ. Microbiol. 1994, 60,
tion and applications of bacterial cellulose. Int. J. Biol. 46424645.
Macromol. 1994, 16, 343347. 191. De Vuyst, L.; De Vin, F.; Vaningelgem, F.; Degeest, B.
187. Yoshinaga, F.; Tonouchi, N.; Watanabe, K. Research Recent developments in the biosynthesis and applications
Progress in production of bacterial cellulose by aeration of heteropolysaccharides from lactic acid bacteria. Int.
and agitation culture and its application as a new Dairy J. 2001, 11, 687707.
industrial material. Biosci. Biotechnol. Biochem. 1997, 192. Laws, A.; Gu, Y.C.; Marshall, V. Biosynthesis, character-
61, 219224. isation and design of bacterial exopolysaccharides from
188. Fontana, J.D.; de Souza, A.M.; Fontana, C.K.; Torriani, lactic acid bacteria. Biotechnol. Adv. 2001, 19, 597625.
I.L.; Moreschi, J.C.; Gallotti, B.J.; de Souza, S.J.; 193. Ricciardi, A.; Parente, E.; Crudele, M.A.; Zanetti, F.;
Narcisco, G.P.; Bichara, J.A.; Farah, L.F.X. Acetobacter Scolari, G.; Mannazzu, I. Exopolysaccharide production
cellulose pellicle as a temporary skin substitute. Appl. by Streptococcus thermophilus SY: production and pre-
Biochem. Biotechnol. 1990, 24, 253264. liminary characterization of the polymer. J. Appl. Micro-
189. Fiechter, A. Biosurfactants: moving towards industrial biol. 2002, 92, 297306.
application. Trends Biotechnol. 1992, 10, 208217. 194. Deasy, P.B.; Quigley, K.J. Rheological evaluation of
190. Elkeles, A.; Rosenberg, E.; Ron, E.Z. Production and deacetylated gellan (Gelrite) for pharmaceutical use. Int.
secretion of the polysaccharide dispersan of Acinetobacter J. Pharm. 1991, 73, 117123.
17
OrderDisorder Conformational Transition of Xanthan Gum
Christer Viebke
The North East Wales Institute, Water Soluble Polymers Group Plas Coch, Wrexham, United Kingdom
I. INTRODUCTION dierent research groups are studying chemical identical

polymer samples, which might explain some of the varia-
Xanthan gum is widely used in food applications, but it tion in experimental data especially regarding the nature of
also extensively used in pharmaceutical, cosmetic, and the ordered conformation.
other technical applications [1]. The practical use of xan- Xanthan gum is not a gelling polysaccharide as, e.g.,
than gum is mainly a result of its ability to induce high some of the carrageenans are, but gel forming has been
viscosity at low polymer concentration in an aqueous reported under very specic conditions [810]. This would
environment. It also has a unique shear thinning behavior typically involve certain cations of higher valence. A good
and is less prone to degradation compared with other general knowledge of the macroscopic properties obtained
polysaccharides [2]. The unique shear thinning behavior through experimental studies has been collected over the
can be observed in a 1% xanthan solution where the last two decades and it is well established that xanthan gum
viscosity at low shear rates are 50100,000 times higher undergoes an orderdisorder conformational transition
than water, but a higher shear rates only a factor of 10 upon heating. It is also well established that the ordered
dierence in viscosity is observed. These properties are conformation is stabilized by adding salt [11]. The molec-
maintained over a wide range of temperature, salt concen- ular mechanism for this orderdisorder conformational
trations, and pH. The behavior can be related to the transition has not yet been fully established, and there is
existence of an ordered rod-like (rigid) conformation with still discussion whether the ordered form is a double- or
a high persistence length [3], which can self-associate single-stranded helix. It is well known that at higher
creating domains of associate chains at low shear rates or concentration xanthan gum solutions will exhibit gel-like
at rest. Xanthan gum is a microbially anionic polysaccha- behavior. This can be attributed to weak intermolecular
ride, soluble in cold and hot water, although quite intensive interactions between the ordered aggregates.
agitation is needed to prevent the formation of lumps. The Several reviews exist where the orderdisorder transi-
polymer is produced by the fermentation with the bacteria tion is addressed [12]. Other reviews regarding the appli-
Xanthomonas campestris. Other bacterial strains would cation of xanthan gum [13], especially in mixtures with
include Xanthomonas phaseoli and Xanthomonas juglandis. polysaccharides such as the galactomannans and gluco-
The polymer is produced in a medium of D-glucose, di- mannans can be found. A number of studies on the
potassium hydrogen phosphate, and trace elements. The rheological behavior of concentrated xanthan solutions
medium is well aerated and a nitrogen source is present. under various ow conditions have also been published
The gum is recovered by precipitation with isopropyl [1416].
alcohol then dried and milled. The characteristics may
vary depending on the nitrogen and carbon source, tem-
perature, and pH [46]. The polymer is usually sold in II. PRIMARY STRUCTURE
powder form and contains about 10% moisture. The ash
content is typically 510%. The diculty to describing the Xanthan gum is an anionic branched polyelectrolyte. The
molecular conformation of xanthan gum to measured repeating unit is displayed in Fig. 1. The backbone consists
solution properties is attributable to the fact that sample of a h-(1 ! 4)-D-glucopyranose glucan. The side chains are
preparation is sensitive both to culture conditions and (3 ! 1)-a-linked D-mannopyranose-(2!)-h-D-glucuronic
isolation methods [7]. This means that it is not certain that acid- (4!)-h-D-mannopyranose [17,18]. Xanthan gum is
459
460 Viebke
Figure 1 Repeating unit for xanthan gum.
ionic and contains up to two carboxyl groups per repeat and other methods [32]. The measured molar mass varies
unit. The degree of acetyl and pyruvic acetal substitution from around a million to tens of millions. This variation in
depends on the fermentation conditions and bacterial measured molar mass values is attributable to the variation
strain [46]. The degree of acetyl and pyruvyl substitution, in processing and production of xanthan gum samples and
DSac and DSp, are dened as the fraction of respective site probably a result of the formation of aggregates (intermo-
substituted. A xanthan gum sample with a DSp=0.5 has an lecular association) [3,33]. In Fig. 2, the dierential weight
average of 1.5 carboxyl groups per repeating pentasaccha- fraction as a function of molar mass is displayed for one
ride unit. The repeat unit has a mass of f1000 Da and a xanthan gum sample at two dierent temperatures. It can
length of f1nm. The ratio of pyruvate and acetate groups be seen that the molar mass distribution is broad, covering
can be determined by using nuclear magnetic resonance a range from around one million to tens of millions. It is
(NMR), which will give a quantitative measure of both also observed that the distribution is dependent on the
species [1921]. A few more recent studies have indicated temperature, which might be attributable to the order
that xanthan gum is not a homogeneous polymer and that disorder transition. However, it shows that xanthan gum
repeating units can contain acetyl, pyruvate, both groups, has a wide molar mass distribution and that the bulk of the
or neither [22]. sample has a large molar mass. Most (weight average)
The acetyl and pyruvate groups can be selectivity molar mass values quoted for xanthan gum for the single
removed [23]. The pyruvate groups can be removed by strand is between 2 and 6 million. Some of the molar mass
heating a xanthan gum solution in presence of oxalic acid data reported in the literature are presented in Table 1. It
for a few hours. To remove the acetyl groups, adding
potassium hydroxide to a solution of xanthan gum at
room temperature for few hours would be sucient [24].
Acetyl groups tend to stabilize the ordered form, presum-
ably by promoting association of the side chains with the
backbone through hydrogen bonding. Pyruvate groups,
in contrast, tend to favor the disordered form because
electrostatic repulsion between these groups is minimized
when the side chains extend away from the backbone [24].
This eect is clearly seen in studies using xanthan gum and
xanthan gum derivatives [25]. The order of thermal stabil-
ity based on experimental data of the ordered confor-
mation is the following, pyruvic-free > native xanthan>
acetyl and pyruvic-free>acetyl-free.
A. Molar Mass and Molar Mass Distribution

Figure 2 Dierential weight fraction vs. molar mass for a
The molar mass and molar mass distribution of several xanthan sample at 25j (dashed line) and 50jC (solid line) in
xanthan samples has been analyzed by various techniques aqueous solution. The refractive index increment is 0.135
such as static light scattering [2628] by Gel Permeation mL/g. Analyzed by asymmetrical FFF combined with
Chromatography (GPC)/light scattering [29], Field Flow MALS. Cross-ow 1 mL/min and channel ow 1 mL/min.
Fractionation (FFF)/multiangle light scattering [30,31], (From Ref. 30.)
Order-Disorder Conformational Transition of Xanthan Gum 461
Table 1 Molar Mass Values Reported for Various Xanthan cycles [40], which is dierent from other polysaccharides
Gum Samples such as carrageenans, where only two conformational
states have been observed. The helical structures of the
Molar mass native and renaturated forms have been reported to be very
106 (g/mol) Solvent Reference similar [11,41]. However, other studies show a distinct
2 01.0 M NaCl 58 dierence. The renaturated conformation (RX) has the
2.2 4 M Urea 46 same molar mass as the native conformation (NX), but a
5.4 1.0 M NaCl mass per unit length of the renatured form is double that of
1.1 4 M Urea 62 the native form. The conformational change at least for
7.4 0.1 M NaCl 37 suciently high molar mass is then an intramolecular
8.7 0.010.3 M NaCl 112 process [42,43].
2.94 0.1 M NaCl 106
3.2 aqueous 30 C. Concentration Regimes of Xanthan Gum
5.5 Phosphate chloride 31
buer (pH=6), I=0.3 As for other polysaccharide systems, it is important to
3.12 0.01 M NaCl 27 dene the concentration regime in which measurements are
made. For xanthan gum, the dilute regime has been
reported to be below 0.025% w/w [3,4447]. Other studies
have indicated that this should be even lower [48]. This
can be seen that the data has been obtained under various probably reects that various xanthan gum batches behave
aqueous solution conditions and that it covers a wide range slightly dierently depending on the manufacturing proce-
in reported values. For the ordered form, molar mass data dure and the chemical composition. However, it shows that
in the order of several millions has been reported [34]. The xanthan gum is a large molecule and, taking the typical
molar mass of Keltrol, one of the most common xanthan molar mass into consideration, a rigid-like polysaccharide.
gum samples studied, has been reported to be 1.9 106 [35], For xanthan gum, a second critical concentration has been
3 106 [36], 3.5 106 [37], 7.4 106 [32]. Various dened [49]. In Fig. 3, the zero-shear rate viscosity vs.
degradation methods have been applied to depolymerize concentration is plotted for a xanthan gum sample. It is
xanthan gum [34,35]. These include sonication, enzymatic clearly observed that the curve has two breaks dividing the
degradation, and hydrolysis. The best way to control the graph into three regions. Below c* is the dilute regime. The
molecular weight is probably by sonication as no major specic viscosity, as a function of the overlap parameter
change in the chemical structure is expected [38,39], where- (c[g]), gives a master curve for various molar mass fractions
as the other methods might alter the chemical composition of xanthan gum [50]. In Fig. 4, this kind of plot is displayed
and thereby the solution behavior. for ve dierent molar mass fractions of xanthan gum. The
B. Xanthan Gum Conformations

Three conformations of xanthan gum are discussed in the
literature. These are the native xanthan gum (NX), dena-
tured (DX), and renatured xanthan gum (RX). The native
xanthan gum refers to xanthan gum dissolved in aqueous
solution containing a moderate electrolyte concentration
[34]. Thus, it has not been exposed to heating or very low
salt concentrations. The denatured state refers to xanthan
gum in solution at high temperature or in an aqueous
solution containing very low electrolyte content. The rena-
tured xanthan gum refers to xanthan gum that has been
heated and subsequently cooled. The samples analyzed in
the literature have usually been preheated by the producer
or during the manufacturing process and thus the native
conformation has been destroyed. The native conforma-
tion is a modication of the ordered conformation nor-
mally observed during analysis. Thus, a special feature of
xanthan gum is that there is evidence that heating a native
xanthan gum sample above the critical transition temper-
ature and then cooling it to the original temperature is not,
from a conformational point of view, reversible. The
conformational transition for xanthan gum would then Figure 3 Zero-shear rate specic viscosity plotted vs. the
be dened as native ! denaturated ! renaturated ! logarithm of concentration. The critical concentrations are
denaturated when going through two heating and cooling labeled c* and c**. (From Ref. 49.)
462 Viebke
coecients indicate the existence of a single coil confor-

mation for the disordered xanthan gum [58]. Thus, a
number of studies using various techniques conrms the
existence of a single stranded coil-like species, which would
correlate with the disordered conformation. All the evi-
dence suggests that the disordered form is a single chain
with a fairly high persistence length. The results found in
the literature also show that, to obtain this form, xanthan
gum should be heated to above 80jC for an extended time
[59]. This carries the risk that degradation might occur,
which could make interpretation regarding the molar mass
or molar mass distribution of the single strand dicult.
IV. ORDERED CONFORMATION OF XANTHAN

GUM
A vast number of studies have been published regarding the
orderdisorder conformational transition and the nature
of the ordered xanthan gum conformation. In these studies
on the solution behavior, a number of various experimental
techniques have been applied. The transition has also been
modeled (molecular modeling) and analyzed by various
theoretical models, such as Mannings polyelectrolyte the-
ory, PoissonBoltzmann equation and, coilhelix theories
(ZimmBragg). Further imaging using, among others, such
techniques as atomic force microscopy, electron microsco-
Figure 4 Specic viscosities of various xanthan gum frac- py, and X-ray studies, has been applied by various research
tions as a function of the overlap parameter, measurements groups. Below, the main ndings from these studies are
performed in 0.1 M NaCl at 25jC. (From Ref. 50.) summarized and discussed.
A. Ordered Conformation in Solution

slope above c[g]f5 gives a slope of f5.5, which is higher
than for the random coil type of polymers where entirely Evidence of a thermally induced orderdisorder transition
physical entanglement occurs, which typically has a value in solution has been reported in a large number of studies
of around 3.3. This data shows that it is important to be by a variety of methods such as optical rotation
aware of self-association between xanthan molecules even [11,23,25,26,34,36,51,54,55,6067], light scattering (static
at very low concentrations. The existence of an ordered ne- and dynamic) [9,26,60,68,69], dierential scanning calo-
matic phase at higher concentration has also been observed. rimetry [36,64,7072], rheology [6,14,15,70,7376], circular
dichroism [77,78], and viscosity [34,50,63,65,66,79,80].
In Figs. 5 and 6 the orderdisorder transition is dis-
III. SOLUTION PROPERTIES OF DISORDERED played as measured by optical rotation and dierential
XANTHAN GUM scanning calorimetry, respectively. It can be seen that the
transition occurs over a broad temperature range. Howev-
A few research groups [34,5153] have studied the confor- er, the transition is still fairly sharp, characteristic of a
mational properties of the disordered xanthan gum chain. sigmoidal cooperative transition. Furthermore, the transi-
It is agreed that the disordered form is only present at high tion is reversible, i.e., no hysteresis is observed as seen for
temperature and/or at very low ionic strength. The disor- other polysaccharides under certain conditions where ag-
dered form has been described as a single-stranded random gregation and gelation occur. It is normal to dene the
coil [34]. The disordered form has also been described as a transition temperature as either the onset of transition (To)
wormlike chain with a moderate persistence length [54]. or as the midpoint of the transition (Tm) [81]. The midpoint
Xanthan gum dissolved in 4 M urea and heated to 95jC for of the transition is when the sample contains equal
3 hr and then cooled to room temperature has a disordered amounts of the ordered and disordered form, whereas the
conformation similar to the one observed in solution with a onset of transition is the temperature where the ordered
very low ionic strength at higher temperatures [55]. Rheo- conformation starts to form. The onset of transition is
logical studies on the disordered conformation show a low easily dened from a number of experimental techniques.
viscosity and no shear thinning [56,57]. From electron The midpoint transition temperature has a specic theo-
microscopy, it is suggested that the denatured state of retical meaning in certain cases, especially when applying
xanthan gum is that of a single strand [53]. The activity coilhelix theory calculations. The midpoint transition
Figure 5 Orderdisorder transition of xanthan gum as monitored by optical rotation. Eect of salt addition and side chain
substitution. The dashed lines represent native xanthan and the full lines pyruvate-free xanthan. The measurements were
performed in a phosphate buer. (From Ref. 23.)
temperature for the dierential scanning calorimetry data by optical rotation, varies depending on the side-chain
shown in Fig. 6 would be f52jC. It can be seen that the substitution. It is clearly observed in Fig. 5 that, by
heating curve is endothermic and fairly broad, covering a removing the pyruvate groups, the orderdisorder transi-
temperature range of approximately 20jC. tion becomes sharper and the transition temperature
As seen in Fig. 5, the orderdisorder transition is increases slightly compared with the native xanthan sam-
dependent on the salt concentration of the solution. The ple. In this case, the data suggest that the presence of
transition temperature increases with increasing salt con-
centration. It has been shown from a number of studies
using various techniques that Tm increases linearly as a
function of log c, where c is the total salt concentration [40].
In Fig. 7, the Tm vs. the logarithm of the total salt
concentration is displayed. The midpoint transition tem-
perature has been measured by various techniques (see
gure legend), and it is clearly observed that all the data
points fall on the same line. This stabilization of the
ordered conformation on addition of electrolyte is in
agreement of screening of a helix structure. It is also
observed that the conformational transition, as measured
Figure 7 The melting temperature (Tm) vs. the logarithm of

Figure 6 Orderdisorder transition of xanthan gum (1% w/w, the ionic strength. Filled circles values from optical rotation,
0.008 M NaCl) monitored by dierential scanning calo- open circles data from circular dichroism, crosses data from
rimetry at a scan rate of 1jC/min. (From Ref. 70.) polarizing microscopy. (From Ref. 11.)
464 Viebke
pyruvate groups in the side chains favor the disordered transition presented by, among others, Liu and coworkers
conformation [82]. The side chains also aect the absolute [60], which will be discussed in more detail below.
value of the measured optical rotation. The correlation Adding electrolyte to an aqueous xanthan gum solu-
between the transition temperature (Tm) and the logarithm tion can also induce the orderdisorder conformational
of the total salt concentration is still linear, and thus is an transition [52]. Salt-induced ordering can be explained due
intrinsic relationship of the secondary structure. It has been to the alignment of trisaccharide (side chains) chains with
implied that the conformational transition observed by the main chain through hydrogen bonding [20,84]. A
optical rotation and/or dierential scanning calorimetry compact, double-stranded structure (NX) in 0.1 M NaCl
can be assigned to conformational changes taking place in was suggested to elongate to a less compact structure upon
the side chain, and not to changes in the backbone [26,64]. exposure to low salt concentration and to dissociate into
Thus, even when the transition is completed as measured single-stranded chains at even lower salt concentrations
by these techniques, a small fraction of the backbone units [85]. The addition of salt aects the conformational tran-
might be in the ordered state, which would prevent a sition by the Coulomb interaction. Thus, there is no
complete separation of the strands. The increased mobility dierence among the monovalent cations, or among diva-
of the side chains as a function of temperature compared lent cations. No specic interaction as found for kappa-
with unwinding of the backbone has been observed from carrageenan has been observed, and in this sense, the
NMR studies [21,83]. In Fig. 8, the orderdisorder transi- orderdisorder conformational transition of xanthan
tion as a function of temperature measured by optical gum is more related to the one observed for iota-carra-
rotation and NMR, respectively, on the same sample is geenan. Gelation is not normally observed although addi-
displayed. From these data, a midpoint transition temper- tions of a few salts give rise to gel-like behaviour [8,9].
ature of Tm=52jC for OR, and Tm= 67jC for NMR was The temperature-induced orderdisorder transition of
calculated. The dierences observed in the measured tran- xanthan gum has been analyzed within the ZimmBragg
sition temperature of the two methods imply that they are theory [81]. This theory has been successfully applied on
monitoring dierent phenomena occurring during the the coilhelix transition of polypeptides and polynucleo-
transition. This clearly demonstrates the complex unwind- tides, but has had less success in describing polysaccharide
ing of the ordered conformation. In an NMR study systems. From this model, r, the nucleation parameter
obtaining 23Na relaxation data, it was shown that even (cooperativity parameter), can be calculated [36,64,86].
after the completion of the orderdisorder transition as The cooperativity parameter can also be calculated from
observed by optical rotation, there were still signs of an dierential scanning calorimetry data by using the rela-
ordered structure, implying that full separation had not tionship r1/2=DHvH/DHcal. Here, DHvH is the vant Ho
occurred on the polymer strands [83]. This agrees with the enthalpy and DHcal is the measured enthalpy from dier-
data and model for the orderdisorder conformational ential scanning calorimetry measurements. The data
reported in the literature shows that the transition has a
fairly high cooperativity (small r value). Norton et al. [36]
concluded that the conformational transition could not be
described as an all-or-none process. Thus, the chain can
simultaneously contain ordered and disordered parts. The
data presented by Christensen et al. [64] gave a higher
cooperativity (smaller r value) compared with the data
from Norton et al. [36]. This was attributed to the fact that
the samples they analyzed had less h-mannose content,
again showing the inuence of the side chains on the optical
rotation and dierential scanning calorimetry data. This
observation would agree with the data shown in Fig. 5. A
problem with these model calculations is that the polydis-
persity of the polymer sample is not accounted for, which
might aect the shape of the transition. Thus, the curve
would not primarily be determined by the cooperativity
parameter. It is also dicult to incorporate any heteroge-
neity in the polymer chain. No study on well-characterized
monodisperse fractions of dierent molar mass of xanthan
gum has been published. This type of study would probably
give the most realistic prediction of the cooperativity of the
orderdisorder conformational transition.
Mannings polyelectrolyte theory [8789] has been
used to analyze the conformational transition. The calcu-
Figure 8 Determination of transition temperature (Tm) by lated data have been compared with experimental results.
NMR and optical rotation measurements at 7.5 mM NaCl. From this type of model studies, the number of strands
(From Ref. 21.) making up the ordered conformation (single- vs. double-
stranded helix) should be possible to evaluate. So far, how-

ever, the results reported in the literature are inconclusive
[12,36,52,64,71,78,9092].
This disagreement in interpretation of data from
model calculations is particularly obvious when comparing
two conductivity studies on the xanthan gum orderdisor-
der transition [91,92], where Mannings theory has been
applied. Jones and coworkers [92] reported that their
experimental data on xanthan gum at 60jC agreed well
with a random coil conformation, whereas the data at
lower temperature agreed with a single helix conformation.
This data is displayed in Fig. 9. It can be seen that the
transition between these two states is smooth. In a similar
study by Bordi et al., it was reported that the Manning
theory failed to distinguish between a double or single helix
structure at low temperature, and between a single helix
and random coil structure at higher temperatures. This
data is displayed in Fig. 10. Again, it can be seen that the Figure 10 Specic conductivity as a function of temperature
transition is smooth; however, it is not possible to draw any for xanthan in salt free solution. Polymer concentra-
conclusion about the conformation either at low (ordered tion=0.25% w/w. Theoretical curves are for random coil,
conformation) or high (disordered conformation) temper- single helix, and double helix conformations. (From Ref. 92.)
atures. The two studies covered more or less the same
temperature range. It has been pointed out that the ap-
proach of using the Manning theory to analyze experimen- py data with theoretical calculated values for the two
tal data on xanthan gum might fail as a result of the models. Paoletti et al. [71] found from their analysis on
following considerations: (1) the theory does not include transition enthalpies that the data best tted a double helix
polymerpolymer interaction, and (2) it represents the structure. Zhang et al. [90] applied the PoissonBoltzmann
polymer as a thin charged line. Xanthan gum in the ordered model to describe titration data; however, they found that
state is a helix with a diameter of f2.4 nm, and then they had to use an unrealistic value on the xanthan helix
the semidilute regime for a xanthan solution is achieved (double helix) radius to t the data. Kawakami and Nor-
at very low polymer concentrations, typically below isuye [93] also observed this in a similar study. Thus, no
0.02% w/w [78,90,91]. Thus, both of the main assumptions consistent picture has developed from polyelectrolyte cal-
in Mannings theory are not fullled. In their study, culation using various electrostatic models on the confor-
Christensen and coworkers [64] found that the best-t lay mational structure of the ordered conformation. Such
between the two models (singlesingle and doublesingle calculations (PoissonBoltzmann model) were proven to
stranded transition). They compared experimental enthal- successfully describe the conformational transition and the
inuence of various electrolytes of other polysaccharide
system such as kappa-carrageenan [94,95].
Norton et al. [51] studied the kinetic of the order
disorder transition by stopped-ow polarimetry. They
induced the ordered conformation by adding salt to a
disordered solution. The experimental data was analyzed
by using a rst-order and a second-order rate equation.
The data is displayed in Fig. 11. The analysis showed that
rst-order kinetics constantly gave the best representation
of the experimental data. This would be in accordance with
an intramolecular orderdisorder transition, and thus
excludes a double-stranded helix. They also reported a
change in molar mass (approximate doubling), but attrib-
uted this to a dimeric association between two single
helices. It was noted that a double-stranded hairpin con-
formation could not be ruled out. In another series of
Figure 9 Average distance b as a function of temperature experiments, Christensen and coworkers [29,61,96] studied
derived from the Manning electrical conductivity theory. the degradation kinetics of ordered xanthan combined with
Each point represents the average value obtained from values Monte Carlo simulations for the depolymerization kinetics
at dierent polymer concentrations from 0.6 to 10 mg/mL. of double-stranded species. They found that the depoly-
The standard deviation is also shown. The values of the merization kinetics agreed with a double-stranded struc-
charge distance for a double stranded helix, a single helix, ture. Their picture of depolymerization is schematically
and a random coil are also indicated. (From Ref. 91.) represented in Fig. 12. The minimum number of glucose
466 Viebke
[28]. Interpretation of these data led to the conclusion that

the structure of the ordered conformation agreed with
dimeric species. Small changes in molar mass can have a
signicant change on the Mark-Houwink exponent (a).
The Mark-Houwink relationship ([g]=kM a) is thus not
valid over a wide molar mass range. For low molar mass
xanthan (<250,000), the exponent [60] has a value of 1.5,
and for high molar mass values, a value of f1.0. A random
coil would give a value of 0.50.8, and a rigid rod a value of
1.8. These studies conrm that xanthan gum is a very rigid
molecule both in the ordered and the disordered confor-
mation [105]. It is this stiness combined with the weak
association between the ordered species that creates the
unique rheological behavior of xanthan gum, which is
the main reason for its use in a variety of technical
applications.
Static light scattering studies on xanthan gum has been
performed by a number of research groups. Many of these
studies support a double-stranded ordered conformation
[27,28,35,37,68,102,106,107]. However, other studies sug-
gest a more complex behavior [43,77,108].
Figure 11 Dynamics of the xanthan orderdisorder tran-
One major concern with light scattering measurements
sition (65jC, 0.5 M KCl) analyzed in terms of rst-order is the sensitivity of the technique to traces of high molar
(open circles) and second-order (lled circles) kinetics. (From mass species. This implies that even a minor presence of
Ref. 51.) incompletely dissolved xanthan gum might aect the mea-
sured molar mass or other parameters derived form light
scattering measurements. When performing measurements
on high molar mass fractions of xanthan gum in the
residues needed to stabilize a double-stranded conforma- presence of added electrolyte, self-association and microgel
tion was estimated to be f1015. From statistical thermo-
dynamic calculations, it is suggested that a population of
double-stranded helices, where each strand forms a helix
with a strand of similar size [26], should exist, but might be
dicult to reach due to kinetic reasons [97]. Igushi et al. [98]
performed kinetic uorescence measurements on various
xanthan gum samples. Their results showed the same
temperature trends as observed for the orderdisorder
transition of kappa- and iota-carrageenan, where it is well
established that a double-stranded helix is formed in
aqueous solution upon cooling or addition of salt [99].
Thus, these data support a double-stranded helix for the
ordered form of xanthan gum.
The persistence length, p, is usually used to describe
the stiness of a polymer chain. For xanthan gum, this
parameter has been determined by using the wormlike
chain model [100103]. The persistence length has also Figure 12 Schematic representation of the depolymerization
of double-stranded xanthan (AB). Side chains attached to
been estimated from electron micrographs [104]. Values
every second glucose residue are omitted for clarity. The
from dierent measurements show that xanthan gum in the
duplex structure is stabilized by segmentsegment interac-
ordered state is a very sti polymer with a persistence
tions between residues in adjacent chains (not depicted).
length of f120 nm. This value is comparable with the Random depolymerization will induce a metastable structure
polysaccharide schizophyllum and DNA. Cellulose deriv- (B) consisting of overlapping chain fragments and a fraction
atives typically have a persistence length of less than 10 nm. of short fragments (DP<DPc) unable to remain incorpo-
Longer chains behave more in the manner of wormlike rated in a duplex. A subsequent heat treatment well above
coils and shorter chains, where the contour length is the Tm results in partial or complete dissociation of the
smaller or comparable with the persistence length more duplexes. Complete dissociation results in dispersed single
like rigid rods. Smaller values for persistence length have strands of various lengths (C). The cooling step may either
been reported ( p = 70 F 15nm). In this study, the poly- induce an aggregated state (D) or perfectly matched duplexes
dispersity of the xanthan gum sample was taken into (E), largely depending on the polymer concentration. (From
consideration when interpreting the light scattering data Ref. 29.)
Figure 13 Schematic representation relating the conformation with the main parameters (charge parameter, molar mass, and
mass per unit length). (From Ref. 12.)
formation might occur. A number of light scattering sponded with a double-stranded helix in both cases. These
studies on xanthan gum have been performed on degraded data only partially agree with another light scattering study
samples, where the solubility, particularly in the presence on a similar system [43]. Milas et al. observed a similar
of salt, is better. From light scattering studies, it should be molar mass for both native and renatured xanthan gum;
possible to obtain data on the molar mass and the mass per however, from their data, they obtained a mass per unit
unit length, and thus if measurements are performed below length of 98 Da/nm for the native form compared with 200
and above the transition temperature, the structure of the Da/nm for the renatured form. A single strand for the
ordered and the disordered conformation could be native state and hairpin structure for the renatured state
revealed. The option regarding the conformational transi- could account for this information. Both studies support a
tion has been summarized by Milas. In Fig. 13, various hairpin structure for the renatured state, but disagree on
transitions and expected changes in the above parameters the conformation for the native state. In a few studies, a
are schematically described. It is seen that a number of slight decrease in molar mass as a function of increasing
options regarding the orderdisorder transition have to be temperature has been observed [28]. This decrease does not
considered. For xanthan gum, the single helix has a mass correspond to halving of the molar mass, which would be
per unit length of f1000 Da nm1, and for the double helix expected for a double-stranded helixcoil transition. The
f2000 Da nm1. decrease in molar mass was modeled [109] by using an all-
The reported light scattering data supporting double- or-none model. In the calculations, the molar-mass depen-
stranded helices do not necessarily detect a change in the dence of the equilibrium constant (for the orderdisorder
molar mass when going through the conformational tran- transition) was included [28]. The model calculations could
sition. However, the observed behavior is consistent with a explain (qualitatively) the temperature dependence of the
single disordered conformation and ordered double helix
formation [68]. In some studies, a reduction in molar mass
is clearly visible when performing measurements above and Table 2 Molar Mass Values of Xanthan Gum in
below the conformational transition temperature [28]. 0.01 M NaCl as a Function of Temperature
Capron et al. [77] studied the native and renatured xanthan
gum and concluded that the native structure probably was Temperature (jC) Molar mass (g/mol)
a double-stranded species, whereas the renatured structure
25 4,200,000
adopted a hairpin conformation. They observed a decrease
40 4,300,000
in molar mass as a function of temperature for the native 50 2,800,000
state; however, no further change in the molar mass was 60 2,200,000
thereafter observed. The data are displayed in Table 2. The
calculated mass per unit length of the polymer corre- Source: Ref. 77.
468 Viebke
Table 3 Values of Weight Average Molar Mass for Na Salt of Xanthan Gum in 0.1 M
NaCl and Cadoxen at 25jC
Mw Mw Mw (0.1 M NaCl)/
Sample (0.1 M NaCl) (Cadoxen) Mw (Cadoxen)
X4-5 7,400,000 3,560,000 2.08

X5-6 3,940,000 1,960,000 2.01
X3-5 1,420,000 743,000 1.91
X9-3 994,000 493,000 2.02
X7-3b 603,000 295,000 2.04
X6-3-7 362,000 177,000 2.05
X6-4-4 240,000 122,000 1.97
X10-4 209,000 110,000 1.90
X8-3-5 112,000 45,300 2.47
X8-3-8 74,000 34,800 2.13
Source: Ref. 82.
molar mass behavior. The most convincing evidence from NaCl and cadoxen. These data are presented in Fig. 14. It
light scattering studies supporting a double-stranded or- can be seen that the slope of the curve (for the ordered
dered conformation was presented by Sato and coworkers state) is unity for molar masses below f300,000, and then
in a series of studies [37,68,101,110]. They measured the decreases with increasing molar mass. This suggests that
molar mass of various fractions of xanthan gum in 0.01 M below 300,000, xanthan gum behaves as a rigid rod and
NaCl at 25jC (ordered conformation) and in cadoxen becomes more exible as the molar mass increases (in
[tris(ethylenediamine) cadium dihydroxide] at 25jC (disagreement with a high persistence length). The slope for
ordered conformation). The ratio of the molar mass be- cadoxen (disordered state) is considerably smaller and
tween the ordered and disordered conformation was, in agrees with a random coil-type conformation. From the
all cases, approximately 2thus supporting a double- radius of gyration vs. molar mass data, it is possible to
stranded structure for the ordered conformation. Results obtain an estimate of the mass per unit length. From the
from these molar mass studies are partly presented in Table data in Fig. 15, this would lead to ML = 1940 F 50 nm1.
3. It can be seen that their study covered a wide range of They compared this result with ML = 1940 F 80 nm1
molar masses from well below 100,000 to several millions. calculated from sedimentation studies. The good agree-
These data also show that the chemical integrity of the ment between the calculated mass per unit length from the
polymer chain is sustained during depolymerization. No two independent techniques plus the data from the cadoxen
time-dependent depolymerization of the xanthan gum studies suggest that in 0.1 M NaCl, xanthan gum is a
sample was observed in the cadoxen solution. However, double-stranded helix. For a single helix, the mass per unit
they observed a gradual decrease in viscosity, which they length is f1000 Da nm1, and for the double helix f2000
credited to a shear-induced degradation [101]. Contrary to Da nm1. Both values can be found in the literature;
these results, Callet et al. [111] showed that xanthan
molecule did degrade in cadoxen solution over a period
of 12 days. They concluded that the dierence between the
weight average molar mass in 0.1 M NaCl and in cadoxen
was a result of the degradation of the xanthan molecule,
and not a dissociation of a double-stranded helix. They
argued that if Sato and coworkers had measured the molar
mass as a function of time, the ratio between data obtained
in 0.01 M NaCl and in cadoxen would continuously
increase. It should be pointed out that the data from Sato
et al. were obtained on a number of xanthan gum samples
(see Table 3) covering a molar mass range from under
100,000 to several millions, and the results were inferential,
whereas the data presented by Callet et al. were obtained on
one sample. It has also been noted that the xanthan
molecule may be deformed by interaction with the cadmi-
um ions present in cadoxen [80], and thus the measure-
ments in cadoxen would not be a true representation of the Figure 14 Molar mass dependence of hS2i1/2 for Na salt
disordered state. Sato and coworkers also measured the xanthan in 0.1 M NaCl aqueous NaCl and cadoxen at 25jC.
radius of gyration as a function of molar mass in 0.1 M (From Ref. 102.)
masses. They concluded that a double-stranded structure

was probably the most realistic species for xanthan gum in
dilute solution. The ordered conformation of xanthan
adopts a helical conformation with a mass per unit length
of 1960 nm1 [42,43,101,112]. This mass per unit length
agrees with a double helix conformation, either a dimer of
two molecules [42,43] or a hairpin structure from a single
chain. The native structure is believed to be a single helix
with a mass per unit length of 980 nm1. Mass per unit data
for native xanthan gum and acetyl-free xanthan gum shows
that they have a double-stranded conformation, whereas
similar data for pyruvate-free xanthan gum supports a
single-chain conformation for the ordered conformation
[106]. This shows that the side chains might inuence the
conformational nature of the ordered conformation, which
might explain some of the contradicting results published
in the literature.
Figure 15 Comparison between the measured and calcu- The model for the orderdisorder transition, as pre-
lated radius of gyration using a worm-like dimer model. sented by Liu and coworkers [60,66], is schematically
(From Ref. 60.) represented in Fig. 16. The ordered conformation (25jC,
0.01 M NaCl) is a double helix similar to the one found at a
higher electrolyte concentration. As the temperature is
however, the latter tends to dominate. Published data on raised to 80jC, the double helix does not dissociate;
the mass per unit length are summarized in Table 4 however, the radius changes. A model (Fig. 16) can repre-
[32,35,93,101]. Variation in the data recorded on the mass sent the disordered conformation at 80jC, where four coils
per unit length (ML) is demonstrated by the results are joined together by a small portion of double helix
obtained on the native xanthan gum (980 Da/nm) and segment. Thus, in this temperature range, the orderdisor-
the renatured form (2000 Da/nm) in one study [43]. In an der transition would represent rst a dissociation of the
early light scattering study by Paradossi and Brant [35], the side chains from the backbone, followed by a partial
mass per unit length were analyzed for a number of melting of the double helix backbone from both ends.
xanthan gum fractions covering a wide range of molar Evidence supporting this kind of process can be found in
a number of studies. In an extensive light scattering study
on depolymerized (sonicated) xanthan gum, Hacche and
coworkers [26] could incorporate the behavior as a func-
Table 4 Mass Per Unit Length Data tion of temperature of the following parameters: the molar
Reported in the Literature mass (almost invariant), the radius of gyration (slight
increase), and the second virial coecient (steep increase),
Mass per unit (nm1) Reference with a similar model for the orderdisorder transition, as
1960 (renatured) 43 reported by Liu and coworkers. The radius of gyration, a
980 (native) function of molar mass, was quantitatively modeled [60] by
2416 27
1940F50 102
f2000 77
2040F80 93
(from light
scattering data)
1920 (sedimentation
equilibrium data)
1900F100 90
1830F80 (NX) 106
1240F160 (PFX)
1623F48 (AFX)
1850 61
1940 63
2000 35
1850 26
For a single helix, the mass per unit length is
f1000 Da nm1, and for the double helix f2000 Figure 16 Wormlike dimer model for the xanthan molecule
Da nm1. in 0.01 M NaCl at 80jC. (From Ref. 60.)
470 Viebke
using the dimeric wormlike chain model displayed in Fig. hydrogen bonds. There are two bonds between the adjacent
16. In Fig. 15, the calculated radius of gyration as a glucoses in the main chain. One between the a-D-mannose
function of molar mass is presented together with the and h-D-glucuronate of the side chain and one between the
experimental data obtained at 80jC and at 25jC. It can a-D-mannose attached directly to the main chain and a
be seen that the model aptly represents the molar mass glucose unit on the main chain. The molecular repeat
dependence measured at 80jC. This would suggest that distance is 4.7 nm [118,120,121]. X-ray analysis (dirac-
even at this temperature, no complete separation of the tion) on xanthan gum shows that, in the solid state, it forms
dimer has occurred. a helical structure with a vefold symmetry. However, X-
ray diraction data are not good enough to obtain a clear
B. Imaging of Xanthan Molecules picture of the molecular structure, i.e., the number of
chains involved in the helix formation. X-ray studies on
A variety of techniques have been used to obtain an image xanthan gum, in which one and two of the side chain sugar
of xanthan gum molecules under dierent conditions. units are absent, form structures with identical helix pitch
These techniques include atomic force microscopy and symmetry to the original xanthan gum sample
(AFM) [41,113116], scanning tunnelling microscopy [118,124,125].
[116,117], X-ray diraction [118121], and electron micros- In extended electron micrograph studies on various
copy [21,104,122,123]. polysaccharides [85,101], including xanthan gum, Stokke
Using AFM, Capron et al. [34] obtained images of et al. obtained images of both single and double helical
xanthan molecules before and after heat treatment in the structures. In a similar study by Milas et al. [123], only
dilute concentration regime. In an aqueous environment, single helix structures were observed. They interpreted the
they found isolated chains with a length of about 0.8 Am, a double helical structure observed by Stokke et al. as a side-
dimension also obtained from electron microscopy studies by-side association of single-stranded helices. This inter-
[104]. In 0.01 M NaCl, it was reported that the unheated pretation would agree with the dimeric structure suggested
samples showed associated molecules, whereas for the by kinetic and molar mass measurements [51].
heated sample, individual polymers could be observed. Direct imaging of molecules such as xanthan gum is
The conformational midpoint transition temperature for dicult to interpret. The substrate and the sample deposi-
a xanthan gum sample in 0.01 M NaCl is approximately tion technique will always aect the results. However, the
55jC. A helical pitch of 4.9 nm could be calculated from the consistency that has evolved in a number of studies indi-
data, in reasonable agreement with the estimates obtained cates the presence of dimeric species in dilute xanthan
from X-ray [120] and modeling studies [103], where a helix solutions. Evidence from electron microscopy show both
pitch of 4.7 nm was found to best represent the data. AFM single-strand, double-strand, and aggregated species, and
[113] has also been used to perform single-molecule force no rm conclusion regarding the conformation of native,
spectroscopy on xanthan gum. These measurements were and renatured xanthan gum can be drawn.
performed in the denatured and native state. By comparing X-ray diraction studies on xanthan gum [120, 121]
the data with measurements performed on carboxymethy- showed that a right-handed ve-folded structure agreed
lated cellulose (CMC), it could be shown that the denatured best with the experimental data. The structure could be
state could be represented by individual single xanthan single or double helices. Model calculation [126] showed
gum polymer chains. The comparison was made with CMC that some of the possible structures they calculated agree
as this polymer has a similar backbone to xanthan gum. with X-ray diraction data. Model calculations also
For the native xanthan molecule, they obtained a force showed that these structures displayed a side chain folding,
double that for the denatured xanthanan indication that again in agreement with models obtained from X-ray
this state consists of two molecules. Further studies using scattering. It should be pointed out that in these calcula-
AFM [114] has reported that a double-stranded xanthan tions, no consideration was taken in regards to long chain
molecule exists in dilute solutions, whereas another study molecules or double helix formation.
has [115] shown that xanthan gum can form intra- and
intermolecular supercoils. As pointed out, it is dicult to
distinguish between supercoil features and double helix V. NATURE OF THE ORDERED
separation. Scanning tunnelling microscopy on xanthan CONFORMATION
in its native ordered conformation has been reported [116],
where images of rodlike molecules with dimensions consis- From the experimental evidence and theoretical analysis, it
tent with a helix conformation have been observed. An- can be concluded that the double helix structure compat-
other group [117], using scanning tunnelling microscopy, ible with X-ray data [120,121] most likely exists in solution.
also obtained images of individual helices. They identied a Modeling studies show that both single- and double-
diameter of 6F1nm for the helix conformation. stranded structures are acceptable from a sterical point of
The X-ray analysis of Okuyama et al. [121] showed view [120,121,126,127]. Data supporting the double helix
that the double-stranded helix, either parallel or antipar- include the vast number of studies indicating a mass per
allel of xanthan in the crystalline state, has four intramo- unit length of f2000 Da/nm, as well as a few studies
lecular hydrogen bonds per repeating unit. The helical indicating doubling of molar mass on measuring above
conformation of the double helix is stabilized by these and below the transition temperature. The data by Sato et al.
[101,102] on measurements performed in cadoxen strongly molar mass. The disassociation of the dimeric structure
support the presence of a dimeric structure in 0.01 M NaCl would then take place at a higher temperature.
at ambient temperature. The contour length per main chain The complex nature of the xanthan gum molecule
glucose residue was estimated. The radii of gyration agreed probably plays a part in the divided observation reported
with the pitch (0.47 nm per glucose unit) of the 51 double- in the literature. The processing and production is not
stranded helix proposed by Okuyama et al. [121] for the performed under identical conditions, which consequently
crystalline structure of xanthan gum. Indirect evidence for gives rise to batch-to-batch variation of produced xanthan
a double helical structure would include the increased gum. Thus, research groups have probably worked on
thermal stability for the ordered conformation and the dierent batches that are not chemically identical when
electron micrograph data [21]. they investigate the structurefunctionality relationships of
Other data indicate that an intramolecular double xanthan gum. It is established that acetyl groups tend to
helix such as hairpin formation [128] (for an antiparallel stabilize the ordered form by promoting the association of
double helix) or cyclization (parallel double helix) might the side chains with the backbone through hydrogen bond-
take place under certain conditions where no change in ing [130,131]. The pyruvate groups, in contrast, tend to
molar mass is expected. favor the disordered form because electrostatic repulsion
However, other models for the ordered conformation between these groups is minimized when the side chains are
have been suggested: the single helix [20] and the dimeric projected away from the backbone. Both the single helix
(side-by-side association of single helix) [51]. The original and the double-stranded helix are supported by experimen-
model of a single helix stabilized by the alignment of the tal evidence. Further studies should concentrate on well-
side chains along the helix backbone was mainly based on characterized samples, both in chemical structure and in
the kinetic studies which showed that the orderdisorder molar mass distribution. Improved X-ray studies should be
transition followed a rst-order rate equation [51]. How- performed to investigate possible helical structures.
ever, these data can be explained within a double helix It can be concluded that xanthan gum in solution
formation if the polymer chain is long enough. Further- exists in its ordered form as a double chain, but a single
more, the cooperativity of the orderdisorder transition chain under certain conditions cannot be ruled out. The
suggests a nucleation and growth process. The initial added complexities of samples, especially regarding side
nucleation would then be the rate-determining step. chain compositions might be the reasons for some of the
Evidence for a single-stranded helix lies in the fact that contradicting data found in the literature regarding the
the spectroscopically observed orderdisorder transition nature of the ordered conformation of xanthan gum.
does not depend on polymer concentration [20]. It was also
found that the weight average molar mass of xanthan gum
in 0.0010.1 M NaCl was independent of the salt concen- REFERENCES
tration [58]. For short chains, the transition might be ex-
pected to be concentration-dependent; however, for long 1. Katzbauer, B. Properties and applications of xanthan gum.
chains, this eect would be reduced [81]which indi- Polym. Degrad. Stab. 1998, 59, 81.
cates that this result could be consistent with a double- 2. Stokke, B.T.; Christensen, B.E. Release of disordered
xanthan oligomers upon partial acid hydrolysis of double-
stranded helix. stranded xanthan. Food Hydrocoll. 1996, 10 (1), 83.
Attempts to analyze the experimental data within the 3. Cuvelier, G.; Launay, B. Concentration regimes in xanthan
framework of Mannings theory or utilizing the Poisson gum solutions deduced from ow and viscoelastic proper-
Boltzmann equation have been less successful. These mod- ties. Carbohydr. Polym. 1986, 6, 321.
els have been successfully used to gain insight in the order 4. Milas, M.; Rinaudo, M. On the existence of 2 dierent
disorder transition for other polysaccharide systems [99]. secondary structure for the xanthan in aqueous solutions.
Polym. Bull. 1984, 12, 507.
The conicting results obtained on xanthan gum probably
5. Muller, G.; Anrhourrache, M.; Lecourtier, J.; Chauveteau,
reect the bulky nature of the repeating unit. In Mannings G. Salt dependence of the conformation of a single-
theory, in particular, the criterion of a charged line is not stranded xanthan. Int. J. Biol. Macromol. 1986, 8, 167.
fullled. The low overlap concentration for the rigid-like 6. Rochefort, W.E.; Middleman, S. Rheology of xanthan
xanthan gum molecule means that polymerpolymer in- gum: salt, temperature, and strain eects in oscillatory and
teraction comes into play in most measurements, which is steady shear experiments. J. Rheol. 1987, 31, 337.
not accounted for in the analysis. 7. Milas, M.; Viehweg, H.; Weiss, A. Structure and properties
of xanthan versus fermentation time in emulsion system.
It is thought that in the ordered state, the side chains Carbohydr. Polym. 1990, 13, 119.
are folded in and associated with the backbone involving 8. Nolte, H.; John, S.; Smidsrd, O.; Stokke, B.T. Gelation of
hydrogen bonds, while in the disordered structure, the side xanthan with trivalent metal ions. Carbohydr. Polym.
chains are not associated and project away from the 1992, 18, 243.
backbone [83,129]. The process where, upon heating, the 9. Rodd, A.B.; Dunstan, D.E.; Boger, D.V.; Schmidt, J.;
side chains rst have to dissociate and the backbone can Burchard, W. Heterodyne and nonergodic approach to
dynamic light scattering of polymer gels: aqueous xanthan
adopt a twofold cellulosic-like structure is in agreement in the presence of metal ions (aluminum(III)). Macro-
with a number of experimental observations. This process molecules 2001, 34, 3339.
would explain the change in optical rotation, the enthalpy 10. Fujiwara, J.; Iwanami, T.; Takahashi, M.; Tanaka, R.;
change, and the NMR data, without a necessary change in Hatakeyama, T.; Hatakeyama, H. Structural change of
472 Viebke
xanthan gum association in aqueous solutions. Thermo- ow fractionation and analytical ultracentrifugation. Anal.
chim. Acta 2000, 352353, 241. Chem. 1998, 70, 3886.
11. Milas, M.; Rinaudo, M. Conformational investigation on 32. Holzwarth, G. Molecular weight of xanthan polysacchar-
the bacterial polysaccharide xanthan. Carbohydr. Res. ide. Carbohydr. Res. 1978, 66, 173.
1979, 76, 189. 33. Southwick, J.G.; Lee, H.; Jamieson, A.M.; Blackwell, J.
12. Milas, M.; Rinaudo, M. On the electrostatic interactions of Self-association of xanthan in aqueous solvent-systems.
ionic polysaccharides in solution. Curr. Trends Polym. Sci. Carbohydr. Res. 1980, 84, 287.
1997, 2, 47. 34. Milas, M.; Rinaudo, M. Properties of xanthan gum in
13. Kang, K.S.; Pettitt, E. In Industrial Gums; Whistler, R.L., aqueous solutions: role of the conformational transition.
DeMiller, J.N., Eds.; Academic: San Diego, 1993; Chap. Carbohydr. Res. 1986, 158, 191.
13. 35. Paradossi, G.; Brant, D.A. Light scattering study of a series
14. Lim, T.; Uhl, J.T.; Prudhomme, R.K. Rheology of self- of xanthan fractions in aqueous solution. Macromolecules
associating concentrated xanthan solutions. J. Rheol. 1984, 1982, 15, 874.
28, 367. 36. Norton, I.T.; Goodall, D.M.; Frangou, S.A.; Morris, E.R.;
15. Tam, K.C.; Tiu, C. Steady and dynamic shear properties of Rees, D.A. Mechanism and dynamics of conformational
aqueous polymer solutions. J. Rheol. 1989, 33, 257. ordering in xanthan polysaccharide. J. Mol. Biol. 1984,
16. Zirnsak, M.A.; Boger, D.V.; Tirtaatmadja, V. Steady shear 175, 371.
and dynamic rheological properties of xanthan gum 37. Sato, T.; Norisuye, T.; Fujita, H. Double-stranded helix of
solutions in viscous solvents. J. Rheol. 1999, 43, 627. xanthan in dilute solution: evidence from light scattering.
17. Jansson, P.-E.; Kenne, L.; Lindberg, B. Structure of the Polym. J. 1984, 16, 341.
extracellular polysaccharide from Xanthomonas campest- 38. Milas, M.; Rinaudo, M.; Tinland, B. Comparative
ris. Carbohydr. Res. 1975, 45, 275. depolymerization of xanthan gum by ultrasonic and
18. Melton, L.D.; Mindt, L.; Rees, D.A.; Sanderson, G.R. enzymic treatments. Rheological and structural properties.
Covalent structure of the extracellular polysaccharide from Carbohydr. Polym. 1986, 6, 95.
Xanthomonas campestris: evidence from partial hydrolysis 39. Rinaudo, M.; Milas, M. Enzymic hydrolysis of the
studies. Carbohydr. Res. 1976, 46, 245. bacterial polysaccharide xanthan by cellulase. Int. J. Biol.
19. Rinaudo, M.; Milas, M.; Lambert, F.; Vincendon, M. 1H Macromol. 1980, 2, 45.
and 13C NMR investigation of xanthan gum. Macro- 40. Rinaudo, M.; Milas, M.; Bresolin, T.; Ganter, J. Physical
molecules 1983, 16, 816. properties of xanthan, galactomannan and their mixtures
20. Morris, E.R.; Rees, D.A.; Young, G.; Walkinshaw, M.D.; in aqueous solutions. Macromol. Symp. 1999, 140, 115.
Darke, E.J. Orderdisorder transition for a bacterial 41. Capron, I.; Alexandre, S.; Muller, G. An atomic force
polysaccharide in solution. A role for polysaccharide microscopy study of the molecular organisation of
conformation in recognition between Xanthomonas path- xanthan. Polymer 1998, 39, 5725.
ogen and its plant host. J. Mol. Biol. 1977, 110, 1. 42. Chazeau, L.; Milas, M.; Rinaudo, M. Conformations of
21. Foss, P.; Stokke, B.T.; Smidsrd, O. Thermal stability and xanthan in solution: analysis by steric exclusion chroma-
chain conformational studies of xanthan at dierent ionic tography. Int. J. polym. Anal. Charact. 1995, 2, 21.
strengths. Carbohydr. Polym. 1987, 7, 421. 43. Milas, M.; Reed, W.F.; Printz, S. Conformations and
22. Kulicke, W.-M.; van Eikern, A. Determination of the exibility of native and re-natured xanthan in aqueous
microstructure of the fermentation polymer xanthan. solutions. Int. J. Biol. Macromol. 1996, 18, 211.
Makromol. Chem. Macromol. Symp. 1992, 61, 75. 44. Tinland, B.; Maret, G.; Rinaudo, M. Reptation in semi-
23. Holzwarth, G.; Ogletree, J. Pyruvate-free xanthan. Carbo- dilute solutions of wormlike polymers. Macromolecules
hydr. Res. 1979, 76, 277. 1990, 23, 596.
24. Tako, M.J. Synergistic interaction between deacylated 45. Carrington, S.; Odell, J.; Fisher, L.; Mitchell, J.; Hartley, L.
xanthan and galactomannan. Carbohydr. Chem. 1991, 10, Polyelectrolyte behaviour of dilute xanthan solutions: salt
619. eects on extensional rheology. Polymer 1996, 37, 2871.
25. Dentini, M.; Crescenzi, V.; Blasi, D. Conformational 46. Jamieson, A.M.; Southwick, J.G.; Blackwell, J. Dynamical
properties of xanthan derivatives in dilute aqueous behavior of xanthan polysaccharide in solution. J. Polym.
solution. Int. J. Biol. Macromol. 1984, 6, 93. Sci. Polym. Phys. Ed. 1982, 20, 1513.
26. Hacche, L.S.; Washington, G.E.; Brant, D.A. Light- 47. Southwick, J.G.; Jamieson, A.M.; Blackwell, J. Quasi-
scattering investigation of the temperature-driven confor- elastic light scattering studies of semidilute xanthan
mation change in xanthan. Macromolecules 1987, 20, 2179. solutions. Macromolecules 1981, 14, 1728.
27. Berth, G.; Dautzenberg, H.; Christensen, B.E.; Harding, 48. Stokes, J.R.; Graham, L.J.W.; Lawson, N.J.; Boger, D.V.
S.E.; Rother, G.; Smidsrd, O. Static light scattering Swirling ow of viscoelastic uids. Part 1. Interaction be-
studies on xanthan in aqueous solutions. Macromolecules tween inertia and elasticity. J. Fluid. Mech. 2001, 429, 67.
1996, 29, 3491. 49. Launay, B.; Cuvelier, G.; Martinez-Reyes, S. In Gums and
28. Gamini, A.; Mandel, M. Physicochemical properties of Stabilisers for the Food Industry2. Phillips, G.O.; Wed-
aqueous xanthan solutions: static light scattering. Biopol- lock, D.J.; Williams P.A. Eds.; Pergamon Press Ltd.:
ymers 1994, 34, 783. Oxford, UK, 1984; 79 pp.
29. Christensen, B.E.; Smidsrd, O.; Stokke, B.T. Metastable, 50. Arendt, O.; Kulicke, W.-M. Determination of the viscoe-
partially depolymerized xanthans and rearrangements lastic properties of a homologous series of the fermentation
toward perfectly matched duplex structures. Macromole- polymer xanthan gum. Angew. Makromol. Chem. 1998,
cules 1996, 29, 2939. 259, 61.
30. Viebke, C.; Williams, P.A. Determination of molecular 51. Norton, I.T.; Goodall, D.M.; Morris, E.R.; Rees, D.A.
mass distribution of n-carrageenan and xanthan using Kinetic evidence for intramolecular conformational order-
asymmetrical ow eld-ow fractionation. Food Hydro- ing of the ertracellular polysaccharide (xanthan) from
coll. 2000, 14, 265. Xanthomonas campestris. J. Chem. Soc. Chem. Commun.
31. Pauck, T.; Colfen, H. Hydrodynamic analysis of macro- 1980, 545.
molecular conformation. A comparative study of ow eld 52. Lambert, F.; Milas, M.; Rinaudo, M. Sodium and Calcium
counterion activity in the presence of xanthan. Int. J. Biol. induced conformational transition of double stranded
Macromol. 1985, 7, 49. xanthan in aqueous salt-solution. Biopolymers 1991, 31,
53. Holzwarth, G.; Prestridge, E.B. Multistranded helix in 1243.
xanthan polysaccharide. Science 1977, 197, 757. 73. Berry, G.C. Crossover behavior in the viscosity of semi-
54. Milas, M.; Rinaudo, M.; Duplessix, R.; Borsali, R.; Linder, exible polymers: intermolecular interactions as a function
P. Small angle neutron scattering from polyelectrolyte of concentration and molecular weight. J. Rheol. 1996, 40,
solutions: from disordered to ordered xanthan chain 1129.
conformation. Macromolecules 1995, 28, 3119. 74. Whitcomb, P.J; Macosco, C.W. Rheology of xanthan gum.
55. Southwick, J.G.; Jamieson, A.M.; Blackwell, J. Confor- J. Rheol. 1978, 22, 493.
mation of xanthan dissolved in aqueous urea and sodium 75. Milas, M.; Rinaudo, M.; Knipper, M.; Schuppiser, J.L.
chloride solutions. Carbohydr. Res. 1982, 99, 117. Flow and viscoelastic properties of xanthan gum solutions.
56. Thurston, G.B.; Pope, G.A. Shear rate dependence of the Macromolecules 1990, 23, 2506.
viscoelasticity of polymer-solutions. 2. Xanthan gum. J. 76. Rodd, A.B.; Dunstan, D.E.; Boger, D.V. Characterisation
Non-Newton. Fluid. Mech. 1981, 9, 69. of xanthan gum solutions using dynamic light scattering
57. Thurstone, G.B. Shear rate dependence of the viscoelas- and rheology. Carbohydr. Polym. 2000, 42, 159.
ticity of polymer solutions. 1. Theoretical model. J. Non- 77. Capron, I.; Brigand, G.; Muller, G. About the native and
Newton. Fluid. Mech. 1981, 9, 57. renatured conformation of xanthan exopolysaccharide.
58. Rinaudo, M.; Milas, M. Polyelectrolyte behavior of a Polymer 1997, 38, 5289.
bacterial polysaccharide from Xanthomonas campestris: 78. Bordi, F.; Cametti, C.; Paradossi, G. Conformational
comparison with carbocymethylcellulose. Biopolymers changes of xanthan in salt-free aqueous solutions: a low
1978, 17, 2663. frequency electrical conductivity study. J. Phys. Chem.
59. Kawakami, K.; Okabe, Y.; Norisuye, T. Dissociation of 1996, 100, 7148.
dimerized xanthan in aqueous solution. Carbohydr. 79. Gravanis, G.; Milas, M.; Rinaudo, M.; Tinland, B.
Polym. 1991, 14, 189. Comparative behavior of the bacterial polysaccharides
60. Liu, W.; Sato, T.; Norisuye, T.; Fujita, H. Thermally xanthan and succinoglycan. Carbohydr. Res. 1987, 160,
induced conformational change of xanthan in 0.01 M 259.
aqueous sodium chloride. Carbohydr. Res. 1987, 160, 267. 80. Bezemer, L.; Ubbink, J.B.; de Kokker, J.A.; Kuil, M.E.;
61. Christensen, B.E.; Smidsrd, O.; Elgsaeter, A.; Stokke, Leyte, J.C. On the conformational transitions of native
B.T. Depolymerization of double-stranded xanthan by xanthan. Macromolecules 1993, 26, 6436.
acid hydrolysis: characterisation of partially degraded 81. Poland, D.; Scheraga, H.A. Theory of HelixCoil Tran-
double strands and single-stranded oligomers released sitions in Biopolymers; Academic Press: New York, 1970.
from the ordered structures. Macromolecules 1993, 26, 82. Callet, F.; Milas, M.; Rinaudo, M. Inuence of acetyl and
6111. pyruvate contents on rheological properties of xanthan in
62. Morris, V.J.; Franklin, D.; IAnson, K. Rheology and dilute solution. Int. J. Biol. Macromol. 1987, 9, 291.
microstructure of dispersions and solutions of the micro- 83. Gamini, A.; de Bleijser, J.; Leyte, J.C. Physico-chemical
bial polysaccharide from Xanthomonas campestris (xan- properties of aqueous solutions of xanthan: an n.m.r.
than gum). Carbohydr. Res. 1983, 121, 13. study. Carbohydr. Res. 1991, 220, 33.
63. Hatakenaka, K.; Liu, W.; Norisuye, T. Stability of xanthan 84. Kitagawa, H.; Sato, T.; Norisuye, T.; Fujita, H. Optical-
in aqueous sodium chloride at elevated temperature. Int. J. rotation behavior of xanthan in mixtures of water and
Biol. Macromol. 1987, 9, 346. cadoxen. Carbohydr. Polym. 1985, 5, 407.
64. Christensen, B.E.; Knudsen, K.D.; Smidsrd, O.; Kita- 85. Lecourtier, J.; Chauveteau, G.; Muller, G. Salt-induced
mura, S.; Takeo, K. Temperature-induced conformational extension and dissociation of a native double-stranded
transition in xanthans with partially hydrolyzed side xanthan. Int. J. Biol. Macromol. 1986, 8, 306.
chains. Biopolymers 1993, 33, 151. 86. Stokke, B.T.; Knudsen, K.D.; Smidsrd, O.; Elgsaeter, A.
65. Nakasuga, M.; Norisuye, T. Charge-induced conformation Optical-rotation of dilute aqueous xanthan solutions at
change of xanthan in 0.01 M aqueous sodium chloride. elevated hydrostatic pressure. J. Appl. Polym. Sci. 1991, 42,
Polym. J. 1988, 20, 939. 2063.
66. Liu, W.; Norisuye, T. Thermally induced conformation 87. Manning, G.S. Limiting laws and counterion condensation
change of xanthan: interpretation of viscosity behaviour in in polyelectrolyte solutions. I. Colligative properties. J.
0.01 M aqueous sodium chloride. Int. J. Biol. Macromol. Chem. Phys. 1969, 51, 924.
1988, 10, 44. 88. Manning, G.S. Limiting laws and counterion condensation
67. Holzwarth, G. Conformation of the extracellular poly- in polyelectrolyte solutions. II. Self-diusion of the small
saccharide of Xanthomonas campestris. Biochemistry 1976, ions. J. Phys. Chem. 1969, 51, 934.
15, 4333. 89. Manning, G.S.; Ray, J. Counterion condensation revisited.
68. Liu, W.; Norisuye, T. Orderdisorder conformation J. Biomol. Struct. Dyn. 1998, 16, 461.
change of xanthan in 0.01 M aqueous sodium chloride: 90. Zhang, L.; Takematsu, T.; Norisuye, T. Potentiometric
dimensional behavior. Biopolymers 1988, 27, 1641. titration of xanthan. Macromolecules 1987, 20, 2882.
69. Coviello, T.; Burchard, W.; Dentini, M.; Crescenzi, V. 91. Bordi, F.; Cametti, C.; Paradossi, G. Radiowave dielectric
Solution properties of xanthan. 2. Dynamic and static light properties of xanthan in aqueous solutions. J. Phys. Chem.
scattering from semidilute solution. Macromolecules 1987, 1995, 99, 274.
20, 1102. 92. Jones, S.A.; Goodall, D.M.; Cutler, A.N.; Norton, I.T.
70. Pelletier, E.; Viebke, C.; Meadows, J.; Williams, P.A. A Application of conductivity studies and polyelectrolyte
rheological study of the orderdisorder conformational theory to the conformation and orderdisorder transition
transition of xanthan gum. Biopolymers 2001, 59, 339. of xanthan polysaccharide. Eur. Biophys. J. 1987, 15, 185.
71. Paoletti, S.; Cesaro, A.; Delben, F. Thermally induced 93. Kawakami, K.; Norisuye, T. Second virial coecient for
conformational transition of xanthan poly-electrolyte. charged rods: sodium xanthan in aqueous sodium chloride.
Carbohydr. Res. 1983, 123, 173. Macromolecules 1991, 24, 4898.
72. Kitamura, S.; Takeo, K.; Kuge, T.; Stokke, B.T. Thermally 94. Nilsson, S.; Piculell, L. Helixcoil transitions of ionic poly-
474 Viebke
saccharides analyzed within the PoissonBoltzmann cell molecule force spectroscopy on xanthan by AFM. Adv.
model. 4. Eects of site specic counterion binding. Mater. 1998, 3, 316.
Macromolecules 1991, 24, 3804. 114. McIntire, T.M.; Brant, D.A. Imaging of individual bio-
95. Nilsson, S.; Piculell, L.; Jonsson, B. Helixcoil transitions polymers and supramolecular assemblies using noncontact
of ionic polysaccharides analyzed within the Poisson atomic force microscopy. Biopolymers 1997, 42, 133.
Boltzmann cell model. 1. Eects of polyion concentration 115. Kirby, A.R.; Gunning, A.P.; Morris, V.J. Imaging xanthan
and valency. Macromolecules 1989, 22, 2367. gum by atomic force microscopy. Carbohydr. Res. 1995,
96. Christensen, B.E.; Smidsrd, O. Hydrolysis of xanthan in 267, 161.
dilute acideects on chemical composition, conforma- 116. Gunning, A.P.; McMaster, T.J.; Morris, V.J. Scanning
tion, and intrinsic viscosity. Carbohydr. Res. 1991, 214, 55. tunneling microscopy of xanthan gum. Carbohydr. Polym.
97. Oviatt, H.W. Jr.; Brant, D.A. Viscoelastic behavior of ther- 1993, 21, 47.
mally treated aqueous xanthan solutions in the semidilute 117. Wilkins, M.J.; Davies, M.C.; Jackson, D.E.; Roberts, C.J.;
concentration regime. Macromolecules 1994, 27, 2402. Tendler, S.J.B. An investigation of substrate and sample
98. Igushi, T.; Kitamura, S.; Kuge, T. Study of polysaccharides preparation eects on scanning tunnelling microscopy
by the uorescence method. 6. Kinetics of the salt-induced studies on xanthan gum. J. Microsc. 1993, 172, 215.
conformational transition of xanthan polysaccharide. 118. Millane, R.P.; Narasaiah, T.V. X-ray ber diraction
Makromol. Chem. Rapid Commun. 1986, 7, 497. studies of a variant of xanthan gum in which the sidechain
99. Piculell, L. In Food Polysaccharides and their Applications; terminal mannose unit is absent. Carbohydr. Polym. 1990,
Stephen, A.M., Ed.; Marcel Dekker: New York, 1995. 12, 315.
100. Yamakawa, H.; Fujii, H. Intrinsic viscosity of wormlike 119. Cairns, P.; Miles, M.J.; Morris, V.J. Intermolecular bind-
chains. Determination of the shift factor. Macromolecules ing of xanthan gum and carob gum. Nature 1986, 322, 89.
1974, 7, 128. 120. Moorhouse, R.; Walkinshaw, M.D.; Arnott, S. In Extrac-
101. Sato, T.; Kojima, S.; Norisuye, T.; Fujita, H. Double- ellular Microbial Polysaccharides; Sandford, P.A., Laskin,
stranded helix of xanthan in dilute solution: further A., Eds.; Washington, DC: ACS, 1977.
evidence. Polym. J. 1984, 16, 423. 121. Okuyama, K.; Arnott, S.; Moorhouse, R.; Walkingshaw,
102. Sato, T.; Norisuye, T.; Fujita, H. Double-stranded helix of M.D.; Atkins, E.D.T.; Wolf-Ullish, C.H. In Fiber Dif-
xanthan: dimensional and hydrodynamic properties in 0.1 fraction Methods; French, A.D., Gardener, K.D., Eds.;
M aqueous sodium chloride. Macromolecules 1984, 17, Washington, DC: ACS, 1980.
2696. 122. Stokke, B.T.; Elgsaeter, A.; Skjak-Braek, G.; Smidsrod, O.
103. Sho, T.; Sato, T.; Norisuye, T. Viscosity behaviour and The molecular size and shape of xanthan, xylinan,
persistence length of sodium xanthan in aqueous sodium bronchial mucin, alginate, and amylose as revealed by
chloride. Biophys. Chem. 1986, 25, 307. electron microscopy. Carbohydr. Res. 1987, 160, 13.
104. Stokke, B.T.; Brant, D.A. The reliability of wormlike 123. Milas, M.; Rinaudo, M.; Tinland, B.; de Murcia, G.
polysaccharide chain dimensions estimated from electron Evidence for a single stranded xanthan chain by electron
micrographs. Biopolymers 1990, 30, 1161. microscopy. Polym. Bull. 1988, 19, 567.
105. Rinaudo, M. On the abnormal exponents ag and aD in 124. Millane, R.P. Chandrasekaran, R.., Ed.; In Frontiers in
Mark Houwink type equations for wormlike chain poly- Carbohydrate Research2; London: Elsevier, 1992.
saccharides. Polym. Bull. 1992, 27, 585. 125. Millane, R.P.; Narasaiah, T.V. X-ray ber diraction
106. Coviello, T.; Kajiwara, K.; Burchard, W.; Dentini, M.; studies of a variant of xanthan gum in which the sidechain
Crescenzi, V. Solution properties of xanthan. 1. Dynamic terminal mannose unit is absent. Carbohydr. Polym. 1990,
and static light scattering from native and modied xan- 12, 315.
thans in dilute solutions. Macromolecules 1986, 19, 2826. 126. Levy, S.; Schuyler, S.C.; Maglothin, R.K.; Staehelin, L.A.
107. Zhang, L.; Liu, W.; Norisuye, T.; Fujita, H. Double- Dynamic simulations of the molecular conformations of
stranded helix of xanthan: rigidity in 0.01 M aqueous wild type and mutant xanthan polymers suggest that
sodium chloride containing 0.01 N hydrochloric acid. conformational dierences may contribute to observed
Biopolymers 1987, 26, 333. dierences in viscosity. Biopolymers 1996, 38, 251.
108. Muller, G.; Lecourtier, J. Temperature-induced extension 127. Millane, R.P.; Wang, B. In Gums and Stabilisers for the
and dissociation of native xanthan. Carbohydr. Polym. Food Industry; Phillips, G.O. Williams, P.A., Wedlock,
1988, 9, 213. D.J., Eds.; IRL Press: Oxford, 1992; Vol. 6, 541.
109. Applequist, J.; Damle, V. Thermodynamics of the helix 128. Cantor, C.R.; Schimmel, P.R. Biophysical Chemistry, Part
coil equilibrium in oligoadenylic acid from hypochromicity III. The Behavior of Biological Macromolecules; Freeman
studies. J. Am. Chem. Soc. 1965, 87, 1450. and company: New York, 1980; Chaps. 23 and 24.
110. Sato, T.; Norisuye, T.; Fujita, H. Double-stranded helix of 129. Annable, P.; Williams, P.A.; Nishinari, K. Interaction in
xanthan: dissociation behavior in mixtures of water and xanthanglucomannan mixtures and the inuence of
cadoxen. Polym. J. 1985, 17, 729. electrolyte. Macromolecules 1994, 27, 4204.
111. Callet, F.; Milas, M.; Tinland, B. Gums and stabilisers, 4th 130. Tako, M.; Nakamura, S. Rheological properties of de-
International Conference, 1987. pyruvated xanthan in aqueous media. Agric. Biol. Chem.
112. Tinland, B.; Rinaudo, M. Dependence of the stiness of the 1988, 52, 1585.
xanthan chain on the external salt concentration. Macro- 131. Tako, M.; Nakamura, S. Evidence for intermolecular
molecules 1989, 22, 1863. associations in xanthan molecules in aqueous media. Agric.
113. Li, H.; Rief, M.; Oesterhelt, F.; Gaub, H.M. Single- Biol. Chem. 1989, 53, 1941.
18
Hemicelluloses: Structure and Properties
Iuliana Spiridon
Petru Poni Institute of Macromolecular Chemistry, Jassy, Romania
Valentin I. Popa
Technical University of Jassy, Jassy, Romania
I. INTRODUCTION Morphologically, the cell wall is a complex laminate

structure that can be divided into three distinct zones: the
The high polymeric carbohydrates in the cell walls, beside middle lamella, primary cell wall, and secondary cell wall.
cellulose and pectin, are called hemicelluloses. The hydro- The primary cell walls are composed of cellulose micro-
lysates of the hemicellulosic wall fraction contain glucose, brils and an interpenetrating matrix of hemicelluloses,
xylose, mannose, galactose, arabinose, fucose, glucu- pectins, and proteins [5]. Cellulose forms the framework of
ronic acid, and mannuronic acid, in various amounts or the cell walls, hemicelluloses cross-link noncellulosic and
traces [1]. cellulosic polymers [6], and pectins provide the structural
Hemicelluloses may function both as framework and support to the cell wall [7].
matrix substances or reserve substances in seeds, where The secondary cell walls are derived from the primary
they form independent wall layers, which are mobilized walls by thickening and inclusion of lignin into the cell wall
when the seed germinates. matrix and occur inside the primary wall [8]. It consists of
In both hardwood and softwood, hemicelluloses frac- three distinct layers (S1, S2, and S3), which are easily
tion in lignied cell walls represents the matrix substance. distinguishable at an ultrastructural level from dierences
In the S-wall, the amorphous matrix is no longer necessary, in the orientation of their cellulose microbrils. The tran-
as in the P-wall, for shaping a semiuid system that extends sition from primary to secondary cell wall synthesis is
growth; it is necessary only for regulating the exibility of marked by the cessation of pectin deposition, and a noted
the cells exoskeleton. increase in the synthesis and deposition of cellulose/hemi-
These polysaccharides occur in close association with cellulose and lignin. The cellulose and noncellulosic
cellulose and lignin. They dier from cellulose by the polysaccharides of the secondary cell wall are qualitatively
composition of their sugar units, length chain, and branch- distinct from those found in the primary cell walls. The
ing of the chain molecules. The content of polysaccharides major dierences are in the hemicellulose component of the
diers from species to species. secondary cell wall, which is composed primarily of xylans
The chemical and thermal stability of hemicelluloses is and mannans.
generally lower than that of cellulose, probably due to lack The middle lamella is shared by two contiguous cells
of crystallinity and lower polymerization degree [2]. They and is composed almost entirely of pectic substances.
occur in nature as highly hydroxylated polymers, usually Study of the polysaccharides deposition process dur-
with three hydroxyl groups for each chain unit. ing the cell wall formation of Cryptomeria japonica
It has been proposed that hemicelluloses could be the evidenced that if cellulose is deposited actively between
primary molecular factor for organizing the orientation of S1 and S3 developmental stages (especially in the middle
the free radical coupling of lignin monomers [3], although part of S2 stage), xylan deposition occurs in the S1 to early
this must be modied in light of the discovery of dirigent S2 and again in the S3 developmental layers. Successive
proteins, which confer specicity in radical coupling [4]. deposition of xylan onto the cell wall increases the micro-
475
476 Spiridon and Popa
bril diameter. The large amounts of xylan that accumu- II. DISTRIBUTION AND STRUCTURE
lated on microbrils appear globular but are covered with OF HEMICELLULOSES
lignin after they are deposited [9]. The synthesis of the
xyloglucans occurs in the Golgi apparatus, by the action of Softwood hemicelluloses consist of glucomannan, galacto-
some glycosyltransferases, which use uridine 5c-diphos- glucomann, arabinan, and a small amount of arabino-(4-
phate (UDP) sugars as substrates (glucose, xylose, galac- O-methylglucurono)-xylan. Softwood xylans do not con-
tose, and fucose). It seems that xyloglucans play a tain acetyl groups; they are separated as a gel from acidic
fundamental role in maintaining spatial organization of solutions, one of them, in methylated form, being presented
the cellulose microbrils, by absorbing onto the surface as a strong lm, indicating xylans linear structure.
(adopting the conformation) of the cellulose microbrils Glucomannans are composed of h-(1,4)-linked D-
and through H-bonding, excluding water, compressing the mannose and h-(1,4)-linked D-glucose residues, presented
backbone into a microcrystalline semirigid structure. Man- in a 3:1 ratio. The mannose units are randomly distributed
nan deposition occurs mainly during secondary wall for- in the chain and have attached various amounts of a-linked
mation [10]. galactose end groups [13]. Using a selective cleavage meth-
Table 1 shows that more than 55% of matrix sub- od, Kenne et al. [14] showed the presence of the O-acetyl
stances in the (P+M) walls are reduced to 40% in the full- groups, distributed irregularly in the pine glucomannan.
grown wall (M+P+S) of birch and even to 20% for the The acetyl groups are eective in preventing molecular
pine wood [11]. orientation [15].
A new molecular model for the cell wall [12], devel- In softwood galactoglucomannans, the a-D-galacto-
oped using the partially lignied cell wall of the woody pyranosyl units are linked as a single-unit side chain to both
tissue of tobacco (Nicotiana tabacum), shows that the D-glucosyl and D-mannosyl units of the main chain by (1,6)
cellulose microbrils are continuous along the cell and bonds. Some of the mannosyl units are substituted by O-
arranged at an angle of about 10j to the long axis. The acetyl groups, equally distributed between C2 and C3 [16].
hemicelluloses and pectins are oriented orthogonal to the Galactoglucomannans are divided into two main fractions:
longitudinal axis of the cell, whereas lignin is randomly totally water-soluble, with a galactose:glucose:mannose
orientated and ll in some of the gaps in the structure (as ratio of 1:1:3, and partially water-soluble, with a galac-
function of how much lignin is present). When the cell is tose:glucose:mannose ratio of 3:1:0.1. Both are constituted
stretched, the helix will open out and the wall surface area by D-glucose and D-mannose units linked by (1!4) glyco-
will be reduced. This reduction will result in the micro- sidic bonds. The ratio of mannose, glucose, and galactose
brils being forced closer together and the matrix mate- depends on the mode of separation.
rial between the microbrils will be compressed. Even A recent study [17] shows that in galactoglucomannan
with a large helical angle to the vertical, the microbrils from spruce (Picea abies), the molar ratio for galactose:-
could still display their full tensile modulus if the gaps in glucose: mannose was approximately 0.1:1:4. About one
the matrix were lled with a sti incompressible material. third of D-mannosyl units is substituted by O-acetyl groups
The hemicelluloses and pectins compartmentalize the almost equally distributed between C2 and C3.
lignin and store elastic strain energy produced by the Arabinogalactan represents 540% by weight of Larix
compression of lignin. This model accounts for experi- wood. It is synthesized in the living ray cell and occurs in
mental mechanical data from cells that are only partial the lumen of the tracheide, ray cells, and epithelial cells. It
lignied. Where the hemicelluloses and pectin chains consists of (1,3)-linked h-D-galactopyranosyl units, each of
are oriented at a large angle to the long axis, the cova- them bearing a substituent to the Ca position. The main
lent connectivity of the lignin is crucial in determining chain contains (1,3)-linked h-D-galactopyranosyl or 3-O-
the properties. h-arabinopyranosyl-a-L-arabinofuranosyl units.
Table 1 Distribution of Polysaccharides in Wood Fibers

Birch Spruce Pine
M+P M+P+S M+P M+P+S M+P M+P+S
Galactan 16.9 1.1 16.4 1.8 20.1 3.1

Cellulose 41.4 53.7 33.4 61.77 35.5 56.2
Glucomannan 3.1 3.6 7.9 22.9 7.7 24.8
Arabinan 13.4 0.7 29.3 0.8 29.4 1.8
Glucuronoxylan 25.2 40.9
Glucuronoarabinoxylan 13.0 13.2 7.3 14.1
Source: Ref. 11.
Hemicelluloses: Structure and Properties 477
Larch arabinogalactan pretreatment induced an in- By hydrolysis of pure glucomannan from hardwood,
creased release of interferon gamma (IFNg), tumor ne- D-glucose and D-mannose have resulted in a ratio of
crosis factor alpha (TNFa), interleukin-1beta (IL-1h), and approximately 1:2. Partial hydrolysis and methylation
IL-6 but, only IFNg was involved in the enhancement of have shown that glucomannan consists of a randomly
natural killer (NK) cell cytotoxicity [18]. linear chain of h-(1,4) linked to D-glucopyranosyl and h-
Other studies have shown that larch arabinogalactan D-mannopyranosyl units.
enhances vascular permeability [19]. At the same time, In annual plants, the commonest hemicelluloses in-
larch arabinogalactan improves liver contrast enhance- cluded in farm crop consist of (1,4)-h-D-xylopyranosyl
ment and has signicant eects on hepatic lesion detection units in the main chain, with side chains of one to several
as assessed by contrast-to-noise ratios (CNR) [20]. a-L-arabinofuranosyl, D-galactopyranosyl, and h-D-glu-
Also, arabinogalactan can be used in pharmaceutical curonopyranosyl units. Also, L-rhamnosyl, L-galactosyl,
dispersions as a tablet binder and as an emulsier for water- and L-fucosyl units, and units of various methylated sugars
in-oil or oil-in-water emulsions. are presented. These hemicelluloses could be partially
Hardwood hemicelluloses by xylans and a small pro- acetylated, with the amount of acetyl groups varying up
portion of glucomannans are represented. Hardwood to 12%. For graminae, every seventh xylose unit from the
xylans are linear polymers, constituted of (1,4)-linked h- main chain of internodes is substituted with L-arabinofur-
xylanopyranosyl units that constitute the main skeleton. anoside residue. For leaves, the xylose:arabinose ratio is
Every tenth D-xylanopyranosyl unit is substituted by a 4- 1:2 [24]. The same arabinose content occurs in nodes. The
O-methyl-D-glucuronic acid residue, linked to the birch acetyl content of graminae represents 12% of dry wall cell;
xylan chain by (1,2) linkages, that has been found to retard also, a small quantity of phenolic acids is linked to C6 of
the alkaline peeling reaction. Analysis of partially hydro- heteroxylans. Arabinoxylans are synthesized from UDP-
lyzed xylan indicates that 4-O-methyl-D-glucuronic acid D-xylose and UDP-L-arabinose by the action of glycosyl
units linked to the C2 position are randomly distributed transferases in the Golgi vesicles [25]. The sugar res-
along the birch xylan backbone [21]. idues are added onto existing acceptor molecules that
Also, acetyl groups could occur as substituents in the can be oligosaccharides or polysaccharides. Cereal arab-
side chain. The acetyl content varies between 8% and 14%, inoxylans also contain ester-linked ferulic and p-cou-
which means that to 10 D-xylose units from a polysaccha- maric acids.
ride chain correspond 3,57 acetyl groups that are linked to As reserve substances, hemicelluloses from monocot-
C2 (sometimes to C3). yledonous seeds are galactoglucuronomannans. They are
The 4-O-methylglucuronoxylan from beechwood [22] present in the seeds of Liliaceae and Iridaceae and consist
presents a xylose-to-uronic acid ratio of 8:1, with the D- of glucosyl and mannosyl units in approximately equal
xylose in mixtures of neutral sugars being 95.5%, amounts, together with 3% galactosyl units. The glucosyl
Mn osmometry = 18,600 and DPcadoxen = 125. By the and mannosyl units are (1,4)-linked, with the galactosyl
electron spin resonance (ESR) method, the radical mech- units forming the polysaccharides end groups. The reserve
anism of radiolysis has been demonstrated as follows: materials in the cell wall of leguminous seeds are mostly
CUH bond cleavage from (1,4) linkage galactomannans. They are deposited outside the P-wall
and S-wall and ll the endosperm cells completely during
seed maturation. In the Caesalpinaceae family, galactosyl
units represent 2025%. From this family, in the seeds of
Ceratonia, a galactomannan with galactosyl-to-mannosyl
units ratio of 20:80 has been evidenced. Powdered endo-
sperm called carrulin is used in the textile and pharmaceu-
tical industries.
Glycosyl bond cleavage, when radicals (R4 and R1)
The puried seed galactomannan of Cassia angustif-
are formed
olis Vahl (from family Caesalpinaceae) contains man-
nose:galactose in a ratio of 2.90. It was found that the
gum from these seeds has the basic structure of legume
galactomannans with a main chain of 1,4-linked h-D-
mannopyranosyl units to which a single a-(1,6)-D-linked
galactopyranosyl unit is attached through block
Gas chromatography has evidenced the presence of pattern [26].
CO2 and H2, which conrms the pyranosyl ring cleavage. Seeds of Cassia laevigata (Caepinaceae family) are
At the C2 (sometimes C3) position of the main chain in used in indigenous medicine (in northern India) with a
hardwood xylans, the acetyl groups are linked to D-xylo- water-soluble galactomannan, with the structure given
pyranosyl units. The amount of acetyl groups is from 9% in Fig. 1.
to 14%. Location of the O-acetyl is determined either by The main chain or backbone of the galactomannan
exchanging specically O-acetyl groups into the methylene polymer is composed of (1,4) glycosidic linkages of D-
groups or by 13C nuclear magnetic resonance (NMR) galactose units. Because the ratio between D-galactose
spectroscopy [23]. and D-mannose is 5:2, the every-second repeating unit is
Figure 1 Tentative structure of C. laevigata seeds galactoglucomannan.
composed of three hexoses with two D-galactose units in helical conformation. The solubility of callose diers from
the main chain [27]. that of cellulose; characteristic for callose is its solubility in
The mannan (80%) of date seeds (Phoenix dactilifera) diluted sulfuric acid and 5% alkali.
is associated with about 10% galactose and 10% glucose. It Another h-1,3-polyglucan is laminarin, which is found
is interesting that ve exoenzymes from the germinated in the brown algae. This hemicellulose might be identical
seeds (a-mannosidase, h-mannosidase, a-galactosidase, with callose.
h-galactosidase and h-glucosidase) are isolated. A specic (1,3)-h-D-glucan, named lentinan, was well
In many seeds, collenchym-like wall thickenings are studied in some details; it contains (1,6)-h-D-Glcp branches
present as xylogalactoglucans, consisting of a main chain and has antitumor activity, preventing cancer recurrence or
of h-(1,4)-glucan, with Xy and Ga as side groups. This metastasis after surgical intervention [29].
hemicellulose appears only later, when the S-wall is From the various species of red-purple seaweeds (the
formed. It is characteristic for species of Primulates, Lina- Rhodophyceae class), a hydrophilic colloid, called agar,
ceae, Fabaceae, Rununculaceae, and Sapotaceae, and has been separated. Agar is unique among polysaccharides
appears in angiosperms as well. in that gelation occurs at a temperature relatively far below
Study on the cell wall polymers of rice grain tissues the gel-melting temperature, the main applications depend-
during dierentiation evidences a major dierence in the ing upon this high hysteresis. It contains two components:
structure of hemicellulosic polysaccharides between the one forming a strong gel (agarose) and a nongelling
preparations obtained from the endosperm cell walls and fraction (agaropectin). Agarose is a linear chain consisting
those from the cell walls of the grains other parts (Table 2). of sequences of (1,3)-linked h-D-galactopyranosyl units
Structural analysis has shown that the endosperm cell joined by (1,4) linkages to 3,6-anhydro-a-D-galactopyra-
wall contains acidic, highly branched arabinoxylans and nosyl units, as Fig. 2 shows.
xyloglucans and h-1,31,4-glucan as minor components. On cooling, followed by aggregation, agarose forms a
Hemicelluloses from bran cell wall contain (over 80% double-helical structure [30,31]. The mechanical properties
by weight) acidic arabinoxylan (dierent from endosper- are directly related to the structure of gel, especially the
mic arabinoxylan structure) as main component and xylo- number of double helices forming the junction zones [32].
glucan as minor component [28]. The double helices of agarose are threefold and left-hand-
A hemicellulose less used as a matrix substance, but ed. It has kinks in their structure, and Smith degradation
mainly as independent wall deposition without a cellulosic results in the obtaining of short blocks or segments.
framework, is callose. It is a h-polyglucan-like cellulose, In its hydrate form, agar provides the smoother non-
consisting of (1,3)-linked glucose molecules and having a irritating bulk necessary for normal peristalsis. Sodium
Table 2 Monosaccharides Composition of Hemicelluloses Obtained from Dierent Cell Wall Preparations
Neutral sugar composition (mol%)
Uronic acid
Fraction Rhamnose Fucose Arabinose Xylose Galactose Glucose content, (wt.%)
Caryopsis coat 1.0 0.4 35.6 43.6 7.4 12.0 13.5
Aleuron layer 1.0 0.4 36.7 43.5 7.4 11.1 12.8
Endosperm 0.9 0.5 26.4 41.1 1.9 29.1 12.1
Germ 1.2 0.5 36.7 38.1 8.8 14.7 13.8
Source: Ref. 28.
Figure 2 Structure of agarose.
agar, agarose, and sodium agarose are used in globulin curdlan are (1,3)-h-D-glucosidic and its average degree of
electrophoresis, immunodiusion diagnostic techniques, polymerization is about 450 (Fig. 3). It is soluble in alkaline
and gel ltration. Hydrophilic properties of agarose make solutions. Optical rotation and viscosimetry indicate a
it a good moistening additive for bread. It binds to proteins reversible change in the conformation of curdlan. Thus,
and is used to clarify wines, juices, and vinegars. For at low concentrations, it presents an ordered conforma-
medicinal tablets and capsules, it is used as binder. tion, whereas at higher concentrations, it consists of ran-
From brown seaweed (Laminaria hyperbores, Macro- dom coils and change in its conformations occurs at about
cystis pyrifera, and Ascophyllum nodosum), alginates are 0.24 M, as conrmed by 13C NMR [34]. On heating (above
extracted. Alginate is made up of the ve-carbon polymers 120jC), a single helix conformation is changed to a triple-
D-mannuronic acid and L-guluronic acid; the distribution stranded helix. Regular or irregular short-branch substitu-
of the uronic acids along the chain is nonrandom and tion on the main-chain O6 hydroxyls does not aect the
involves relatively long sequences of each uronic acid. In triplex structure as scleroglucan, which retains a triplex
the presence of divalent cations (such as calcium ion), it structure even in aqueous solution.
forms irreversible gels. The ratio of calcium over which The NMR studies show that curdlan undergoes gela-
thixotropic gels are formed depends on the alginate type, tion by forming a pseudo-cross-linking domain composed
pH, and solid content of the system [33]. Alginates must be of single-helical chains associated hydrophobically after
regarded as a family of polymers with a wide range in swelling at lower temperatures and, subsequently, by in-
chemical composition, molecular size, and, hence, func- creasing the triple-helix fraction at elevated temperature.
tional properties. They are compatible with a wide variety The triple-helical conformation of the anhydrous form
of materials including other thickeners, lattices, oils, sug- occurs at the initial stage of annealing and a transforma-
ars, fats, pigments, various surfactants, and alkalimetal tion is possible from anhydrous to hydrous form [35].
solutions. The incompatibility occurs as result of a reaction Curdlan gels are stable over a wide pH range. They
with divalent cations (except magnesium) or other heavy might be useful for removing tannin from various foods
metal ions, cationic quaternary amines, or chemical agents and also for improving texture, stability of water binding,
that cause alkaline degradation or acid precipitation. and holding (at concentrations between 0.05% and 3%).
Alginates have a hemostatic function and are able A derivative of curdlan, carboxymethylcurdlan
to absorb specic solutes, whereas calcium alginates are proved to have antitumor activity [36]. Another derivative,
used as hydrogel dressings due to large pore size and high curdlan sulfate [37,38], proved to have a blocking eect on
water absorbency. human immunodeciency virus (HIV) infection.
Such alginate gel capsules should ideally have high Pullulan is a polysaccharide resulting from fermenta-
mechanical and chemical stability; controllable swelling tion of sugars (such as maltose, xylose, arabinose, glucose,
properties; low content of toxic, pyrogenic, and immuno- and sucrose) by Aureobasidum pullulans. It is a linear
genic materials; dened pore size; and narrow pore size glucan consisting of repeating three glucose units joined
distribution. These characteristics could be obtained by by a-D-(1,6) linkages (Fig. 4).
selection and purication of alginates from seaweeds or
bacteria, followed by further renement (by chemical
fractionation and modication, or by enzymatic modica-
tion) and control of the gelling process, in combination
with other biopolymers.
Strains of dierent microorganisms are capable of
producing various polysaccharides with important appli-
cations. Thus, curdlan is produced by Alcaligenes faecalis
var. myxogenes or some strains of Agrobacterium and
Rhizobium by secondary metabolism (associated with the
stationary phase of growth) of sugars, especially D-glucose
at low concentrations of inorganic salts and pH between 6
and 7. Curdlan is a h-(1,3)-linked D-glucan and a thermo-
regulatable polysaccharide. More than 99% of linkages in Figure 3 Structure of curdlan (n f 450).
Figure 4 Structure of pullulan.
Its solubility and degradability can be modied by backbone is identical to that of cellulose, with trisaccha-
esterication, etherication, or cross-linking reactions. rides branches on alternating D-glucopyranosyl units.
It is important in medicine in obtaining dierent Based on a ber pattern of high quality, xanthan
vaccines. Caboxymethylpullulan is expected to be a prom- conformation in the crystal was analyzed. Thus, it was
ising carrier for targeting immune tissues with an im- found that xanthan chain takes an antiparallel right-hand-
munosuppressant to enable treatment of autoimmune ed vefold (5/1) double helix that is stabilized by four
diseases [39]. intramolecular bonds and one intermolecular hydrogen
Scleroglucans represent a class of branched polysac- bond [43].
charides that, on complete hydrolysis, gives only D-glucose. Light scattering and transition dynamics analysis con-
Their chemical structure consists of h-1,3-glucose residues rmed the helix conformation of xanthan [44]. The stabil-
with one h-1,6-D-glucose side chain every three main ity of its conformation is determined by noncovalent
residues. These glucans are produced by the fungi of bonds (such as hydrogen bonds), electrostatic interactions,
Sclerotium genus. X-ray investigations have indicated a and steric eects. The rigidity of the helical conformation
triple-helical backbone conformation, similar to that of is responsible for the xanthan behavior to ionic strength
curdlan [40]. Scleroglucan solutions may form gels, as some and pH.
of the glycosidic bonds are broken, which determines a less
highly branched molecule, whereas the helixes may aggre-
gate and form gels. Scleroglucan has high thermal stability,
and also high yield and viscosity. Because of the high yield
value, it is very eective in holding particles in suspension,
in static, as well as in dynamic conditions, without the risk
of sedimentation.
Remarkable is its compatibility with most thickeners
(such as gums, alginate, carrageenans, and cellulose deriv-
atives) and surfactants (sulfates, sulfonates, and quater-
nary ammonium salts), and it has medical applications
(modulates the immune response and shows antineoplastic
activity) [41].
Xanthan, a microbial polysaccharide produced by
Xanthomonas bacterium, presents a great scientic and
industrial interest due to its properties. It is a heteropoly-
saccharide with a high molecular weight, estimated from 15
to 50 106.
Xanthan biosynthesis starts with the assembly of
pentasaccharide repeating units, and these are then poly-
merized to produce the macromolecule. The oligosaccha-
ride repeating units of xanthan are formed by the
sequential addition of monosaccharides from energy-rich
sugar nucleotides, involving acetyl-CoA and phosphoenol-
pyruvate. A polyisoprenol phosphate from the inner mem-
brane functions as an acceptor [42].
The primary structure of xanthan (Fig. 5) consists of
two D-glucopyranosyl units, two D-mannopyranosyl units.
and one D-gluco-pyranosyluronic acid unit. The polymer Figure 5 Structure of xanthan.
The stable helix conformation is resistant at the inu- transfer, easier pumping, and more accurate lling. Gums
ence of temperature, thus explaining the moderate slope of also produce, under controlled conditions, gels of dierent
the viscosity-vs.-temperature curve. Xanthan solutions strength and stability.
have very high viscosity at low concentration. Polysaccha- There are exudate gums, seaweed gums, seed gums,
ride polyelectrolytes assume a random coil conformation, and microbial gums. Also, starch and cellulose (there are
where the variation of the electrolyte levels important easy modied by heat, oxidation, or derivatization) are
eects determines the solution viscosity. available as starting materials for obtaining gums.
Xanthan can form gels either by cross-linking with When gums are produced by pathogenic degradation
certain metal ions or by interaction with other polysac- of certain cells or whole plant tissues (cherry tree gum),
charides. These gels are used in the oil industry to modify certain cell groups derived from cambium are not dieren-
the permeability proles of heterogeneous oil reservoirs. tiated into xylem cells but remain in the parenchymatous
High solution viscosity, compatibility with salts, and state. Gum production starts in these cells and then spreads
stability to shear and heat make xanthan solution well to the adjacent dierentiated xylem tissues where the
suited to meet the requirements of polymer ooding sys- walls are autolyzed so that the pocked gum is formed in-
tems. In the food industry, xanthan is used in products side the cambium. The gums swelling capacity may break
such as beverages, desserts, and dressing. Also, it can the cortex of the cherry tree and the gum exudes through
stabilize emulsied creams with pharmaceutical and cos- this split.
metic applications. Industrial gums are available as powders with dierent
Dextrans represent (1,6)-linked h-D-glucans with the degrees of nes. They will be mixed in water, or in water
side chain attached to O3 of the backbone chain units. and other food components for hydration and dissolution.
Dextran adopts a ribbonlike conformation with the unit To prevent partial gums gelation, it is necessary to add the
cell containing two antiparallel chains, each of the two D- dry powdered gums to a highly turbulent stream of water.
glucopyranosyl units. Clinical dextrans are produced on Another method for gum dissolution in water begins with
cultures of Leuconostoc strain in the presence of sucrose; mixing of the gum with a rapidly dissolving material (such
also, they can be obtained by controlled degradation of as sucrose or alcohol), followed by separation of nes gum
native dextrans and have lower molecular weights. The particles and maintenance of their separation for a short
characteristics of branching, length, and frequency are period of high shear necessary for their dissolution.
dependent upon synthesis temperature and molecular Agaragar is a polysaccharide derived from various
weight [45]. Dextrans are readily soluble in water and species of red algae such as Sphaerococucus, Euchema, and
electrolytes, and very concentrated aqueous solutions of Gelidium and contains sulfated galactose monomers.
dextrans fractions may be prepared. Chia gum of Salvia sp. from the Labiatae family
By conjugating dextran with biologically active sub- appears either in the seed coat or in the adjacent layer.
stances, an increase in its stability has been obtained, which The exudate is either partially cross-linked or bound to the
facilitates drug targeting. Conjugates between dextran and seed surface, as it is not easily separated from the seed. It is
cytostatics display high cellular uptake and less toxicity. composed of a-D-xylopyranosyl, h-D-glucopyranosyl, and
The tumor targeting of immunoconjugate was found to be 4-O-methyl-h-D-glucopyranosyluronic acid units in the
superior to that of the drug itself and host toxicity was 2:1:1 ratio.
reduced [46,47]. Okra gum presents a rhamnogalacturonan as major
Dextrans were designed as volume expanders to component, whereas the main constituent of acacia gum is
treat hypovolemia or shock because they are osmotically arabic acid. It contains D-galactose, L-arabinose, L-rham-
active and too large to pass through the uninjured ves- nose, and D-glucuronic acid in a 3:3:1:1 ratio.
sel wall. Dextran sulfate has an inhibitory eect on the Plantago ovata gum contains 63.6% D-xylose, 20.4%
AICS virus (HIV) by inhibiting the activity of its reverse L-arabinose, 6.5% L-rhamnose, and 9% D-glucuronic
transcriptase [48]. acid [49].
The emulsion polymerization of dextran with epichlo- Guar gum, known for its thickening properties, is
rhydrin leads to insoluble cross-linked dextran beads derived from the seed bean plant Cyamopsis tetragonolo-
(Sephadex). These beads swell in water, and their ability bus. The main chain consists of (1,4)-linked h-D-mannose
to separate molecules according to size leads to their use in residues and the side chain consists of (1,6)-linked a-D-
biological applications. galactose. It has an overall mannose-to-galactose ratio of
Some of the cell wall plant hemicelluloses, which are around 2:1, the galactose substituents being regularly
extractable with hot water, are mostly characterized by an distributed along the mannose chain. The galactose units
essential part of polyuronans. These substances present a solubilize the polymer through steric eects, whereas ga-
high hydration capacity and, in contact with water, they lactose-poor regions, on the contrary, are less soluble and
form soluble colloidal glue or gel with a very high water can associate both intramolecularly and intermolecularly
content; they are called mucilages (slimes) or gums. The to form partially crystalline complexes. Because of these
main property of gums is their easy hydration to produce associations, guar has remarkable rheological properties
aqueous solutions with high viscosity at low gum concen- that are used in food, personal care, and oil recovery
trations. Gums can give thinner process viscosity, especial- industries. To fully control the properties of guar solutions
ly at higher temperatures. This can oer better heat in applications, it is essential to characterize the structural
changes induced by the dierent additives and to correlate They are negatively charged at neutral pH and approach
them with the modication of rheological behavior. Thus, zero charge at low pH. Galacturonic acid is the major
it was found that isopropyl alcohol promotes the formation constituent of all natural pectins. Pectins also contain
of a network of large-scale structures via intermolecular varying quantities of neutral sugars, mainly arabinose,
associations, thus increasing dramatically the elastic re- galactose, and rhamnose. Galacturonic acid molecules
sponse of guar solutions. On the other hand, salts (NaCl, polymerize by (1,4) bonds into linear chains. Because the
Na2CO3, and NaSCN, tested at a concentration of 1 M) position of OH at C(4) is exchanged as compared with
aect the guar on a local scale, leading to a more collapsed glucose, this bond is a-glucosidic. The a-(1,4)-polygalac-
chain conguration, thus to a lower eective volume turonic acid is termed pectic acid. Yet, its degree of
fraction and reduced viscosity [50]. dissociation is too small to make pectic acid water-soluble.
Analysis of corn ber gum hydrolysates shows 48 Its salts are called pectates.
54% D-xylose, 3335% L-arabinose, 711% D,L-galactose, In the cell wall, a considerable part (up to 50%, of the
and 3% D-glucuronic acid. The gum structure appears to carboxyl groups) is esteried with methanol. Such methyl-
consist of highly branched xylan, with attached branching ated pectic acids are pectins (Fig. 6). Because a certain
containing one to three sugar units. Corn ber gum so- number of the carboxyl groups is not esteried, pectins still
lutions produce lms of high strength. After conditioning behave as polyanions. In contrast to pectic acids, they are
at 65% relative humidity (RH), the tensile strength of the water-soluble and therefore extractable with hot water.
unplasticized lms is high, elongation is low, and elastic Their solubility increases with the degree of esterication.
modulus is high. Pectins dissolved in water can be precipitated with ethanol
Almost all gums have medicinal applications. or Ca ions. The pectin of the middle lamella in the cell walls
Mucilages appear on the surface of root hairs, leaves, is insoluble. Therefore, it is considered as a Ca-pectate.
and stalks of water plants and seeds. By hydrolysis, uronic Biogenesis of pectins proceeds during deposition on
acid, galacturonic acid, galactose, arabinose, and xylose the P-wall. At the same time, labeled representatives of this
are obtained. Glucuronic acid, mannose, and fucose are series (glucose, glucuronic acid, and xylose) appear, indi-
less frequent components. cating that the epimerase in the Golgi vesicles no longer
Mucilage functions as a regulator of seeds hydration, quantitatively transforms the resorbed glucose into galac-
or as glue that xes the seed to its substrate or in seeds of tose. Later on, deposition of the S-wall follows and the
zoochores to the bodies of animals for their dispersion. activity of the epimerase is stopped, so that the pectic
Mucilages can be stained either with a chlorozinc iodine or substances (indispensable for the extension growth of the
with pectin dyes (rhutenium red) and callose dyes (anilline P-wall) are replaced by hemicellulosic substances such as
blue). Therefore, classical histologists distinguished be- polyglucuronic acids and xylans.
tween cellulose slimes and pectin slimes. Conversion of hexose into uronic acid and pentose
Cellulose slimes are cell walls with a matrix swollen up units of cell wall polysaccharides in higher plants may
to such an extent that their ultrastructural cellulose brils occur not only by the sugar nucleotide oxidation pathway,
become separated from each other. Parallel-textured brils
confer birefringence to cellulose slimes. In the epidermis of
the seeds of Cobaea scadens, the S-wall consists of a helical
ribbon. When the seed is moisturized, the swelling sub-
stances that ll the cells extrude the helically textured S-
wall as a whole. Outside the broken epidermal cell, the helix
may be reeled o as ribbons many millimeters long and
only 1 Am in width.
Pectic slimes function as a hydration regulator in the
same (although on a larger scale) in the plasmalemma.
Plant slimes do not contain any protein and perform this
analogous function with polysaccharides. The source of
slimes is a layered S-wall, which completely lls the lumen
of the epidermal cells. They contain brils included in a
matrix with a spectacular hydration capacity. By swelling,
the increase in volume is enormous. The activated outer
wall is pushed away and the cell content is extruded. At the
same time, original layering is still visible even outside the
cell. Incorporation of water into the slime continues until
the faintly marked layer boundaries disappear completely
and a voluminous amorphous jelly is left.
Pectins function together with hemicelluloses as ma-
trix substances for the brillar network. They exist in the
middle lamella of the cell walls and in the pectic lamella of
the colenchyma walls. Pectins are polycarboxylic acids. Figure 6 Synthesis of pectic acid from protopectin.
which leads to UDP-uronate, but also by the myoinositol are most abundant. However, primary hydroxyl groups
oxidation pathway. In this case, the homocyclical inositol may be absent, as in xylan. Acetal linkages and backbone
ring is split, oxidized at C(6), and concurrently replaced by end groups correspond to those in cellulose. In addition,
a heterocycle with an O bridge between C(1) and C(5), as the hemicelluloses contain ester bound in the form of acetyl
shown by label experiments. Glucuronic acid can be gen- groups. Uronic acid groups contribute both to an acetal
erated in this way. linkage and a carboxyl group. Methyl ethers are also
Pectins are more recycled than cellulose. Several exam- present. Lignincarbohydrates covalent bonds may also
ples of active pectin degradation are known. The technical exist as esters, ethers, or acetals.
pectins extracted from apples, sugar beet, or citrus fruits
are water-soluble, due to the initiation of protopectins A. Hydrolysis Reactions
enzymic degradation. In overripe fruits of pear or snow
berries, this process results in a complete maceration of The two functional groups subjected to hydrolysis in plant
their tissues. polysaccharides are the ester and acetal linkages; of them,
Pectins are well known for their gelling, thickening, the acetals are more important because of their greater
and stabilizing properties. For these reasons, new applica- abundance and role in linking the monosaccharides in the
tions are found for pectins in the food industry and also in polymers. Hydrolysis may take place under both acidic and
the pharmaceutical and cosmetic elds. alkaline conditions. The extent and consequences of acid
The relative stability of pectin molecules depends on hydrolysis are greater. Ester groups in wood polysaccha-
the temperature and pH. Pectins have optimal stability at rides are, for the most part, acetyl substituents on the
pH 3.54. The modication of pH determines the loss of hemicellulose components. In the presence of alkali, they
stability, especially at elevated temperatures. High-esteri- are hydrolyzed to liberate the free hydroxyl groups on the
ed pectins are vulnerable to a high pH. Thus, even at pH 5, hemicellulose and acetate ions in solution. The hydroxyl
the depolymerization process is considerable; this means ions in solution are replaced by acetate, leading to a
that is dicult to raise pectin solutions pH without causing reduction in alkalinity. In conjunction with the free car-
a decline in the degree of polymerization. If depolymeriza- boxyl groups present in the cell wall components and
tion at low pH is due to hydrolysis of glycosidic bonds [51], extractives, this consumption of alkali can neutralize sig-
high-pH depolymerization is determined by h-elimination nicant quantities of an alkaline reagent and possibly limit
of methyl ester groups at the anhydrogalacturonic residue, further alkaline hydrolysis.
which has its C4 attached to the bond being split [52]. On the other hand, under acidic condition, liberation
Factors that aect gelation and gel characteristics of the free acetic acid during esters hydrolysis may increase
include temperature, pectin concentration, pH, concentra- acidity and further enhance hydrolysis of, not only addi-
tion of cosolutes (sugars), and concentrations of ions such tional ester groups, but also of acetal linkages and lignin
as Ca2+. Thus, a pectin gel is prepared, in most cases, hot bonds as well. A prime example is the so-called autohy-
and then solidied by cooling, and a pH of about 3.03.1 is drolysis reaction, in which the acetic acid liberated by
typical for high-sugar jams (with a concentration of ap- steam can cause complete hydrolysis of the hemicelluloses
proximately 65% soluble solids). Most divalent cations and convert the lignin into soluble fragments.
could be eective in pectin gelation, but only Ca2+ is used Acid hydrolysis of the acetal linkages in plant poly-
in food applications. Increasing the cation concentration saccharides follows the normal hydrolysis of glycopyrano-
determines increase of both gel strength and gelling tem- sides with ssion of the glycosyl oxygen bond between the
perature. Also, it must be mentioned that if some of the rings. The mechanism involves protonation of this oxygen
galacturonic acid subunits in the galacturonan chain con- atom, followed by slow breakdown of the conjugate acid to
tain acetyl groups at O2 or O3, gelation will be hampered. the cyclical carbonium ion, which is then rapidly attached
to water.
Because the formation of the cyclical carbonium ion is
III. REACTIONS OF HEMICELLULOSES the determining rate step, the hydrolysis rate is markedly
aected by the crystallinity of the polysaccharide. The ion
Hemicelluloses are polysaccharides that are shorter, or must assume a partially planar half-chain conguration,
branched ve-carbon sugars such as xylose or six-carbon but the intermolecular hydrogen bonds in the crystalline
sugars other than glucose. They are readily hydrolyzable regions as in cellulose would tend to keep the pyranose ring
predominantly to xylose in the case of hardwood, and to in its puckered chair form and thus retard the formation of
mannose in the case of softwood. Mannose and other the planar intermediate required for hydrolysis. For the
hexoses can be fermented to ethanol, whereas xylose and heterogeneous rate of cellulose hydrolysis, several orders of
other pentoses are readily converted to furfural, in high magnitude have been found, which are less compared with
yield, by acids. Hemicelluloses dier from cellulose in that those of simple glycosides or noncrystalline polysacchar-
they consist, for the most part, of pentose and hexose ides. Thus, the initial attack by acids involving acetal
sugars others than glucose; they are usually branched and hydrolysis in cellulose would overwhelmingly involve the
have much lower degrees of polymerization. They are not amorphous regions up to the almost complete exclusion of
crystalline so that they do not present the same barriers to the crystalline regions (Fig. 7). In the noncrystalline hemi-
accessibility as cellulose does. Again, the hydroxyl groups celluloses, all acetals are susceptible to an initial attack.
dioxide to the primary hydroxyl group, other oxidizing

agents are less specic and may aect all these groups as
well as any of the secondary hydroxyl groups. Stronger
oxidants such as nitric acid, potassium dichromate, and
potassium permanganate cause extensive oxidative degra-
dation to a series of dicarboxylic acids.
Carbonyl groups are introduced into polysaccharides
by the action of chlorine, hypochlorite, and ozone. Under
alkaline conditions, glycosidic bonds may be cleaved by h-
alkoxy elimination. Hydrogen peroxide and chlorine diox-
ide react much more slowly with polysaccharides and,
consequently, they are less degrading agents.
C. Peeling
Peeling is the term applied to the stepwise depolymeriza-
tion of polysaccharides from the reducing end groups
under alkaline conditions. In conjunction with the forma-
tion of new end groups by alkaline hydrolysis of glycosidic
Figure 7 Acid-catalyzed hydrolysis of glucopyronosides. bonds, polysaccharide degradation can be extensive.
Besides glucose (G), also small amounts of disaccharides (Di) The mechanism involves alkali-catalyzed rearrange-
are formed. ment of the aldose end group to a 2-ketose. Elimination of
the h-alkali group from the C(4)-position generates a new
aldohexose end group and the process continues down the
Under alkaline conditions, hydrolysis of glycopyranosides chain. Under alkaline pulping conditions, as many as 50
is much slower and proceeds only at higher temperatures. glucose units may be peeled from a single cellulose mole-
The mechanism involves intramolecular displacement of cule before the reaction is stopped by direct h-alkoxy
the exocyclical glycosydic oxygen with formation of cycli- elimination from the C(3) position.
cal 1,6 or 1,4 anhydroglycopyranose. Usually, after methylation by the Hakamori method,
methylated and esteried polysaccharides are obtained. By
treating with methylsulfunyl sodium in methylsulfoxide,
B. Oxidative Reactions the 4-linked substituent is eliminated and a hex-4-enopy-
Mild oxidation agents such as chlorine, bromine, or iodine ranosiduronate residue is formed.
readily convert the aldehyde end groups from the plant When a 4-O-methylglucuronoxylan is subjected to
polysaccharides to aldonic acid end groups. In cellulose, these reactions, the acid-labile hex-4-enopyranosiduronate
nitrogen dioxide selectively converts the primary hydroxyl [54] may be hydrolyzed in mild conditions without hydro-
groups on C(6) to carboxyl groups. Periodic acid is a lysis of the xylosidic linkages. Base-catalyzed h-elimination
specic oxidant for vicinal diols, yielding formaldehyde of 4-O-methyl-D-glucuronic acid residue in methylated
from primary hydroxyl groups and aldehydes from the xylan is presented in Fig. 9.
secondary ones. Wood and other lignocellulosic materials are trans-
Periodate oxidation indicates the number of a-glycol formed to pulp and paper by using dierent technologies,
group present. For each oxidized glycol unit, 1 mol of
periodate is reduced to iodate and the carbon chain is
cleaved, with the formation of two carbonyl groups.
Oxidation could occur in a-hydroxyhemiacetals. Formal-
dehyde results from the oxidation of primary hydroxyls,
and formic acid from an aldehyde or carbinol unit
between two other hydroxyl-bearing carbons [53]. Oxida-
tions are conducted in a dilute solution of sodium meta-
periodate in the dark, at low temperature and pH 33.5.
Comparison of the amount of periodate consumption and
the amount of formic acid and formaldehyde produced
oers information on the molecular structure, nature of
the end group, and points of linkage between the constit-
uent monosaccharides.
An example of periodate oxidation for reduced xylan
in Fig. 8 is presented.
Whereas the oxidation of polysaccharides by halogens
leads to the aldehydic end group and that by nitrogen Figure 8 Oxidation of reduced xylan with periodate.
cellulose dissolved in the liquor. The adsorbed xylan was

found to be resistant to heterogeneous acid hydrolysis and
the uronic acid content of the adsorbed xylan is much lower
than that of the native polysaccharide. So, it is concluded
that, upon elimination of uronic acid branches and short-
ening of the polymer, xylan is adsorbed and possibly
crystallized on the surface of cellulosic brils. The uronic
acid content from glucuroxylans and arabinoglucuroxy-
lans is greatly reduced.
The hydrolysis of hemicelluloses by sulte liquor
proceeds faster than that of cellulose because of their
amorphous nature and higher accessibility and the nature
of glycosidic bonds involved. Although partial hydrolysis
of polysaccharides determines recrystallization and de-
Figure 9 Base-catalyzed h-elimination of 4-O-methyl-D- creasing accessibility, or debranching and readsorption of
glucuronic acid residue in methylated xylan. (1) Base
hemicelluloses, it does not entirely counter the progress of
treatment; (2) mild acid hydrolysis.
the degradation. During sulte pulping, arabinogalactan,
galactoglucomannan, and pectin are completely hydro-
lyzed. The O-methylglucuronoarabinoxylan of softwood
which dier as a function of chemical or biochemical is converted to O-methylglucuronoxylan (losing the units
agents used for lignin removing. The structure of wood, arabinofuranose), which is further degraded by hydrolytic
especially distribution of cell wall components, their sur- cleavage of xylosexylose glycosidic bonds into units of
face area, and porosity, have great importance for the rate shorter chain length and largely dissolved in the cooking
of delignication process. The chemical agents concentra- liquor. The glycosidic bonds, which are closer to hexuronic
tion in the cooking liquor, the temperature, and the acid branch, are more resistant to acid hydrolysis and may
reaction time inuence the chemical reactions that are persist into the sulte spent liquor.
developed during digestion process. It must be mentioned The acetyl groups of both softwoods and hardwoods
that the hydrolytic degradation of wood polysaccharides split o by alkaline sulte liquors more easily than in acidic
lowers the yield of carbohydrates in the pulp. But strength sulte liquors. This phenomenon explains the better ad-
properties and other papermaking characteristics are sorption or retention of mannan.
markedly aected by the quantity of hemicelluloses pre- In the sulte acid process, hemicellulose degradation is
sented in pulp. Generally, a high content of hemicelluloses acid-catalyzed hydrolysis and this process permits to ob-
improves the bonding of bers in the papermaking process. tain pulps with little loss in hemicelluloses at low temper-
Also, pulps with a high hemicelluloses amount produce atures and high bisulte ion concentration.
papers of decreased opacity and increased tensile and
bursting strength. Extremely signicant amounts of hemi-
celluloses may result in a decrease in tensile and bursting IV. SOME APPLICATIONS OF HEMICELLULOSES
strength indexes because individual ber strength may be
reduced as a consequence of the decrease in the number- The chemical structure of hemicelluloses is the most im-
average molecular weight of the polymer system. That is portant factor that inuences their properties. That is why,
why an optimum hemicellulose content is very important polysaccharides present large dierences in solubility in
for the papermaking properties. solution and gel properties. Their chemical structure deter-
Two of the most used digestion processes are alkaline mines the shapes of the molecules adopted both in aqueous
pulping process and sulte process [5557]. system and in the solid state.
During alkaline cooking, the polysaccharides partici- In industrial applications, hemicelluloses are used to
pate at two dierent types of degradation: one is the peeling control water and the rheology of aqueous phases. Thus,
o of the monomers at the reducing end by oxidative they may be used as food additives, thickeners, emulsiers,
degradation and the other is the breaking of glucosidic gelling agents, adhesives, and adsorbents.
linkages by alkaline hydrolysis. The reaction rate of the Thus, derivatives of xylans (acetate, butyrate, and
hemicelluloses like xylan, glucomannan, and galactoglu- benzoate) are used as extruders for fatty acids. Carbox-
comannan depends on the accessibility, branching, types of ymethylxylans are used as detergents, occulants, and
sugars, and glycosidic bonds involved. During kraft diges- adhesives in coating paper. Due to their antitumoral and
tion, the xylan is retained by transglycosidation involving lypolemic activity, xylan sulfate derivatives have applica-
ligninxylan glycosidic linkages, where xylan chains are tions in medicine. Arabinoxylans are used as emulsiers,
grafted on another by glycosidic bond. Studies on the thickeners, or stabilizers in the food, cosmetic, or pharma-
absorption xylan on cellulose bers during cooking dem- ceutical industries. Recently, it was proved that a modied
onstrated that the adsorption of xylan increases with arabinoxylan from rice bran can be used as a safe alterna-
decreasing alkali concentration in the black liquor and tive or as an adjuvant to the existing immunotherapeutic
with increasing degradation of the xylose-containing hemi- modalities for human NK cell function [58].
Some red algal polysaccharides may control cold sore penetration along the ber axis. Rapid solubilization of
herpes. They are active compounds in Zovirax by interfer- hemicelluloses opens up the pore structure of biomass,
ing with virus binding to human cell membranes. similar to what occurs in dilute acid hydrolysis. Explosive
Glucomannans are used in the food industry (as caviar decompression phenomenon increases the specic surface
substituent), whereas arabinogalactans have applications area of the material. Thus, steam explosion increases the
in the mining (for processing of iron and copper ores) or susceptibility of biomass to enzymatic hydrolysis.
pharmaceutical industry (as a tablet binder or emulsier).
4-O-methylglucuronoxylan is a water absorption
agent and presents antitumoral activity. V. OTHER DIRECTIONS OF HEMICELLULOSE
Other hemicelluloses such as gums are stabilization TREATMENT
agents in medicine (e.g., stabilization of barium sulfate
suspensions for X-ray diagnostic preparations) or food Enzymatic hydrolysis of hemicelluloses presents important
industry (stabilization of fruit drinks) in obtaining clays practical applications. The enzymatic system acting upon
used as coatings for printing and writing. The gels obtained hemicelluloses contains hydrolase (hemicellulase) that
from dierent hemicelluloses nd applications in the cos- degrades the polyoses main chain and hemicellulases: h-
metic, pharmaceutical, and photographic industries. D-galactosidase, h-D-mannosidase, and h-D-xylosidase.
Hemicelluloses can be successfully used as binders or These enzymes hydrolyze both glycosides with low molec-
adhesives in papermaking, and as retention aids for bers. ular weight and short chains. The glycosidases action
Addition of hemicelluloses along with clay in the pulp performs the total hemicelluloses hydrolysis up to mono-
improves the retention yield, as seen in Table 3. saccharides, as the enzymes mentioned act synergistically
Bioconversion of hemicellulose to useful chemicals with the hemicellulases [60]. Similarly to most enzymes that
rst requires hydrolysis of the polysaccharides to their degrade polysaccharides, hemicellulases act upon the sub-
sugars components. A possibility is represented by strate both exohydrolytically and endohydrolytically.
steam explosion. An exoenzyme degrades polyoses through successive
Steam explosion represents a variant of the high- separation of the monosaccharide or oligosaccharide
temperature dilute acid hydrolysis technique in which units, whereas splitting occurs stepwise, usually from the
biomass is treated with saturated steam in a pressure vessel, nonreducing end of the polysaccharidic chain. The endo-
which is then ashed, causing explosive disruption of the enzyme acts randomly, inducing multiple hydrolysis,
biomass by the liberated steam. Steam explosion treatment which is accomplished by a pronounced decrease of the
is carried out at 200250jC and at a residence time of substrates polymerization degree. The polymer is degrad-
approximately 1 min. The reactor is stopped instantly by ed to monosaccharides and oligosaccharides that cannot
means of a pneumatic valve to depressurize the material, be further split.
which can then be expelled through a nozzle. The process Although the paper industrys use of enzymes in pitch
permits the separation of the three main constituents: control, dewatering, and deinking is in its infancy, pulp
cellulose is depolymerized to the required extent (the prebleaching using xylanase is a reality. Application of
amorphous regions of cellulose crystals), hemicelluloses enzymatic hydrolysis in the pulp and paper industry
are hydrolyzed to low-molecular-weight sugars, and lignin includes modications of bers to improve bleachability,
becomes soluble in organic or alkaline solvents. When drainage, or deinking. The eect of hemicellulolytic
steam explosion is carried out using high temperature or enzymes, mainly xylanases, on the bleachability of conven-
long reaction times, this treatment causes extensive degra- tional kraft pulp is reduction of bleaching chemical con-
dation of hemicellulose and lignin modications. Impreg- sumption or increased brightness.
nation of biomass with H2SO4 improves selectivity of the The concept of hemicellulase use to improve the
process, permitting a more complete degradation, hemicel- bleachability of kraft pulp was introduced by Viikari et al.
lulose solubilization, and reduced sugar degradation. Tem- [61]. The use of hemicellulases in combination with other
perature studies [59] demonstrate the importance of steam bleaching agents has led to a substantial decrease in the
Table 3 Retention of Claya by Softwood Pulp in the Presence of Hemicelluloses

Rice straw Retention value Bagasse Retention value
hemicellulosesb of clay (%) hemicellulose of clay (%)
None 25.4 None 25.4
5 37.0 5 41.2
10 30.8 10 35.6
15 33.6 15 36.0
a
Added amount of clay20% based on pulp.
b
Gram of hemicellulose per 100 g of pulp.
Source: Ref. 81.
bleaching chemicals consumption and discharge of toxic at a given apparent density. Other authors [79] reported
compounds into the environment (AOX levels in euents that xylanase treatment of commercial P. radiata selective-
have been substantially reduced). ly increased handsheet tear index relative to apparent
Although xylanase-aided bleaching treatments have density and tensile index, with retention of ber strength.
been developed and several studies have been published Selective removal of xylan during enzymatic hydrolysis
[6265], there are many unanswered questions concerning aects ber bonding.
its reaction mechanism during the enzymatic process. A It is clear that utilization of enzymes in the pulp
number of theories have been developed to explain the industry permitted important reduction of energy and
consequences of enzymatic treatment. chemical agents, and the continuous eorts of researchers
One of them is based on structural studies that indi- concerning the establishment of optimal conditions for
cated the presence of ligninhemicelluloses bonds [66,67]. enzymatic treatments will lead to improved enzymatic
According to this theory, by enzyme treatment, xylan is treatment eciency and partial resolution of environmen-
hydrolyzed into small fragments, allowing the lignin asso- tal problems.
ciated with these small fragments to be more rapidly With the awareness of the limitations anticipated in
removed by extraction. the worlds supplies of petroleum came an increased inter-
A second theory proposes that xylanase treatments est in gasoline extenders and additives. The oxygenated
remove reprecipitated xylan from the ber surface. This additives tert-butyl ether, methanol, and ethanol were
reprecipitation phenomenon occurs late in kraft cooking, found to have additional advantages of octane enhance-
when the liquor cooking is at about pH 13. Xylan reprecip- ment and lowered air pollution. Thus, by hydrolysis of
itated onto the bers is further enhanced by hydrolysis of lignocellulosic materials, saccharides are obtained. In the
acetyl and glucuronic acid groups, which determines case of acid and enzymatic hydrolysis, the main product is
straight chains of xylan. Thus, removal of reprecipitated glucose, whereas xylose results from partial hydrolysis.
xylan could increase ber permeability and lignin accessi- These compounds represent the source for the production
bility, and, nally, improve lignin extractability [68,69]. of ethanol, through fermentation of the C(6) biomass
Because removal of xylan was accompanied by lignin sugars by yeast; polyols (butanol, butanediol) could be
release (evidenced by measure of ultraviolet absorbance obtained by fermentation of biomass hydrolysate with an-
at 280 nm), it appears that xylanases contribute to increase aerobic bacteria. In birch hemicellulose hydrolysate liquor,
the extractability of lignin from softwood kraft pulp [70]. when the sugar content of the liquor was 41 g/L [80], Lac-
It is known that the high hemicellulose content of the tobacillus pentosus produced 27 g/L lactic acid and 11 g/L
pulp results in high yield and assures interber bonding acetic acid. Through fermentative synthesis, using natural
properties. The removal of hemicellulose by enzymatic or recombinant yeast strains, which express the enzyme
treatment is not expected to improve the papermaking aldose reductase (EC 1.1.1.21), xylitol was produced. Also,
properties of the pulp. Our studies concerning the use of furfural, the basic material in the production of furan and
xylanases in the bleaching of alkaline and/or acid-treated furfurilyc alcohol, could be obtained from pentosans.
wheat straw have demonstrated the inuence of enzymes Due to the abundance of literature concerning the
on the lignincarbohydrate complex, with improving structure, biogenesis, and applications of natural poly-
strength and optical properties [71,72]. Biobleaching of mers, this review was not intended to be comprehensive
wheat straw with a chlorine-free sequence (enzymeoxy- in nature. Instead, interpretation, speculation, and analysis
genhydrogen peroxide), obtaining an 11-point increase in of recent progress in this eld have been oered in an
brightness compared with OP bleached pulp and improv- attempt to stimulate new ideas and discussion.
ing some papermaking properties (folder endurance, ten-
sile, and burst index), has also been reported [73].
One of the most important theories that explains the
enzyme mechanism involved in ber modication suggests REFERENCES
that the brils and ber bundles are attacked on the 1. Popa, V.I. Hemicelluloses. In Polysaccharides in Medicinal
surface, peeling o subsequent layers and eventually lead- Applications; Dumitriu, S., Ed.; Marcel Dekker, Inc.: New
ing to disintegration of the ber [74,75]. This cleavage may York, 1996; 107 pp.
improve pulp freeness [76] and ber exibility [77] and also 2. Moroishi, N. In Wood and Cellulosic Chemistry; Hon,
determine the decrease of yield and ber strength. Fiber D.W., Shiraishi, N., Eds.; Marcel Dekker, 1991; 3.
3. Houtman, C.J.; Atalla, R.H. Celluloselignin interactions.
strength loosing depends on used enzymes. Thus, xylanase Plant Physiol. 1995, 107, 977.
treatment (Pulpzyme HC) on Pinus radiata commercial 4. Lewis, N.G. A 20th century roller coaster ride: a short
kraft pulp resulted in approximately 50% savings in energy account of lignication. Curr. Opin. Plant Biol. 1999, 2,
required to achieve a given apparent density using the 153.
EscherWyss rener. In this case, the primary eect of 5. Goodwin, T.W.; Mercer, E.I. The plant cell wall. Intro-
xylanase treatment was an increase in the apparent density duction to Plant Biochemistry; Pergamon Press: New York,
1983; 5591.
brought about by enhanced ber exibility, but it was not 6. Keegstra, K.; Talmadge, K.W.; Bauer, W.D.; Albersheim,
associated with an increase of cell wall dislocation [78]. P. The structure of plant cell walls: I. The hemicelluloses of
After rening, the enzyme-treated pulp had freeness in- the walls of suspension-cultured sycamore cells. Plant
creasing, with minimal changes in the tear and tensile index Physiol. 1973, 51, 174.
7. Fry, S.C. Cross-linking of matrix polymers in the growing obtained from dierent parts of rice grains. In Plant Cell
cell walls of angiosperms. Annu. Rev. Plant Physiol. 1986, Wall Polymers; Lewis, N.G., Paice, M.G., Eds.; ACS
37, 165. Symposium, 1989; 333.
8. Theander, J.O.; Aman, P. Anatomical and chemical 29. Stone, B.A.; Clark, A.E. Chemistry and Biology of (1,3) h-
characteristics. In Straw and other Fibrous by-products as Glucans; Victoria: La Trobe University Press, 1992; 355.
feed; Sundstol, F., Owen, E., Eds.; Elsevier: Amsterdam, 30. Rinaudo, M. Gelation of polysaccharides. J. Intell. Mater.
Holland, 1984; 4578. Syst. Struct. 1993, 4, 210.
9. Awano, T.; Takabe, K.; Fujita, M. Xylan deposition on 31. Simionescu, C.I.; Rusan, V.; Popa, V.I. Chimia Algelor
secondary wall of Fagus crenata ber. Protoplasma 2002, Marine (Seaweeds Chemistry); Publishing House of the
219, 106. Romanian Academy, 1974; 95.
10. Takabe, K.; Fukuzuma, K. Deposition of Cell Wall 32. Dumitriu, S.; Vidal, P.F.; Chornet, E. Hydrogels based on
Components in Coniferous Tracheides. In Plant Cell Wall polysaccharides; In Polysaccharides in Medicinal Applica-
Polymers; Lewis, N.G., Paice, M.G., Eds.; ACS Symp., tions; Dumitriu, S., Ed.; Marcel Dekker, Inc., 1996; 183.
1989; 49. 33. Sime, W.J. Alginates. In Food Gels; Harris, P., Ed.; Elsevier
11. Frey-Wyssling, A. Biochemistry of the cell wall. The Plant Applied Science: London, 1989; 53 pp.
Cell Wall; Gebruder Borntraeger: Berlin-Stuttgart, 1976; 34. Saito, H.; Ohki, T.; Sasaki, T. A 13C-nuclear magnetic
114 pp. resonance study of gel-forming (13)-h-D-glucans: evidence
12. Vincent, J.F.V. Survival of the cheapest. Mater. Today of the presence of single-helical conformation in a resilient
2002, 5, 38. gel of curdlan type polysaccharides 13140 from Alcaligenes
13. Dutton, G.G.S.; Josseleau, J.-P. Hemicelluloses of redwood faecalis var. myxogenes IFO 13140. Biochemistry 1977, 16,
(Sequoia sempervires). Cellul. Chem. Technol. 1977, 11, 313. 908.
14. Kenne, L.; Rossel, K.G.; Svensson, S. Studies on the 35. Saito, H.; Yoshioka, Y.; Yokoi, M.; Yamada, J. Distinct
distribution of the O-acetyl groups in pine glucomannan. gelation mechanism between linear and branched (1,3)h-D-
Carbohydr. Res. 1975, 44, 69. glucans as revealed by high-resolution solid-state 13C
15. Katz, G. Location and signicance of the O-acetyl groups NMR. Biopolymers 1990, 29, 1689.
in a glucomannan from Parana pine. Tappi J. 1965, 48, 34. 36. Sasaki, T.; Abiko, N.; Uchida, A.; Tanaka, M.; Nitta, K.;
16. Lindberg, B.; Rossel, K.G.; Svensson, S. Position of O- Yoshida, Y.; Nomura, H. Antitumor activity of tetrahy-
acetyl groups in pine glucomannan. Sven. Papp.tidn. 1973, dro-2-furanyl-and tetrahydro-2-pyranyl-glucans obtained
76, 383. from Alcaligenes faecalis var. myxogenes IFO 13140 and its
17. Lundqvist, J.; Teleman, A.; Junel, L.; Zacchi, G.; Dahlman, low molecular weight glucans. Cancer Treat. Rep. 1983, 67,
O.; Tjerneld, F.; Stalbrand, H. Isolation and character- 275.
ization of galactoglucomann from spruce (Picea abies). 37. Naito, T.; Takeda-Hirokawa, N.; Kaneko, H.; Sekigawa,
Carbohydr. Polym. 2002, 48, 29. I.; Matsumoto, T.; Hashimoto, H.; Kaneko, Y. Role of
18. Hauer, J.; Anderer, F.A. Mechanism of stimulation of curdlan sulfate in the production of beta-chemokines and
human natural killer cytotoxicity by arabinogalactan from interleukin-16. Med. Microbiol. Immunol. (Berl.) 1998,
Larix occidentalis. Cancer Immunol. Immunother. 1993, 187, 43.
36, 237. 38. Naito, T.; Okada, M.; Ogasawara, H.; Kaneko, H.;
19. Kind, L.S.; Macedo-Sobrinho, B.; Ako, D. Enhanced Hishikawa, T.; Matsumoto, T.; Sekigawa, I.; Hashimoto,
vascular permeability induced in mice by larch arabinoga- H.; Kaneko, Y. Curdlan sulfate induces the downmodula-
lactan. Immunology 1970, 5, 799. tion of chemokine receptors leading to suppression of HIV
20. Wisner, E.R.; Amparo, E.G.; Vera, D.R.; Brock, J.M.; infection. J. Acquir. Immune Dec. Syndr. 2001, 26, 512.
Barlow, T.W.; Griey, S.M.; Drake, C.; Katzberg, R.W. 39. Masuda, K.; Sakagami, M.; Horie, K.; Nogusa, H.;
Arabinogalactan-coated supermagnetic iron oxide: eect of Hamana, H.; Hirano, K. Evaluation of carboxymethylpul-
particle size in liver MRI. J. Comput.-Assist. Tomogr. lulan as a novel carrier for targeting immune tissues.
1995, 19, 211. Pharm. Res. 2001, 18, 217.
21. Rossel, K.G.; Svensson, S. Studies on the distribution of 40. Brigand, G. Scleroglucan. In Industrial Gums Polysacchar-
the 4-O-methyl-D-glucuronic acid residues in birch xylan. ides and their Derivatives; Whistler, L., Miller, J.N.B., Eds.;
Carbohydr. Res. 1975, 42, 297. Academic Press, 1993; 461.
22. Ershov, V.E.; Suhov, N.L.; Ebringerova, A. Study of the 41. Falch, B.H.; Espevik, T.; Ryan, L.; Stokke, B.T. The
radicalic products in the radiolysis of xylan through the cytokine stimulating activity of (1,3)-beta-D-glucans is
ESR method. Cellul. Chem. Technol. 1990, 24, 351. dependent on the triple helix conformation. Carbohydr.
23. Reicher, F.; Corecca, J.B.; Gorin, P.A.J. Location of O- Res. 2000, 329, 587.
acetyl groups in the acidic D-xylan of Mimosa scabrella 42. Ielpi, L.; Corso, R.; Daukert, M. Xanthan gum biosyn-
(bracatinga), a study of O-acetyl group migration. Carbo- thesis acetylation occurs at the prenyl-phopho-sugar stage.
hydr. Res. 1984, 135, 129. Biochem. Int. 1983, 6, 323.
24. Wilkie, K.C.B. The hemicelluloses of grass and cereals. 43. Okuyama, K.; Arnott, S.; Moorhouse, R.; Walkinshaw,
Adv. Carbohydr. Chem. Biochem. 1959, 36, 215. M.D.; Atkins, D.; Wolf-Ullish, C. Fiber diraction studies
25. Porchia, A.; Scheller, H.V. Arabinan biosynthesis: identi- of bacterial polysaccharides. In Fiber Diraction Methods;
cation and partial characterization of xylan-4-h-xylosyl- French, A.D. Gardner, K.-C.H., Eds.; ACS Symposium
transferase from wheat. Physiol. Plant 2000, 110, 350. Series 141; American Chemical Society: Washington, DC,
26. Chaubey, M.; Kappor, V.P. Structure of a galactomannan 1980; 411 pp.
from seeds of Cassia angustifolia Vahl. Carbohydr. Res. 44. Goodall, D.M.; Frangon, S.A.; Morris, E.D.; Rees, D.A.
2001, 332, 439. Mechanism and dynamic of conformational ordering in
27. Singh, R.B.; Jindal, V.K. Structure of galactomannan from xanthan polysaccharide. J. Mol. Biol. 1984, 175, 371.
Cassia laevigata seeds: methylation, periodate oxidation 45. Kuge, T.; Kobayashi, K.; Kitamura, S.; Tanahashi, H.
and Smith degradation studies. Cellul. Chem. Technol. Degrees of long-chain branching in dextrans. Carbohydr.
1990, 24, 453. Res. 1987, 160, 205.
28. Shibuya, N. Comparative studies on the cell wall polymers 46. Brich, Z.; Ravel, S.; Kissel, T.; Fritsch, J.; Schoman, A.
Preparation and characterisation of a water soluble dextran 64. Yang, L.; Lou, G.; Eriksson, K.-E.L. The impact of xylanase
immunoconjugate of doxorubicin and monoclonal anti- on bleachability of kraft pups. Tappi J. 1992, 75, 95.
body. J. Control. Release 1992, 19, 245. 65. Clark, T.A.; Kibblewhite, R.P.; Allison, R. Eect of
47. Hagiwara, A.; Sakakura, C.; Yamasaki, J.; Togawa, T.; enzymatic modication on radiata pine kraft ber wall
Sonoyama, Y.; Fujiyama, J.; Yamagishi, H. Dextran chemistry and physical properties. Appita J. 1997, 50, 329.
sulfate inhibits injured abdominal wall-specic tumor 66. Kantelinen, A.; Hortling, B.; Sundquist, J.; Linko, M.;
implantation in mice. Anticancer Drugs 2000, 11, 873. Viikari, L. Proposed mechanism of the enzymatic bleaching
48. Flexner, G.; Barditch-Crovo, P.A.; Kornhauser, D.M.; of kraft pulp with xylan. Holzforschung 1993, 47, 318.
Farzodegan, H.; Nerhood, L.J.; Chaisson, R.E.; Bell, 67. Kantelinen, A.; Rato, M.; Sundquist, J.; Rama, M.;
K.M.; Lorentsen, K.J.; Hendrix, C.W.; Petty, B.G.; Liet- Viikari, L.; Linko, M. Hemicellulases and their potential
man, P.S. Pharmacokinetics, toxicity and activity of role in bleaching. Proceedings of the Tappi International
intravenous dextran sulphate in human immunodeciency Pulp Bleaching Conference, Orlando, FL, USA, 1998; 1.
virus infection. Antimicrob. Agents Chemother. 1991, 35, 68. Hortling, B.; Korhonen, M.; Buchert, J.; Sundquist, J.;
2544. Viikari, L. The leachability of lignin from kraft pulps after
49. BeMiller, J.N.; Chen, C.; Whistler, R.L. Alloe and other xylanase treatment. Holzforschung 1994, 48, 441.
gums. In Industrial Gums Polysaccharides and Their 69. Kantelinen, A.; Sundquist, J.; Linko, M.; Viikari, L. The
Derivatives; Whistler, L.R., BeMiller, J.N., Eds.; Academic role of reprecipitated xylan in the enzymatic bleaching of
Press, 1993; 228. kraft pulp. Proceedings of the 6th International Symposium
50. Gittings, M.R.; Cipelletti, L.; Trappe, V.; Weitz, D.A.; In, on Wood and Cellulosic Chemistry, Melbourne, Australia
M.; Lal, J. The eect of solvents and ions on the structure 1991; Vol. 1, 493.
and rheological properties of guar solutions. J. Phys. 70. Wang, J.; Jiang, Z.-H.; Argyropoulos, D.S. Isolation and
Chem. A 2001, 105, 9310. characterization of lignin extracted from softwood kraft
51. BeMiller, J.N. Acid-catalysed hydrolysis of glycosides. pulp after xylanase treatment. J. Pulp Paper Sci. 1997, 23,
Adv. Carbohydr. Chem. 1967, 22, 25. J 47.
52. Albersheim, P.; Neukom, H.; Deuel, H. Splitting of pectin 71. Popa, V.I.; Spiridon, I.; Bobu, E. Study of enzymatic
chain molecules in neutral solution. Arch. Biochem. treatments inuence in obtaining pulp from wheat straw.
Biophys. 1960, 90, 46. Proceedings of the 8th International Symposium on Wood
53. Zimbo, M.; Timmel, T.E. The degree of branching of and Pulping Chemistry, 1995; 461.
hardwood xylans. Sven. Papp.tidn. 1965, 68, 647. 72. Popa, V.I.; Spiridon, I. Inuence of the treatment with
54. Lindberg, B.; Lonngren, J.; Thomson, J.L. Degradation of hemicellulase enzymes on the bleaching capacity of dierent
polysaccharides containing uronic acid residues. Carbo- lignocellulose materials. Proceedings of the Tappi Interna-
hydr. Res. 1993, 28, 351. tional Pulp Bleaching Conference, Washington, 1996; 579.
55. Yllner, S.; Ostberg, K.; Stockman, L. A study of the 73. Jimenez, L.; Navarro, E.; Garcia, J.C.; Perez, I. Bleaching
removal of the constituents of pine wood in the sulphate of wheat straw pulp with enzymes in an oxygenhydrogen
process using a continuous liquor ow methods. Sven. peroxide sequence. Tappi J. 2000, 83, 65.
Papperstidn. 1957, 60, 695. 74. Kim, B.-H.; Jun, Y. Eect of enzyme (endo-h-1,4-
56. Simonson, R. The hemicellulose in the sulphate pulping glucanase, exo-h-1,4-glucanase) treated rening on ber
process. Sven. Papperstidn. 1971, 43, 691. surface and paper physical properties. J. Korea Tappi 1994,
57. Sjostrom, E. Pulping chemistry. Wood Chemistry, Funda- 26, 23.
mentals and Applications; Academic Press, Inc.: New York, 75. Bhardwaj, N.K.; Bajpai, P.; Bajpai, P.K. Use of enzymes in
1981; 104144. modication of bers for improved beatability. J. Bio-
58. Ghoneum, M.; Jewett, A. Production of tumor necrosis technol. 1996, 51, 21.
factor-alpha and interferon-gamma from human peripheral 76. Pommier, J.C.; Fuentes, J.L.; Goma, G. Using of enzymes
blood lymphocytes by MGN-3, a modied arabinoxylan to improve the process and the product quality in the
from rice bran, and its synergy with interleukin-2 in vitro. recycled paper industry. Tappi J. 1989, 72, 187.
Cancer Detect. Prev. 2000, 24, 314. 77. Manseld, S.D.; Wong, K.K.Y.; de Jong, E.; Saddler, J.N.
59. Brownell, H.H.; Saddler, J.N. Steam pretreatment of Modication of Douglas r mechanical and kraft pulps by
lignocellulosic materials for enhanced enzymatic hydroly- enzyme treatments. Tappi J. 1996, 79, 125.
sis. Biotech. Bioeng. 1987, 21, 228. 78. Dickson, A.R.; Wong, K.K.Y.; Manseld, S.D. Proceed-
60. Dekker, R.F. Biodegradation of hemicellulose. In Biosyn- ings of the 10th ISWPC, EscherWyss and PFI Rening of
thesis and Biodegradation of Wood Components; Higuchi, Kraft Pulp Treated with a Commercial Xylanase, Japan,
T., Ed.; Academic Press: New York, 1985; 506. Yokohama 1999; Vol. III, 252.
61. Viikari, L.; Ranua, M.; Kantelinen, A.; Sundquist, J.; 79. Kibblewhite, R.P.; Wong, K.K.Y. Modication of a
Linko, M. Applications of enzymes in bleaching. Proceed- commercial radiata pine kraft pulp using carbohydrate
ings of 3rd International Conference of Biotechnology degrading enzymes. Appita J. 1999, 52, 300.
in the Pulp and Paper Industry, Stockholm, 1986; 67. 80. Perttunen, J.; Sohlo, J.; Maentausta, O. The fermentation
62. Li, J.; Paice, M.G.; MacLeod, J.M.; Jurasek, L. The of birch hemicellulose hydrolyzate liquor to lactic acid.
bleachability improvements of kraft pulps by alkaline Proceedings of the Sixth International Conference in the
leaching and xylanase treatment. J. Pulp Paper Sci. 1996, Pulp and Paper Industry, Vienna, 1995; 295 pp.
22, J207. 81. Mobarak, F.; El-Lalyoulu, S.F.; Shukry, N. Hemicelluloses
63. Wong, K.; de Jong, E.; Saddler, N.; Alisson, R. Mechanism as additives in papermaking. Rice straw and bagasse
of xylanase aided bleaching of kraft pulp: Part 2. Target hemicelluloses as retention aid for bers. Cellul. Chem.
substrates. Appita J. 1997, 50, 509. Technol. 1992, 25, 125.
19
Chemical Modication of Hemicelluloses and Gums
Margaretha Soderqvist Lindblad and Ann-Christine Albertsson
Royal Institute of Technology, Stockholm, Sweden
I. INTRODUCTION hemicelluloses are also present between the cellulose brils

and the matrix [3]. Pores in the matrix are small, and permit
Hemicelluloses constitute 2030% of the total bulk of the transfer of water, salts, and small organic molecules
annual and perennial plants, and they are consequently only. Although the hemicelluloses are at least partly water-
among the most abundant native polymers in the world. soluble, they cannot diuse out of the cell wall because of
According to the classical denition, hemicelluloses are cell their molecular size.
wall heterogeneous polysaccharides that are extractable by Because the hemicelluloses are embedded in the cell
aqueous alkaline solutions [1]. Because hemicelluloses have wall, their isolation is dicult. Methods for the isolation of
up to now hardly been utilized for industrial production, hemicelluloses from wood are too numerous to be com-
they represent an extensive raw material resource. For their pletely reported here, but the most important methods will
industrial utilization, it is necessary rst to isolate the be described. They can be roughly divided into methods
hemicelluloses from plants or process waters and secondly using a high-energy input, which involve the formation of
to chemically modify them to increase their reactivity. organic acids from the wood itself to dissolve the hemi-
celluloses, and methods where chemicals are added for the
II. ISOLATION OF HEMICELLULOSES FROM dissolution and the temperature is then usually only mod-
estly raised.
WOOD FOR CHEMICAL MODIFICATION
Wood is in general composed of the linear polymer cellu- A. Steam Treatment
lose, dierent mainly linear hemicelluloses, and the net-
work polymer lignin. Dierent kinds of extractives such as When wood is treated with steam, the temperature is raised
terpenoids, fats, and waxes are also present. The wood and water is added. The increase in temperature lead to the
polymers are organized in a highly ordered pattern during formation of organic acids as a result of the cleavage of
the biogenesis of the cell wall. Wood consists of dead cells ester bonds in the hemicelluloses. Mainly acetic acid is
and several layers build up the cell wall. There is general formed by cleavage of O-acetyl groups. The organic acids
agreement that the layers in a typical wood cell are orga- released give rise to an acidic environment that catalyses
nized as illustrated in Fig. 1 [2]. These cell wall layers are the the hydrolysis of the glycosidic bonds in the hemicelluloses,
primary wall, the outer layer of the secondary wall, the a process called autohydrolysis. The result is a partial
middle layer of the secondary wall, and the inner layer of depolymerization of the hemicelluloses into water-soluble
the secondary wall. Some cells also have a warty layer low molecular weight hemicelluloses. These acids also have
toward an open lumen. These layers dier from one some eect on the lignin and on the cellulose, and lead
another with respect to their morphology as well as their to some dissolution of these substances, particularly the
chemical composition. Between the cells is the middle lignin. With this technique, it is therefore dicult to
lamella, which is also built up by polymers. achieve a good yield of hemicelluloses without extensive
The study of the secondary cell wall, which is the most degradation and contamination with dissolved lignin
extensive part of the wood ber, showed that the cellulose and cellulose.
brils consist of highly ordered cellulose chains, which are It has been reported that 8 g of hemicellulose oligo-
embedded in a matrix of hemicelluloses and lignin. Pure mers can be isolated from 100 g dry spruce wood after
491
492 Lindblad and Albertsson
charides. A schematic illustration of the experimental

procedure is given in Fig. 2. The yield of hemicellulose
could be increased to 10 g from 100 g dry wood after 5-min
treatment at 200jC, but the molecular weight was lower.
The remaining solid can be used for ethanol fermentation
of the cellulose, which was one important motive for
developing this process. These two processes could be
combined as an ecient way of utilizing all the carbohy-
drates in the wood, as one problem during ethanol fermen-
tation is that sugars from hemicellulose and cellulose
exhibit their maxima under dierent conditions [5].
Other related methods involving the heat treatment
of wood are autohydrolysis and rapid steam hydrolysis
(RASH) [6] and also wet oxidation [6,7]. All this tech-
niques suer from contamination with other wood con-
stituents, especially lignin, and degradation of the
hemicellulose chains.
B. Microwave Irradiation
Microwave irradiation of wood in the presence of water
leads to autohydrolysis in the same way as when wood is
Figure 1 Schematic organization of the wood cell wall. ML treated with steam. The diculty with this technique is on
= middle lamella or intercellular layer; P = primary wall; S1, how to achieve a good yield without extensive degradation
S2, S3 = outer, middle, and inner layers of the secondary wall, of the hemicelluloses and contamination with dissolved
respectively; W = warty layer [2]. (Courtesy of University of lignin and cellulose. An advantage of microwave treatment
Washington Press.) is the uniform heating of the wood. A disadvantage is that it
is not feasible on a large scale because the microwaves
penetrate only a few centimeters into the material. Another
disadvantage is the long heating and cooling times required.
2-min steam treatment at 200jC with a mean molecular Microwave treatment gave 10 g of hemicellulose
weight of O-acetyl-galactoglucomannan of 3,400 g/mol oligomers from 100 g of dry wood after 2 min at 200jC.
(range 60010,200 g/mol) determined by size exclusion The range of molecular weights for the O-acetyl-gluco-
chromatography [4]. To obtain a suciently pure hemicel- mannan was 87024,000 g/mol determined by fast protein
lulose for chemical modication, the raw product was rst liquid chromatography [4]. About one-third of the D-
ltered to eliminate solids and then fractionated by size mannose units were substituted by O-acetyl groups, which
exclusion chromatography using water as the eluent to were distributed almost equally between C2 and C3 in a
separate salts and monomers from the desirable oligosac- fraction of O-acetyl-galactoglucomannan [8].
Figure 2 Fractionation of spruce for the production of hemicellulose. (Acknowledgement to Magnus Palm, LTH for the
outline). (Reprinted with permission from Ref. 4a. Copyright 2003 American Chemical Society.)
Chemical Modification of Hemicelluloses and Gums 493
C. Alkali Extraction III. CHEMICAL MODIFICATION

Hemicelluloses can be isolated from holocellulose by ex- Hemicelluloses are, as mentioned earlier, classically dened
traction with aqueous alkali. This method was developed as cell wall heterogeneous polysaccharides extractable by
for characterization purposes, but it can also be used as a alkaline solutions [1]. However, in this section, the chem-
preparative method [9]. The isolation is made as a gradient ical modication of gums is included because the scientic
extraction with dierent alkali concentrations to give a literature contains so few works concerning chemical mod-
rough fractionation of the hemicelluloses. The soluble ications with hemicelluloses as the polysaccharide. Fur-
hemicellulose fractions are then precipitated with acidied thermore, acceptable denitions for the dierence between
ethanol. Borohydride can be added to minimize degrada- hemicelluloses and gums would be almost impossible to
tion of the reducing end groups but the cleavage of O-acetyl devise. They are entrenched in the chemical literature and
groups cannot be avoided. Already, at pH 10, all acetyl serve as functional expressions for the purposes of infor-
groups are split o [10]. Cleavage of the O-acetyl groups mation retrieval [15].
signicantly reduces the solubility of the galactogluco- The following gives a brief review of most of the
mannans in water [11]. Galactoglucomannans with dier- polysaccharides used in the reported investigations:
ent compositions and xylan are obtained in separate
Arabinogalactan: Two types exist. Type I are arabi-
fractions. The total yield of hemicelluloses from eastern
nose-substituted derivatives of the linear (14)-
hemlock is reported to be between 19% and 25% of extrac-
linked h-D-galactans in which the arabinose
tive-free wood [9]. The yield of hemicelluloses decreased
content varies from about 25% to 43%. Type II
with increasing degree of delignication of the holocellu-
are heavily branched and may carry a consider-
lose because of the degradation of the hemicelluloses by the
able proportion of dierent complex arrange-
acid chlorite used for holocellulose preparation.
ments of sugar and uronic acid residues [16].
Arabinoxylan: Branched heteropolysaccharide in
D. Extraction with Dimethyl Sulfoxide which a homopolysaccharide chain of uniform
linkages carries second sugar constituents as
Spruce holocellulose was extracted with dimethyl sulfoxide
single-unit side chains. The backbone consists of
(DMSO) at room temperature, followed by cold and hot
(1-4)-linked h- D -xylopyranos units and the
water and nally 18% sodium hydroxide solution [12]. A
branches are (1-3)-linked a-L-arabinofuranose
considerable fraction, approximately 6% of holocellulose,
units [17].
was obtained by the extraction with DMSO, with xylose as
the dominating saccharide monomer. In the subsequent Cassia tora gum: Composed of galactose and mannose
extraction with water, approximately 6% of holocellulose units [18].
was obtained with mannose as the dominating saccharide Cassia obovata gum: Composed of galactose and
monomer. The acyl groups (acetyl and formyl) survived mannose units [18].
both extractions. Fenugreek gum: Composed of galactose and mannose
units [18].
Galactoglucomannan: The backbone is a linear or
E. Extraction with a Mixture of Methanol possibly slightly branched chain built up of (1-4)-
and Water linked h-D-glucopyranose and h-D-mannopyra-
Pulping processes using organic solvents allow a sequenced nose units. Galactoglucomannans can be divided
separation of the wood polymers. When spruce wood was into two fractions having dierent galactose
treated with a watermethanol mixture at high tempera- contents. In the fraction with a low galactose
ture, 190jC, hemicellulose associated with lignin was content, the galactose/glucose/mannose ratio is
obtained as a colloidal suspension when the methanol about 0.1:1:4, whereas in the galactose-rich
was eliminated. A separation process using strong acid fraction, the ratio is 1:1:3. The a-D-galactopyr-
and weak basic ion exchangers led to a highly puried anose residue is linked as a single-unit side chain
hemicellulose representing 28% of the dry wood with DP to the backbone by (16)-bonds [19].
of 43. However, the resulting hemicellulose showed only a Glucuronoxylan: The backbone consists of h-D-xylo-
weak solubility, 0.8 g/L, in water [13]. pyranose units liked by (14)-bonds. Most of the
xylose residues contain an O-acetyl group at C2 or
C3. The xylose units also carry (12)-linked 4-O-
F. Hemicellulose from Process Water methyl-a-D-glucuronic acid residues [20].
Hemicelluloses can also be found in dierent process Guar gum: Linear (14)-h-D-mannan backbone with a
waters in, e.g., the hardboard industry, and in pulp and D-galactose side chain on approximately every
paper mills. 1.3 g/L of oligosaccharides, mainly O-acetyl other mannose unit. The molecular weight has
galactoglucomannan, have been found [14]. The disadvan- been estimated to be about 220,000 [21].
tage is the low concentration, but otherwise the dissolved Gum arabic: Complex calcium, magnesium, and
hemicelluloses will be a waste material and its disposal will potassium salt of Arabic acid. It has a backbone
involve expenses. of (13)-linked h-D-galactopyranose units, some
of which are substituted at the C6 position with

various side chains consisting of D-galactopyr-
anose, D-glucuronic acid, L-arabinofuranose, and
L-rhamnopyranose units. It has a molecular
weight of 250,000 [22].
Gum tragacanth: Complex mixture of acidic polysac-
charides containing D-galacturonic acid, D-galac-
tose, L-fucose, D-xylose, and L-arabinose. The
molecular weight has been estimated to be 840,000
[23].
Konjac galactoglucomannan: Neutral polysaccharide
derived from the tuber of Amorphophallus konjac.
Konjac glucomannan consists of h-1,4-linked
glucose and mannose units. The presence of some
branching points at the C3 position of the
mannoses is suggested. The glucomannan back-
bone possesses 510% acetyl substitution [24].
Locust bean gum: Smooth regions of unsubstituted or
sparingly substituted (14)-linked h-D-manno-
pyranose residues and hairy regions that are
heavily substituted with a-D-galactopyranose
residues attached by (16)-linkages [25].
It should be observed that the structure information given
relates to the native biopolymers. The structure can be
altered during isolation of the biopolymers.
A. Esterification
The complete utilization of the renewable raw materials,
hemicelluloses and gums, is facilitated if they are made
thermoplastic for easier processing and better thermal
stability, and esterication is a possible way of achieving Scheme 1 Overview of some ester structures made by modi-
a thermoplastic hemicellulose-based material. Other reas- cation of hemicelluloses.
ons for esterication are to reduce the water absorbency of
the hydrophilic hemicelluloses, to increase solubility, and
to reduce crystallinity. Hemicelluloses can be esteried
under either heterogeneous or homogeneous conditions. loses on the properties after acetylation under simulated
Heterogeneous esterication can be performed in water, commercial conditions was studied [27]. Acetic acid and
but it gives, e.g., poor uniformity of substitution, low yield, acetic anhydride were used as acetylating agents under
by-product formation, and unfavorable reaction kinetics. conditions commercially used for cellulose. It was found
On the other hand, homogeneous reaction conditions that clear acetate solutions were obtained when branched
require specic organic solvents or alkaline solutions. It hemicelluloses (from, e.g., kraft glucomannans, sulte
is dicult to esterify hemicelluloses of the xylan type, the xylans) were used, but that crystalline acetates and hazy
most frequently occurring hemicellulose, because xylan has solutions were obtained from linear hemicelluloses (from,
only two hydroxyl groups available for esterication com- e.g., kraft xylans, sulte glucomannans).
pared to cellulose, which has three. Some ester structures Acetylation of (4-O-methyl-D-glucurono)-D-xylan iso-
are shown in Sch. 1. lated from beech sawdust was performed in organic sol-
vents (CH2Cl2, CHCl3) with acetic anhydride and catalytic
amounts of inorganic acids (HClO4) [28]. The reactions
1. Acetylation and Preparation of Other Carboxylic were initially heterogeneous but dissolution occurred as the
Acid Esters reaction proceeded and the product became fully acetylat-
In early work, esters of hemicelluloses were prepared for ed when all of the substrate had dissolved. Acetylation of
analytical reason, e.g., for the determination of molecular the same substrate with acetic anhydride in triuoroacetic
weights. For example, the acetate, propionate, and buty- acid, in which the substrate was dissolved within 5 min,
rate of lima bean pod hemicellulose with xylan as the resulted in acetylation up to a maximum of 30% (with 40%
predominant hemicellulose were prepared, and their solu- as the theoretical value), which indicated that some hy-
bility characteristics were studied [26]. In another early droxyl groups were not accessible to the reagent [29]. The
work, the inuence of structural parameters in hemicellu- acetylation was performed during 15 min in room temper-
ature. From measurements of the degree of polymeriza- and highly acetylated samples were soluble only in aprotic
tion, it was concluded that the degradation of the substrate solvents such as chloroform and dimethyl sulfoxide.
during the procedure was not greatly inuenced by the A heterogeneous method for the preparation of esteri-
quantity of triuoroacetic acid used. ed biopolymers has been applied to konjac glucomannan
Another possible way to achieve homogeneous acety- [38]. Konjac glucomannan was immersed in benzene/pyr-
lation is by using the system N,N-dimethylacetamide idine, and palmitoyl chloride was added (70jC, 4.5 hr). A
(DMAc)/lithium chloride (LiCl), which was rst reported degree of substitution between 0.5 and 2.7 was achieved
as a dissolving system for cellulose in 1979 [30]. It has been depending on the added amount of palmitoyl chloride.
found that N,N-dimethylformamide (DMF) is more eec- With increasing degree of substitution, the crystalline state
tive for dissolving hemicelluloses [31]. Hemicelluloses of the konjac glucomannan disappeared and thermal anal-
extracted from poplar chips with xylose as the predominant ysis revealed that the palmitoylated konjac glucomannan
sugar were esteried with various acyl chlorides [C2, C3, C4, adopted a new crystalline state. It was demonstrated that
C6, C8, C10, C14, C16, C18 (saturated and unsaturated)] in palmitoylated konjac glucomannan was a good water-in-
DMF/LiCl using 4-dimethylaminopyridine as a catalyst oil emulsier at a degree of substitution between 1.0 and
and triethylamine (TEA) as a neutralizer [31,32]. Compar- 1.7. Acetylation of konjac glucomannan was performed
ative reactions were performed with dierent molar ratios with acetic anhydride in the presence of sodium hydroxide
of acyl chloride/anhydroxylose units, dierent concentra- as catalyst [39]. Fully acetylated konjac glucomannan with
tions of TEA, various reaction times between 25 and 45 a degree of substitution of 3.0 was achieved by reuxing for
min, and temperatures between 45jC and 75jC. Under 12 hr. This fully acetylated product exhibited 1.0 g/g water
optimum conditions (molar ratio 3:1, TEA 280% based on absorbency in contrast to the 105 g/g absorbency of native
hemicellulose weight, 45 min, 75jC), 75% of the hydroxyl konjac. In view of the exponential decrease of water
groups in the hemicelluloses was esteried. Molecular absorbency with increasing degree of substitution, it was
weight measurements showed minimal degradation of the found that a relatively low degree of substitution was
hemicelluloses during this short reaction time. When the sucient to signicantly suppress the absorption of water.
same system was applied to wheat straw hemicelluloses
with xylose as the predominant sugar [33], about 95% of 2. Succinoylation
the hydroxyl groups were esteried under optimum con-
The introduction of carboxylic functionalities into poly-
ditions (molar ratio 1:3, TEA 160% based on hemicellulose
saccharides is known to increase the hydrophilicity of the
weight, 30 min, 75jC). Only slight degradation with respect
material. To obtain hemicellulose esters containing car-
to molecular weight was detected and the thermal stability
boxylic groups, a method of succinoylation has been
of the products was increased by acetylation. When the
developed. Wheat straw hemicelluloses were dissolved in
DMF/LiCl system was used for acetylation of wheat straw
the DMF/LiCl system with pyridine and/or 4-dimethyla-
hemicellulose with 4-dimethylaminopyridine as catalyst
minopyridine (DMAP) as catalyst [40]. The succinoylation
but acetic anhydride as the esterifying agent, the reaction
reaction was studied as a function of the molar ratio,
time was prolonged to about 60 hr even when the temper-
succinic anhydride/anhydroxylose units from 1:1 to 5:1,
ature was raised to 85jC [34]. About 75% of the free
dierent DMAP concentrations, temperature from 40jC
hydroxyl groups in the hemicellulose were acetylated and,
to 140jC and time from 2 to 12 hr. Under optimum
in spite of the high reaction temperature and long reaction
reaction conditions, a high degree of substitution of 1.5
time, the degradation was stated to be controllable.
was obtained, which means that 78% of the free hydroxyl
Water-soluble arabinoxylan isolated from corn ber, a
groups in the hemicellulose were succinoylated. The ther-
complex and highly branched hemicellulose composed of
mal stability of the products slightly decreased upon
xylose, arabinose, galactose, and glucose, was easily esteri-
chemical modication, but no signicant further decrease
ed with C2C4 aliphatic anhydrides using methanesul-
in thermal stability was observed for degrees of substitu-
fonic acid as catalyst [35]. These arabinoxylan esters were
tion z0.7. Another method for succinoylation giving a low
soluble in a range of organic solvents and could tolerate
degree of substitution was to use an aqueous alkaline
signicant amounts of water as a co-solvent. Typical
system [41]. The eects on the degree of substitution of
reaction conditions to reach arabinoxylan acetate with a
reaction times of 0.516 hr, temperatures of 2545jC, and
degree of substitution of 2.1 were 1 hr at 50jC, and for
molar ratios of succinic anhydride to anhydroxylose units
arabinoxylan butyrate with a degree of substitution of 2.0
in wheat straw hemicelluloses of 1:5 to 1:1 were studied.
were 14.5 hr at 50jC. The arabinoxylan esters were ther-
The highest degree of substitution (0.2) in the pH range
mally stable up to almost 200jC.
8.59.0 was obtained in the temperature range 2528jC, at
To obtain fully acetylated aspen glucuronoxylan, acet-
times of 12 hr, and a 1:1 molar ratio. The thermal stability
ylation was performed in formamide/pyridine with acetic
of the esteried hemicellulose increased as a result of this
anhydride as acetylating agent [36], adapted from an elderly
chemical modication.
technique [37]. The maximum degree of substitution, 1.9,
was obtained after 4 hr. The acetylation resulted in a
thermoprocessable glucuronoxylan, which was conrmed 3. Sulfation
by making lms under elevated temperature and pressure. Xylan from beechwood has been sulfated in the dinitrogen
The acetylation strongly aected the solubility properties, tetroxide-N,N-dimethylformamide (N2O4-DMF) system
as a model substrate devoid of primary OH groups [42]. and instability of the xanthate but that they sometimes
The N2O4-DMF system is a derivatizing solvent system serve as an indicator of the reactivity of the bers.
acting via the formation and solvation of an unstable
cellulose derivative and the subsequent introduction of
B. Etherification
the desired acyl groups, coupled with the elimination of
the primary unstable substituents. NOSO4H, the active Etherication of gums and hemicelluloses is important for
sulfating agent, can be formed by a redox process between increasing solubility, stability against microorganisms to
SO2 and N2O4 via HNO3 as an intermediate. prevent fermentation on storing, lm-forming ability, and
Xyl OH N2 O4 f Xyl ONO HNO3 increasing viscosity. A higher viscosity is important espe-
cially for various kinds of gums because they are commer-
Xyl ONO NOSO4 H f Xyl OSO3 H N2 O3 cially used in industries such as the mining, paper, and
With xylan, a partial derivatization of the secondary OH textile industries. Etherication can be performed under
groups to a rather low degree of substitution was achieved. both heterogeneous and homogeneous conditions. An
It was concluded that on sulfation with SO2, secondary OH overview of reported ether structures is given in Sch. 2.
groups reacted only if no primary groups were available.
There seems to be no signicant dierences in reactivity 1. Carboxymethylation
between the two OH groups of xylan with respect to sulfate Carboxymethylation of hemicelluloses was rst reported
ester formation. in 1957. Xylan from beechwood was then carboxymethy-
To study biological activities such as anticoagulation lated to degrees of substitution from 0.1 to 0.9 [47].
and brinolysis, dierent seed galactomannans were sul- Sodium chloroacetate in alkaline solution was used for
fated using chlorosulfonic acid [43]. It was found that the carboxymethylation. It was found that higher degrees
sulfation of these polysaccharides increased their biologi- of substitution could be obtained by increasing the reac-
cal activity. tion time (e.g., from 48 to 96 hr) and also by a stepwise
carboxymethylation process. An oxygen-free atmosphere
did not inuence the degree of substitution. Fractionation
4. Tosylation
showed that the product was polydisperse but that it was
Konjac glucomannan is soluble in aqueous solutions but essentially homogeneous with respect to the degree of
insoluble in common organic solvents. For the eective substitution. The products were soluble in water and alkali
introduction of many types of functional groups, it is
therefore desirable to convert konjac glucomannan to a
derivative soluble in organic solvents. The synthesis of a
precursor, tosylated konjac glucomannan, has therefore
been studied [44]. The tosyloxy group is also a good leaving
group in nucleophilic substitution and O-tosyl derivatives
are used as precursors in saccharide chemistry. Samples
with a degree of substitution of up to 2.3 were obtained,
and samples with a degree of substitution higher than
about 0.5 were soluble in many organic solvents.
5. Nitrition
Nitrite esters of, e.g., guar gum, locust bean gum, and
hemicellulose have been prepared [45]. The esters were
obtained by reaction with dinitrogen tetroxide or nitrosyl
chloride in a medium containing a suitable proton accep-
tor. The esters were unstable and could be isolated only at
low temperatures as wet, brous materials.
6. Xanthation
Xanthation of spruce glucomannan, birch glucuronoxylan,
spruce arabinoglucuronoxylan, ivory nut mannan B, and
esparto xylan has been investigated to study whether
hemicellulose components in viscose pulps cause dicul-
ties in the viscose process for chemical reasons [46]. How-
ever, it was found that all polysaccharides were xanthated
relatively easily. The rate of dexanthation was very dier-
ent for dierent hemicelluloses. It was concluded that Scheme 2 Overview of some ether structures made by mod-
hemicelluloses are not the direct cause of the low reactivity ication of hemicelluloses.
but insoluble in organic solvents. Detergent properties was repeated two to four times to reach dierent degrees of
were investigated and it was found that a soil-suspending substitution up to 1.4. The methylated konjac gluco-
ability was exhibited although to a lesser extent than by mannan was soluble in water, but the viscosity varied
carboxymethylcellulose. according to the degree of substitution. The highest vis-
Guar gum has been carboxymethylated according to cosity was observed for the sample having a degree of
an aqueous method [48]. Sodium hydroxide solution was substitution of 0.45, which indicated a stronger solvation
rst added and then monochloroacetic acid. The tem- with water and a better stability of the solution than was
perature was held at 80jC for 1.5 hr and a degree of achieved with the other samples. It was also shown that the
substitution from 0.3 to 0.8 was achieved by varying the solubility of the samples was dependent on the distribution
concentration of the etherifying agent. The starting mate- of the methyl groups. When methyl groups were uniformly
rial was partly soluble in both water and ethanol but after distributed throughout the molecule, a high degree of
carboxymethylation, the guar gum was completely soluble substitution was not necessarily essential for the konjac
in water regardless of the degree of substitution. Non- glucomannan to be soluble in water.
aqueous carboxymethylation in medium was carried out in
a mixture of ethanol and toluene [49]. Sodium hydroxide 3. Hydroxyalkylation
was rst added and then monochloroacetic acid. The
Hydroxyethylated guar gum was prepared by dispersing
reaction took place at room temperature overnight. To
guar gum in tertiary butyl alcohol in the presence of sodium
obtain highly substituted derivatives, the procedure was
hydroxide and ethylene oxide (added at 0jC) [55]. The
repeated several times. Performing the reaction only once
reaction mass was then heated to 70jC and maintained at
resulted in a degree of substitution of 0.8, whereas a
that temperature for 3 hr. The rheological properties of
fourfold procedure gave a degree of substitution of 2.1.
aqueous solutions of guar gum and hydroxyethylated guar
All the carboxymethylated samples were soluble in water
gum were then investigated. Master viscosity curves indi-
but insoluble in ethanol. Carboxymethylated derivatives
cated that the molecular weight distribution of the native
obtained by both methods were characterized by a non-
guar gum was changed by the hydroxyethylation.
Newtonian behavior regardless of the degree of substitu-
Xylan from barley husks has been reacted homoge-
tion or of the measuring temperature. All the derivatives
neously with propylene oxide in an aqueous alkaline
also showed an increased stability toward microorgan-
solution for 24 hr at room temperature [56] giving hydroxy-
isms. The same results were, in principle, obtained for the
propylated xylan. Derivatives with dierent degrees of
carboxymethylation of Leucaena gum [50] and Cassia
substitution, 0.21.9, were prepared by varying the pH of
obovata gum [51]. When Leucaena glauca seed gum was
the reaction mixture from 10.5 to 13.0. A higher pH
carboxymethylated in tert-butanol with optimal concen-
resulted in a higher degree of substitution. The derivatives
tration of sodium monochloroacetate and sodium hydrox-
with a low degree of substitution were completely soluble in
ide solution, the highest degree of substitution obtained
water and they formed tough and transparent lms. The
was 1.8 after 3 hr at 70jC [52]. Carboxymethylation of
hydroxypropylated xylan was further peracetylated by
konjac glucomannan with monochloroacetic acid in meth-
dissolving the derivative in formamide and reacting it with
anol and aqueous sodium hydroxide for 40 hr at 50jC
acetic anhydride at room temperature, giving a water-
gave, at most, a degree of substitution of 0.3 [53]. Carbox-
insoluble thermoplastic acetoxypropyl xylan (T g =
ymethylated konjac glucomannan was characterized by
160jC at degree of substitution 0.2; Tg = 70jC at degree
dynamic viscoelasticity measurements and it was found
of substitution 2.0). These materials were potentially bio-
that both the storage modulus ( GV) and the loss modulus
degradable and they were found to be suitable as thermo-
( GW) decreased and became more frequency-dependent
plastic additives in melt-processed plastics. Blend
with increasing degree of substitution. The introduction
characteristics with polystyrene revealed a shear-thinning
of carboxymethyl groups into konjac glucomannan sup-
eect in the melt and a plasticization eect in the solid state.
pressed the mutual interaction of konjac glucomannan
Carboxymethylation and hydroxypropylation of guar
and tended to give a more liquid-like rheological behavior.
gum were simultaneously carried out using monochloro-
acetic acid and propylene oxide in the presence of a
2. Methylation hydrophilic solvent, e.g., 2-propanol having an alkaline
pH [57]. Two hours at 6085jC were the recommended
Methylation of xylan was used already in the 1960s as a
reaction conditions. Applications in cosmetics and as a
means of making xylan solvent-soluble for the determina-
binder and thickening agent in adhesives and as a viscosi-
tion of nonreducing end groups, to obtain information
er in fracturing uids were proposed.
about the branching of hemicelluloses [37].
Konjac glucomannan has been partially methylated
with dimethyl sulfate to prepare a stable aqueous solution 4. Sulfoalkylation
for the purpose of macromolecular chemical research by a Sulfopropylation of hemicelluloses obtained from hard-
moderate chemical procedure, which may not develop wood sulte and sulfate pulps has been carried out in a
depolymerization [54]. To an aqueous solution of konjac homogeneous dimethyl sulfoxide solution [58]. Various
glucomannan, sodium hydroxide solution was added with quantities of dimsyl and propanesultone were added. The
an adequate amount of dimethyl sulfate. This treatment reaction time was varied between 1 and 24 hr and the
reaction was carried out at room temperature. The degree In the rst works [65,67], either living polystyrenes or
of substitution ranged between 0.5 and 1.1 depending on living poly(2-vinylpyridine) was grafted onto methylated 4-
the experimental conditions. Molecular measurements O-methylglucuronoxylan from aspen wood. As the active
showed a controllable degradation of hemicellulose chains hydrogens can act as terminators for the carbanion end
during the reaction. A signicant regioselectivity for the group of the living polystyrene, the xylan was converted to
OH2-hydroxyl groups was observed only for degrees of its methyl derivative while the carboxyl of the 4-O-methyl
substitution V0.5. glucuronic acid moiety was esteried. The reaction was
Long alkyl chains (C12) have been introduced onto performed in tetrahydrofuran (THF) because the methyl-
glucuronoxylan and its sulfoethyl derivative via O-alkylated xylan was soluble in THF and the living polystyrene
ation using 1-bromododecane in dimethyl sulfoxide [59]. was prepared in THF. The grafting site was located on the
The alkylation time and temperature were 3 hr and 70jC, ester grouping of the methylated glucuronate moiety,
respectively. The addition of long alkyl chains to the xylan which occurs randomly along the backbone as a side
molecules bearing ionic substituents imparted amphiphilic group. Most of the samples were stated to involve two
characteristics that lowered the surface tension and ex- graftings per ester, provided the hydroxyl functions were
hibited emulsifying and protein foam stability properties. inactivated.
The rst study in water was reported in 1967 [66].
5. Cyanoethylation Aspen 4-O-methylglucuronoxylan was grafted with sodi-
Guar gum has been cyanoethylated under a variety of um acrylate in 3.4 N NaOH with the ammonium persul-
conditions [60]. Guar gum was mixed with aqueous sodi- fate/sodium thiosulfate redox initiator system at 2530jC.
um hydroxide, acrylonitrile was added and the reaction The starting point for this study was an initiatormono-
time was 4 hr. It was shown that the extent of reaction mer combination, which was soluble in an alkaline xylan
increased with increasing acrylonitrile concentration, and solution at 25jC and in which radicals capable of abstract-
decreased with increasing reaction temperature. Cyano- ing hydrogen atoms from carbohydrates would be gener-
ethylated guar gum was characterized by a non-Newto- ated. The persulfate-thiosulfate redox couple and acrylic
nian pseudoplastic behavior. acid (as acrylate ion in NaOH solution) met these require-
ments, and the formation of a true graft copolymer was
6. Benzylation indicated by fractional precipitation and light-scattering
studies. A grafted fraction containing no ungrafted xylan
A benzylation method was developed with benzylbromide
but possibly some poly(sodium acrylate) homopolymer
as benzylating agent. It was added after swelling of the
was isolated.
xylan in dimethyl sulfoxide with 18-crown-6 ether, and
potassium hydroxide as catalysts [61]. The reaction took
place at 70jC for 20 hr and at 8590jC for 3 hr. Substitu- 1. Grafting Parameters
tion was conrmed by infrared (IR) spectrum. The product Many papers have been published describing grafting in
was soluble in most organic solvents and presented ther- water slurries using redox initiators. Most papers [68
moplastic properties that allowed processing in a softened 79,8190] investigated reaction conditions inuences on
state between 205jC and 225jC without degradation of the one or more of the grafting parameters:
polymer chains.
Xylan has been reacted with p-carboxybenzyl bromide Grafted polymer
Grafting ratio%G 100
[62]. Xylan was rstly dispersed in water containing sodium Weight of substrate
hydroxide, and p-bromomethylbenzoic acid was then Grafted polymer
added. The reaction continued until the bromomethylben- Grafting Efficiency%E 100
Polymer formed
zoic acid had disappeared. Degrees of substitution between
0.05 and 0.25 were obtained, the higher values being Synthetic polymer
Add on%A 100
reached when the reaction was performed in room temper- Graft copolymer
ature instead of at 40jC or 60jC. The xylan chains did not Polymer formed
degrade up to a reaction temperature of 40jC. Conversion%C 100
Monomer charged
Homopolymer%H 100 E
C. Grafting
Anhydroglucose units mol
Grafting frequency
Vinyl monomers can be graft-copolymerized onto many Grafted polymer mol
natural substrates in aqueous slurry under a wide range of
reaction conditions. Graft copolymers of cellulose [63,64] The reaction conditions investigated were generally the
have been extensively studied but graft copolymerization concentration of initiator, monomer, and substrate; tem-
with hemicellulose substrates has received comparatively perature; and time. The purpose was to nd optimal
little attention. Greater attention has been given to the conditions for one or more of the grafting parameters.
graft copolymerization of gums, especially guar gum. An The eects of adding organic solvents, salts, surfactants,
overview of substrates, monomers, and initiators is given in and complexing agents have also been investigated [72].
Table 1 [6590]. The dierent reaction variables inuenced the grafting
Table 1 Overview of substrates, monomers, and initiators [6590]
Substrate Grafted monomer Initiator Reference
Methylated 4-O Living polystyrene Polystyrylpotassium 65

-methylglucuronoxylan
from aspen wood
4-O-methylglucuronoxylan Sodium acrylate Ammonium persulfate/sodium 66
from aspen wood thiosulfate
Methylated 4-O Living polystyrene Polystyrylpotassium and 67
-methylglucoronoxylan and living poly corresponding potassium ion
from aspen wood (2-vinylpyridine) for poly(2-vinylpyridine)
Hemicellulose from Acrylonitrile and Ceric ammonium nitrate/nitric 68
larchwood and methylacrylate acid and ferrous
wheat straw sulfate/hydrogen peroxide
Hemicellulose from Acrylonitrile Ceric ammonium nitrate/nitric 69
bagasse acid
Guar gum Acrylamide Potassium permanganate/oxalic 70
acid
Guar gum Methyl methacrylate Hydrogen peroxide 71
Guar gum Acrylamide Potassium persulfate/ascorbic 72
acid
Leucaena glauca Acrylonitrile Hydrogen peroxide 73
seed gum
Leucaena glauca Acrylonitrile, Hydrogen peroxide 74
seed gum, and methylmethacrylate,
guar gum methylacrylate
acrylamide, styrene,
methacrylic acid,
and acrylic acid
Leucaena glauca Methyl methacrylate Hydrogen peroxide 75
seed gum
Guar gum Acrylonitrile Potassium persulfate/ascorbic 76
acid
Guar gum Methylacrylate Hydrogen peroxide 77
Guar gum Acrylamide Potassium bromate/thiomalic 78
acid
Hemicellulose from Acrylonitrile Potassium 79
corn cobs permanganate/sulfuric acid
Fenugreek gum Methacrylic acid Potassium persulfate 80
Guar gum Acrylamide Copper sulfate/mandelic acid 81
Guar gum Acrylic acid Peroxydiphospate-silver(I) 82
Guar gum Acrylamide Ceric ammonium nitrate/nitric 83
acid
Guar gum Acrylamide Peroxydiphosphate/metabisulte 84
Hydroxypropyl guar Acrylamide Ceric ammonium nitrate 85
gum
Guar gum Methacrylamide Potassium chromate/malonic 86
acid
Guar gum Methyl methacrylate Ceric ammonium 87
sulfate/dextrose
Guar gum 4-vinylpyridine Potassium 88
monopersulfate/thioacetamide
Cassia tora gum Acrylamide Ceric ammonium nitrate/nitric 89
acid
Gum arabic and Acrylonitrile Ceric ammonium nitrate/nitric 90
tragacanth gum acid
parameters in dierent ways, and an add on up to 92% has vinyl polymerization is faster than H abstraction by pri-
been reported [82]. mary radicals.
Many redox pair systems have been used, as can be
seen in Table 1. The following advantages have been
2. Initiating and Grafting emphasized: potassium bromate/thiomalic acid works in
Vinyl graft copolymerization through chemical initiation is the presence of oxygen and can thus make the whole system
easier than thermal, photochemical, and other initiation cost-eective [78], copper sulfate/mandelic acid preferably
methods because the activation energy of the redox initia- gives a graft copolymer rather than homopolymer [81], and
tion is quite low in comparison to that of the other methods peroxydiphosphate/metabisulte is also said to give a graft
[70]. However, the choice of redox initiating system has a copolymer rather than homopolymer [84] as well as the
great inuence on the grafting result. potassium chromate/malonic acid system [86]. The ceric
Free radicals are generated in the initiating redox ion is widely used as an initiator for grafting natural
system. The overall mechanism of the redox system ascor- polymers [92]. Cerium ammonium nitrate/nitric acid has
bic acid (A) and persulfate initiating guar gum (G) is given also been found to be an eective initiator for graft
as an example here [76]. The reaction between persulfate polymerization on hemicelluloses, but it was found to be
and ascorbic acid involves a chain mechanism [91] with the inert with crude wheat straw hemicellulose containing 11%
formation of sulfate ion radicals, which are well-known lignin [68]. The ferrous sulfatehydrogen peroxide redox
chain carriers. system could be used instead with this high-lignin material.
Formation of free radicals:
3. Grafting Site
S
S2 O2
8 H2 O ! 2SO4 The site where the grafting takes place on the biopolymer
SOS 2 S
4 H2 O ! SO4 OH H

has been discussed with respect to the graft polymerization
S S of acrylamide onto C. tora gum with ceric ammonium
OH AH ! AH OH
S nitratenitric acid as the redox initiator [89]. The formation
S
AH S2 O2 2
8 ! A SO4 SO4 H of free radicals on biopolymers by cerium(IV) has been
S
AH S OH ! A OH H demonstrated by electron spin resonance [93]. The mech-
S
AH SOS 2 anism by which cerium(IV) generates free radicals is be-
4 ! SO4 H A
lieved to involve the formation of a coordination complex
Mechanism for graft copolymerization: between the oxidant and the hydroxyl group of the bio-
ki
RS M Monomer ! RMS polymer. The ceric(IV)polysaccharide complex then dis-
proportionates, forming a free radical on the biopolymer
k
RMS nM !p RMSn1 chain and cerium(III) [94]. Model compound studies of the
S k cerium(IV) oxidation of monohydric alcohol and 1,2-gly-
GOH RMn !p GOS RMn H Homopolymer cols support the postulated mechanism and suggest that the
k C2C3 glycol and the C6 hydroxyl of the anhydro-D-glucose
GOH RS !i GOS RH
kV
unit may be the preferred sites for radical generation [95].
GOS M !i GOMS The relative reactivities of the C6 hydroxyl and C2C3
kV glycol (cis- and trans-) have been estimated by comparing
GOMS nM !p GOMn
reactivities of models. The results showed that the relative
kt
2GOMSn ! Graft copolymer rate of the ceric ion oxidation of the glycol in the models
was higher than that of the primary hydroxyl [96]. It was
It is apparent that SO S
4 and/or A H radicals may initiate then proposed that the oxidation of C. tora gum occurred
graft copolymerization by H abstraction from the guar preferably at the C2C3 glycol unit and, to a much less
gum backbone. However, the generation of the guar gum extent, at the C6 hydroxyl. The proposed reaction mecha-
macroradical (GOS) was proposed, because the initiation of nism is shown in Schs. 3 and 4.
Scheme 3 Complex between ceric ion and C. tora seed gum [89]. (Courtesy of John Wiley & Sons, Inc.)
Scheme 4 Proposed reaction mechanism for ceric ammonium nitrate-initiated graft copolymerisation of acrylamide onto C. tora
seed gum [89]. (Courtesy of John Wiley & Sons, Inc.)
Guar gum was also cross-linked with dierent

amounts of trisodium metaphosphate to reduce its swelling
properties for use in drug delivery systems [98]. Swelling in
gastrointestinal uids was reduced from 100- to 120-fold
for native guar gum to 10- to 35-fold depending on the
amount of cross-linker used. As a result of the cross-link-
ing, guar gum lost its nonionic nature and became nega-
tively charged. Measurements showed that the cross-link
density of the new products was linearly dependent on the
amount of trisodium metaphosphate used.
The copolymer of guar gum grafted with acryl amide
was cross-linked with glutaraldehyde to form hydrogel
microspheres by the water-in-oil emulsication method
[99]. The structure of the cross-linked graft copolymer is
shown in Sch. 5. The grafted copolymer showed a better
thermal stability than guar gum. A further description of
this material is given in Hydrogels and Drug Delivery
Systems under Applications Section. Guar gum in the
native state has also been cross-linked with glutaraldehyde
[100]. Cross-linking led to the introduction of new covalent
bonds. Glutaraldehyde substitutes part of the hydroxyl
groups, and the cross-linking causes a change in the
polymer structure. These physicochemical changes are
reected in the thermal behavior of the cross-linked guar
gum. The amount of glutaraldehyde used in cross-linking
Scheme 5 Copolymer of guar gum grafted with acrylamide was found to inuence the thermal stability of the cross-
and cross-linked with glutaraldehyde [99]. (Courtesy of Elsevier.) linked guar gum, so that an excess of the cross-linker
reduces the stability of the cross-linked product. The
reaction of guar gum with glutaraldehyde occurred either
by inter- or intra-cross-linking, depending on the amount
4. Characterization of glutaraldehyde used. Cross-linking resulted in the for-
In early works, graft copolymer was conrmed by frac- mation of new regions in the polysaccharide characterized
tional precipitation and light-scattering studies [66]. Infra- by modied structures whose abundance and density were
red was used to estimate the degree of grafting and to cross-linker-dependent.
determine where the grafting sites occurred. Intrinsic vis-
cosity measurements on well-dened copolymer fractions E. Various Modifications
were evaluated in terms of branch density, branch length,
and backbone molecular weight [67]. Nowadays, various Aromatic carbamates have been prepared by the reaction
experimental techniques such as viscosimetry, elemental between linear xylan from oat spelts and aromatic mono-
analysis, IR, dierential scanning calorimetry, thermogra- isocyanates [101]. Linear xylan was prepared by treating
vimetric analysis, x-ray diraction (XRD), and scanning xylan extracted from oat spelts with oxalic acid. The
electron microscopy (SEM) are used for the characteriza- carbamates, xylan-2,3-bis (phenyl carbamate), and xylan-
tion of grafted copolymers [85]. 2,3-bis (tolyl carbamate) were prepared in pyridine. The
reagents were added to the suspensions and the tempera-
ture was raised to 110jC for 20 hr. The structure is shown
D. Cross-Linking in Sch. 6. Fully substituted derivatives were obtained, as
Polysaccharides are cross-linked, e.g., to alter their swelling shown by nitrogen analysis and spectroscopic data (13C
properties to prepare hydrogels. Interpenetrating networks NMR). These chemical derivatives were easily soluble in
and semi-interpenetrating networks are sometimes alter-
natives to cross-linking, but they are not discussed here.
Guar gum has been reacted with dierent amounts of
borate, a common cross-linker used with polymers con-
taining hydroxyls, in aqueous alkali at pH 9 to see whether
this chemical modication enhances the enzymatic degra-
dation of the material [97]. Typical reactions between the
cis-positioned hydroxyls of the galactose subunits of the
guar gum occurred. The modied products were found to
possess better swelling properties and, as a result, a great Scheme 6 Structures of xylan-2,3-bis(phenyl carbamate)
susceptibility to enzymatic degradation. and xylan-2,3-bis(tolyl carbamate) [101].
Scheme 7 Proposed structure for sponges prepared of arabinogalactan and chitosan [102]. (Courtesy of Elsevier.)
polar organic solvents and they were also thermoplastic OR linkages was shown by infrared spectroscopy. The
between 240jC and 300jC. This means that it is possible to products from the reactions between xylan and monoha-
spin bers either from their highly concentrated solutions loorganostannanes as well as from dibutyl-tin dichloride
or from their softening state. and xylan were soluble in dipolar solvents such as dimethyl
Sponges based on arabinogalactan from the Larix tree sulfoxide. Roughly one stannane per three xylopyranose
have been prepared by reductive amination [102]. Dierent units was substituted. The proposed main stannane struc-
sponges were prepared and of these, arabinogalactanchi- ture when the reaction agent was a dichloroorganostan-
tosan was chosen as the most suitable to serve as a scaold nane is shown in Sch. 8.
for cell growth in tissue engineering. The reaction pathway Hemicellulose from spruce, a mixture of galactoglu-
is shown in Sch. 7. Arabinogalactan was rst oxidized to comannan and glucuronoxylan, was modied with well-
obtain the aldehyde form by reaction with potassium dened amounts of methacrylic functions [104]. Soluble
periodate in water. A solution of chitosan in 2% acetic acid
was prepared and the oxidized arabinogalactan was added.
The reaction time and temperature were 72 hr and 37jC,
respectively, and the pH was gradually raised to 5. Imine
bonds in the sponge were reduced by addition of sodium
borohydride to the more stable amine form.
Xylan from larchwood has been condensed with orga-
nostannanes [103]. Xylan and triethylamine dissolved in
water were added to a CCl4 solution containing organo- Scheme 8 Proposed stannane structure obtained when
stannane chloride at 25jC for 60 sec. The presence of Sn xylan was reacted with dichloroorganostannane [103].
polymers that can be used as biomaterials. Arabinogalac-

tan-based sponges were prepared and proposed as scaolds
for possible use in tissue engineering [102]. The evaluation
of their biocompatibility showed an inammatory re-
sponse. However, it was conned to the implant site and
decreased with time, indicating a healing process.
A. Flocculants
Organic occulants for the treatment of wastewater are
essentially synthetic and natural water-soluble polymers.
Synthetic polymers are very ecient occulants because of
their tailored molecular weights and chemical structure,
but they are not shear resistant. Natural polymers, partic-
ularly polysaccharides, are shear-stable, biodegradable,
and available from renewable resources. Their biodegrad-
ability reduces their shelf life and the required dosage is
high. Both groups of polymers consequently have disad-
vantages. One way to overcome them is by grafting poly-
Scheme 9 Modication of hemicellulose with 2-[(1- acrylamide branches onto polysaccharides. The dangling
imidazolyl)formyloxy]ethyl methacrylate [104]. grafted chains have good approachability to the contam-
inants. It has been shown that such occulants can be made
from grafted guar gum, but occulants made from grafted
hemicellulose was modied in dimethyl sulfoxide with 2- amylopectin have a better performance [83,105].
[(1-imidazolyl)formyloxy]ethyl methacrylate and triethyl-
amine as catalyst. Typical reaction conditions were 3 hr at
45jC resulting in approximately 13% substitution per sugar B. Film Former
unit. The reaction pathway is shown in Sch. 9. The product Guar gum has been tested as a lm former, but lms
was easily soluble in both water and dimethyl sulfoxide. formed from guar gums lack clarity and tensile strength.
The preparation of hemicellulose-based hydrogels is fur- Sodium carboxymethylated guar gum has thus been made
ther described in the section entitled Hydrogels and drug and evaluated as a lm former by casting lms from its
delivery systems. aqueous solution [106]. The type and amount of plasticizers
were optimized by evaluating the breaking strength and
water vapor transmission rate. Dummy tablets were coated
IV. APPLICATIONS
with aqueous lms and the performance was compared
This section presents a very brief summary of some of the with that of commercially available hydroxypropyl meth-
more interesting applications of chemically modied hemi- ylcellulose. It was concluded that sodium carboxymethy-
celluloses and gums. Esterication and some of the ether- lated guar gum could be employed as a lm former for
ication reactions are generally adopted to achieve tablet coating.
thermoplastic materials that can be thermoprocessed. Ar-
omatic carbamates are also thermoplastic materials, which C. Hydrogels and Drug Delivery Systems
is suggested may be spun into bers [101]. One paper
suggested that esters be used as emulsiers [38]. Emulsify- Hydrogels are three-dimensional network polymers, which
ing activity has also been demonstrated in the case of xylan swell in aqueous solutions. In the swollen state, the hydro-
alkylated with long alkyl chains bearing ionic substituents, gels become soft and rubbery, resembling a living tissue,
which imparted amphiphilic characteristics [59]. Etheri- and some possess excellent biocompatibility. Two major
cation by carboxymethylation is said to give detergent and approaches for preparing saccharide hydrogels are de-
thickening agents suitable for, e.g., textile printing. The scribed in the literature. In the rst, mono-, di-, oligo-, or
grafting of acrylonitrile onto hemicelluloses has been per- polysaccharides are reacted or mixed with synthetic poly-
formed with the aim to nd applications as thickening mers to create a biodegradable matrix or lm, e.g., acrylic
agents for water systems [68] and as additives for reducing copolymers containing cellobiose. In the second approach,
the time and power required to brillate pulp bers [79]. polysaccharides are modied to reduce their solubility in
Grafting of gum with vinyl and acrylic monomers may water without aecting their ability to be degraded by
remove the disadvantages associated with unmodied colonic enzymes. Examples of the second approach are
gumsdiculty in controlling viscosity and short stability cross-linked chondroitin sulfate and dextran hydrogels.
time as a result of hydrolysis by microorganisms. Fenu- Hemicellulose-based hydrogels have been formed
greek gum grafted with methacrylic acid can be used as a from xylan/chitosan lms by immersing them in water
thickening agent in, e.g., printing cotton fabrics with [107]. Films with 520% chitosan formed hydrogels while
reactive dyes [80]. Today, there is a great interest in lms with more than 20% chitosan dissolved in water. It
was suggested that cohesive forces of the hydrogels were a V. CONCLUDING REMARKS
result of the crystalline arrangement of the polymers and
the electrostatic interaction between acidic groups in the Hemicelluloses and gums have been chemically modied
hemicellulose and amino groups in the chitosan. Hemicel- for more than 50 years for dierent reasons. In the early
lulose-based hydrogels have also been prepared by radical years, the purpose of the modications was often to make it
polymerization of hemicellulose modied with well-dened possible to analyze the materials or to investigate whether it
amounts of methacrylic functions and 2-hydroxyethyl was hemicelluloses that caused problems in derivatizing
methacrylate using a redox initiator system [104]. The cellulose. Later, the purpose was often to make polymeric
hydrogels obtained after a 30-min reaction at 40jC were materials from renewable resources. Many scientic stud-
homogeneous, soft, elastic, and transparent materials. ies have been carried out, but unfortunately all the studies
Targeting of drugs to the colon, after oral adminis- used the traditional experimental strategychanging the
tration, can be accomplished by the use of modied, level of one factor and keeping the other factors xed and
biodegradable polysaccharides. One example is low swell- then changing another factor and keeping the other factors
ing guar gum synthesized by reacting it with trisodium xed. A limitation of this strategy is that it may not give the
trimetaphosphate [98,108]. The behavior of these cross- global optimum because dierent starting points can give
linked guar gums as possible colon-specic drug carriers dierent local optima. Another limitation is that variability
has been characterized by the release kinetics of preloaded and noise are dicult to estimate using this traditional
hydrocortisone with and without guar gum-degrading experimental strategy. A statistical experimental design
enzymes and by direct measurement of polymer degrada- and modeling would probably give more reliable results,
tion in the cecum of conscious rats. It was shown that guar because the inuences of all factors are then investigated
gum cross-linked with trisodium trimetaphosphate was together. Especially for grafting investigations, this would
able to maintain 80% of the drug load in buer solutions be an attractive strategy.
and also to retard the release of a low water-soluble drug. Particularly in the early works, the same reactions
This product, which can be biodegraded enzymatically, were often used for hemicelluloses and gums as for cellu-
could thus potentially be used as a vehicle for colon-specic lose, but there seems now to be a trend toward developing
drug delivery. reactions adapted to these specic materials and their
A copolymer of guar gum grafted with acrylamide was specic properties. Our expectation is that this work will
cross-linked with glutaraldehyde to form hydrogel micro- continue and that these materials will eventually be used to
spheres by the water-in-oil emulsication method [99]. a greater extent.
These microspheres were loaded with a soluble (verapamil
hydrochloride) and a water-insoluble drug (nifedipine) to
investigate their controlled release characteristics. Two REFERENCES
ways to incorporate the drugs were used: during cross-
linking by dissolving the drug in the reaction medium or 1. Schulze, E. Zur Kenntniss der chemischen Zusammenset-
after cross-linking by a soaking technique. In vitro drug zung der panzlichen Zellmembranen. Ber. Dtsch. Chem.
release showed a dependence on both the nature of the drug Ges. 1891, 24, 2277.
2. Cote, W.A., Jr. Wood ultrastructureAn Atlas of
molecule and the method of drug loading. The drug release Elektron Mikrographs; University of Washington Press:
also showed a dependence on the extent of cross-linking Seattle, 1967. plate 1.
and on the drug loading. The release of drugs was swelling- 3. Kerr, A.J.; Goring, D.A.I. The ultrastructural arrange-
controlled in the initial stages, but in the later stage ment of the wood cell wall. Cellul. Chem. Technol. 1975, 9,
diusion of the solute dominated. On the whole, it was 563.
concluded that these microspheres might serve as good 4. Palm, M.; Zacchi, G. Extraction of hemicellulosic oligo-
saccharides from spruce using microwave oven or steam
biomaterials for the controlled release of both water-
treatment, Biomacromolecules 2003, 4, 617.
soluble and water-insoluble bioactive materials. 4a. Lindbland, M.S.; Albertson, A.C.; Ranucci, E. In Hemi-
Another type of drug delivery system is based on the celluloses: Science and Technology; Gatenholm, P., Tenka-
ionic cross-linking of sodium carboxymethylated guar gum nen, M., Eds.; ACS Sympossium Series; 2003, 867: 347.
with microencapsulation of sensitive drugs [109]. When a 5. Galbe, M.; Zacchi, G. A review of the production of
solution of sodium salt of carboxymethylated guar gum ethanol from softwood. Appl. Microbiol. Biotechnol.
containing a model drug was added as droplets to dierent 2002, 59, 618.
6. Schultz, T.P.; McGinnis, G.D.; Biermann, C.J.; Drawer,
multivalent metal ions solutions, they cross-linked to form P.O. Similarities and dierences in preheating woody bio-
microbeads. Trivalent ions such as Al3+ and Fe3+ were mass by steam explosion, wet oxidation, autohydrolysis,
found to be superior to divalent metal ions such as Ca2+. and rapid steam hydrolysis/continuous extraction. Energy
Beads cross-linked with trivalent ions released the protein From Biomass and Wastes VIIISymposium Papers, Lake
over a longer period of time in enzyme-free simulated Buena Vista, Florida, 1984, 1171.
intestinal uid than those cross-linked with divalent ions. 7. Schmidt, A.S.; Mallon, S.; Thomsen, A.B.; Hvilsted, S.;
Lawther, J.M. Comparison of the chemical properties of
All the metal salts provided protection for the loaded wheat straw and beech bers following alkaline wet oxi-
protein from the acidic environment of the stomach. It dation and laccase treatments. J. Wood Chem. Technol.
was concluded that it should be possible to engineer a 2002, 22 (1), 39.
desirable release prole in a formulation. 8. Lundqvist, J.; Teleman, A.; Junel, L.; Zacchi, G.; Dahl-
man, O.; Tjerneld, F.; Stalbrand, H. Isolation and char- 32. Sun, R.C.; Fang, J.M.; Tomkinson, J.; Geng, Z.C.; Liu,
acterization of galactoglucomannan from spruce (Picea J.C. Fractional isolation, physico-chemical characteriza-
abies). Carbohydr. Polym. 2002, 48, 29. tion and homogeneous esterication of hemicelluloses
9. Timell, T.E. Isolation galactoglucomannans from the from fast-growing poplar wood. Cabohydr. Polym. 2001,
wood of gymnosperms. Tappi J. 1961, 44 (2), 88. 44, 29.
10. Lundqvist, J.; Jacobs, A.; Palm, M.; Zacchi, G.; Dahlman, 33. Fang, J.M.; Sun, R.C.; Fowler, P.; Tomkinson, J.; Hill,
O.; Stalbrand, H. Characterization of galactoglucoman- C.A.S. Esterication of wheat straw hemicelluloses in the
nan extracted from spruce (Picea abies) by heat-fraction- N,N-dimethylformamide/lithium chloride homogeneous
ation at dierent conditions. Carbohydr. Polym. 2003, 51, system. J. Appl. Polym. Sci. 1999, 74, 2301.
203. 34. Fang, M.; Sun, R.C.; Tomkinson, J.; Fowler, P. Acetyla-
11. Laend, K.B.; Swenson, H.A. Eect of acetyl content of tion of wheat straw hemicellulose B in a new non-aqueous
glucomannan on its sorption onto cellulose and on its swelling system. Carbohydr. Polym. 2000, 41, 379.
beater additive properties. I. Eect on sorption. Tappi J. 35. Buchanan, C.M.; Buchanan, N.L.; Debenham, J.S.; Gaten-
1968, 51 (3), 118. holm, P.; Jacobsson, M.; Shelton, M.C.; Watterson, T.L.;
12. Hagglund, E.; Lindberg, B.; McPherson, J. Dimethyl- Wood, M.D. Preparation and characterization of arabi-
sulphoxide, a solvent for hemicelluloses. Acta Chem. noxylan esters and ester/cellulose ester polymer blends.
Scand. 1956, 10, 1160. Carbohydr. Polym. 2003, 52, 345.
13. Bennani, A.; Rigal, L.; Gaset, A. Rening of lignocellulose 36. Grondahl, M.; Teleman, A.; Gatenholm, P. Eect of
by organosolv processes. Part I: Isolation, characterization acetylation on the material properties of glucuronoxylan
and utilization of hemicellulose extracted from Norway from aspen wood. Carbohydr. Polym. 2003, 52, 359.
spruce. Biomass Bioenergy 1991, 1, 289. 37. Zinbo, M.; Timell, T.E. The degree of branching of
14. Palm, M. Extraction and separation of hemicellulose hardwood xylans. Sv. Papperstid 1965, 68, 647.
oligomers from lignocellulosic materials, Licentiate The- 38. Tian, B.; Dong, C.; Chen, L. Preparation of konjac
sis, Lund University, Lund, Sweden, 2003; 1719. glucomannan ester of palmitic acid and its emulsication.
15. Stephen, A.M. In Molecular Biology; Aspinall, G.O., Ed.; J. Appl. Polym. Sci. 1998, 67, 1035.
New York: Academic Press, 1983; Vol. 2, 99. 39. Koroskenyi, B.; McCarthy, S.P. Synthesis of acetylated
16. Stephen, A.M. In Molecular Biology; Aspinall, G.O., Ed.; konjac glucomannan and eect of degree of acetylation on
Academic Press: New York, 1983; Vol. 2, 124127. water absorbency. Biomacromolecules 2001, 2, 824.
17. Aspinall, G.O. In Molecular Biology; Aspinall, G.O., Ed.; 40. Sun, R.C.; Sun, X.F.; Zhang, F.Y. Succinoylation of
Academic Press: New York, 1982; Vol. 1, 2223. wheat straw hemicelluloses in N,N-dimethylformamide/
18. Dierckx, S.; Dewettinck, K. In Biopolymers, Polysacchar- lithium chloride systems. Polym. Int. 2001, 50, 803.
ides II; Vandamme, E.J., De Baets, S., Steinbuchel, A., 41. Sun, R.C.; Sun, X.F.; Bing, X. Succinoylation of wheat
Eds.; Wiley-VCH: Weinheim, 2002; Vol. 6, 323. straw hemicelluloses with a low degree of substitution in
19. Sjostrom, E. Wood ChemistryFundamentals and Appli- aqueous systems. J. Appl. Polym. Sci. 2002, 83, 757.
cations. 2nd Ed.; Academic Press: New York, 1993; 6768. 42. Philipp, B.; Nehls, I.; Wagenknecht, W.; Schnabelrauch,
20. Sjostrom, E. Wood ChemistryFundamentals and Appli- M. 13C NMR spectroscopic study of the homogeneous
cations, 2nd Ed.; Academic Press: New York, 1993; 63. sulphation of cellulose and xylan in the N2O4DMF
21. Sandford, P.A.; Baird, J. In Molecular Biology; Aspinall, system. Carbohydr. Res. 1987, 164, 107.
G.O., Ed.; Academic Press: New York, 1983; Vol. 2, 462. 43. Hussein, M.M.-D.; Helmy, W.A.; Salem, H.M. Biological
22. Sandford, P.A.; Baird, J. In Molecular Biology; Aspinall, activities of some galactomannans and their sulfated
G.O. Ed.; Academic Press: New York, 1983; Vol. 2, 458 derivatives. Phytochemistry 1998, 48, 479.
459. 44. Takechi, K.; Furuhata, K.-i. Synthesis and nucleophilic
23. Sandford, P.A.; Baird, J. In Molecular Biology; Aspinall, substitution of tosylated konjac glucomannan. Seni
G.O., Ed.; Academic Press: New York, 1983; Vol. 2, 461. Gakkaishi 1999, 55, 315.
24. Huang, L.; Takahashi, R.; Kobayashi, S.; Kawase, T.; 45. Schweiger, R.G. Nitrite esters of polyhydroxy polymers. J.
Nishinari, K. Gelation behavior of native and acetylated Org. Chem. 1976, 41, 90.
konjac glucomannan. Biomacromolecules 2002, 3, 1296. 46. Croon, I.; Donetzhuber, A. Xanthation and dexanthation
25. Rees, D.A.; Morris, E.R.; Thom, D.; Madden, J.K. In of some hemicellulose fractions. Tappi J 1963, 46, 648.
Molecular Biology; Aspinall, G.O. Ed.; Academic Press: 47. Schmorak, J.; Adams, G.A. The preparation and proper-
New York, 1982; Vol. 1, 268 pp. ties of carboxymethylxylan. Tappi J. 1957, 40, 378.
26. Carson, J.F.; Maclay, W.D. Esters of lima bean pod and 48. Kamel, M.; El-Thalouth, I.A.; Amer, M.A.; Ragheb, A.;
corn cob hemicelluloses. J. Am. Chem. Soc. 1948,70, 293. Nassar, S.H. Chemical modication of guaran gum. Part
27. Gardner, P.E.; Chang, M.Y. The acetylation of native and 1: Carboxymethylation in aqueous medium. Starch-Starke
modied hemicelluloses. Tappi J. 1974, 57 (8), 71. 1992, 44, 433.
28. Ebringerova, A.; Simkovic, I.; Pastyr, J. Hemicellulose 49. Ragheb, A.A.; Kamel, M.; El-Thalouth, I.A.; Nassar, S.H.
acetate. Chem. Abstr. 1983, 98, 36356h. Chemical modication of guran gum. Part 3: Carboxyme-
29. Simkovic, I.; Alfoldi, J. Acetylation of (4-O-methyl-D- thylation in non-aqueous medium. Starch-Starke 1994, 46,
glucurono)-D-xylan under homogeneous conditions using 443.
triuoroacetic acid-acetic anhydride. Carbohydr. Res. 50. Kamel, M.; Ragheb, A.; Haggag, K.; El-Thalouth, I.A.
1990, 201, 346. Isolation, chemical modication and rheological charac-
30. McCormick, C.L.; Lichatowich, D.K. Homogeneous terization of Leucaena gum. Starch-Starke 1992, 44, 374.
solution reactions of cellulose, chitin, and other poly- 51. El-Molla, M.M. Preparation and characterization of
saccharides to produce controlled-activity pesticide sys- carboxymethyl Cassia obovata gum and their utilization
tems. J. Polym. Sci., Polym. Lett. Ed. 1979, 17, 479. in textile printing. Macromol. Mater. Eng. 2000, 282, 51.
31. Sun, R.C.; Fang, J.M.; Tomkinson, J.; Hill, C.A.S. 52. Raval, D.K.; Patel, S.P.; Patel, R.G.; Patel, V.S. A study
Esterication of hemicelluloses from poplar chips in on reaction inuencing factors in carboxymethylation of
homogeneous solution of N,N-dimethylformamide/lith- Leucanena glauca seed gum. Starch-Starke 1994, 46, 399.
ium chloride. J. Wood Chem. Technol. 1999, 19, 287. 53. Kobayashi, S.; Tsujihata, S.; Hibi, N.; Tsukamoto, Y.
Preparation and rheological characterization of carboxy- 75. Patel, M.V.; Raval, D.K.; Patel, R.G.; Patel, V.S. Synthesis,
methyl konjac glucomannan. Food Hydrocoll. 2002, 16, optimization and characterization of graft copolymers
289. from Leucaena glauca seed gum and methylmethacrylate.
54. Kishida, N.; Okimasu, S. Preparation of water-soluble Carbohydr. Polym. 1992, 17, 115.
methyl konjac gluco-mannan. Agric. Biol. Chem. 1978, 42, 76. Bajpai, U.D.N.; Mishra, V.; Rai, S. Grafting of poly(-
669. acrylonitrile) onto guar gum using potassium persulfate/
55. Patel, S.P.; Patel, R.G.; Patel, V.S. Rheological properties ascorbic acid redox initiating system. J. Appl. Polym. Sci.
of guar gum and hydroxyethyl guar gum in aqueous 1993, 47, 717.
solution. Int. J. Biol. Macromol. 1987, 9, 314. 77. Raval, D.K.; Patel, S.P.; Patel, R.G.; Patel, V.S. Studies on
56. Jain, R.K.; Sjostedt, M.; Glasser, W.G. Thermoplastic the graft copolymerization of methylacrylate onto guar
xylan derivatives with propylene oxide. Cellulose 2001, 7, gum by hydrogen peroxide initiation. Cellul. Chem.
319. Technol. 1993, 27, 489.
57. Khan, W.U.; Pitochumani, B. Carboxymethylation and 78. Bajpai, U.D.N.; Jain, A. Grafting of polyacrylamide on to
hydroxypropylation of guar gum and the eect of various guar gum with the redox system potassium bromate/
process parameters on the yield and quality of carbox- thiomalic acid. Polym. Int. 1993, 31, 1.
ymethyl hydroxypropyl guar gum. Res. Ind. 1995, 40, 42. 79. Soliman, A.A.A. Graft copolymerization of acrylonitrile
58. Focher, B.; Marzetti, A.; Naggi A.; Torri, G. Sulfopropy- onto hemicellulose and its eect on paper sheets. J. Sci.
lated hemicellulose: Synthesis and NMR characterization. Ind. Res. India 1997, 56, 545.
Macromol. Chem. 1989, 190, 129. 80. El-Molla, M.M.; El-Thalouth, I.A. Technological evalua-
59. Ebringerova, A.; Srokova, I.; Talaba, P.; Kacurakova, M.; tion of methacrylic acid/fenugreek gum grafted products
Hromadkova, Z. Amphiphilic beechwood glucuronoxylan as thickener in textile printing. Polym. Polym. Compos.
derivatives. J. Appl. Polym. Sci. 1998, 67, 1523. 1999, 7, 501.
60. Ragheb, A.; El-Thalouth, I.A.; Amer, M.A.; Nassar, S.H.; 81. Behari, K.; Taunk, K.; Tripathi, M. Cu2+/mandelic acid
Kamel, M. Chemical modication of guaran gum. Part 2: redox pair initiated graft copolymerization acrylamide
Cyanoethylation. Starch-Starke 1993, 45, 244. onto guar gum. J. Appl. Polym. Sci. 1999, 71, 739.
61. Vincendon, M. Xylan derivatives: Benzyl ethers, synthesis, 82. Taunk, K.; Behari, K. Graft copolymerization of acrylic
and characterization. J. Appl. Polym. Sci. 1998, 67, 455. acid onto guar gum. J. Appl. Polym. Sci. 2000, 77, 39.
62. Ebringerova, A.; Novotna, Z.; Kacurakova, M.; Machova, 83. Singh, R.P.; Karmakar, G.P.; Rath, S.K.; Karmakar,
E. Chemical modication of beechwood xylan with p-car- N.C.; Pandey, S.R.; Tripathy, T.; Panda, J.; Kanan, K.;
boxybenzyl bromide. J. Appl. Polym. Sci. 1996, 62, 1043. Jain, S.K.; Lan, N.T. Biodegradable drag reducing agents
63. Ranby, B.; Sundstrom, H. Graft copolymerization onto and occulants based on polysaccharides: Materials and
native cellulosic bres using Mn3+ initiation. Eur. Polym. applications. Polym. Eng. Sci. 2000, 40, 46.
J. 1983, 19, 1067. 84. Behari, K.; Tripathi, M.; Taunk, K.; Kumar, R. Studies of
64. Arthur, J.C. Jr. Cellulose, Graft Copolymers. Encycl. graft copolymerization of acrylamide onto guar gum using
Polym. Sci. Eng., Wiley-Interscience: New York, 1985; Vol. peroxydiphosphatemetabisulphite redox pair. Polym. Int.
3, 68 pp. 2000, 49, 153.
65. OMalley, J.J.; Marchessault, R.H. A polysaccharide graft 85. Nayak, B.R.; Singh, R.P. Synthesis and characterization
copolymer prepared by anionic coupling. J. Polym. Sci. B, of grafted hydroxypropyl guar gum by ceric ion induced
Polym. Phys. 1965, 3, 685. initiation. Eur. Polym. J. 2001, 37, 1655.
66. Church, J.A. A redox-initiated xylan-poly(sodium acryl- 86. Behari, K.; Kumar, R.; Tripathi, M.; Pandey, P.K. Graft
ate) graft copolymer. J. Polym. Sci. A-1, Polym. Phys. copolymerization of methacrylamide onto guar gum using
1967, 5, 3183. a potassium chromate/malonic acid redox pair. Macro-
67. OMalley, J.J.; Marchessault, R.H. Reaction of living mol. Chem. Phys. 2001, 202, 1873.
polystyrene with some natural polymers and their analogs. 87. Chowdhury, P.; Samui, S.; Kundu, T.; Nandi, M.M. Graft
J. Polym. Sci. C, Polym. Symp. 1968, 24, 179. polymerization of methyl methacrylate onto guar gum
68. Fanta, G.F.; Burr, R.C.; Doane, W.M. Graft polymer- with ceric ammonium sulfate/dextrose redox pair. J. Appl.
ization of acrylonitrile and methyl acrylate onto hemi- Polym. Sci. 2001, 82, 3520.
cellulose. J. Appl. Polym. Sci. 1982, 27, 4239. 88. Taunk, K.; Behari, K. Studies on graft copolymerization
69. El-Shinnawy, N.A.; El-Kalyoubi, S.F. Graft copolymer- of 4-vinylpyridine onto guar gum. J. Appl. Polym. Sci.
ization of acrylonitrile onto hemicellulose using ceric 2002, 84, 2380.
ammonium nitrate. J. Appl. Polym. Sci. 1985, 30, 2171. 89. Sharma, B.R.; Kumar, V.; Soni, P.L. Ceric ammonium
70. Bajpai, U.D.N.; Rai, S. Grafting of acrylamide onto guar nitrate-initiated graft copolymerization of acrylamide onto
gum using KMnO4/oxalic acid redox system. J. Appl. Cassia tora gum. J. Appl. Polym. Sci. 2002, 86, 3250.
Polym. Sci. 1988, 35, 1169. 90. Pourjavadi, A.; Zohuriaan-Mehr, M.J. Modication of
71. Raval, D.K.; Patel, R.G.; Patel, V.S. Grafting of methyl carbohydrate polymers via grafting in air. 2. Ceric-
methacrylate onto guar gum by hydrogen peroxide initiated graft copolymerisation of acrylonitrile onto
initiation. J. Appl. Polym. Sci. 1988, 35, 2201. natural and modied polysaccharides. Starch-Starke
72. Bajpai, U.D.N.; Jain, A.; Rai, S. Grafting of polyacryla- 2002, 54, 482.
mide on to guar gum using K2S2O8 ascorbic acid redox 91. Mehrotra, U.S.; Mushran, S.P. Kinetics and mechanism of
system. J. Appl. Polym. Sci. 1990, 39, 2187. the reduction of peroxydisulphate by ascorbic acid. J.
73. Patel, M.V.; Raval, D.K.; Patel, R.G.; Patel, V.S. Indian Chem. Soc. 1970, 47 (1), 41.
Modication and characterization of Leucaena glauca 92. Mino, G.; Kaizerman, S. A new method for the
seed gum by graft copolymerization with acrylonitrile. preparation of graft copolymers. Polymerization initiated
Starch-Starke 1990, 42, 226. by ceric ion redox systems. J. Polym. Sci. 1958, 31, 242.
74. Raval, D.K.; Patel, M.V.; Patel, R.G.; Patel, V.S. 93. Arthur, J.C., Jr.; Baugh, P.J.; Hinojosa, O. ESR study of
Perspective study of vinyl grafting onto Leucaena glauca reactions of cellulose initiated by the ceric ion method. J.
seed gum and guar gum by hydrogen peroxide initiation. Appl. Polym. Sci. 1966, 10, 1591.
Starch-Starke 1991, 43, 483. 94. McCormick, C.L.; Park, L.S. Water-soluble copolymers.
III. Dextran-g-poly(acrylamides) control of grafting sites T.; Golenser, J.; Domb, A.J. Synthesis and biodegradation
and molecular weight by Ce(IV)-induced initiation in of arabinogalactan sponges prepared by reductive amina-
homogeneous solutions. J. Polym. Sci., Polym. Chem. Ed. tion. Biomaterials 2002, 23, 4621.
1981, 19, 2229. 103. Naoshima, Y.; Shudo, H.; Uenishi, M. Structural analysis
95. Mino, G.; Kaizerman, S.; Rasmussen, E. The oxidation of of the condensation products of xylan with organotin
pinakol by ceric sulfate. J. Am. Chem. Soc. 1959, 81, 1494. halides. J. Macromol. Sci., Chem. 1988, A25, 895.
96. Hintz, H.L.; Johnson, D.C. The mechanism of oxidation 104. Soderqvist Lindblad, M.; Ranucci, E.; Albertsson, A.-C.
of cyclic alcohols by cerium(IV). J. Org. Chem. 1967, 32, Biodegradable polymers from renewable sources. New
556. hemicellulose-based hydrogels. Macromol. Rapid Com-
97. Rubenstein, A.; Gliko-Kabir, I. Synthesis and swelling- mun. 2001, 22, 962.
dependent enzymatic degradation of borax-modied guar 105. Singh, R.P.; Tripathy, T.; Karmakar, G.P.; Rath, S.K.;
gum for colonic delivery purposes. STP Pharma, Sci. 1995, Karmakar, N.C.; Pandey, S.R.; Kannan, K.; Jain, S.K.;
5, 41. Lan, N.T. Novel biodegradable occulants based on
98. Gliko-Kabir, I.; Yagen, B.; Penhasi, A.; Rubenstein, A. polysaccharides. Curr. Sci. India 2000, 78, 798.
Phosphated crosslinked guar for colon-specic drug 106. Baweja, J.M.; Misra, A.N. Modied guar gum as lm
delivery. I. Preparation and physiochemical character- former. Pharmazie 1999, 54, 678.
ization. J. Control. Release 2000, 63, 121. 107. Gabrielii, I.; Gatenholm, P.; Glasser, W.G.; Jain, R.K.;
99. Soppirnath, K.S.; Aminabhavi, T.M. Water transport and Kenne, L. Separation, characterization and hydrogel-
drug release study from cross-linked polyacrylamide formation of hemicellulose from aspen wood. Carbohydr.
grafted guar gum hydrogel microspheres for the controlled Polym. 2000, 43, 367.
release application. Eur. J. Pharm. Biopharm. 2002, 53, 87. 108. Gliko-Kabir, I.; Yagen, B.; Baluom, M.; Rubenstein, A.
100. Gliko-Kabir, I.; Penhasi, A.; Rubenstein, A. Character- Phosphated crosslinked guar for colon-specic drug
ization of crosslinked guar by thermal analysis. Carbo- delivery. II. In vitro and in vivo evaluation in the rat. J.
hydr. Res. 1999, 316, 6. Control. Release 2000, 63, 129.
101. Vincendon, M. Xylan derivatives: Aromatic carbamates. 109. Reddy, T.; Tammishetti, S. Gastric resistant microbeads of
Macromol. Chem. 1993, 194, 321. metal ion cross-linked carboxymethyl guar gum for oral
102. Ehrenfreund-Kleinman, T.; Gazit, Z.; Gazit, D.; Azzam, drug delivery. J. Microencapsul. 2002, 19, 311.
20
Role of Acetyl Substitution in Hardwood Xylan
Maria Grondahl and Paul Gatenholm
Biopolymer Technology, Department of Materials and Surface Chemistry, Chalmers University of Technology,
Goteborg, Sweden
Abstract and is one of our major nutrients. Starch has recently been
successfully converted into plastic materials by conven-
Hemicelluloses are biosynthesized by the majority of plants. tional thermoplastic processing [1]. Hemicelluloses are
The most abundant hemicelluloses, found mainly in hard- biosynthesized by the majority of plants and act as a matrix
wood and annual plants, comprise a 1,4-h-D-xylopyranosyl material that is present between the cellulose microbrils
main chain with a varying number of side chains. In hard- and as a linkage between cellulose and lignin in the cell wall
woods such as aspen, beech, and birch, the hemicellulose [2]. In contrast to cellulose and starch, hemicelluloses have
consists chiey of O-acetyl-(4-O-methylglucurono)xylan, not yet been used commercially for the preparation of
often simply referred to as xylan. In contrast to other poly- polymeric materials.
saccharides, such as cellulose and starch, hemicelluloses One of the possible reasons for the lack of utilization of
have not yet been used commercially for the preparation hemicellulose as a material has been the shortage of access
of polymeric materials. The understanding of the eect of to high molecular weight hemicellulose on an industrial
molecular architecture on material properties is crucial. We scale. For example, in traditional pulping processes
have isolated xylan from aspen through extraction with designed for isolation of cellulose from wood, hemicellu-
alkali and DMSO. Extraction with alkali leads to deacety- loses are degraded to a low degree of polymerization [3].
lation of the xylan, whereas the DMSO-extracted xylan has Only recently was a pilot plant for the isolation of hemi-
a degree of acetyl substitution of 0.45, determined using 1H cellulose from plant tissue built [46]. Other processes for
NMR. The alkali-extracted xylan aggregates in aqueous isolation of high molecular weight hemicellulose from
solution to a much higher extent than the DMSO-extracted agricultural sources are currently being developed [78].
xylan. This dierence can be a result not only of the presence There is a general lack of knowledge on the structure
of acetyl groups, but also of the lower lignin content of the property relationships of hemicellulose. The molecular
DMSO-extracted xylan. Both samples have a large water architecture of hemicellulose is complex in comparison to
sorption. The alkali-extracted xylan crystallizes when cast cellulose and starch, with dierent monosaccharides as
from water, whereas the DMSO-extracted xylan is totally monomer units, various substitution patterns, and branch-
amorphous. The irregularity caused by the acetyl groups ing. A general denition of hemicellulose is polysaccha-
impedes crystallization. rides that can be extracted by water or aqueous alkali from
plant tissue [5,9]. Hemicelluloses are heteropolysaccha-
I. INTRODUCTION rides whose composition and amount vary between dier-
ent plant species. In wood, the hemicellulose comprises
Polysaccharides such as cellulose, starch, and hemicellu- between 20% and 30%, and the hemicellulose content in
loses are produced by plants in vast quantities by the brans and hulls from annual plants such as maize can be as
conversion of carbon dioxide and water using solar energy, high as 4050 wt.% [9,10]. The most common monosac-
which leads to a better carbon dioxide balance in our charides are D-xylose, L-arabinose, D-glucose, D-galactose,
ecosystem. Cellulose, which is the reinforcing component D-mannose, D-glucuronic acid, 4-O-methyl-d-glucuronic
of the cell wall, is used in many applications, such as paper, acid, and D-galacturonic acid (Fig. 1).
textile bers, plastics, membranes, food additives, and The most abundant hemicelluloses, found mainly in
medicines. Starch is produced as an energy reserve in plants hardwood and annual plants, comprise a 1,4-h-D-xylopyra -
509
510 Grondahl and Gatenholm
Figure 1 The most common building blocks in hemicelluloses.
nosyl main chain with a varying number of side chains generally xylose and arabinose, and they are thus termed
based on L-arabinofuranosyl, 4-O-methyl-D-glucurono- arabinoxylans (Fig. 3).
pyranosyl, D-galactopyranosyl, or D-glucurono pyranosyl Xylan can be isolated from the cell wall using alkaline
units. Hemicelluloses isolated from hardwood and annual extraction. This method involves hydrolysis of ester link-
plants dier from one another. ages and thus deacetylation of the xylan. In order to avoid
In hardwoods such as aspen, beech, and birch, the saponication of ester linkages, delignication can be done
hemicellulose consists chiey of O-acetyl-(4-O-methylglu- followed by extraction with dimethylsulphoxide (DMSO).
curono)xylan [11], often simply referred to as xylan (Fig. 2). Xylan is generally considered to be amorphous in the
The backbone consists of h-(1!4)-linked D-xylopyranosyl native state but can crystallize after separation. The rst
residues substituted with one a-(1!2)-linked 4-O-methyl- report on crystalline xylan came in 1951. Spherocrystals of
D-glucuronic acid per approximately every 10th such res- xylan were isolated from barley straw after the isolation of
idue. The xylopyranosyl residues are partially acetylated in a fraction with no uronic acid groups [15]. Xylan single
the C-2 and/or C-3 positions [11]. The degree of acetylation crystals were also successfully prepared from rye and wheat
in native aspen glucuronoxylan has been reported to be after cleaving the hemicellulose chains and removing the
between 0.6 and 0.7 [1213]. side groups [16]. The acid groups are not an integral part of
The hemicelluloses found in annual plants, such as the unit cell [17], but it has been observed that the inter-
maize, rice, oats, sunower, rye, barley, and wheat, are planar spacings are enlarged with an increasing amount of
generally more structurally diverse and complex. These uronic acid and that samples with a great deal of uronic
plant hemicelluloses have a 1,4-h-D-xylopyranosyl main acid are not crystalline [18].
chain that can be heavily branched with xylopyranosyl, Xylan from beech and birch with a degree of polymer-
arabinofuranosyl, and galactopyranosyl side chains and ization of about 80 has a crystallinity of a macromolecular
can also contain 4-O-methyl-D-glucuronopyranosyl or D- nature that diers from that of isolated crystalline particles
glucuronopyranosyl substituents. The two predominant of degraded polysaccharides [19]. The crystallinity and the
monosaccharides in these annual plant hemicelluloses are interplanar distances increase with an increasing amount of
Figure 2 Partial structure of hardwood xylan.

Role of Acetyl Substitution in Hardwood Xylan 511
chosen to start with hardwood xylan, as its structure can

be considered the least complex among hemicelluloses. The
backbone of hardwood xylan contains only one monosac-
charide type, xylose, and the main chain linkage is of a h-
1,4 structure as in cellulose.
Figure 3 Proposed structure for maize bran arabinoxylan II. EXPERIMENTAL

[14]. A: arabinofuranose, X: xylopyranose, G: galactopy-
ranose, GlcA: glucuronic acid, FA: ferulic acid. A. Materials
The aspen xylan used was isolated by alkali extraction,
resulting in deacetylation of the material. The separation
water. The hydrated form has a layer spacing of about 14.84
procedure is described elsewhere [56]. The isolated material
A and a threefold screw axis along the chain direction [20
contained 83 wt.% xylose, 14 wt.% methyl glucuronic acid, 2
21]. In the left-handed structure, an intramolecular hydro-
wt.% mannose, and less than 1 wt.% of other sugars. The
gen bond O5: : : O3V is possible, whereas there is a steric
weight average molecular weight (Mw) was 15,000 g/mol.
conict for a right-handed helix between O2 and H4V [22].
Isolation with DMSO was done to obtain aspen xylan
The O5 : : :O3V intramolecular hydrogen bond is weaker in
with the acetyl groups intact. The rst step was to delignify
xylan than in cellulose and the xylan helix is stabilized
the wood. Aspen wood chips were ground and dried in a
mainly by van der Waals forces. The absence of strong in-
vacuum oven at 45jC overnight, and 20 g was placed in a
tramolecular hydrogen bonds makes the xylan less rigid in
three-necked round-bottomed ask. A solution of 50 mL of
comparison with cellulose [23]. Arabinoxylan from rice also
120 g/L sodium chlorite, 50 mL of 136 g/L sodium acetate,
has an extended left-handed threefold helical structure [24].
and 2 mL of acetic acid was prepared and diluted to 400 mL
There are six monomer units in two antiparallel chains
with 70jC deionized water. The solution was poured onto
and six water molecules in the unit cell. The empty lattice
the aspen wood meal under magnetic stirring and temper-
site is able to accommodate water as well as side groups
ature control at 70jC. After 30 min another addition of 50
such as glucuronic acid. Xylan seems to depend on the
mL sodium chlorite and 2 mL acetic acid was made, and
presence of water to stabilize the structure [25]. The O2H
additions were then made at an interval of 24 hr another six
and O3 atoms of each xylose residue point toward one of
times. In order to remove the remaining chlorine gas, nitro-
the unoccupied lattice sites in the structure, the expected
gen gas was ushed through the ask for 2 hr, after which
locations of the water molecules. In the proposed water
the ask was cooled in an ice bath until the slurry had a
chain, each water molecule accepts from the xylose O2H
temperature of 4jC. The solids were ltered o on a
and donates to an O3. The same water molecule also
Buchner funnel, washed with 1 L of 50jC deionized water
accepts and donates to its neighboring water molecules,
and dried under vacuum at 45jC overnight. This procedure
giving two strong hydrogen bonds in addition to the weak
gave 12.7 g of holocellulose, which is 63% of the initial
intramolecular hydrogen bond O3V: : : O5 [26].
weight. Nine grams of holocellulose and 180 mL of DMSO
The commercial utilization of isolated hemicellulose
were heated to 60jC overnight and the solids were ltered
has as yet been very limited. In the pulp and paper industry,
o on a glass frit funnel. The solution was freeze dried and
retaining the hemicellulose in the pulp has been shown to
the solid obtained was dissolved in 180 mL of deionized
improve both the mechanical properties of the paper and
water at 60jC over a period of 5 hr. After ltration, the solu-
the yield [27]. Hemicelluloses have also been used as
tion was freeze dried. The sample was redissolved in 60 mL
viscosity modiers and emulsiers in food [2,9]. There
of deionized water and the solution was freeze dried again.
has been interest in the use of hemicellulose as a nutraceu-
The total amount of dry xylan extracted in this fraction was
tical [28], in chiral separations [29], and as an HIV inhibitor
1.7 g, corresponding to 19 wt.% of the holocellulose. Size
[30]. The hemicellulose can also be hydrolyzed to mono-
exclusion chromatography in DMSO revealed that the
saccharides that are converted to chemicals such as furfural
product had a Mw of 9000 and a polydispersity of 1.4.
and xylitol or are used as fermentation feedstock for
making ethanol or lactic acid [31].
The aim of this paper is to improve the understanding B. Methods
of the eect of molecular architecture on the material
properties of hemicellulose, focusing on acetylation. In 1. Nuclear Magnetic Resonance Spectroscopy
order to utilize the enormous renewable resource that For 1H NMR analysis, a portion of the dried samples (1.6
hemicellulose constitutes, the availability must be im- 2.7 mg) was dissolved in 0.6 mL of dimethyl-d6 sulfoxide. A
proved by developing ecient processes for isolation. small amount (2 AL) of D2O was added to exchange
However, these processes will not be developed until we hydroxyl protons. 1H NMR spectra were obtained at
see potential applications. The understanding of the eect 400.13 MHz using a Bruker DPX400 spectrometer. The
of molecular architecture on material properties is crucial. spectra were acquired at a probe temperature of 80jC. The
Hemicelluloses are intimately connected with other cell typical acquisition parameters employed were a 90j pulse,
wall polymers and have complex structures. We have a spectral width of 6000 Hz and a repetition time of 17 sec.
2. Size Exclusion Chromatography ppm indicates acetylation [13,33,34]. DSAc was calculated
The size exclusion chromatography system consisted of a by comparing the relative intensities of the signals of the
Waters 2690 with on-line degasser, autosampler, and col- acetyl groups at 2.0 ppm and those of all carbohydrate
umn oven (Waters, Milford, MA, USA) and two serially signals following the equation:
connected columns (TSK gel G6000 WXL and TSK gel Integral for acetyl groups at 2:0 ppm=3
GMPWXL, TosoHaas, Stuttgart, Germany). The used DSAc
Sum of integrals for carbohydrate
detectors were refractive index (RI) (Optilab DSP, Wyatt
signals in the region 3:2 5:6ppm=6
Technology Corp., Santa Barbara, CA, USA), ultraviolet
(UV) monitor (Shimadzu SPD 10A/vp, Shimadzu Corp., The alkali-extracted xylan had no acetyl peak, but was
Kyoto, Japan), and multiangle laser light scattering totally deacetylated, whereas the DMSO-extracted mate-
(MALLS) (Dawn DSP equipped with a HeNe laser at rial was partially acetylated with a DSAc of 0.45.
632.8 nm, Wyatt Technology Corp., Santa Barbara, CA, The samples were analyzed with size exclusion chro-
USA). The columns and the RI detector were kept at 50jC. matography in aqueous media, using both light scattering
The eluent was 0.1 M sodium nitrate containing 0.01% and refractive index detectors. The LS detector gave a peak
sodium azide. The eluent ow was 0.5 mL/min. The at low elution volume (around 14 mL) as a result of light
DMSO-extracted xylan was injected at 10 mg/mL and scattering from aggregated material. This peak was con-
the alkali-extracted xylan at 1 mg/mL. siderably larger for the alkali-extracted xylan than for the
DMSO-extracted xylan despite the fact that the concen-
3. Water Vapor Sorption Isotherms tration of alkali-extracted xylan was 10 times lower than
that of the DMSO-extracted xylan (Fig. 4). The DMSO-
The samples were conditioned in a dynamic vapor sorption
extracted xylan aggregates to a much smaller extent than
(DVS) system (Surface Measurement Systems v. 9.0.) at
the alkali-extracted xylan. The RI signal shows the con-
30jC. The samples were rst conditioned at the lowest RH
centration, and it is clear from the chromatogram that the
and then moved successively to the highest RH. The
amount of soluble DMSO-extracted xylan is higher than
desorption curves were also obtained. First, the samples
the amount of soluble alkali-extracted xylan. The concen-
were conditioned at 0% RH for 500 min, then at 11.3% RH
tration of xylan in the aggregated state is, however, larger
for 600 min, 33.1% RH (600 min), 54.4% RH (500 min),
for the alkali-extracted xylan. This dierence is not neces-
75.5% RH (500 min), 95% RH (900 min), 75.5% RH (500
sarily completely due to the presence of acetyl groups in the
min), 54.4% RH (500 min), 33.1% RH (600 min), 11.3%
DMSO-extracted xylan, but can also be an eect of the
RH (600 min), and nally at 0% RH for 800 min. The
lignin content of the samples. The UV signal at 280 nm
equilibrium water content was measured gravimetrically
could hardly be detected for the DMSO-extracted xylan,
and calculated as the weight of water in the sample
but was clear for the alkali-extracted xylan, indicating that
compared to the total weight.
the alkali-extracted xylan contains higher amounts of
lignin than the DMSO-extracted xylan.
4. Wide Angle X-ray Scattering
The crystallinity of the samples was determined with wide
angle x-ray scattering (WAXS). The ratio of crystalline
material to amorphous material can be measured by
integrating the intensity of the crystal diraction peaks
and dividing it with the intensity of the amorphous dirac-
tion [32]. The samples were investigated with a Siemens
D5000 diractometer. CuKa radiation with a wavelength
of 1.54 A was used, and 2h was varied between 5j and 30j
at a rate of 1j (2h) per minute and a step size of 0.1j (2h).
III. RESULTS AND DISCUSSION

Intriguing features of the molecular architecture of xylan in
hardwood are the substitution with acetyl groups and
glucuronic acid. The reason for acetylation in the native
state is not fully understood. Some possible explanations
are that the acetyl groups control solubility and/or inter-
actions with water or are present to make the xylan
amorphous. In this study, we isolated xylan from the same
raw material with two dierent methods: extraction with Figure 4 Size exclusion chromatograms of the samples; A:
alkali or DMSO. LS of DMSO extracted xylan, B: LS of alkali extracted xylan,
1
H NMR analysis was used to determine the degree of C: RI of DMSO extracted xylan and D: RI of alkali extracted
acetyl substitution (DSAc) of the samples. A signal at 2.0 xylan.
Role of Acetyl Substitution in Hardwood Xylan 513
Figure 5 Water vapor sorption isotherms of the alkali- Figure 7 X-ray diractogram of the alkali-extracted aspen
extracted and DMSO-extracted aspen xylan at 30jC. Circles: xylan.
alkali-extracted xylan; triangles: DMSO-extracted xylan.
The water vapor sorption isotherms show that these the case of DMSO-extracted xylan, no crystalline peaks
materials interact with water to a great extent (Fig. 5). This could be observed, but the material was totally amorphous
has been observed before for hemicelluloses [3536]. The (Fig. 6). The alkali-extracted xylan, however, was semi-
acetyl groups of the DMSO-extracted xylan do not make crystalline (Fig. 7). The irregularity introduced with the
the material more hydrophobic, as is the case for chemi- acetyl groups impedes crystallization of the DMSO-
cally acetylated xylan [36]. extracted xylan.
Another interesting dierence was observed with
WAXS analysis of ground lms from water casting. In
IV. CONCLUSION
Aspen xylan extracted with alkali is deacetylated, whereas
DMSO-extracted aspen xylan contains acetyl groups. The
degree of acetyl substitution of the DMSO-extracted xylan
was 0.45. The alkali-extracted xylan aggregates in aqueous
solution to a much higher extent than the DMSO-extracted
xylan. This dierence can be a result not only of the
presence of acetyl groups, but also of the lower lignin
content of the DMSO-extracted xylan. Both samples have
a large water sorption. One very interesting dierence
between the two samples is that the alkali-extracted xylan
crystallizes when cast from water, whereas the DMSO-
extracted xylan is totally amorphous. The irregularity
caused by the acetyl groups impedes crystallization.
ACKNOWLEDGMENT
We would like to express our gratitude to the following
persons:
Anita Teleman at STFI, the Swedish Pulp and
Paper Research Institute, for the NMR analysis.
Figure 6 X-ray diractogram of the DMSO-extracted Bodo Saake at BFH, the Federal Research Centre
aspen xylan. of Forestry and Forest Products in Hamburg,
Germany, for determination of the molecular wall polysaccharide interactions in maize bran. Carbohydr.
weight of DMSO-extracted xylan. Polym. 1995, 26, 279287.
Lennart Salmen and Anne-Mari Olsson at STFI, 15. Yundt, A.P. Crystalline hemicelluloses. Tappi 1951, 34, 89
95.
for instructions on the DVS measurements. 16. Bishop, C.T. Crystalline xylans from straws. Can. J. Chem.
Johannes Roubroeks for assistance on the SEC 1953, 31, 793800.
measurements. 17. Marchessault, R.H.; Timell, T.E. The x-ray pattern of
crystalline xylans. J. Phys. Chem. 1960, 64, 704.
This work was funded by Vinnova, the Swedish Agency for 18. Horio, M.; Imamura, R.; Tani, K. Characterization of alkali
Innovation Systems, through the Prohem Program. solubles of cellulose by x-ray diraction analysis. Tappi
1960, 43, 769775.
19. Horio, M.; Imamura, R. Crystallographic study of xylan
from wood. J. Polym. Sci., Part A, Gen. Pap. 1964, 2, 627
REFERENCES 644.
20. Marchessault, R.H.; Liang, C.Y. The infrared spectra of
1. Shogren, R.L.; Fanta, G.F.; Doane, W.M. Development of crystalline polysaccharides: VIII. Xylans. J. Polym. Sci.
starch-based plasticsA reexamination of selected polymer 1962, 59, 357378.
systems in historical perspective. Starch/Starke 1993, 45, 21. Marchessault, R.H.; Settineri, W.J. Some comments on the
276280. crystallography of xylan hydrate. Polym. Lett. 1964, 2,
2. Popa, V.I., Dumitriu, S., Ed.; In Hemicelluloses, Polysac- 10471051.
charides in Medicinal Applications; Marcel Dekker: New 22. Settineri, W.J.; Marchessault, R.H. Derivation of possi-
York, 1996; 107124. ble chain conformations for poly-h-1,4-anhydroxylose. J.
3. Sjostrom, E. Wood Chemistry: Fundamentals Applications, Polym. Sci. Part C 1965, 11, 253264.
2nd Ed.; Academic Press Inc.: San Diego, CA, 1993. 23. Sundararajan, P.R.; Rao, V.S.R. Conformational studies of
4. Glasser, W.G.; Jain, R.K.; Sjostedt, M.A. Thermoplastic h-D-1,4V-xylan. Biopolymers 1969, 8, 305312.
Pentosan-Rich Polysaccharides from Biomass; The Center 24. Yui, T.; Imada, K.; Shibuya, N.; Ogawa, K. Conformation
for Innovative Technology: USA, 1995. of an arabinoxylan isolated from the rice endosperm cell wall
5. Gabrielii, I.; Gatenholm, P.; Glasser, W.G.; Jain, R.K.; by x-ray diraction and a conformational analysis. Biosci.
Kenne, L. Separation, characterization and hydrogel- Biotechnol. Biochem. 1995, 59, 965968.
formation of hemicellulose from aspen wood. Carbohydr. 25. Nieduszynski, I.; Marchessault, R.H. Structure of h-D-
Polym. 2000, 43, 367374. (1!4V) xylan hydrate. Nature (London) 1971, 232, 4647.
6. Gustavsson, M.; Bengtsson, M.; Gatenholm, P.; Glasser, 26. Nieduszynski, I.A.; Marchessault, R.H. Structure of
W.G.; Teleman, A.; Dahlman, O. Isolation, Characterisation h,D(1!4V)-xylan hydrate. Biopolymers 1972, 11, 13351344.
and Material Properties of 4-O-Methylglucuronoxylan From 27. Lima, D.U.; Oliveira, R.C.; Buckeridge, M.S. Seed storage
Aspen, Biorelated Polymers: Sustainable Polymer Science hemicelluloses as wet-end additives in papermaking. Carbo-
and Technology; Chiellini, H.G.E., Braunegg, G., Buchert, hydr. Polym. 2003, 52, 367373.
J., Gatenholm, P., van der Zee, M., Eds.; Kluwer Aca- 28. Sugawara, M.; Suzuki, T.; Totsuka, A.; Takeuchi, M.; Ueki,
demic/Plenum Publishers: New York, N.Y., 2001; 4152. K. Composition of corn hull dietary ber. Starch/Starke
7. Buchanan, C.M.; Buchanan, N.L.; Debenham, J.S.; Shel- 1994, 46, 335337.
ton, M.C.; Wood, M.D.; Visneski, M.J.; Arumugam, 29. Okamoto, Y.; Kawashima, M.; Hatada, K. Useful chiral
B.K.; Sanders, J.K.; Lingerfelt, L.R.; Blair, L. Corn ber packing materials for high-performance liquid chromato-
for the production of advanced chemicals and materials: graphic resolution of enantiomers: phenylcarbamates of
separation of monosaccharides and methods thereof. US polysaccharides coated on silica gel. J. Am. Chem. Soc. 1984,
6,352,845, 2002. 106, 53575359.
8. Buchanan, C.M.; Buchanan, N.L.; Debenham, J.S.; Gate- 30. Magerstaedt, M.; Meichsner, C.; Schlingmann, M.; Schrin-
nholm, P.; Jacobsson, M.; Shelton, M.C.; Watterson, T.L.; ner, E.; Walch, A.; Wiesner, M.; Winkler, I.; Bader, H.;
Wood, M.D. Preparation and characterization of arabinox- Paessens, A. Preparation of polysaccharide ester sulfates as
ylan esters and arabinoxylan ester/cellulose ester polymer HIV inhibitors. DE 3,921,761, 1991..
blends. Carbohydr. Polym. 2003, 52, 345357. 31. Heikkila, H.; Alen, R.; Kauko, S.; Lindroos, M.; Nurmi, J.;
9. Whistler, R.L. Hemicelluloses, Industrial Gums; Academic Sarmala, P.; Tylli, M. Methods of producing polyols from
Press: New York, N.Y., 1993; 295308. arabinoxylan-containing material. US 6,262,318, 2001.
10. Chanliaud, E.; Saulnier, L.; Thibault, J.F. Alkaline 32. Hermans, P.H.; Weidinger, A. Quantitative x-ray inves-
extraction and characterisation of heteroxylans from maize tigations on the crystallinity of cellulose bers. A back-
bran. J. Cereal Sci. 1995, 21, 195203. ground analysis. J. Appl. Phys. 1948, 19, 491506.
11. Timell, T.E. Recent progress in the chemistry of wood 33. Hirano, S. Acetate-methyl signals in the nuclear magnetic
hemicelluloses. Wood Sci. Technol. 1967, 1, 4570. resonance spectra of peracetylated derivatives of some oligo-
12. Khan, A.W.; Lamb, K.A.; Overend, R.P. Comparison of and polysaccharides. Org. Magn. Reson. 1971, 3, 353360.
natural hemicellulose and chemically acetylated xylan as 34. Teleman, A.; Tenkanen, M.; Jacobs, A.; Dahlman, O.
substrates for the determination of acetylxylan esterase Characterization of O-acetyl-(4-O-methylglucurono)xylan
activity in Aspergilli. Enzyme Microb. Technol. 1990, 12, isolated from birch and beech. Carbohydr. Res. 2002, 337,
127131. 373377.
13. Teleman, A.; Lundqvist, J.; Tjerneld, F.; Stalbrand, H.; 35. Christensen, G.N.; Kelsey, K.E. The sorption of water
Dahlman, O. Characterization of acetylated 4-O-methyl- vapour by the constituents of wood: Determination of
glucuronoxylan isolated from aspen employing 1H and sorption isotherms. Aust. J. Appl. Sci. 1958, 9, 265282.
13C NMR spectroscopy. Carbohydr. Res. 2000, 329, 807 36. Grondahl, M.; Teleman, A.; Gatenholm, P. Eect of
815. acetylation on the material properties of glucuronoxylan
14. Saulnier, L.; Marot, C.; Chanliaud, E.; Thibault, J.F. Cell from aspen wood. Carbohydr. Polym. 2003, 52, 359366.
21
AlginateA Polysaccharide of Industrial Interest
and Diverse Biological Functions
Wael Sabra
Microbiology Department, Faculty of Science, Alexandria University, Alexandria, Egypt
Wolf-Dieter Deckwer
Biochemical Engineering, GBFNational Research Center for Biotechnology,
Braunschweig, Germany
I. INTRODUCTION A. Sources of Alginate

As early as 600 B.C., seaweed was used as food by man, but Alginate is a gelling polysaccharide found in great abun-
algin, a component of seaweed, was rst discovered in 1880 dance in brown seaweed. As part of the cell wall and
by the Britisch chemist Stanford. Pure alginic acid was rst intracellular material in the form of a gel containing
prepared in 1896 by Akrefting, and in 1929, Kelco Com- sodium, calcium, magnesium, strontium, and barium ions,
pany began commercial production of alginate and intro- it accounts for up to 40% of the dry algal masses. Its main
duced milk-soluble alginate as an ice cream stabilizer. function is believed to be skeletal, giving both strength and
Furthermore, its unique physical properties enable it to exibility to the algal tissue. Most of the commercially used
be used as a stabilizer, viscosier, and gelling agent in the alginate is mainly obtained from three genera: Macro-
food and beverage, paper and printing, biomaterials, and cystis; Laminaria, and Ascophyllum [3]. The chemical
pharmaceutical industries. Still, the high cost of this structures of alginates vary greatly between dierent algae
product and the environmental impact associated with species and dierent regions settled by the same alga.
seaweed harvesting and processing have prevented a major Additionally, alginic acids from dierent sources vary with
breakthrough in various applications. It was only at the regard to the arrangement of uronic acids within the
middle of the 19th century that an interesting possible polymer molecule.
commercial avenue for a microbial polysaccharide Extracellular polymeric material resembling alginates
appeared through the discovery that strains of Pseudomo- from brown algae is also produced by two families of
nas aeruginosa [1] and Azotobacter vinelandii [2], elaborate heterotrophic bacteria. Species of Pseudomonas and Azo-
extracellular polyuronides that closely resemble the alginic tobacter are the only prokaryotic sources for this algal-
acid recovered from the brown algae. Since that time, like polymer. P. aeruginosa (a human pathogen causing
interest in microbial alginate has been chiey medically chronic respiratory infections of cystic brosis [CF]
oriented owing to its association with the pathogenicity of patients) was rst reported to produce this polysaccha-
strains of P. aeruginosa. However, the rapid development ride, which plays an important role in the virulence of this
of pharmaceutical applications of this polymer as well as strain and its survival in the lung [4]. Also, several species
the discovery of its unique immunological properties in the of the genus Pseudomonas (Pseudomonas mendocina and
past few years aroused the interest of industrial biochem- Pseudomonas syringae [5]) have the ability to produce
ists in developing a microbially optimized production alginate under several conditions. Many strains of A.
process of this useful compound. vinelandii (a nitrogen-xating soil bacterium) were also
515
516 Sabra and Deckwer
found to produce this polymer in complex and synthetic II. STRUCTURAL AND PHYSICAL PROPERTIES
media [68]. The presence of the synthetic machinery of OF ALGINATES
this unique polymer, normally associated with marine
algae, in members of two largely diverse prokaryotic Polysaccharide utility is based on a broad spectrum of
genera is not yet claried. Indeed, it is interesting to functional characteristics. Among the performance char-
speculate about a possible taxonomic relationship among acteristics of polysaccharides, the ability to modify the
these two genera. properties of aqueous solutions or dispersions is of utmost
importance, e.g., their capacity to thicken, suspend, gel,
stabilize, swell emulsify, chelate and to form lms and
membrane. Many other applications are based on the often
B. Industrial Production of Alginate
unique interactions of polysaccharides with biological and
The extraction of alginate from algal material is schemat- synthetic materials, as well as on their ubiquitous role in
ically illustrated in Fig. 1. In the rst step, acidication of various biological processes.
the insoluble counterion alginate (Na+, Mg2+, Ca2+,
Sr2+, etc. via ion exchange equilibrium with the seawater)
by extracting the milled algal tissue with 0.10.2 M mineral A. Monomeric and Polymeric Structure
acid. Alginic acid is brought into solution by neutralization of Alginate
with alkali such as sodium carbonate or sodium hydroxide
from water-soluble sodium alginate. Removal of algal In molecular terms, the monomeric units of alginate, a-L-
materials is carried out by various separation methods guluronic acid (G), and h-D-mannuronic acid (M) are C5
such as sifting, otation, centrifugation, and ltration. epimers of each other. The orientation of the carboxyl
Finally, sodium alginate is precipitated by addition of group (COOH) on the C5 carbon of the six-membered
alcohol, calcium chloride, or mineral acid, reconverted to saccharide ring is above the plane of the ring in the M unit
the sodium form if needed, and nally dried and milled. and below the plane in the G unit. Alginate may be
Almost 20 years ago, Young [9] suggested a similar process organized in three ways: (1) homopolymeric G blocks,
for the microbial alginate production, as shown in Fig. 2. polyguluronate [poly(G)]; (2) homopolymeric M blocks,
Nevertheless, the commercial production of microbial polymannuronate [poly(M)]; and (3) heteropolymeric G
alginate is still under development. M blocks, in a randomly arranged G and M sequence,
Figure 1 Flow diagram for the manufacture of sodium alginate.

Alginate 517
Figure 2 Processes for the production of the microbial alginate (as proposed by Young [9]).
either as alternating MG or as short interchanging poly(M) tata, and Ascophyllum nodosum, and, to a lesser extent,
and poly(G) blocks interspersed with individual G and M from Laminaria japanica and Sargassum spp. The sequen-
unit. Although some alginates may exist predominantly as tial structure and composition of alginate vary consider-
one type of block, all three blocks may be present within a ably between dierent species, or even between dierent
single alginate molecule (Fig. 3). part of the same alga. Generally, the highest content of
Alginates do not have any regular repeating unit and guluronic acid is found in alginate prepared from stipes of
when 1!4O-linked randomly, the six-membered ring old laminaria hyperborean plants. Alginates from A.
structure of the monomeric units, each in its own chair nodosum, on the other hand, are characterized by the
conformation, can contribute to many possible three-di- low content of G-blocks and a low gel strength. Addi-
mensional macromolecular structures. The viscosity and tionally, the composition and sequential structure of
the gel forming capacities are the most important charac- alginate may also vary according to seasonal and growth
teristics of this polymer and these, in turn, are largely conditions [10].
aected by the block structure and its chain length. Poly(G) Alginate with more extreme compositions can be
blocks form a buckled chain conformation that binds isolated from bacteria. The bacterial alginates are all
calcium ions avidly and produces rm but brittle gels. The composed of the same two uronic acids as algal alginate,
egg-box model [10] has been proposed to explain this but in addition, many of them are highly acetylated. The
phenomenon (Fig. 3). On the other hand, regions rich in acetyl groups are present solely on D-mannuronosyl resi-
MM or MG blocks do not discriminate between binding of dues in the polymers. Interestingly, bacterial alginates,
calcium or other metal ions and form elastic gels. Poly(M) despite their similar composition, vary considerably in
blocks form an extended ribbon conformation, whereas their structure. The material from A. vinelandii bears the
randomly alternating M and G polymers may form chains closest resemblance to algal alginate, although unlike the
with links and disrupted ribbons. Regularly alternating latter, it does contain O-actyl groups. Both A. vinelandii
poly(MG)n, on the other hand, has a sinusoidal conforma- and algal alginates are composed of the three types of
tion. Physicochemical studies and structurefunction anal- block structures (GG, MM, and GM blocks). The in-
ysis are important to understand the biological role of this teresting feature of the Pseudomonas alginate is that there
polymer in dierent organisms. are no continuous sequence of L-guluronic acid resides.
Whereas the properties of A. vinelandii polysaccharide can
B. Source Dependence therefore be expected to have much in common with the
commercial algal products, the other bacterial alginate
Commercial alginates are produced mainly from Lami- possess dierent and perhaps unique properties, as will
naria hyperborea, Macrocystis pyrifera, Laminaria digi- be discussed below.
Figure 3 Structure of Azotobacter alginate (A) block structure, (B and C) the Ca+2-dependent epimerization process, and the
formation of the so-called egg-box model (B) forming hard gel.
C. Ion Binding Properties as a function of the degree of acylation. The acyl substitu-
tion appears to reduce the number of junction zones and
The ion binding properties of alginates are the basis for their strength, thus permitting the polymer network to
their gelling properties. Alginates show characteristic ion- swell greatly when it is rehydrated [15].
binding properties in that their anity for multivalent
cations depends on their composition. Characteristic an-
ities were shown to be a property exclusive to polygulu- D. Rheology of Alginate Solution
ronate, while polymannuronate was almost without
selectivity. The anity of alginates for alkaline earth met- Alginate solutions are in general highly viscous. This,
als increases in the order Mg<<Ca<Sr<Ba. The high however, is caused by the extended conformation of the
selectivity between ions as similar as the alkaline earth alginate molecule, giving alginate a large hydrodynamic
metals indicates that the mode of binding could not be by volume and high ability to form viscous solutions. The
nonspecic electrostatic binding only, but that some che- intrinsic viscosity of alginates is shown to be dependent on
lation caused by structural features in the G-blocks must the conformation (their molecular weight, compositions,
contribute to the selectivity. This characteristic property and sequence of M and G units) and on the ionic strength
was explained by the so-called egg-box model based of the solution [10,11]. Furthermore, any change of ionic
upon the linkage conformations of the guluronate residues strength in an alginate solution has a profound eect on
studies, suggesting a possible binding sites for Ca2+ ions in polymer behavior, especially on polymer chain extension
a single alginate chain, as shown in Fig 3 [10]. and hence the solution viscosity. In fact, at high ionic
The selectivity of alginate for multivalent cations is strength, the solubility is also aected. The polymer can
also dependent on the ionic composition of the alginate gel, be precipitated and fractionated by high concentrations of
as the anity toward a specic ion increases with increas- inorganic salts such as potassium chloride.
ing content of the ion in the gel. Thus a Ca alginate gel has a Alginate solutions, in general, are pseudoplastic and
markedly higher anity toward Ca2+ ions than a Na thus the apparent viscosity of such solutions depends on
alginate gel. alginate concentrations, shear rate (a 2.5% medium vis-
In bacterial alginate, the presence of acetyl groups in cosity sodium alginate solution is pseudoplastic over a wide
the polymers modies its binding capacity. The capacity range of shear rates (1010,000 sec1), whereas a 0.5%
for Ca2+ binding and the selectivity of this ion are greatly solution is newtonian at low shear rates (1100 sec1), and
reduced. In chemically acetylated alginates, the elastic pseudoplastic only at high shear rates (100010,000 sec1))
modulus of the acetylated alginate in the gel form decreases and temperature [12]. At constant alginate concentrations,
Alginate 519
the eect of temperature on the consistency coecient (K) vinelandii. They revealed the existence of an enzyme that
was better described by using the power law model [12] was able to introduce L-guluronic acid residues into the
alginate chain. The existence of the same pathway in brown
s Kcn algae was conrmed by Madgwick et al. [19].
where K=K(T) is the temperature-dependent consistency
index (apparent viscosity at a shear rate of 1 sec1), c is the B. In Bacteria
rate of shear, and n is the ow behavior index. The higher
the value of K, the more viscous the uid. The ow The A. vinelandii and P. aeruginosa alginate-biosynthesis
behavior index varies from 1 to 0 for pseudoplastic uids; pathways are very similar [20,21]. The interest in Pseudo-
the lower its value, the more pronounced is the pseudomonas alginate has traditionally been medically oriented.
plasticity of the uid. Solution of bacterial alginate was However, polysaccharide production is not common in P.
reported to be more pseudoplastic than that of algal aeruginosa, an opportunistic pathogen in cystic brosis.
alginate [13]. Furthermore, the viscosity of the puried Mucoid strains, isolated from the lung, seem to revert to
bacterial alginate reached as much as 11,200 cps at a low nonmucoid variants when grown on liquid or solid medium
concentration (0.6%). A greater than vefold concentra- in vitro.
tion of algal alginate was required to reach the same In contrast to P. aeruginosa, A. vinelandii is a stable
viscosity at a low shear rate [13]. producer of acetylated alginates under various culture
conditions and is regarded as a potential source of alginates
for technical uses. Exopolysaccharide (EPS) is produced
E. Alginate as a Gel from a range of mono- and disaccharides including glu-
cose, fructose, lactose, maltose, and mannitol [7].
In general, the type of alginate gel depends on the number
Besides the production of alginate, A. vinelandii and
and strength of the cross links and on the length and
P. aeruginosa produce an intracellular polyester, poly-
stiness of the chains between cross links. The modulus of
h-hydroxybutyrate (PHB). PHB is a biodegradable ther-
Ca-alginate gel strongly depends on the composition and
moplastic and is accumulated by a large variety of bacteria
sequence of the monomers in the alginate molecule. Algi-
as carbon and energy storage compound during unbal-
nates rich in guluronate residues form strong and dense gels,
anced growth.
while M-rich alginates form softer and elastic gels [14]. For
NMR studies [22] showed that the metabolism of
this reason, only alginates having G blocks (algal and
sugar follows the EntnerDoudoro pathway. Fructose
Azotobacter alginates) can bind calcium to form rigid gels,
can be metabolized either to fructose-1,6-biphosphate or to
while Pseudomonads alginate form only soft gels because of
mannose-6-phosphate, which is further converted into
the lack of G blocks in the polymer. Therefore, the potential
GDP-mannuronic acid and then incorporated into the
for bacterial alginates may reside in specialized applications
polymannuronic acid polymer.
for polymers of D-mannuronic acid, totally devoid of
On a molecular basis, the initial steps in the alginate
guluronic acid residues, or for polymers in which the
biosynthesis pathway are essentially those of the general
percentage of guluronic acid has been greatly increased [15].
carbohydrate metabolism and the intermediates are widely
utilized. In particular, these steps up to, and including,
GDP mannose are common to alginate and LPS biosyn-
III. BIOSYNTHESIS OF ALGINATE thesis. The nal steps of alginate biosynthesis, however,
A. In Brown Algae (Phaeophyta) remain unclear, especially for Azotobacter. These steps
include polymerization, transacetylation, epimerization,
In a classical work by Lin and Hassid [16,17], the biosyn- and nally, the export of alginate.
thetic route in the brown algae from mannose to the The polymerization process occurs through proteins
incorporation of D-mannuronic acid into a polymer frac- localized in the cytoplasmic membrane. The products of
tion was elucidated. In a cell-free system made from Fucus the genes alg8, alg44, algX, and algK are candidates as
gardneri, they were able to detect enzymes responsible for subunits of the alginate polymerase. The proteins Alg8,
the alginate biosynthesis. These enzymes, which include Alg44, and AlgX were suggested to be localized in the
hexokinase phosphomannose isomerase and D-mannose- cytoplasmic membrane because the amino acid sequences
1-phosphate guanylyltransferase, catalyze the formation of of these proteins contain hydrophobic regions. However,
the sugar nucleotide GDP-D-mannose. They further dem- evidence was obtained that AlgK is entirely periplasmic
onstrated the activity of GDP-mannose dehydrogenase and may also be involved in the export of alginate [23]. Jain
oxidizing GDP-D mannose into GDP-D-mannuronic acid, and Ohman [24] reported that deletion of algK in mucoid P.
and nally the transferase incorporating mannuronic acid aeruginosa stops alginate formation and resulted in uronic
residues into a growing chain of alginate. The authors also acid secretion. They suggested that AlgK plays an impor-
isolated and identied GDP-mannuronic acid and traces of tant role in the polymerization of mannuronate to alginate.
GDP-guluronic acid; however, they failed to detect any It is still not clear as to what stage the acetyl groups are
enzyme with the capacity to epimerize GDP-D-mannuronic introduced into the polymer. This is actually the most
acid into its epimer. The explanation followed shortly [18], conspicuous feature which distinguishes the bacterial from
after they had studied the alginate biosynthesis in A. the algal product. Acetyl groups are always associated with
the mannuronic acid residues, and they are suggested to 3-hydroxybutyryl-CoA, and this is further incorporated by
protect some of these residues from the action of the PHB synthase into the polymer chain [27].
epimerase enzyme [25,26]. It is clear that these bacteria convert the carbon source
The precursor of PHB biosynthesis is acetylCoA to alginate or PHB. The potential of using A. vinelandii as a
formed from catabolizing the carbohydrate through the source of alginate or PHB has been considered [28,29].
EntnerDoudoro pathway, as shown in Fig. 4. PHB is Martinez et al. [30] reported that it is dicult to separate
synthesized by the sequential action of three enzymes: 3- the synthesis of these two polymers in the wild-type organ-
ketothiolase reversibly combines two acetyl CoA into ism. A mutation in the algK gene in A. vinelandii, the gene
acetoacetylCoA, followed by a NADPH-dependent ace- thought to be responsible for alginate polymerization,
toacetylCoA reductase, which reduces its substrate to (R)- reduces alginate production but signicantly enhances
Figure 4 Dierent pathways in relation to alginate biosynthesis from sucrose.

Alginate 521
PHB accumulation (to about 50%). The turnover between 66% sequence identity with the mannuronan-5-C-epimer-
alginate and PHB was also observed in oxygen limited ase gene algG found in P. aeruginosa and therefore, it seems
culture of A. vinelandii UWD strain [31]. This interplay probable that the two deduced enzymes have a similar
between these biopolymers was also observed in pathogen- activity, leading to the production of alginate lacking G-
ic cystic brosis strains. Williams et al. [32] reported that blocks. Generally, epimerization occurs extracellularly in
the progressive loss of alginate production in P. aeruginosa A. vinelandii (Alg E1E7), although there is also evidence
was accompanied by an increased production of PHA. that it might occur to some extent in the periplasm through
the action of epimerase encoded by algG gene [37].
C. Role of Mannuronan C5 Epimerases and The genome of alginate synthesizing organisms encode
their Application of in vitro Alginate alginases that degrade the polymer at specic uronic acid
Tailoring sequences, but most bacterial lyases display low activity
against the native highly acetylated polymers [38]. The
Mannuronan C5 epimerases are produced extracellularly alginases associated with A. vinelandii are reported to
or in the periplasm in A. vinelandii and P. aeruginosa, cleave M-rich alginates, even though the periplasmic AlgL
respectively. This two-compartment biosynthesis pro- enzyme degrades MG bonds with similar eciency as M
cess provides the cells with means to control the function- M bonds. Interestingly, AlgE7 protein displays both epim-
ality of their exopolymer. The primary product synthesized erase and lyase activity [38].
in the cells and secreted is a soluble, partly acetylated The ability of alginate to form a wide variety of
mannuronan. Outside the cell wall, or in the periplasmic ordered structures in an aqueous environment allows the
space, this polymer is processed by the epimerase into specic structurefunction properties which are of para-
molecules with high selectivity for the binding of cross- mount importance for their biological function. Indeed, the
linking calcium ions and hence, a protective gel is formed ability to manipulate the polymer characteristics, through
around the cells, as will be discussed below. In A. vinelandii, the epimerase action, which is a key enzyme in generating
the epimerase is further ingeniously controlled by the level the gel-forming properties, will facilitate the engineering of
of calcium ion in such a way that when the level of cross biopolymers to meet specic functional criteria. The tai-
linking ions is low, the epimerase will increase the calcium loring of various types of alginate structures has been
anity of the alginate by introducing long homopolymeric evaluated [39].
sequences of guluronic acid.
The alginate modifying enzymes, acetylase (algI, algJ,
and algF), epimerase (algG), and lyase (algL), are mainly IV. BIOLOGICAL ROLE OF ALGINATES
found in the periplasm of P. aeruginosa. It is worth noting
A. In Brown Algae
that transacetylation of the mannuronic acid residues
prevents these residues from being epimerized to guluronic The biological signicance of alginate in brown algae
acid residues. Thus the periplasmic acetylase indirectly (phaeophceae) is generally believed to be a structure-form-
controls the periplasmic epimerase activity on the alginate ing component providing both mechanical strength and
polymer in P. aeruginosa [33]. exibility. This relation between structure and function is
The epimerization process in A. vinelandii is much reected in the compositional dierence of alginates be-
more complicated. A set of seven strongly related Ca2+- tween dierent parts of the same plant. The stipe and
dependent epimerases (encoded by algE1algE7) was holast of L. hyperborea have a very high content of
found, compared to only one mannuronan C-5 epimerase guluronic acid, imparting mechanical rigidity to the tissue.
in P. aeruginosa (algG). The epimerization patterns gener- On the other hand, the young tissue and the blades of the
ated by all seven AlgE epimerases can be divided into two same algae have a lower G-content, resulting to more
main groups: those that almost exclusively generate MG exible tissue [40,41,10].
blocks and those that form G blocks. NMR spectroscopy
analysis of the reaction product of all epimerases indicated B. In Bacteria
that only AlgE4 belongs to the rst group [34,35]. All the
other Ca2+-dependent epimerases are involved in the Many microorganisms synthesize exopolysaccharides
formation of G blocks [36]. However, in the same study, (EPS), which either remain attached to the cell surface or
it was also reported that G-block-forming enzymes do not are found in the extracellular medium in the form of amor-
generate the same epimerization patterns (the mean G- phous slime. In natural environments in which the micro-
block length diers with dierent enzymes). This may organisms are found, such polysaccharides may either be
reect the need of the bacterium to generate an alginate associated with virulence factorsas in the case of plant
polymer of special structure required for the survival under or animal pathogensor even protect the microbial cell
conditions of environmental stress or during the formation against desiccation, attack by bacteriophages and proto-
of metabolically dormant cysts. Besides this family of zoa, or protection against macrophagesas in the case of
highly related genes, Rehm et al. [37] identied another human pathogens. Such polymers vary considerably in
mannuronan C5 epimerase gene (algG) which encodes an their chemical structures, and some microorganisms can
enzyme that catalyzes epimerization in the absence of synthesize more than one type of EPS in accordance to
Ca2+. The algG gene from A. vinelandii was found to share dierent environmental stimuli.
1. A. vinelandii A. vinelandii is a gram-negative bacterium, which xes

dinitrogen nonsymbiotically and aerobically. Among bac-
Alginate Production and Cyst Formation teria, aerobic nitrogen xing microbes are rare. They are
A. vinelandii is distinguished from the rest of the mainly found in the family Azotobacteriaceae, which com-
Azotobacteriaceae family by the presence of a characteristic prises the genera Azotobacter, Azomonas, Beijerinchia, and
life cycle that includes the formation of spherically dor- Derxia. Pseudomonas methanitricans, a methane-oxidiz-
mant cyst. For a successful formation of cyst, both PHB ing organism, also xes N2 aerobically. Mycobacterium
and alginate are essential. Alginate is an essential compo- avum and possibly one or two related species are also
nent of the cyst. This polysaccharide coating protects the aerobic nitrogen xers. The remainder of the aerobic
cyst from desiccation and unfavorable conditions. The bacteria that can x N2 are facultative anaerobes which
intine (inner coat) and exine (outer coat) layers of the cyst x N2 only when growing anaerobically [45,46]. The ma-
contain dierent types of alginates. The exine alginate is of jority of nitrogen xers belong to the group of strictly
an unusually high G-block content polysaccharide result- anaerobic microorgansms [47,48].
ing in a much higher stiness. The intine alginate was found The nitrogenase enzyme complex, catalyzing the re-
to be rich in polymannuronic acid residue. The divalent duction of dinitrogen to ammonia, is highly sensitive to
cations Ca2+ and Mg2+ are generally thought to serve as oxygen. However, members of the diazotrophic Azotobac-
structural elements in the cyst coat, and omission of either ter are able to grow under fully aerated conditions [4952].
mineral in the encystment medium has been reported to They do not x nitrogen optimally at high pO2 values, but
result in an absence of exine and intine materials, and, tend to be microaerophilic in the sense that their N2
hence, a lack of resistance against unfavorable conditions xation is most eective at low pO2 levels, although they
[42]. The existence of a large number of alginate modifying are obligate aerobes. Obligate aerobes such as A. vinelandii
enzymes (epimerases, lyases, and acetylase) in the dieren- are known to use two mechanisms for protecting the
tiation process was reported for A. vinelandii [43]. Further- nitrogenase system against oxygen damage (Fig. 5) [53
more, the correlation between encystment and the release 56,50,57]. The hypothesis of respiratory protection postu-
of epimerases indicate that epimerases were, additionally, lates that the unusually high activities of cellular oxygen
cell- and cyst-associated. Cysts have been reported to consumption, characteristic for azotobacters, prevent the
survive in dry soil for several years. Under favorable diusion of O2 into the cells and thus to the nitrogenase.
conditions, the alginate coating will swell and the cyst will Whenever this steady state becomes disturbed by an in-
germinate [41,44,43]. crease in the dissolved O2 concentration or phosphate
limitation, a conformational protection, i.e., the switch-
Alginate Capsule and Nitrogenase Protection o of nitrogenase activity, protects nitrogenase proteins.
The association of alginate production with cyst for- Additional protection of the nitrogenase may also be
mation and its structural signicance does not explain the provided by the decrease in the cellular surface area per
abundant production of this polymer during vegetative cell volume, as observed by Post et al. [58].
growth. Under conditions permitting vegetative growth, Recently, we observed that in a phosphate limited
alginate is produced as extracellular materials, which play continuous culture, the specic rate of oxygen consump-
dierent roles depending on environmental conditions. tion ( qO2) increased as the dissolved oxygen tension ( pO2)
Figure 5 Proposed mechanisms for the protection of nitrogenase against the damage imposed by oxygen.
Alginate 523
was elevated from 1% to 5% air saturation [52]. However, Theoretically, when qO2 is constant and to maintain a
qO2 remained essentially constant when pO2 was above 5%. low intracellular O2 concentration suitable for nitrogenase
These results cannot be interpreted in terms of the respi- and with increasing the DOT in the medium, any of the
ratory protection concept. However, both the molecular following may occur: (1) a decrease of the diusion coef-
weight and the guluronic acid content of alginate formed in cient of oxygen (DO2) through the dense capsule, (2) a
these cultures monotonically increased with pO2. Transmis- decrease in the specic surface area (A) as described by Post
sion electron microscopy of negatively stained A. vinelandii et al. [58], (3) an increase in the capsule thickness (d). A
showed that cells at lower and higher pO2 values formed theoretical dierence between a normal aerobic microor-
capsules with signicant dierences in the thickness and ganism with constant permeability and a nitrogen-xing
compactness of the polysaccharide (Fig. 6). These results bacteria with varied permeability of the cell wall is shown
revealed that under nitrogen xing conditions, the bacte- in Fig. 7.
rium tends to build alginate capsules with variable compo- The involvement of the rst and third possibilities as
sition according to the external dissolved oxygen protection mechanisms for the nitrogenase was suggested
concentration, implying that its production may contribute by Sabra et al. [52]. Studies on the continuous culture of A.
to a protection of nitrogenase against oxygen damage. vinelandii growing diazotrophically with dierent oxygen
The results obtained in our laboratory [59,51,52] and concentrations exhibited that alginate capsules are formed
those obtained by Post et al. [60] and Boiardi [61] indicate even in the presence of high shear rate in bioreactor.
that respiration, at least above a certain O2 concentration, Moreover, the alginate capsule was more compact and
is not the prevailing mechanism for nitrogenase protection. dense at higher dissolved oxygen concentration (Fig. 6). It
Because cells grown at their maximum specic oxygen was therefore postulated that the compact alginate layer
consumption rate (over 5% air saturation in A. vinelandii formed around the cell also acts as a diusion barrier for
case) form compact capsules (Fig. 6), and if one considers oxygen that limits its transfer to the extremely oxygen
that the intracellular oxygen level (CO2, int) in Azotobacter sensitive nitrogenase enzyme. Indeed, measurements of
cells should be very low (near the anaerobic condition) to intracellular oxygen concentrations and a quantitative
stimulate the nitrogenase function [50], the permeability of knowledge about the sensitivity of nitrogenase toward
the cell wall to oxygen should then be decreased with oxygen are necessary.
increasing the dissolved oxygen tension (DOT, CO2,ext) In general, for the vegetative cells, it is reasonable to
after reaching qO2 max. believe that alginate has no single function, but rather
At the maximum specic respiration rate (here above provides the cell with a multitude of protective properties
5% air saturation), the specic oxygen consumption rate under various environmental conditions. Alginate acts as a
( qO2) is represented by protective barrier against heavy metal toxicity, as an ion
exchange system with enhanced selectivity for Ca2+, or
qO2 qO2 ;max PCO2 ;ext CO2 ;int constant provides the bacterium with a hydrophilic, negatively
charged coating which causes a barrier against attack and
where P is the permeability and is equal to adverse environmental conditions [25]. Recent studies
revealed that alginate-encapsulated Azotobacter chrococ-
P DO2 A=d:
cum is better protected against the depressive eect of
DO2 is the oxygen diusion coecient through the cell phages than nonimmobilized cells [62]. The presence of
membrane (cm2/sec), A is the specic cell surface area eight dierent types of epimerases, together with lyases and
(cm2/g), and d is the cell wall thickness (cm). acetylase, represent excellent tools for the cell to manufac-
Figure 6 Thin-section electron micrograph of Azotobacter cells grown at lower (2.5%) and higher (20%) pO2 values.
isolates that appear nonmucoid are truly nonmucoid but,

instead, may belong to the stress condition-dependent
category of mucoid mutants. Recently, it was shown that
in a nonmucoid PAO1 strain of P. aeruginosa, alginate
formation can be triggered in chemostat culture under
oxidative stress conditions [70]. Interestingly, and despite
the lack of the oxygen-sensitive nitrogenase in P. aerugi-
nosa, this bacterium exhibits better growth at the micoaer-
ophilic range as A. vinelandii [52,71,70]. The preference of
P. aeruginosa for microaerophilic growth is in accordance
with ndings from genomic analysis for this strain [72].
Analysis of the genomic DNA sequences of P. aeruginosa
PAO1 showed that this strain possesses a 15-kb cluster of
genes encoding a micro-oxic respiration system, similar to
that used by nitrogen-xing bacteria, including genes
encoding homologues of nitrogen xation/micro-oxic reg-
ulatory cascade proteins (xL, xJ, and xK/anr). These
genes enable the preferred microaerobic growth of nitrogen
xing bacteria such as A. vinelandii. For P. aeruginosa, they
might be a contributing factor to its unusual survival under
the conditions in the biolm [4].
Sabra et al. [70] further investigated the mechanisms
by which P. aeruginosa protects itself against oxidative
stress. The P. aeruginosa strain PAO1 can create micro-
arophilic growth conditions by at least two mechanisms:
(1) blockage of transfer rate of oxygen (kLa) and (2)
formation of a polysaccharide capsule on the cell surface
Figure 7 Theoretical presentation of the dierence in [70]. It is postulated that the blockage of oxygen transfer
permeability of the cell membrane between aerobic hetero- may play an important role in the defense of this pathogen
troph and aerobic nitrogen xer. against reactive oxygen intermediates. Fig. 8 shows the
dierence between Pseudomonas cells grown under nearly
anaerobic conditions, in asks, and in a pO2-controlled
chemostat culture. The dierence in the capsule morphol-
ture polymers of dierent and unique properties to suit its ogy of P. aeruginosa from that of A. vinelandii may reect
external environment. the variations in capsule structures, in particular the oc-
currence of GG block in the latter, and hence the produc-
2. Pseudomonads tion of compact alginate layer around the nitrogen xing
bacterium (Figs. 6 and 8).
Role of Alginate in the Surface Attachment
of P. aeruginosa The Antibiotic Therapy and the Mucoidy
Alginate is a key component of biolm of P. aerugi- Prevalence in P. aeruginosa
nosa and certain other pseudomonads. Expression of algC, Over the years, a prime target for antimicrobial ther-
the gene responsible for phosphomannomutase, and algD, apy has been P. aeruginosa. However, despite constant
the gene responsible for the guanosine-diphosphomannose improvements in antibiotic research, P. aeruginosa remains
dehydrogenase, are upregulated following the adherence to impossible to eradicate. The association of mucoid strains
solid substrata [63], with a concomitant increase in alginate of P. aeruginosa with chronic pulmonary infection in CF
production. Alginate biosynthesis and biolm formation patients is well recognized. Evidence for their emergence in
are triggered by a number of environmental factors includ- vivo was rst proven by Doggett et al. [73]. However, in
ing high osmolarity, ethanol, nitrogen, or phosphate lim- contrast to their emergence in vivo, when cultured in vitro,
itation [64]. It is recognized that oxygen plays an important mucoid strains of P. aeruginosa tend to revert to the non-
role in the formation of alginate and mucoidy appearance mucoid form. The particular cause for the conversion to
in this bacterium [6569]. Oxygen-dependent upregulation mucoidy is not fully understood in P. aeruginosa. In the
of transcription of alginate genes was normally found in course of phage typing, Martin [74] observed mucoid
highly mucoid strains of P. aeruginosa [68]. In an experi- growth of P. aeruginosa immediately around the areas of
ment with a mixture of mucoid and nonmucoid strains phage lysis and obtained mucoid variants after subcultur-
(1:1), Krieg et al. [66] found that aeration favorably selects ing from such areas. Prolonged antibiotic therapy has been
the growth of only mucoid phenotypes. Nonmucoid strains proven as a causative factor in the emergence of mucoidy in
were reported to be sensitive to oxygen. In this connection, patients with cystic brosis [75]. Indeed, isolation of algi-
it should also be mentioned that not all cystic brosis (CF) nate producing mutants of Pseudomonas uorescens, Pseu-
Alginate 525
Figure 8 Transmission electron microscopy of ultrathin sections of negatively stained vegetative cells grown at dierent
conditions: (a) cells from shake ask culture ( pO2=0 ); (b) cells grown under controlled oxygen tension ( pO2=10% of air
saturation).
domonas putida, and P. mendocina was carried out via involvement. It is dicult to be precise about the number of
treatment with antibiotics. Moreover, in vivo, aged bio- genes that are solely associated with alginate biosynthesis
lms with high concentration of alginate materials are as it is now clear, for example, that some of the control
signicantly less susceptible to antibiotic therapy [76]. genes act globally and encode proteins such as alternative
Oxidative burst in neutrophils in contact with P. aeruginosa factor [79]. Other alg genes, e.g., alg A,C, are also
in the lung and the release of oxidative radicals was essential for lipopolysaccharide (LPS) and rhamnolipid
reported to be another reason for the emergence of biosynthesis in P. aeruginosa [80].
mucoidy [77,70]. The genetics of alginate biosynthesis was rst studied
Other Pseudomonads in Pseudomonas and the genes involved in the synthesis of
GDP-mannuronic acid have all been identied and char-
For nonpathogenic alginate-producing pseudomo-
acterized (Fig. 9). Interestingly, algL, a gene encoding an
nads, no natural ecological niche was reported. This sug-
alginate lyase, is located within the biosynthetic operon
gests that in the majority of natural habitats, alginate
[81,82]. It is not clear why a degradative enzyme should be
biosynthesis confers no advantage to the organism. Non-
concurrently expressed with the biosynthetic enzymes. It is
mucoid pseudomonads are routinely isolated from natural
clear, however, that the overexpression of algL gene prod-
environments. Because exopolysaccharides are known to
uct results in the release of planktonic bacteria from
chelate heavy metals, it was also reported that alginate
biolms, and this helps the colonization of other sites [83].
formation was induced in plant-parasitic pseudomonads by
The regulation of alginate biosynthesis is complex and
treatment with a bacteriocidal spray containing copper.
involves specic gene products and those that act more
Thus the secretion of alginate may contribute to some
globally. The ve genes algU (the sigma-like factor-algT),
plant-bacterial diseases. Although alginate is an important
mucA (algS), mucB (algN), mucC (algM), and mucD (algW)
virulence factor in the infection of plants by some phyto-
comprise the main switch controlling the conversion be-
pathogenic pseudomonads, the precise purpose of this
tween nonmucoid and mucoid forms of P. aeruginosa (Fig.
polysaccharide remains unclear, particularly for some
9). AlgU causes an increase in the alginate biosynthesis by a
pseudomonads producing more than one polysaccharide.
direct action on the algD promotor, and indirectly by
For example, P. syringae pv. glycinea produces levan and
upregulating transcription of another regulatory gene,
acetylated alginates in vitro when growing on sucrose or
algR [84]. This environmentally induced transcription of
glucose as the carbon source, respectively, yet alginate is
the alg cluster and the resulting production of alginate only
the only polysaccharide isolated from the infected soya
occur at a low level. However, the copious amounts of
bean leaves [78]. Sodium chloride and ethanol were shown
alginate, found in patients with cystic brosis, are only
to signicantly increase alginate production in a variety of
produced in combination with mutation in the negative
uorescent pseudomonads, suggesting that osmolarity and
regulators of the AlgU (antisigma factors), mucA (algS) or
dehydration may be general signals for the production of
mucB (algN) [85,86]. An analogous set of genes associated
this polysaccharide [5].
with the control of alginate production, the negative
regulation of algU by mucA and mucB, together with mucC
V. GENETIC CONTROL AND REGULATION and the transcriptional regulation of the algD gene, have
OF ALGINATE BIOSYNTHESIS been described for A. vinelandii [87,88].
AlgR (algR1), algQ (algR2), algP (algR3), and algB are
The genes for alginate biosynthesis are all located on the also genes involved in alginate biosynthesis. The best
bacterial chromosome, with no evidence to date of plasmid characterized of these regulators is algR, which is tran-
Figure 9 Model of alginate biosynthesis, polymerization, modication, and export, as well as the organization and control of
alginate genes in P. aeruginosa. (From Refs. 23 and 79.)
scribed in response to the protein algU. AlgR binds to polyguluronic acid and, hence, the oxygen-dependent
three sites upstream of the algD promotor and, in con- upregulation of epimerase enzymes, especially the gene
junction with algU, upregulates transcription of algD and algE2 and algE5, which are responsible for introducing
the downstream genes. AlgR also promotes the expression guluronic acid blocks into alginate. Interestingly, Ramstad
of algC. The eciency of algR is increased via phosphoryl- et al. [92] showed that the same enzyme algE2 had a
ation by the cognate kinase algQ. AlgB also modulates temperature optimum of about 55jC, in reference to its
algD expression [79]. A number of additional genes for, important function during encystment and the prevalence
e.g., algP and algZ, are also involved in alginate regulation of the unfavorable conditions.
[89], and a two-component signal transduction pathway
comprising the putative sensor proteins algQ (kinase) and
algZ, interacting with regulator protein such as algR and
VI. CONDITION FOR OPTIMAL ALGINATE
algB, was identied [89]. These positive regulators bind,
upstream of the algD promotor, presumably leading to the PRODUCTION
formation of a superhelical structure with the aid of the A. Fermentation Conditions and Alginate
histone-like algP protein, causing activation of transcrip- Production in A. Vinelandii
tion [90,91].
Although the properties of alginate are crucial for its There have been many attempts to increase alginate pro-
commercial application, the regulation of epimerases has ductivity through medium formulation [68,13,9397].
not been well studied so far, in particular, for A. vinelandii. The addition of xed nitrogenous compounds to the
Holdal et al. [43] postulated that the release of epimerases is culture medium, based on a controversial report [28], con-
regulated by the oxygen level and by the nature and cludedthat varyingthe sourceof peptone usedin the medium
concentration of the carbon source. In their work, they could alter alginate yield by up to 30%, suggesting a more
showed that under oxygen limitation, both agE6 and algE4 specic role for nitrogenous nutrients. Furthermore, growth
enzymes are released in signicant amounts. Furthermore, in the presence of ammonium inhibits alginate formation in
Sabra et al. [52] showed that the oxidative stress applied to A. vinelandii [28,59]. To understand the precise role of
the phosphate-limited chemostat culture of A. vinelandii nitrogenous compounds on alginate production, the dis-
grown diazotrophically stimulates the production of high- solved oxygen tension must be accurately controlled. How-
molecular-weight alginate gel with high proportion of ever, most of the work in this respect was performed in
Alginate 527
uncontrolled shake ask cultures. This may explain the The eect of other growth limitations on alginate
contradictory results obtained with A. vinelandii. production has also been thoroughly investigated. Iron
The crucial eect of controlling the dissolved oxygen and/or molybdate limitation yielded the highest specic
tension also accounts for the controversially reported rates for alginate production [6,101,102]. Ca2+ limitation,
results with respect to phosphate [6,7,13,51]. On one hand, on the other hand, did not enhance alginate and biomass
Brivonese and Sutherland [28] reported that phosphate formation in diazotrophically grown cells [100,102]. This
might simply act to buer the medium, and used a phos- may reect the importance of epimerase activity that is
phate-rich medium (7.5 g K2HPO4). On the other hand, it known to be dependent on the Ca2+ level for the survival of
was recently observed that a drop in alginate leads to a these cells. The chelation of Ca2+ through the addition of
medium with excess phosphate, and for their continuous ethylenediaminetetraacetic acid (EDTA) to the fermenta-
culture studies, a phosphate limitation gave maximum tion medium resulted in the production of alginate with a
alginate production [51]. Moreover, a correlation between preponderance of mannuronic acid residues [103,104].
phosphate limitation, respiratory requirement of the cell in Surprisingly, under sucrose limitation in a fed batch cul-
term of respiratory quotient (RQ), and alginate production ture, conditions where the cell would be expected to make
by diazotrophically grown cells of A. vinelandii was recent- the most ecient use possible of its available carbon and
ly demonstrated by Sabra et al. [51,59]. They showed that energy substrate, alginate was produced at similar rates to
in phosphate-limited batch or continuous culture, the other limitations [59,70]. This was also observed in carbon-
highest specic alginate production was accompanied by limited chemostat culture by Deavin et al. [6]. The forma-
a value of RQ about 0.8 at an intermediate value of pO2 (2 tion of alginate under C limitation may indicate how
5% air saturation). This RQ value was, indeed, near to the important alginate biosynthesis is for the survival of this
theoretical optimal RQ value (0.8) calculated on the basis diazotrophically grown bacterium in controlled pO2 cul-
of the stoichiometry leading to alginate formation from ture. This means that the protection of nitrogenase by
sucrose. This optimal RQ appears to be a useful parameter forming a compact capsule around the cell under C limi-
for the control of alginate production by A. vinelandii. tation is the predominating mechanism as a respiratory
It is generally recognized that the control of oxygen protection that wasting of the limiting carbon source is not
supply is of critical importance for alginate production by advantageous for cell survival. In agreement with this
A. vinelandii, especially when grown diazotrophically. Re- assumption, the guluronic acid content and the relative
cently, it was reported that under controlled microaero- molecular weight of alginate produced after the onset of C
philic conditions, a very narrow range of dissolved oxygen limitation in a fed batch culture increased [59]. Although
tension exists for both optimal alginate and biomass the carbon-limited culture produced alginate of high qual-
production under diazotrophic conditions [51]. This ex- ity (higher molecular weight and higher guluronic acid
treme oxygen sensitivity renders the studies on cell physi- content), a lower yield was obtained in comparison to that
ology with respect to the eects of dierent nutritional of a normal batch process (3 and 4.9 g/L for fed batch and
conditions dicult and sometimes controversial (see batch culture, respectively) [59].
above). Indeed, it was shown that under phosphate-limited
conditions, results from controlled microaerophilic condi- B. Kinetics of Alginate Production
tions signicantly diered from those of ask cultures
under the same phosphate concentrations [51]. The eect Alginate production by A. vinelandii in phosphate-limited
of increasing the viscosity of the culture broth due to the batch cultures was found to parallel cell growth and to
polymer production and cell growth on the eective pO2 on cease when growth stopped [51]. This was in agreement
one hand and the decrease in pH due to alginate secretion with results of Deavin et al. [6]. The rst attempt to describe
on the other make such heterogeneous environments un- the kinetics of alginate production was made by Klimek
suitable for investigating the physiology of such a process. and Ollis [105]. They used the experimental data from
Several studies on Azotobacter showed that for optimal Deavin et al. [6] in batch cultures to describe the rate of
alginate formation, the dissolved oxygen tension must be alginate production by the well-known Luedeking and
accurately controlled in a microaerophilic range. Oxygen Piret kinetics [106], and a growth-associated form allowed
limitation in this bacterium leads to an increased accumu- this fermentation processes to be described well. However,
lation of PHB [98,99,52]. However, the narrow microaero- in continuous culture, they found that the same strains
philic range of alginate production by diazotrophically showed a specic alginate production rate that was rela-
growing culture of Azotobacter remains unexplained [98 tively independent of growth rate [6,7]. These controversial
99]. Recently, it was concluded that alginate production results between batch and continuous cultures was later
does not increase linearly with increasing pO2, because the explained by the fact that alginate synthesis might have
dense alginate capsule around the cell (at high pO2 values) been inhibited in the late stage of the batch culture by
exhibits not only a mass transfer barrier against oxygen, oxygen limitation that did not occur in continuous culture,
but also to other media constituents, and hence lowers the as a result of DOT control [107]. The contribution of the
biomass and alginate productivity. On the contrary, the nongrowing cell metabolism to alginate production was
soft alginate slimy layer at intermediate pO2 value exhibits observed by Clementi et al. [8] using A. vinelandii DSMZ
lower mass transfer resistance to other nutrients and thus 576, and alginate production continued after the organisms
permits the continuous production of alginate [52]. entered the stationary phase. They also conrmed that
alginate production was only partially growth associated, strong turbulence in local zones where damaging of the
with a 7580% contribution of nongrowth-associated pro- sensitive cell membrane occurs [109].
duction. Recently, we showed that for strain of A. vine- Changing the rheology of the uid will obviously aect
landii DSMZ 93-541b at a xed growth rate, the specic the ow pattern created by the impeller. Around the
alginate production rate ( qalg) was dependent on the pO2 impeller the uid is turbulent, and turns to be laminar or
applied (Fig. 10). Furthermore, a general trend was ob- stagnant when the shear stresses fall below the yield stress
servedqalg peaked at intermediate pO2 values (25% air of the polymer. The impeller creates a cavern in which the
saturation). Therefore, it may be concluded that the kinet- uid is moving relatively fast and where the ow is turbu-
ics of alginate production depends not only on the strain lent. However, in the bulk of the uid, where the shear
[28], but also on the growth conditions. stresses are below the yield stresses, the uid is not moving
at all [109].
C. Non-Newtonian Alginate Fluids and the Role Preliminary experiments in laboratory bioreactors
of Fermentor Hydrodynamics used for the production of microbial alginate were typically
restricted to aerated, mechanically agitated vessels
In aerobic submerged exocellular microbial polysaccha- [6,52,110]. This produced the turbulence necessary for
rides fermentations such as xanthan, dextran, and algi- small bubble formation and mixing and hence, good mass
nate, oxygen supply to the media and mass transfer of O2 transfer. However, for the oxygen-sensitive A. vinelandii,
to growing microbial cells is still a major technical the necessity of accurately controlling the dissolved oxygen
problem aecting microbial productivity, because the concentrations in large-scale bioreactors remains a chal-
solution becomes highly viscous and non-Newtonian lenging problem. The pseudoplastic behavior of alginate
during fermentation owing to the presence of polysac- solution results in an exponential increase in viscosity as
charides in the culture medium. The relationship between the uid slows down after it leaves the impeller and hence
the introduced mixing energy and the oxygen transfer the reduction in the pO2. On the other hand, the relatively
rate in solutions with pseudoplastic behavior is still low viscosity in the region near the impeller tends to
dicult to establish [108]. In the case of alginate produc- promote channeling of gas up the center of the vessel,
tion by A. vinelandii, the low oxygen solubility in fer- creating a highly oxygenated zone. To our knowledge,
mentation media coupled with high O2 consumption rate experiments with other types of reactors, especially bubble
(reported for Azotobacter) make the task of O2 supply column and airlift reactors, should be tested. Basically, if it
(aeration) dicult. On the other hand, increasing me- is possible to maintain a continuous and uniform motion of
chanical stirring to enhance the aeration may cause the broth, the viscosity will decrease because of its pseduo-
Figure 10 The dependence of the specic alginate production rate ( qalg) on the dissolved oxygen tension ( pO2) at the same
growth rate. (From Ref. 52.)
Alginate 529
plasticity, thereby allowing a much easier mixing and hence treatment processes because they help in increasing the
the accurate control of DOT [111]. aggregate sizes in occulation processes [23].
The use of alginate as an immobilization matrix has
been reported many years ago. Calcium alginate gel has
VII. THE APPLIED POTENTIAL OF ALGINATES become the most widely used technique for immobilizing
living cells (bacteria, cyanobacteria, algae, fungi, yeast,
The most widely used from of alginate is the readily water- plant protoplasts, plant and animal cells). Alginate immo-
soluble sodium salt, although other salts, such as calcium, bilized cell systems are used as biocatalysts in several
potassium, and ammonium alginate, are commercially industrial processes ranging from ethanol production by
available and have been approved for food use. In the food yeast cells to the production of monoclonal antibodies
industry, which currently consumes about 50% of the from hybridoma cells [41,117,118]. The use of immobilized
alginate produced, they are widely used as additives capa- cell technology processes have been proposed, mainly with
ble of viscosifying, stabilizing, emulsifying, and gelling the aim of shortening production time and hence reducing
aqueous solutions [10]. The most important application is the production costs, or establishing a continuous system.
probably in ice cream production, where alginates are used The applications of immobilized cells in food and nonfood
to prevent crystallization and shrinkage, thereby giving a systems have been reviewed by Groboillot et al. [119].
more homogenous product. Alginates used for food indus- The current industrial interest in the microbial alginate
tries are currently being produced by harvesting of brown production and the use of recombinant techniques may
seaweeds, and the global market for this polysaccharide is lead to an improved understanding of their physiology, and
about 30,000 tons. Selling prices are in the range US$ 520/ should also extend our knowledge about the production
kg for most applications [23]. process. Probably, it will become more evident that the
As the prices of such alginates are generally low, it microbial production of high-quality alginate is technically
seems to be a dicult task to establish a competitive more exible than the algal route.
bacterial production process in this price range. However,
environmental concern associated with seaweed harvest-
ing and processing, and the possibility of producing high-
quality alginate make it probable that the bacterial algi- VIII. CONCLUDING REMARKS
nate may become a commercial product in the future. Interesting commercial opportunities seem to exist for
Pharmaceutical industries commonly use highly puried bacterial alginates, especially in the biomedical and phar-
forms of alginates and alginic acid, the former as stabi- maceutical elds. The high price of alginates applied for
lizers in solution and dispersions of solid substances and these elds may open a new market for polymers with high
the latter as a binder and disintegrating agent in tablets degree of purity and dened chemical compositions. The
[112]. Moreover, Ca- and/or Na alginate bers are used in good knowledge of the correlation between alginate struc-
the manufacture of surgical guts, wound dressings, and as tures and function will help the biotechnologist to synthe-
dental impression material [113]. One of the most inter- size the required polymer for specic applications. Current
esting application of such high-quality alginate samples knowledge on the epimerase gene system, especially in A.
involves the reversal of type I diabetes by immobilizing vinelandii, would help in achieving this purpose. However,
insulin-producing cells in alginate capsules. These capsules there are still areas that require further investigations. For
have been implanted into the body of whole animals and A. vinelandii, the problem of oxygen sensitivity represents a
even humans, and encapsulated langerhans islets are challenging task for the biochemical engineerin particu-
currently being evaluated as a bioarticial endocrine lar, the appropriate bioreactor design is critical. An alter-
pancreas. This high-quality alginate of extreme purity native solution for the biochemists is decreasing the
used in the pharmaceutical eld are sold for up to extreme oxygen sensitivity of this bacterium. Achieving
US$40,000/kg [23]. this feat will give a chance for the commercialized produc-
Alginate is active in stimulating immune cells to secrete tion of this bacterial polymer. Simultaneously, understand-
cytokines, e.g., tumor necrosis factor-a (TNF-a), interleu- ing the mechanisms of epimerization, acetylation, degra-
kin-1 (IL-1), and interleukin-6 (IL-6) [114]. Surprisingly, dation, as well as its regulation in A. vinelandii are crucial
the response of the immune system appears to depend on for obtaining high-quality products for an emerging market.
the sequential structures of alginates, giving the highest
response with M-rich polymers, while the G-blocks appear
to be nonstimulating. In fact, guluronic acid residues
cannot be accepted in therapeutic preparations because it REFERENCES
triggers unwanted eects such as antibody generation
[41,115]. 1. Linker, A.; Jones, R.S. A polysaccharide resembling alginic
Textile and paper industries use this polymer along acid from a Pseudomonas microorganism. Nature 1966,
204, 187188.
with other materials as sizes to improve the surface 2. Gorin, J.P.A.; Spencer, T.J.F. Exocellular alginic acid from
properties of cloths and paper. This is important prior to Azotobacter vinelandii. Can. J. Chem. 1966, 44, 993998.
printing to enable the deposition and adherence of dyes and 3. Skjak-Braek, G.; Martinsen, A. Application of some algal
ink substances [116]. Alginates are also used in water polysaccharide in biotechnology. Guiry, MD Blundenn, G
Eds.; Seaweed Resources in Europe: Uses and Potential; and applications. Appl. Microbiol. Biotechnol. 1997, 48,
New York: Wiley and Sons, 1991, 219257. 281288.
4. Boyd, A.; Chakrabarty, A.M. Pseudomonas aeruginosa 24. Jain, S.; Ohman, D.E. Deletion of algK in mucoid
biolms: Role of the alginate exopolysaccharide. J. Ind. Pseudomonas aeruginosa blocks alginate polymer forma-
Microbiol. 1995, 15, 162168. tion and results in uronic acid secretion. J. Bacteriol. 1998,
5. Kidambi, P.S.; Sundin, G.W.; Palmer, A.D.; Chakrabarty, 180 (3), 634641.
M.A.; Bender, L.C. Copper as a signal for alginate 25. Fyfe, J.A.M.; Govan, J.R.W. Synthesis, regulation and
synthesis in Pseudomonas syringae pv. syringae. Appl. biological function of bacterial alginate. Prog. Ind. Micro-
Environ. Microbiol. 1995, 61 (6), 21722179. biol. 1983, 18, 4583.
6. Deavin, L.; Jarman, T.R.; Lawson, C.J.; Richelato, R.C.; 26. Anderson, A.J.; Hacking, A.J.; Dawes, E.A. Alternative
Slocombe, S. The production of alginic acid by Azotobacter pathway for the biosynthesis of alginate from fructose and
vinelandii in batch and continuous culture. In Extracellular glucose in Pseudomonas mendocina and Azotobacter
Microbial Polysaccharides; Sanford, P.A., Laskin, A., Eds.; vinelandii. J. Gen. Microbiol. 1987, 133, 10451052.
American Chemical Society: Washington, 1977; 1426. 27. Page, W.J.; Bhanthumnavin, N.; Manchak, J.; Ruman, M.
7. Jarman, T.R.; Deavin, I.; Slocombe, S.; Righelato, R.C. Production of poly(h-hydroxybutyrate-h-hydroxyvaler-
Investigation of the eect of environmental conditions on ate) copolymer from sugars by Azotobacter salinestris.
the rate of exopolysaccharides synthesis in Azotobacter Appl. Microbiol. Biotechnol. 1997, 48, 8893.
vinelandii. J. Gen. Microbiol. 1978, 107, 5964. 28. Brivonese, A.; Sutherland, W.I. Polymer production by a
8. Clementi, F.; Fantozzii, P.; Mancini, F.; Moresi, M. Opti- mucoid strain of Azotobacter vinelandii in batch culture.
mal conditions for alginate production by Azotobacter vine- Appl. Microbiol. Biotechnol. 1989, 30, 97102.
landii. Enzyme Microb. Technol. 1995, 17 (11), 983988. 29. Page, W.J.; Knosp, O. Hyperproduction of PHB during
9. Young, M. Microbial polysaccharide. In Comprehensive exponential growth of Azotobacter vinelandii UWD. Appl.
biotechnology; Pergamon Press: Toronto, 1983; Vol. 3, Environ. Microbiol. 1989, 55 (6), 13341339.
10051044. 30. Martinez, P.; Guzman, J.; Espin, G. A mutation impairing
10. Moe, S.T.; Draget, K.I.; Skjak-Braek, G.; Smidsrod, O. alginate production increased accumulation of poly-h-
Alginates. In Food Polysaccharide and Application; Marcel hydroxybutyrate in Azotobacter vinelandii. Biotechnol.
Dekker: New York, 1995; Vol. 9, 245286. Lett. 1997, 19 (9), 909912.
11. Lebrun, L.; Junter, G.A.; Jouenne, T.; Mignot, L. Exo- 31. Page, W.J.; Tindale, A.; Chandra, M.; Kwon, E. Alginate
polysaccharide production by free and immobilized micro- formation in Azotobacter vinelandii UWD during sta-
bial cultures. Enzyme Microb. Technol. December 1994, tionary phase and the turnover of poly-h-hydroxybutyrate.
16, 10481054. Microbiology 2001, 147, 483490.
12. Mancini, M.; Moresi, M.; Sappino, F. Rheological 32. Williams, G.S.; Greenwood, A.J.; Jones, W.C. Physiolog-
behaviour of aqueous dispersions of algal sodium algi- ical and biochemical changes accompanying the loss of
nates. J. Food Eng. 1996, 28, 238295. mucoidy by Pseudomonas aeruginosa. Microbiology 1996,
13. Chen, W.P.; Chen, J.Y.; Chang, S.C.; Su, C.L. Bacterial 142, 881888.
alginate produced by a mutant of Azotobacter vinelandii. 33. Franklin, M.J.; Chitnis, C.E.; Gacesa, P.; Sonesson, A.;
Appl. Environ. Microbiol. 1985, 49 (3), 543546. White, D.C.; Ohman, D.E. Pseudomonas aeruginosa AlgG
14. Matsumoto, T.; Kawal, M.; Masuda, T. Inuence of is a polymer level alginate C5-mannuronan epimerase. J.
concentration and mannuronate/guluronate ratio on Bacteriol. 1994, 176, 18211830.
steady ow properties of alginate aqueous systems. 34. Ertesvag, H.; Doseth, B.; Larson, B.; Skjak-Braek, G.;
Biorheology 1992, 29, 411417. Valla, S. Cloning and expression of an Azotobacter
15. Sutherland, L.W. Extracellular polysaccharides. In Bio- vinelandii mannuronan C-5 epimerase gene. J. Bacteriol.
technology Metabolism, 2nd Ed.; Rehm, H.J., Redd, G., 1994, 176, 28462853.
Eds.; VCH: Weinheim, 1996; Vol. 16, 614657. 35. Ertesvag, H.; Hoidal, H.K.; Hals, I.K.; Rian, A.; Doseth,
16. Lin, T.Y.; Hassid, W.Z. Isolation of guanosine diphos- B.; Valla, S. A family of modular type mannuronan C-5
phate uronic acids from a marine brown alga, Fucus epimerase genes control alginate structure in Azotobacter
gardneri silva. J. Biol. Chem. 1966a, 241, 32823293. vinelandii. Mol. Microbiol. 1995, 9, 719731.
17. Lin, T.Y.; Hassid, W.Z. Pathway of alginic acid synthesis in 36. Ertesvag, H.; Hoidal, K.H.; Schjerven, H.; Svanem, B.I.G.;
the marine brown alga Fucuc gardneri silva. J. Biol. Chem. Valla, S. Mannuronan C-5 epimerases and their applica-
1966b, 241, 52845297. tion for in vitro and in vivo design of new alginates useful in
18. Larsen, B.; Haug, A. Biosynthesis of alginate: Part 1. biotechnology. Metab. Eng. 1999, 1, 262269.
Composition and structure of alginate produced by 37. Rehm, B.H.A.; Ertesvag, H.; Valla, S. A new Azotobacter
Azotobacter vinelandii. Carbohydr. Res. 1971, 17, 287296. vinelandii mannuronan C-5 epimerase gene (algG) is part of
19. Madgwick, J.C.; Haug, A.; Larsen, B. Acta Chem. Scand. an alg gene cluster physically organized in a manner similar
1973, 27, 35923594. to that in Pseudomonas aeruginosa. J. Bacteriol. 1996, 178,
20. Lynn, N.A.; Sokatch, J.R. Incorporation of isotope from 58845889.
specially labelled glucose into alginates of Pseudomonas 38. Svanem, B.I.G.; Strand, W.I.; Ertesvag, H.; Skjak-Braek,
aeruginosa and Azotobacter vinelandii. J. Bacteriol. 1984, G.; Hartmann, M.; Barbeyron, T.; Valla, S. The catalytic
158 (3), 11611162. activities of the bifunctional Azotobacter vinelandii man-
21. Lioret, L.; Barreta, R.; Leon, R.; Moreno, S.; Matinez- nuronan C-5 epimerase and alginate lyase AlgE7 probably
Saazer, J.; Espin, G.; Soberon-Chavez, G. Genetic analysis originate from the same active site in the enzyme. J. Biol.
of the transcriptional arrangement of Azotobacter vinelandii Chem. 2001, 276 (34), 3154231550.
alginate biosynthetic genes: identication of two independ- 39. Skjak-Braek, G.; Smidsrad, O.; Larsen, B. Tailoring of
ent promoters. Mol. Microbiol. 1996, 21 (3), 449457. alginates by enzymatic modication in vitro. Int. J. Biol.
22. Beale, J.R.J.M.; Foster, J.L. Carbohydrate uxes into Macromol. 1986, 8, 330336.
alginate biosynthesis in Azotobacter vinelandii NCIB 8789: 40. Haug, O.; Larsen, A.; Smidsrod, B. Uronic acid sequence in
NMR investigations of the triose pools. Biochemistry 1996, alginate from dierent sources. Carbodydr. Res. 1974, 32,
35, 44924501. 217225.
23. Rehm, B.H.A.; Valla, S. Bacterial alginate; Biosynthesis 41. Skjak-Braek, G. Alginate: biosynthesis and some structure
Alginate 531
function relationships relevant to biomedical and biotech- 60. Post, E.; Kleiner, D.; Oelze, J. Whole cell respiration and
nological applications. Biochem. Soc. Trans. 1992, 20 (1), nitrogenase activities in Azotobacter vinelandii growing in
2733. oxygen controlled continuous culture. Arch. Microbiol.
42. Parker, L.T.; Socolofsky, M.D. Central body of the 1983, 134, 6872.
Azotobacter cyst. J. Bacteriol. 1966, 91, 297303. 61. Boiardi, J.L. Metabolic cost of nitrogen incorporation by
43. Holdal, H.K.; Svanem, I.G.; Gimmestad, M.; Valla, S. N2-xing Azotobacter vinelandii is aected by the culture
Mannuronan C-5 epimerases and cellular dierentiation pH. Biotechnol. Lett. 1994, 16 (11), 11951198.
of Azotobacter vinelandii. Environ. Microbiol. 2000, 2 (1), 62. Hammad, A.M.M. Evaluation of alginate encapsulated
2738. Azotobacter chroococcum as a phage-resistant and an
44. Nunez, C.; Moreno, S.; Soberon-Chavez, G.; Espin, G. The eective inoculum. J. Basic Microbiol. 1998, 1, 916.
Azotobacter vinelandii response regulator AlgR is essential 63. Davies, G.D.; Chakrabarty, M.A.; Geesey, G.G. Exopo-
for cyst formation. J. Bacteriol. 1999, 181 (1), 141148. lysaccharide production in biolms: substratum activation
45. Gottschalk, G. Fixation of molecular nitrogen. In Bacterial of alginate gene expression by Pseudomonas aeruginosa.
Metabolism; Gottschalk, G., Ed.; Springer Verlag: Berlin, Appl. Environ. Microbiol. 1993, 59 (4), 11811186.
1988 (10), 318326. 64. Govan, J.R.; Deretic, V. Microbial pathogenesis in cystic
46. Lichtl, R.J.; Bazin, M.O.; Hall, D. The biotechnology of brosis: mucoid Pseudomonas aeruginosa and Burkholderia
hydrogen production by Nostoc aelliforme grown under cepacia. Microbiol. Rev. 1996, 60 (3), 539574.
chemostat conditions. Appl. Microbiol. Biotechnol. 1997, 65. Sato, S.S.; Mukataka, H.; Kataoka; Takahashi, J. Eects
47, 701707. of pressure and dissolved oxygen concentration on growth
47. Postgate, J.R. Nitrogen xation by free-living microbes. In of Pseudomonas aeruginosa. J. Ferment. Technol. 1984, 62
The Chemistry and Biochemistry of Nitrogen Fixation; (1), 7175.
Postgate, J.R., Ed.; Plenum Press: London, 1971; Vol. 5, 66. Krieg, D.P.; Bass, J.A.; Mattingly, S.J. Aeration selects for
161187. mucoid phenotype of Pseudomonas aeruginosa. J. Clin.
48. Postgate, J.R. Evolution within nitrogen xing systems. Microbiol. 1986, 24 (6), 986990.
Symp. Soc. Gen. Microbiol. 1974, 24, 242263. 67. Bayer, A.S.; Eftekhar, F.; Tu, J.; Nast, C.C.; Speert, D.P.
49. Linkerhagner, K.; Oelze, J. Cellular ATP level and nitro- Oxygen-dependent up-regulation of mucoid exopolysac-
genase switcho upon oxygen stress in chemostat cultures charide (alginate) production in Pseudomonas aeruginosa.
of Azotobacter vinelandii. J. Bacteriol. 1995, 177 (18), 5289 Infect. Immun. 1990, 58 (5), 13441349.
5293. 68. Leitao, J.H.; Sa-Correia, I. Oxygen dependent upregula-
50. Linkerhagner, K.; Oelze, J. Nitrogenase activity and tion of transcription of alginate genes algA, algC and algD
regeneration of the cellular ATP pool in Azotobacter in Pseudomonas aeruginosa. Res. Microbiol. 1997, 148, 37
vinelandii adapted to dierent oxygen concentrations. J. 43.
Bacteriol. 1997, 179 (4), 13621367. 69. Xu, K.D.; Stewart, P.S.; Xia, F.; Huang, C.-T.; McFeters,
51. Sabra, W.A.; Zeng, A.-P.; Sabry, S.; Omar, S.; Deckwer, G.A. Spatial physiological heterogenicity in Pseudomonas
W.-D. Eect of phosphate and oxygen concentrations on aeruginosa biolm is determined by oxygen availability.
alginate production and stoichiometry of metabolism of Appl. Environ. Microbiol. 1998, 64 (10), 40354039.
Azotobacter vinelandii under microaerobic conditions. 70. Sabra, W.A.; Kim, E.-J.; Zeng, A.-P. Physiological
Appl. Microbiol. Biotechnol. 1999, 52, 773780. responses of Pseudomonas aeruginosa PA01 to oxidative
52. Sabra, W.A.; Zeng, A.-P.; Lunsdorf, H.; Deckwer, W.-D. stress in controlled microaerobic and aerobic cultures.
Function and variation of alginate production in Azoto- Microbiology 2002, 148, 31953202.
bacter vinelandii under nitrogen xation conditions. Appl. 71. Sabra, W.A.; Zeng, A.-P.; Deckwer, W.-D. Bacterial
Environ. Microbiol. 2000, 66 (9), 40374044. alginate: physiology, product quality and process aspects.
53. Haddock, B.A.; Jones, C.W. Bacterial respiration. Bacter- Appl. Microbiol. Biotechnol. 2001, 56, 315325.
iol. Rev. 1977, 41 (1), 4799. 72. Croft, L.; Beatson, S.A.; Whitchurch, C.B.; Huang, B.;
54. Kuhla, J.; Oelze, J. Dependence of nitrogenase switch-o Blakeley, R.L.; Mattick, J.S. An interactive web-based
upon oxygen stress on the nitrogenase activity in Pseudomonas aeruginosa genome database: discovery of
Azotobacter vinelandii. J. Bacteriol. 1988, 170 (11), new genes, pathways and structures. Microbiology 2000,
53255329. 146, 23512364.
55. Liu, J.K.; Lee, F.T.; Lin, C.S.; Yao, X.T.; Davenport, J.W.; 73. Doggett, R.G.; Harrison, G.M.; Stillwell, R.M.; Wallis,
Wong, T.Y. Alternative function of the electron transport E.S. An atypical Pseudomonas aeruginosa associated with
system in Azotobacter vinelandii: removal of excess cystic brosis of the pancreas. J. Pediatr. 1966, 68, 215221.
reductant by the cytochrome d pathway. Appl. Environ. 74. Martin, D.R. Mucoid variation in Pseudomonas aeruginosa
Microbiol. 1995, 61 (11), 39984003. induced by the action of phage. J. Med. Microbiol. 1973, 6,
56. Moshiri, F.; Crouse, B.R.; Johnson, M.K.; Maier, R.J. The 111118.
nitrogenase protective FeSII protein of Azotobacter 75. Govan, J.R.; Fyfe, M.J.A. Mucoid Pseudomonas aerugio-
vinelandii: Over expression, characterisation and crystal- nosa and cystic brosis: resistance of the mucoid form to
lisation. Biochemistry 1995, 34, 1297312982. carbencillin, ucloxacillin and tobramycin and the iso-
57. Duyvis, M.; Wassink, H.; Haaker, H. Nitrogenase of lation of mucoid variants in vitro. J. Antimicrob. Chemo-
Azotobacter vinelandii: kinetic analysis of the Fe protein ther. 1978, 4, 233240.
redox cycle. Biochem 1998, 37, 1734517354. 76. Anwar, H.; Strap, J.L.; Costerton, J.W. Establishment of
58. Post, E.; Golecki, R.J.; Oelze, J. Morphological and aging biolms: possible mechanisms of bacterial resistance
ultrastructural variations in Azotobacter vinelandii growing to antimicrobial therapy. Antimicrob. Agent Chemother.
in oxygen-controlled continuous culture. Arch. Microbiol. 1992, 36, 13471351.
1982, 133, 7582. 77. Mathee, K.; Ciofu, O.; Sternberg, C.; Lindum, P.W.;
59. Sabra W.A. Microaerophilic production of alginate by Azo- Campbell, J.I.A.; Jensen, P.; Johnsen, A.; Givskov, M.;
tobacter vinelandii. Dissertation, Technische Universitat Ohman, D.E.; Molin, S.; Hoiby, N.; Kharazmi, A. Mucoid
Carolo-Wilhelmina zu Braunschweig, Germany (Electronic conversion of Pseudomonas aeruginosa by hydrogen per-
publication address: http://www.biblio.tu-bs.de/ediss/ oxide: a mechanism for virulence activation in the cystic
data/19990415a/19990415a.html), 1998. brosis lung. Microbiology 1999, 145, 13491357.
78. Osman, S.F.; Fett, W.F.; Fishman, M.L. Exopolysaccha- 94. Okabe, E.; Nakayima, M.; Murooka, H.; Nisizawa, K.
rides of the phytopathogen Pseudomonas syringae pv. Investigation of carbon and phosphorous sources in cul-
glycinea. J. Bacteriol. 1986, 166, 6671. tural media of a selected strain of alginate producing Azo-
79. Gacesa, P. Bacterial alginate biosynthesis-recent progress tobacter vinelandii. J. Ferment. Technol. 1981, 59 (1), 17.
and future prospects. Microbiology 1998, 144, 11221143. 95. Savalgi, V.; Savalgi, V. Alginate production by Azotobacter
80. Olvera, C.; Goldberg, J.B.; Sanchez, R.; Soberon-Chavez, vinelandii in batch culture. J. Gen. Appl. Microbiol. 1992,
G. The Pseudomonas aeruginosa algC gene product 38, 641645.
participates in rhamnolipid biosynthesis. FEMS Micro- 96. Pena, C.; Campos, N.; Galindo, E. Evolution of alginate
biol. Lett. 1999, 179, 8590. molecular weight distributions, broth viscosity and mor-
81. Boyd, A.; Gosh, M.M.; May, T.B.; Shinabarger, D.; phology of Azotobacter vinelandii cultured in shake asks.
Keogh, R.; Chakrabarty, A.M. Sequence of the algL gene Appl. Microbiol. Biotechnol. 1997, 48, 510515.
from Pseudomonas aeruginosa and purication of its 97. Clementi, F.; Crudele, M.A.; Parente, E.; Mancini, M.;
alginate lyase products. Gene 1993, 131, 18. Moresi, M. Production and characterisation of alginate
82. Boyd, A.; Chakrabarty, A.M. Role of alginate lyase in cell from Azotobacter vinelandii. J. Sci. Food Agric. 1999, 79,
detachment of Pseudomonas aeruginosa. Appl. Environ. 602610.
Microbiol. 1994, 60 (7), 23552359. 98. Horan, N.J.; Jarman, T.R.; Dawes, E.A. Studies on some
83. May, B.T.; Chakrabarty, A.M. Isolation and assay of enzymes of alginic acid biosynthesis in Azotobacter
Pseudomonas aeruginosa alginate. Meth. Enz. 1994, 235, vinelandii grown in continuous culture. J. Gen. Microbiol.
295304. 1983, 129, 29852990.
84. Martin, D.W.; Schurr, M.J.; Mudd, M.H.; Govan, J.R.W.; 99. Parente, E.; Crudele, M.A.; Aquino, M.; Clementi, F.
Deretic, V. Analysis of promoters controlled by the Alginate production by Azotobacter vinelandii DSM576 in
putative sigma-factor AlgU regulating conversion to batch fermentation. J. Ind. Microbiol. Biotechnol. 1998,
mucoidy in Pseudomonas aeruginosa-relationship to sigma 20, 171176.
and stress response. J. Bacteriol. 1994, 176, 66886696. 100. Annison, G.; Couperwhite, I. Eect of limiting substrate
85. Martin, D.W.; Schurr, M.J.; Mudd, M.H.; Deretic, V. concentration, growth rate and aeration on alginate com-
Dierentiation of Pseudomonas aeruginosa into the algi- position and production by Azotobacter vinelandii in con-
nate producing form: inactivation of the mucB causes tinuous culture. Food Hydrocolloid. 1986b, 1 (2), 101111.
conversion to mucoidy. Mol. Microbiol. 1993, 9, 495506. 101. Ferrala, N.F.; Westervelt, P.; Mabbott, G.A.; Fekete, F.A.
86. Martin, D.W.; Schurr, M.J.; Mudd, M.H.; Govan, J.R.W.; Relation between extracellular polysaccharide production
Holloway, B.W.; Deretic, V. Mechanisms of conversion to and medium iron concentration in nitrogen xing Azoto-
mucoidy in Pseudomonas aeruginosa infecting cystic bacter chrococcum B-8. Abstr. Annu. Meet. Am. Soc.
brosis patients. Proc. Natl. Acad. Sci. USA 1993, 90, Microbiol. 1986, 86, K-143.
83778381. 102. Obika, H.; Sakakibara, J.; Kobayashi, Y. Direct control of
87. Martinez-Salazar, J.M.; Moreno, S.; Najera, R.; Bucher, the constituent ratio in a wide range in alginate produced by
J.C.; Espin, G.; Soberon-Chavez, G.; Deretiv, V. Charac- Azotobacter vinelandii. Biosci. Biotechnol. Biochem. 1993,
terization of genes coding for the putative sigma factor 57 (2), 332333.
algU and its regulators MucA, MucB, MucC and MucDIn 103. Couperwhite, I.; McCallum, M.F. The inuence of EDTA
Azotobacter vinelandii and evaluation of their roles in on the composition of alginate synthesized by Azotobacter
alginate biosynthesis. J. Bacteriol. 1996, 178, 18001808. vinelandii. Arch. Microbiol. 1974, 97, 7380.
88. Nunez, C.; Leon, R.; Guzman, J.; Espin, G.; Soberon- 104. Annison, G.; Couperwhite, I. Inuence of calcium on
Chavez, G. Role of Azotobaceter vinelandii mucA and alginate production and composition in continuous cul-
mucC gene products in alginate production. J. Bacteriol. tures of Azotobacter vinelandii. Appl. Microbiol. Biotech-
2000, 182 (23), 65506556. nol. 1986a, 25, 5561.
89. Yu, H.; Mudd, M.; Boucher, J.C.; Schurr, M.J.; Deretic, V. 105. Klimek, J.; Ollis, D.F. Extracellular microbial polysac-
Identication of the algZ gene upstream of the response charides: kinetics of Pseudomonas sp., Azotobacter vine-
regulator algR and its participation in control of the landii and Aureobasidium pullulans batch fermentations.
alginate production in Pseudomonas aeruginosa. J. Bacter- Biotechnol. Bioeng. 1980, 22, 23212342.
iol. 1997, 179, 187193. 106. Luedeking, R.; Piret, E.L. A kinetic study of lactic acid
90. Deretic, V.; Churr, M.J.; Boucher, J.C.; Martin, D.W. fermentation. Batch process at controlled pH. J. Biochem.
Conversion of Pseudomonas aeruginosa to mucoidy in Microbiol. Technol. Eng. 1959, 1, 393431.
cystic brosis: environmental stress and regulation of 107. Margaritis, A.; Pace, G.W. Microbial polysaccharides. In
bacterial virulence by alternative sigma factors. J. Bacter- Comprehensive Biotechnology; Blanch, H.W., Drew, S.,
iol. 1994, 176, 27732780. Wang, D.I.C., Eds.; Pergamon: Oxford, 1985; Vol. 3,
91. Ma, S.; Selvaraj, U.; Ohman, D.E.; Quarless, R.; Hassett, 10051044.
D.J.; Wazniak, D.J. Phosphorylation-independent activity 108. Dussap, C.-G.; Gros, J.-B. Power input, KLa values in
of the response regulators AlgB and AlgR in promoting pneumatically agitated fermentors with extracellular mi-
alginate biosynthesis in mucoid Pseudomonas aeruginosa. J. crobial polysaccharides. Biotechnologie (Europe), 1985, 85,
Bacteriol. 1998, 180 (4), 956968. 691692.
92. Ramstad, M.V.; Markussen, S.; Ellingsen, T.E.; Skjak- 109. McNeil, B.; Harvey, L.M. Viscous fermentation products.
Braek, G.; Levine, D.W. Inuence of environmental con- Crit. Rev. Biotechnol. 1993, 13 (4), 275304.
ditions on the activity of the recombinant mannronan 110. Trujillo-Roldan, M.; Pena, C.; Ramirez, O.T.; Galindo, E.
C-5 epimerase AlgE2. Enzyme Microb. Technol. 2001, 28, Eect of oscillating dissolved oxygen tension on the
5769. production of alginate by Azotobacter vinelandii. Biotech-
93. Horan, N.J.; Jarman, T.R.; Dawes, E.A. Eects of carbon nol. Prog. 2001, 17, 10421048.
source and inorganic phosphate concentration on the 111. Deckwer, W.-D. Bubble Column Reactors; Wiley: Chi-
production of alginic acid by a mutant of Azotobacter chester, 1992.
vinelandii and on the enzyme involved in its biosynthesis. J. 112. Hagstam, H. Alginates and heartburn-evaluation of a
Gen. Microbiol. 1981, 127, 185191. medicine with a mechanical mode of action. In Gums and
Alginate 533
Stabilizers for the Food Industry; Philipps, G.O., Wedlock, 116. Sutherland, I.W.; Ellwood, D.C. Microbial exopolysac-
D.J., Williams, P.A., Eds.; Elsevier Applied science charides-industrial polymers of current and future poten-
publishers: London, 1985; 363370. tial. Symp. Soc. Gen. Microbiol. 1979, 29, 107150.
113. Picquadio, D.; Nelson, D.B. Alginates a new dressing 117. Crescenzi, V. Microbial polysaccharides of applied interest:
alternative. J. Dermatol. Surg. Oncol. 1992, 18, 992998. ongoing research activities in Europe. Biotechnol. Prog.
114. Otterlei, M.; Ostgaard, K.; Skjak-Braek, G.; Smidsrod, O.; 1995, 11, 251259.
Soon-Shiong, P.; Espevik, T. Induction of cytokine 118. Clementi, F. Alginate production by Azotobacter vinelan-
production from human monocytes stimulated with dii. Crit. Rev. Biotechnol. 1997, 17 (4), 327361.
alginate. J. Immunother. 1991, 10, 286291. 119. Groboillot, A.; Boadi, D.K.; Poncelet, D.; Neufeld, R.J.
115. Espevik, T.; Otterlei, M.; Skjak-Braek, G.; Ryan, L.; Immobilization of cells for application in the food in-
Wright, S.D.; Sundan, A. The involvement of CD14 in dustry. Crit. Rev. Biotechnol. Special issue on Immobi-
stimulation of cytokine production by uronic acid poly- lized Cell Technology in Food Processing 1994, 14 (2),
mers. Eur. J. Immunol. 1993, 23, 255261. 75107.
22
Characterization and Properties of Hyaluronic Acid
(Hyaluronan)
Michel Milas and Marguerite Rinaudo
Centre de Recherches sur les Macromolecules Vegetales (CERMAV), CNRS,
and Joseph Fourier University, Grenoble, France
I. INTRODUCTION from the fermentation broth. Hylan A is a loosely cross-

linked HA extracted from rooster combs pretreated with
Hyaluronan (also called hyaluronate, hyaluronic acid, or formol as proposed by Balazs and Leshchiver. Some
HA) was previously extracted from bovine vitreous humor, Hylans were also chemically cross-linked and called Hylan
roster combs, or umbilical cords; then, it was very expen- B [5].
sive and certainly associated with some proteins. Now, the
same polysaccharide was shown to be produced by the
bacteria Streptococcus zooepidemicus on a large scale with a B. Purification
good yield and a large degree of purity. Then, the price
decreased, allowing the development of its applications. The bacterial polysaccharide is isolated from the broth
The main uses of HA are ophthalmic surgery [1,2], arthritic following the usual method adopted in our laboratory [6].
treatment, and, more recently, in cosmetics [3,4]. The work Some comments on this procedure are necessary: the
developed for a few years in our laboratory concerned solution (the broth or diluted broth) after centrifugation to
mainly the bacterial hyaluronan under the native form; remove the solids in suspension must be ltered through
some chemical modications were also performed and are porous membranes with decreasing porosity down to 0.45
in progress in our laboratory; our main results will be given Am to suppress not only solid material but also aggregated
and discussed in this chapter. Loosely cross-linked HA molecules. The rst precipitation with ethanol is performed
(named Hylan) is also produced for viscosupplementation in the presence of a salt excess (usually NaCl) to exchange
in arthrosis treatment [5]; some characteristics are also the divalent counterions and recover the sodium salt of HA,
presented in the following. which must be perfectly water soluble in these conditions.
The presence of divalent counterions modies the hyaluro-
nan properties in solution [7,8] and they must be removed.
II. PRODUCTION AND CHARACTERIZATION If necessary, a second precipitation can be achieved.
At the end, the polysaccharide is dried by solvent
A. Production exchange with ethanolwater mixtures up to 100% in
The work we have developed concerns mainly bacterial ethanol and dried under low pressure at moderate temper-
hyaluronan. It is produced in France by the ARD Com- ature (30jC).
pany (Pomacle). The bacteria S. zooepidemicus was select-
ed by ARD after a screening based on the yield of C. Chemical Structure and Conformation
production (better than 5 g/L in the broth) and on the
molecular weight of HA (larger than 2 106 g/mol on the The chemical structure of hyaluronan is presented in Fig. 1;
laboratory scale). The samples used are directly isolated it is a linear polyelectrolyte based on h1-4 D-glucuronic acid
535
536 Milas and Rinaudo
Figure 1 Tetramer unit of HA (in the acid form) showing the conformational parameters used in molecular modeling. GlcNAc
represents the acetylated D-glucosamine unit and GlcA, the D-glucuronic acid unit.
(GlcA) and h1-3 N-acetyl-D-glucosamine (GlcNAc) repeat The control of the structure of the polysaccharides can
unit. It can be controlled by conductimetric or pH-metric also be performed by 1H nuclear magnetic resonance
titration of the acid form of HA; for this determination, a (NMR) (Fig. 2) in the presence of a standard to calibrate
known quantity of sodium hyaluronan is dissolved in water, the signal corresponding to the CH3 of the N-acetylglu-
passed through an ion exchanger (Amberlite-SO3H), and cosamine unit. The standard usually used is sodium succi-
titrated with NaOH. The ionic capacity (COOH equiv/g nate 5 mM (or DMSO) in D 2 O when the polymer
dried material) must be compared with the theoretical value concentration for NMR is around 5 mg/mL in D2O. But
of 2.5103 equiv/g (Na form). This method can also be the NMR signals of specic groups, especially in 1H NMR,
used to control the sample purity. are quantitative only when they are mobile, i.e., not implied
Figure 2 1H NMR spectrum of HA in the presence of a standard for quantitative determination (Mw=8.5104, T=85jC, C=5
g/L; standard: DMSO). N is related to GlcNAc and G to GlcA units. (From Ref. 16.)
Characterization and Properties of Hyaluronic Acid 537
Figure 3 Secondary structure of HA showing the intramolecular H-bonds. The counterion is Na+ or H+ depending on the pH.
(From Ref. 16.)
in an ordered secondary structure such as a helical struc- In fact, for hyaluronan, some authors propose the
ture which can be stabilized by H-bonds in stereoregular existence of an ordered conformation in solution which is
polymers or by specic interactions (Fig. 3). stabilized by H-bonds [9,10]. These bonds can be released
13
C NMR is also a good technique to control the in the presence of NaOH [11] or urea [12]. An example of
structure of the polymer and the absence of organic the local structure proposed is given in Fig. 3. However,
impurities. An example is given in Fig. 4. Sicinska et al. [13] concluded from a detailed 1H and 13C
On this spectrum, the two carbonyl groups around 175 NMR study of the repeating hyaluronan disaccharide that
ppm are clearly identied as well as the CH3 of the N- interactions with water predominated, and they found no
acetylated glucosamine around 23 ppm. The 11 carbon evidence for long-lived intramolecular hydrogen bonds
signals of the two sugar units are well distinguished. in aqueous solution for this disaccharide. On the opposite,
13
Figure 4 C NMR spectrum of a HA (10 g/L) in D2O at 85jC. (From Ref. 16.)
Table 1 Integral of the NMR Proton Signals as a Function

of Temperature
T (jC) 25 40 55 70 85
H1 (%) 42 83
H (cycle) (%) 56.4 49 55 79 84
H (acetyl) (%) 41 42 52.5 70 74
% of signal using sodium succinate signal as standard refers to the
total protons of the dierent species present in the solution.
Source: From Ref. [37]; Copyright 2003 Marcel Dekker.
on hyaluronan, the apparent intensity of the signal cor-

responding to the protons of the acetyl groups as well as Figure 6 Evolution of the chain mobility reected by the H
those of the sugar units depends on the NaOH concen- signal amplitude in NMR spectrum as a function of temper-
tration and on the temperature adopted for 1H NMR ature. (From Ref. 16.)
measurements. The percentage of mobile protons from
NMR as a function of temperature is given in Table 1. As
for the disaccharide unit, the 1H NMR spectrum changes when the temperature increases due to the dissociation of
with temperature and the fraction of mobile protons for the H-bonds (Fig. 5).
hyaluronan never reaches the theoretical values in the This allows us to justify the evolution of the NMR
range of temperature tested, which means that interactions spectra of such polysaccharide when the temperature
still exist at 85jC. Another parameter may also interfere, increases (Table 1). In Fig. 6, the ratio between the eective
such as the viscosity of the solution. The protons of the intensity of 1H NMR signals and the value of the standard
acetyl groups are more aected than those of the sugar based on the polymer concentration (X) is represented as a
units. Consequently, it is not possible to use 1H NMR to function of temperature.
test the HA structure (acetyl content). The change ob- In the solid state, dierent helical conformations were
served by 1H NMR seems not to be due to the presence of a demonstrated in which intramolecular H-bonds stabilize
helix-coil conformational transition, as suggested by Ghosh extended single- or double-helical conformation, depend-
et al. [11] or as demonstrated in concentrated urea [12], ing on the counterion type (H+, Na+, K+, Ca2+). For
because we never found this cooperative conformational example, in the presence of residual Ca2+ ions, a single
change using hyaluronan from bacterial source. The exact helix is described with an axial projection of the disaccha-
conformation in aqueous solution is not yet established, ride unit b equal to 9.5 A [17,18]. In a pure sodium
but it may be possible that for the polymeric chain, hyaluronan, a more compact helical structure with b=8.5
dynamic H-bonded regions exist, controlling the average A is given [19]. The pH during the lm preparation is
stiness of the molecule. This hypothesis was proposed in important too. At low pH (pH lower than 4), the x-ray
the literature [14]; the fraction of H-bonded domains
depends on the temperature.
Molecular modeling was developed on HA molecule
allowing to characterize the intrinsic persistence length
(Lp) of the disordered chain [15,16]; it was shown that
the stiness of the chain characterized by Lp decreases
Figure 7 X-ray diraction pattern obtained on an extended

Figure 5 Evolution of the intrinsic persistence length as a lm of HA-sodium form. ID2 beamline, ESRF (Grenoble).
function of temperature obtained from molecular modeling. k=0.99 A, 10 sec irradiation; ambient relative humidity and
(From Ref. 16.) temperature. (From Ref. 16.)
patterns correspond to a double helix obtained in the

presence of NH4+, Ca2+, and Rb+ counterions [20]. The
conformation of hyaluronans in the solid state needs to be
claried using well-characterized samples from bacterial
source. In Fig. 7, the diraction pattern obtained by
synchrotron radiation on a pure extended Na form HA
sample is given. This diractogram corresponds to a 43
helical conformation, which was one of the conformations
obtained by molecular modeling [15].
III. POLYELECTROLYTIC PROPERTIES

The hyaluronan is a charged acidic polysaccharide. It can
be characterized by a charge parameter k=e2/(DbkT),
where e is the proton charge, D is the bulk dielectric
constant of the solvent, k is the Boltzmann constant, and
T is the absolute temperature. Then, in an aqueous solution
at 25jC, taking b as the distance between two ionic groups Figure 9 Variation of the ratio I1/I3 characterizing the uo-
equal to 9.8 A from the chemical structure in the extended- rescence of pyrene in the presence of increasing concentration
coil conformation, k=0.71, but from the helical structure of hyaluronan and dextran in water at 25jC. n and 5
with b=8.5 A, k=0.82 for hyaluronan. Hyaluronan; w dextran.
One characteristic of a polyacid is its intrinsic pK value
(pK0). This value can be obtained from the pH-metric
neutralization curves of a solution of the acid form of
HA (obtained from cation-exchange resin). This measure-
ment allows the determination of the apparent pK (pKa) of trapolation to a null dissociation degree aT gives pK0=
the carboxylic group, taking into account the degree of 2.9F0.1 as usually found on polycarboxylic acid in the
autodissociation aH, and the degree of neutralization aN: absence of the specic interaction involving the carboxylic
pKa pH log1 aT =aT groups. The theoretical variation of pKa(aT)pK0=DpK
1 (e.s.) can be obtained from the model of Alexandrowicz
pK0 DpK e:s: and Katchalsky [21]. As the pK variation vs. aT follows the
where aT = aN + aH equals the fraction of ionized theoretical one, we can also exclude a large conformational
carboxylic groups. From our experimental data, the exchange during titration.
In addition, for our hyaluronan samples, the potenti-
ometric titration gives the capacity of the polymer; the
values obtained (2.5103 equiv/g) from the sodium hyal-
uronan are identical to the theoretical values given from
chemical structure showing the high purity of these sam-
ples. Then, the polyelectrolyte properties of hyaluronan
show the classic behavior corresponding to a charge pa-
rameter k close to 0.82 [22]. No specic intermolecular or
intramolecular interactions are detected.
But a specic behavior was obtained when the viscos-
ity of a hyaluronan solution is plotted as a function of the
pH (Fig. 8).
When the pH of the solution is decreased by progres-
sive addition of HCl, a gel is formed around pH=2.5 due to
the decrease of the carboxylate dissociation, favoring
intermolecule interactions. Further decrease of the pH
causes the gelsol transition; this transition may be related
to the protonation of the acetamido groups causing an
electrostatic repulsion. Over the critical overlap concentra-
tion, it was also demonstrated that a fraction of associated
molecules exists in solution of HA [16]; it seems that it may
be related to cooperative loose interactions between semi-
rigid molecules and found also in other polysaccharide
Figure 8 Evolution of the viscosity of a solution as a func- solutions [23,24]. From uorescence measurements in the
tion of the pH. HA Mw=8105 g/mol, C=10 g/L in 0.1 M presence of pyrene as probe, domains having some hydro-
NaCl at 25jC. phobicity are proved (Fig. 9). In this gure, it is shown that
the behavior of HA is independent of the molecular weight

but quite dierent from the very exible neutral dextran.
IV. WATER RETENTION

An important role of hyaluronan is the moisture retention
recommended for cosmetic applications [25]. Dierent
types of interacting water molecules are dierentiated using
dierent techniques [2529]: the unfreezing bound water,
the freezing bound water, and the free water. We have
recently determined the isotherms of water retention. The
hyaluronans were investigated under dierent ionic forms
and dierent morphologies (lm, powder, freeze-dried, or
precipitated with ethanol). These results were recently
discussed [30]. The total water retention was determined Figure 10 Freezing (Ec) and nonfreezing (ENC) water (ex-
by thermogravimetry and the freezing water by dierential pressed in gram of water per gram of dried HA) as a function
scanning calorimetry (DSC) on samples previously equili- of total water (ET) adsorbed on sodium salt form HA at
brated at dierent relative humidities (RH). 25jC. (From Ref. 54b.)
Some results are given in Table 2; as usual, the total
water content increases when the samples are equilibrated
in increasing relative humidities. The freezing water deter-
mined by DSC appears when the relative humidity is From Fourier transform infrared (FTIR) spectroscopy, it
greater than 80%; it corresponds to looser interaction was recently suggested that molecular weight does not
which is also the limit over which the modulus of a HA aect the hydration of sodium hyaluronate [31], as already
lm dropped severely (in relation with plasticizing eect noted [25]. These results show no evidence of a specic
of water). water retention compared to other polysaccharides. It is
Some results are also given in Fig. 10, in which the approximately equal to the additivity of the contribution
nonfreezing water and the freezing water content are of the dierent polar groups present in the hyaluronan
represented as a function of the total water adsorption; molecule.
up to 0.75 g water adsorbed per gram of dried Na-HA, the New experimental data were recently obtained to
water is strongly interacting and corresponds to nonfreez- analyze the progressive hydration of HA from the solid
ing water. This nonfreezing water which is rmly interact- state; the technique used is FTIR and the papers are just in
ing with the polar sites of the polymer goes to a limit of 0.75 press [32].
g/g (i.e., 16 mol of water per disaccharide unit). These
results agree with those given in the literature [2628].
It was also shown that the counterions have some V. MOLECULAR-WEIGHT DISTRIBUTION AND
eect on the hydration of hyaluronan as given in Fig. 11. CHAIN DIMENSIONS
Up to 76% RH, only nonfreezing water exists and the
content is nearly independent of the counterions; over One of the most important characteristics of a polymer is
this hydration, the order of hydration is given by the fol- its molecular weight (M) related to its dimensions in
lowing sequence: solution (i.e., the radius of gyration, Rg). The viscosity of
a polymeric solution depends not only on M and stiness,
H < Mg2 fCa2 < Na fK but also on the polymer and ionic concentrations and
temperature. This property is certainly the most important
for the applications when HA is used to modify solvent
properties (i.e., to increase its viscosity).
Absolute molecular-weight determination is dicult
Table 2 Water Retention Content for Na Hyaluronate because the sample may contain aggregates which overes-
Stabilized at Dierent Relative Humidities (T=25jC) timate the value of M. Nevertheless, the polymer has to be
characterized by a molecular-weight distribution and av-
RH% 22 44 76 88 94 98 100 erage molecular weights. This M distribution can be
Total 0.12 0.17 0.33 0.56 0.83 1.56 2.06 obtained directly by steric exclusion chromatography
content (SEC). Analysis of the chromatogram gives the M distri-
in H2O
bution and the dierent average molecular weights (Mn,
adsorbed
Mw, etc.). In our laboratory, one develops a SEC connected
(g/g)
Freezing 0.04 0.12 0.92 1.27
with three detectors online: a dierential refractometer
H2O (g/g) giving the polymer concentration, a viscometer giving the
viscosity (g) of the corresponding elution volume, and a
Source: From Ref. [37]; Copyright 2003 Marcel Dekker. multiangle laser light scattering (MALLS) giving the mo-
Figure 11 Inuence of the counterion on the freezing (Ec) and nonfreezing (ENC) water content (expressed in g/g) on HA
powder stabilized at dierent relative humidities at 25jC. (From Ref. 54b.)
lecular weight and the Rg of the polymer eluted for the Steric exclusion chromatography appears to be a good
same elution volume [33]. Fig. 12 gives the molecular- technique to characterize hyaluronan samples and their
weight distribution of a hyaluronan sample; this technique purity [3436].
allows a very complete characterization of the polymer The parameters K and a are known as the Mark
from which we also determine the relations Houwink parameters in the relation [g]=KM a; in the range
4105<M<1.5 106 and in the SEC conditions (0.1 M
g KM a and Rg KVMm 2 NaNO3, 30jC), one gets K=0.0336 and a=0.79, respec-
tively, when [g] is expressed in mL/g. The parameters K V
in the solvent used for SEC (0.1 M NH4NO3 or NaNO3) at and m relate the Rg to the molecular weight in the relation
ambient temperature; the set of constants depends on the Rg=K VMm; one found in the same conditions K V=0.049
range of molecular weights covered. and m=0.55, with Rg expressed in nanometers (Fig. 13).
These parameters are directly connected with the confor-
mation of the chain in solution; the models used to predict
them were developed previously [37,38].
The viscometric-average molecular weight can also be
estimated from the intrinsic viscosity measurements [39,40]
using the MarkHouwink relation [g]=KMa with, for
example, the K and a values given in the range of molecular
weight and conditions previously described. The Mark
Houwink relation is obtained either from dierent frac-
tions of well-characterized hyaluronate samples with dif-
ferent molecular weights in the absence of aggregates and
with low polydispersities [38a] or directly from SEC
experiments with a viscometer, a light-scattering equip-
ment, and a refractometer as detectors as seen previously
[33]. Thereby, a simple measurement of intrinsic viscosity
under the same conditions leads to a viscosity-average
molecular weight Mv. The determination of the intrinsic
viscosity must be achieved in the Newtonian plateau (see
Rheological Behavior Section), where the viscosity of the
solution does not depend on the shear rate used. As an
example for a sodium hyaluronate with Mw=2.4 106, we
Figure 12 Molecular-weight distribution obtained by SEC of nd [g]=2575 mL/g using a capillary viscometer (capillary
a HA sample (Mw=3.7 105; I=1.67). Temperature: 30jC; diameter equal to 0.5 mm) and [g]=3500 mL/g from a low-
eluent: 0.1 M NaNO3. (From Ref. 37.); Copyright 2003 Marcel shear Contraves viscometer. In this last case, the values are
Dekker. taken in the Newtonian plateau. These measurements were
account the contribution of the polyelectrolyte (Cp

expressed in equiv/L). Then, we can dene a total persis-
tence length Lt=Lp+Le. We also assumed that extrapo-
lation of Lt to innite salt concentration, such as to screen
all the electrostatic contributions, represents the h condi-
tions. We also take into account the electrostatic excluded
volume which is important at low salt concentration. When
Lp is unknown, the values of Rg and [g] are calculated for
dierent imposed Lp values and compared with the exper-
imental values; the best t allows the determination of the
local stiness of the molecule characterized by Lp. From
the results obtained and data from the literature [42], it
comes Lpc8090 A at 25jC.
As soon as Lp is known, the dimension Rg is directly
predicted from M if we admit that the polydispersity is low
enough. An example of the comparison between experi-
mental data and theoretical predictions is given in Fig. 13.
Concerning the intrinsic viscosity in h conditions, we
Figure 13 Variation of the radius of gyration with the mole-
adopted the YamakawaFujii treatment [43]; to take into
cular weight (SEC determination) (Mw=8.9105; I=1.33) in
account the electrostatic forces causing the polyelectrolyte
0.1 M NaNO3 at 30jC. (From Ref. 37.); Copyright 2003
swelling, Odijk [44] proposed, at a given ionic concentra-
Marcel Dekker.
tion, a modied relation. This relation introduces the total
persistence length and a draining factor / which varies with
the swelling of the chain mainly imposed by the salt
concentration. The use of steric exclusion chromatography
carried out on the same hyaluronate solutions in 0.1 M
coupled with three detectors (refractometer, viscometer,
NaCI at 25jC, in the concentration range from 0.03 to
and MALLS) allows directly the determination of [g](M )
0.125 g/L. Using the proposed MarkHouwink law,
and Rg(M ) relations, and by using the previous theoretical
Mv=2.3 106 and 1.6106 should be found from the
treatments, we are able to obtain directly Lp in the molec-
low-shear and capillary [g] values, respectively; these values
ular weight range covered without any correction for the
demonstrate the importance of controlling the shear rate
polydispensity.
for measurements. It is also necessary to take into account
The persistence length can be predicted by molecular
the degree of hydration of the hyaluronate samples which
modeling separately as soon as the exact chemical structure
can be determined from thermogravimetry, for instance.
is known [15]. The HA molecule has a regular structure
As hyaluronan is a charged polymer, the molecular
based on (AB) linear copolymer (Fig. 1) which may be
characterization must be done in the presence of an excess
modeled in the absence of long-range electrostatic inter-
of salt in order to screen the electrostatic interactions. A 1
actions. In a rst step the conformational analysis of
1 electrolyte concentration equal to 0.1 M appears to be the
most convenient in reducing these interactions without
leading to aggregate formations which may appear by
using a 12 electrolyte, such as CaCl2, or higher salt
concentrations.
The most useful characteristics of polymers in dilute
solution (polymer concentration lower than the overlap
concentration C*) are the Rg and the intrinsic viscosity {[g]
(mL/g)} reecting the specic hydrodynamic volume of the
molecule. As a rst approach, C*c[g]1, but more pre-
cisely, it is given by:
1
C* 4=3pRg3 Na=M 3
with Na the Avogadro number. C* decreases when M

increases with C*fM (13m).
Recently [41], it was proposed to predict the dimen-
sions of hyaluronan, assuming a semirigid chain model
characterized by a persistence length Lp. In this wormlike
chain model, an electrostatic contribution in the persis-
tence length, Le, was introduced, depending on the charge Figure 14 Snapshot of the disordered chain of HA at 25jC.
parameter and the total ionic concentration, taking into (From Ref. 15; Copyright 2003. Oxford University Press.)
two components of the stress: in phase with the

strain, GV(x), the storage modulus, related to
the elastic behavior and out of phase related to the
viscous behavior GW(x), the loss modulus. An
example of the curves is shown in Fig. 17. From
these two values, one can calculate the complex
dynamic viscosity:
jg*jx GV2 GVV2 1=2 x1 4
For all the experiments described in the following,

an excess of NaCl is added to screen the electro-
static long-range interactions which play a role on
the dimensions of the chains but also on
interchain interactions (known as electroviscous
eects). This behavior is characteristic of poly-
electrolytes and was discussed separately [4649].
A. Flow Experiments
Figure 15 Determination of the persistence length (Lpx) at
dierent temperatures from molecular modeling. Lp is ob- For viscosity measurements, the polymeric solutions are
tained for a degree of polymerization X larger than 500. (From subjected to a shear. From a critical shear rate (c c), the
Ref. 15; Copyright 2003. Oxford University Press.) characteristic time imposed by the shear rate becomes
lower than the relaxation time of the solution. Then, some
modications appear in the structure of the solutions
(alignment of the molecules in the ow, disentanglement,
disaccharides up to tetrasaccharides was performed to get etc.) or in the molecule conformation (stretching, etc.),
the low-energy conformations used to access the confor- leading to a decrease of the solution viscosity. The critical
mational statistics of the corresponding polysaccharides shear rate (c c) decreases when the polymer concentration,
represented in Fig. 14. molecular weight, or rigidity of the chain increases. There-
From modeling, the persistence length Lpx was deter- fore the ow curves can be characterized by three param-
mined as a function of the degree of polymerization X for eters [5052]:
dierent temperatures as shown in Fig. 15. It goes to an as-
ymptotic value when X > 500; one gets: Lp=75 A at 25jC 1. The constant viscosity (g0) in the Newtonian
in good agreement with the experimental determination [45]. plateau, which corresponds to the viscosity for
This modeling also demonstrated that H-bonds stabi- lower than (cc)
shear rate (c)
lized the conformation of oligasaccharides and are impor- 2. The critical shear rate (c c) over which the viscosity
tant to control the stiness. decreases with the shear rate, usually following a
power law
VI. RHEOLOGICAL BEHAVIOR g kc n1 5
For usual applications, it is important to determine the 3. The value of n1=p, equal to the slope in loglog
viscosity of the solution (g) and especially how a polymer which characterizes the
representation of g vs. c,
increases the viscosity of the solvent (gs); the problem is pseudoplastic or viscoelastic behavior of the
then to relate (ggs), with the polymer concentration C, its polymeric solution [50]
molecular weight, and/or its intrinsic viscosity [g], which
includes the solvent interaction and the conditions of 1. Behavior of the Newtonian Viscosity
measurements.
Two types of measurements are presented: By plotting in a loglog representation the specic viscos-
ity in the Newtonian plateau as a function of the hyalur-
1. Flow experiments which allow the determination onan concentration in 0.1 M NaCl, three parts can be
of g (c) curves as a function of temperature, considered which characterize the dilute, semidilute, and
hyaluronan, and salt concentrations (c is the shear concentrated regimes [50]. The end of the dilute regime is
rate). An example of the curves is given in Fig. 16. given by the value of the overlap concentration C* which
2. Dynamic experiments. These experiments were corresponds to the entrance in the semidilute regime and
carried out on the more concentrated solutions for progressive entanglements; the beginning of the concen-
which a strain of dened amplitude and frequency trated regime is obtained for hyaluronate concentrations
is applied. For a viscoelastic uid, this gives the higher than about 50 g/L.
and polymer concentration (Mw=1.3 106 ) in

Figure 16 Variation of the viscosity of Na-hyaluronate with the shear rate (c)
0.1 M NaCI at 25jC. (From Ref. 54b.)
Figure 17 GV and GU moduli as a function of the pulsation and polymer concentration for HA Mw=8 105, [g]=1600 mL/g,
25jC, 0.1 M NaCl. (From Ref. 54b.)
Especially, hyaluronan ts well in this representation

[56,57].
2. Critical Shear Rate of the Onset of

Viscoelastic Behavior
The cc can be related to the longest relaxation time of the
solution sr [5052] with
cco fs1
r0
fp2 RT 6gs gM1 8
in the dilute regime, with gs as the solvent viscosity and
cc fs1
r
fp2 C RT 6g0 gs M1 9
in the more concentrated solutions.

Figure 18 Dependence of gsp at zero shear rate as a function In the dilute regime, cco is expected to be independent
of the overlap parameter C[g] for hyaluronan solutions in 0.1 of C and changes as ([g]M)1. A rough estimation of c co
M NaCl at 25jC. (From Ref. 60.) leads to c cof1000 sec1 for a hyaluronate sample of
Mwf1 106. This c co value corresponds approximately
to the shear rate in a capillary viscometer. Then, [g] cannot
be determined from measurements carried out with a
capillary viscometer for hyaluronate sample with a molec-
A master curve is expected at zero shear rate, for gsp as
ular weight higher than about 106.
a function of C[g] for dierent samples having dierent
molar masses as given by the general development:
3. Viscoelastic Behavior
2
gsp 0 Cg kVCg BCg n
6 From the ow curves, the viscoelastic behavior can be
characterized from the (n1)=p values. The p values are
a function of the overlap parameter C[g]. For the lowest
in which n equals 4.1 in the semidilute regime but decreas- C[g] values, C[g]<10, (1n) increases with C[g]
ing to 3.4 over C[g]c100 (limit of the concentrated do- corresponding to the decrease of hydrodynamic interac-
main) (Fig. 18). tions. At higher C[g], (n1) values reach a plateau near
The rst two terms on the right are valid in the 0.8, which is of the same order as for synthetic
dilute solution and are known as the Huggins relation, polymers and close to the value (0.818) predicted by
with k V the Huggins constant (k Vc0.4 for perfectly
soluble polymers) [53]. The departure from the linearity
in the Huggins plot gsp=C[g]+k V(C[g])2 can be consid-
ered as an estimation of the overlap concentration C*
deduced from hydrodynamic behaviour. kV, B, and n
depend on the ionic strength for low salt concentration,
but they are not very sensitive to the molecular weight.
Then, Eq. (6) can be used to estimate, at a rst approx-
imation, (gsp)0 for a polymeric solution at a given
polymer concentration C, knowing its intrinsic viscosity
[g]. This relation can be applied for a mixture of
hyaluronans with dierent molecular weights with
[g]mix=wi [g]i; wi is the weight fraction of the HA sample
having an intrinsic viscosity [g]i [54]. The rheological
behavior of hyaluronan was recently discussed by Krause
et al. [55].
It was recently demonstrated that a general relation-
ship can be proposed for perfectly soluble polymers:
gsp 0 Cg
n o
1 k1 Cg k2 Cg2 k3 Cg3
7 Figure 19 |g*| (x) and g0(c) curves for hyaluronan solutions
at 25jC (Mw=8 105, C=88.1 g/L in 0.1 M NaCl). (From
with k1=0.4; k2=k12/2!; k3=k13/3! Ref. 37; Copyright 2003 Marcel Dekker.)
Graessley [50] from the entanglement concept applied to values, it is possible to obtain GN0, the plateau modulus and
exible chains. its dependence with the hyaluronate concentration in a
large range of concentration:
B. Dynamic Experiments
G0N fC2:26F0:1 for C < 50 g=L
A series of experimental curves obtained in dynamic G0N fC1:9F0:1 for G > 50 g=L
mode is given in Fig. 17; the crossover of GV and GU
characterized by xp determines two domains: at low The role of the molar mass was also examined and it was
frequency, GU>GV is typical of solution behavior and, found GN0 is independent of M at C=40 g/L of HA in 0.1
over xp, GV>GU when the elastic character of the tem- M NaCl, 25jC.
porary network becomes signicant. The exponent obtained in the semidilute regime cor-
From the GV and GU dependencies with the radial responds very well with that predicted by de Gennes [58],
frequency, we can calculate the complex dynamic viscosity from the reptation concept, in good solvent ( GN0fC9/4).
(Eq. 4) which according to the CoxMerz rule, can be
compared with the steady shear viscosity when plotted
against radial frequency and shear rate, respectively. The VII. CHEMICAL MODIFICATIONS
curves are superposed (Fig. 19), indicating that no strong
interchain interactions exist at least for bacterial hyaluro- An important derivative is obtained by chemical cross-
nan solutions at a concentration such as used in this work. linkage of HA allowing to modify deeply the rheological
It is seen that xp decreases in frequency but increases behavior of the solutions. This derivative was intro-
in modulus progressively as a function of the polymer duced by Balazs for medical applications in viscosup-
concentration. From the series of dynamic measurements plementation in arthrosis treatment [5]. The presence of
obtained for dierent polymer concentrations, it is possible large aggregates is demonstrated in solution of Hylan
to get a master curve by shifting of each curve to a reference A, a loose cross-linked hyaluronan, but a rheological
one, from horizontal (ax) and vertical (ay) translations of solution behavior is generally obtained. This product
GV(x) and GU(x) curves (Fig. 20). ax and ay represent the has an apparent Mw around 810 106 g/mol and by
coecient of translation of the frequencies and moduli, successive ltrations on calibrate membranes, we can
respectively, taking GV(x) and GU(x) curves obtained at a determine the yield and dimensions of the aggregates
given concentration as reference. From these curves and ay [59,60]. In Fig. 21, the dynamic characteristics of a 10 g/L
Figure 20 Master curves of GV and GU vs. x obtained by translation using as reference GV (x) and GU (x) curves at the
hyaluronan concentration 39.9 g/L (Mw=1.3 106; T=25jC). (From Ref. 54b.)
Figure 21 Rheological behavior of linear ( GV 5, GU n) and

loosely cross-linked hyaluronans ( GV w , GU ). Polymer
concentration 10 g/L in 0.15 M NaCl at 37jC.
solution of SynviscR and of a linear HA (Mw=1.3 106) in

0.15 M NaCl are compared. SynviscR is in fact a mixture of
Hylan A (80% w/w) and Hylan B (20% w/w), a more cross-
linked HA. Dierent authors are interested in the charac-
terization of HA for medical applications [61,62].
Specic oxidation of HA was realized using TEMPO Figure 23 13C NMR of hyaluronan (a) and oxidized hyalu-
reactant; 2,2,6,6-Tetramethyl-1-piperidinyloxy radical ronan (b). (From Ref. 63; Copyright 2003. Elsevier Science.)
(TEMPO) mediated oxidation on the C-6 position of
the N-acetyl unit of hyaluronan was studied at pH 10.2
and temperature of 0jC with NaOCl as the primary
oxidant (Fig. 22). The molecular weight of the polymer
is not altered by the primary oxidant (NaOCl) and the carboxylic group around 176 ppm increases. New methods
oxidation process itself is responsible for the degradation for chemical derivatization of HA are now in progress in
of the polymer [63]. The kinetics of the oxidation our laboratory and also in others [64].
depends on several factors, among which the attraction
of the nitrosonium cation by the ionic hyaluronan chains VIII. CONCLUSION
is important.
The 13C NMR spectra of the initial HA and the This chapter discussed a series of new experimental data
oxidized form are given in Fig. 23. From these spectra, it obtained on bacterial sodium hyaluronate in the absence of
is shown that the amplitude of the C-6 signal of the N- protein impurities and polymeric aggregates.
acetyl D-glucosamine unit is reduced due to specic oxida- One of the properties discussed here concerns the
tion on that position; at the same time, the signal of the polyelectrolytic behavior playing a role on the activity
Figure 22 Schematic representation of the specic oxidation of HA. (From Ref. 63; Copyright 2003. Elsevier Science.)
coecient of counterions and dimensions of the chains due 9. Heatley, F.; Scott, J.E. A water molecule participates in
to the electrostatic expansion. the secondary structure of hyaluronan. Biochem. J. 1980,
254, 489.
From the analysis of dimensions, the persistence
10. Scott, J.E.; Heatley, R.; Hull, W.E. Secondary structure of
length of the corresponding wormlike chain was deter- hyaluronate in solution. A 1H NMR investigation in
mined and one found its intrinsic value equal to Lpc90 A. dimethylsulfoxide solution. Biochem. J. 1984, 220, 197.
Water retention is also predicted independently of the 11. Ghosh, S.; Kobal, I.; Zanette, D.; Reed, W.F. Conforma-
molecular weight, but it is controlled mainly by the pres- tional contraction and hydrolysis of hyaluronate in sodium
ence of ionic sites and the nature of the counterions. Water hydroxide solution. Macromolecules 1993, 26, 4685.
retention is related to the chemical structure and follows an 12. Hirano, S.; Kondo, S. Molecular conformational transition
of hyaluronic acid in solution. J. Biochem. 1973, 74, 861.
additivity of the contribution of the dierent polar groups 13. Sicinska, W.; Adams, B.; Lerner, L. A detailed VH and UC
in the molecule. The rheological behavior of hyaluronan is n.m.r. study of a repeating disaccharide of hyaluronan:
developed and discussed; the dierent domains of polymer The eects of temperature and counterion type. Carbo-
concentration are examined. In some respect, hyaluronan hydr. Res. 1993, 242, 29.
behaves as other polysaccharides tested previously in our 14. Darke, A.; Filner, E.G.; Moorhouse, R.; Rees, D.A. Studies
laboratory and not very dierent from synthetic polymers. of hyaluronate solutions by nm.r. relaxation measurements.
Detection of covalently dened sti segments within the
Signicant dierences are observed compared with hyal- exible chains. J. Molec. Biol. 1975, 99, 477.
uronan samples from animal sources. They are attributed 15. Haxaire, K.; Braccini, I.; Milas, M.; Rinaudo, M.; Perez,
to the presence of aggregates in these samples, most S. Conformational behavior of hyaluronan in relation to
probably due to protein contamination. Loosely cross- its physical properties as probed by molecular modelling.
linked hyaluronan is presented and its particular rheolog- Glycobiology 2000, 10, 587.
ical behavior is shown. 16. Haxaire, K. Conformation du hyaluronane et interactions
en solution et a` letat solide. Ph.D. Thesis. Grenoble Uni-
In conclusion, the linear hyaluronan behaves as pre-
versity: France, 2000.
dicted for a wormlike polyelectrolyte with an intrinsic 17. Sheehan, J.K.; Atkins, E.D.T.; Nieduszynski, I.A. X-ray
persistence length equal to about 90 A in the absence of diraction studies on the connective tissue polysacchar-
electrostatic intrachain interactions and a charge parame- ides. Two dimensional packing schemes. J. Molec. Biol.
ter compatible with that of a single extended chain, as 1975, 91, 153.
described in the solid state. 18. Winter, W.T.; Arnott, S. Hyaluronic acid: The role of
divalent cations in conformation and packing. J. Molec.
Biol. 1977, 117, 761.
19. Guss, J.M.; Hukins, D.W.L.; Smith, P.J.C.; Winter, W.T.;
ACKNOWLEDGMENTS Arnott, S.; Moorhouse, R.; Rees, D.A. Hyaluronic acid:
Molecular conformations and interactions in two sodium
E. Fouissac, I. Roure, N. Berriaud, and K. Haxaire are salts. J. Molec. Biol. 1975, 95, 359.
acknowledged for their technical cooperation during the 20. Sheehan, J.K.; Gardner, K.H.; Atkins, E.D.T. Hyaluronic
long time we have studied hyaluronan and derivatives acid: A double-helical structure in the presence of potassium
physical properties. at low pH and found also with the cations ammonium,
rubidium and caesium. J. Molec. Biol. 1977, 117, 113.
21. Alexandrowicz, Z.; Katchalsky, A. Colligative properties
of polyelectrolyte solutions in excess of salt. J. Polym. Sci.
REFERENCES Al 1963, 3231, 32.
22. Manning, G.S. Limiting laws for equilibrium and transport
1. Hammer, M.E.; Burch, T.G. Viscous corneal protection properties of polyelectrolyte solution. In Polyelectrolytes;
by sodium hyaluronate chondroitin sulfate and methyl- Selegny, E. Mandel, M., Strauss, U.P., Eds.; D. Reidel
cellulose. Invest. Ophthalmol. Iris. Sci. 1984, 25, 1329. Publishing Company: Dordrecht, 1972; 9.
2. Balazs, E.A. The development of sodium hyaluronate as a 23. Buhler, E.; Rinaudo, M. Structural and dynamical
viscosurgical material in ophthalmic surgery. In Ophthal- properties of semi-rigid polyelectrolyte solutions: A
mic Viscosurgery: A Review of Standards, Techniques and light-scattering study. Macromolecules 2000, 33, 2098.
Applications; Eisner, G., Eds.;Medicopea: Montreal, 1988. 24a. Buhler, E.; Guetta, O.; Rinaudo, M. Characterization of
3. Boudet, D.; Voskamp, K. Lacide hyaluronique: pro- semi-exible polyelectrolyte solutions in the presence of
prietes et applications. Parfums, Cosmet. Aromes 1986, excess salt: From dilute to semidilute regime. Int. J.
68, 53. Polym. Anal. Charact. 2000, 6, 155.
4. Balazs, E.A.; Band, P. Hyaluronic acid: its structure and 24b. Philippova, O.E.; Volkov, E.V.; Sitnikova, N.L.; Khokhlov,
use. Cosmet. Toilet. 1984, 99, 65. A.; Desbrie`res, J.; Rinaudo, M. Dierent types of hydro-
5. Balazs, E.A.; Leshchiver, E. US Patent 4,582,865, 1984. phobic aggregates in aqueous solutions of chitosan and its
6. Villain-Simonnet, A.; Milas, M.; Rinaudo, M. A new hydrophobic derivative. Biomacromolecules 2001, 2, 483.
bacterial exopolysaccharide (YAS34). I: Characterization 25. Davies, A.; Gormaly, J.; Wyn-Jones, E.; Wedlock, D.J.;
of the conformations and conformation transition. Int. J. Phillips, G.O. A study of hydration of sodium hyaluro-
Biol. Macromol. 2000, 27, 65. nate from compressibility and high precision densitomet-
7. Chang, N.S.; Broackle, R.J. Hyaluronic acid-complement ric measurements. Int. J. Biol. Macromol. 1982, 4, 436.
interactions: II. Role of divalent cations and gelatin. 26. Yoshida, H.; Hatakeyama, T.; Hatakeyama, H. Charac-
Molec. Immunol. 1985, 22, 843. terization of water in polysaccharide hydrogels by DSC. J.
8. Sipos, P.; Veber, M.; Burger, K.; Illaes, J.; Machula, G. Therm. Anal. 1993, 40, 483.
Hyaluronatemetal ion interactions: Correlations between 27. Joshi, H.N.; Topp, E.M. Hydration in hyaluronic acid
viscometric, potentiometric, polarographic and electro- and its esters using dierential scanning calorimetry. Int.
phoretic data. Acta Chim. Hung. 1992, 129, 671. J. Pharm. 1992, 80, 213.
28. Ikada, Y.; Suzuki, M.; Iwata, H. Water in mucopolysac- 44. Odijk, T. On the ionic-strength dependence of the intrinsic
charides. ACS Symp. Ser. 1980, 127, 287. viscosity of DNA. Biopolymers 1979, 18, 3111.
29. Takigami, S.; Takigami, M.; Phillips, G.O. Hydration 45. Haxaire, K.; Buhler, E.; Milas, M.; Perez, S.; Rinaudo, M.
characteristics of the cross-linked hyaluronan derivative Predictive and Experimental behaviour hyaluronan in
hylan. Carbohydr. Polym. 1993, 22, 153. solution and solid state. In Hyaluronan; Kennedy, J.F.,
30. Berriaud, N.; Rinaudo, M.; Milas, M.; Desbrieres, J. Phillips, G.O., Williams, P.A., Eds.; Woodhead Publ.:
Hydration of hyaluronic acid as a function of the Cambridge, U.K., 2002; Vol. 1, 37.
counterion type and relative humidity. Carbohydr. Polym. 46. Roure, I.; Rinaudo, M.; Milas, M. Viscometric behavior
1995, 26, 69. of dilute polyelectrolytes. Role of electrostatic interac-
31. Gilli, R.; Kacurakova, M.; Mathlouthi, M.; Navarini, L.; tions. Ber. Bunsenges. Phys. Chem. 1996, 100, 703.
Paoletti, S. FTIR studies of sodium hyaluronate and its 47. Rinaudo, M.; Roure, I.; Milas, M.; Malovikova, A.
oligomers in the amorphous solid phase and in aqueous Electrostatic interactions in aqueous solutions of ionic
solution. Carbohydr. Res. 1994, 263, 315. polysaccharides. Int. J. Polym. Anal. Charact. 1997, 4, 57.
32. Haxaire, K.; Milas, M.; Rinaudo, M.; Marechal, Y. Hy- 48. Roure, I.; Rinaudo, M.; Milas, M.; Frollini, E. Visco-
dration of polysaccharide hyaluronan observed by IR metric behaviour of polyelectrolytes in the presence of low
spectrometry. IPreliminary experiments and band as- salt concentration. Polymer 1998, 39, 5441.
signment. Biopolymers/Biospectroscopy. Biopolymers 49. Milas, M.; Rinaudo, M. On the electrostatic interactions
2003, 72, 10; Haxaire, K.; Mare chal, Y.; Milas, M.; of ionic polysaccharides in solution. Curr. Trends Polym.
Rinaudo, M. Hydration of polysaccharide hyaluronan ob- Sci. 1997, 2, 47.
served by IR spectrometry. II: Denition and Quantitative 50. Graessley, W.W. The entanglement concept in polymer
Analysis of Elementary Hydration Spectra and Water Up- rheology. Adv. Polym. Sci. 1974, 16, 1.
take. Biopolymers 2003, 72, 149; Marechal Y.; Milas, M.; 51. Barnes, H.A.; Hutton, J.F.; Walters, K. An Introduction to
Rinaudo, M. Hydration of polysaccharide hyaluronan ob- Rheology; Elsevier Science; Amsterdam, 1989.
served by IR spectrometry. III: Structure and Mechanism 52. Ferry, J.D. Viscoelastic Properties of Polymers, 3rd ed.;
of Hydration. Biopolymers 2003, 72, 162. John Wiley and Sons: New York, 1980.
33. Rinaudo, M.; Roure, I.; Milas, M. Use of steric exclusion 53. Milas, M.; Rinaudo, M.; Tinland, B. The viscosity depen-
chromatography to characterize hyaluronan, a semi-rigid dence on concentration, molecular weight and shear rate of
polysaccharide. Int. J. Polym. Anal. Charact. 1999, 5, 277. xanthan solutions. Polym. Bull. 1985, 14, 157.
34. David, M.; Blandel, H. Contribution analytique a` la 54a. Berriaud, N.; Milas, M.; Rinaudo, M. Rheological study
determination de la purete et du degre de polymerisation on mixtures of dierent molecular weight hyaluronates.
de lacide hyaluronique. Int. J. Cosmet. Sci. 1986, 8, 55. Int. J. Biol. Macromol. 1994, 16, 137.
35. Terbojevick, M.; Cosani, A.; Palumbo, M. Molecular 54b. Berriaud, N. Contribution a` letude des proprietes du
weight distribution of hyaluronic acid by high-perform- hyaluronane: Comportement rheologique, hydratation.
ance gel permeation chromatography. Carbohydr. Res. Ph.D. Thesis, Grenoble: France, 1994.
1986, 157, 269. 55. Krause, W.E.; Bellomo, E.G.; Colby, R.H. Rheology of
36. Kery, V.; Orvisky, E.; Stancikova, M. An approach for a sodium hyaluronate under physiological conditions. Bio-
rapid identication and characterization of hyaluronic macromolecules 2001, 2, 65.
acid produced by fermentation of Streptococcus equi. 56. Rinaudo, M. Relation between the molecular structure of
Carbohydr. Polym. 1992, 17, 227. some polysaccharides and original properties in sol and
37. Berriaud, N.; Milas, M.; Rinaudo, M. Characterization gel states. Food Hydrocoll. 2001, 15, 433.
and properties of hyaluronic acid (hyaluronan). In 57. Kwei, T.K.; Nakazawa, M.; Matsuoka, S.; Cowman,
Polysaccharides. Structural Diversity and Functional Ver- M.K.; Okamoto, Y. The concentration dependence of
satility. Dumitriu, S., Ed.; Marcel Dekker: New York, solution viscosities of rigid rod polymers. Macromolecules
USA, 1998. 2000, 33, 235.
38a. Fouissac, E.; Milas, M.; Rinaudo, M.; Borsali, R. 58. De Gennes, P.G. Dynamics of entangled polymer solu-
Inuence of the ionic strength on the dimensions of tions. I. The Rouse model and II. Inclusion of hydro-
sodium hyaluronate. Macromolecules 1992, 25, 5613. dynamic interaction. Macromolecules 1976, 9, 587, 594.
38b. Fouissac, E. Contribution dacide hyaluronique par voie 59. Milas, M.; Rinaudo, M.; Roure, I.; Al-Assaf, S.; Phillips,
fermentaire et etude de ses proprietes physicochimiques. G.O.; Williams, P.A. Comparative rheological behavior of
Ph.D. Thesis, Grenoble, France, 1992. hyaluronan from bacterial and animal sources with cross-
38c. Fouissac, E.; Milas, M.; Rinaudo, M. Shear-rate, concen- linked hyaluronan (hylan) in aqueous solution. Biopoly-
tration, molecular weight, and temperature viscosity mers 2001, 59, 191.
dependencies of hyaluronates, a wormlike polyelectrolyte. 60. Roure, I. Rheologie et dimensions des cha nes du hyal-
Macromolecules 1993, 26, 6945. uronane en solution. Ph.D. Thesis. Grenoble University,
39. Shimada, E.S.; Matsumura, G. Viscosity and molecular France, 1997.
weight of hyaluronic acids. J. Biochem. 1975, 78, 513. 61. Mendichi, R.; Soltes, L. Hyaluronan molecular weight
40. Barth, H.G.; Mays, J.W. Modern Methods of Polymer and polydispersity in some commercial intra-articular
Characterization; John Wiley and Sons: New York, 1991. injectable preparations and in synovial uid. Inamm.
41. Reed, W. Light scattering results on polyelectrolyte Res 2002, 51, 115.
conformations, diusion and interparticle interactions 62. Maheu, E. Lacide Hyaluronique dans le traitement de la
and correlations. In Macro-Ion Characterization from gonarthrose, Le Journal Francais de lOrthopedie. http://
Dilute Solutions to Complex Fluids; Schmitz, K.S., Eds.; maitrise-orthop.com.
ACS Symp. Ser. 548, 1993; 297. 63. Jiang, B.; Drouet, E.; Milas, M.; Rinaudo, M. Study on
42. Ghosh, S.; Li, X.; Reed, C.E.; Reed, W.F. Apparent TEMPO-mediated selective oxidation of hyaluronan and
persistence lengths and diusion behavior of high molec- the eects of salt on the reaction kinetics. Carbohydr. Res.
ular weight hyaluronates. Biopolymers 1990, 30, 1101. 2000, 327, 455.
43. Yamakawa, H.; Fujii, M. Intrinsic viscosity of worm-like 64. Lapcik, L., Jr.; Lapcik, L.; Smedt, S.D.E.; Demeester, J.;
chains. Determination of the shift factor. Macromolecules Chabrecek, P. Hyaluronan: preparation, structure, prop-
1974, 7, 128. erties, and applications. Chem. Rev. 1998, 98, 2663.
23
Chemical Functionalization of Cellulose
Thomas Heinze
Center of Excellence for Polysaccharide Research at the Friedrich Schiller University of Jena, Jena, Germany
I. INTRODUCTION biocompatibility, stereoregularity, multichirality, reactive

hydroxyl groups (polyfunctionality), and the ability to
The application of the polyglucane cellulose as a precursor
form superstructures (e.g., helix formation, cholesteric
for chemical modications has been exploited extensively
mesophases, and LangmuirBlodgett layers) are very at-
even before its polymeric nature was determined and well
tractive for the design of smart materials.
understood. Cellulose nitrate (commonly misnamed nitro-
The advantages and limitations of alternative syn-
cellulose) of higher nitrogen content was one of the most
thesis path and concepts, product design, peculiarities in
important explosives. Its partially nitrated ester was
product structures, and briey suitable analytical tools
among the rst polymeric materials used as a plastic
for the determination of structural features will be dis-
well known under the trade name of Celluloid. Today,
cussed. The object is not to supplement or replace any of
cellulose nitrate is the only cellulose ester of commercial
the several review articles, but rather to describe the topic
interest [1]. Furthermore, cellulose products like methyl
comprehensively considering adequately recent results
ethers, ethyl ethers, or hydroxyalkyl ethers or cellulose
of research.
acetate, and, in addition, products with combinations of
functional groups, e.g., ethylhydroxyethyl cellulose as well
as hydroxypropylmethyl cellulose, cellulose acetopropio- II. CELLULOSE SOURCES, CONSTITUTION,
nates, and cellulose acetobutyrates, are still commercially AND PROPERTIES
important many decades after their discovery. In addition,
ionic cellulose derivatives are known for a long time. Cellulose constitutes the most abundant, renewable poly-
Carboxymethylcellulose, up to now the most important mer resource available today worldwide. It has been esti-
cellulose ether, was rst prepared in 1918 and was pro- mated that by photosynthesis, 10111012 t are synthesized
duced commercially in the early 1920s in Germany [2]. annually in a rather pure form, e.g., in the seed hairs of
They are produced in large quantities for diversied appli- cotton plant, but mostly are combined with lignin and
cations. Mainly, the type of the functional group deter- other polysaccharides (so-called hemicellulose) in the cell
mines properties of the cellulose derivative. In addition, wall of woody plants [3].
they can be modied signicantly by adjusting the degree A rather new approach to pure cellulose is the lab-scale
of functionalization and the molecular weight of the production of the polymer by acetic acid-producing bac-
polymer backbone (Table 1). teria, such as Gluconoacetobacter xylinum and Acantha-
Although there is an established or even expanding moeba castellani [4]. Algae form another origin of cellulose,
worldwide market for the classical cellulosics men- e.g., Valonia ventricosa and Chaetamorpha melagonicum.
tioned, recent research and development elds are focused The cellulose obtained is highly crystalline and was useful
on new polymers based on cellulose. This review is intended for studying polymorphs of the polymer [58]. Cellulose of
to demonstrate the growing willingness of the development the Valonia type is found in fungal cell walls as well. In
of new cellulose derivatives, which results from the fact that addition, there are several celluloses of animal origin, of
cellulose, as the main constituent of cell wall of woody which tunicin, a cell wall component of ascidians, has been
plants, is renewable. Moreover, from the polymer chemists extensively studied.
point of view, the unique structure of the polymer com- Commercial cellulose production, however, concen-
bined with such promising properties like hydrophilicity, trates on easily harvested sources such as wood or on the
551
552 Heinze
Table 1 Important Cellulose Esters and Ethers Commercially Produced
Product Functional group Degree of substitution Examples of solubility

Cellulose acetate C(O)CH3 0.60.9 Water
1.21.8 2-Methoxyethanol
2.22.7 Acetone
2.83.0 Chloroform
Cellulose C(O)CH3/C(O)CH2CH3 2.4/0.2 Acetone, methyl ethyl ketone
acetopropionate
Cellulose acetobutyrate C(O)CH3/C(O)(CH2)2CH3 0.2/2.7 Acetone, diisobutylketone
1.1/1.6 Acetone
Cellulose C(O)CH3/C(O)C6H5COOH 0.8/1.6 Acetone
acetophthalate
Cellulose nitrate NO2 1.82.0 Ethanol
2.02.3 Methanol, acetone, methyl ethyl
ketone
Cellulose xanthatea C(S)Sna 0.50.6 Aqueous NaOH
Carboxymethylcellulose CH2COONa 0.52.9 Water
Methylcellulose CH3 0.40.6 4% Aqueous NaOH
1.32.6 Cold water
2.53.0 Benzene, chloroform, cyclohexane
Ethylcellulose CH2CH3 0.50.7 4% Aqueous NaOH
0.81.7 Cold water
2.42.8 Toluene, xylene, methylene
chloride
Hydroxyethylcellulose CH2CH2OHb 0.10.5 4% Aqueous NaOH
0.61.5 Water
Methylhydroxypropyl- CH3/CH2CH2CH2OH 1.22.3 Cold water
cellulosec 0.10.8 Mixtures of methylene
chloride/methanol
a
Key intermediate in cellulose ber spinning.
b
The average number of functional groups per repeating unit is called degree of substitution (DS) arriving at a level of three after complete
functionalization of all hydroxy groups. In some case, e.g., in synthesis of hydroxyethylcellulose, the hydroxyethyl functions may react further to
extended polyethylene glycol like side chains. This value is termed molar substitution (MS) with DSVMS.
c
For example, Culminal 20000; Hercules.
naturally highly pure source as cotton. Other cellulose- deprotection procedures that generally are required in
containing materials include agricultural residues, water polysaccharide chemistry.
plants, grasses, and other plant substances (Table 2) [9]. Up to now, the cellulose obtained via these paths does
not have any importance for functionalization of the
A. Synthetic Cellulose polymer. However, it is an interesting part of basic research
in this eld and the regioselectively substituted cellulose
The chemosynthesis of cellulose by ring-opening poly- intermediates may be valuable, e.g., in structure character-
merization of 3,6-di-O-benzyl-a-D-glycopyranose 1,2,4- ization as model compounds.
orthopivalate [10], or by stepwise reactions of selectively From a chemists point of view, cellulose is a polydis-
protected h-D-glucoses [11], has been experimentally perse linear homopolymer consisting of regioselectively
realized. Complete deprotection of the polymers yields and enantioselectively h-1,4-glycosidic linked D-glucose
cellulose with a rather low degree of polymerization (DP) units (so-called anhydroglucose units, Fig. 1). Taking the
in the range from 9 to 55, dependent on the applied pro- dimer cellobiose as a basic unit, cellulose can be considered
tecting groups. as an isotactic polymer of cellobiose, which sometimes
The nonbiosynthetic preparation of cellulose (Mw of appears appropriate to describe the reactivity of the poly-
6300 g/mol) was described involving an enzymatic polymer, e.g., in degradation reactions.
merization using h-D-cellobiosyl uoride as a substrate for The polymer contains three reactive hydroxy groups at
puried cellulase [12,13]. This approach has interesting the C-2, C-3, and C-6 atoms, which are, in general,
potential for the control of molecular weight and dispersity accessible to the typical conversions of primary and sec-
of the polymer formed. Furthermore, the enzyme-con- ondary alcoholic OH groups. It is worth mentioning that
trolled stereospecicity and regioselectivity may minimize the vicinal secondary hydroxy groups may undergo typical
or even avoid the laborious, multistep chemical protection glycol reactions. Based on the molecular structure, i.e., the
Chemical Functionalization of Cellulose 553
Table 2 Composition of Some Typical Cellulose-Containing as well as in common organic liquids, which stimulated and
Materialsa still stimulates the search for solvents appropriate for
homogeneous phase reactions [17,18].
Composition
Source Cellulose Hemicellulose Lignin Extract
III. REACTION MEDIA FOR CELLULOSE
Bagasse 40 30 20 10
FUNCTIONALIZATION
Cotton 95 2 1 0.4
Flax (retted) 71 21 2 6 A. Heterogeneous Systems
Hardwood 4347 2535 1624 28
Hemp 70 22 6 2 The commercial synthesis of cellulose derivatives is carried
Jute 71 14 13 2 out exclusively under heterogeneous reaction conditions,
Ramie 76 17 1 6 at least at the beginning of the conversion. In some cases,
Sisal 73 14 11 2 the cellulose derivative formed may dissolve in the reaction
Softwood 4044 2529 2531 15 mixture at a certain degree of substitution (DS), e.g., by
a
acetylation of cellulose. Conventionally, cellulose acetates
Adopted from Hon [9].
(CA) are prepared by conversion of cellulose with an excess
of acetic anhydride (1040% above the amount needed for
cellulose triacetate formation) in the presence of sulfuric
acid or perchloric acid as catalyst. Two dierent methods
tacticity and the uniform distribution of the hydroxy are applied today. The majority of the CAs (about 900,000
groups, ordered hydrogen bond systems form various types t per year) is produced using a route that includes the
of supramolecular semicrystalline structures (cf. Chaps. 4 dissolution of the products formed. A mixture of about 2
and 5). Both intramolecular and intermolecular hydrogen parts methylene chloride and 1 part glacial acetic acid is
bonding occur in cellulose. The detailed structures of the employed as the medium for swelling and dissolving the
hydrogen-bond networks are still a subject of discussion, as CA formed. On the other hand, it is possible to carry out
nicely summarized by Gilbert and Kadla [14] recently. the acetylation on the intact ber (ber acetylation). The
The order of the macromolecules in a cellulose ber is products obtained in the rst step are fully esteried.
not uniform throughout the whole structure, which is an Usually, they are partially deacetylated in a one-pot hy-
important structural feature with regard to chemical func- drolysis to give the widely applied acetone soluble 2.5-
tionalization. There exist regions of low order (so-called acetate (acetyl content ca. 40%; DS 2.42.6). This syn-
amorphous regions) as well as of high crystalline order. The thetic detour is necessary because CA samples with equal
experimental evidence available today is adequately inter- DS synthesized directly from cellulose are not soluble in
preted by a two-phase model, the fringed bril model, acetone [1921]. Structure analysis to elucidate the reasons
assuming low-order (amorphous) and high-order (crystal- for this behavior and the search for new synthesis tools for
line) regions and neglecting the rather small amount of a more eective acetylation are still among the major
matter with an intermediate state of order [15]. stimulants for the work in this eld. A comprehensive
The signicance of the accessibility factor in aecting review about the present understanding for structure
the cellulose reactivity is generally accepted today. Not property relationships of various cellulose carboxylic acid
only the crystallinity, but also hydrogen bonding patterns esters including mixed products of commercial importance
have a strong inuence on the general chemical behavior of appeared recently [22].
cellulose [16]. It is important to emphasize that the acces- In any case, a limited swelling of the cellulose moiety,
sibility of cellulose is by no means a constant structural i.e., a loosening of cellulose supramolecular structure with
parameter, as it depends decisively on the interactions principal preservation of the original solid state of the
considered. A further consequence of the supramolecular sample, is an indispensable prerequisite for a subsequent
structure is the insolubility of the macromolecule in water functionalization. The swelling of cellulose in aqueous so-
Figure 1 Molecular structure of cellulose with end groups.

554 Heinze
lutions of sodium hydroxide (f1040% aqueous NaOH) B. Cellulose Solvents

forming alkali cellulose is of high practical relevance for
commercial cellulose chemistry. Large-scale production of Dissolution of cellulose destroys the highly organized
carboxymethylcellulose (CMC, as it is usually called) is hydrogen-bonding system surrounding the polyglucan
exclusively carried out by slurry processes, i.e., by conver- chain. It has to be mentioned that not only physical
sions of alkali cellulose swollen in aqueous NaOH and an dissolution of the polysaccharide (nonderivatizing sol-
organic liquid (e.g., ethanol, isopropanol, and acetone) vents), but also dissolution by partial functionalization of
with monochloroacetic acid or its sodium salt. Common the polymer (derivatizing solvents) is known [32]. In the
CMC in the DS range from 0.5 to 1.5 is subject of several latter case, cellulose intermediates are formed in situ by
review papers [2326]. introducing new functional groups via covalent bonds
Cyanoethylation is the classical example of cellulose especially ester moieties of rather low hydrolytic stability.
etherication by Michael-type addition of CC bond to the It is possible to isolate these intermediates and to use them
hydroxy groups of the swollen polymer in aqueous alkaline in a subsequent homogeneous modication starting from
medium [27]. The cyanoethyl cellulose, an organo-soluble the polymers dissolved in an inert organic solvent. A clear
product, can be subsequently transformed to carboxyethyl line between these cellulose intermediates (or transient
cellulose (CEC) by hydrolysis or oxidation of the nitril derivatives as they are some times called) and true cellu-
groups [28]. It is worth mentioning that the most ecient lose derivatives cannot be drawn. However, it seems
method for preparing CEC, namely, the addition of acrylic reasonable to dene cellulose intermediates as hydro-
acid, does not proceed suciently rapid. Moreover, the lytically unstable cellulosics that can be either obtained by
synthesis of CEC by etherication of alkali cellulose with isolation from solutions of derivatizing solvents or as
the corresponding halogen carboxylic acid is not possible. specically synthesized cellulosics with substituents which
are easily cleaved o during a common workup proce-
dure, e.g., in aqueous media [18]. For example, cellulose
1. Nonaqueous Media triuoroacetates formed by the reaction of the polymer
Swelling of cellulose in aqueous systems, especially in with triuoroacetic acid (TFA)/triuoroacetic anhydride
aqueous alkaline media, has been investigated thoroughly (TFAA, toxic) can be isolated and redissolved in common
for many decades in connection with the use of cellulosic organic solvents. On the other hand, formation of cellulose
bers in textiles and with papermaking. In contrast, data acetate followed by a separate dissolution in acetone would
on swelling of cellulose in organic liquids are still rather not be classied as a solution of cellulose in a derivatiz-
scarce but are of relevance for cellulose functionalization in ing solvent.
nonaqueous media. The equilibrium swelling value
depends on the liquid and on the structure of the cellulose 1. Aqueous Nonderivatizing Solvents
sample. Compared to water, some protic as well as aprotic Aqueous solvents for cellulose-like solutions of inorganic
organic liquids show a signicantly higher swelling power, salts and complex compounds (Table 4) were widely used
e.g., formamide, dimethyl sulfoxide, and ethanolamine, for cellulose regeneration. The best-known solvents of this
while alcohols lead to a much smaller swelling only group are cuprammonium hydroxide (Cuam) and cupri-
(Table 3) [29,30]. A detailed discussion about the swelling ethylenediamine hydroxide (Cuen). Regeneration of mem-
of cellulose of dierent origin in water and a broad variety branes from Cuam solution provides high-quality products
of aqueous and organic liquids is given in Ref. 31. for hemodialysis [33]. Cellulose of rather low degree of
Table 3 Swelling of Cellulose in Organic Liquids

Liquid retention value (%)
Spruce sulte pulp

Hydrolyzed
Medium Cotton cotton linters Untreated Decrystallized Mercerized Rayon staple
Ethanolamine 106 71 163 189 192 256
DMSO 90 72 121 168 170 186
Formamide 71 58 88 158 106 105
Water 51 45 63 87 82 86
DMF 49 25 63 113
Acetic acid 36 13 45 92 33 30
Ethanol 21 14 32 22 29 20
n-Hexane 12 7 15 14 13
DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide.
Table 4 Selected Cellulose Solvents Useful for Characterization, Regeneration, and Homogeneous Phase Reactions of the
Polymer
Type of solvent Compounds Examples

Aqueous, Transition metal complexes/amine or NH3 Cadoxen, [Cd(H2N(CH2)2NH2)3](OH)2
nonderivatizing Cuam, [Cu(NH3)4](OH)2
Cuen, [Cu(H2N(CH2)2NH2)2](OH)2
Inorganic salt Pure salt ZnCl2 4H2O
hydrates LiClO4 3H2O
LiSCN 2H2O
Salt mixtures LiI 2H2OLiClO4 3H2O/MgCl2 6H2O
LiCl/ZnCl2/H2O
NaSCN/KSCN/LiSCN/H2O
Nonaqueous, N-Alkylpyridinium halogenides N-Ethylpyridinium chloride
nonderivatizinga Oxides of tertiary amines N-Methylmorpholine-N-oxide
Triethylamine-N-oxide
Dimethyl sulfoxide (DMSO) DMSO/methylamine
containing solvents DMSO/KSCN
Liquid ammonia/sodium or NH3/NaI (NH4I)
ammonium salts NH3/NaSCN (NH4SCN)
Dipolar aprotic solvents/LiCl N-N-Methylpyrrolidone/LiCl
N-Methylpyrrolidone/LiCl
Liquid SO3/secondary or SO3/triethylamine
tertiary amines
NH3 or amine/salt/dipolar Ethylenediamine/NaI/N,N-dimethylformamide
aprotic solvent
NH3 or amine/SO2 or Diethylamine/SO2/DMSO
SOCl2/dipolar aprotic solvent
a
In most cases, a preactivation of the cellulose is required.
polymerization (DP) of up to 200 is soluble in 10% dissolution of the polysaccharide lead to an almost even
aqueous NaOH as recently reinvestigated [34]. Moreover, accessibility, and hence there is no particular advantage
some inorganic acids can act as cellulose solvents of for carboxymethylation of the dissolved polymer [25].
limited solvent power. The dissolution is accompanied
by severe chain degradation especially at higher tempera-
ture limiting further the practical relevance of these sol- 2. Inorganic Salt Hydrates and Ionic Liquids
vents. Due to the high tendency of these solvents toward Molten inorganic salt hydrates have gained attention as
hydrolysis of reagents applied for modication, problems new solvents and media for cellulose modication. Com-
concerning the purication of the derivatives obtained, pounds of the general formula LiX H2O (X=I, NO3,
and their tendency to become inhomogeneous during the CH3COO, and ClO4) in the molten state were found to
reaction, their application as reaction medium is just of dissolve cellulose with DP values as high as 1500 (Table 4)
limited interest. [3941]. LiClO4 3H2O yields transparent cellulose solu-
Nevertheless, a number of aqueous solvents, e.g., the tions within a few minutes and mixtures of LiClO4 3H2O
aqueous solution of Ni(tren)(OH)2 [tren=tri(2-amino- with Mg(ClO4)2H2O or the eutectic mixture of NaSCN/
ethyl)amine], were studied as medium for homogeneous KSCN/H2O with dierent amount of LiSCN 2H2O were
etherication reactions [35,36]. It is possible to convert proved to dissolve cellulose as well. It is possible to acquire
cellulose dissolved in Ni(tren) in a fully homogeneous NMR spectra in these systems and to regenerate cellulose
process to CMC [37]. Structure investigations by means II from them. The carboxymethylation of the dissolved
of 1H NMR and HPLC analysis after complete depolym- cellulose in molten LiClO4 3H2O (110jC) yields prod-
erization revealed that the products show a statistic ucts of a rather high DS of up to 2 after a short reaction
content of the dierent repeating units and a distribution time of 4 hr, however, applying a high excess of
of the carboxymethyl functions within the AGU in the monochloroacetate [42].
order O-2 z O-6 > O-3; that is, they possess the same Ionic liquids containing 1-butyl-3-methylimidazolium
functionalization pattern as ethers prepared from alkali cations ([C4mim]+) and Cl, Br, or SCN dissolve cel-
cellulose which is applied for commercial production of lulose on heating to 100110jC [43]. Due to the interac-
CMC [38]. These results clearly show that both simple tions of the various cations and anions with the repeating
activation of cellulose with aqueous NaOH and complete units of the polymer, control of the functionalization
556 Heinze
pattern may be expected. One of the most essential ques- with the general composition polar organic liquid/SO2/
tions is the search for appropriate reaction conditions and primary, secondary, or tertiary aliphatic amine of second-
recycling to take full advantage of the new solvents in ary alicyclic amine. From the wide variety of possible
cellulose chemistry and in producing bers, beads, and mixtures, DMSO/SO2/diethylamine is the most versatile.
other particles of the polymer. Whereas acylation reactions succeed just to a limited
extent, etherication with a number of dierent reagents
3. Nonaqueous Nonderivatizing Solvents was very ecient. It was shown that degradation occurring
The mixture of N,N-dimethylacetamide (DMA)/LiCl during dissolution and modication reaction can be dras-
shows an enormous potential for the analysis of cellulose tically diminished by utilization of protective gas atmo-
and for the preparation of a wide variety of derivatives. Its sphere [5660].
usefulness in analysis is due to the fact that the solvent is N-alkyl pyridinium halides and N-oxides of tertiary
colorless and dissolution succeeds without or at least with amines are worth mentioning as single-component solvents.
negligible degradation even in case of high molecular The most powerful solvents in this regard are N-ethyl-
weight samples, e.g., cotton or bacterial cellulose. Experi- pyridinium chloride and N-methylmorpholine-N-oxide
mental details for simple dissolution procedures are given (NMNO). NMNO has gained quite an enormous attention
in Refs. 44 and 45. The solvent system proved to be useful for cellulose regeneration, and a new ber spinning process
for the investigation of the dissolved cellulose by means of based on this solvent is already industrially realized form-
13
C NMR spectroscopy [46,47], electron spray mass spec- ing the so-called Lyocell bers. Recent results were pub-
troscopy (ESI-MS) [48], SEC [4951], and light scattering lished dealing with the investigation of the interaction of
techniques [52]. Although it is the solvent of choice for cellulose with the solvent by means of 13C CPMAS NMR
these applications, a dissolution mechanism has still not and microscopy [61].
been clearly postulated [53]. Dierent solventpolymer A novel and powerful solvent for cellulose consists in
structures were proposed [54]. the mixture DMSO/tetrabutylammonium uoride trihy-
A number of modied compositions were investigat- drate (TBAF). The advantage of DMSO/TBAF is that
ed. DMA can be substituted in the solvent mixture with N- cellulose with a DP as high as 650 dissolves without
methyl-2-pyrrolidone (NMP), N,N-dimethylformamide pretreatment within some minutes. Highly resolved 13C
(DMF), DMSO, N-methylpyridine, or hexamethylphos- NMR spectra of cellulose can be obtained showing all the
phoric triamide, but only NMP, the cyclic analog of ring carbons of the AGU and giving no hints for
DMA, was found to dissolve the polysaccharides without derivatization during the dissolution process (Fig. 2). It
major degradation. The mixture of 1,3-dimethyl-2-imida- should be mentioned that not only cellulose, but also
zolidinone (DMI) and LiCl also dissolves cellulose [55]. other polysaccharides like the hemicellulose xylan are
DMI is commercially available and shows thermal stabil- soluble [62,63].
ity and low toxicity. DMI/LiCl is able to dissolve cellulose
samples with DP values of up to 1200 and concentration of 4. Aprotic Derivatizing Solvents
210% applying the same procedures as used for DMA/ All the solvents previously discussed show physical disso-
LiCl; that is, an activation of the polymer by a heat treat- lution of cellulose without derivatization of any hydroxy
ment or a stepwise solvent exchange is absolutely neces- group. The application of so-called derivatizing solvents or
sary. DMI/LiCl is capable both for esterication and the utilization of hydrolytically unstable, organo-soluble
etherication of cellulose. cellulose intermediates is an alternative route to dissolve
Other nonaqueous, nonderivatizing solvents (Table 4) the polymer. Representative examples of such solvents and
usable for homogeneous modications are mixtures the derivatives formed are given in Table 5.
Figure 2 13C NMR spectrum of cellulose dissolved in DMSO-d6/tetrabutylammonium uoride trihydrate. (From Ref. 62.
Copyright 2000.)
Table 5 Examples of Derivatizing Solvents and Subsequent Reactions on Cellulose Intermediates Isolated Under Aprotic
Conditions
Intermediate
CellUORDS
j
OH3-DS Cellulosic
Solvent Subsequent reaction products
mixture R DS reagents (after workup) Ref.
N2O4/DMF NO 3.0 Pyridine/SO3 Cellulose sulfate [141]
HCOOH/H2SO4 O 2.2 Pyridine/SO3 Cellulose sulfatea [77]
O DMF/SO3 Cellulose sulfatea [70,71]
CUH NaOH/ sodium CMC [72,189]
monochloroacetate (SMCA)
CF3COOH O 1.5 Pyridine/SO3 Cellulose sulfateb [333]
O N,N-carbonyldiimidazole/ Cellulose-4- [77]
CUCUF3 4-nitrobenzoic acid nitrobenzoatea
4-Nitrobenzoic acid/ Cellulose-4- [77]
tosyl chloride nitrobenzoatec
Palmitoyl chloride/ Cellulose [77]
tosyl chloride palmitatea
NaOH/SMCA CMC [72,189]
Phenyl isocyanate Cellulose [77]
phenylcarbamatea
ClSi(CH3)3/ Si(CH3)3 1.6 3,4-Dinitrobenzoyl Cellulose-3,4- [334]
DMF chloride/triethylamine dinitrobenzoatea
2.0 4-Bromobenzoyl chloride/ Cellulose-4- [334]
4-dimethylamino pyridine bromobenzoate
2.5 4-Nitrobenzoyl chloride Cellulose-4- [141]
nitrobenzoateb
1.1 NaOH/SMCA CMC [72,189]
a
Inverse pattern of functionalization.
b
Preferred O-3 functionalization.
c
Partial or total removal of functional groups of the intermediate during the reaction.
The major disadvantage of the derivatizing solvents is synthesis of esters via homogeneous conversion with
the occurrence of side reactions during dissolution and the anhydrides [69].
formation of undened structures. In turn, this leads to
products hardly reproducible. Despite its highly toxic
nature, DMF/N2O4 solvent, yielding cellulose nitrite as 5. Soluble Intermediates
intermediate, has found considerable interest in the syn- A much higher predictability compared to the application
thesis of inorganic cellulose esters, in particular, cellulose of the derivatizing solvents can be realized if the intermedi-
sulfates [6466]. Today, it is accepted that dissolution ates formed resulting from dissolution are isolated prior to
under strictly anhydrous conditions succeeds by formation the conversion into the nal derivative. Thus structure
of the cellulose trinitrite. analysis of the intermediates is possible. They can be
The mixture DMSO/paraformaldehyde (PF) dis- dissolved in a variety of common organic solvents, which
solves cellulose rapidly and almost without degradation decreases the tendency toward side reactions (especially
even in case of high-molecular weight polymers forming degradation). Therefore, reactive intermediates can be the
the hemiacetal; that is, the so-called methylol cellulose is starting material for a variety of highly engineered deriv-
obtained (Table 5). 13C NMR spectroscopy revealed that atives (Table 5).
the acetalization occurs preferentially at position 6 of the Cellulose dissolves in a surplus of formic acid without
AGU [67,68]. The methylol structure remains intact in catalyst over periods of 415 days or with sulfuric acid as
nonaqueous media resulting in a pronounced derivatiza- catalyst within 15 min yielding fairly degraded polymers
tion of the secondary OH groups. In contrast, the meth- with DS values of about 2.5. Cellulose formate (CF) can be
ylol functions can be easily removed by a treatment with isolated; however, the DP of the starting cellulose is
water. The solvent was exploited for the preparation of a reduced from 600 to 200. They are soluble in DMF at
whole number of ethers yielding almost completely func- DS values of 1.2 [70,71]. In case of the application of
tionalized nonionic cellulose derivatives and for the partially hydrolyzed POCl3 as swelling and dehydrating
558 Heinze
agent, it is possible to increase the DS of up to 2.2 yielding workup procedure. Especially, the CTFA is a promising
CF samples soluble in DMSO, DMF, and pyridine with intermediate because of its easy preparation combined with
DP values of 280 (starting from spruce sulte pulp, the highest DP accessible, its solubility in various common
DP=680) [72]. organic solvents, its fast cleavage under aqueous condi-
Solutions of cellulose in TFA studied by means of tions, and its stability under aprotic conditions. Neither
NMR spectroscopy show that the primary OH groups transesterication nor migration under typical acylation
are almost completely functionalized [73,74]. The disso- conditions occurs [77].
lution of cellulose in mixtures of TFA and dierent
organic liquids strongly depends on the electron acceptor
behavior of the liquid. During dissolution of the polymer IV. SYNTHESIS PATHS AND TYPES OF
in TFA/CH2Cl2, triuoroacetylation occurs only to a CELLULOSE CARBOXYLIC ACID ESTERS
limited extent and the polysaccharide is rather slowly
Although the susceptibility of the glycosidic linkages to
degraded [75].
hydrolytic cleavage resulting in chain degradation and the
Pure cellulose triuoroacetates (CTFA) soluble in
supramolecular order (as discussed) inuence the chemical
DMSO, pyridine, and DMF can be easily prepared by
properties of the polymer, the reactivity is mainly deter-
treating cellulose with mixtures of TFA and its anhydride
mined by the three hydroxyl groups per repeating unit. The
[76]. These intermediates have DS values of 1.5 and were
polyfunctionality oers additional freedom for a tailored
completely substituted at position 6 as can be concluded
chemical functionalization and also gives rise to additional
from 13C NMR spectroscopy (Fig. 3) and from HPLC after
problems with regard to uniformity of the reaction prod-
methylation, saponication, and complete depolymeriza-
ucts. Moreover, cellulose sulfonates, halodeoxy cellulose,
tion. Thereby, the inverse pattern of functionalization of
and other cellulose derivatives of inversed reactivity
the methyl ether is determined. If the triuoroacetylation is
provide the possibility to modify the polymer with donor
carried out in the presence of chlorinated hydrocarbons as
reagents serving as leaving groups in nucleophilic displace-
cosolvents (e.g., chloroform), the DS can be increased upto
ment reactions, i.e., to design cellulose materials by reac-
2.2 yielding products soluble in THF. The CTFA samples
tions directed toward the C atoms as well [78]. Typical
can be saponied completely within 6 min.
reactions of cellulose and cellulose derivatives are dierent
All intermediates described can be applied in subse-
types of cross-linking, which were recently described [79].
quent functionalization reactions in homogeneous phase
(Table 5). Thus, a wide variety of organic and inorganic
esters, carbamates, and ethers with a specic distribution of A. New Methods for Heterogeneous Esterification
functional groups were prepared. Applying special
reagents (e.g., N,N-carbonyldiimidazole, see below) and Heterogeneous esterication processes (at least at their
aprotic conditions, these reactions yield nal products with initial stages) are still frequently used. All large-scale
inverse pattern of functionalization with negligible side processes are exclusively carried out heterogeneously
reactions; that is, the primary substituent acts as a protec- which, however, is not a topic of this subchapter. Compre-
tive group and is usually simply cleaved o during the hensive review articles appeared elsewhere [14,22,80].
Figure 3 13C NMR spectrum of cellulose triuoroacetate (DS=1.5; index V means substituted and W means inuenced by a
functionalization of the adjacent position).
A very ecient lab-scale method for cellulose esteri- methylstilbene-5-carboxylate functions [93], and p-phenyl-
cation under heterogeneous reaction conditions is the so- benzoate functions [94] were prepared.
called impeller method. The carboxylic acids are con-
verted to reactive mixed anhydrides. Chloroacetyl moieties, B. Homogeneous Esterification of Cellulose
methoxyacetyl moieties, and, most importantly, triuor-
oacetyl moieties are used as impellers [81]. Tri-O-propio- From the various solvents discussed above, DMA or NMP
nates and tri-O-butyrates can be obtained [82], which were in combination with LiCl have gained special importance.
part of series of DSC studies including also regioselectively Moreover, DMI/LiCl and DMSO/TBAF are of interest
substituted mixed cellulose esters (e.g., 2,3-di-O-acetyl-6- from the authors point of view.
O-propionyl cellulose). On the other hand, new catalysts
were developed, e.g., titanium-(IV)alkoxide compounds 1. Acylation in Nonderivatizing Solvents
like titanium-(IV)isopropoxide (Table 6) [83]. The catalyst The esterication of cellulose in DMA/LiCl using car-
can be applied for the preparation of partially esteried boxylic acid anhydrides and chlorides was among the rst
cellulose derivatives if an appropriate solvent is used. attempts of chemical modication of polysaccharide un-
Furthermore, the esterication of cellulose with acetyl der totally homogeneous conditions [44,95,96]. It was
chloride at elevated temperatures and in vacuum using observed that cellulose dissolved in DMA/LiCl with
1,4-dioxan/pyridine as swelling and activating medium is polymer concentrations higher than 10% forms meso-
known. Vacuum is applied to remove the liberated HCl phases. Therefore, the conversion of the polysaccharide
during the reaction [84]. may be combined with irreproducible eects because in
Acylation is also possible by conversion of cellulose the region of usable solutions of up to 15%, these
suspended in pyridine or DMF (both swelling media for anisotropic systems are not fully stable [97]. Nevertheless,
cellulose) using sulfonic acid chlorides as activating agent. the advantages of acylation in homogeneous phase in
Thus, it was demonstrated that CA could be obtained in a DMA/LiCl are an excellent control of the DS values and
wide range of DS values by treatment of the polysaccharide a uniform distribution of the functional groups along the
with acetic acid in the presence of p-toluenesulfonyl chlo- polymer chains. Moreover, a selectivity of the function-
ride (Tos-Cl) or methanesulfonyl chloride [85]. Highly alization reactions within the repeating units may appear.
ecient is the reaction with the alkali or alkaline earth salt Thus, the reaction of cellulose with acetyl chloride in the
of acetic acid in combination with Tos-Cl [86,87]. These presence of pyridine as base gives CA with complete
methods were extended for the preparation of aromatic functionalization of the primary hydroxy groups at DS
cellulose esters, e.g., nitro-, chloro-, methyl-, methoxy- values starting from 1.6. Especially for esters with aro-
benzoyl-esters [88,89], and 4-azidobenzoyl esters [90]. matic functions, the use of acyl chlorides is still a conve-
The classical method to prepare cellulose esters, i.e., nient and eective path. 4-Phenylbenzoyl cellulose was
converting the polymer suspended in pyridine with car- obtained from the homogeneous esterication yielding
boxylic acid chlorides, is still an important procedure even polymers with DS values as high as 2.4 [98]. Phenyl-
to synthesize cellulosics with unconventional functional acetoxy cellulose, 4-methoxyphenylacetoxy cellulose, and
groups and hence with new properties. For example, p-toloylacetoxy cellulose with DS values of 1.81.9 could
products containing photoreactive moieties like stilbene- be prepared according to this procedure. Solid-state 13C
4-carboxylate functions [91], cinnamoyl functions [92], 2- NMR spectroscopic studies were carried out, and the
Table 6 Long-Chain Cellulose Esters Synthesized by Titanium (IV) Isopropoxide-Catalyzed Reaction

in N,N-Dimethylacetamide [83]
Carboxylic acid Equivalent Time Temp. DS
anhydride per AGU (hr) (jC) (1H NMR) Mn 103a Mw 103a Tg (jC)
Acetic/hexanoic 2.0/2.0 9 155 1.91/0.75 40 164 149
Acetic/hexanoic 1.0/3.0 9 155 1.38/1.36 35 113 122
Acetic/hexanoic 0.0/4.5 6 155 0.12/1.36 33 245 119
Acetic/nonanoic 2.0/2.0 11 145 2.03/0.70 44 177 129
Acetic/lauric 3.5/1.0 12 140 2.40/0.20 96 295 165
Acetic/palmitic 2.0/2.0 12 145 2.06b/0.42 33 125 156
Aceticc/hexanoic 0.0/3.0 7 140 0.00/2.73 23 61 104
Acetic/nonanoic 3.0/1.0 8 145 2.44/0.26 43 220 161
Acetic/nonanoic 1.0/3.0 13 155 1.59/1.11 44 182 118
Acetic/nonanoic 0.0/4.0 13 160 1.11/1.35 31 200 110
a
Determined by GPC in NMP.
b
Partly insoluble.
c
N,N-Dimethylimidazolidinone (DMI) used as solvent instead of DMA.
560 Heinze
derivatives were investigated toward their liquid crystal- results were compared with those of the acetylation in
line properties [99,100]. The introduction of bioactive DMA/LiCl. CA with a DS of 1.4 was accessible, showing
compounds for controlled release systems was achieved the same distribution of substituents as products obtained
with acid chlorides of 2,4-dichlorophenoxyacetic acid in DMA/LiCl (reactivity: C-6>C-2>C-3) [55]. The sol-
(2,4-D) and 2,2-dichloropropionic acid (dalapon) [95]. vent is especially ecient for etherication (see below).
In terms of its usefulness as protective group, the The DMSO/TBAF solvent was studied in terms of its
adamantoyl moiety was investigated [101]. Cellulose furo- usefulness for homogeneous acylations. It was possible to
ate samples synthesized in DMA/LiCl were used to make obtain CA with a DS of 0.83, which is quite remarkable
lms and bers. As the DS decreases, the Tg shifted toward because the solvent contains a rather high amount of water.
higher temperature. The success of wet-spun bers from An alternative to the reaction with an acid anhydride is the
DMA/LiCl solution of cellulose and cellulose furoate transesterication with carboxylic acid esters. Especially
substantiated that cellulose furoate is compatible with the use of vinyl acetate and its higher homologues is a very
cellulose [102104]. Besides the use of acyl chlorides for interesting path because the formation of the acetic alde-
the synthesis of the simple fatty triesters, mixed triesters hyde during the reaction shifts the equilibrium to the
were obtained by conversion of cellulose in DMA/LiCl product site. Thus, CA with DS of 2.7 (10 molar excess of
with fatty acid and acetic acid anhydride in the presence of reagent) was obtained. Even long chain alkyl esters can be
HClO4 [105]. Comparable results were published for the synthesized with DS values as high as 2.6 (Table 7) [62,63].
reaction of cellulose with acyl chlorides, e.g., stearyl chlo-
ride in combination with triethylamine (TEA) [106]. The
advantage of TEA in comparison with pyridine as base is 2. In Situ Activation of Carboxylic Acids
the lower acidity of the hydrochloride formed resulting in a A number of very ecient reagents for the in situ activation
decreased tendency toward chain degradation and split-o of carboxylic acids were applied. Starting from the free acid
of the ester functions introduced. Even cellulose methacry- overcomes disadvantages connected to the acetylation with
lates with DS values of up to 1.3 were accessible which the derivatives discussed above. Whereas acid chlorides are
jellify when irradiated by UV due to the cross-linking most reactive, they are collectively insoluble (except acetyl
reaction of lateral double bonds [107]. Anhydrides of chloride) in the solvent when TEA is present. Carboxylic
dicarbonic acids were applied for the synthesis of water- acid anhydrides, which permit the homogeneous conver-
soluble carboxylic acid half-esters of cellulose [108]. sion, possess problems due to the limited commercial
New and eective procedures for homogeneous acyl- availability and the inherent ineciency because only half
ation were studied which could be employed in large scale of the reagent becomes incorporated into the product. This
as well. Acetates, propionates, butyrates, and mixed ace- makes the use of the free acid combined with suitable
tates/propionates with a stoichiometric control of the activation a desirable tool.
acetyl content can be obtained by reacting dissolved cellu- In situ activation of carboxylic acids is possible with
lose with acid anhydrides, without catalyst, at 110jC for 4 Tos-Cl rst applied for the heterogeneous preparation of
hr. The acylation occurs without degradation of the poly- CA samples [85]. The mixed p-toluenesulfonic/carboxylic
mer [109111]. A preferred acylation of position 6 was acid anhydride, the carboxylic acid anhydride, and the acyl
determined by means of 13C NMR spectroscopy. Both the chloride are formed, which represent the reactive species
negligible eect on the DP and the stoichiometric conver-
sion make this synthesis path a very reliable and reproduc-
ible process.
A highly ecient and sophisticated method is the
conversion of cellulose in DMA/LiCl with diketene or with Table 7 Acylation of Cellulose Dissolved in Dimethyl
a mixture of diketene/carboxylic acid anhydrides [112]. It is Sulfoxide/Tetrabutylammonium Fluoride Trihydrate at 40jC
possible to prepare both pure acetoacetates and mixed for 70 hr (DS Calculated from 1H NMR Spectra)
acetoacetate/carboxylic acid ester of cellulose (especially
with acetyl and propionyl moieties). The reaction with Cellulose ester
diketene is a very useful alternative to the conversion of Acylation mixture Partial DS
cellulose with tert-butylacetoacetate [113,114], which does
a
not yield products of high DS in predictable processes. Compound Molar ratio DS O-6 O-2/3
Acetoacetylation with diketene occurs very rapidly at 100
110jC yielding a complete derivatization within 30 min. Vinyl acetate 2.3 1.04 0.49 0.55
Conversion with mixtures of diketene/carboxylic acid anhy- Vinyl acetate 2.3 1.07 0.52 0.55
drides reveals the same eciency and predictability as dis- Vinyl acetate 1.5 0.63 0.39 0.24
Vinyl acetate 10.0 2.72 0.98 1.74
cussed for the pure esters. This derivatization imparts the
Vinyl butyrate 2.3 0.86
polymer solubility ranging from water to THF depending
Vinyl laurate 2.3 1.47
on the DS. The Tg of the cellulose acetoacetates shows no Vinyl laurate 10 2.60
correlation with the DP but is strongly inuenced by the DS. Vinyl benzoate 2.3 0.95
Very recently, the conversion of cellulose with acetic
a
anhydride/pyridine in DMI/LiCl was studied and the Mol acylation agent per mol anhydroglucose unit.
Figure 4 In situ activation of carboxylic acid with p-toluenesulfonic acid useful for cellulose acylation.
(Fig. 4). The extension of this path on the homogeneous derivatives with low DS eciently. Among the advantages
derivatization of cellulose with waxy carboxylic acids, of the method are the high reactivity of the intermediately
having alkyl substituents in the range from C12 (laurylic formed mixed anhydride with PP and a completely homo-
acid) to C20 (eicosanic acid), yields products with almost geneous reaction in DMA/LiCl. If the reaction is carried
complete functionalization of the OH groups (DS 2.82.9) out with anhydrides of carboxylic acids, forming the mixed
[115]. By means of DSC and dynamic mechanical thermal anhydride with PP recycles the acid liberated. The highly
analysis (DMTA), it was shown that the transition tem- toxic DCC can be recycled as well (Fig. 5). DCC/PP was
perature is increased by 10jC per C-atom of the ester suitable for the preparation of bulky esters like fatty acid
moieties. The method was applied for the introduction of esters of up to eicosan acid with a wide range of DS and
uorine-containing substituents, e.g., 2,2-diuoroethoxy even with complete functionalization of all hydroxy
functions, 2,2,2-triuoroethoxy functions, and 2,2,3, groups. These polymers were prepared as thermoplastic
3,4,4,5,5-octauoropentoxy functions, to increase stepwise one-component compounds [83,123,124]. Moreover,
the hydrophobicity of the products [116,117]. The bulky unsaturated esters (methacrylic acid ester, cinnamic acid
anthracene-9-carboxylate of cellulose could be prepared ester, and vinylacetic acid ester) and esters of aromatic
with DS as high as 1.0 [118]. The amazing result was that carboxylic acids including ( p-N,N-dimethylamino)ben-
the uorescence spectra of the esters and the free acid are zoate were synthesized using N,N-dimethylaminopyridine
identical. The derivatization of cellulose with oxocarbonic (DMAP) as catalyst. The amino group containing ester
acids, e.g., 3,6-dioxahexanoic and 3,6,9-trioxadecanoic acid provides a site for the conversion to the quaternary ammo-
(TODA), yields nonionic cellulose esters with DS values in nium derivative, which imparts water solubility [125,126].
the range from 0.4 to 3.0 [119]. These cellulosics start to
dissolve in water at a DS as low as 0.4. In addition, they are
soluble in organic solvents like acetone or ethanol and 3. Esterication in Derivatizing Solvents
resist a thermal treatment of up to 325jC. Various methods were studied for the homogeneous
A powerful condensation agent is N,N-dicyclohexyl- esterication of cellulose in DMSO/PF. The polymer
carbodiimide (DCC), which was rst exploited in combi- could be converted with dierent carboxylic acid anhy-
nation with 4-pyrrolidinopyridine (PP) and free carboxylic drides/pyridine at low temperature [127130]. Among the
acids by Samaranayake and Glasser [120122] to prepare acylating reagents are aliphatic compounds, e.g., acetic
Figure 5 Synthesis of cellulose esters with carboxylic acids activated with N,N-dicyclohexylcarbodiimide, 4-pyrrolidinopyridine.
(From Ref. 83. Copyright 1998.)
562 Heinze
and butyric anhydride, aromatic acid derivatives like esterication including caprionyl chloride, caproyl chlo-
phthalic anhydride, and even unsaturated species like ride, lauryl chloride, palmitoyl chloride, and stearyl chlo-
methacrylic and maleinic anhydrides. The DS values ride [132,133]. The reaction succeeds via transesterication
reached are usually in the range from 0.2 to 2.0, except of the cellulose nitrite formed during dissolution of the
acetylation where DS of up to 2.5 were realized. By means polymer. The conversion with acetic anhydride leads to
of 1H and 13C NMR spectroscopy, it was shown that the products with DS of up to 2. In case of derivatives with DS
hydroxy groups of the methylol chains are preferentially values of about 0.5, the transesterication is rather selective
acylated. Reactions with acetyl chloride or with the free of position 2 of the AGU as can be concluded from 13C
acids succeed to a limited extent only. Furthermore, NMR spectra.
introduction of acetyl groups by transesterication has Cellulose dissolved in TFA forms the partially triuoro-
been achieved with methylene diacetate and ethylene acetylated product (DS about 11.5), which is used for
diacetate in the presence of sodium acetate at 90jC esterication [134]. Anhydrides of acetic acid, propionic
yielding products with high DS values (acetyl content acid, and 3-nitrophthalic acid and a variety of aliphatic,
22%). An interesting observation was that DMSO in aromatic, and unsaturated acid chlorides (acetyl chloride,
the solvent mixture could be substituted with DMF or acryl chloride, cinnamoyl chloride, benzoyl chloride, and
DMA [131]. Quite recently, esterication of cellulose with 4-nitrobenzoyl chloride) as well as free acids like benzoic
trimethylacetic, trimellitic, and phthalic anhydrides in acid in combination with TFAA were applied as acylation
DMSO/PF was described [69]. DS values between 2.4 reagents [135,136].
and 2.6 are accessible. The esters are versatile compounds
because of their elastomeric and thermoplastic properties
and can be used for the preparation of lms, membranes, 4. Esterication of Organo-Soluble Cellulose
and enteric lm coatings of medical tablets. Intermediates
Although highly toxic, the solvent DMF/N2O4 was An important advantage of soluble, regenerable inter-
widely used for the preparation of cellulose esters. Note- mediates for the functionalization reactions compared to
worthy is its extensive exploitation for the synthesis of derivatizing solvents is that acylation with highly reactive
cellulose phosphates and sulfates [6466] not discussed reagents can be performed in inert common organic sol-
here. A variety of acid chlorides were applied for the vents avoiding side reactions. Important intermediates
Figure 6 Acylation of organo-soluble cellulose triuoroacetate with carboxylic acid imidazolide obtained by in situ activation.
(From Ref. 101.)
are the triuoroacetates [76], formates [7072], and tri- trimethylsiloxy groups [142]. For the conversion of TMSC
alkylsilyl derivatives of cellulose of various DS values with sulfating reagents, a replacement reaction was con-
[137139]. These compounds can be subsequently esteri- cluded where the SO3 attacks the SiO bond yielding
ed in homogeneous phase with carboxylic acid chlorides polymers with the same substitution pattern as the starting
and anhydrides or with the free acid using in situ activa- intermediate [143,144].
tion with N,N-carbonyldiimidazole (Fig. 6). The substitu-
ents, which impart solubility of the polymer, remain
usually at the polymer backbone during the subsequent
functionalization and are cleaved-o during the workup V. SYNTHESIS AND SUBSEQUENT REACTIONS
procedure in protic media eventually catalyzed by chang- OF SULFONIC ACID ESTERS OF CELLULOSE
ing the pH values to acidic or basic conditions. In case of AND DEOXYCELLULOSES
trimethylsilyl cellulose (TMSC), the application of uoride
ions for deprotection is a suitable method as well [140]. Heterogeneous reactions of cellulose with sulfonyl chlo-
Typical examples of homogeneous esterication reactions rides in the presence of NaOH or tertiary amines are usu-
carried out via intermediates are given in Table 8. As ally combined with a variety of side reactions including
already discussed, the partial derivatization does not only undesired nucleophilic displacement reactions caused es-
impart solubility, but also causes selectivity in the subse- pecially by the long reaction times and high temperatures
quent modication. required. In contrast, homogeneous mesylation [145,146]
As mentioned, trimethylsilylation of cellulose with and tosylation [147149] in the solvent system DMA/LiCl
trimethylsilyl chloride is also considered as a solvent, which yield uniform and well-dened products. The tosylation
again shows clearly the problems to dene derivatizing found considerable interest and was extensively studied
cellulose solvents. Anyhow, TMSC was applied for the [45,150]. Products within a wide DS ranging from 0.4 to
esterication with substituted benzoyl chlorides (Table 8). 2.3, which are well soluble in a variety of organic solvents,
It was stated that depending on the reaction conditions, it is could be synthesized (Table 9). They contain only traces of
possible to obtain subsequent derivatives with inverse halogen and no nitrogen at all. Cellulose tosylates form
distribution of the new functional groups or with replace- membranes showing high benzene permselectivity for ben-
ment of the silyl functions. A treatment of TMSC with acid zene/cyclohexane mixtures in pervaporation [151]. The
chlorides, especially 3,4-dinitrobenzoyl chloride and 4- thermal behavior in argon of homogeneously synthesized
bromobenzoyl chloride in the presence of tertiary amines, cellulose tosylate samples within a wide range of DS (from
leads to an acylation of the remaining OH groups [141]. On 0.4 to 2.3) was studied by means of thermogravimetry from
the other hand, the reaction with acid chlorides at elevated ambient temperatures of up to 500jC. Compared to cel-
temperature (in nitrobenzene at 160jC) without catalyst is lulose, the thermal degradation was initiated at a lower
selective to trimethylsiloxy groups while the OH groups are temperature and proceeds in two main stages [152].
unaected [141]. In contrast, the acylation of partially The isolation of highly reactive cellulose triats was
silylated TMS-polyvinylalcohol in nitrobenzene at 160jC not possible up to now because of their sensitivity to even
in the absence of catalysts and proton acceptors resulted, mild nucleophils. An interesting preparation path is the
however, in an unselective conversion of both hydroxy and application of soluble TMSC, which was partially desilyl-
Table 8 Synthesis of Cellulose Esters by Acylation of Trimethylsilyl Cellulose (TMSC) of

Dierent DS with Carboxylic Acid Chloride/Triethylamine [138]
Cellulose ester
TMSC
CDSSi R Solvent DSSi DSEster
1.55 UCHMCHUC6H5 DMF 1.31 0.98
1.55 U(CH2)14UCH3 DMF 1.27 1.05
1.55 UCH2Cl DMF 1.28 1.55
1.55 UC6H3U(3,5)U(NO2)2 DMF 1.43 0.59
1.55 UC6H3U(3,5)U(NO2)2 DMF 1.05 0.85
1.99 UC6H4U(4)UNO2 Benzene 1.95 0.46
1.99 UC6H4U(4)UNO2 Benzene >0.02a 0.43
1.99 UC6H4U(4)UBr Benzene >0.02a 0.39
a
Treatment of the cellulose ester with 1N HCl.
564 Heinze
Table 9 Homogeneous Reaction of Cellulose (Cotton Linters, DP 850) with Tosyl Chloride in DMA/LiCl [45]
Tosyl cellulose
Molar ratio
Tosyl chloride/repeating unit DSTosa S (%) Cl (%) Soluble in
0.6 0.38 5.51 0.35 DMSO
0.9 0.46 6.34 0.40 DMSO, DMF, DMA
1.2 0.89 9.50 0.50 DMSO, DMF, DMA
1.5 1.12 10.70 0.50 DMSO, DMF, DMA
1.8 1.59 12.47 0.30 DMSO, Acetone, THF, Dioxane
2.1 1.74 12.90 0.40 DMSO, Acetone, THF, Dioxane
3.0 2.04 13.74 0.50 DMSO, Acetone, THF, CHCl3
a
DSTos: Degree of substitution calculated on the basis of sulfur analysis.
ated with TBAF and simultaneously treated with triuoro- in nonaqueous solvents is the most ecient route to pure
methanesulfonic anhydride in THF [145]. Novel, soluble 5- and well-dened products, in particular, chloro- and bro-
dimethylamino-1-naphthalenesulfonyl (dansyl) esters of modeoxy derivatives. Earlier attempts were performed in
cellulose with DS from 0.7 to 1.6 were prepared. The the solvent chloral/DMF. Thionyl chloride [154] and mesyl
remaining OH groups could be fully acetylated in pyridine chloride [155,156] were applied as chlorinating agents. DS
with DMAP as catalyst. The polymers show typical uo- values of up to 2.8 were accessible, but the reactions were
rescence and adsorption spectra, which are in good agree- combined with signicant depolymerization.
ment with quantum chemical calculations [153]. A mild and selective conversion is the chlorination
of cellulose in DMA/LiCl with N-chlorosuccinimide-
A. Deoxycellulose triphenylphosphine. At the early state, the reaction suc-
ceeds only under substitution of the 6 hydroxy groups. The
Deoxycelluloses are products in which the hydroxy groups maximum DS achieved is 1.86 [157]. It was proved that an
of the AGU are partially or completely replaced by other inversion at C-3 occurs [158]. Homogeneous synthesis of
functional groups; that is, a nucleophilic substitution (SN) bromodeoxycellulose (Cell-Br) was carried out in dierent
reaction at the carbon atoms was involved (Fig. 7). Espe- media (DMF, NMP, and DMA) with N-bromosuccini-
cially halodeoxycellulose derivatives have found consider- mide-triphenylphosphine and LiBr. The derivatives have
able interest. Again, the application of dissolved cellulose DS values of up to 0.9 and are selectively functionalized at
Figure 7 Typical structures of repeating units with inverse reactivity and unusual structures found by SN reactions.
C-6 [159]. An alternative bromation reagent consists of a iminodiacetic acid. The insoluble products possess high
mixture of tribromoimidazole, triphenylphosphine, and water retention values of up to 11,000% [169]. A number of
imidazole [160]. Reactions in DMA/LiBr yield DS values aminodeoxycelluloses were accessible. Water-soluble 6-
of up to 1.6 combined with inversion at C-3. trialkylammonium-6-deoxycellulose could be prepared by
Synthesis of iododeoxycellulose is still most conve- SN reactions with various amines [170]. Conversion of
nient by displacement of tosylate functions with NaI in cellulose tosylate with 1,4-phenylenediamine yields a poly-
acetylacetone. The reaction is selective yielding 6-iodo-6- mer, which can be used for the immobilization of glucose
deoxycellulose [45,161]. SN reactions at cellulose tosylate oxidase by diazo coupling. The products are applied as
with uoride ions are possible to a limited extent only. The biosensors (e.g., Refs. 171173). The synthesis of 6-amino-
reasons are the bad solubility of uorides and their pure 6-deoxycellulose via the azido derivative was throughout
nucleophilic character. An elegant way to 6-uoro-6-deoxy- reinvestigated. The reaction conditions for a complete
cellulose is a conversion starting from trityl cellulose. The functionalization at C-6 were optimized, as well as various
uorinating agent applied was diethylaminosulfur triuo- subsequent reactions of the product were studied [174].
ride (DAST) [162]. The water-soluble 6-S-thiosulfate-6-deoxycelluloses
[175] can be oxidized by H2O2in analogy to nonpoly-
B. Subsequent Reactions meric compounds of this type [176]under the formation
of SS bridges leading to new water-born coatings. The
A wide variety of subsequent reactions on halodeoxycellu- water-soluble cellulose derivative 6-O-(2,3-bis(thiosulfa-
lose derivatives are possible, including conversions with to)propyl-oxy-2-hydroxypropyl)cellulose was synthesized
thiourea and iminodiacetic acid [163] and with thiols [164] by addition of tetrathionate to 6-O-(allyl-oxy-2-hydroxy-
yielding unusual polymers. Cell-Br is mostly used due to propyl)cellulose. The polymers form dense monolayers on
the fact that it can be synthesized very selectively, bromide gold surfaces by chemisorption [177]. Further details on the
is a better leaving group than chloride, and Cell-Br shows preparation of deoxycelluloses with carbonnitrogen, car-
higher thermal stability than Cell-I [156]. Thus 6-sulfonate- bonsulfur, carbonphosphorus, and carboncarbon
6-deoxycelluloses [165] and 6-mercapto-6-deoxycellulose bonds are recently reviewed [178]. Moreover, related struc-
were synthesized, which were exploited as S-containing tures like cellulosenes and anhydrocelluloses are critically
derivative for metal ion adsorption [166]. discussed. Applications for the deoxycelluloses are numer-
Cellulose sulfonates are usable for a variety of nucleo- ous and characterized by biological, chemical, and physical
philic substitutions summarized by Belyakova et al. [167] end uses (Table 10).
and Hon [168]. Novel water-soluble or swellable cellulosics The mechanism (SN1 vs. SN2) of nucleophilic displace-
were obtained by SN reactions of cellulose tosylates with ment reaction of cellulose derivatives is still a matter of
Table 10 Selected Applications of Deoxycelluloses (Adopted from Vigo and Sachinvala) [178]
Application area Functionalization Ref.
Biological
Bacteriostatic Dierent cellulosics including deoxycellulose [335,336]
treated with 5-nitrofuroyl chloride
Immobilization of enzymes Aminodeoxy cellulose [172,173]
Anticoagulants Sulfated aminodeoxy cellulose [337]
Enzyme purication Aminodeoxy cellulose [338]
Chemical
Removal of heavy metals, e.g., Chlorodeoxy cellulose + ethylenediamine [339]
Hg thiourea, thiosemicarbazide, thioacetamide
Cu, Mn, Co, Ni 6-Chlorodeoxy cellulose + diamines [340]
Hg, Ag S-substituted 6-deoxy-6-mercapto cellulose [166]
Flocculent Cellulose tosylate + amines [170]
Preconcentration of trace elements Tosyl cellulose treated with piperazine and CS2 [341]
(cellulose piperazinedithiocarboxylate)
Sorption of uranium Cellulose piperazinedithiocarboxylate (see above) [342]
Chelating and complexing agents Chlorodeoxy cellulose + iminodiacetic acid [343,344]
Schi base formation from aminodeoxy cellulose
Physical
Flame retardants Chlorodeoxy cellulose [345]
Propellants Azidodeoxy cellulose [346]
Electrostatic printing Dierent cellulosics including aminodeoxy derivatives [347]
Electron transfer catalysts Viologens from ethyl cellulose and deoxy derivatives [348]
566 Heinze
Table 11 Homogeneous Sulfation of Tosyl Cellulose with SO3Pyridine Complex (SPC) [180]
Tosyl cellulose Tosyl cellulose sulfatea Solubilityb

Mol SPC/mol free
DSTosyl OH groups Time (hr) DSSulfate H2O DMSO
0.46 3.0 2.5 0.80 +

0.89 2.0 2.5 0.85 + +
1.43 2.0 2.0 0.57 + +
1.43 4.0 2.0 0.71 + +
1.43 4.0 4.0 0.85 + +
1.43 4.0 6.0 0.85 + +
2.02 2.6 2.0 0.34 +
a
Calculated from sulfur and sodium analysis.
b
Determined at a concentration of 1 g/100 mL+: soluble: : insoluble: DMSO=dimethyl sulfoxide.
discussion. A remarkable nding is that a treatment of NMNO is a suitable medium for various etherication
partially substituted cellulose tosylates (DS 1.5) with a reactions including hydroxyethylation, mercaptoalkyla-
strong nucleophile-like azide ions leads to a substitution tion, cyanoethylation, and carboxymethylation (up to DS
of both primary and secondary tosylates. It is assumed that 1.8 in a one-step synthesis) [37,181,182]. The solvent DMA/
an epoxide-type intermediate is formed during the conver- LiCl is useful for the preparation of methylcellulose, car-
sion. If a perpropionated cellulose tosylate, i.e., a product boxymethylcellulose, benzyl cellulose, and hydroxyethyl
without any free OH group, is used as starting material, the cellulose of rather high DS (up to 2.5) [96,147], for the
reaction does not exceed DS of 1 and the substitution tailored synthesis of cellulose ethers with low DS (0.10.5)
occurs exclusively at the primary C atoms [146]. In order [183], and for the dened conversion of cellulose into ethers
to gain regioselective conversions, the reaction conditions having an unconventional distribution of functional groups
employed in our own work guarantee a pronounced reac- as discussed below.
tion at the primary carbon atoms. The etherication in DMA/LiCl, which is the medium
It is worth to mention that cellulose tosylates can be of choice for acylation reactions, is generally combined
modied at the remaining hydroxy groups as well. Hence, with a number of diculties, e.g., a high excess of reagents
novel cellulosic polymers were obtained by both acylation and long reaction times. Isogai et al. [5760] compared the
[179] and sulfation [180] of the OH groups. Sulfation with etherication of cellulose in DMA/LiCl, N2O4/DMSO,
SO3pyridine complex in DMA for 2 hr at room temper- and DMSO/SO2/TEA. It was found that benzylation is
ature aorded novel amphiphiles having both reactive most ecient in the latter solvent, and fully functionalized
tosylate and ionic sulfuric acid half-ester functions. These cellulose ethers are accessible in a one-step reaction
polymers are soluble both in water and DMSO at an appro- (Table 12).
priate balance of DS values, and they are promising start-
ing materials for self-organization systems (Table 11) [180].
A. Carboxymethylation
VI. ETHERIFICATION OF CELLULOSE Although a number of highly engineered ionic cellulose

derivatives were prepared in lab-scale procedures during
The most successful alternatives to the commercially the last decade [184], the most important and widely
exploited slurry processes for etherication are homoge- applied ionic ether is still CMC with an estimated produc-
neous conversions or modications starting from the dis- tion volume >300,000 t/year worldwide. CMC is usually
solved polymer. In addition, reactions under partial produced in the slurry process; that is, cellulose is sus-
retaining or after new formation of cellulose superstruc- pended in a mixture of isopropanol/water (or ethanol/
tures were investigated. It is basically possible to apply the acetone) [26], treated with aqueous NaOH solution and
same solvents for the etherication of cellulose as described converted with monochloroacetic acid or its sodium salt as
for acylation reactions. discussed [25,185]. This process and its potential for the
Table 12 Examples of Cellulose Ethers solubility, as samples obtained in the slurry reaction [35].
Prepared in DMSO/SO2/DEA [59] Consequently, new synthesis path has to be developed in
order to control the distribution of functional groups. A
Reagent DS special state of activation was achieved by using the so-
CH3I 3.0 called concept of reacting structural feature including
CH3CH2I 3.0 stepwise etherication after activation with aqueous
CH3CH2CH2I 2.9 NaOH of comparatively low concentration [186,187]. Car-
(CH3)2CHCH2I 0.5 boxymethylation is thereby mainly achieved in noncrystal-
CH3CH2CH(CH3)I 0.5 line regions of the cellulose structure.
CH3(CH2)xCH2I, x=36 3.0
ICH2COONa 0.6
CH3CH2CH2Br 3.0 1. Induced Phase Separation prior to Etherication
CH3(CH2)xCH2Br, x=2, 3 3.0 A dierent concept for the synthesis of CMC with new
CH3(CH2)xCH2Br, x=58 3.0 structural features is the activation of cellulose by dis-
C6H5OCH2CH2CH2Br 3.0 solution (e.g., in DMA/LiCl) and a subsequent addition
BrCH2COOH 0.8 of solid water-free NaOH particles [188]. By means of
BrCH2CH2COOH 0.4 FTIR and polarizing light microscopy, it was shown that
BrCH2CH2SO3Na 1.8 a gelation occurred due to the regeneration of cellulose
BrCH2CH2NH2*HBr 0.1 II on the interface solid particle/solution. This process is
BrC6H11 0.5 called induced phase separation leading to reactive
BrCH2CH2C6H5 0.5
microstructures [36,72,189]. The reaction of cellulose
BrCH2CH(OC2H5)2 0.5
with sodium monochloroacetate yields CMC with DS
values as high as 2.2 in a one-step procedure (Table 13).
The etherication at the 6 and 3 positions is more
variation of structural features of CMC prepared were eective compared to the conventional synthesis. It is
extensively studied. An important conclusion is that a possible to completely depolymerize the polymer back-
treatment of cellulose with aqueous NaOH of appropriate bone (preferably with HClO4) and to separate the re-
concentration is a very ecient activation method. Addi- peating units of the CMC chain by HPLC [190]. A
tional evidence for the high activation power of aqueous comparison of the mol fractions measured with values
NaOH prior to etherication was obtained from a homo- calculated by statistics [191], which simulated the con-
geneous carboxymethylation carried out. The conversions version along the polymer chain without preference of
in Ni(tren)(OH)2, which is the rst example for a totally any of the OH groups and without the inuence of the
homogeneous carboxymethylation, yield polymers with DS already reached, leads to very interesting results.
the same pattern of functionalization and properties, e.g., While the statistic data meet the mol fractions of con-
Table 13 Carboxymethylation of Cellulose Dissolved in DMA/LiCl and of Cellulose Triouroacetate

(CTFA), Cellulose Formate (CF), Cellulose Acetate (CA), and Trimethylsilyl Cellulose (TMSC) in DMSO
via Induced Phase Separation with NaOH Particles (Size<0.25 mm) at 70jC
Carboxymethylcellulose
Starting material Reaction
DS Molar ratioa time (hr) DS Solubility in waterb
Cellulose/DMA/ 1:2:4 48 1.13
LiCl 1:4:8 48 1.88 +
1:5:10 48 2.07 +
CTFA/DMSO 1:5:10 2 0.11
(1.5) 1:10:20 4 1.86 +
1:10:20 16 1.54 +
CF/DMSO 1:10:20 2 1.46 +
(2.2) 1:10:20 4 1.91 +
1:15:30 4 1.36
1:20:40 2 2.21 +
CA/DMSO 1:10:20 2 0.36
(0.8) 1:10:20 4 0.45
TMSC/DMSO 1:10:20 0.5 2.04 +
(1.1)
a
Molar ratio: repeating unit/ClCH2COOH(Na)/NaOH.
b
: Insoluble; +: soluble.
568 Heinze
ventionally obtained CMC completely, in case of poly- ing with areas of limited substitution. The highly carboxy-
mers synthesized via induced phase separation, signi- methylated fragments were dominated by 2,3,6-tri-O-
cant dierences between the data sets appear, as can be seen carbyoxymethyl glc units [192].
in Fig. 8. The comparably high amount of glc and 2,3,6-tri-
O-carboxymethylated glc units is a rst evidence for a B. Synthesis of Nonionic Cellulose Ethers
gradient-like distribution of ether functions along the
polymer backbone. It may be concluded that after an A wide variety of completely functionalized cellulose ethers
induced phase separation, the etherication is more or less bearing alkyl and aryl substituents as well as functions
limited to the area directly at the interface polymer-solid containing double bonds were synthesized starting from
NaOH particle. cellulose dissolved in DMSO/SO2/DEA with solid NaOH
Applying organo-soluble cellulose intermediates like and the corresponding alkyl and aryl halides (cf. Table 12)
CTFA (DS 1.5) dissolved in DMSO for the induced [5760]. Tri-O-isopentyl cellulose is capable of forming ul-
phase separation technique (Table 13), it was revealed trathin layers and supramolecular architectures [193195].
by polarizing light microscopy and FTIR spectroscopy After an appropriate activation, cellulose may dis-
that by addition of solid NaOH, the primary substituents solve in DMSO/LiCl, which was found to be a medium
(triuoroacetate) are cleaved o and cellulose II is regen- for etherication using dimsyl sodium solution obtained
erated on the solid particles. Additionally, the deacetyla- from sodium hydride and DMSO to initiate the reaction
tion was nicely observed by the growth of sodium [196]. Iodomethane, iodoethane, bromoethane, 1-bro-
triuoroacetate on the NaOH and in the medium. It is mopropane, and bromobutane could be used as ether-
worthy to note that the decrease of the size of NaOH ifying agents.
particles from 1 to <0.25 mm increases the DS from 0.64 to Recently, the complete methylation of cellulose was
1.12 but does not inuence the distribution of the func- described using DMI/LiCl as reaction medium. In this
tional groups [189]. solvent, tri-O-methyl cellulose is obtained in a one-step
Endoglucanase fragmentation of CMC samples fol- conversion. In comparison to reactions in DMSO/SO2/
lowed by analytical and preparative SEC proved that DEA, the amount of alkylating reagents is drastically
samples of DS of up to 1.9 are intensively degraded, diminished [55].
supporting the proposed blocklike pattern of functional- The preparation of tri-O-allyl cellulose and tri-O-
ization. The detailed analysis of the fragments obtained crotyl cellulose in large scale using DMA/LiCl, allyl chlo-
was carried out by preparative SEC, hydrolysis, and anion ride and crotyl chloride, and NaOH powder as base was
exchange chromatography with pulsed amperometric de- published [197]. The products were studied in terms of their
tection. From these studies, a structure can be proposed thermal and mechanical properties and were investigated
with chains containing segments of very high DS alternat- by dierent NMR methods including Double-Quantum
Figure 8 Representative examples of the mole fractions of (. ) glucose; (n) mono-O-carboxymethyl glucoses; (D) di-O-
carboxymethyl glucoses; (q) 2,3,6-tri-O-carboxymethyl glucose in hydrolyzed carboxymethylcellulose samples (polymers were
synthesized via induced phase separation starting from cellulose acetate, CA, trimethylsilyl cellulose, TMSC, cellulose
triuoroacetate, CTFA, cellulose formate, CF, and cellulose dissolved in DMA/LiCl) plotted as function of degree of sub-
stitution (DSHPLC) determined by means of HPLC. (From Ref. 189. Copyright 1998.)
Filtered-Correlated Spectroscopy (DQF-COSY), Hetero- compared to both diphenylmethyl chloride and trityl chlo-
nuclear Single-Quantum Coherence (HSQC), Total-Cor- ride homogeneously in DMA/LiCl. The increased reactiv-
related Spectroscopy (TOCSY), and Heteronuclear ity results from the insertion of electron-donating methoxy
Multiple Bond Correlation (HMBC). The tri-O-allyl ether functions as well as from the small steric hindrance [198].
may cross-link in air to generate branched products if not The reaction shows no pronounced regioselectivity as can
stabilized with, for example, butylated hydroxytoluene. be determined by means of 13C NMR spectroscopy. A
photosensitive cellulose structure can be built up by reac-
ting cellulose in DMA/LiCl with 4,4V-bis(dimethylamino)-
1. Diphenylmethyl Ethers diphenylmethyl chloride at 50jC for 24 hr yielding
The etherication of cellulose with dierently substituted polymers with DS between 0.54 and 1.05 soluble in a
diphenylmethyl functions was investigated as summarized variety of solvents including DMSO, DMF, THF, and
in Table 14. It was observed that the 4,4V-bis(dimethoxy)- 1,4-dioxane (Table 14) [199]. It is possible to cast lms from
diphenylmethyl chloride reacts much faster with cellulose these derivatives which show photoconductivity.
Table 14 Homogeneous Conversion of Cellulose with Dierent Diphenylmethyl

Chlorides for 24 hr (DS Determined by Elemental Analysis)
Reagent
Temperature
Type Mol/mol AGU (jC) DS
3 70 0.02
3 70 0.33
3 70 0.01
3 70 1.00
2 50 0.54
4 50 1.05
570 Heinze
2. Silylation of Cellulose Trimethylsilyl cellulose can be desilylated with stoi-

The polymer can be silylated in a mixture consisting of chiometric amounts of water in the presence of ammonia,
THF or o-xylene/pyridine and trimethylsilyl chloride and the partially substituted derivatives can be selectively
yielding the soluble trimethylsilyl cellulose (TMSC). The acetylated [206]. TMSC was exploited for the continuous
reaction mixture becomes homogeneous starting from a polymer fractionation (CPF) [207]. The cellulose deriva-
DS of about 2 [200,201]. Pure TMSC was rst described by tives can be applied to build up well-dened supramolec-
Schuyten et al. [202] and was extensively studied because ular architectures because they are composed of sti
of the potential to regenerate cellulose simply by a treat- backbone decorated with exible side chains; that is, they
ment with acids and to obtain, thereby, cellulose bers, belong to the class of so-called hairy rod molecules. Ultra-
particles, or lms [203]. Homogeneous synthesis in DMA/ thin lms were prepared by the LangmuirBlodgett (LB)
LiCl using hexamethyldisilazane gives products with an technique [208]. The advantage of TMSC lms on a silicon
almost complete silylation (DS 2.72.9) [137]. TMSC substrate is their in situ conversion to thin cellulose lms by
within a broad range of DS values were synthesized, exposure of the material to HCl gas. In turn, these lms can
showing solubility in organic solvents dependent on DS be modied by adsorption processes, e.g., dye adsorption,
(Fig. 9) [138,141]. and by chemical reactions like the ring opening esterica-
An interesting path for the silylation of cellulose is the tion with dicarboxylic acid anhydrides [209]. In addition,
conversion with hexamethyldisilazane (HMDS) in liquid these layered structures were applied as basis for the
ammonia carried out in an autoclave at elevated temper- immobilization of proteins and used for the detection of
ature [139,204,205]. One of the major advantages of the single antigen molecules [210].
method consists in the fact that during silylation with
HMDS, only NH3 is generated as by-product. SEC mea-
surements of the TMSC samples obtained revealed that VII. REGIOSELECTIVE FUNCTIONALIZATION
the reaction succeeds without notable depolymerization
In general, all chemical modication reactions of cellulose
[204]. Furthermore, it was shown that a partial silylation
show at least some regioselectivity; that is, in no case, an
of the polymer is possible under stoichiometric control via
equal reactivity of position 2, 3, and 6 appears. The re-
the amounts of reagent applied and even products with a
activity of the free hydroxy groups depends on many factors
complete derivatization of all hydroxy groups are acces-
including the state of activation and dissolution, the com-
sible (DS=3.0) [205] which was not possible by silylation
position of the reaction mixture, the reagent in question, etc.
in DMA/LiCl. Complete conversion of all OH groups
For instance, homogeneous acylation in DMA/LiCl yields
imparts the polymers new properties. They are not fully
products with a preferred functionalization at position 6,
soluble in solvents like THF, toluene, DMA, tetraline, or
while the carboxymethylation in 2-propanol/aqueous
cyclohexane compared to the products with a DS of 2.9. It
NaOH proceeds faster at position 2 compared to 6 and 3.
is assumed that this insolubility is due to aggregation ef-
An important area in cellulose chemistry is the close
fects of the fully functionalized homopolymer. DSC mea-
control of the substitution site within the repeating units.
surements show no glass transition.
For the synthesis of new cellulose derivatives with a well-
dened primary structure, polymer analogous reactions
are in the center of interests. However, enzymatically
catalyzed regioselective functionalization, e.g., the lipase
catalyzed acylation [211,212], the enzyme-catalyzed poly-
merization of cellulose derivatives [213], and the chemical
polymerization (e.g., by ring-opening polymerization of
glucose derivatives [10,11]) are alternative routes. It is
assumed that they will be increasingly studied in the future.
The regioselective introduction of new functions into
cellulose via sulfonic acid esters and halodeoxy derivatives
of the polymer and subsequent reactions of these deriva-
tives were already described.
A. Protective Group Technique

The most widely used protective group is the trityl moiety.
A number of homogeneous procedures were developed for
its controlled introduction. Husemann and Siefert [214]
described rst attempts exploiting N-ethylpyridinium chlo-
ride melts as reaction medium. Furthermore, tritylation
reactions of cellulose in DMSO/N2O4, DMA/LiCl, and
Figure 9 Solubility of trimethylsilyl cellulose dependent on DMSO/SO2/DEA were carried out yielding polymers with
DSSi. DS values of 1.0 [5658,215]. The 6-mono-O-trityl cellulose
Table 15 Tritylation of Cellulose with Methoxy-Substituted Trityl Chlorides (3 mol Reagent/mol AGU, DMA/
LiCl, 70jC) and Detritylation (37% HClaq in THF, 1:25 v/v at 25jC) After Subsequent Permethylation [219]
Tritylation
Detritylation
Protecting group Time (hr) DSa Relative rate Relative rate
Trityl 4 0.41 1 1
24 0.92
48 1.05
4-Monomethoxytrityl 4 0.96 2 18
24 0.92
48 0.89
4,4V-Dimethoxytrityl 4 0.97 2 105 100
24 1.05
48 0.90
4,4V,4W-Trimethoxytrityl 4 0.96 6 106 590
24 0.92
48 0.93
a
DS calculated from elemental analysis.
was widely applied for the regioselective functionalization trityl function. Moreover, the detritylation succeeds 20
of cellulose. The synthesis of 2,3-di-O-substituted methyl- times faster.
cellulose and benzyl cellulose as well as of 6-mono-O- A procedure for the synthesis of 2,3-O-CMC via 6-O-
methyl and benzyl ethers was carried out via various triphenylmethyl cellulose or more eciently via the 4-mono-
protectiondeprotection steps [216218]. One of the major methoxylated derivative was established. The synthesis
disadvantages is the deprotection step, which is usually path and the product properties were investigated in detail
carried out with HCl resulting in a signicant degradation [220222]. The complete determination of the molecular
of the polymer chains. Therefore, alternative protecting structure of 2,3-O-CMC was carried out by enzymatic and
groups are still of interest. chemical methods. As a result of endoglucanase treatment,
Methoxysubstituted triphenylmethyl compounds are an intensive depolymerization of the samples occurred
new eective protective groups in cellulose functionaliza- which was more pronounced for derivatives of com-
tion (Table 15) [219]. The reaction of cellulose dissolved in paratively low DS. The degraded samples can be separated
DMA/LiCl with 4-monomethoxytriphenylmethyl chloride into 18 fractions by preparative SEC, and the proportion of
is 10 times faster than the conversion with unsubstituted each individual repeating unit can be analyzed by AEC
trityl chloride. Complete functionalization of the primary following complete hydrolytic chain degradation. These
hydroxy groups is possible within 4 hr and 70jC. Even after analytical studies indicate a homogeneous distribution of
long reaction times, excess of the reagent, and elevated the CM functions within the polymer chain [223].
temperatures, alkylation of the positions 2 and 3 was less Trialkyl and triarylsilyl groups were investigated to
than 11%, which is the same range as for the unsubstituted protect the primary groups of cellulose. Among the deriv-
Figure 10 Important organo-silyl protecting groups useful for regioselective acylation of secondary positions of cellulose.
572 Heinze
methylation reactions, which are usually applied for struc-

ture analysis [226].
On the other hand, the partial degree of TDMS groups
introduced at position 6 by silylation in DMA/LiCl (total
DS=0.99) was determined to be 85% only (GC/MS anal-
ysis). However, homogeneous reaction in DMA/LiCl can
be used to synthesize a 2,6-di-O-TDMS derivative of
cellulose useful for the subsequent reaction at position 3
[227]. The residual OH groups in position 3 can be com-
pletely etheried without loss of any of the protecting
functions. Treatment with TBAF leads to novel com-
pounds 3-O-allyl cellulose and 3-O-methylcellulose. The
structures of these polymers were revealed by means of
Figure 11 13C NMR spectrum of 3-O-allyl cellulose prepared one- and two-dimensional [COSY and Heteronuclear Mul-
via 2,6-di-O-TDMSC (index s means substituted). (From tiple-Quantum Coherence (HMQC)] NMR techniques
Ref. 228. Copyright 2001.) (Figs. 11 and 12) [228].
Regioselective esterication of the secondary hydroxy
groups is also approached after protection of the primary
atives, the 6-O-thexyldimethylsilyl (TDMS) cellulose was functions with the bulky triphenylmethyl moiety. 2,3-Di-
the most suitable product (Fig. 10). It can be synthesized O-acetyl cellulose was obtained after detritylation. More-
using a heterogeneous phase reaction in ammonia-saturat- over, regioselectively substituted mixed cellulose acetate
ed polar-aprotic liquids at 15jC. The conversion of propionates were prepared by subsequent acylation of the
cellulose with TDMS chloride in N-methylpyrrolidone/ generated free hydroxy groups [229232]. These esters were
NH3 does not exceed a total DS of 1.0 and gives a product utilized as model compounds for NMR spectroscopic
of a remarkable high O-6 selectivity (96% silylation at O-6) studies, in DSC measurements, and for the direct imaging
[224]. Various subsequent products, e.g., 6-O-acetyl-2,3-di- of single crystals by AFM [82,233]. Furthermore, the
O-methylcellulose, were prepared, and their molecular dynamic structures formed in polar solvents of regioselec-
structures were proved by means of 1H1H-COSY NMR tively substituted CA samples were compared with those of
spectroscopy [225]. An interesting nding was that silylated commercial products. Dierences in the chain conforma-
polysaccharides show rearrangement phenomena during tion, solubility, and clustering mechanism were found
Figure 12 1H1H correlated spectrum (a) and 1H13C HBQC measurement (b) of 3-O-allyl-2,6-di-O-acetyl cellulose in CDCl3.
(From Ref. 228. Copyright 2001.)
Table 16 Homogeneous Deacetylation of Cellulose Triacetate in Amine-Containing Media at 80jC

Pattern of functionalizationa
Molar ratio
Amine (mol/mol AGU) Time (hr) DSAca C-2 C-3 C-6
NH2(CH2)6NH2 2.3 2.5 2.65 0.80 0.85 1.00
4.5 2.40 0.65 0.75 1.00
9.0 1.95 0.45 0.55 0.90
14.0 1.50 0.20 0.45 0.85
24.0 0.75 0.05 0.10 0.60
HN(CH3)2 4.5 5.0 2.55 0.75 0.80 1.00
11.0 2.00 0.50 0.50 1.00
15.0 1.80 0.35 0.50 0.95
20.0 1.60 0.30 0.40 0.90
24.0 1.20 0.20 0.30 0.70
a
Calculated from NMR spectra.
[234]. Synthesis of 6-O-acetyl-2,3-di-O-methylcellulose is a,h-diol units with periodate, which can be further oxidized
possible, applying the protection with TDMS function as to give 2,3-dicarboxy cellulose (Fig. 13). The tendency of
well [225,227]. these oxidations depends substantially on the nature of the
oxidants. It is rather complicated to gain both a selective
B. Selective Cleavage of Functional Groups and complete transformation of a desired position. Most of
the oxidants known from low molecular chemistry produce
A special method for regioselective functionalization carbonyl and carboxyl functions to a varying extent
includes the selective deacetylation of cellulose acetates. depending on the experimental conditions. Moreover,
Thus, cellulose was partially acetylated in DMA/LiCl many oxidation reactions will result in a depolymerization
with pyridine/acetic anhydride to give acetate with a of the macromolecules [240,241]. Although oxidative deg-
preferred substitution at O-6. In a subsequent step, this radation is important in cellulose chemistry and process-
CA was carefully deacetylated at 30jC in DMSO with 80% ing, it is not discussed here. Recent reviews are available
aqueous solution of hydrazine monohydrate yielding a [242,243].
polymer with functionalization at position 6 only (DS
0.6). Using such a sample with DP of 96, it was possible
to determine the sequence of substituted repeating units A. Oxidation of Primary Hydroxy Groups
along the polymer chain by means of 13C NMR spectro-
scopic studies [229]. Nitrogen dioxide, gaseous or dissolved in tetrachlorome-
A selective deacetylation of the secondary functions thane, reacts rather specic with cellulose yielding 6-car-
was realized by the hydrolysis of cellulose triacetates using boxy cellulose rst found by Yackel and Kenyon [244].
amines (Table 16) [66,235,236]. The selectively substituted Surface oxidation via this method gives products (degree of
cellulose acetates obtained were applied for the prepara- oxidation 1218%), which are interesting fabrics in wound
tion of cellulose sulfuric acid half-esters with heparinoid healing. They undergo fast biodegradation in serum [245].
activity [237,238]. Besides the industrially applied hydro- If higher degrees of oxidation are required, depolymeriza-
lysis of cellulose triacetate, this process was used for the tion occurs and the products contain nitrogen because of
selective deacetylation of acetyl-1-13C-labeled cellulose the low swelling power of the reaction medium leading to
acetate applying methanol and magnesium acetate tetra- less accessibility of the reaction sites in the polymer matrix.
hydrate. These samples were investigated by means of 2D- An improved procedure of the nitrogen dioxide method
COSY and One Dimensional-Insensitive Nuclei Assign- has been developed using phosphoric acid and sodium
ment by Polarization (1D-INAPT) techniques [239]. nitrite, which generates the oxidizing agent N2O3 [246].
The selective desilylation of TMSC was studied as well Although signicant polymer degradation in the acidic
(see above). medium occurs as well, the products were useful to study
typical polyelectrolytic eects [181,247249]. Important
problems of all reaction systems applying nitrogen oxides
VIII. OXIDATION REACTIONS arise from its toxicological hazards.
deNooy et al. [250252] succeeded in the selective
In principle, cellulose can be oxidized to 6-aldehyde cellu- oxidation of unprotected water-soluble polysaccharides
lose and 6-carboxy cellulose, as well as to 2-ketocellulose, like starch, inulin, and pullulan using sodium hypochlorite
3-ketocellulose, or 2,3-diketocellulose neglecting the trans- in the presence of a catalytic amount of 2,2,6,6-tetra-
formation of the end groups. 2,3-Dialdehyde cellulose may methyl-1-piperidyloxy free radical (TEMPO). This oxida-
be obtained by the well-known glycol cleavage oxidation of tion system was recently extended for the preparation of
574
13
Figure 13 Important paths of cellulose oxidation including C NMR spectra of 6-carboxy cellulose and 2,3-dicarboxy cellulose (DO=degree
of oxidation).
Heinze
6-carboxy cellulose [253], which might be a suitable alter- IX. MODIFICATION OF CELLULOSE
native to the oxidation with nitrogen oxides. In case of the DERIVATIVESSELECTED EXAMPLES
oxidation of cellulose with TEMPO/NaBr/NaClO, the
polymer needs to be converted to an amorphous form by Subsequently modied water-soluble cellulosics have
dissolving it in 9% aqueous sodium alkali lye followed by attracted considerable attention due to their outstanding
regeneration with anhydrous ethanol. Oxidation of native solution properties and potential practical applications.
cellulose is combined with a number of diculties The modication of water-soluble cellulosics, in particular,
[254,255]. The oxidation of hydroxyethyl cellulose with hydroxyethyl cellulose, hydroxypropyl cellulose, and
TEMPO/NaClO shows some selectivity yielding either a methylcellulose, as well as CMC can be done by reacting
sodium carboxymethyl 6-carboxy cellulose where all ac- the remaining OH groups of the AGU with long-chain ali-
cessible primary OH groups are oxidized or a compound phatic compounds carrying reactive functions as epoxide,
modied at the hydroxyethyl moieties only [256,257]. chloride, isocyanate, acid chloride, and acid anhydride
moieties. On the other hand, the functional groups of the
substituents introduced (except methylcellulose) can be
1. Oxidation under Ring Cleavage functionalized. This idea was rst realized by Landoll
The treatment of cellulose with periodic acid and its salts [265] and studied by various other groups [266,267]. The
under aqueous conditions forming 2,3-dialdehyde cellulose solutions of the hydrophobically modied cellulosics pos-
(2,3-DAC) is a very selective conversion. The degree of sess enhanced viscosity eciency, improved shear and salt
oxidation may be controlled by the reaction time and may stability, and shear-thickening rheology compared to the
even be conducted in a quantitative fashion [258,259]. starting polymers [268271].
Synthesis should be carried out in the dark and in the pres- Compared to the hydrocarbon containing water-sol-
ence of radical scavengers like propan-1-ol [260] to avoid uble cellulosics, the corresponding uorocarbon analogs
radically induced depolymerization. A homogeneous peri- show lower critical micelle concentrations. Low amounts
odate oxidation was performed via methylol cellulose ob- of uorocarbon moieties can be detected by means of 19F
tained by dissolution of the polymer in PF/DMSO to NMR spectroscopy. 1,1-Dihydroperuorobutyl, 1,1-dihy-
minimize polymer degradation [261]. The oxidation level droperuorooctyl, and octyl derivatives of HEC were
reaches almost 100% within 10 hr, while the DP remains prepared [272]. The content of the hydrophobic chains is
unchanged. in the range from 0.023 to 1.20 mol% determined by 19F
A stereoregular polycarboxylic acid derivative, 2,3- NMR spectroscopy. The Brookeld viscosity plotted
dicarboxy cellulose (2,3-DCC), is obtained by subsequent against peruorocarbon content goes through a maximum
oxidation of 2,3-DAC with sodium chlorite/hydrogen per- at about 42 mol%. The solutions of the new polymers are
oxide (see Fig. 13) [258,259]. The sodium salt of 2,3-DCC highly pseudoplastic, and the viscosity increases upon
shows typical polyelectrolytic properties such as continu- addition of NaCl indicating a very strong hydrophobic
ous increase in reduced viscosity with decreasing concen- association of the uorocarbon moieties.
tration below 0.2 mg/100 mL water, occulation of Polymeric surfactants from HEC and CMC were
multivalent metal cations, and high calcium-binding ca- obtained by partial hydrophobization with higher fatty
pacity as well as biodegradability [262264]. acid chlorides (C10C18) showing reduced surface tension
Figure 14 Structure of a hydrophobically modied cellulose derivative with pyrene moieties as uorophor for analytical purposes.
576 Heinze
13
Figure 15 C NMR spectrum of CMC (DS = 0.95) dissolved in formic acid yielding CMC formate (DSF = 0.50). (From
Ref. 285.)
of water and good emulsication eciency [273,274]. By N,N-dimethyl-N-dodecylammonium moieties) dissolve to-
etherication of CMC with diethylaminoethyl (DEAE) gether in water at high temperature. Careful cooling of the
chloride in aqueous NaOH solution, the corresponding mixed system aords a solution with a viscosity higher than
CM-DEAE cellulose was obtained [275]. The polyampho- that of the corresponding single component solutions [282].
lyte (DSCM 0.64, DSDEAE 0.19) with an isoelectric point at In addition, it was possible to convert cellulose ethers
pH 3 shows a dierent reduced viscosity dependent on pH including HPMC with aromatic carboxylic acid anhydrides,
value compared to the parent CMC. Further amphoteric e.g., anhydrides of phthalic and pyromellitic acids, to obtain
cellulose products such as CM-O-2-hydroxy-3-(trimethyl- pH-sensitive polymers. They are especially useful for the
amino)propyl cellulose [275] and CM-xanthate [276] were design of pH-detecting systems based on ber optics [283].
prepared homogeneously in aqueous NaOH. Esterication The subsequent reaction of CMC in a nonaqueous
of water-soluble HEC and HPC with methoxytriethoxy- swelling system with ClSO3H or SO3pyridine complex
acetic and related carboxylic acids using the corresponding yields the corresponding CMC sulfuric acid half-esters of
acid chlorides as reactive species leads to novel water- high total degree of functionalization. Even a regioselective
soluble derivatives, which were investigated with regard 2,3-di-O-CMC-6-sulfate may be synthesized [284]. The
to their special interactions with the anionic surfactant nonaqueous derivatizing solvent, formic acid, oers op-
dodecylsulfate by means of rheological ow and oscillatory portunity of homogeneous modication of CMC including
measurements as well as electrolytic conductivity in aque- reaction of the free OH groups or of the formate functions
ous solution [277]. by transesterication (Fig. 15) [285].
Fluorescence labeling technique has proved to be use-
ful for the investigation of structure in solution of hydro-
phobically modied cellulosics [278,279]. The commercial X. STRUCTURE ANALYSIS OF CELLULOSE
polymer JR400 (Amerchol Union Carbide Chemicals and DERIVATIVES
Plastics Comp., U.S.A.) is a hydroxyethyl cellulose modi-
ed with cationic side chains. The uorophore pyrene was The goal of this chapter is a very brief review of modern
attached by ether formation between 4-(1-pyrenyl)-butyl routes for the structure analysis of cellulose and cellulose
tosylate and OH groups of the polymer to study solvolysis derivatives. Methods concerning the characterization of
processes and aggregation in solution (Fig. 14) [280,281]. properties, e.g., solution properties like viscosity, thermo-
The formation of mixed micelles between the polymer and reversible gelation, mesophase formation, and colloidal or
an anionic surfactant was conrmed. polyelectrolytic behavior (in case of ionic derivatives)
It was reported that mixtures of amylose with hydro- which are important from a practical point of view, are
phobically modied, water-soluble cationic ethers (e.g., not discussed here but were subject of a great number of
HPC bearing additional N,N,N-triethylammonium or studies during the last decade which are nicely reviewed
[97,286290] (cf. Chap. 7). Important tools in structure methods for the determination of the distribution of the
analysis of cellulose and cellulose derivatives are NMR CM functions within the AGU (Fig. 17) [72,293,294,299
spectroscopy and chromatography. 303]. A valuable alternative to the spectroscopy in solution
after degradation is the application of 13C-CP/MAS NMR
A. NMR Spectroscopy spectroscopy [186,304].
In addition, the total DS and the distribution of the
Highly resolved 13C NMR spectra of cellulose could be functional groups can be determined from the NMR
obtained from the dissolved polymer. A spectrum acquired spectra of cellulose esters [74,235,305,306]. In case of
of cellulose in DMSO/TBAF is very well resolved and partially substituted CA samples, it is possible to calculate
shows all the ring carbons of the AGU typical for a the partial DS values from 1H NMR spectra after perace-
nonderivatizing solvent (cf. Fig. 2) [62]. tylation of the derivatives with acetyl-d3-chloride [307,308].
The application of NMR techniques was among the Comparable results can be obtained after perpropionyla-
rst attempts for the structure analysis of cellulose ethers tion of cellulose acetates [62,230]. This path is also very
both on the level of the repeating units and the polymer suitable for the characterization of starch derivatives [309].
backbone [74,191,291,292]. A rather comprehensive as- The exact signal assignment has to be done in previous
signment of all ring carbons was achieved for 13C NMR experiments in 2-D NMR spectroscopy (COSY, HMBC).
spectra [293295]. Furthermore, it is possible to peracylate By applying 13C NMR experiments, the determination
the free hydroxy groups of cellulose derivatives and to use and quantication of the distribution of ester functions are
either the methyl protons of 1H NMR spectra [119] (Fig. 16) possible using the signals of the ring carbons or of the
or the carbonyl functions introduced as sensitive probes for carbonyl carbon atoms. The specic 13C NMR techniques,
13
C NMR spectroscopic investigations [296]. which need to be applied (inverse gated-decoupled), result
High eld NMR analysis was carried out on CMC in time-consuming experiments [74,229,296].
showing that 2-D experiments including TOCSY, HMQC, A complex problem is the determination of the distri-
and gated decoupled 13C NMR spectra can be used for the bution of functional groups along the polymer chain,
determination of the partial DS values on the free reactive which was successfully carried out by NMR investigations
sites [297]. Nevertheless, a disadvantage is the high viscos- of CA samples labeled with 13C acetyl groups [239]. Fur-
ity of solutions of cellulose ethers. Therefore, the polymers thermore, NMR spectroscopy after enzymatic treatments
should be partially or completely degraded prior to the of cellulose esters is capable of giving an insight in the
NMR measurements by acidic, ultrasonic, or enzymatic composition of the polymer with regard to the eight
treatment [298]. Thus, 1H and 13C NMR spectroscopy of dierent repeating units [310]. On the other hand, specic
hydrolytically degraded CMC is one of the most ecient NMR techniques, e.g., Two Dimensional-Nuclear Over-
Figure 16 1H NMR spectrum of peracylated 3,6,9-trioxadecanoic acid ester of cellulose (DS = 0.62). The DS and distribution
of function can be determined using the peaks of methyl moieties (H-18). (From Ref. 119.)
578 Heinze
Figure 17 1H NMR spectrum of hydrolyzed CMC (DS = 1.31) including peak assignment. The calculation of DS and partial
DS can be carried out according to Refs. 188 and 349.
hauser Eect Spectroscopy (2D-NOESY)-NMR, can be functionalization pattern is determined after permethyl-
used to study supramolecular structures like helixes and ation and depolymerization [314,315]. It was possible to
domain formation, which may give drawbacks on the form the methyl ether of the OH groups in CA and to
pattern of substitution [311,312]. Recent developments in exchange the acetate functions completely with ethyl
structure characterization of cellulose esters are solid-state groups [316]. The mixed ethers were depolymerized by
NMR spectroscopy and its combination with other ana- reduction and the repeating units were separated by
lytical tools like Raman spectroscopy, as nicely reviewed by GLC. A comparable technique was studied using HPLC
Lowman [313]. for the separation. In this case, the derivatives were meth-
ylated with methyl triate/2,6-di-tert-butylpyridine and
B. Chromatography hydrolyzed by treatment with TFA (Fig. 18). Besides
cellulose esters, this technique can be used for dierent
An important alternative to NMR spectroscopy is the de- unstable derivatives, e.g., trialkylsilyl celluloses [317].
termination of the functionalization pattern by chromato- Cellulose acetate samples have been investigated with
graphic techniques after depolymerization. In case of more regard to the functionalization patterns both within the
or less hydrolytically unstable cellulose esters, the inverse AGU and the polymer chain by permethylation, deacety-
Figure 18 Functionalization of cellulose esters prior to the analyses of the inverse functionalization pattern by chromatography.
(From Ref. 314.)
lative deuteromethylation under alkaline conditions, ran- mentation of the polysaccharide followed by preparative
dom cleavage, remethylation with methyl-d3, FAB-MS SEC. Each SEC fraction is subsequently converted into
analysis, and comparison of the experimental data with monosaccharides by acid hydrolysis, followed by quanti-
those calculated for a random distribution of the acetate tative HPLC. A number of CMC samples with dierent
groups (Fig. 19) [318]. This route was extended to a variety patterns of substitution both within the AGU and along
of cellulose esters, e.g., cellulose sulfuric acid half-esters the polymer chain were investigated [192,223,330,331].
[319,320]. Problems may be both under methylation and Noteworthy is a review dealing with the analysis of sub-
migration or cleavage of the primary functional groups stituent pattern of cellulose ethers [332].
during the reaction yielding incorrect results.
A very useful tool for structure analysis of cellulose
ethers is the complete or partial degradation of the poly-
mer and subsequent analysis of the fragments [321]. Thus XI. CONCLUSIONS AND OUTLOOK
MC was investigated by GLC-MS after complete degra-
From the authors point of view, cellulose chemistryas
dation with TFA [322]. Furthermore, MC and HEC
the dominant route in improving the overall utilization of
samples can be analyzed after degradation by GC and
the polymerhas been able to deliver tools for the struc-
FAB-MS. Comparison of these analytical results with
ture design of cellulose derivatives. Worldwide research
statistic calculations yields information about the distri-
and development activities with regard to new synthesis
bution of the ether groups along the chain [323,324]. This
concepts and paths will lead to a broader use of this
path was also applied for the investigation of the distribu-
renewable resource, and cellulose may become one of the
tion of CM functions along the chain for a number of
key intermediates for the chemical industry in the future.
carboxymethylated polysaccharides. The depolymeriza-
In particular, activation of cellulose by:
tion was carried out with perchloric acid [188,190,325].
An interesting alternative for the analysis of nonionic applying a certain degree of swelling starting from the
cellulose ethers, e.g., MC, HEC, HPC, and HEPC, is the native polymer (without dissolution) and hence a
pyrolysis ammonia chemical ionization mass spectroscopy controlled accessibility of the hydroxy groups
(PyCIMS) [326]. both within the repeating units and along the
Because of the selective depolymerization of nonsub- polymer chains,
stituted or low-substituted parts of cellulose ethers by dissolution without covalent blocking of any of the
cellulases, it was concluded that they cut out highly sub- OH groups within the AGU (physical activation),
stituted regions. Therefore, this analysis yields information dissolution after partial functionalization without or
about the length of these regions, i.e., about the distribu- with isolation of the reactive intermediate formed
tion of functional groups along the polymer chains including the subsequent modication of the iso-
[327,328]. In earlier studies, the degradation was followed lated intermediates in common solvents, and
by viscosity and measurements of the reducing sugars. In creation of reactive domains exploiting phase separa-
case of HEC, the fragments were analyzed by 13C NMR tion phenomena leading to reactive microstruc-
spectroscopy [329]. An ecient method was developed for tures, starting from a swollen or dissolved
CMC, which includes an endoglucanase-catalyzed frag- polymer
will open up new avenues for the design of products.
Moreover, the control of the reactivities of low-molecular
reagents especially in situ formation of reactive species and
application of new catalysts, promoters, and impeller
agents are important tools for cellulose chemistry. Last
but not least, chemical activation of the hydroxy groups for
direct (in situ) or subsequent substituent replacement by
nucleophilic attack is a suitable path to new cellulose-based
materials.
The synthesis of cellulose derivatives by homogeneous
phase reaction or starting at least from the dissolved state
is appropriate to control the distribution of functional
groups, especially when an even distribution of the func-
tions along the polymer chains is guaranteed. Moreover,
applying a specic molar ratio reagent to AGU can control
the total DS values and the homogeneous synthesis creates
more options to introduce novel substituents including
Figure 19 Gas chromatogram of the 4-O-acetyl-1,5-anhy- bulky and really exotic functions. At present, functional
dro- (a) and 5-O-acetyl-1,4-anhydro- (b) O-methyl-O-pentyl- groups of interest are chromophores and uorophores,
D-glucitol derivatives obtained from cellulose acetate by redox active groups, substituents with special magnetic or
methylation, acetylpentyl exchange, reductive cleavage, and optical properties, biological activity, and cationic func-
acetylation. (From Ref. 315.) tions. Very interesting and new cellulose derivatives are
580 Heinze
products with more than one functional group (double and tain pure and uniform pulps suitable for cellulose deriva-
triple substitution) including combinations of hydrophilic tization also in the commercial scale, as well as cellulose of
and lipophilic ones (amphiphiles), reversible functionaliza- very uniform chain length (DP) are important goals of
tion with special supramolecular structures obtained after research and development and may contribute to the
cellulose regeneration, products of complete functionaliza- improvement of the properties of cellulose derivatives.
tion of all hydroxy groups, and, on the other hand, polymer All structural levels of the polymer, i.e., the molecular,
derivatives of comparatively low DS. An enormous supramolecular, and morphological levels, have to be
amount of work is needed to explore and exploit the full considered concerning the reactivity of cellulose.
benets of homogeneous phase reactions of polysaccha- The outstanding and unique structure of cellulose
rides in the future. makes polymers available by functionalization reactions,
The preparation of the fairly exotic (and hence costly) which are an interesting and valuable class of polymeric
cellulose products can be justied only if these highly materials whose attractiveness is expected to rise in the
engineered derivatives are useful in molecular recognition future.
concepts as self-organizing supramolecular systems, in
nanostructures, in environmentally responsive (smart) ABBREVIATIONS
materials, in sensors, and in chiral templates as well as
due to their biological eects. Application seems to be AEC anion exchange chromatography
possible also in elds where they substantially inuence AGU anhydroglucose unit
material properties in low concentrations in mixtures or CA cellulose acetate
blends with biopolymers including common cellulose CEC carboxyethyl cellulose
derivatives and synthetic polymers. This remains true for Cell-Br bromodeoxycellulose
the modication of the polymer by nucleophilic displace- Cell-I iododeoxycellulose
ment reactions. Chemical functionalization reactions of CF cellulose formate
polysaccharides by reaction at the C atoms are a very CMC carboxymethylcellulose
promising path within the eorts to design the tailored CTFA cellulose triuoroacetate
molecular structure of the products. Cuam cuprammonium hydroxide
Most of the synthesis methods, paths, and concepts Cuen cupriethylene diamine hydroxide
described can be used to control the distribution of the DAC 2,3-dialdehyde cellulose
functional groups within the AGU and along the polymer DCC 2,3-dicarboxy cellulose
chain to a certain extent. In this context, i.e., to modify a DCC N,N-dicyclohexylcarbodiimide
preset site of the AGU, it is important to evaluate the whole DEA diethylamine
reaction system. Bulky moieties can be selectively intro- DEAE diethylaminoethyl
duced both under homogeneous and heterogeneous reac- DMA N,N-dimethylacetamide
tion conditions, while for functions with lower steric DMAP N,N-dimethylaminopyridine
hindrance, a strong inuence of the reactivity of the low- DMF N,N-dimethylformamide
molecular reagent and the state of activation (degree of DMI 1,3-dimethyl-2-imidazolidinone
swelling and state of dissolution) of the polymer on the DMSO dimethyl sulfoxide
regioselectivity may appear. DMTA dynamic mechanical thermal analysis
Protecting group techniques applied on cellulose have DP degree of polymerization
some signicant drawbacks. Thus, it is not possible to carry DS degree of substitution
out the introduction of the protecting group as well as the DSC dierential scanning calorimetry
deprotection and their complete removal from the product Glc glucose
in a strictly polymer-analogous manner. These techniques HEC hydroxyethyl cellulose
are still very useful in designing uniform polymers neces- HMDS hexamethylene disilazane
sary for understanding the inuence of the pattern of HPC hydroxypropyl cellulose
functionalization on properties. However, from the present HPLC high-performance liquid chromatography
knowledge, it seems possible to apply protecting group HPMC hydroxypropylmethyl cellulose
techniques in lab- or small-scale synthesis only. MC methylcellulose
Despite the discussed progress in the eld of cellulose Ni(tren) nickel tri(2-aminoethyl)amine
research, a large number of challenges remain to take full NMNO N-methylmorpholine-N-oxide
advantage of celluloses great assets. In addition to the NMP N-methyl-2-pyrrolidone
products and synthesis paths described, cellulosics with PF paraformaldehyde
ionic functions should be mentioned. It is still a matter of PP 4-pyrrolidinopyridine
research to obtain regioselectively oxidized products, e.g., SEC size exclusion chromatography
6-carboxy cellulose, with high content of carboxy groups as SN nucleophilic displacement
well as cationic cellulosics with high DS and controlled TBAF tetrabutylammonium uoride trihydrate
distribution of the functions. TEA triethylamine
Regarding the starting material cellulose, high reactive TEMPO 2,2,6,6-tetramethyl-1-piperidyloxy
samples (never-dried pulp), new pulping processes to ob- TFA triuoroacetic acid
TFAA triuoroacetic acid anhydride 14. Gilbert, R.D.; Kadla, J.F. PolysaccharidesCellulose. In
Tg glass transition temperature Biopolymers from Renewable Resources; Kaplan, D.L., Ed.;
Springer Verlag: Berlin, 1998; 47 pp.
THF tetrahydrofuran
15. Hearle, J.W.S. Fringed-bril theory of structure in
TMSC trimethylsilyl cellulose crystalline polymers. J. Polym. Sci. 1958, 28, 432.
Tos-Cl p-toluenesulfonyl chloride 16. Lai, Y.-Z. Reactivity and accessibility of cellulose, hemi-
cellulose, and lignins. In Chemical Modication of Ligno-
cellulosic Materials; Hon, D.N.-S., Eds.; Marcel Dekker:
New York, 1996; 35 pp.
ACKNOWLEDGMENTS 17. Philipp, B. Organic solvents for cellulose as a biodegrad-
able polymer and their applicability for cellulose spinning
The author is indebted to Dr. U. Heinze, Dr. T. Liebert, and derivatization. J. Macromol. Sci. A Pure Appl. Chem.
Dr. A. Koschella, and C. Grote for helpful discussions 1993, 30, 703.
and technical assistance in preparing the manuscript. 18. Heinze, T.; Liebert, T. Organic solvents and sophisticated
derivatives of cellulosepromising tools in cellulose
chemistry. Cellul. Chem. Technol. 1998, 32, 3.
19. Bogan, R.T.; Brewer, R.J. Cellulose esters, organic, survey.
REFERENCES In Encyclopedia of Polymer Science and Engineering;
Mark, H.F., Bikales, N.M., Overberger, C.G., Menges, G.,
1. Balser, K.; Hoppe, L.; Eicher, T.; Wendel, M.; Astheimer, Kroschwitz, J.I., Eds.; John Wiley & Sons: New York,
A.-J. Cellulose esters. In Ullmanns Encyclopedia of 1985; 158 pp.
Industrial Chemistry, 5th ed.; Gerhartz, W., Yamamoto, 20. Serad, G.A. Cellulose esters, organic, bres. In Encyclopedia
Y.S., Champbell, F.T., Pfeerkorn, R., Rounsaville, J.F., of Polymer Science and Engineering; Mark, H.F., Bikales,
Eds.; VCH, Weinheim: New York, 1986; 419 pp. N.M., Overberger, C.G., Menges, G., Kroschwitz, J.I.,
2. Balser, K.; Hoppe, L.; Eicher, T.; Wendel, M.; Astheimer, Eds.; John Wiley & Sons: New York, 1985; 200 pp.
A.-J. Cellulose ethers. In Ullmanns Encyclopedia of 21. Muller, F.; Leuschke, C. Organic cellulose esters, thermo-
Industrial Chemistry, 5th Ed.; Gerhartz, W., Yamamoto, plastic molding compounds. In Engineering Thermoplastics,
Y.S., Champbell, F.T., Pfeerkorn, R., Rounsaville, J.F., Polycarbonates, Polyacetates, Polyesters, Cellulose Esters;
Eds.; VCH, Weinheim: New York, 1986; 461 pp. Bottenbruch, L., Ed.; Hanser Publ.: Munich, 1996; 385 pp.
3. Krassig, H.A. Cellulosestructure, accessibility, and 22. Edgar, K.J.; Buchanan, C.M.; Debenham, J.S.; Rundquist,
reactivity. Gordon & Breach: Amsterdam, 1993. P.A.; Seiler, B.D.; Shelton, M.C.; Tindall, D. Advances in
4. Tarchevsky, J.A.; Marchenko, G.N. Cellulose: Biosyn- cellulose ester performance and application. Prog. Polym.
thesis and structure; Springer Verlag: Heidelberg, 1991. Sci. 2001, 26, 1605.
5. VanderHart, D.L.; Atalla, R.H. Studies of microstructure 23. Dahlgren, L. Cellulose ethersproperties and applica-
in native celluloses using solid-state 13C NMR. Macro- tions. In Wood and Cellulosics; Kennedy, J.F., Phillips,
molecules 1984, 17, 1465. G.O., Williams, G.A., Eds.; Horwood Publ.: Chichester,
6. Isogai, A.; Usuda, M.; Kato, T.; Uryu, T.; Atalla, R.H. 1987; 427 pp.
Solid-state CP/MAS 13C NMR study of cellulose poly- 24. Nicholson, M.D.; Merritt, F.M. Cellulose ethers. In
morphs. Macromolecules 1989, 22, 3168. Cellulose Chemistry and Its Application; Nevell, T.,
7. Sugiyama, J.; Okano, T.; Yamamoto, H.; Horii, F. Zeronian, P., Haig, S., Eds.; Horwood Publ.: Chichester,
Transformation of valonia cellulose crystals by an alkaline 1989; 363 pp.
hydrothermal treatment. Macromolecules 1990, 23, 3196. 25. Feddersen, R.L.; Thorp, S.N. Sodium carboxymethylcel-
8. Yamamoto, H.; Horii, F.; Odani, H. Structural changes of lulose. In Industrial Gums, Polysaccharides and Their
native cellulose crystals induced by annealing in aqueous Derivatives; Whistler, R.L., BeMiller, J.N., Eds.; Academic
alkaline and acidic solutions at high temperature. Macro- Press: San Diego, 1993; 537 pp.
molecules 1989, 22, 4130. 26. Olaru, N.; Olaru, L.; Stoleriu, A.; Timpu, D. Carboxyme-
9. Hon, D.N.-S. Functional polymers: A new dimensional thylcellulose synthesis in organic media containing ethanol
creativity in lignocellulosic chemistry. In Chemical Mod- and/or acetone. J. Appl. Polym. Sci. 1998, 67, 481.
ication of Lignocellulosic Materials; Hon, D.N.-S., Ed.; 27. Bikales, N.M. Cyanoethylcellulose. Macromol. Synth.
Marcel Dekker: New York, 1996; 1 pp. 1974, 5, 35.
10. Nakatsubo, F.; Kamitakahara, H.; Hori, M. Cationic ring- 28. Sefain, M.Z.; Fadl, M.H.; Elwakil, N.A.; Naoum, M.M.
opening polymerisation of 3,6-di-O-benzyl-alpha-D-glu- Kinetics of heterogeneous cyanoethylation of cellulose.
cose 1,2,4-orthopivalate and the rst chemical synthesis of Polym. Int. 1993, 32, 251
cellulose. J. Am. Chem. Soc. 1996, 118, 1677. 29. Philipp, B.; Schleicher, H.; Wagenknecht, W. Time-
11. Nishimura, T.; Takano, T.; Nakatsubo, F.; Murakami, K. dependence of the swelling of regenerated cellulose bers
Synthetic studies of cellulose. X. Selection of suitable and lms in organic liquids. Faserforsch. Text.Tech. 1973,
starting materials for the convergent synthesis of cello- 24, 106.
oligosaccharides. Mokuzai Gakkaishi 1993, 39, 40. 30. Zeronian, S.H. Intracrystalline swelling of cellulose. In
12. Kobayashi, S.; Kashiwa, K.; Kawasaki, T.; Shoda, S. Cellul. Chem. Its Appl.; Nevell, P.T., Zeronian, S.H., Eds.;
Novel method for polysaccharide synthesis using an Ellis Horwood: Chichester, 1985; 159 pp.
enzyme: The rst in vitro synthesis of cellulose via a 31. Klemm, D.; Philipp, B.; Heinze, T.; Heinze, U.; Wagen-
nonbiosynthetic path utilizing cellulase as catalyst. J. Am. knecht, W. Comprehensive Cellulose Chemistry, Fundamen-
Chem. Soc. 1991, 113, 3079. tals and Analytical Methods; Wiley-VCH: Weinheim, 1998;
13. Kobayashi, S.; Shoda, S.; Lee, J.H.; Okuda, K.; Brown, Vol. 1, 43.
R.M., Jr.; Kuga, S. Direct visualization of synthetic 32. Philipp, B. Organic solvents for cellulose. Polym. News
cellulose formation via enzymatic polymerization using 1990, 15, 170.
transmission electron-microscopy. Macromol. Chem. 33. Hoenich, N.A.; Wondin, C.; Stamp, S.; Roberts, S.J.;
Phys. 1994, 195, 1319. Turnbull, J. Synthetically modied cellulose: An alterna-
582 Heinze
tive to synthetic membranes for use in hemodialysis. 53. Striegel, A. Theory and applications of DMAc/LiCl in the
Biomaterials 1997, 18, 1299; and references cited therein. analysis of polysaccharides. Carbohydr. Polym. 1997, 34,
34. Isogai, A.; Atalla, R.H. Dissolution of cellulose in aqueous 267.
NaOH solutions. Cellulose 1998, 5, 309. 54. Spange, S.; Reuter, A.; Vilsmeier, E.; Keutel, D.; Heinze,
35. Burger, J.; Kettenbach, G.; Klufers, P. Coordination T.; Linert, W. Application of solvatochromic probe
equilibria in transition metal based cellulose solvents. molecules on the cellulose solvent N,N-dimethylaceta-
Macromol. Symp. 1995, 99, 113. mide/LiCl. J. Polym. Sci. A Polym. Chem. 1998, 36, 1945;
36. Liebert, T.; Heinze, T. Synthesis path versus distribution of and references cited therein.
functional groups in cellulose ethers. Macromol. Symp. 55. Takaragi, A.; Minoda, M.; Miyamoto, T.; Liu, H.Q.;
1998, 130, 271. Zhang, L.N. Reaction characteristics of cellulose in LiCl/
37. Heinze, T.; Liebert, T.; Klufers, P.; Meister, F. Carboxy- 1,3-dimethyl-2-imidazolidinone solvent system. Cellulose
methylation of cellulose in unconventional media. Cellu- 1999, 6, 93.
lose 1999, 6, 153. 56. Hagiwara, I.; Shiraishi, N.; Yokota, T. Homogeneous tri-
38. Heinze, T.; Pfeier, K. Studies on synthesis and character- tylation of cellulose in a sulfur dioxidediethylamine
ization of carboxymethyl cellulose. Angew. Makromol. dimethylsulfoxide medium. J. Wood Chem. Technol. 1981,
Chem. 1999, 266, 37. 1, 93.
39. Fischer, S.; Voigt, W.; Fischer, K. The behavior of cellulose 57. Isogai, A.; Ishizu, A.; Nakano, J. Preparation of tri-O-
in hydrate melts of the composition LiX nH2O. Cellulose benzylcellulose by the use of non-aqueous cellulose
1999, 6, 213. solvents. J. Appl. Polym. Sci. 1984, 29, 2097.
40. Fischer, S.; Leipner, H.; Brendler, E.; Voigt, W.; Fischer, 58. Isogai, A.; Ishizu, A.; Nakano, J. Preparation of tri-O-
K. Molten inorganic salt hydrates as cellulose solvents. In substituted cellulose ethers by the use of non-aqueous
Polysaccharide Applications, Cosmetics and Pharmaceut- cellulose solvents. J. Appl. Polym. Sci. 1984, 29, 3873.
icals; El-Nokaly, M.A., Soini, H.A., Eds.; ACS Sympo- 59. Isogai, A.; Ishizu, A.; Nakano, J. Preparation of tri-O-
siums Series 737; American Chemical Society: Washington, alkylcelluloses by the use of a non-aqueous cellulose
DC, 1999; 143 pp. solvent and their physical characteristics. J. Appl. Polym.
41. Leipner, H.; Fischer, S.; Brendler, E.; Voigt, W. Structural Sci. 1986, 31, 341.
changes of cellulose dissolved in molten salt hydrates. 60. Isogai, A.; Ishizu, A.; Nakano, J. Dissolution mechanism
Macromol. Chem. Phys. 2000, 201, 2041. of cellulose in SO2aminedimethylsulfoxide. J. Appl.
42. Fischer, S.; Thummler, K.; Pfeier, K.; Liebert, T.; Heinze, Polym. Sci. 1987, 33, 1283.
T. Evaluation of molten inorganic salt hydrates as reaction 61. Michael, M.; Ibbett, R.N.; Howarth, O.P. Interaction of
medium for the derivatization of cellulose. Cellulose 2002, cellulose with amine oxide solvents. Cellulose 2000, 7, 21.
9, 293. 62. Heinze, T.; Dicke, R.; Koschella, A.; Kull, A.H.; Klohr, E.-
43. Swatloski, R.P.; Spear, S.K.; Holbrey, J.D.; Rogers, R.D. A.; Koch, W. Eective preparation of cellulose derivatives
Dissolution of cellulose with ionic liquids. J. Am. Chem. in a new simple cellulose solvent. Macromol. Chem. Phys.
Soc. 2002, 124, 4974. 2000, 201, 627.
44. McCormick, C.L.; Dawsey, T.R. Preparation of cellulose 63. Ciacco, G.T.; Liebert, T.; Frollini, E.; Heinze, T. Applica-
derivatives via ring-opening reactions with cyclic reagents tion of the solvent dimethylsulfoxide/tetrabutylammonium
in lithium chloride/N,N-dimethylacetamide. Macromole- uoride trihydrate as reaction medium for the acylation of
cules 1990, 23, 3606. sisal cellulose. Cellulose 2003, 10, 125.
45. Rahn, K.; Diamantoglou, M.; Berghmans, H.; Heinze, T. 64. Philipp, B.; Nehls, I.; Wagenknecht, W. 13C NMR spec-
Homogeneous synthesis of cellulose p-toluenesulfonates in troscopic studies of the homogeneous sulfation of cellulose
N,N-dimethylacetamide/LiCl solvent system. Angew. and xylan in N2O4-DMF system. Carbohydr. Res. 1987,
Makromol. Chem. 1996, 238, 143. 164, 107.
46. Nehls, I.; Wagenknecht, W.; Philipp, B. 13C NMR spec- 65. Wagenknecht, W.; Nehls, I.; Philipp, B. Studies on the
troscopic studies of cellulose in various solvent systems. regioselectivity of cellulose sulfation in an N2O4-N,N-
Cellul. Chem. Technol. 1995, 29, 243. dimethylformamide cellulosesystem.Carbohydr.Res.1993,
47. El-Kafrawy, A. Investigation of the cellulose/lithium 240, 245.
chloride/dimethylacetamide and cellulose/LiCl/N-methyl- 66. Klemm, D.; Heinze, T.; Philipp, B.; Wagenknecht, W. New
2-pyrrolidone solutions by 13C NMR spectroscopy. J. approaches to advanced polymers by selective cellulose
Appl. Polym. Sci. 1982, 27, 2435. functionalization. Acta Polym. 1997, 48, 277; and refer-
48. Striegel, A.M.; Timpa, J.D.; Biotrowiak, P.; Cole, R.B. ences cited therein.
Multiple neutral alkali halide attachments onto oligosac- 67. Johnson, D.C.; Nicholson, M.D.; Haigh, F.G. Dimethyl-
charides in electrospray ionization mass spectrometry. Int. sulfoxide/paraformaldehyde: A non-degrading solvent for
J. Mass Spectrom. Ion Process. 1997, 162, 45. cellulose. J. Appl. Polym. Sci., Appl. Polym. Symp. 1976, 28,
49. Striegel, A.M.; Timpa, J.D. Molecular characterization of 931.
polysaccharides dissolved in N,N-dimethylacetamide-lith- 68. Baker, T.J.; Schroeder, L.R.; Johnson, D.C. Formation of
ium chloride by gel-permeation chromatography. Carbo- methylol cellulose and its dissolution in polar aprotic
hydr. Res. 1995, 267, 271. solvents. Cellul. Chem. Technol. 1981, 14, 311.
50. Silva, A.A.; Laver, M.L. Molecular weight character- 69. Saikia, C.N.; Dutta, N.N.; Borah, M. Thermal behavior of
ization of wood pulp cellulose: Dissolution and size ex- some homogeneously esteried products of high a-cellu-
clusion chromatographic analysis. Tappi J. 1997, 80, 173. lose pulps of fast growing plant species. Thermochim. Acta
51. Hasegawa, M.; Isogai, A.; Onabe, F. Size-exclusion 1993, 219, 191.
chromatography of cellulose and chitin using lithium 70. Schnabelrauch, M.; Vogt, S.; Klemm, D.; Nehls, I.;
chloride-N,N-dimethylacetamide as a mobile phase. J. Philipp, B. Readily hydrolysable cellulose esters as
Chromatogr. 1993, 635, 334. intermediates for the regioselective derivatization of
52. Sjoholm, E.; Gustafsson, K.; Eriksson, B.; Brown, W.; cellulose. Angew. Makromol. Chem. 1992, 198, 155.
Colmsjo, A. Aggregation of cellulose in lithium chloride/ 71. Philipp, B.; Wagenknecht, W.; Nehls, I.; Ludwig, J.;
N,N-dimethylacetamide. Carbohydr. Polym. 2000, 41, 153. Schnabelrauch, M.; Rim, K.H.; Klemm, D. Untersuchun-
gen zur Sulfatierung von Celluloseformiat im Vergleich zu procedure for the preparation of cellulose esters with
Celluloseacetat unter homogenen Reaktionsbedingungen. aromatic carboxylic acids. Chem. Pap. 1996, 50, 365.
Cellul. Chem. Technol. 1990, 24, 667. 91. Arai, K.; Sano, S.; Satoh, H. Preparation of cellulose
72. Liebert, T.; Klemm, D.; Heinze, T. Synthesis and charac- stilbene-4-carboxylate and its application to thin-layer
terization of organo-soluble triuoroacetates and formates chromatography. J. Mater. Chem. 1992, 2, 1257.
of cellulose. J.M.S. Pure Appl. Chem. A 1996, 33, 613. 92. Arai, K.; Satoh, H. Liquid crystalline phase-formation
73. Hasegawa, M.; Isogai, A.; Onabe, F.; Usuda, M. Dissolv- behavior of cellulose cinnamate. J. Appl. Polym. Sci. 1992,
ing states of cellulose and chitosan in triuoroacetic acid. J. 45, 387.
Appl. Polym. Sci. 1992, 45, 1857. 93. Arai, K.; Sano, S. Preparation of cellulose 2-methylstil-
74. Nehls, I.; Wagenknecht, W.; Philipp, B.; Stscherbina, D. bene-5-carboxylate and photoregulation of its properties.
Characterization of cellulose and cellulose derivatives in J. Mater. Chem. 1994, 4, 275.
solution by high resolution 13C NMR spectroscopy. Prog. 94. Arai, K.; Udagawa, H. Inuence of DP of cellulose
Polym. Sci. 1994, 19, 29. backbone on liquid crystalline phase of cellulose p-phenyl-
75. Hawkinson, D.E.; Kohout, E.; Fornes, R.E.; Gilbert, R.G. azobenzoate. Seni Gakkaishi 1990, 46, 491.
Some further observations on the system cellulose/triuoro- 95. McCormick, C.L.; Lichatowich, D.K. Homogeneous
acetic acid/CH2Cl2 and cellulose triacetate/TFA/CH2Cl2. solution reactions of cellulose, chitin, and other polysac-
J. Polym. Sci., Part B, Polym. Phys. 1991, 29, 1599. charides to produce controlled-activity pesticide systems. J.
76. Liebert, T.; Schnabelrauch, M.; Klemm, D.; Erler, U. Polym. Sci. Polym. Lett. Ed. 1979, 17, 479.
Readily hydrolysable cellulose esters as intermediates for 96. McCormick, C.L.; Shen, T.S. Cellulose dissolution and
the regioselective derivatization of celluloseII: Soluble derivatization in lithium chloride/N,N-dimethylacetamide
highly substituted cellulose triuoroacetates. Cellulose solutions. In Macromolecular Solutions, SolventProperty
1994, 1, 249. Relationships in Polymers; Symor, R.B., Stahl, G.A., Eds.;
77. Liebert, T. Celluloseester als hydrolytisch instabile Inter- Pergamon Press: New York, 1982; 101 pp.
mediate und als pH-sensitive Trgermaterialien, Ph.D. The- 97. Guo, J.X.; Gray, D.G. Lyotropic cellulosic liquid crystals.
sis, University of Jena, Germany, 1995.. In Cellulosic Polymers, Blends and Composites; Gilbert,
78. Klemm, D.; Schmauder, H.-P.; Heinze, T. Cellulose. In R.D., Ed.; Hanser Publ.: Munich, 1994; 25 pp.
Biopolymers: Biology, Chemistry, Biotechnology, Applica- 98. Terbojevich, M.; Cosani, A.; Focher, B.; Gastaldi, G.; Wu,
tions; Vandamme, E., DeBaets, S., Steinbchel, A., Eds.; W.; Marsano, E.; Conio, G. Solution properties and
Wiley-VCH: Weinheim, 2002; 275 pp. mesophase formation of 4-phenyl-benzoyl cellulose. Cellu-
79. Klemm, D.; Philipp, B.; Heinze, T.; Heinze, U.; Wagen- lose 1999, 6, 71.
knecht, W. Comprehensive Cellulose Chemistry, Funda- 99. Pawlowski, W.P.; Sanakar, S.S.; Gilbert, R.D. Synthe-
mentals and Analytical Methods; Wiley-VCH: Weinheim, sis and solid state 13C NMR studies of some cellulose
1998; Vol. 1, 130 pp. derivatives. J. Polym. Sci. A Polym. Chem. 1987, 25, 3355.
80. Jagur-Grodzinski, J. Heterogeneous modication of poly- 100. Pawlowski, W.P.; Gilbert, R.D.; Fornes, R.; Purrington, S.
mers; J. Wiley & Sons: Chichester, 1997; 64 pp. The liquid-crystalline properties of selected cellulose
81. Morooka, T.; Norimoto, M.; Yamada, T.; Shiraishi, N. derivatives. J. Polym. Sci., B, Polym. Phys. 1988, 26, 1101.
Dielectric properties of cellulose acylates. J. Appl. Polym. 101. Grbner, D.; Liebert, T.; Heinze, T. Synthesis of novel
Sci. 1984, 29, 3981. adamantoyl cellulose using dierently activated carboxylic
82. Iwata, T.; Fukushima, A.; Okamura, K.; Azuma, J. DSC acid derivatives. Cellulose 2002, 9, 193.
study on regioselectively substituted cellulose heteroesters. 102. Hon, D.N.-S.; Yan, H. Cellulose furoate, I. Synthesis in
J. Appl. Polym. Sci. 1997, 65, 1511. homogeneous and heterogeneous systems. J. Appl. Polym.
83. Edgar, K.J.; Pecorini, T.J.; Glasser, W.G. Long-chain Sci. 2001, 81, 2649.
cellulose esters: Preparation, properties, and perspective. In 103. Hon, D.N.-S.; Yan, H. Cellulose furoate, II. Character-
Cellulose Derivatives: Modication, Characterization and ization. J. Appl. Polym. Sci. 2001, 82, 243.
Nanostructures; Heinze, T. Glasser, W.G. ACS Symp. 104. Yan, H.; Hon, D.N.-S. Cellulose furoate, III. Properties
Series 688, Washington, DC, 1998; 38 pp. and applications. J. Appl. Polym. Sci. 2001, 82, 253.
84. Wang, P.; Tao, B.Y. Characterization of plasticized and 105. Vaca-Garcia, C.; Thiebaud, S.; Borredon, M.W.; Gozze-
mixed long-chain fatty cellulose esters. In Biopolymers, lino, G. Cellulose esterication with fatty acids and acetic
Utilizing Natures Advanced Materials; Imam, S.H., anhydride in lithium chloride/N,N-dimethylacetamide me-
Greene, R.V., Zaidi, B.R., Eds.; ACS Symp. Series 723, dium. JAOCS 1998, 75, 315.
Washington, DC, 1999; 77 pp. 106. Diamantoglou, M.; Kundinger, E.F. Derivatisation of
85. Shimizu, Y.; Hayashi, J. A new method for cellulose ace- cellulose in homogeneous reaction. In Cellulose and
tylation with acetic acid. Seni Gakkaishi 1988, 44, 451. Cellulose Derivatives, Physicochemical Aspects and Industri-
86. Shimizu, Y.; Nakayama, A.; Hayashi, J. Acylation of al Applications; Kennedy, J.F., Phillips, G.O., Williams,
cellulose with carboxylate salts. Cellul. Chem. Technol. P.A., Picullel, L., Eds.; Woodhead Publisher: Cambridge,
1991, 25, 275. 1995; 111 pp.
87. Brewster, J.H.; Ciotti, C.J., Jr. Dehydrations with aromatic 107. Marsano, E.; De Paz, L.; Tambuscio, E.; Bianchi, E. Cel-
sulfonyl halides in pyridine. A convenient method for the lulose methacrylate: synthesis and liquid crystalline behav-
preparation of esters. J. Am. Chem. Soc. 1955, 77, 6214. ior of solutions and gels. Polymer 1998, 39, 4289.
88. Shimizu, J.; Nakayama, A.; Hayashi, J. Preparation of 108. Diamantoglou, M.; Kuhne, H. Reaktionen von Cellulose
cellulose esters with aromatic carboxylic acids. Seni in homogener Losung. Papier (Darmst.) 1988, 42, 690.
Gakkaishi 1993, 49, 352. 109. Regiani, A.M.; Frollini, E.; Marson, G.A.; Arantes, G.M.;
89. Shimizu, J.; Nakayama, A.; Hayashi, J. Preparation of El Seoud, O.A. Some aspects of acylation of cellulose under
cellulose esters with aromatic carboxylic acids. In Cellulo- homogeneous solution conditions. J. Polym. Sci. A Polym.
sics, Chemical, Biochemical, and Material Aspects; Ken- Chem. 1999, 37, 1357.
nedy, J.F., Phillips, G.O., Williams, P.A., Eds.; Ellis 110. Marson, G.A.; El Seoud, O.A. A novel, ecient procedure
Horwood: New York, 1993; 369 pp. for acylation of cellulose under homogeneous solution
90. Talaba, P.; Srokova, I.; Hodul, P.; Ebringerova, A. New conditions. J. Appl. Polym. Sci. 1999, 74, 1355.
584 Heinze
111. El Seoud, O.A.; Marson, G.A.; Giacco, G.T.; Frollini, E. solutions of cellulose. J. Polym. Sci. Polym. Chem. Ed.
An ecient, one-pot acylation of cellulose under homoge- 1978, 16, 1.
neous reaction conditions. Macromol. Chem. Phys. 2000, 131. Leoni, R.; Baldini, A. The acetylation of cellulose in
201, 882. dimethylacetamideparaformaldehyde system. Carbo-
112. Edgar, K.J.; Arnold, K.M.; Blount, W.W.; Lawniczak, hydr. Polym. 1982, 2, 298.
J.E.; Lowman, D.W. Synthesis and properties of cellulose 132. Mansson, M.; Westfeld, L. Homogeneous acetylation of
acetoacetates. Macromolecules 1995, 28, 4122. cellulose via the nitrite ester. Cellul. Chem. Technol. 1980,
113. Witzemann, J.S.; Nottingham, W.D.; Rector, F.D. Com- 14, 13.
parison of methods for the preparation of acetoacetylated 133. Shimizu, Y.; Nakayama, A.; Hayashi, J. Preparation of
coating resins. J. Coat. Technol. 1990, 62, 101. cellulose esters with aromatic carboxylic acids. Seni
114. Witzemann, J.S.; Nottingham, W.D. Transacetoacetyla- Gakkaishi 1993, 49, 352.
tion with tert-butylacetoacetate: Synthetic applications. J. 134. Emelyanov, Y.G.; Grinshpan, D.D.; Kaputskii, E.F.
Org. Chem. 1991, 56, 1713. Homogeneous synthesis of cellulose esters in nonaqueous
115. Sealey, J.E.; Samaranayake, G.; Todd, J.G.; Glasser, W.G. cellulose solventsIII. Low substituted cellulose triuor-
Novel cellulose derivativesIV. Preparation and thermal oacetate acetylation in triuoroacetic acid and dimethyl-
analysis of waxy esters of cellulose. J. Polym. Sci. B Polym. formamide. Khim. Drev. 1988, 1, 23.
Phys. 1996, 34, 1613. 135. Salin, B.N.; Cemeris, M.; Mirnov, D.P.; Zatsepin, A.G.
116. Sealey, J.E.; Frazier, C.E.; Samaranayake, G.; Glasser, Triuoroacetic acid as solvent for the synthesis of cellulose
W.G. Novel cellulose derivativesV. Synthesis and estersI. Synthesis of triesters of cellulose and aliphatic
thermal properties of esters with triuoroethoxy acetic carboxylic acids. Khim. Drev. 1991, 3, 65.
acid. J. Polym. Sci. B Polym. Phys. 2000, 38, 486. 136. Salin, B.N.; Cemeris, M.; Malekova, O.L. Triuoroacetic
117. Glasser, W.G.; Becker, U.; Todd, J.G. Novel cellulose acid as solvent for the synthesis of cellulose estersI.
derivativesVI. Preparation and thermal analysis of two Synthesis of mixed cellulose esters. Khim. Drev. 1993, 5, 3.
novel cellulose esters with uorine-containing substituents. 137. Schempp, W.; Krause, T.; Seifried, U.; Koura, A.
Carbohydr. Polym. 2000, 42, 393. Herstellung hochsubstituierter Trimethylsilylcellulosen
118. Koschella, A.; Haucke, G.; Heinze, T. New uorescence im System Dimethylacetamid/Lithiumchlorid. Papier
active cellulosics prepared by a convenient acylation (Darmst.) 1984, 38, 607.
reaction. Polym. Bull. 1997, 39, 597. 138. Klemm, D.; Schnabelrauch, M.; Stein, A.; Philipp, B.;
119. Heinze, T.; Schaller, J. New water-soluble cellulose esters Wagenknecht, W.; Nehls, I. Neuere Ergebnisse zur
synthesized by an eective acylation procedure. Macromol. homogenen Veresterung von Cellulose ber lsliche Zwi-
Chem. Phys. 2000, 201, 1214. schenprodukte. Papier (Darmst.) 1990, 44, 624.
120. Samaranayake, G.; Glasser, W.G. Cellulose derivatives 139. Mormann, W.; Demeter, J. Silylation of cellulose with
with low DSI. A novel acylation system. Carbohydr. hexamethyldisilazane in liquid ammoniaFirst examples
Polym. 1993, 22, 1. of completely trimethylsilylated cellulose. Macromolecules
121. Samaranayake, G.; Glasser, W.G. Cellulose derivatives 1999, 32, 1706.
with low DSII. Analysis of alkonates. Carbohydr. 140. Greene, T.W.; Wuts, P.G.M. Protective groups in organic
Polym. 1993, 22, 79. synthesis; J. Wiley Sons: New York, 1991; 71 pp.
122. Glasser, W.G.; McCartney, B.K.; Samaranayake, G. 141. Stein, A.; Klemm, D. Syntheses of cellulose derivatives via
Cellulose derivatives with low DSIII. The biodegrad- O-triorganosilyl cellulosesI. Eective synthesis of organ-
ability of cellulose esters using a simple enzyme assay. ic cellulose esters by acylation of trimethylsilyl celluloses.
Biotechnol. Prog. 1994, 10, 214. Makromol. Chem. Rapid Commun. 1988, 9, 569.
123. Dave, V.; Glasser, W.G. Cellulose-based bers from liquid 142. Mormann, W.; Wagner, T. Acylation of (partially)
crystalline solutionsII. Processing and morphology of silylated poly(vinyl alcohol)s with acyl chlorides. Vinyl
cellulose and cellulose hexanoate esters. J. Appl. Polym. chloride/vinyl ester copolymers by polymer analogous
Sci. 1993, 48, 683. reaction. Macromol. Chem. Phys. 1996, 197, 3463.
124. Glasser, W.G.; Samaranayake, G.; Dumay, M.; Dave, V. 143. Klemm, D.; Stein, A.; Erler, U.; Wagenknecht, W.; Nehls,
Novel cellulose derivativesIII. Thermal analysis of mixed I.; Philipp, B. New presecures for regioselective synthesis
esters with butyric and hexanoic acid. J. Polym. Sci. B and modication of trialkylsilylcelluloses. In Cellulosics:
Polym. Phys. 1995, 33, 2045. Materials for Selective Separation and Other Technologies;
125. Zhang, Z.B.; McCormick, C.L. Structopendant unsatu- Kennedy, J.F., Phillips, G.O., Williams, P.A., Eds.; Ellis
rated cellulose esters via acylation in homogeneous lithium Horwood: New York, 1993; 221 pp.
chloride/N,N-dimethylacetamide solutions. J. Appl. Pol- 144. Wagenknecht, W.; Nehls, I.; Stein, A.; Klemm, D.; Philipp,
ym. Sci. 1997, 66, 293. B. Synthesis and substituent distribution of Na-cellulose
126. Williamson, S.L.; McCormick, C.L. Cellulose derivatives sulphates via O-trimethylsilyl cellulose as intermediate.
synthesized via isocyanates and activated esters pathways Acta Polym. 1992, 43, 266.
in homogeneous solutions of lithium chloride/N,N-dime- 145. Frazier, C.E.; Glasser, W.G. Attempts at the synthesis of
thylacetamide. J.M.S. Pure Appl. Chem. A 1998, 35, 1915. uorine containing cellulose derivative. Polym. Prepr.
127. Arai, K.; Ogiwara, Y. Homogeneous butyration of (Am. Chem. Soc. Div. Polym. Chem.) 1990, 31, 634.
cellulose dissolved in dimethyl sulfoxideformaldehyde 146. Siegmund, G. Untersuchungen zur Reaktivitt von Cellulo-
system. Seni Gakkaishi 1980, 36, T82. sesulfonaten in nucleophilen Substitutionsreaktionen,
128. Morooka, T.; Norimoto, M.; Yamada, T.; Shiraishi, N. Ph.D. Thesis, University of Jena, Germany, 2000.
Viscoelastic properties of (cellulose oligo oxymethylene 147. McCormick, C.L.; Callais, P.A. Derivatization of cellulose
ether) acylates. J. Appl. Polym. Sci. 1982, 27, 4409. in lithium chloride and N,N-dimethylacetamide solutions.
129. Miyagi, Y.; Shiraishi, N.; Yokota, T.; Yamashita, S.; Polymer 1987, 28, 2317.
Hayashi, Y. Preparation and thermal properties of acetates 148. Dawsey, T.R.; Newman, J.K.; McCormick, C.L. Tosyla-
derived from cellulose dissolved in DMSO-PF. J. Wood tion of cellulose in lithium chloride and N,N-dimethylace-
Chem. Technol. 1983, 3, 59. tamide. Polym. Prepr. (Am. Chem. Soc. Div. Polym.
130. Seymour, R.B.; Johnson, E.L. Acetylation of DMSO:PF Chem.) 1989, 30, 191.
149. McCormick, C.L.; Dawsey, T.R.; Newman, J.K. Compet- substituted derivatives as sorbents for metal ions. React.
itive formation of cellulose p-toluenesulfonate and chlor- Funct. Polym. 1999, 42, 223.
odeoxycellulose during homogeneous reactions of 167. Belyakova, M.K.; Galbraik, L.S.; Rogovin, Z.A. Synthesis
p-toluenesulfonyl chloride with cellulose in N,N- of cellulose stereoisomers by nucleophilic substitution
dimethylacetamide-lithium chloride. Carbohydr. Res. reactions. Cellul. Chem. Technol. 1971, 5, 405.
1990, 208, 183. 168. Hon, D.-N.S. Chemical modication of cellulose. In
150. Heinze, T.; Rahn, K. Synthese und Folgechemie von Chemical Modication of Lignocellulosic Materials; Hon,
Cellulose-p-toluensaulfonsureesternPool fur neuartige D.-N.S., Ed.; Marcel Dekker: New York, 1996; 114 pp.
Funktionspolymere. Papier (Darmst.) 1996, 50, 721. 169. Rahn, K. Neue Funktionspolymere durch regioselektive
151. Uragami, T.; Tsukamoto, K.; Miyata, K.; Heinze, T. Synthese und Folgechemie der p-Toluensulfonsureester
Permeation and separation characteristics for benzene/ der Cellulose, Ph.D. Thesis, University of Jena, Germany,
cyclohexane mixtures through tosylcellulose membranes in 1997.
pervaporation. Cellulose 1999, 6, 221. 170. Koschella, A.; Heinze, T. Novel regioselectively 6-func-
152. Heinze, T.; Rahn, K.; Jaspers, M.; Berghmans, H. Thermal tionalized cationic cellulose polyelectrolytes prepared via
studies on homogeneously synthesized cellulose p-toluene- cellulose sulfonates. Macromol. Biosci. 2001, 1, 178.
sulfonates. J. Appl. Polym. Sci. 1996, 60, 1891. 171. Tiller, J. Mageschneiderte Aminoderivate zum Aufbau
153. Heinze, T.; Camacho Gomez, J.A.; Haucke, G. Synthesis supramolekularer Cellulosearchitekturen mit Analyt-
and characterization of the novel cellulose derivative Erkennungsfunktion und optischer Signalgruppe, Ph.D.
dansyl cellulose. Polym. Bull. 1996, 37, 743. Thesis, University of Jena, Germany, 1999.
154. Nakao, T.; Yamazaki, S.; Amano, T. Japanese Patent 72 172. Tiller, J.; Berlin, P.; Klemm, D. Soluble and lm-forming
39, 951, 1972. cellulose derivatives with redox-chromogenic and enzyme
155. Ishi, T.; Ishizu, A.; Nakano, J. Chlorination of cellulose immobilizing 1,4-phenylenediamine groups. Macromol.
with methanesulphonyl chloride in N,N-dimethylforma- Chem. Phys. 1999, 200, 1.
mide and chloral. Carbohydr. Res. 1977, 59, 155. 173. Tiller, J.; Berlin, P.; Klemm, D. Novel matrices for
156. Sato, T.; Koizumi, J.; Ohno, Y.; Endo, T. An improved biosensor applications by structural design of redox-
procedure for the preparation of chlorinated cellulose with chromogenic aminocellulose esters. J. Appl. Polym. Sci.
methanesulfonyl chloride in a dimethylformamidechlo- 2000, 75, 904.
ralpyridine mixture. J. Polym. Sci. A Polym. Chem. 1990, 174. Liu, C.; Baumann, H. Exclusive and complete introduction
28, 2223. of amino groups and their N-sulfo and N-carboxymethyl
157. Furuhata, K.-I.; Chang, H.-S.; Aoki, N.; Sakamoto, M. groups into the 6-position of cellulose without the use of
Chlorination of cellulose with N-chlorosuccinimide-triphe- protecting groups. Carbohydr. Res. 2002, 337, 1.
nylphosphine under homogeneous conditions in lithium 175. Camacho Gomez, J.A. Untersuchungen zur Synthese und
chloride-N,N-dimethylacetamide. Carbohydr. Res. 1992, Folgechemie von O-Arylmethylethern und S-Alkylthio-
230, 151. sulfaten (BUNTE-Salze) der Cellulose, Ph.D. Thesis, Uni-
158. Furuhata, K.-I.; Aoki, N.; Suzuki, S.; Arai, N.; Sakamoto, versity of Jena, Germany, 1996.
M.; Saegusa, Y.; Nakamura, S. Reaction of dichloroallose 176. Milligan, B.; Swan, J.M. BUNTE salts (RSSO3Na). Rev.
units in a chlorodeoxycellulose with lithium chloride under Pure Appl. Chem. 1962, 12, 72.
homogeneous conditions in N,N-dimethylacetamide. Car- 177. Petri Siqueira, D.F.; Choi, S.; Beyer, H.; Schimmel, T.;
bohydr. Res. 1994, 258, 169. Bruns, M.; Wenz, G. Synthesis of a cellulose thiosulfate
159. Furuhata, K.-I.; Koganai, K.; Chang, H.U.; Aoki, N.; and its immobilization on gold surfaces. Polymer 1999, 40,
Sakamoto, M. Dissolution of cellulose in lithium bromide 1593.
organic solvent system and homogeneous bromination 178. Vigo, T.L.; Sachinvala, N. Deoxycelluloses and related
of cellulose with N-bromosuccinimide-triphenylphosphine structures. Polym. Adv. Technol. 1999, 10, 311.
in lithium bromide-N,N-dimethylacetamide. Carbohydr. 179. Heinze, T.; Rahn, K.; Jaspers, M.; Berghmans, H. p-
Res. 1992, 230, 165. Toluenesulfonyl esters in cellulose modications: Acyla-
160. Furuhata, K.-I.; Aoki, N.; Suzuki, S.; Sakamoto, M.; tion of remaining hydroxyl groups. Macromol. Chem.
Saegusa, Y.; Nakamura, S. Bromination of cellulose with Phys. 1996, 197, 4207.
tribromoimidazole, triphenylphosphine and imidazole 180. Heinze, T.; Rahn, K. The rst report on a convenient
under homogeneous conditions in LiBr-dimethylaceta- synthesis of novel reactive amphiphilic polysaccharides.
mide. Carbohydr. Polym. 1995, 26, 25. Macromol. Rapid Commun. 1996, 17, 675.
161. Heinze, T.; Rahn, K. New polymers from cellulosesulfo- 181. Kotz, J.; Philipp, B.; Nehls, I.; Heinze, T.; Klemm, D. Zum
nates. J. Pulp Pap. Sci. 1999, 25, 136. Polyelektrolytverhalten einer C-6-substitutierten Carbox-
162. Kasuya, N.; Iiyama, K.; Meshitsuka, G.; Ishizu, H. ycellulose im Vergleich zu Carboxymethylcellulose. Acta
Preparation of 6-desoxy-6-uorocellulose. Carbohydr. Polym. 1990, 41, 333.
Res. 1994, 260, 251. 182. US Patent 3447939.
163. Mentasti, E.; Sarzanini, C.; Gennora, M.C.; Porta, V. 183. Diamantoglou, M.; Kundinger, E.F. Derivatisation of cel-
Nitroacetic acid, thiourea and cycteine ligands immobilized lulose in homogeneous reaction. In Cellulose and Cellulose
on cellulose for the uptake of trace metal ions. Polyhedron Derivatives: Physico-chemical Aspects and Industrial Appli-
1987, 6, 1197. cation; Kennedy, J.F., Phillips, G.O., Williams, P.O., Eds.;
164. Aoki, N.; Koganei, K.; Chang, H.-S.; Furuhata, K.; Wood Head Publishing Ltd.: Cambridge, 1995; 141 pp.
Sakamoto, M. Gas chromatographic-mass spectrometric 184. Heinze, T. Feature article: New ionic polymers by cellulose
study of reactions of halodeoxycelluloses with thiols in functionalization. Macromol. Chem. Phys. 1998, 199, 2341.
aqueous solutions. Carbohydr. Polym. 1995, 27, 13. 185. Brandt, L. Cellulose ethers. In Industrial Polymers Hand-
165. Furuhata, K.; Ikeda, H. Ionic cellulose derivatives: Syn- book; Wilks, S.E., Ed.; Wiley-VCH: Weinheim, 2001; 1569
thesis of sodium 6-deoxycellulose-sulfonate with high pp.
degree of substitution. React. Funct. Polym. 1999, 42, 103. 186. Mann, G.; Kunze, J.; Loth, F.; Fink, H.-P. Cellulose ethers
166. Aoki, N.; Fukushima, K.; Kurakata, H.; Sakamoto, M.; with a block-like distribution of substituents by structure-
Furuhata, K. 6-Deoxy-6-mercaptocellulose and its S- selective derivatization of cellulose. Polymer 1998, 39, 3155.
586 Heinze
187. Fink, H.-P.; Dautzenberg, H.; Kunze, J.; Philipp, B. The 203. Weigel, P.; Gensrich, J.; Wagenknecht, W.; Klemm, D.;
composition of alkali cellulose: A new concept. Polymer Erler, U.; Philipp, B. Modelluntersuchungen zum Einuss
1986, 27, 944. einer Zwischenderivatisierung auf Struktur und Eigen-
188. Heinze, T. Ionische Funktionspolymere aus Cellulose: schaften von Regeneratcellulosefaden. Papier (Darmst.)
Neue Synthesekonzepte, Strukturaufklarung und Eigen- 1996, 50, 483.
schaften, Habilitations Thesis, University of Jena, Ger- 204. Mormann, W.; Wagner, T. Silylation of cellulose and low-
many, 1997, Shaker Verlag, Aachen, 1998. molecular-weight carbohydrates with hexamethyldisila-
189. Liebert, T.; Heinze, T. Induced phase separationa new zane in liquid ammonia. Macromol. Rapid Commun.
synthesis concept in cellulose chemistry. In Cellulose 1997, 18, 512.
Derivatives: Modication, Characterization, and Nano- 205. Mormann, W.; Demeter, J.; Wagner, T. Reaktionen von
structures; Heinze, T., Glasser, W.G., Eds.; ACS Symp. Cellulose in AmmoniakSilylierung und Desilylierung.
Series 688, 61 Washington, DC, 1998; 61 pp. Papier (Darmst.) 1998, 52, 725.
190. Heinze, T.; Erler, U.; Nehls, I.; Klemm, D. Determination 206. Mormann, W.; Demeter, J.; Wagner, T. Selectivity in the
of the substituent pattern of heterogeneously and homoge- acylation of partially silylated hydroxy polymersA study
neously synthesized carboxymethyl cellulose by using high- with trimethylsilyl cellulose and model compounds. Acta
performance liquid chromatography. Angew. Makromol. Polym. 1999, 50, 20.
Chem. 1994, 215, 93. 207. Stoehr, T.; Petzold, K.; Wolf, B.; Klemm, D. Continuous
191. Reuben, J.; Conner, H.T. Analysis of the carbon-13C NMR polymer fractionation of polysaccharides using highly
spectrum of hydrolyzed O-(carboxymethyl)cellulose: substituted trimethylsilylcellulose. Macromol. Chem. Phys.
Monomer composition and substitution patterns. Carbo- 1998, 199, 1895.
hydr. Res. 1983, 115, 1. 208. Schaub, M.; Wenz, G.; Wegner, G.; Stein, A.; Klemm, D.
192. Saake, B.; Horner, S.; Kruse, T.; Puls, J.; Liebert, T.; Ultrathin lms of cellulose on silicon wavers. Adv. Mater.
Heinze, T. Detailed investigations of the molecular 1993, 5, 919.
structure of carboxymethyl cellulose with unusual sub- 209. Buchholz, V.; Wegner, G.; Stemme, S.; Oedberg, L.
stitution pattern by means of an enzyme-supported Regeneration, derivatization, and utilization of cellulose
analysis. Macromol. Chem. Phys. 2000, 201, 1996. in ultrathin lms. Adv. Mater. 1996, 8, 399.
193. Schaub, M.; Fakirov, C.; Schmidt, A.; Lieser, G.; Wenz, 210. Loscher, F.; Ruckstuhl, T.; Seeger, S. Ultrathin cellulose
G.; Wegner, G.; Albouy, P.-A.; Wu, H.; Foster, M.D.; based layers for detection of single antigen molecules. Adv.
Majrkzak, C.; Satiya, S. Ultrathin layers and supra- Mater. 1998, 10, 1005.
molecular architectures of isopentylcellulose. Macromole- 211. Therisod, M.; Klibanov, A.M. Facile enzymatic prepara-
cules 1995, 28, 1221. tion of monoacylated sugars in pyridine. J. Am. Chem. Soc.
194. Fakirov, C.; Lieser, G.; Wegner, G. Chain conformation 1986, 108, 5683.
and packing of isopentylcellulose in thin lms. Macromol. 212. Riva, S.; Chopineau, J.; Kieboom, A.P.G.; Klibanov, A.M.
Chem. Phys. 1997, 198, 3407. Protease-catalyzed regioselective esterication of sugars
195. DAprano, G.; Henry, C.; Godt, A.; Wegner, G. Design, and related compounds in anhydrous dimethylformamide.
characterization and processing of cellulose-S-acetyl: A J. Am. Chem. Soc. 1988, 110, 584.
precursor to an electroactive cellulose. Macromol. Chem. 213. Kobayashi, S.; Shoda, S.; Uyama, H. Enzymic polymer-
Phys. 1998, 199, 2777. ization and oligomerization. Adv. Polym. Sci. 1995, 121, 1.
196. Petrus, L.; Gray, D.G.; BeMiller, J.N. Homogeneous 214. Husemann, E.; Siefert, S. N-thyl-pyridinium-chlorid als
alkylation of cellulose in lithium chloride/dimethyl sulf- Losungsmittel und Reaktionsmedium fur Cellulose. Mak-
oxide solvent with dimsyl sodium activation. A proposal romol. Chem. 1969, 128, 288.
for the mechanism of cellulose dissolution in LiCl/Me2SO. 215. Kasuya, N.; Sawatari, A. A simple and facile method for
Carbohydr. Res. 1995, 268, 319. triphenylmethylation of cellulose in a solution of lithium
197. Sachinvala, N.D.; Winsor, D.L.; Hamed, O.A.; Maskos, chloride in N,N-dimethylacetamide. Seni Gakkaishi 2000,
K.; Niemczura, W.P.; Tregre, G.J.; Glasser, W.G.; 56, 249.
Bertoniere, N.R. The physical and NMR characterizations 216. Noyiri, M.; Kondo, T. Application of regioselectively
of allyl- and crotylcellulose. J. Polym. Sci. A Polym. Chem. substituted methylcellulose to characterize the reaction
2000, 38, 1889. mechanism of cellulase. Macromolecules 1996, 29, 2392.
198. Erler, U.; Klemm, D.; Nehls, I. Homogeneous synthesis of 217. Harkness, B.R.; Gray, D.G. Preparation and chiroptical
diphenylmethyl ethers of cellulose in N,N-dimethylaceta- properties of tritylated cellulose derivatives. Macromole-
mide/LiCl solvent system. Makromol. Chem. Rapid cules 1990, 23, 1452.
Commun. 1992, 13, 195. 218. Kondo, T.; Gray, D.G. The preparation of O-methyl- and
199. Heinze, T.; Erler, U.; Heinze, U.; Camacho, J.A.; Grummt, O-ethyl cellulose having controlled distribution of sub-
U.-W.; Klemm, D. Synthesis and characterization of pho- stituents. Carbohydr. Res. 1991, 220, 173.
tosensitive 4,4V-bis(dimethylamino)diphenyl-methyl ethers 219. Camacho Gomez, J.A.; Erler, U.; Klemm, D. 4-Methoxy
of cellulose. Macromol. Chem. Phys. 1995, 196, 1937. substituted trityl groups in 6-O protection of cellulose:
200. Philipp, B.; Wagenknecht, W.; Nehls, I.; Klemm, D.; Homogeneous synthesis, characterization, detritylation.
Schnabelrauch, M.; Stein, A. Homogeneous derivatization Macromol. Chem. Phys. 1996, 197, 953.
of cellulose via reactive organo-soluble intermediates. In 220. Heinze, T.; Rottig, K.; Nehls, I. Synthesis of 2,3-O-
Cellulose Sources and Exploitation; Kennedy, J.F., Phillips, carboxymethylcellulose. Macromol. Rapid Commun.
G.O., Williams, P.A., Eds.; Ellis Horwood: New York, 1994, 15, 311.
1990; 258 pp. 221. Li, H.-Q.; Zhang, L.-N.; Takaragi, A.; Miyamoto, T.
201. Aelenei, N.; Bontea, D.; Ioan, C. Synthesis and character- Water solubility of regioselectively 2,3-O-substituted car-
ization of trimethylsilylcellulose in solution. J.M.S.-Pure boxymethylcellulose. Macromol. Rapid Commun. 1997,
Appl. Chem. 1998, A35, 1667. 18, 921.
202. Schuyten, A.A.; Weaver, J.W.; Reid, J.D.; Jurgens, J.F. 222. Heinze, U.; Heinze, T.; Klemm, D. Synthesis and
Trimethylsilylcellulose. J. Am. Chem. Soc. 1948, 70, characterization of 2,3-O-carboxymethylcellulose. Macro-
1919. mol. Chem. Phys. 1999, 200, 896.
223. Heinze, U.; Schaller, J.; Heinze, T.; Horner, S.; Saake, B.; 242. Lewin, M. Oxidation and aging of cellulose. Macromol.
Puls, J. Characterization of regioselectively functionalized Symp. 1997, 118, 715.
2,3-O-carboxymethyl cellulose by enzymatic and chemical 243. Fengel, D.; Wegener, G. Wood, chemistry, ultrastructure,
methods. Cellulose 2000, 7, 161. reactions; W. DeGruyter: Berlin, 1989.
224. Klemm, D.; Stein, A. Silylated cellulose materials in design 244. Yackel, E.C.; Kenyon, W. Oxidation of cellulose by
of supramolecular structures of ultrathin cellulose lms. nitrogen dioxide. J. Am. Chem. Soc. 1942, 64, 121.
J.M.S.-Pure Appl. Chem. 1995, A32, 899. 245. Dan Dimitrijevech, S.; Tatarko, M.; Gracy, R.W.; Linsky,
225. Stein, A.; Klemm, D. Selektive Cellulose Derivatisierung C.B.; Olsen, C. Biodegradation of oxidized regenerated
unter heterogenen Reaktionsbedingungen und Struktur- cellulose. Carbohydr. Res. 1990, 195, 247.
aufklrung mit mehrdimensionaler NMR-Spektroskopie. 246. Painter, T.J. Preparation and periodate oxidation of C-6-
Papier (Darmst.) 1995, 49, 732. oxycellulose: Conformational interpretation of hemiacetal
226. Mischnick, P.; Lange, M.; Gohdes, M.; Stein, A.; Petzold, stability. Carbohydr. Res. 1977, 55, 95.
K. Trialkylsilyl derivatives of cyclomaltoheptaose, cellu- 247. Cesaro, A.; Delben, F.; Flaibani, A.; Paoletti, S. Polyelec-
lose, and amylose: Rearrangement during methylation trolytic eects in carboxylic derivatives of natural poly-
analysis. Carbohydr. Res. 1995, 277, 179. saccharides. Carbohydr. Res. 1987, 160, 355.
227. Koschella, A.; Klemm, D. Silylation of cellulose regiocon- 248. Heinze, T.; Klemm, D.; Schnabelrauch, M.; Nehls, I.
trolled by bulky reagents and dispersity in the reaction Properties and following reactions of homogeneously
media. Macromol. Symp. 1997, 120, 115. oxidized cellulose. In Cellulosics: Chemical, Biochemical
228. Koschella, A.; Heinze, T.; Klemm, D. First synthesis of 3- and Material Aspects; Kennedy, J.F., Phillips, G.O.,
O-functionalized cellulose ethers via 2,6-di-O-protected Williams, P.A., Eds.; Ellis Horwood: New York, 1993;
silyl cellulose. Macromol. Biosci. 2001, 1, 49. 349 pp.
229. Kamide, K.; Saito, M. Recent advances in molecular and 249. Nehls, I.; Heinze, T.; Philipp, B.; Klemm, D.; Ebringerova,
supramolecular characterization of cellulose derivatives. A. 13C-NMR-spektroskopische Untersuchungen zur Oxi-
Macromol. Symp. 1994, 83, 223; and references cited dation von Cellulose im System H3PO4/NaNO2. Acta
therein. Polym. 1991, 42, 339.
230. Iwata, T.; Azuma, J.; Okamura, K.; Muramoto, M.; Chun, 250. deNooy, A.E.J.; Besemer, A.C.; van Bekkum, H. Highly
B. Preparation and nmr assignments of cellulose mixed selective TEMPO mediated oxidation of primary alcohol
esters regioselectively substituted by acetyl and propanoyl groups in polysaccharides. Recl. Trav. Chim. Pays-Bas
groups. Carbohydr. Res. 1992, 244, 277. 1994, 113, 165.
231. Iwata, T.; Okamura, K.; Azuma, J.; Tanaka, F. Molecular 251. deNooy, A.E.J.; Besemer, A.C.; van Bekkum, H. Selective
and crystal structure of cellulose propanoate diacetate oxidation of primary alcohols mediated by nitroxyl radical
(CPDA, 2,3-di-O-acetyl-6-O-propanoyl cellulose). Cellu- in aqueous solution, kinetics and mechanism. Tetrahedron
lose 1996, 3, 91. 1995, 51, 8023.
232. Iwata, T.; Okamura, K.; Azuma, J.; Tanaka, F. Molecular 252. deNooy, A.E.J.; Besemer, A.C.; van Bekkum, H.; van Dijk,
and crystal structure of cellulose acetate dipropanoate J.A.P.P.; Smit, J.A.M. TEMPO mediated oxidation of
(CADP, 6-O-acetyl-2,3-di-O-propanoyl cellulose). Cellu- pullulan and inuence of ionic strength and linear charge
lose 1996, 3, 107. density on the dimensions of the obtained polyelectrolyte
233. Iwata, T.; Doi, Y.; Azuma, J. Direct imaging of single chains. Macromolecules 1996, 29, 6541.
crystals of regioselectively substituted cellulose esters by 253. Chang, P.S.; Robyt, J.F. Oxidation of primary alcohol
atomic force microscopy. Macromolecules 1997, 30, 6683. groups of naturally occurring polysaccharides with 2,3,6,6-
234. Tsunashima, Y.; Hattori, K. Substituent distribution in tetramethyl-1-piperidine oxoammonium ion. J. Carbo-
cellulose acetates: Its control and the eect of structure hydr. Chem. 1996, 15, 819.
formation in solution. J. Colloid Interface Sci. 2000, 228, 254. Isogai, A.; Kato, Y. Preparation of polyuronic acid from
279. cellulose by TEMPO-mediated oxidation. Cellulose 1998,
235. Deus, C.; Friebolin, H.; Siefert, E. Partiell aktivierte 5, 153.
CelluloseSynthese und Bestimmung der Substituenten- 255. Tahiri, C.; Vignon, M.R. TEMPO-oxidation of cellulose:
verteilung mit Hilfe der 1H-NMR-Spektroskopie. Makro- Synthesis and characterization of polyglucuronans. Cellu-
mol. Chem. 1991, 192, 75. lose 2000, 7, 177.
236. Wagenknecht, W. Regioselektive Cellulosederivate durch 256. Vieira, M.; Liebert, T.; Heinze, T. New ionic polymers by
Modizierung technischer Cellulosederivate. Papier subsequent functionalization of cellulose derivatives. In
(Darmst.) 1996, 50, 712. Recent Advances in Environmentally Compatible Polymers;
237. Klemm, D.; Heinze, T.; Wagenknecht, W. Properties of Kennedy, J.F., Phillips, G.O. Williams, P.A., Eds.; Wood-
regioselectively substituted anionic cellulose derivatives. head Publishing: Cambridge, U.K., 2001; 5360.
Ber. Bunsenges. Phys. Chem. 1996, 100, 730. 257. Vieira, M. Studien zur selektiven Oxidation von Cellulosee-
238. Schuldt, U.; Wagenknecht, W.; Richter, A. Electrosorption thern und Funktionalisierung cellulosereicher Fasern,
of sodium cellulose sulfates with dierent substitution Ph.D. Thesis, University of Jena, Germany, 2000.
patterns. Cellulose 2002, 9, 271. 258. Maekawa, E.; Koshijima, T. Properties of 2,3-dicarboxy
239. Buchanan, C.M.; Edgar, K.J.; Hyatt, J.A.; Wilson, A.K. cellulose combined with various metallic ions. J. Appl.
Preparation of cellulose 1-13C acetates and determination Polym. Sci. 1984, 29, 2289.
of monomer composition by NMR spectroscopy. Macro- 259. Rahn, K.; Heinze, T. New cellulosic polymers by sub-
molecules 1991, 24, 3050. sequent modications of 2,3-dialdehyde cellulose. Cellul.
240. Nevell, T.P. Oxidation of cellulose. In Cellulose Chemistry Chem. Technol. 1998, 32, 173.
and Its Application; Nevell, T.P., Zeronian, S.H., Eds.; Ellis 260. Painter, T.J. Control of depolymerisation during the
Horwood: Chichester, 1985; 243 pp. preparation of reduced dialdehyde cellulose. Carbohydr.
241. Engelskirchen, K. Umwandlung von Cellulose. In Res. 1988, 179, 259.
Houben Weyl, Methoden der organischen Chemie; Bartl, 261. Morooka, T.; Norimoto, N.; Yamada, T. Periodate
H., Falbe J., Eds.; G. Thieme: Stuttgart: New York 1987; oxidation of cellulose by homogeneous reaction. J. Appl.
E20/3, 2047 pp. Polym. Sci. 1989, 38, 849.
588 Heinze
262. Floor, M.; Hofsteede, L.P.M.; Groenland, W.P.T.; Ver- water: Fluorescence studies with labeled (hydroxypropyl)-
haar, L.A.T.; Kieboom, A.P.G.; van Bekkum, H. Prepa- cellulose. Macromolecules 1989, 22, 734.
ration and calcium complexation of oxidized poly- 280. Winnik, F.M.; Regismond, S.T.A.; Goddard, E.D. Inter-
saccharides. II. Hydrogen peroxide as coreactant in the actions of anionic surfactants with uorescent-dye-labeled
chlorite oxidation of dialdehyde glucans. Recl. Trav. Chim. hydrophobically modied cationic cellulose ether. Lang-
Pays-Bas 1989, 108, 384. muir 1997, 13, 11.
263. Matsumura, S.; Nishioka, M.; Shigeno, H.; Tanaka, T.; 281. Alexandrides, P.; Sungsook, A.; Tsianou, M. Interactions
Yoshikawa, S. Builder performance in detergent formula- between cyclodextrins and a mixed cationic cellulose ether:
tions and biodegradability of partially dicarboxylated Anionic surfactant gelling system. In Polysaccharide
cellulose and amylose containing sugar residues in the Applications, Cosmetics and Pharmaceuticals; El-Nokaly,
backbone. Angew. Makromol. Chem. 1993, 205, 117. M.A. Soini, H.A. Eds.; ACS Symposium Series 737, Wash-
264. Varma, A.J.; Chavan, V.B. A study of crystallinity changes ington, DC, 1999; 185 pp.
in oxidized celluloses. Carbohydr. Polym. 1995, 27, 63. 282. Gruber, J.V.; Konish, P.N. Aqueous viscosity enhance-
265. Landoll, L.M. Nonionic polymer surfactants. J. Polym. ment through helical inclusion complex cross-linking of a
Sci., Polym. Chem. Ed. 1982, 20, 443. hydrophobically-modied, water-soluble, cationic cellu-
266. Schulz, D.N.; Bock, J.; Valint, P.L. Synthesis and lose ether by amylose. Macromolecules 1997, 30, 5361.
characterization of hydrophobically associating water- 283. Liebert, T.; Walt, D.R. Synthesis of pH-sensitive modied
soluble polymers. In Macromolecular complexes in chem- cellulose ether halfesters and their use in pH detecting
istry and biology; Dubin, P., Bock, J., Davis, P.M., Schulz, systems based on ber optics. J. Controlled Rel. 1995, 35,
D.N., Thies, C., Eds.; Springer Verlag: Heidelberg, 1994; 155.
3 pp. 284. Vogt, S.; Heinze, T.; Rottig, K.; Klemm, D. Preparation of
267. McCormick, C.L. Structural design of water-soluble carboxymethyl cellulose sulfates of high degree of sub-
copolymers. In Water-Soluble Polymers: Synthesis, Sol- stitution. Carbohydr. Res. 1995, 266, 315.
ution Properties, and Application; Shalaby, S.W., 285. Heinze, T.; Heinze, U. The rst approach to non-aqueous
McCormick, C.L., Butler, G.B., Eds.; ACS Advances in solutions of carboxymethyl cellulose. Macromol. Rapid
Chemistry Series 467; American Chemical Society: Wash- Commun. 1997, 18, 1033.
ington, DC, 1989; 10 pp. 286. Clasen, C.; Kulicke, W.M. Determination of viscoelastic
268. Tanaka, R.; Meadows, J.; Williams, P.A. Interaction of and rheo-optical material functions of water-soluble
hydrophobically modied (hydroxyethyl)cellulose with cellulose derivatives. Prog. Polym. Sci. 2001, 26, 1839.
various added surfactants. Macromolecules 1992, 25, 1304. 287. Kastner, U.; Homann, H.; Donges, R.; Hilbig, J.
269. Tanaka, R.; Meadows, J.; Phillips, G.O.; Williams, P.A. Structure and solution properties of sodium carboxymeth-
Viscometric and spectroscopic studies on the solution yl cellulose. Colloids Surf. 1997, 123124, 307.
behavior of hydrophobically modied cellulosic polymers. 288. Donges, R. Non-ionic cellulose ethers. Br. Polym. J. 1990,
Carbohydr. Polym. 1992, 12, 443. 23, 315.
270. Um, S.-U.; Poptoshev, E.; Pugh, R.J. Aqueous solutions of 289. Kotz, J.; Bogen, I.; Heinze, T.; Heinze, U.; Kulicke, W.M.;
ethyl (hydroxyethyl) cellulose and hydrophobic modied Lange, S. Peculiarities in the physico-chemical behavior of
ethyl (hydroxyethyl) cellulose polymer: Dynamic surface non-statistically substituted carboxymethyl cellulose. Col-
tension measurements. J. Colloid Interface Sci. 1997, 193, loids Surf. A Physicochem. Asp. 2001, 183185, 621.
41. 290. Polysaccharides, Structural Diversity and Functional Versa-
271. Stainer, G.A.; Gelman, R.A. Cellulosics utilization: Re- tility; Dumitriu, S., Ed.; Marcel Dekker, Inc.: New York,
search and rewards in cellulosics. In New Cellulosic 1998; 1, 57, 131, 925.
Surfactants; Inagaki, H., Phillips, G.O., Eds.; Elsevier 291. Parfondry, A.; Perlin, A.S. Carbon-13 NMR spectroscopy
Applied Science: London, 1989; 132 pp. of cellulose ethers. Carbohydr. Res. 1977, 57, 39.
272. Hwang, F.S.; Hogen-Esch, T.E. Fluorocarbon-modied 292. Reuben, J. NMR analysis and description of cellulose
water-soluble cellulose derivatives. Macromolecules 1993, ethers. Polym. Mater. Sci. Eng. 1984, 51, 263.
26, 3156. 293. Bach Tuyet, L.T.; Iiyama, K.; Nakano, J. Preparation of
273. Talaba, P.; Srokova, I.; Hodul, P. Polymeric surfactants carboxymethyl cellulose from rener mechanical pulp.
prepared from water-soluble O-(2-hydroxyethyl)cellulose. Mokuzai Gakkaishi 1985, 31, 14.
Chem. Pap. 1996, 50, 369. 294. Gronski, W.; Hellmann, G. NMR-spektroskopische Char-
274. Srokova, I.; Talaba, P.; Hodul, P.; Balazova, A. Emulsify- akterisierung von Carboxymethylcellulose. Papier (Darmst.)
ing agents based on O-(carboxymethyl) cellulose. Tenside 1987, 41, 668.
Surfactants Deterg. 1998, 35, 342. 295. Abdel-Malik, M.M.; Yalpani, M. Determination of sub-
275. Zheng, G.-Z.; Meshitsuka, G.; Ishizu, A. Chain behavior of stituent distribution in cellulose ethers by means of 13C-
an amphoteric cellulose derivative: O-Carboxymethyl-O-2- NMR spectroscopy. In Cellulose: Structural and Functional
hydroxy-3-(trimethylammonio)propyl cellulose. J. Polym. Aspects; Kennedy, J.F., Phillips, G.O., Williams, P.A.,
Sci. Part B Polym. Phys. 1995 33, 867 and 2211. Eds.; Ellis Horwood: New York, 1989; 263 pp.
276. Zeng, H.; Li, W.; Li, Z. Studies on a new water-soluble 296. Tezuka, Y. 13C NMR structural study on cellulose
sulfur-containing cellulose derivative. J. Appl. Polym. Sci. derivatives with carbonyl groups as a sensitive probe. In
1994, 54, 1989. Cellulose Derivatives: Modication, Characterization, and
277. Aust, N.; Zugenmaier, P. Formation of supramolecular Nanostructures; Heinze, T. Glasser, W.G. Eds.; ACS
structures of polysaccharidesurfactant complexes in Symposiums Series 688; American Chemical Society:
aqueous solutions. Inuence of the polymer backbone. Washington, DC, 1998; 163 pp. and references cited
Makromol. Chem. 1993, 194, 1583. therein.
278. Winnik, F.M.; Winnik, M.A.; Tazuke, S.; Ober, C.K. 297. Capitani, D.; Purro, F.; Segre, A.L. High eld NMR
Synthesis and characterization of pyrene-labeled hydrox- analysis of the degree of substitution in carboxymethyl
ypropyl cellulose and its uorescence in solution. Macro- cellulose sodium salt. Carbohydr. Polym. 2000, 42, 283.
molecules 1987, 20, 38. 298. Kulicke, W.M.; Otto, M.; Baar, A. Improved NMR
279. Winnik, F.M. Association of hydrophobic polymers in characterization of high-molecular-weight polymers and
polyelectrolytes through the use of preliminary ultrasonic 1995. Challenges in structure analysis of polysaccharide
degradation. Makromol. Chem. 1993, 194, 751. deriviations. Cellulose 2001, 8, 245.
299. Ho, F.F.L.; Klosiewicz, D.W. Proton nuclear magnetic 316. Lee, C.K.; Gray, G.R. Analysis of positions of substitution
resonance spectrometry for determination of substituents of O-acetyl groups on partially O-acetylated cellulose by
and their distribution in carboxymethylcellulose. Anal. the reductive-cleavage method. Carbohydr. Res. 1995, 269,
Chem. 1980, 25, 913. 167.
300. Baar, A.; Kulicke, W.M.; Szablikowski, K.; Kiesewetter, 317. Erler, U.; Mischnick, P.; Stein, A.; Klemm, D. Determi-
R. Nuclear magnetic resonance spectroscopic character- nation of the substitution pattern of cellulose methyl ethers
ization of carboxymethylcellulose. Macromol. Chem. by HPLC and GLCcomparison of methods. Polym. Bull.
Phys. 1994, 195, 1483. 1992, 29, 349.
301. Gautier, S.; Lecourtier, J. Structural characterization by 318. Heinrich, J.; Mischnick, P. Determination of the substitu-
13
C nuclear magnetic resonance of hydrolyzed carboxyme- tion pattern in the polymer chain of cellulose acetates. J.
thylcellulose. Polym. Bull. 1991, 26, 457. Polym. Sci., A, Polym. Chem. 1999, 37, 3011.
302. Chaudhari, S.N.K.; Gouden, K.C.; Srinivasan, G.; Ek- 319. Gohdes, M.; Mischnick, P.; Wagenknecht, W. Methylation
kundi, V.S. High resolution 13C NMR spectroscopy of analysis of cellulose sulphates. Carbohydr. Polym. 1997,
sodium carboxy methyl cellulose. J. Polym. Sci. A Polym. 33, 163.
Chem. 1987, 25, 337. 320. Gohdes, M.; Mischnick, P. Determination of the sub-
303. Kragten, E.A.; Leeang, B.R.; Kamerling, J.P.; Vliegen- stitution pattern in the polymer chain of cellulose sulfates.
thart, J.F.G. H-NMR spectroscopy of O-carboxymethyl Carbohydr. Res. 1998, 309, 109.
derivatives of D-glucose. Carbohydr. Res. 1992, 228, 321. Kragten, E.A.; Kamerling, J.P.; Vliegenthart, J.F.G.
433. Composition analysis of carboxymethylcellulose by high-
304. Kuper, P.; Kulicke, W.M.; Horn, S.; Saake, B.; Puls, J.; pH anion-exchange chromatography with pulsed ampero-
Kunze, J.; Fink, H.P.; Heinze, U.; Heinze, T.; Klohr, E.A.; metric detection. J. Chromatogr. 1992, 623, 49; references
Thielking, H.; Koch, W. Development and evaluation of cited therein.
new approaches of determination of functionalization 322. Rosell, K.-G. Distribution of substituents in methylcellu-
patterns of carboxymethyl cellulose. Angew. Makromol. lose. J. Carbohydr. Chem. 1988, 7, 525.
Chem. 1998, 260, 53. 323. Arisz, P.W. Mass spectrometric analysis of cellulose ethers,
305. Hikichi, K.; Kakuta, Y.; Katoh, T. H NMR study on Ph.D. Thesis, University of Amsterdam, Netherlands,
substituent distribution of cellulose diacetate. Polym. J. 1995.
1995, 27, 659. 324. Arisz, P.W.; Kauw, H.J.J.; Boon, J.J. Substituent distribu-
306. Nehls, I. 13C-NMR-spektroskopische Untersuchungen tion along the cellulose backbone in O-methylcelluloses
von Cellulose und Cellulosederivaten in Losung, Habilita- using GC and FAB-MS for monomer and oligomer
tions Thesis, University of Potsdam, Germany, 1993. analysis. Carbohydr. Res. 1995, 271, 1.
307. Goodlett, V.W.; Dougherty, J.T.; Patton, H.W. Character- 325. Heinze, T.; Pfeier, K.; Liebert, T.; Heinze, U. Eective
ization of cellulose acetates by nuclear magnetic resonance. approaches for estimating the functionalization pattern of
J. Polym. Sci. A-1 1971, 9, 155. carboxymethyl starch of dierent origin. Starch/Starke
308. Shiraishi, N.; Katayama, T.; Yokota, T. Acetylation of 1999, 51, 11.
cellulose in paraformaldehydedimethylsulfoxide solvent 326. Arisz, P.W.; Boon, J.J. Pyrolysis chemical ionization mass
system and its application to the chemical processing of spectrometry of cellulose ethers. J. Polym. Sci. A7 Polym.
wood. Cellul. Chem. Technol. 1978, 12, 429. Chem. 1995, 33, 2855.
309. Dicke, R.; Rahn, K.; Haack, V.; Heinze, T. Starch 327. Gelman, R.A. Characterization of carboxymethylcellulose:
derivatives of high degree of functionalization. 2. Deter- Distribution of substituent groups along the chain. J. Appl.
mination of the functionalization pattern of p-toluenesul- Polym. Sci. 1982, 27, 2957.
fonyl starch by peracylation and NMR spectroscopy. 328. Ma, Z.; Zhang, W.; Li, Z. Study on the characterization of
Carbohydr. Polym. 2001, 45, 43. distribution of substituents along the chain of carboxyme-
310. Iijima, H.; Kowsaka, K.; Kamide, K. Determination of thylcellulose. Chin. J. Polym. Sci. 1989, 7, 45.
sequence distribution of substituted and unsubstituted 329. Martinez-Richa, A.; Munoz-Alarcon, H.; Joseph-Nathan,
glucopyranose units in water-soluble cellulose acetate chain P. Studies on enzymatic resistance on molecular structure
as revealed by enzymatic degradation. Polym. J. 1992, 24, by 13C-NMR of cellulosic ethers. J. Appl. Polym. Sci. 1992,
1077. 44, 347.
311. Buchanan, C.M.; Hyatt, J.A.; Lowman, D.W. Supra- 330. Horner, S.; Puls, J.; Saake, B.; Klohr, E.A.; Thielking, H.
molecular structure and microscopic conformation of Enzyme-aided characterization of carboxymethylcellulose.
cellulose esters. J. Am. Chem. Soc. 1989, 111, 7312. Carbohydr. Polym. 1999, 40, 1.
312. Nunes, T.; Burrows, H.D.; Bastos, M.; Feio, G.; Gill, M.H. 331. Puls, J.; Horner, S.; Kruse, T.; Saake, B.; Heinze, T.
13
C nuclear magnetic resonance studies of cellulose ester Enzymuntersttzte Charakterisierung von Carboxymethyl-
derivatives in solution, powder, and membranes. Polymer cellulose mit herkommlicher und neuartiger Verteilung
1995, 36, 479. der funktionellen Gruppen. Papier (Darmst.) 1998, 52,
313. Lowman, D.W. Characterization of cellulose esters by 743.
solution-state and solid-state NMR spectroscopy. In 332. Urbanski, J. Analysis and characterization of cellulose and
Cellulose Derivatives: Modication, Characterization and its derivatives. Appl. Polym. Anal. Char. 1992, 2, 345.
Nanostructures; Heinze, T. Glasser, W.G., Eds.; ACS 333. Klemm, D.; Heinze, T.; Stein, A.; Liebert, T. Polyglucane
Symp. Series 688, Washington, DC, 1998; 131 pp. derivatives with regular substituent distribution. Macro-
314. Mischnick, P. Determination of the substituents pattern of mol. Symp. 1995, 99, 129.
cellulose acetates. J. Carbohydr. Chem. 1991, 10, 711. 334. Stein, A. Untersuchungen zur Synthese und Veretherung
315. Mischnick, P. Analytische Charakterisierung von Polysac- von O-Trialkylsilylcellulose, Ph.D. Thesis, University of
chariden und Polysaccharidderivaten mittels chemischer, Jena, Germany, 1991.
enzymatischer und massenspektrometrischer Methoden, 335. Krkoska, P.; Hostomosky, J. Czechoslovakian Patent
Habilitations Thesis, University of Hamburg, Germany, 138,087, 1970.
590 Heinze
336. Krkoska, P.; Hostomosky, J. Czechoslovakian Patent 343. Priksane, A.; Prikulis, A.; Isaeva, D.A. Modication of
145,910,15, 1972. cellulose with iminodiacetic acid chelating agents. Latv.
337. Teshirogi, T.; Yamamoto, H.Y.; Sakamoto, M.; Tonami, PSR Zinat. Akad. Vestis, Kim. Ser. 1990, 4, 494.
H. Preparation of sulfated aminodeoxycelluloses. Seni 344. Strizhakov, O.D. Synthesis of N,O-chelating complexons
Gakkaishi 1980, 36, 560. based on aminodeoxycellulose and hydroxyaldehydes.
338. Hou, K.C.; Zaniewski, R. Endotoxin removal by anion- Vestsj. Akad. Nauk. Belarusi, Ser. Khim. Navuk 1996, 1, 68.
exchange polymeric matrix. Biotechnol. Appl. Biochem. 345. Inagaki, N.; Hamajima, K.; Katsuura, K. Flame-retardant
1990, 12, 315. action of chlorine compounds and antimony trioxide on
339. Tashiro, T.; Shimura, Y. Removal of mercuric ions by cellulose fabric. J. Appl. Polym. Sci. 1978, 22, 3283.
systems based on cellulose derivatives. J. Appl. Polym. Sci. 346. Gilbert, E.E. US Patent 4,849,514, 1989.
1982, 27, 747. 347. Mahorta, S.L.; Jones, A.Y. European Patent Application
340. Nakamura, S.; Amano, M.; Saegusa, Y.; Sato, T. EP 546,750, 1993.
Preparation of aminoalkyl celluloses and their adsorption 348. Liu, F.-T.; Song, N.; Yu, X.-D.; Li, S.-B. The electron
and desorption of heavy metal ions. J. Appl. Polym. Sci. transfer behavior of some new polymers with viologen
1992, 45, 265. structure on the reduction of azobenzene in heterophases.
341. Imai, S.; Muroi, M.; Hamaguchi, A. Preconcentration of Macromol. Chem. Phys. 1994, 195, 2169.
trace elements in natural water with cellulose piperazine- 349. Heinze, T.; Pfeier, K.; Lazik, W. Starch derivatives with
dithiocarboxylate and determination by neutron activation high degree of functionalization. III: Inuence of reaction
analysis. Anal. Chem. 1983, 55, 1215. conditions and starting materials on molecular structure
342. Muroi, M.; Imai, S.; Hamaguchi, A. Sorption of uranium of carboxymethyl starch. J. Appl. Polym. Sci. 2001, 81,
by cellulose derivatives. Analyst 1985, 110, 1083. 2036.
24
The Physical Chemistry of Starch*
R. Parker and S. G. Ring
Institute of Food Research, Norwich Research Park, Norwich, United Kingdom
I. INTRODUCTION II. THE MACROMOLECULES

Starch is the main reserve polysaccharide of higher plants Before discussing the physical chemistry it is necessary to
where it occurs as water insoluble granules. Starch is readily rst consider macromolecular structure. There are many
isolated from plant crops, and major commercial sources excellent recent reviews on starch structure and biosynthe-
include maize, wheat, potato, and rice. The isolated starch sis, so these aspects will only briey be considered [1,2].
nds a very wide range of commercial application, includ- Starch consists of two main polysaccharides, amylose and
ing use in the food, pharmaceutical, paper and packaging amylopectin. Both polysaccharides are based on chains of
industries. The performance of starch in these various 1 ! 4 linked a-D-glucose (Fig. 1a) but whereas amylose is
applications often depends on the macromolecular behav- essentially linear, amylopectin is highly branched contain-
ior of starch, and how various macromolecular interactions ing on average one branch point which is 1 ! 4 ! 6 linked
aect its functional behavior. The functional behavior for every 2025 straight chain residues. Linear regions of
could include its use as a texturizing agent, as an adhesive the amylose chain form a dark blue complex with poly-
or its behavior as a macronutrient during passage down our iodide ions in aqueous solution at room temperature [3]. In
gastrointestinal tract. One motivation for examining the this complex, the amylose chain adopts a helical confor-
physical chemistry of starch is to attempt to understand the mation with the polyiodide ion occupying the central
relationship between the macromolecular behavior of cavity. This interaction is a basis for dening amylose as
starch and its functionality. This understanding should that starch polysaccharide which, under standardized con-
enable the rational modication of material behavior ditions, binds 20% of its weight of iodine while under the
through changing macromolecular characteristics, or same conditions amylopectin generally binds <1% w/w.
through changing the interaction of the macromolecule This iodine binding allows a distinction to be made be-
with other components in the product. Such an approach is tween amylose and amylopectin, and permits the determi-
well developed in the synthetic polymer area and in recent nation of the amylose content of native starch [3]. Most
years has been extended to include a polymeric description starches contain between 20% and 25% w/w amylose
of the behavior of a biopolymer such as starch. In this although some waxy starches contain very little, if any,
review we will focus on the physical chemistry of starch and amylose (<1%) and other starches, such as amylomaize,
its relationship to material characteristics. contain in the region of 65% amylose. Although a fraction
of the amylose population is linear and is quantitatively
hydrolyzed by the exo-acting enzyme h-amylase, a fraction
is lightly branched [48]. For example, puried wheat
amyloses were hydrolyzed from 79% to 85% by h-amylase.
Isoamylolysis allowed quantitation of a short chain frac-
* Extended review reprinted from Journal of Cereal Science, Vol. 34, tion representing between 3.2% and 7.6% w/w of the
Parker, R. and Ring, S.G. Aspects of the Physical Chemistry of amylose depending on the variety of wheat examined [9].
Starch, pp. 117, Copyright 2001, with permission from Elsevier Thus the fraction of amylose that is branched contains
Science. short chain branches on a main chain. Typical molecular
591
592 Parker and Ring
give rise to signicant iodine binding behavior [15,16].

Classication of the constituent chains can also be based
on how the chains are linked to the rest of the molecule. The
C chain carries the sole reducing end of the molecule, the A
chain is only linked to the rest of the molecule through its
potential reducing end, while the B chain is linked in this
way and also carries one or more A chains. Typical
estimates of the ratio of A/B chains is in the region of 1:1
to 1.5:1 [10]. The ne structure of amylopectin is dependent
on botanical origin, with variations reported in the A/B
chain ratio, the length and abundance of short and long
chains, and the form of distribution of the constituent
chains. The structure of dierent amylopectins is generally
characteristic of a particular species, although small vari-
etal dierences are also observed [9,11,17]. Current models
[1] of amylopectin structure depict short linear chains, 10 to
20 units long, arranged in clusters on longer chains, with
the longer chains spanning more than one cluster (Fig. 1b).
Examination of the products of a limited a-amylolysis of
amylopectin reveals the presence of branched clusters of
short chains, lending support to the model [18,19]. The
cluster model is a two-dimensional representation of a
three-dimensional structure. The product of starch biosyn-
thesis is a starch granule that is formed by the successive
addition of small units to a granular surface rather than the
in vivo self-assembly of the granule from the high molec-
ular weight polymer. Even starches that are wholly amy-
lopectin have a partially crystalline granule, therefore the
way that the constituent chains are arranged on the amy-
lopectin molecule must permit the formation of an ordered
crystalline structure with its associated dimensional con-
straints. In addition, the dry starch granule is a relatively
dense structure with a measured density of about 1500 kg
m3, the branching of the amylopectin chain therefore
must permit this density of packing.
Figure 1 The glycosidic linkage of amylose showing the pre-

ferred chair conformations, and the possibility for confor-
III. AQUEOUS SOLUTION BEHAVIOR OF THE
mational exibility about the glycosidic linkage (a); the
two-dimensionalclustermodelofamylopectinwithshortchains STARCH POLYSACCHARIDES
arranged in clusters on longer chains (b); the arrangement of For polysaccharides in general, water at room temperature
double helices in the A and B crystalline forms of starch (c). is a rather indierent solvent, and the starch polysaccha-
rides are no exception. Even dilute solutions are metastable
and if left for a sucient time will eventually precipitate
from solution. While D-glucose is very soluble in water,
weights of extracted amylose are in the region of 105 to increasing the chain length of 1 ! 4 linked a-D-glucose
106 g mol1. chains lowers solubility. Short amylose chains with a DP
The branching within amylopectin is not random greater than 9 readily crystallize from aqueous solution in
[2,10]. Debranching amylopectin with a debranching en- the temperature range 2jC to 20jC [2022]. With longer
zyme such as isoamylase followed by size exclusion chro- chains, the precipitates isolated from aqueous solution at
matography [11], or high pressure anion exchange room temperature are generally poorly crystalline [23],
chromatography [12] reveals an essentially bimodal popu- indicating that the driving force for crystallization has
lation of chains with two main populations with peak increased to the extent that there is insucient time for
degree of polymerization (DP) of around 1214 and f45 the chains to form ordered crystalline structures, and that
[11]. The short chain fractions of many amylopectins also the amylose chains just precipitate from solution. What
have shoulders of around DP 1820 [13,14]. The short then are the physicochemical origins of this poor solubil-
chain fraction is the most abundant by weight and number. ity? It is well known that D-glucose has a favorable
There is also the possibility that amylopectin can contain energetic interaction with water that can be measured
some longer chains, of sucient uninterrupted length to calorimetrically. Experiments on maltooligomers reveal a
The Physical Chemistry of Starch 593
similar favorable energetic interaction [24]. It may be the is to suciently rene the modeling of amylose chains so
case that this clustering of water around the glucan chain that insight may be obtained on its interaction with amy-
becomes increasingly unfavorable entropically as the chain losic enzymes. Natural amyloses, extracted from starches,
length increases. are polydisperse with weight average molecular weights
The dilute aqueous solution behavior of amylose has typically ranging from 105 to 106 gmol1, and a hydrody-
been extensively characterized using a range of physico- namic radius of 3050 nm.
chemical techniques, including light scattering [2530]. In Any branching of the amylose chain will reduce coil
the use of these techniques it is essential to demonstrate dimensions. A consequence of the more extensive branch-
that true molecular solution has been achieved, and that ing of amylopectin is that for its molecular weight it is much
the interpretation of the data is not aected by the presence more compact. Amylopectin is one of the largest biopoly-
of macromolecular aggregates. The studies that are easier mers known with typical molecular weights being in the
to interpret are those that have used synthetic amyloses. region of 107 to 108 gmol1. Estimates of molecular size
The amylose is synthesized using a potato phosphorylase, a from light scattering experiments vary somewhat and range
primer molecule such as maltohexaose, and glucose-1- from 100 to 300 nm [29]. Measurements of the intrinsic
phosphate as a substrate. Glucose units are successively viscosity of amylopectin in aqueous solution indicate a
added to the primer with the length of the amylosic chain molecular size similar to that of amylose [33]. The reasons
being controlled by the relative amounts of primer and for these dierences are not clear, a macromolecule as large
substrate. The advantage of this approach is that the as amylopectin might be very susceptible to degradation by
synthesized chains are linear and have a very limited mechanical shear. Additionally, a consequence of the
polydispersity. For amylose in solution, total intensity light highly branched nature of amylopectin is that in the
scattering permits the measurement of molecular weight, extraction of amylopectin from the native granule it might
M, and molecular size through the determination of a be very dicult to isolate individual molecules as they
radius of gyration, RG. The dependence of RG on M gives might be very heavily entangled.
information on the conformation of a polymer in solution
through the determination of the exponent v
RG / Mv IV. GRANULE ORGANIZATION
For essentially monodisperse amylose in aqueous solution Starch occurs naturally as water-insoluble granules whose
at room temperature, the radius of gyration, RG is propor- form is characteristic of its botanical origin. When viewed
tional to molecular weight, M, to the exponent 0.5. This under polarized light the granules are birefringent. As the
observation indicates that under these conditions amylose radial refractive index is larger than the tangential refrac-
behaves as a exible coil. In dilute solution, the data show a tive index a preferred radial distribution of chains is
very weak dependence on concentration, indicating that indicated [1]. The starch granule is also partially crystalline
under these conditions water is eectively behaving as an with crystallinities in the region of 30% being reported. A
ideal solvent. In other solvents such as 0.1 M KOH, or number of crystalline forms are known (Fig. 1c), the A
dimethyl sulfoxide, exible coil behavior is also observed, form [34], which is found in most cereal starches, including
although the coil has expanded and the exponent has wheat, consists of starch double helices packed into a
increased from 0.5 to f0.6. monoclinic array. The B form [35,36], which is found in
It is also of interest to characterize the stiness of the some tuber starches and high amylose cereal starches, is a
amylose chain. One approach is to calculate a characteris- more highly hydrated and open structure, consisting of
tic ratio, Cl, which, for a high molecular weight polymer, double helices packed in a hexagonal array. As waxy
is the ratio of the observed mean-square end-to-end sepa- starches, containing only amylopectin, still have crystalline
ration of the polymer coil, hr 2i0, and that calculated from granules the participation of amylopectin chains within the
the degree of polymerization, n, and the length of the crystalline domains is indicated. Acid etching of the gran-
monomeric segment, l. ules, or lintnerization as it is known, leads to a preferential
erosion of the amorphous fraction. Examination of the
Cl hr 2 i0 =nl 2 product has shown that the length of chain participating in
the crystalline domains is comparable to the short chain
For amylose in water, a value of f6 has been found, this fraction of amylopectin, hence the suggestion that it is the
compares with values of f6.7 for polymethylene and f10 short chains of amylopectin that form the double helices
for atactic polystyrene [31]. This would indicate that the [37,38]. Using high-intensity x-ray sources it is now possi-
amylose coil is relatively compact. These values of Cl can ble to map the crystallinity of dierent regions of the starch
be compared with those produced by simulation tech- granule [39,40]. This has given information on the orien-
niques such as molecular dynamics [32]. At present these tation of crystalline domains within individual granules.
simulations are restricted to relatively short amylose For example, for potato starch the B-type domains showed
chains, although the agreement between prediction and a preferred radial orientation whereas in wheat starch no
experiment (in this case x-ray scattering in order to obtain such orientation was observed of the A-type crystallites.
information on the size of a small amylosic fragment) The approach has also been used to probe the distribution
appears promising. One motivation for these experiments of A and B crystalline domains. For legume starch granules
594 Parker and Ring
[41] it was found that the A crystalline domains were Substantial solubilization of high molecular weight amy-
concentrated in the granule periphery [42]. Other types of lopectin is not observed although there are reports for some
order can be probed by other techniques. Solid-state NMR starches of the solubilization of a low molecular weight
experiments have been used to complement x-ray measure- amylopectin [56]. Although the gelatinization temperature
ments [43,44]. The spectra of the A and the B forms of is relatively easy to determine experimentally it is impor-
starch are characteristically dierent, and dierent to tant to bear in mind that the gelatinization process occurs
amorphous starch. over a limited temperature range for a single granule and a
X-ray and neutron scattering experiments [45] on somewhat wider temperature range for a population of
starch granules have revealed an intriguing peak in the granules. As heating is continued, past the gelatinization
scattering prole representing a periodicity in the range 8.9 temperature, the granule continues to swell. At temper-
to 10.9 nm [4649]. Through analysis of the whole prole, atures less than 100jC and in the absence of mechanical
in terms of a model of repeating amorphous and crystalline shear, the swollen granules, enriched in amylopectin,
layers, it was found that, for a range of starches of dierent maintain their integrity. The increase in volume fraction
botanical origin, within experimental error, this periodicity of starch granules in the suspension leads to an increase in
had a constant value of 9 nm. The constancy of this spacing viscosity of the paste. If the temperature is such that the
is surprising given that the starches examined had dierent solubilized amylose is slow to aggregate, the viscosity of
crystalline forms and dierent amylopectin structures. A these pastes, in common with other particulate suspen-
recent interpretation of this proposed lamellar structure is sions, is dominated by the volume fraction occupied by the
that the amylopectin forms a side chain liquid crystal starch granules [57]. At concentrations greater than about
structure [5052]. It is further proposed that the lamellae 6% w/w for cereal starches, the gelatinized granules ll the
are tilted as in a proposed superhelical structure [50,53]. available volume producing a viscoelastic material, the
Another aspect of granule structure is the shells [1] properties of which are additionally inuenced by the
observed directly by light microscopy and by scanning deformability of the swollen starch granule and hence the
electron microscopy of granules which have been etched amylopectin concentration in the granule. The swelling of
by acid and enzyme treatment. These structures have a the granule on gelatinization clearly has a major impact on
radial repeat length of between 120 and 400 nm and are the rheology of starch pastes. What molecular features
considered to consist of repeating amorphous and semi- aect swelling? One approach for gaining insight into the
crystalline layers. Other structures [54] observed on the swelling properties of starch might be to consider it as a
surface of starch granules by atomic force and other network structure [58]. For neutral, chemically cross-
microscopies are blocklets of about 30 nm in diameter linked, polymer networks the extent of equilibrium swell-
[55]. One interpretation is that these blocklets represent the ing observed is dependent on the extent of cross-linking
amylopectin cluster, another is that the blocklets are and the anity of the network for the solvent [59]. The
some surface contamination. If all this information is network will swell until the osmotic pressure generated by
considered as a whole then clearly the starch granule is a the network, as a result of its anity for the solvent, is
very complex macromolecular assembly, and it is perhaps balanced by the restorative stiness of the network resist-
useful to distinguish between that which has received ing swelling. Clearly, neutral amylopectins will largely have
widespread acceptance and that which is still debated. similar anities for water and it might be proposed that it is
Areas which remain a focus of discussion, largely because the eective cross-linking of the entangled amylopectin
they represent recent research, are the blocklet concept and network which might limit swelling. Changing the solvent
proposed models relating to the lamellar structures having characteristics, for example by increasing temperature, or
a 9-nm spacing. Models of granule assembly should rec- by mixing with other compounds that have a high anity
ognize the diversity of chemical structures that make up the for the starch chain, would lead to increased swelling. For
granule, for example, how is the amylose arranged? what is polyelectrolyte networks there is an additional contribu-
the signicance of the variability of amylopectin structure, tion to the osmotic pressure driving swelling, coming from
and the lengths of the constituent chains of amylopectin, to the mobile ions which are required to neutralize the charge
its organization in the granule? on the polymer [59]. The pressure generated by this
Donnan eect is greatest at low ionic strength. Some
amylopectins, e.g., potato amylopectin, are weakly phos-
V. GELATINIZATION, MELTING, phorylated and therefore behave as polyelectrolytes. Pota-
AND DISSOLUTION to starch is also a high swelling starch whose swelling is
reduced if ionic strength is increased. Clearly, there is a
Starch is usually processed by heating in the presence of polyelectrolyte eect in operation and one potential way of
water which disrupts the native crystalline structure, a modifying the swelling properties of these starches would
phenomenon known as gelatinization. In an excess of water be to alter their polyelectrolyte characteristics. While the
(>90% w/w), above a characteristic temperature, known as consideration of the gelatinized granule as a network
the gelatinization temperature, the starch granule loses its structure might be useful, it is important to bear in mind
native crystalline order and swells irreversibly to many that the foregoing discussion is based on the equilibrium
times its original size. At the same time the starch polysac- swelling of polymer networks, i.e., there is a reversibility in
charide amylose (if present) is preferentially solubilized. swelling if the conditions are changed. The swelling of the
starch granule after gelatinization is profoundly nonequi- range of temperature, those that gelatinize rst have access
librium as illustrated by the observation that starches such to the available water. On gelatinization the granules that
as wheat starch which have been subjected to dierent swell reduce the amount of water available for the swelling
thermal histories, such as slow vs. rapid heating, swell to of other granules [65]. At intermediate starch concentra-
dierent extents and as a consequence the starch pastes tions, of around 4050% w/w, gradients in starch concen-
have dierent rheologies [57,60]. tration are observed in a macroscopic sample. As reducing
As the starch granule is a complex structure it is the water content elevates the temperature of gelatiniza-
perhaps not surprising that the description of the processes tion, one eect is to broaden the transition. Double peaks
occurring during gelatinization is similarly complex. One are observed at these concentrations in plots of order loss,
approach has been to study the loss of various types of as assessed by x-ray diraction or dierential scanning
order with a range of techniques during the gelatinization calorimetry, as a function of temperature [66]. This is at
process. As the dierent techniques probe dierent aspects least in part due to the heterogeneous nature of the sample.
of the gelatinization process it should not be surprising that As the physical descriptions of the gelatinization process
they do not exactly map onto each other. Ideally, experi- are becomingly increasingly sophisticated, it is worthwhile
ments should be carried out during the gelatinization posing the questionhow might the gelatinization charac-
process; often, for practical necessity, they are carried out teristics of a starch be changed through modifying aspects
on starch materials that have been quenched to room of granule organization? To attempt to answer this ques-
temperature, allowing a potential reordering of structure. tion some physical insight is needed into the underlying
One such study used solid-state NMR, x-ray diraction physical processes. During gelatinization crystalline order
and dierential scanning calorimetry [61]. Although these is lost. From comparison with synthetic polymer systems it
techniques probe dierent aspects of the loss of order of the can be proposed that the temperature dependence of this
granule, it was found that the extent of loss of order as a loss in crystallinity is dependent on the crystalline poly-
function of temperature was broadly similar. This conclu- morph present, its degree of perfection, the length of chain
sion was consistent with a more recent study [62] which involved in the crystalline unit, and the diluent content
followed the loss of crystallinity of maize starch, as a func- (with increasing diluent content reducing the temperature
tion of temperature, using a synchrotron source of x-rays. of the transition) [67]. As an individual amylopectin mol-
In this case, in addition to the disappearance of the native ecule can participate in a number of crystalline domains or
crystalline structure, it was also possible to monitor the lamellae it is the local diluent content which is important.
crystallization of starch as a lipid complex at higher tem- There are a number of models of gelatinization [46] that
peratures. In addition to the loss of crystalline order, the give somewhat dierent emphasis to the relative impor-
loss of lamellar order has also been followed as a function tance of these factors. For example, it has been proposed
of temperature [46,52]. In the initial stages of gelatinization that the hydration of the amorphous regions of the granule
there is no marked shift in the peak in the scattering prole, is strongly temperature dependent, and that at a certain
and therefore no marked change in lamellar spacing. The temperature there is a large change in the water content of
feature does, however, become weaker. Finally, another these regions, with a result that the local diluent content is
study examined order loss by birefringence measurements, suciently high that gelatinization can occur. It has been
dierential scanning calorimetry, and x-ray diraction. It suggested that this hydration is suciently strong to over-
was found that the loss of birefringence occurred over a come the forces holding the crystalline lattice together. The
narrower temperature range than the loss of crystalline physical origin of this marked change in hydration has been
order [63,64]. In conclusion, there is a broad consensus attributed to exceeding a glass transition [68] (see later), or
that, for a population of granules, all the forms of order simply that the anity of the amorphous regions of starch
(crystalline, lamellar, orientational) are lost or modied for water shows a strong temperature dependence. Esti-
over a broadly similar temperature range. To further assess mates of the average water content of a hydrated starch
order loss as a function of temperature it is necessary to granule are in the region of 2733% [65]. For this average
consider heterogeneity in structure over the population composition the glass transition occurs below ambient
studied. For example, in pea starches that give a C-type temperatures [69]. Furthermore, solid-state NMR studies
diraction pattern (a mixture of the A and B forms) it was on granular starch have shown that the amorphous frac-
found that the B form was present in the interior of the tion is plasticized into a rubbery state at ambient temper-
granule and the A form in the periphery [41]. On gelatini- atures [52]. Generally, water is a poor solvent for the starch
zation, birefringence is initially lost from the interior of the polysaccharides and experiments which have measured the
granule followed by the periphery. On examination of the anity of the starch chain for water show a weak temper-
loss in crystallinity it is found that the B-type crystallinity is ature dependence [70]. These observations suggest that the
lost rst. A population of granules might be expected to amorphous fraction of starch may not have a dominant
show further heterogeneity. For example, in wheat starch role in controlling gelatinization.
the distribution in granule sizes is bimodal and the dierent One of the essential elements of the gelatinization
sizes might display somewhat dierent gelatinization be- process is the loss of crystalline order [66]. To gain insight
havior. A further source of heterogeneity during gelatini- into the factors aecting this loss of order one approach is
zation occurs as a result of concentration gradients. As the to examine the behavior of highly crystalline materials,
population of starch granules gelatinizes over a limited preferably single crystals of macroscopic size. Short linear
596 Parker and Ring
chains of amylose crystallize readily and under appropriate 50% to 5% w/w water is remarkably similar [75] (Fig. 2),
conditions highly crystalline materials of the dierent suggesting that dissolution experiments on very crystalline
polymorphic forms of starch are obtained [2022,71]. The materials provide useful insight into the very much more
eects of diluent content, and polymorphic form, on the complex phenomenon of gelatinization. A further indica-
temperature dependence of dissolution have been exam- tion of usefulness is gained from experiments on smooth-
ined [72]. It is also possible to examine the eect of chain seeded pea starches [42] the granules of which contain a
length on the process, with the expectation that the longer mixture of the A and B crystalline forms of starch. The
the length of chain involved in the crystalline unit the prediction that the dissolution of the B domains should
higher the melting temperature [73]an expectation which occur at a lower temperature than the A domains was
was conrmed experimentally [74]. A schematic diagram of conrmed by x-ray diraction experiments on the gelati-
the expected dependence of the melting of A-type crystal- nization process.
lites on water content is shown in Fig. 2. Also included is an Although the spherulites formed from the short amy-
estimate of how the dissolution temperature varies with losic chains are somewhat analogous to the starch granule,
increasing chain length. The melting temperature of the particularly for the B spherulites which are formed from
low water content materials is experimentally inaccessible the stacking of crystalline lamellae [76], they dier in at
due to thermal degradation. In common with polymeric least one important respect. In the case of the spherulite,
crystalline materials [59,67], the addition of a diluent, in disruption of the crystalline lattice leads to a solubilization
this case water, depresses the observed melting tempera- of the amylosic chain. In the granular case, disruption of a
ture, Tm. For an A-type starch crystallite formed from crystalline domain will only lead to a partial solubilization
chains 12 units in length, the expected peak melting tem- because the amylopectin molecule spans dierent domains
perature at high water contents is about 75jC. As a or lamellae. The initial partial solubilization of a crystal-
comparison, the predicted high molecular weight limit of line domain increases the local water content of the
Tm in excess water is 150jC. With decreasing water con- granule facilitating the subsequent dissolution of crystal-
tent, the melting temperature rises, reaching a predicted line material.
150jC for chains 12 units in length at a water content of For native starches, the length of chain of the short
16% w/w. The polymorphic form of starch also aects the chain fraction and the crystalline form of the granule are
dissolution behavior. At a xed water content, the melting associated [11,77]. It is found that starches that have a
and dissolution of the B-form occurs at f20jC lower relatively short short-chain fraction, e.g., wheat and maize
temperature [72]. For the crystallites in wheat starch the starch, have an A crystalline form whereas starches that
gelatinization/dissolution temperature in excess water is in have a longer short-chain fraction, e.g., potato starch, have
the region of 60jC rather than the 75jC of the highly a B crystalline form. In vitro experiments on short amylosic
crystalline material, probably indicating an eect of crystal chains reveal the same type of behavior with the shorter
perfection/size on the observed dissolution. The form of chains crystallizing in the A form and longer chains in the B
the increase with decreasing water content in the range form [20,21]. Crystallization at small undercoolings, below
the equilibrium dissolution temperature of the crystallites,
favors the production of A form whereas a large under-
cooling favors the production of the B form. This suggests
that the B form is a kinetic product. The associations
between amylopectin chain length and crystalline form
suggest that these considerations remain relevant to gran-
ule assembly. Both crystal type and chain length involved
in the crystal aect dissolution. One strategy to design a
cereal starch (A-type crystalline form) with a reduced
gelatinization temperature might be to reduce the chain
length of the short chain fraction of amylopectin. If a
higher gelatinization temperature was required, then in-
creasing the chain length of the short chain fraction might
initially lead to a depression in gelatinization temperature,
as the crystalline form changed from A to B, followed by
a subsequent increase as the chain length eect started
to dominate.
Figure 2 The eect of water content on the melting and

VI. INTERACTIONS WITH OTHER MOLECULES
glass transitions of starch. Key: solid line, estimates of the
melting temperature of A-type short-chain amylose crystals A. Low Molecular Weight Species
with degree of polymerization (DP) 10, 12, 14 [61,63]; o,
.
glass transition of pregelatinized starch [55]; , melting To start to understand the role of water as a diluent, it is
transition of granular wheat starch [64]. useful to examine predictive relationships describing the
composition dependence of melting [59,66]. The classical procedure for its purication from molecularly disperse
description of the compositional dependence of polymer solutions of amylose and amylopectin.
melting in the presence of a diluent is given by The crystalline material consists of single helices of
amylose which are packed in a hexagonal array if prepared
1=Tm 1=Tm0 R=DHu
Vu =V1
v1 vv12 1 from aqueous solution [83]. The single helices may be both
parallel or antiparallel. Under more dehydrating condi-
tions an orthorhombic unit cell is produced [84], which can
where Tm0 is the melting temperature of the pure polymer, be converted to the more hydrated hexagonal form (VH). It
Vu and V1 are the molar volumes of polymer repeating unit is interesting to note that a cycloamylose (cyclomaltohexa-
and diluent, respectively, and v1 is the diluent volume icosaose, CA26) readily forms the V-amylose helices which
fraction. DHu is the enthalpy of fusion per repeating unit fold back on themselves to generate an antiparallel pack-
and v is the FloryHuggins interaction parameter [78] ing. This conformational exibility of the single helical
characterizing the interaction energy per solvent molecule. conformation helps explain why this form of amylose
This relationship predicts that the smaller the diluent readily forms chain-folded crystals [85].
size relative to that of the polymer repeating unit, in this The interior of the amylose single helix is somewhat
case an anhydroglucose unit, and the stronger the favor- hydrophobic as it is lined with the CH groups of carbons
able interaction between the diluent and polymer, the 2, 3, and 5 of the D-glucose unit. Molecular modeling
stronger the depression in Tm. It is well known that the studies show that the interior of the helix is suciently
starch polysaccharides precipitate from aqueous solution large to be able to accommodate a linear hydrocarbon
at room temperature, indicating that water interacts weak- chain, although charged headgroups can disrupt the helical
ly with the starch chain and at room temperature it can be conformation [8688].
considered a poor solvent. Physicochemical measurements
on water sorption behavior can provide more quantitative
estimates of solvent quality. This requires equilibrium C. Biopolymer/Biopolymer Interactions
behavior which can be achieved if the behavior of oligo- A further aspect of phase behavior that should be consid-
saccharides is examined [74]. For polysaccharides nonequi- ered is the miscibility of biopolymer mixtures. The immis-
librium eects generally dominate [79], particularly for cibility of concentrated solutions of chemically dierent
compositions that are glassy at the temperature of mea- synthetic polymers is a well-described phenomenon and
surement. The values of the parameter v obtained for can result from either an unfavorable energetic interaction
concentrated aqueous mixtures of maltooligomers were between the polymers [59], or the solvent having very
in the range 0.7 to 0.8 at room temperature [74], conrming dierent interactions with the dierent polymers [89]. Bio-
that water is a poor solvent under these conditions. To polymers show the same general type of behavior with
rene this approach more information is needed on the aqueous mixtures of proteins and polysaccharides [90], and
temperature and composition dependence of v. The rela- dierent polysaccharides [91,92] exhibiting immiscibility.
tively small molecular size of water is predicted to lead to a At equilibrium, macroscopic phase separation is observed
strong depression in Tm. It has been found that the Flory with the formation of separate layers enriched in each of
Huggins approach ts the available experimental data for the biopolymers. This macroscopic phase separation can
starch crystallite dissolution reasonably well. The predic- be arrested, for example by gelation of one of the compo-
tion that replacement of water by other larger water- nents, and as a result useful textures can be produced. Even
soluble solutes that interact weakly with the starch chain, the two chemically similar starch biopolymers amylose and
such as low molecular weight carbohydrates, would lead to amylopectin, diering primarily in their extent of branch-
an elevation of Tm is also conrmed experimentally [80,81]. ing, phase separate from concentrated solution [91]. This is
an example of quite a small dierence in molecular struc-
B. Hydrophobic Species ture producing a potentially large eect on the microstruc-
ture of the mixed biopolymeric material. The way that
If a hot aqueous solution of amylose is saturated with 1- solvent distributes itself between phases as a result of
butanol prior to cooling, a precipitate is formed which will their relative anity for water can have an important
dissolve on heating to 100jC. This precipitate consists of impact on material properties. For example, if the same
amylose in a single helical form and can contain the 1- amounts of two glucan polymers amylose and dextran are
butanol as a complexed guest molecule3. On the basis of mixed in concentrated solution, at a temperature where
x-ray diraction experiments, the complex may be either crystallization is not observed on practical timescales,
crystalline or amorphous [82]. The amorphous material is phase separation is observed with the formation of a
formed on rapid cooling whereas slow cooling favors the dextran-rich and amylose-rich phases [92]. The phase
formation of crystalline material. Depending on the hy- volume of the dextran phase is much larger than that of
drophobic compound added, (e.g., if it is charged) it is also the amylose phase, i.e., water has a preferred interaction
possible to induce a conformational change to a helical with the dextran. The material properties of this biphasic
form which does not readily crystallize or precipitate. The system will depend on the phase volumes and polymer
precipitation of amylose with 1-butanol is a standard concentrations in each phase. There will also be an eect on
598 Parker and Ring
stability. For example, consider a concentrated solution with low molecular weight hydroxy compounds such as
containing 40% w/w water with 30% w/w each of dextran glycerol [98101] or sorbitol [102], often in combination
and amylopectin at room temperature. If the water was with water. Mixing 29% w/w glycerol with amorphized
distributed evenly then storage at room temperature for barley starch containing a minimal quantity of water (1%)
this composition would represent a quench of 80jC for depressed the glass transition to 70jC [103]. Glycerol is
chains 12 units in length (Tmf100jC), and the amylopec- therefore a less eective plasticizer than water. Initial
tin in the amylopectin-rich phase would tend to crystallize. additions of glycerol to starch resulted in a depression of
If the amylopectin phase was phase-concentrated, by the a single glass transition. As water was added this glass
presence of dextran, then the resulting increase in amy- transition was further depressed and an additional lower
lopectin concentration would increase the driving force glass transition appeared, suggesting that the ternary
for crystallization. starch/glycerol/water mixture phase separated. The lower
Tg was close to the glass transition of pure glycerol. There
are a number of relationships which describe the way that
VII. GLASS TRANSITION BEHAVIOR the Tg of mixed binary systems should vary as a function of
composition, and these have found use in describing the
Included in the schematic of Fig. 2 is another transition behavior of starch mixtures. One such is due to Couchman
which has an important inuence on material properties [104,105] and is of the form
[93,94]the glass transition which is characterized by the
glass transition temperature Tg. Crystalline anhydrous h-
Tgm w1 DCp1 Tg1 w2 DCp2 Tg2
D-glucose melts at 150jC, if this melt is cooled at a rate that 2
is rapid compared to the rate of crystallization, then the = w1 DCp1 w2 DCp2
viscosity of the liquid will progressively increase until at
7jC the viscosity reaches about 1012 Pa sec [95]. At these The glass transition temperature of the mixture, Tgm, is
enormous viscosities the material behaves as a brittle solid. related to the glass transition temperatures of the individ-
Glasses may be stable to crystallization for many years, the ual components [106,107] and the heat capacity increment
enormous viscosity of the glass arresting crystal nucleation at the glass transition, DCpi, and wi is the mass fraction of
and growth. At the calorimetric Tg a sharp change in heat the ith component. Replacing water that has a relatively
capacity is observed, indicative of a change from solid-like low Tg (139jC) with glycerol (Tg 80jC) [108] would
to liquid-like behavior within the timescale of the calori- lead to an elevation in the Tg of the mixture. Recently,
metric experiment. This provides an experimentally con- experiments were reported where the development of the x-
venient method for the determination of Tg as the midpoint ray diraction pattern of granular starches, on solvation in
of the step change. The calorimetric approach is useful for the presence of ethylene glycol and glycerol, was measured
the determination of the glass transition behavior of simple as a function of time [109]. It was found that the develop-
amorphous biopolymer mixtures. As the degree of poly- ment of crystalline order took several hours at room
merization of the glucan chain increases there is an increase temperature for ethylene glycol, and several days for
in glass transition temperature, reaching 173jC for malto- glycerol. As the relative ability of the diluents to depress
hexaose [96]. Addition of water to the carbohydrates Tg would be in the order water > ethylene glycol >
depresses the glass transition, as illustrated in Fig. 2, which glycerol, the observations are consistent with the view that
shows the composition dependence of the Tg of a starch/ replacement of water by glycerol would elevate the Tg of
water mixture [69]. The Tg of the dry polymer is experi- the mixture, and as a consequence, at a xed temperature
mentally inaccessible for familiar reasonsthermal degra- slow the rate of development of crystalline order.
dation intervenes. The addition of water has a strong At low water contents, the Tg of the starch/water
plasticizing eect, causing a marked depression in Tg, until mixture is very sensitive to small changes in water content
at 20% w/w water, Tg reaches room temperature. More and it follows that some of the material properties are
recently, a more detailed study on the glass transition equally sensitive. At Tg the structural relaxation time is of
behavior of dierent glucan polymers including amylose, the order of 100 sec. This structural relaxation time is
amylopectin, pullulan, and dextran has been reported [97]. relevant to mechanical behaviorhow quickly a material
While all the glucans behave in the same general way, in should relax after a mechanical perturbationand poten-
that water is a very eective plasticizing agent, there are tially transport properties within the material [110]. As a
subtle dierences which lead to a glass transition range result there is interest in knowing not only Tg but also how
approaching 30j at a water content of 10% w/w. The the associated structural relaxation time will depend on
branching of polymeric materials is thought to depress Tg temperature and composition. Fortunately for most amor-
as a result of an internal plasticization from the short chain phous organic materialslow molecular weight liquids,
branches. Although water is a ubiquitous plasticizer of synthetic polymers, and biopolymers (including exible
starchy materials, it is volatile, and small changes in its proteins such as gluten and polysaccharides), the expected
content can lead to large changes in mechanical behavior. dependence of structural relaxation time on temperature is
For this reason, the use of other, nonvolatile, plasticizers broadly similar. An expression which has been shown to be
has been examined. Most studies have been carried out widely applicable in describing this temperature depen-
dence for synthetic polymers is the WilliamsLandelFerry motion from the shear viscosity with diusion being much
relationship [111] more rapid than expected [112114]. The examination of
the dynamics of these mixtures reveals other relaxations
log aT c1;0 T T0 =c2;0 T T0 3 as well as the main structural relaxation [115,116]. Their
eect on material properties of starchy materials remains
where aT is the ratio of relaxation times at the temperature, to be established.
T, and a reference temperature T0. If Tg becomes the
reference temperature then,
log aT c1g T Tg =c2g T Tg 4 VIII. TIME-DEPENDENT CHANGE IN AQUEOUS
with values of the coecients, c1g and c2g, obtained from STARCH SYSTEMS
tting data on a range of synthetic polymers, being 17.44
A. Structural Relaxation
K1 and 51.6 K, respectively. At the calorimetric glass
transition the shear viscosity, g, is of the order of 1012 Pa sec If the structural relaxation is suciently slow, it can lead to
and the shear stress relaxation time, s, is of the order of 100 time-dependent changes in the properties of glassy materi-
sec, and is given by s = g/Gl, where Gl is the high- als over practical timescales of hours to weeks [117
frequency limit of the shear modulus which is 1010 Pa for 119,119125] and is potentially relevant to the observed
many materials. The temperature and composition depen- ageing of low water content carbohydrate products [126
dence of the relative relaxation time, aT, is shown schemat- 128]. The equilibrium structure of an undercooled liquid
ically in Fig. 3 for an amorphous starch/water mixture. As is temperature dependent. For example it is expected that
the glass transition is approached, either through reducing reducing temperature would increase the density of a
the temperature or the water content, there is predicted to material. As a liquid is undercooled toward the glass
be a very marked change in relaxation behavior. Structural transition its viscosity increases as does the associated
relaxation and molecular mobility slows and, as a result, structural relaxation time. If an amorphous material is
there is a very marked change in material properties for a rapidly quenched into the glass state, the structural relax-
relatively small change in either water content or temper- ation time may be so high that the amorphous structure,
ature. For a more detailed discussion on the use of Eq. 3 to and resulting density, is eectively frozen. The structure
describe the behavior of synthetic polymers the reader is will gradually evolve and at very long times it will have a
referred to the work of Ferry [111]. The use of this fully relaxed equilibrium structure. If it is cooled again,
approach to predict temperature-dependent mechanical further structural relaxations and rearrangements within
behavior is useful. It could also be extended to make some the undercooled liquid will occur until, given sucient
prediction of how transport properties should vary with time, a new equilibrium structure is obtained. The
temperature but care is needed in the application of this structure of the undercooled amorphous polymer liquid
approach. For example, there is an expectation that the can therefore show a dependence on time and thermal
increase in viscosity/structural relaxation time as an under- history. This relaxation behavior has been extensively
cooled liquid approaches the glass transition would slow studied, particularly for polymers and inorganic glasses.
diusion. While this is the case for a macroscopic particle The temperature-dependent changes in bonding between
embedded in a highly viscous matrix, for smaller molecules molecules and their conguration are associated with
such as water there can be a marked uncoupling of its changes in volume, enthalpy, heat capacity, and material
properties, including mechanical behavior and diusivity.
The observed changes in mechanical behavior of amor-
phous glasses with time are often described as embrittle-
ment, as the material becomes stier and less compliant. At
Tg there is a sharp change in behavior as the liquid structure
becomes arrested over the timescale of the cooling. If the
cooling is slower, there is more time available for the
structure to relax and the appearance of Tg will occur at
a lower temperature. Liquid structure can be characterized
on a temperature scale through the notion of a ctive
temperature, Tf, the temperature at which a particular
structure would be fully relaxed.
Although the increase in density of a liquid with time is
one way of probing this structural relaxation, its use is
rather restricted. As densication also aects the energetics
of interaction between molecules, and the accessibility of
Figure 3 Eect of temperature and water content upon the liquid congurations, it can be probed in a calorimetric
relaxation time calculated from the glass transition curve [55] experiment where structural relaxation is observed as a
and assuming WLF behavior [93]. peak in heat capacity preceding Tg or an overshoot at Tg.
600 Parker and Ring
There is often a requirement to be able to predict the consist of assemblies of many individual polymer strands
change in the material properties of a glassy product with [135]. Subsequent to this precipitation, a slow crystalliza-
time. Common questions might be at what temperature tion of the amylose is observed by x-ray diraction over the
do I need to store the product to minimize this time- course of a few days. This crystallization does not have a
dependent change?, how might uctuations in water marked eect on material properties; at the end of this
content aect the observed behavior?. Fortunately, there time, these materials are still poorly crystalline. One pro-
are various phenomenological approaches for describing posal for the structure of the initial aggregate is that it is
the observed time-dependent behavior; a widely applied formed from assemblies of double helices [134] with a
one, which has a useful predictive capability, is the Tool typical dimension, orthogonal to the helix axis, of 1020
Narayanaswamy method [122], which has been applied to nm [136]. These structures further aggregate to form a
polymeric systems. The dependence of structural relaxa- fractal structure. The aggregation process has also been
tion on time, t, can be described by an empirical relaxation likened to a phase separation in which polymer-rich and
function, /, of the form polymer-decient domains are formed and if the amylose is
suciently concentrated the polymer-rich domains will
/t expt=s0 b form an interconnected network [131]. The amylose gel is
far from equilibrium and its structure will be very depen-
and b (0 < b V 1) is a measure of its nonexponentiality. s0 is dent on the condition of its preparation.
a characteristic time which is dependent on both tempera- The eective quench for the formation of crystallites
ture, T, and, to an extent, liquid structure (characterized by from short linear chains is much more modest. If concen-
Tf) and has been successfully obtained using the expression trated amylopectin/water mixtures are quenched to room
temperature or below, a slow crystallization, as assessed by
s0 A expxDh*=RT 1 xDh*=RTf
x-ray diraction, of the amylopectin is observed. In this
where A, x (0 < x V 1), and Dh* are constants. Dh* can be case the crystallization is associated with the development
determined from the dependence of the calorimetric Tg on of stiness of the material and typically, at high water
scanning rate. These relationships can be used to calculate contents, takes days or weeks to approach a plateau value
the time dependence of Tf following a temperature step and [33,137]. At the end of this time the extent of crystallinity of
from this the heat capacity change with temperature can be the amylopectin is comparable to that found in the native
predicted. By appropriate selection of the constants A, x, starch granule, i.e., in the region of 30%. The length of
and b the experimentally observed behavior may be mod- chain involved in the crystalline unit is relatively short. On
eled. For a simple carbohydrate, such as maltose [129], it examination of the behavior of amylopectins from dierent
was found that a single set of constants described the botanical sources it was observed that the longer the length
dependence of ageing on time and temperature and had a and abundance of the short chain fraction of amylopectin
useful predictive utility. More recently, it was found that the greater the tendency to retrogade and crystallize from
the above approach was useful in describing the ageing of a aqueous solution [137139]. Wheat amylopectin, which has
plasticized starchy material. a relatively short, short chain fraction, generally shows a
reduced tendency to retrograde, even so it can lead to very
B. Retrogradation of Starch signicant time-dependent changes in material properties.
In a detailed examination of the retrogradation of maize
If a gelatinized starch/water mixture is cooled to room amylopectins it was found that retrogradation was propor-
temperature there is a strong driving force favoring crys- tional to the amount of short chains having a DP of 1630
tallization. The processes that occur on cooling are gener- and inversely proportional to the level of short chains with
ally known as retrogradation [33,130134]. In the case of a DP of 611 [139]. Treatment of starch with h-amylase (an
the high molecular weight linear polymer, amylose, the exo-acting enzyme) shortens the short chain fraction and
driving force is strong, with an eective quench of at least reduces the rate of retrogradation [140].
120jC if the material is held at room temperature. Such a The rate of retrogradation is expected to be at a
strong quench is not conducive to the formation of very maximum approximately midway between the melting
crystalline materials. In fact, if a concentrated aqueous temperature of the crystallites, Tm, and the glass transition
amylose solution is cooled to room temperature there is a temperature, Tg. This reects a balance between the eect
very rapid precipitation/phase separation process. The of temperature on the driving force favoring crystalliza-
clear amylose solution rapidly becomes opaque indicating tion, and the slowing of mobility as the glass transition is
the formation of polymer aggregates. To produce the approached. Bell-shaped curves of extent of retrograda-
opacity the aggregates are at least the order of the wave- tion vs. temperature are observed supporting this sugges-
length of light in size. At the same time, for suciently tion [141].
concentrated solutions (typically greater than 1% to 2% For materials prepared by gelatinizing starch and then
w/w), network formation is observed through the forma- cooling to room temperature the precipitation of amylose
tion of an elastic gel. Network formation, as assessed by the is a very rapid process. The addition of a hydrophobic
development of stiness, is generally complete within a few compound prevents the formation of the B-crystalline form
hours at room temperature. Electron microscopic exami- of amylosethe single helical complex is the preferred
nation of the network reveals coarse network strands which product [86,88,142]. A further indication of this preference
is the observation that the amylose complex forms imme- Molecular-structures of some wheat starches. Carbohydr.
diately after gelatinization [62]. As the B form of amylose Polym. 1994, 25, 111.
10. Manners, D.J. Recent developments in our understanding
is thermally more stable than the single helical V form
of amylopectin structure. Carbohydr. Polym. 1989, 11, 87.
this might again indicate the role of kinetics in inu- 11. Hizukuri, S. Relationship between the distribution of the
encing crystallization. chain-length of amylopectin and the crystalline-structure
The crystallization of amylopectin leads to an increase of starch granules. Carbohydr. Res. 1985, 141, 295.
in stiness of the gelatinized granule and a slow rming of 12. Koizumi, K.; Fukuda, M.; Hizukuri, S. Estimation of the
the material. Amylopectin crystallization in gels and the distributions of chain-length of amylopectins by high-
associated rming can be abolished by reheating to 60jC. performance liquid-chromatography with pulsed ampero-
metric detection. J. Chromatogr. 1991, 585, 233.
13. Hizukuri, S.; Maehara, Y. Fine-structure of wheat
amylopectinthe mode of A-chain to B-chain binding.
IX. CONCLUSION Carbohydr. Res. 1990, 206, 145.
14. Hanashiro, I.; Abe, J.; Hizukuri, S. A periodic distribu-
Although starch has long been studied from a polymeric tion of the chain length of amylopectin as revealed by
perspective, recent research has added to this knowledge high-performance anion-exchange chromatography. Car-
more particularly in terms of its phase behavior and bohydr. Res. 1996, 283, 151.
dynamics, and how they are modied by the presence of 15. Takeda, Y.; Maruta, N.; Hizukuri, S.; Juliano, B.O.
other compounds. This information is relevant to the Structures of indica rice starches (ir48 and ir64) having
intermediate anities for iodine. Carbohydr. Res. 1989,
practical usage of processed starch materials which are 187, 287.
generally metastable. To devise strategies for the control of 16. Takeda, Y.; Hizukuri, S.; Juliano, B.O. Structures of rice
material properties, and their stability, it is practically amylopectins with low and high anities for iodine.
useful to know where the system is going (its underlying Carbohydr. Res. 1987, 168, 79.
phase behavior) and how quickly it is going to get there 17. Schulman, A.H.; Tomooka, S.; Suzuki, A.; Myllarinen,
(inuenced by its dynamics). Important advances are also P.; Hizukuri, S. Structural-analysis of starch from normal
and shx (shrunken endosperm) barley (hordeum-vulgare).
being made on the levels of organization of the granule. Carbohydr. Res. 1995, 275, 361.
During the same period our understanding of starch 18. Bertoft, E.; Manelius, R. A method for the study of the
biosynthesis has also increased markedly. The processabil- enzymatic-hydrolysis of starch granules. Carbohydr. Res.
ity of starch is strongly inuenced by granule organization. 1992, 227, 269.
An important problem for the future is the biochemical 19. Bertoft, E. Investigation of the ne-structure of amylo-
control of granule assembly which will involve research pectin using alpha-amylase and beta-amylase. Carbohydr.
Res. 1989, 189, 195.
from both biological and physical perspectives.
20. Pfannemuller, B. Inuence of chain-length of short mono-
disperse amyloses on the formation of A-type and B-type x-
ray-diraction patterns. Int. J. Biol. Macromol. 1987, 9,
ACKNOWLEDGMENT 105.
21. Gidley, M.J.; Bulpin, P.V. Crystallization of malto-
The authors acknowledge the support of the core strategic oligosaccharides as models of the crystalline forms of
grant of the BBSRC. starchminimum chain-length requirement for the for-
mation of double helices. Carbohydr. Res. 1987, 161, 291.
22. Ring, S.G.; Miles, M.J.; Morris, V.J.; Turner, R.; Colonna,
P. Spherulitic crystallization of short chain amylose. Int. J.
REFERENCES
Biol. Macromol. 1987, 9, 158.
1. Buleon, A.; Colonna, P.; Planchot, V.; Ball, S. Starch 23. Miles, M.J.; Morris, V.J.; Ring, S.G. Gelation of amylose.
granules: structure and biosynthesis. Int. J. Biol. Macro- Carbohydr. Res. 1985, 135, 257.
mol. 1998, 23, 85. 24. Moates, G.K.; Noel, T.R.; Parker, R.; Ring, S.G. The eect
2. Thompson, D.B. On the non-random nature of amylo- of chain length and solvent interactions on the dissolution
pectin branching. Carbohydr. Polym. 2000, 43, 223. of the B-type crystalline polymorph of amylose in water.
3. Banks, W.; Greenwood, C.T. Starch and its Components; Carbohydr. Res. 1997, 298, 327.
Edinburgh University Press: Edinburgh, 1975. 25. Roger, P.; Axelos, M.A.V.; Colonna, P. SEC-MALLS
4. Takeda, Y.; Tomooka, S.; Hizukuri, S. Structures of and SANS studies applied to solution behavior of linear
branched and linear-molecules of rice amylose. Carbo- alpha-glucans. Macromolecules 2000, 33, 2446.
hydr. Res. 1993, 246, 267. 26. Roger, P.; Bello-Perez, L.A.; Colonna, P. Contribution of
5. Takeda, Y.; Maruta, N.; Hizukuri, S. Structures of amy- amylose and amylopectin to the light scattering behaviour
lose subfractions with dierent molecular sizes. Carbo- of starches in aqueous solution. Polymer 1999, 40, 6897.
hydr. Res. 1992, 226, 279. 27. Roger, P.; Colonna, P. Molecular weight distribution of
6. Hizukuri, S. Properties of hot-water-extractable amylose. amylose fractions obtained by aqueous leaching of corn
Carbohydr. Res. 1991, 217, 251. starch. Int. J. Biol. Macromol. 1996, 19, 51.
7. Takeda, Y.; Shitaozono, T.; Hizukuri, S. Structures of 28. Roger, P.; Colonna, P. Evidence of the presence of large
subfractions of corn amylose. Carbohydr. Res. 1990, 199, aggregates contaminating amylose solutions. Carbohydr.
207. Polym. 1993, 21, 83.
8. Takeda, Y.; Shirasaka, K.; Hizukuri, S. Examination of 29. Aberle, L.B.; Kleemeier, M.; Hennemann, O.D.; Burch-
the purity and structure of amylose by gel-permeation ard, W. Selection of single scattering from multiple
chromatography. Carbohydr. Res. 1984, 132, 83. scattering systems by 3D cross-correlation: 2. Concen-
9. Shibanuma, K.; Takeda, Y.; Hizukuri, S.; Shibata, S. trated polymer solutions. Macromolecules 2002, 35, 1877.
602 Parker and Ring
30. Aberle, T.; Burchard, W. Starches in semidilute aqueous 52. Waigh, T.A.; Gidley, M.J.; Komanshek, B.U.; Donald,
solution. Starch-Starke 1997, 49, 215. A.M. The phase transformations in starch during
31. Flory, P.J. Statistical Mechanics of Chain Molecules; Inter- gelatinisation: a liquid crystalline approach. Carbohydr.
science Publishers: New York, 1969. Res. 2000, 328, 165.
32. Shimada, J.; Kaneko, H.; Takada, T.; Kitamura, S.; 53. Oostergetel, G.T.; Vanbruggen, E.F.J. The crystalline
Kajiwara, K. Conformation of amylose in aqueous domains in potato starch granules are arranged in a
solution: Small-angle X-ray scattering measurements and helical fashion. Carbohydr. Polym. 1993, 21, 7.
simulations. J. Phys. Chem. B 2000, 104, 2136. 54. Gallant, D.J.; Bouchet, B.; Baldwin, P.M. Microscopy of
33. Ring, S.G.; Colonna, P.; Ianson, K.J.; Kalichevsky, M.T.; starch: Evidence of a new level of granule organization.
Miles, M.J.; Morris, V.J.; Orford, P.D. The gelation and Carbohydr. Polym. 1997, 32, 177.
crystallization of amylopectin. Carbohydr. Res. 1987, 162, 55. Ohtani, T.; Yoshino, T.; Ushiki, T.; Hagiwara, S.;
277. Maekawa, T. Structure of rice starch granules in nano-
34. Imberty, A.; Chanzy, H.; Perez, S.; Buleon, A.; Tran, V. metre scare as revealed by atomic force microscopy. J.
The double-helical nature of the crystalline part of a- Electron Microsc. 2000, 49, 487.
starch. J. Mol. Biol. 1988, 201, 365. 56. Mizukami, H.; Takeda, Y.; Hizukuri, S. The structure of
35. Wu, H.-C.H.; Sarko, A. The double-helical molecular the hot-water soluble components in the starch granules of
structure of crystalline B-amylose. Carbohydr. Res. 1978, new Japanese rice cultivars. Carbohydr. Polym. 1999, 38,
61, 7. 329.
36. Imberty, A.; Perez, S. A revisit to the 3-dimensional 57. Ellis, H.S.; Ring, S.G.; Whittam, M.A. A comparison of
structure of b-type starch. Biopolymers 1988, 27, 1205. the viscous behavior of wheat and maize starch pastes.
37. Guilbot, A.; Mercier, C. Starch. In The Polysaccharides; J. Cereal Sci. 1989, 10, 33.
Aspinall, G.O., Ed.; Academic Press: New York, 1985. 58. Lelievre, J. Eects of sugars on the swelling of crosslinked
38. French, D. Organization of starch granules. In Starch potato starch. J. Colloid Interface Sci. 1984, 101, 225.
Chemistry and Technology; Whistler, R.L., Paschall, E.F., 59. Flory, P.J. Principles of Polymer Chemistry; Cornell
BeMiller, J.N., Eds.; Academic Press: New York, 1984. University Press: Cornell, 1953.
39. Buleon, A.; Pontoire, B.; Riekel, C.; Chanzy, H.; Helbert, 60. Doublier, J.L.; Llamas, G.; Lemeur, M. A rheological
W.; Vuong, R. Crystalline ultrastructure of starch investigation of cereal starch pastes and gelseect of
granules revealed by synchrotron radiation microdirac- pasting procedures. Carbohydr. Polym. 1987, 7, 251.
tion mapping. Macromolecules 1997, 30, 3952. 61. Cooke, D.; Gidley, M.J. Loss of crystalline and molecular
40. Waigh, T.A.; Hopkinson, I.; Donald, A.M.; Butler, M.F.; order during starch gelatinizationorigin of the enthalpic
Heidelbach, F.; Riekel, C. Analysis of the native structure transition. Carbohydr. Res. 1992, 227, 103.
of starch granules with X-ray microfocus diraction. 62. Le Bail, P.; Bizot, H.; Ollivon, M.; Keller, G.; Bourgaux,
Macromolecules 1997, 30, 3813. C.; Buleon, A. Monitoring the crystallization of amylose
41. Buleon, A.; Gerard, C.; Riekel, C.; Vuong, R.; Chanzy, H. lipid complexes during maize starch melting by synchro-
Details of the crystalline ultrastructure of C-starch tron x-ray diraction. Biopolymers 1999, 50, 99.
granules revealed by synchrotron microfocus mapping. 63. Liu, H.; Lelievre, J. A model of starch gelatinization
Macromolecules 1998, 31, 6605. linking dierential scanning calorimetry and birefringence
42. Bogracheva, T.Y.; Morris, V.J.; Ring, S.G.; Hedley, C.L. measurements. Carbohydr. Polym. 1993, 20, 1.
The granular structure of C-type pea starch and its role in 64. Liu, H.; Lelievre, J. A dierential scanning calorimetry
gelatinization. Biopolymers 1998, 45, 323. study of glass and melting transitions in starch suspen-
43. Gidley, M.J.; Bociek, S.M. Molecular-organization in sions and gels. Carbohydr. Res. 1991, 219, 23.
starchesa C-13 CP MAS NMR-study. J. Am. Chem. 65. Liu, H.; Lelievre, J.; Ayoungchee, W. A study of starch
Soc. 1985, 107, 7040. gelatinization using dierential scanning calorimetry, X-
44. Gidley, M.J. Quantication of the structural features of ray and birefringence measurements. Carbohydr. Res.
starch polysaccharides by nmr-spectroscopy. Carbohydr. 1991, 210, 79.
Res. 1985, 139, 85. 66. Donovan, J.W. Phase transitions in the starchwater
45. Blanshard, J.M.V.; Bates, D.R.; Muhr, A.H.; Worcester, system. Biopolymers 1979, 18, 263.
D.L.; Higgins, J.S. Small-angle neutron scattering studies of 67. Prasad, A.; Mandelkern, L. Equilibrium dissolution
starch granule structure. Carbohydr. Polym. 1984, 4, 427. temperature of low-molecular weight polyethylene frac-
46. Jenkins, P.J.; Donald, A.M. Gelatinisation of starch: a tions in dilute-solution. Macromolecules 1989, 22, 914.
combined SAXS/WAXS/DSC and SANS study. Carbo- 68. Slade, L.; Levine, H. Non-equilibrium melting of native
hydr. Res. 1998, 308, 133. granular starch: 1. Temperature location of the glass
47. Jenkins, P.J.; Donald, A.M. Application of small-angle transition associated with the gelatinization of A-type
neutron scattering to the study of the structure of starch cereal starches. Carbohydr. Polym. 1988, 8, 183.
granules. Polymer 1996, 37, 5559. 69. Zeleznak, K.J.; Hoseney, R.C. The glass transition in
48. Jenkins, J.P.J.; Cameron, R.E.; Donald, A.M. A universal starch. Cereal Chem. 1987, 64, 121.
feature in the structure of starch granules from dierent 70. Benczedi, D. Statistical Thermodynamic Investigations on
sources. Starch-Starke 1993, 45, 417. Amorphous StarchWater Systems; Swiss Federal Institute
49. Cameron, R.E.; Donald, A.M. A small-angle X-ray of Technology: Zurich, 1995; 194.
scattering study of the annealing and gelatinization of 71. Helbert, W.; Chanzy, H.; Planchot, V.; Buleon, A.; Colonna,
starch. Polymer 1992, 33, 2628. P. Morphological and structural features of amylose sphe-
50. Waigh, T.A.; Donald, A.M.; Heidelbach, F.; Riekel, C.; rocrystals of a-type. Int. J. Biol. Macromol. 1993, 15, 183.
Gidley, M.J. Analysis of the native structure of starch 72. Whittam, M.A.; Noel, T.R.; Ring, S.G. Melting behavior
granules with small angle x-ray microfocus scattering. of a-type and b-type crystalline starch. Int. J. Biol.
Biopolymers 1999, 49, 91. Macromol. 1990, 12, 359.
51. Waigh, T.A.; Perry, P.; Riekel, C.; Gidley, M.J.; Donald, 73. Mandelkern, L.; Prasad, A.; Alamo, R.G.; Stack, G.M.
A.M. Chiral side-chain liquid-crystalline polymeric prop- Melting temperature of the n-alkanes and the linear
erties of starch. Macromolecules 1998, 31, 7980. polyethylenes. Macromolecules 1990, 23, 3696.
74. Moates, G.K.; Noel, T.R.; Parker, R.; Ring, S.G. The application of the food polymer science approach to
eect of chain length on the dissolution of the B- structurefunctionrelationships of sucrose in cookie and
crystalline polymorph of starch. Carbohydr. Res. 1997, cracker systems. J. Sci. Food Agric. 1993, 63, 133.
298, 327. 94. Slade, L.; Levine, H. Beyond water activityrecent
75. Burt, D.J.; Russel, P.L. Gelatinization of low water advances on an alternative approach to the assessment
content wheat starch mixturesa combined study by of food quality and safety. Crit. Rev. Food Sci. Nutr.
dierential scanning calorimetry and light microscopy. 1991, 30, 115.
Starke 1983, 35, 354. 95. Parks, G.S.; Barton, L.E.; Spaght, M.E.; Richardson,
76. Buleon, A.; Duprat, F.; Booy, F.P.; Chanzy, H. Single J.W. The viscosity of undercooled liquid glucose. Physics
crystals of amylose with a low degree of polymerization. 1934, 8, 193.
Carbohydr. Polym. 1984, 4, 161. 96. Orford, P.D.; Parker, R.; Ring, S.G.; Smith, A.C. Eect
77. Gerard, C.; Planchot, V.; Colonna, P.; Bertoft, E. of water as a diluent on the glass-transition behavior of
Relationship between branching density and crystalline malto-oligosaccharides, amylose and amylopectin. Int. J.
structure of A- and B-type maize mutant starches. Biol. Macromol. 1989, 11, 91.
Carbohydr. Res. 2000, 326, 130. 97. Bizot, H.; Lebail, P.; Leroux, B.; Davy, J.; Roger, P.;
78. Orwoll, R.A. The polymersolvent interaction parameter, Buleon, A. Calorimetric evaluation of the glass transition
c. Rubber Chem. Technol. 1977, 50, 452. in hydrated, linear and branched polyanhydroglucose
79. van den Berg, C. Vapour Sorption Equilibria and other compounds. Carbohydr. Polym. 1997, 32, 33.
WaterStarch Interactions; A Physico-Chemical Approach; 98. Lourdin, D.; Ring, S.G.; Colonna, P. Study of plasticizer
Agricultural University: Wageningen, 1981; 1185. oligomer and plasticizerpolymer interactions by dielec-
80. Lelievre, J. Theory of gelatinization in a starchwater tric analysis: maltoseglycerol and amyloseglycerol
solute system. Polymer 1976, 17, 854. water systems. Carbohydr. Res. 1998, 306, 551.
81. Moates, G.K.; Parker, R.; Ring, S.G. Preferential solvent 99. Lourdin, D.; Bizot, H.; Colonna, P. Antiplasticization in
interactions and the dissolution of the B-type crystalline starchglycerol lms? J. Appl. Polym. Sci. 1997, 63, 1047.
polymorph of starch in aqueous solutions. Carbohydr. 100. vanSoest, J.J.G.; Knooren, N. Inuence of glycerol and
Res. 1998, 313, 225. water content on the structure and properties of extruded
82. Whittam, M.A.; Orford, P.D.; Ring, S.G.; Clark, S.A.; starch plastic sheets during aging. J. Appl. Polym. Sci.
Parker, M.L.; Cairns, P.; Miles, M.J. Aqueous dissolution 1997, 64, 1411.
of crystalline and amorphous amylose alcohol complexes. 101. Hulleman, S.H.D.; Helbert, W.; Chanzy, H. Single-
Int. J. Biol. Macromol. 1989, 11, 339. crystals of v-amylose complexed with glycerol. Int. J.
83. Brisson, J.; Chanzy, H.; Winter, W.T. The crystal and Biol. Macromol. 1996, 18, 115.
molecular-structure of vh amylose by electron-diraction 102. Gaudin, S.; Lourdin, D.; Forssell, P.M.; Colonna, P.
analysis. Int. J. Biol. Macromol. 1991, 13, 31. Antiplasticisation and oxygen permeability of starch
84. Helbert, W.; Chanzy, H. Single-crystals of v-amylose com- sorbitol lms. Carbohydr. Polym. 2000, 43, 33.
plexed with n-butanol or n-pentanolstructural features 103. Forssell, P.M.; Mikkila, J.M.; Moates, G.K.; Parker, R.
and properties. Int. J. Biol. Macromol. 1994, 16, 207. Phase and glass transition behaviour of concentrated
85. Gessler, K.; Uson, I.; Takaha, T.; Krauss, N.; Smith, barley starchglycerolwater mixtures, a model for
S.M.; Okada, S.; Sheldrick, G.M.; Saenger, W. V-amylose thermoplastic starch. Carbohydr. Polym. 1997, 34, 275.
at atomic resolution: X-ray structure of a cycloamylose 104. Couchman, P.R. Composition-dependent glass-transition
with 26 glucose residues (cyclomaltohexaicosaose). Proc. temperatures and copolymers. Nature 1982, 298, 729.
Nat. Acad. Sci. U.S.A. 1999, 96, 4246. 105. Couchman, P.R.; Karasz, F.E. A classical thermodynamic
86. Godet, M.C.; Tran, V.; Colonna, P.; Buleon, A.; Pezolet, discussion of the eect of composition on glass-transition
M. Inclusion exclusion of fatty-acids in amylose com- temperatures. Macromolecules 1983, 11, 117.
plexes as a function of the fatty-acid chain-length. Int. J. 106. Sugisaki, M.; Suga, H.; Seki, S. Calorimetric study of the
Biol. Macromol. 1995, 17, 405. glassy state: IV. Heat capacity of glassy water and cubic
87. Godet, M.C.; Bizot, H.; Buleon, A. Crystallization of amy- ice. Bull. Chem. Soc. Jpn. 1968, 41, 2591.
losefatty acid complexes prepared with dierent amylose 107. Johari, G.P.; Hallbrucker, A.; Mayer, E. The glass liquid
chain lengths. Carbohydr. Polym. 1995, 27, 47. transition of hyperquenched water. Nature 1987, 330, 552.
88. Godet, M.C.; Tran, V.; Delage, M.M.; Buleon, A. 108. Ould-Kaddour, F.; Barrat, J.L. Molecular-dynamics
Molecular modeling of the specic interactions involved investigation of tracer diusion in a simple liquid. Phys.
in the amylose complexation by fatty-acids. Int. J. Biol. Rev. A 1992, 45, 2308.
Macromol. 1993, 15, 11. 109. Perry, P.A.; Donald, A.M. The role of plastization in
89. Altena, F.W.; Smolders, C.A. Calculation of liquidliquid starch granule assembly. Biomacromolecules 2000, 1,
phase separation in a ternary system of a polymer in a 424.
mixture of a solvent and a non-solvent. Macromolecules 110. Arvanitoyannis, I.; Kalichevsky, M.; Blanshard, J.M.V.
1982, 15, 1491. Study of diusion and permeation of gases in undrawn
90. Durrani, C.M.; Donald, A.M. Compositional mapping of and uniaxially drawn lms made from potato and rice
mixed gels using FTIR microspectroscopy. Carbohydr. starch conditioned at dierent relative humidities. Carbo-
Polym. 1995, 28, 297. hydr. Polym. 1994, 24, 1.
91. Kalichevsky, M.T.; Ring, S.G. Incompatibility of amylose 111. Ferry, J.D. Viscoelastic Properties of Polymers; John Wiley
and amylopectin in aqueous-solution. Carbohydr. Res. and Sons, Inc.: New York, 1980.
1987, 162, 323. 112. Cicerone, M.T.; Blackburn, F.R.; Ediger, M.D. Anom-
92. Kalichevsky, M.T.; Orford, P.D.; Ring, S.G. The alous diusion of probe molecules in polystyrene
incompatibility of concentrated aqueous-solutions of evidence of spatially heterogeneous segmental dynamics.
dextran and amylose and its eect on amylose gelation. Macromolecules 1995, 28, 8224.
Carbohydr. Polym. 1986, 6, 145. 113. Tromp, R.H.; Parker, R.; Ring, S.G. Water diusion in
93. Slade, L.; Levine, H.; Ievolella, J.; Wang, M. The glassy glasses of carbohydrates. Carbohydr. Res. 1997, 303, 199.
state phenomenon in application for the food industry 114. Parker, R.; Ring, S.G. Diusion in maltosewater
604 Parker and Ring
mixtures at temperatures close to the glass-transition. 129. Noel, T.R.; Parker, R.; Ring, S.M.; Ring, S.G. A
Carbohydr. Res. 1995, 273, 147. calorimetric study of structural relaxation in a maltose
115. Noel, T.R.; Parker, R.; Ring, S.G. A comparative study of glass. Carbohydr. Res. 1999, 319, 166.
the dielectric relaxation behaviour of glucose, maltose and 130. Miles, M.J.; Morris, V.J.; Orford, P.D.; Ring, S.G. The
their mixtures with water in the liquid and glassy states. roles of amylose and amylopectin in the gelation and
Carbohydr. Res. 1996, 282, 193. retrogradation of starch. Carbohydr. Res. 1985, 135,
116. Butler, M.F.; Cameron, R.E. A study of the molecular 271.
relaxations in solid starch using dielectric spectroscopy. 131. Miles, M.J.; Morris, V.J.; Ring, S.G. Gelation of amylose.
Polymer 2000, 41, 2249. Carbohydr. Res. 1985, 135, 257.
117. Hodge, I.M. Physical aging in polymer glasses. Science 132. Clark, A.H.; Gidley, M.J.; Richardson, R.K.; Rossmur-
1995, 267, 1945. phy, S.B. Rheological studies of aqueous amylose gels
118. Hodge, I.M. Enthalpy relaxation and recovery in amor- the eect of chain-length and concentration on gel
phous materials. J. Non-Cryst. Solids 1994, 169, 211. modulus. Macromolecules 1989, 22, 346.
119. Hodge, I.M.; Berens, A.R. Eects of annealing and prior 133. Gidley, M.J.; Bulpin, P.V. Aggregation of amylose in
history on the enthalpy relaxation in glassy polymers: 5. aqueous systemsthe eect of chain-length on phase-
Mathematical modeling of nonthermal preageing pertur- behavior and aggregation kinetics. Macromolecules 1989,
bations. Macromolecules 1985, 18, 1980. 22, 341.
120. Hodge, I.M. Eects of annealing and prior history on 134. Gidley, M.J. Molecular mechanisms underlying amy-
enthalpy relaxation in glassy polymers: 4. Comparison of lose aggregation and gelation. Macromolecules 1989, 22,
5 polymers. Macromolecules 1983, 16, 898. 351.
121. Berens, A.R.; Hodge, I.M. Eects of annealing and prior 135. Leloup, V.M.; Colonna, P.; Ring, S.G.; Roberts, K.;
history on enthalpy relaxation in glassy polymers: 1. Wells, B. Microstructure of amylose gels. Carbohydr.
Experimental study on polyvinyl-chloride. Macromole- Polym. 1992, 18, 189.
cules 1982, 15, 756. 136. Putaux, J.L.; Buleon, A.; Chanzy, H. Network formation
122. Hodge, I.M.; Berens, A.R. Eects of annealing and prior in dilute amylose and amylopectin studied by TEM.
history on enthalpy relaxation in glassy polymers: 2. Macromolecules 2000, 33, 6416.
Mathematical modeling. Macromolecules 1982, 15, 762. 137. Kalichevsky, M.T.; Orford, P.D.; Ring, S.G. The retro-
123. Hodge, I.M.; Berens, A.R. Calculation of the eects of gradation and gelation of amylopectins from various
annealing on sub-Tg endotherms. Macromolecules 1981, botanical sources. Carbohydr. Res. 1990, 198, 49.
14, 1598. 138. Shi, Y.C.; Seib, P.A. The structure of 4 waxy starches
124. Lee, M.; Saha, S.K.; Moynihan, C.T.; Schroeder, J. Non- related to gelatinization and retrogradation. Carbohydr.
exponential structural relaxation, anomalous light scatter- Res. 1992, 227, 131.
ing and nanoscale inhomogeneities in glasses. J. Non-Cryst. 139. Shi, Y.C.; Seib, P.A. Fine-structure of maize starches 4
Solids 1997, 222, 369. WX-containing genotypes of the W64A inbred line in
125. Moynihan, C.T.; Crichton, S.N.; Opalka, S.M. Linear and relation to gelatinization and retrogradation. Carbohydr.
non-linear structural relaxation. J. Non-Cryst. Solids Polym. 1995, 26, 141.
1991, 131, 420. 140. Wursch, P.; Gumy, D. Inhibition of amylopectin retro-
126. Lammert, A.M.; Lammert, R.M.; Schmidt, S.J. Physical gradation by partial beta-amylolysis. Carbohydr. Res.
aging of maltose glasses as measured by standard and 1994, 256, 129.
modulated dierential scanning calorimetry. J. Therm. 141. Farhat, I.A.; Blanshard, J.M.V.; Mitchell, J.R. The
Anal. Calorim. 1999, 55, 949. retrogradation of waxy maize starch extrudates: Eects
127. Schmidt, S.J.; Lammert, A.M. Physical aging of maltose of storage temperature and water content. Biopolymers
grasses. J. Food Sci. 1996, 61, 870. 2000, 53, 411.
128. Shogren, R.L. Eect of moisture content on the melting 142. Buleon, A.; Delage, M.M.; Brisson, J.; Chanzy, H. Single-
and subsequent physical aging of cornstarch. Carbohydr. crystals of v-amylose complexed with isopropanol and
Polym. 1992, 19, 83. acetone. Int. J. Biol. Macromol. 1990, 12, 25.
25
Starch: Commercial Sources and Derived Products
University of Birmingham Research Park, Birmingham, United Kingdom
I. INTRODUCTION II. STARCH COMPOSITION AND STRUCTURE

Starches are the principle food reserve polysaccharides in A. Amylose and Amylopectin
plants and are therefore the major source of carbohydrates
in the human diet, traditionally supplying f7080% of the Starch is a mixture of two D-glucan homopolymers, com-
calories consumed by humans with approximately two- posed of a-D-glucopyranosyl (a-D-Glcp) units, namely,
thirds of these calories coming from starch [13]. Starch is amylose and amylopectin. Amylose is essentially a linear
present in all staple foods, e.g., wheat (Triticum vulgare), polymer consisting of (1!4)-linked a-D-Glcp units (Fig. 1),
maize (Zea mays), rice (Oryza sativa), potato (Solanum although some (1!6)-linkages are thought to exist about
tuberosum), sago (Metroxylon palm species), tapioca/cas- every 180400 units [5,7,12,1931]. The abundance of hy-
sava (Manihot esculenta), rye (Secale cereale), barley (Hor- droxyl groups makes amylose hydrophilic, and its uniform
deum vulgare), oats (Avena sativa), etc. [414]. Other less linear-chain nature permits crystallization from solution
common sources of starch include sorghum (Sorghum as well as in a semisolid state in lms and coatings. On
bicolor), yam/sweet potato (Dioscorea species), and arrow- cooling, amylose readily crystallizes and precipitates. How-
root (Maranta arundinacea) [4,5,7,12,15]. ever, if the concentration is too high (steric hindrance) or
The breakdown of consumed starch molecules to the temperature is too low, low diusivity may hamper
component glucose is somewhat slow, thus providing a crystallization.
sustained source of energy. Starch, taken from grains, Both the linearity and the hydroxyl groups cause
tubers, and roots, has been consumed as food and feed amylose to orient in parallel, thus permitting molecular
for centuries and is of great economic importance, being reassociation and hydrogen bonding between hydroxyl
isolated on an industrial scale from many sources. Wheat is groups of adjacent chains forming a network of junction
the leading cereal grain produced, consumed, and traded in zones between molecules. With time, this may result in
the world, followed closely by rice and maize [16]. Food is formation of a three-dimensional gel network held together
the major use for wheat, accounting on average for ap- by hydrogen bonding wherever close alignment has oc-
proximately two-thirds of total consumption [17]. No other curred, resulting in opacity/cloudiness in amylose solutions
single food ingredient compares with starch in terms of known as retrogradation or setback [7,14,22,23,29,
sheer versatility of application in the food industry. 30,3234]. Ultimately, under favorable conditions, a crys-
The industrial uses of starch arise from its unique talline order appears [20]. Thus amylose solutions are
character since it can be used directly as intact granules, unstable and rm resilient gels can result, and crystalline
in the swollen granular state, in the dispersed form, as a lm regions can thus develop. These provide additional gel
dried from a dispersion, as an extrudate powder, after strength (rigidity) and stability and may require signicant
controlled partial hydrolysis to a mixture of oligosaccha- heating to reverse gelation [35,36].
rides, after hydrolysis and isomerization to glucose and The degree of polymerization (DP) of amylose is
fructose syrups, or after chemical modication [18]. Many f20020, 000, giving a molecular weight of f303200
of these aspects will be discussed during the course of kDa [3,5,7,12,19,22,23,30,37,38]. The molecular weight of
this chapter. rye amylose was determined as being f218 kDa, which is
605
606 Knill and Kennedy
Figure 1 Basic structure of amylose.
somewhat lower than for maize starch amylose (f250 kDa) pectin to account for its physicochemical properties. These
and wheat and triticale starch amylose (f260285 kDa) include the laminated structure, the herringbone model,
[39]. Some potato starch amylose fractions are of the order the randomly branched structure, the tassel-on-a-string
of 1000 kDa [5,35]. representation, and the cluster model (also known as the
Amylopectin is a highly branched form of amylose, racemose or grape structure) [5,12,21,23,37,41,42].
consisting of (1!4)-linked a-D-Glcp units and a signicant Amylopectin chains can be classied into three types,
proportion of 1,4,6-tri-O-substituted residues acting as namely, A, B, and C chains. A chains are linked to the
(1!6)-linkage branch points (Fig. 2) [5,7,12,19,21,23,25, molecule only through their potential reducing end, while B
2931,40]. Several models have been proposed for amylo- chains also carry other chains and C chains carry the single
Figure 2 Basic structure of amylopectin.

Starch: Commercial Sources and Derived Products 607
reducing end-group (Fig. 3) [3,23,31,37,43]. The linear amylopectin or a slightly branched amylose are also pres-
outer branches of amylopectin (A chains) can also partic- ent. This intermediate is typical for many cereal starches
ipate in gel formation since they are essentially like small but is not found in potato starch [37,49,50]. Besides
amylose fragments. For relatively large molecular weights, water, amylose, and amylopectin, natural starches also
each model has a characteristic ratio of A/B chains [37]. contain some minor constituents (e.g., protein, lipids,
The initial determination of the A/B ratio favored the etc.). In potato starch, esteried phosphate groups are
randomly branched structure [44], with most natural present in the amylopectin fraction, attached to f1 in every
starches having an A/B chain ratio in the range of 1.1 215560 anhydroglucose units, with most (f6070%) be-
1.5 [45]. ing attached via the C6 hydroxyl group and the other third
The degree of branching and chain lengths between via the C3 hydroxyl, and f90% of them attached to B
branch points in amylopectin varies not only with the chains [3,44].
source of the sample, but also within each sample [22].
Rye amylopectin has been shown to have branch points B. Starch Granules and Crystallinity
about every 1921 glucose units [39,46,47], which is similar
to wheat, triticale, and other amylopectins (every f2025 Nature has chosen the starch granule as an almost univer-
glucose units) [26,39]. Hydrolysis of starch with a sal form for packaging and storing carbohydrates in green
debranching enzyme (such as isoamylase) and measure- plants. Starch granules are quasicrystalline and cold-
ment of the molecular weight of the resulting linear chains water-insoluble. The shape of the granules is somewhat
by gel permeation chromatography (GPC) can give an characteristic of the source of the starch, and they range in
excellent prole of branch lengths. Amylopectin molecules size from submicron elongated granules to oval granules
are thus very large, with molecular weights ranging from well over 100 Am [5,14,30]. A summary of starch granule
10,000 to over 500,000 kDa [3,7,29,35,37,38]. dimensions is provided in Table 1 [3,7,1214,21,29,42,43].
The amylose/amylopectin ratio varies according to the The shapes of the granules include nearly perfect
source of the starch and its maturity, but is f1:3 for most spheres typical for small wheat starch granules; disks,
general starches. One unusual genetic variety of maize typical for the large granules of wheat and rye; polyhedral
arose in China, whose starch granules contained essentially granules, as in rice and maize; oyster shell (oval, egg-
only amylopectin (<2% amylose). When the maize kernel shaped, ellipsoidal) irregular granules as often found in
was cut with a knife, the cut surface appeared shiny as potato starch; and highly elongated irregular lamentous
though it contained wax, and the maize was referred to as granules as in high-amylose maize starch [51]. In the case of
waxy maize. This was developed into a high-yielding potato starch, there is a gradual change from oval toward
hybrid in the United States [1,31]. Waxy sorghum and spherical shape with decreasing granule diameter. As in
glutinous rice are similar. Oppositely, high-amylose maize wheat and barley, rye starch consists of populations of
(amylomaize) was also genetically developed in the United large and small granules [47]. The large granules increase in
States with starch amylose contents of 6080% produced size during maturation and, at maturity, vary in size from
commercially [31,44]. High amylose content mutant culti- 10 to >35 Am in diameter. The distribution curve is broader
vars of pea and barley also exist. The majority of starches and the maximum diameter is larger than in wheat starch.
contain f1535% amylose, e.g., the amylose content of The small starch granules accumulate relatively late in
potato, rice, and rye starches are f1823%, f1630%, development and have diameters <10 Am [52]. Wheat
and f2426%, respectively [39,46,48]. starch granules are lenticular and have a bimodal or
Most natural starches contain both amylose and am- trimodal size distribution (>14, 514, and 15 Am) [3].
ylopectin; however, in some starches, signicant amounts Amylose is found naturally in three crystalline mod-
of an intermediate that seems to be either a less-branched ications designated A (cereal), B (tuber), and C (smooth
pea and various beans) [25,31,37]. Precipitated starch
complexes (with iodine, long-chain alcohols, and fatty
acids) adopt the so-called V structure (Verkleisterung)
[31]. The so-called linearity is further complicated by a
twisting of the chain into a helix, and it is the dierent
degrees of hydration of the helix that give rise to the A, B,
and C forms [38]. B-amylose helices contain six a-D-Glcp
units per unit cell (helical turn) and three to four molecules
of water of hydration, with left-handed helices being
slightly more energetically favorable [44]. Proof for amy-
lose chains adopting left-handed double helical structures
has been obtained [53].
Starch granules are made up of amylose and/or amy-
lopectin molecules arranged radially. They contain both
crystalline and amorphous (noncrystalline) regions in al-
Figure 3 Representation of A, B, and C chains in amylo- ternating layers. The clustered branches of amylopectin
pectin. occur as packed double helices. It is the packing together of
Table 1 Dimensions of Starches of Various Botanical Origin
Diameter Gelatinization Pasting Amylose Cooked

Starch Type (Am) Morphology temp. (jC) temp. (jC) (%) properties
Wheat Cereal 155 Round, lenticular 5285 77 2528 Opaque gel

Maize Cereal 230 Round, polygonal 6272 80 2528 Opaque gel
Waxy maize Cereal 230 Round, polygonal 6372 74 <2 Clear cohesive
Amylomaize Cereal 230 Polygonal, irregular, 63170 >90 5090 Opaque,
elongated strong gel
Rice Cereal 19 Polygonal, spherical 6878 81 19 Opaque gel
Potato Tuber 5100 Oval, spherical 5868 64 2021 Clear, cohesive
Sago Pith 1565 Oval, truncated 6974 74 26 Opaque gel
Tapioca Root 435 Oval, truncated 5273 63 17 Clear, cohesive
Source: Refs. 3, 7, 1214, 21, 29, 38, 42, and 43.
these double helical structures that forms the many small Double helical structures are found for part of the
crystalline areas comprising the dense layers in starch amylopectin component within granules (and are formed
granules that alternate with less dense amorphous layers. from both amylose and amylopectin after gelatinization).
Amylose molecules occur among the amylopectin mole- A minimum of 10 glucose residues is required for double
cules. The origin of the growth of the granule is called the helix formation [55], although chains as short as six residues
hilum [3]. A range of single- and double-stranded helices can cocrystallize [56]. The outer branch length of amylo-
have been identied and characterized from native starches pectin is the major determinant of which double helical
[50]. Starch chains usually show a high proportion of local polymorph is found in native starch granules. Relatively
glycosidic conformations characteristic of V-type struc- short branches lead to A-type with longer branches giving
tures [54] that can be stabilized by complexation with B-type order [57]. A- and B-type polymorphs have very
iodine, fatty acids, monoglycerides, etc. Only the cereal similar individual helical structures but dier in packing
starches contain endogenous lipids in the granule, primar- arrangements [58].
ily free fatty acids and lysophospholipid [3]. An example of Amylose can be leached from many starches leaving
the arrangement/interactions of amylose/amylopectin in the amylopectin as well as the granule crystallinity
starch is provided in Fig. 4. largely intact, which has led to the conclusion that amy-
lose is mainly in the amorphous phase [21,45,50]. Con-
versely, amylopectin is the main component of the
crystalline fraction. However, some amylopectin is ho-
mogeneously mixed with the amylose in the amorphous
phase [5,59]. There is no sharp demarcation between the
crystalline and amorphous phases of starch granules, and
it is generally believed that some or all of the starch
molecular chains run continuously from one phase to
another [60].
C. Starch Gelatinization/Pasting
Undamaged starch granules are insoluble in cold water but
can imbibe water reversibly; that is, they can swell slightly
and then return to their original size on drying. As the
temperature is increased, the process becomes irreversible
and eventually the granule bursts to form a starch paste,
and a rapid onset in the development of viscosity is seen.
In the initial phase of this heating process, swelling begins
in the least organized amorphous, intercrystalline regions
of the granule [7,12,21,33,43,60]. This results in porous
amylopectin-based granules suspended in a hot amylose
solution [25]. Further heating leads to uncoiling or disso-
ciation of double helical regions and disappearance of the
amylopectin crystallite structure, and an increase in vis-
cosity occurs.
Figure 4 Example of amylose/amylopectin interactions in a Upon continued heating and hydration, the granule
starch granule. (From Ref. 21.) weakens to the point where it can no longer resist mechan-
Figure 5 Gelatinization curves for starches of dierent botanical origins.
ical or thermal shearing, and a starch paste results [30]. of dierent botanical origin as a function of temperature
Pasting is dened as the phenomenon following gelatiniza- (determined by measurement of Congo red stain uptake by
tion in the dissolution of starch. It involves granular swell- gelatinized amyloplasts) are presented in Fig. 5.
ing, exudation of molecular components from the granule,
and eventually, total disruption of the granules [12,20,34].
A starch paste consists of a continuous phase of
solubilized amylose and/or amylopectin and a discontinu- III. HYDROLYSIS
ous phase of granule remnants (granule ghosts and frag- A. Enzymolysis
ments). A granule ghost consists of the outer insoluble
envelope of the granule (but is not a membrane). Complete The most diverse and numerous enzymes for carbohydrate
molecular dispersion can only be accomplished under hydrolysis and modication are those that act on starch.
conditions of high temperature, high shear, and excess Enzymes that are capable of catalyzing the hydrolysis of
water, which are seldom, if ever, encountered in most the (1!4)-linkages in amylose/amylopectin are called
applications [3]. However, in many instances of processing, amylases and are widely produced by plants, bacteria,
what is required is the breakdown of the granule structure fungi, and animals [67]. In mammals, amylases are mainly
via gelatinization [61,62]. produced by the salivary glands and the pancreas [68]. a-
Not all starch granules in a sample burst at the same Amylase (1,4-a-D-glucan glucanohydrolase, EC 3.2.1.1) is
temperature, but the range of temperature of gelatinization an endoglycosidase, attacking glucans away from the chain
is characteristic of starch from a particular source. Starch ends at an internal glycosidic bond and producing a rapid
gelatinization can be dened as the collapse (disruption) of drop in viscosity [3,37,43,67,6972,76]. Varying types of
molecular orders within the starch granule manifested in oligosaccharides are produced, characteristic of the type of
irreversible changes in properties such as granular swelling, a-amylase. Traditionally, a-amylase has been obtained
native crystalline melting, and starch solubilization. The from Aspergillus oryzae, but the enzymes obtained from
point of initial gelatinization and the range over which it various thermophilic Bacillus species (e.g., Bacillus amylo-
occurs is governed by starch concentration, method of liquefaciens and Bacillus licheniformis) have the advantage
observation, granular type, and heterogeneities within the of temperature stability [5,7174].
granule population under observation [2022,34]. The Dextrins produced by a-amylase action can be further
length of double helices is proposed to be a determinant processed by a variety of enzymes including h-amylases for
of gelatinization temperature. Amylose double helices in, the production of maltose syrups and glucoamylases for
for example, gels probably occur over a length scale of the production of glucose syrups. h-Amylase (1,4-a-D-
f4080 residues and melt at f150jC [63,64]. Typical glucan maltohydrolase, EC 3.2.1.2) attacks amylose/amy-
amylopectin-based double helices occur over f1520 res- lopectin in an exo fashion from the nonreducing ends,
idues and melt at f6080jC. Gelatinized starches/starch releasing h-maltose and a high molecular weight limit
pastes can undergo retrogradation on cooling. dextrin when the enzyme reaches a (1!6)-linkage in amy-
Detailed information on rye starch [47] and wheat lopectin [5,37,43,6872,76]. h-Amylases occur widely in
starch [65] physical properties (e.g., gelatinization/pasting) many plants, with barley, wheat, sweet potatoes, and
has been published. The gelatinization temperature of rye soybeans being common sources [70].
starch has been determined as being f5560jC (by DSC Glucoamylase (g-amylase; amyloglucosidase; glucan
measurements) [66]. Examples of gelatinization of starches 1,4-a-glucosidase; 1,4-a-D-glucan glucohydrolase, EC
3.2.1.3) occurs almost exclusively in fungi and attacks sources [68]. 1,4-a-D-Glucan maltotriohydrolase (EC 3.2.
amylose/amylopectin in an exo fashion from the nonre- 1.116), 1,4-a- D -glucan maltotetraohydrolase (EC
ducing ends, releasing h-D-glucopyranose. It does not 3.2.1.60), and 1,4-a-D-glucan maltohexaohydrolase (EC
produce limit dextrins as it can catalyze hydrolysis of both 3.2.1.98) remove successive maltotriose, maltotetraose,
(1!4)- and (1!6)-linkages (although at very dierent and maltohexaose residues from nonreducing chain ends
rates) and can therefore convert starch to glucose in amylaceous polysaccharides, respectively [69,76,77].
[5,37,43,6872,76]. Glucoamylases are produced by a va- Enzymic biochemical conversions of starch to alcohols
riety of organisms, although commercial products are [70,78], organic acids [79], and polyols [8082] do exist.
obtained principally from Aspergillus or Rhizopus [75]. Processes are in operation that convert starch enzymically
a-D-Glucosidase (a-D-glucoside glucohydrolase; EC into sugars (monosaccharides and disaccharides), which
3.2.1.20) is produced as an intracellular, cell-bound, or are then fermented by yeast or Zymomonas bacteria to
extracellular enzyme by many fungi (Aspergillus niger), alcohol [70]. Simultaneous starch saccharication and
yeasts (Saccharomyces cerevisiae), and bacteria (Bacillus ethanol fermentation has been carried out using immobi-
species) [69,70]. a-D-Glucosidases act by exo-hydrolysis on lized Zymomonas mobilis and amyloglucosidase [83].
the terminal (1!4)- and (1!6)-linkages of disaccharides to The use of genetically engineered yeast strains can also be
liberate a-D-glucose. Polysaccharides are attacked very used for the direct conversion of starch to ethanol. In-
slowly, if at all; however, the enzyme can work on starch creased production of ethanol from starch has been
in conjunction with other enzymes that liberate oligosac- obtained by a recombinant yeast secreting Aspergillus
charides [69,70,76]. awamori glucoamylase [84,85]. A recombinant S. cerevisiae
Isoamylases or debranching enzymes hydrolyze the strain expressing rice a-amylase from Pichi pastoris alcohol
(1!6)-linkages in amylopectin. These include amylo-1,6- oxidase promoter has been reported to convert starch to
glucosidase (dextrin 6-a-D-glucosidase; EC 3.2.1.33), pul- ethanol [86].
lulanase (amylopectin 6-glucanohydrolase; a-dextrin Starch-hydrolyzing enzymes are often added to the
endo-1,6-a-glucosidase; EC 3.2.1.41), and isoamylase (gly- fermentation stage of the brewing process to increase the
cogen 6-glucanohydrolase; EC 3.2.1.68) [5,37,43,69 level of fermentable saccharides utilizable by yeast. In this
72,76]. The precise location of the (1!6)-linkages in the way, a greater degree of fermentation is achieved and
substrate is of utmost importance since it aects the ability residual carbohydrate level is reduced resulting in a lower
of the various strains of the enzymes to act on dierent calorie, or light beer [67]. Typically, a soluble gluco-
substrates. For example, pullulanase from Bacillus macer- amylase is used to convert residual dextrins to glucose that
ans has very little action on amylopectin but is able to is then fermented by the yeast. The enzyme must then be
degrade its h-limit dextrin, while the pullulanase from inactivated by a pasteurization process to prevent the
Aerobacter aerogenes almost totally debranches amylopec- possibility of further action during beer storage.
tin and its h-limit dextrin [70]. Amylo-1,6-glucosidase
hydrolyzes terminal (1!6)-linkages in limit dextrins [70]. B. Maltodextrins
A visual overview of the major starch-hydrolyzing
enzymes is provided in Fig. 6 [69]. Information on the Commercial starch hydrolysates are classied in terms of
mechanism of action of such enzymes is detailed in many dextrose equivalent (DE). Maltodextrins are dened as
Figure 6 Visual overview of the major starch hydrolyzing enzymes: (i) a-amylase; (ii) pullulanase; (iii) isoamylase; (iv) cyclodextrin
D-glucotransferase; (v) exo-(1,4)-a-D-glucanase; (vi) a-D-glucosidase; (vii) glucoamylase; (viii) h-amylase. (From Ref. 69.)
nonsweet starch hydrolysates that consist of a-D-glucose Two types of maltodextrin are in commercial use:
units linked primarily by (1!4) glycosidic linkages with a those ranging from about 10 to 14 DE and those ranging
DE of 320 [3,71]. DE is dened as the percentage of from about 15 to 19 DE. The compositions of these
reducing sugar calculated as dextrose (glucose) on a dry products depend not only on DE, but also on the method
weight basis. Therefore, in terms of molecular size, malto- of hydrolysis employed in their manufacture. The saccha-
dextrins bridge the gap between starch and monosaccharide component proles of maltodextrins obtained by acid-
rides/disaccharides [87]. Starch hydrolysates with a DE catalyzed hydrolysis are somewhat dierent from those
greater than 20 are designated as various kinds of syrups obtained by enzyme- or acid/enzyme-catalyzed hydrolysis,
depending on their source and composition. although they can possess the same DE. Acid hydrolysates
In general, maltodextrins are fully water-soluble (some tend to contain greater proportions of higher molecular
are even cold-water-soluble) carbohydrates of low bulk weight dextrins and glucose. The former makes them less
density and are metabolized similarly to starch. They have water-soluble and they easily retrograde.
very little or no sweetness and a bland, not starchy avor The high viscosity of maltodextrins, an important
that does not mask other avors [88]. Due to these prop- property in many applications, is due to high levels of
erties, maltodextrins have considerable application in higher-molecular weight saccharides. Since maltodextrins
the food industry, particularly in convenience and pro- exhibit virtually no sweetness, their main contribution is a
cessed foods. bodying eect resulting from their relatively high vis-
The production of maltodextrins is, by denition, cosity. The varying DE and/or bulk density give maltodex-
achieved by hydrolysis of starch down to glucose poly- trins their varying characteristics and functionality. For
mers with an average chain length of 510 glucose units/ example, high DE maltodextrins have solubility, bulking,
molecule. The properties of maltodextrins are controlled and bodying characteristics more similar to corn syrup
by their DE and DP, which change with the degree of sweeteners. Low DE maltodextrins have binding proper-
hydrolysis [71,87,89]. Theoretically, they can be pro- ties of starch and can function more eectively as fat
duced by controlled hydrolysis by either enzymic or binders than high DE maltodextrins. Low DE maltodex-
acidic means; however, in practice, acid hydrolysis pro- trins are eective in preventing formation of coarse crys-
duces too much free glucose (and large fragments) and tals. The variety of sugar polymers in maltodextrins
maltodextrins, thus produced have a strong tendency to prevents the formation of large, gritty crystals, as sugar
retrograde. Linear starch fragments in low DE acid mixtures will not crystallize as readily as pure compounds.
hydrolysates large enough to reassociate form insoluble The ne particle size of maltodextrins contributes to a
aggregates causing hazy solutions, which are undesirable smooth, creamy mouthfeel in food products [89].
for some applications. Therefore, on a commercial level, Chromatography is one of the best techniques for the
maltodextrins are invariably prepared from starch by characterization of oligosaccharides and polysaccharides.
controlled enzymic hydrolysis. The physicochemical Much of the earlier work on oligosaccharide analysis has
characteristics of dextrins have been extensively reviewed been reviewed and the theory, separation modes, and ins-
[90,91]. trumentation have been described [88,93]. Anion-exchange
A starch slurry is initially liqueed by heating (70 HPLC has exceptional resolving power for complex oligo-
90jC) at neutral pH in the presence of a bacterial a- saccharides. Such analyses are carried out at high pH
amylase to a DE of 215. The liquid starch hydrolysate is coupled with pulsed amperometric detection (PAD), allow-
then autoclaved (110115jC) to gelatinize any remaining ing separation of oligosaccharides and polysaccharides up
insoluble starch and, on cooling, subjected to further to DPz50 [94,95]. A complete study of linear and branched
enzymic treatment to reach the desired DE. Some hydrol- glucose oligomers using anion exchange HPLC and PAD
ysis schemes employ both acid- and enzyme-catalyzed has been performed to look at variations in detector re-
hydrolysis. Initial acid-catalyzed hydrolysis of a starch sponse at dierent degrees of polymerization [96]. A con-
slurry to a DE of 515 is followed by neutralization and siderable amount of work outlining the characterization of
further hydrolysis with a bacterial a-amylase (e.g., from starch oligosaccharides has been published previously
Bacillus subtilis or Bacillus mesentericus) to produce mal- [97,98]. An example of an HPAEC-PAD chromatogram
todextrins that are haze-free and exhibit no retrograda- of a commercial maltodextrin is provided in Fig. 7, detail-
tion upon storage [88,92]. Such systems overcome the ing the oligosaccharide prole.
problem of hazy solutions and result in maltodextrins Maltodextrin properties and cost make them ideally
with low hygroscopicity and high water solubility. The suited to widespread application throughout the food
source and variety of the starch are relatively unimportant industry. They perform multifaceted functions in food
for these processes. The nal product is concentrated in systems as bulking/lling/carrier agents, sweetness reduc-
vacuum evaporators to give nished syrups containing ers, oxygen barriers, and lms. The purposes for which they
about 75% solids, or more regularly, is spray-dried to a have been put to use in such forms are to save costs by
white powder containing f35% moisture. The dextrini- replacing (or partially replacing) a higher cost material
zation reaction is allowed to progress until the required [88,89]. Maltodextrin addition is by far the most eective
DE product is obtained. Low DE value maltodextrins will method for reducing sweetness without altering properties.
tend to retrograde in solutions, whereas those with higher Many products can be made more palatable by lowering
DE values will form less viscous solutions and will exhibit their levels of sweetness without reducing their total solids
increasing sweetness. content. This is of use in foods and drinks containing high
Figure 7 HPAEC-PAD chromatogram of a commercial maltodextrin.
sugar concentrations (for example, confectionery, pre- in the sugar confectionery industry. In products where
serves, llings, and fondants). Replacement of sugar often lower degrees of hydrolysis are required to give thickening,
requires a combination of two or more bulking agents in viscosity, body, etc. without appreciable sweetness, it is not
order to directly duplicate the functional properties of possible to use acid hydrolysis to produce materials in
that sugar (e.g., freezing point depression, ice crystal which every starch molecule has been reduced in molecular
control, and structure/bulk contribution). Frozen desserts weight suciently to give a nonretrograding, clear, stable
use maltodextrin because its water holding capacity com- solution. Enzymic hydrolysis is therefore utilized, particu-
bined with low molecular weight give it freezing point larly a-amylase, glucoamylase, and often pullulanase
depressant qualities. [71,88,104].
Maltodextrin substituted directly into a food product Judicious use of fungal a-amylase and glucoamylase
provides 4 kcal of energy per gram of solid material [89,99]. can give rise to syrups with various ratios of glucose,
However, when maltodextrins are being used as oil or fat maltose, and higher oligosaccharides. These high conver-
replacers, they are generally utilized in a 1:3 maltodextrin/ sion syrups generally contain about 3543% Glc, 3040%
water ratio, thus giving a solution or gel (depending on DE) maltose, and 815% maltotriose. It is important that these
which provides only 1 kcal/g. Paselli SA2, a fully digestible syrups have a high DE and yet be suciently stable not to
enzymically derived low DE potato starch maltodextrin, crystallize at temperatures down to 4jC at f8082% dry
has been used to replace in excess of 50% fat in a variety of substance [74]. An HPAEC-PAD chromatogram detailing
products [88,99101]. Other applications include the use of the oligosaccharide prole of a commercial glucose syrup is
Stellark, a fat mimicking microparticulate acid-hydro- provided in Fig. 8. Glucoamylases are also used to convert
lyzed cornstarch gel, which slows down the staling process acid liqueed starch hydrolysates of a moderate degree of
in baked goods [88,89,102], and is used in the production of hydrolysis under mild conditions into products which have
reduced fat salad dressings [103]. DE levels >90. A signicant amount of information on the
enzymic production [3,70,71,105108], physicochemical
C. Glucose and Fructose Syrups properties [71,107,109], and food applications, e.g., in con-
fectionery, preserves, frozen desserts, and beverages [71,
Glucose syrup is a rened, concentrated aqueous solution 110,111], of glucose syrups is readily available. Likewise,
of D-glucose, maltose, and other polymers of D-glucose maltose syrups can be produced using h-amylase [112].
obtained by controlled partial hydrolysis of edible starch, A signicant development in the production of syrups
with a DE of 20100 [22,71,88,104,105]. Acid conversion is was the introduction of advanced enzyme engineering to
reproducible, although random in its attack on starch, and convert high-purity D-glucose (derived from starch) using
although little or no inuence can be made on the individ- glucose isomerase (EC 5.3.1.18) to a mixture of D-glucose
ual sugar composition/spectrum, it is possible by control- and D-fructose equivalent to invert sugar from sucrose,
ling temperature, time, and acid level to make products of thereby opening to the starch industry the previously
reasonably constant composition for a given degree of unavailable but enormous sweeteners market [76,81,88,
hydrolysis. Acid conversion still occupies a signicant 105,113]. High fructose syrups (HFS) are produced com-
place in the family of glucose syrups, especially those used mercially in many countries due to their extensive use in the
Figure 8 HPAEC-PAD chromatogram of a commercial glucose syrup.
processed food and beverage industries. HFS are the best commonly isolated from B. macerans and Bacillus mega-
alternative to sucrose where starchy resources are abun- terium is capable of hydrolyzing starch to a series of
dant and cheap. An overview of the sweeteners that can be nonreducing cyclomalto-oligosaccharides called cy-
manufactured from starch is presented in Fig. 9. clodextrins (previously referred to as Schardinger dex-
trins or cycloamyloses) [37,43,70,114]. The structure of
D. Cyclodextrins a-cyclodextrin, and the molecular dimensions of a-, h-,
and g-cyclodextrins, composed of 6, 7, and 8 gluco-
Cyclodextrin D-glucotransferase {1,4-a-D-glucan 4-a-D- pyranose units, respectively, are presented (Fig. 10) [3,
[1,4-a-D-glucano]-transferase (cyclizing), EC 2.4.1.19}, 71,115].
Figure 9 An overview of the sweeteners that can be produced from starch. (From Ref. 82.)
Figure 10 Cyclodextrin structures and cavity dimensions. (From Ref. 71.)
As a consequence of cyclodextrin conformation, all complex formation, and easily oxidizable substances can
secondary hydroxyl groups are located on one side of the be protected from atmospheric oxidation [88,116].
toruslike cyclodextrin molecule, while all primary hydroxyl Essential oils extracted from well-known spices (dill,
groups are on the other side. The lining of the internal tarragon, marjoram, etc.) form stable crystalline complexes
cavity is formed by hydrogen atoms and glycosidic oxygen- with cyclodextrins, making them stable to oxygen, light,
bridge atoms, making this surface slightly apolar. Mole- and heat. However, in aqueous media, e.g., in the mouth,
cules, or functional groups of molecules, having molecular they dissociate instantly and their avoring action is readily
dimensions that correspond to the cyclodextrin cavity, experienced [88].
being less hydrophilic than water, can be included into
the cyclodextrin cavity if both components are dissolved in
water. In solution, the slightly apolar cyclodextrin cavity is IV. STARCH MODIFICATION
occupied by water molecules, which is energetically unfa-
vorable (polarapolar interactions) and are therefore read- The manufacture of starch employs a variety of extraction
ily substituted by appropriate guest molecules which are processes that isolate puried starch from the other con-
less polar than water. This often advantageously modies stituents of the raw material, the objective being to recover
the various physical and chemical properties of the includ- the insoluble starch granules as undamaged or intact as
ed/encapsulated molecules and is simpler and cheaper than possible. Unprocessed native starches are generally too
many other methods of encapsulation, and powderlike weak and functionally too restricted for application in
crystalline inclusion complexes can be isolated todays advanced technologies. Processing/modication
[88,116,117]. is therefore generally necessary to engender a range of
Since covalent bonds do not form between the com- desired functionalities. Physical, chemical, or biochemical
ponents, under physiological conditions, the complexes are modications mean that numerous highly functional deriv-
easily dissociated. In the pharmaceutical industry, cyclo- atives have been developed, which continue to result in the
dextrins are utilized as an auxiliary substance for improv- evolution of new processing technologies and market
ing stability and absorption qualities of active ingredients trends. Speciality starches are tailored to create competitive
in tablets. This makes the production and introduction of advantage in a new product, enhance product aesthetics,
numerous active substances possible whose stability, com- reduce recipe/production costs, ensure product consisten-
patibility, or absorption features prevented their use. Vol- cy, and extend shelf life [38]. Commercial interest can arise
atile compounds can be stabilized without losses through whenever it becomes economically feasible to meet a
evaporation, liquid compounds can be transformed into particular need as a result of interest in a unique property,
crystalline, compressible forms, solubility in water as well a new, lower-cost production method, increased demand
as the rate of dissolution of poorly soluble substances can allowing economies of scale, or a new or unforeseen appli-
be increased, bad taste and smell can be masked by cation [118].
Polysaccharides represent a diverse group of renew- is used to prepare higher DS, cold-water-dispersible deriv-
able bioresources that provide a complex and essentially atives in granule form [118].
inexhaustible substrate library for chemical derivatization. Nonfood applications of modied starches are in the
Modication of their physicochemical properties by chem- paper (surface sizing and paper coating) [120], textile (as
ical derivatization is an important factor in their industrial adhesive, stiener, printing, and polishing agents) [121],
production and application. Chemical modication of soap, laundry, cosmetic, and pharmaceutical industries, in
polysaccharides is based upon hydroxyl group chemistry reproong preparations, as explosive- and fuel-binding
and a modied polysaccharide can be dened as one whose agents, as occulating agents for water treatment, and in
hydroxyl groups have been altered by chemical reaction the building industry [122]. In the United Kingdom, ap-
(e.g., by oxidation, esterication, etherication, cross-link- proximately a quarter of the total starch consumption is for
ing, etc.) [118,119]. The physicochemical properties of such nonfood purposes, mainly in the paper, packaging, and
a modied polysaccharide are largely dependent on the textile industries [2]. However, more than half of world
degree of substitution (DS), which is dened as the average starch production goes into the manufacturing of food and
number of substituted hydroxyl groups per anhydroglu- feed products, either as such or in pregelatinized and
cose unit. In the case of amylose, there are three hydroxyl chemically modied form, the main proportion being
groups available for substitution, attached to the C2, C3, transformed into syrups and sugars. An overview of the
and C6 carbon atoms, the C1 and C4 hydroxyl groups eect of modications on starch properties is provided in
being involved in the glycosidic linkages that form the Table 2.
polysaccharide backbone. Thus the maximum theoretical
average DS value, and maximum DS value for a single A. Pregelatinization, Thermal Treatment,
anhydroglucose monomer unit, is 3 [118]. Obviously, this and Acid Thinning
value is reduced in the case of amylopectin, since a
branched anhydroglucose unit has a maximum theoretical Certain starches require cooking to develop their function.
DS value of 2 (on the C2 and C3 hydroxyl groups since the The process of pregelatinization is designed to remove the
C6 hydroxyl group is part of the glycosidic linkage of necessity for cooking, providing a versatile range of cold
the branch); thus the higher the degree of branching, the thickening starches [3]. Pregelatinized or precooked
lower the maximum theoretical average DS value. With starches are manufactured by drum drying, extrusion, or
higher degrees of branching, steric factors are also signif- spray drying. In drum drying, an aqueous starch slurry is
icant as is the size of the derivatizing agents and substit- fed onto the surface of steam-heated rollers. The starch gels
uent group. rapidly and is dried and then removed as a thin lm by a
Such chemical modications aect hydrogen bonding, scraper. The product is then crushed and sieved. Drum-
charge interactions, and hydrophobic character, thereby dried starches have slightly lower viscosities than their
altering the nature of the interactions between the polysac- parent native starches due to grinding of the drum-dried
charide chains. Since starch is inherently water-soluble akes to the required particle size grades (coarse or ne)
after disruption of the granular structure, a relatively high [20,123]. The ner the particle size required, the greater the
DS is not required to impart solubility or dispersibility; damage, and hence dierent grades perform dierently in
therefore, the majority of industrial applications require terms of dispersion, smoothness, and rate of viscosity
only a low DS value (<0.2) to dramatically change poly- development. Spray-dried starches are nding increased
saccharide physicochemical properties [118]. application [38]. Such starches are mainly used in the food
Low DS derivatives are generally manufactured by industry (for instant puddings and baby foods). They are
reacting starch in an aqueous suspension/slurry of f30 also used as beater sizes in the papermaking industry. They
45% solids, usually at pH 712. Sodium hydroxide is disperse and swell evenly in water, thus coating the bers
commonly employed to produce the alkaline pH. Condi- uniformly with starch gel.
tions are adjusted to prevent gelatinization of granular In the last decade, the search for physical modication
starch and to allow recovery of the starch derivative in techniques that are greener and more natural alter-
granular form by ltration or centrifugation and drying. natives to chemical modication has resulted in the devel-
The derivative may be washed to remove unreacted opment of proprietary technologies for the production of
reagents, by-products, salts, and other solubles before thermally treated or functionally native starches, which
drying. To prevent the swelling of the starch under strongly possess the process tolerance of chemically modied
alkaline conditions, sodium chloride may be added to a starches, while importantly remaining classied as native
concentration of f1030%. There is a limit to the level of starches [20,38,123125].
substitution that can be made in aqueous slurry while Acid-thinned or thin-boiling starches are those that
maintaining the starch in granular form, because the have been subjected to mild aqueous acid hydrolysis (at a
granule gelatinization temperature is lowered as the DS temperature <50jC), which predominantly results in at-
increases. If the level of hydrophilic substituents becomes tack and depolymerization of the amorphous regions of the
high enough, the starch derivative gelatinizes, becomes granule. Thus, when the starch is heated beyond its gela-
dispersible at room temperature, and is said to be cold- tinization temperature, the granules rupture quickly. This
water-dispersible. A nonswelling solvent, such as acetone, results in a lower hot viscosity and development of a
Table 2 Eect of Modications on Starch

Modication Objective Benets Applications
Pregelatinization Cold-water thickening Eliminates need to cook Instant soups, sauces,

properties convenience and energy dressings, desserts, etc.
saving
Thermal Strengthen granules and Functional native starch Ambient stable products,
treatment retard swelling/viscosity with improved tolerance bottled sauces, and soups
increase to heat, acid, and shear
Acid thinning Lower viscosity and Enhance textural properties Gums, pastilles, and jellies
increase gel strength at higher concentrations
Enzyme Varied viscosity and Contributes texture Fat mimetics, avor carriers,
conversion gel strength, and rheology dry mix llers
thermoreversibility,
and sweetness
Oxidation Carbonyl and carboxyl Improves adhesion of Confectionery and batter
groups, better clarity, coatings, soft stable gels
less retrogradation at higher dosage
Stabilization Prevent granule shrinkage, Chill and freeze/thaw Chilled and frozen processed
lower gelatinization stability extends shelf life foods, high brix llings
temperature
Cross-linking Strengthen granules Improved tolerance to heat, Ambient stable products,
and retard swelling/ acid, and shear bottled sauces, and soups
viscosity increase
Source: Refs. 2 and 38.
stronger gel on cooling (setback) due to the increase in the colors. Large quantities of oxidized starch are used for
number of smaller linear fragments [7,20,38,119,126128]. paper and paperboard surface sizing to seal pores, tie down
loose surface bers, improve surface strength, and provide
B. Oxidation holding of printing inks. Oxidized starches are also used to
provide yarn abrasion protection, in laundry nishing and
Although many reagents oxidize starch, alkaline hypochlo- in the fabrication of construction materials, such as insu-
rite is the most commonly utilized commercial reagent [7]. lation and wall boards; to provide adhesive, binding, and
Starch is oxidized to obtain low-viscosity, high-solid dis- sizing properties; and in batters and breadings for food-
persions and resistance to viscosity increases or gelling in stus, e.g., sh, where it is claimed to give good adhesion to
aqueous dispersion. The oxidation causes some depolym- the food [118].
erization, reducing viscosity, and introduces relatively
bulky carboxylic acid (COOH) and aldehyde (CHO) C. Stabilization
groups. The steric hindrance of the bulky groups disrupts
any retrogradation tendency, thus reducing gel strength Esterication or etherication with monofunctional
and providing viscosity stability, which is advantageous in reagents can be used to substitute bulky groups onto the
many applications [20,38,128]. An overview of oxidation starch in order to take up space and sterically hinder any
chemistry is provided in Fig. 11; more detailed information tendency for dispersed (cooked), linear fragments to re-
on oxidation procedures and mechanisms is available align and retrograde. The eectiveness of stabilization
[118,119,129131]. depends on the number and nature of the substituted
Since hypochlorite oxidation results in scission of group. This type of modication is therefore often called
some glycosidic linkages, some of the starch is solubilized stabilization, and the products are called stabilized starches
and removed during the separation and washing process. [3]. The primary objective of stabilization is to prevent
In general, oxidized starches are sensitive to heat, tending retrogradation and thereby enhance shelf life through
to yellow when exposed to heat and on prolonged storage. tolerance to temperature uctuations such as freezethaw
They gelatinize at a lower temperature than native starches cycles. There are two food-approved types of stabilized
and produce aqueous dispersions of greater clarity and starches, namely, acetylated and hydroxypropylated
lower viscosity, with less tendency to retrograde; thus the [23,38].
pastes are more uid [118]. A relatively low DS is adequate to achieve this stabi-
About 8085% of hypochlorite-oxidized starch pro- lization eect; thus, many commercial starch acetates are
duced is used in the paper industry. It is primarily a paper- generally granular with a DS of <0.2 [23,119]. Starch
coating binder where its high uidity and adhesive prop- acetates of higher DS are of interest because of their
erties make it eective in high-solid pigmented coating organic solvent solubility, thermoplasticity, and lm prop-
Figure 11 Examples of starch oxidation chemistry.
erties. A wide variety of reagent/catalyst/solvent systems linking can produce starches with the required properties.
have been utilized for the production of starch acetate. Ready dispersibility, viscosity stability, and the noncon-
Reagents include acetic acid, acetic anhydride, vinyl ace- gealing character of pastes make low DS starch acetates
tate, acetyl phosphate, etc. [118,132134]. In many cases, convenient for preparation, storage, handling, and appli-
activation treatment is used to improve reactivity, which cation in textile and paper manufacture. In textiles, the
involves disruption of intermolecular hydrogen bonds major market for starch acetates is in warp sizing where
[135]. Glacial acetic acid can be used for the production they have good yarn adhesion, tensile strength, and exi-
of partially degraded low DS starch acetates, but is not bility. For fabric sizing, good penetration into the fabric or
ecient for production of DS 23 starch acetates. Acetic yarn is required to stick the piles of fabric together without
anhydride is generally used for the production of starch too much starch remaining on the surface [22,119].
acetates, either alone, or in conjunction with acetic acid, Other commercially utilized starch esters are produced
pyridine, dimethyl sulfoxide (DMSO), or in aqueous alka- by reaction with sodium orthophosphate (starch phos-
line solution [132,134139]. Polysaccharide esters are gen- phate) [7,20] and a range of cyclic anhydrides, such as
erally prepared in two ways. Aqueous reactions under succinic anhydride (starch succinates) [140]. Hydrophobic
controlled pH generally produce low DS value esters character is added to starch via production of starch 1-
(<2), whereas nonaqueous processes can produce higher octenylsuccinate [23]. This derivative provides emulsion
DS value esters (up to f3). The maximum DS attainable stabilization by concentrating at the interface of an oil-
without gelatinization varies with the particular starch, but in-water emulsion because of the hydrophobicity of the
the upper limit is f0.5. Microscopic examination of the alkenyl group, and it can also be used as a partial re-
granule of DS up to 0.2 reveals no discernible dierence placement for fats as it gives a sensory perception of fat-
from native granules. Acetylated starches are more readily tiness [20].
dispersed on cooking (have lower gelatinization temper- Stabilization is also achieved by starch etherication.
atures as DS increases) than the corresponding native Hydroxyalkyl derivatives are produced by reacting starch
starches [7,118,119]. with alkyl epoxides, the most common being hydroxypro-
The major use of starch in foods is for thickening, pylstarch produced using propylene oxide and carboxy-
which requires the starch to have a bland taste and impart methylstarch produced using sodium chloroacetate [20,22,
appealing texture. In food preparations and sterilization, 42,119,141]. Hydroxypropyl starches have a stronger de-
starch may be exposed to a pH as low as 3, high shear in creasing eect on gelatinization temperature than acetate
mixing and pumping, high temperatures, and temperatures groups and have a remarkable stabilizing eect over a wide
of 4jC (refrigerator) or 20jC (freezer). Acetylation alone pH range, especially at low temperature, making them
or in combination with other treatments such as cross- useful in cold storage applications [7,118,119]. They also
have application in paper (coating) and textiles (sizing). action also takes place. This is not signicant in the usual
More detailed information on the manufacturing, proper- granular reactions because the close packing of starch
ties, and uses of hydroxyalkyl starches is available molecules favors intermolecular cross-linking. Reaction
[20,42,118,141,142]. of starch with multifunctional agents can also be used to
Overviews of the chemistry of acetylation, succinyla- bind starch to another substrate. Cross-linking granular
tion, and hydroxypropylation are provided in Fig. 12, and starch reinforces hydrogen bonds holding the granule
more detailed information on esterication and etherica- together. This produces considerable changes in the gela-
tion mechanisms is available [131]. The polysaccharide tinization and swelling properties of the starch granule with
esters of greatest commercial value are those that provide cross-linking as low as 1 cross-link per 1003000 anhydro-
enhanced functional properties compared with the native glucose units [7,38,119]. This toughening of the granule
polysaccharides, such as improved water resistance, stabil- leads to restriction in the swelling of the granule during
ity, and improved lm-forming ability [118]. gelatinization, the degree of which is related to the amount
of cross-linking.
D. Cross-Linking Very low degrees of cross-linking are dicult to
determine directly; therefore, characterization of cross-
When starch is treated with multifunctional reagents, linked starches is dependent on measurement of physical
cross-linking occurs. The reagent introduces intermolecu- properties, such as viscosity, swelling power, solubility, and
lar bridges or cross-links between molecules, thereby mark- resistance to shear [143]. The viscosity of cooked starch
edly increasing the average molecular weight. Since starch pastes is primarily dependent on the size of swollen,
contains many hydroxyl groups, some intramolecular rehydrated starch granules, which are quite fragile and tend
Figure 12 Starch stabilization chemistry: (i) general esterication; (ii) succinylation; (iii) hydroxypropylation.
Figure 13 Production of distarch phosphate.
to be fragmented by continuing heating or agitation (par- for surgical gloves. Low cross-linking levels minimize or
ticularly potato, tapioca, and waxy maize starches). Cross- eliminate the rubbery, cohesive, stringy nature of the
linking provides covalent bonds, which are not readily aqueous dispersions of waxy maize, tapioca, and potato
disrupted by cooking and hold the granule together. Thus, starch and the accompanying clinging mouthfeel texture
cross-linked granules are less fragile and more resistant to that is unpalatable in foods. This viscoelasticity of native
fragmentation by shear, high temperature, and low pH and root type starches is the result of interactions between
thus will maintain higher working viscosities and show less highly swollen, fragile, hydrated granules, which interpen-
viscosity breakdown than untreated starches. As the etrate, become entangled, and hence generate resistance to
amount of cross-linking increases, viscosity breakdown ow. Cross-linked granules result in little or no interpen-
becomes less and the high peak viscosity stabilizes, and etration giving a noncohesive structure.
the rate of gelatinization and swelling of the granules Cross-linking reagent is generally added to an aqueous
decreases, resulting in a continuous gradual increase in alkaline suspension of starch. After reaction for the re-
viscosity with prolonged cooking [20,119]. quired length of time, the starch is recovered by ltration,
At high cross-linking levels, granules no longer gelati- washed, and dried. A wide range of derivatizing agents can
nize in boiling water and have been used as dusting powder be used for cross-linking, such as bifunctional etherifying
Figure 14 Cross-linking of starch with epichlorohydrin. (From Ref. 118.)

and/or esterifying agents, e.g., epichlorohydrin, bisepox- propyl starch (E1440), hydroxypropyl distarch phosphate
ides, dibasic organic acids, anhydrides, or acid chlorides, (E1442), starch sodium octenyl succinate (E1450), and
e.g., adipic acid, adipic anhydride, or adipoyl chloride [23]. acetylated oxidized starch (E1451) are permitted in foods
Phosphorous oxychloride and sodium trimetaphosphate at levels no greater than 5 wt.% (E1440 is not permitted in
are used to produce phosphate cross-linked starch, as infant foods) [38,150].
outlined in Fig. 13 [20,23]. A wide range of other cross- Starch usage has shown a steady, rapid rate of growth
linking reagents have been investigated [118,128]. The over an extended period of time. The increasing price of
cross-linking of starch with epichlorohydrin has been energy will contribute to the demand for starch as low-cost
studied extensively and is outlined in Fig. 14 [144]. starch is utilized for the production of alcohols, compo-
The need for three-dimensional matrices with con- nents for plastics, absorbents, paper extenders, etc. Chang-
trolled release properties for pharmaceutical and agro- ing world economics is also making it more practical to
chemical uses has increased interest in unmodied and obtain chemicals from agricultural crops and wastes;
modied starch gelation [145148]. Cross-linked starches therefore, more attention is focusing on conversion/mod-
have the required biodegradability and a relatively high ication of starch and cellulose. Starch is the lowest priced
mechanical and chemical stability required for application and most abundant commercial commodity, is produced
as hydrogel medical devices [149]. in most countries, and is available at low cost in all coun-
Cross-linking is often used in combination with other tries [1].
derivatization treatments to maintain dispersion viscosity
upon exposure to high-temperature cooking, high shear, or
acid. Food starches, especially those made from waxy REFERENCES
maize, potato, and tapioca starches, are usually phos-
1. Whistler, R.L. History and future expectation of starch
phates, acetates, or hydroxypropyl ethers that are cross-
use. In Starch, Chemistry and Technology, 2nd Ed.; Whist-
linked to provide appropriate gelatinization, viscosity, and ler, R.L., BeMiller, J.N., Paschall, E.F., Eds.; Academic
textural properties. Cross-linked starches are also needed Press: New York, 1984; 19.
for salad dressings to provide the thickening without 2. Galliard, T. Starch availability and utilization. In Starch:
allowing viscosity breakdown by low pH and the high Properties and Potential. Galliard, T., Ed.; John Wiley &
shear of the homogenization process. Cross-linked Sons: Chichester, 1987; 115.
starches are used in soups, gravies, sauces, baby foods, 3. BeMiller, J.N.; Whistler, R.L. Carbohydrates. In Food
Chemistry, 3rd Edition, Food Science and Technology 76;
fruit pie lings and puddings, and batter mixes for deep- Fenema, O.R., Ed.; Marcel Dekker, Inc.: New York, 1996;
fried foods. Cross-linked starches with low amylose con- 157223.
tents, as in waxy sorghum or waxy maize, are claimed to 4. Corbishley, D.A.; Miller, W. Tapioca, arrowroot, and sago
improve cake volume, crumb softness, and keeping qual- starches: Production. In Starch, Chemistry and Technology,
ities of cakes [7,118]. 2nd Ed.; Whistler, R.L., BeMiller, J.N., Paschall, E.F.,
Eds.; Academic Press: New York, 1984; 469478.
5. Zobel, H.F.; Stephen, A.M. Starch: Structure, analysis and
application. In Food Polysaccharides and Their Applica-
V. CONCLUSIONS tions; Stephen, A.M., Ed.; Marcel Dekker, Inc.: New York,
1995; 1966.
The technical attributes of a native or modied starch are 6. Knight, J.W.; Olson, R.M. Wheat starch: production,
brought to the fore through cooking. Depending on the modication, and uses. In Starch, Chemistry and Technol-
origin or modication, phases such as gelatinization, past- ogy, 2nd Ed.; Whistler, R.L., BeMiller, J.N., Paschall, E.F.,
ing, and retrogradation can be achieved within temper- Eds.; Academic Press: New York, 1984; 491506.
atures and times that are suited to the product in which the 7. BeMiller, J.N. Starch-based gums. In Industrial Gums:
Polysaccharides and Their Derivatives, Whistler, R.L.
starch is an ingredient [20,38]. With respect to food appli-
BeMiller, J.N., Eds.; Academic Press, Inc.: San Diego,
cations, chemically modied starches are, in most cases, 1993; 579600.
labeled as modied starch. Physically treated starches 8. Juliano, B.O. Rice starch: Production, properties, and uses.
(without any additional chemical modications) carry a In Starch, Chemistry and Technology, 2nd Ed.; Whistler,
simple starch label, and enzymatically modied starches R.L., BeMiller, J.N., Paschall, E.F., Eds.; Academic Press:
can be labeled as starch or hydrolyzed starch [38]. New York, 1984; 507528.
Derivatives permitted in food in the United States are 9. Bushuk, W. History, world distribution, production, and
marketing. In Rye: Production, Chemistry, and Technology,
acetate, succinate, acetate adipate, 1-octenyl succinate, 2nd Ed.; Bushuk, W., Ed.; American Association of Cereal
phosphate, hydroxypropyl, hypochlorite oxidized, and Chemists (AACC): St. Paul, 2001; 17.
various combinations [3]. The maximum DS permitted 10. Mitch, E.L. Potato starch: Production and uses. In Starch,
for starch in foods in the U.S. foods is 0.09 for starch Chemistry and Technology, 2nd Ed.; Whistler, R.L., BeMil-
acetate, 0.002 for starch phosphate, and 0.2 for hydroxy- ler, J.N., Paschall, E.F., Eds.; Academic Press: New York,
propyl starch. In the United Kingdom, oxidized starch 1984; 479490.
11. DAppolonia, B.L.; Rayas-Duarte, P. Wheat carbohy-
(E1404), monostarch phosphate (E1410), distarch phos- drates: Structure and functionality. Wheat: Production,
phate (E1412), phosphated distarch phosphate (E1413), Properties and Quality; Blackie Academic & Professional:
acetylated distarch phosphate (E1414), acetylated starch Glasgow, 1994; 107127.
(E1420), acetylated distarch adipate (E1422), hydroxy- 12. Swinkels, J.J.M. Sources of starch, its chemistry and
physics. In Starch Conversion Technology, Food Science and drocolloids: Structures, Properties, and Functions; Nishi-
Technology 14; van Beynum, G.M.A., Roels, J.A., Eds.; nari, K., Doi, E., Eds.; Plenum Press: New York, 1993;
Marcel Dekker, Inc.: New York, 1985; 1546. 179182.
13. Dziedzic, S.Z.; Kearsley, M.W. The technology of starch 33. Slade, L.; Levine, H. A food polymer science approach to
production. In Handbook of Starch Hydrolysis Products and selected aspects of starch gelatinisation and retrogradation.
their Derivatives; Kearsley, M.W., Dziedzic, S.Z., Eds.; Frontiers in Carbohydrate Research1: Food Applications;
Blackie Academic & Professional: Glasgow, 1995; 125. Elsevier Applied Science: London, 1989; 215270.
14. Singh, N.; Singh, J.; Kaur, L.; Sodhi, N.S.; Gill, B.S. Mor- 34. Svegmark, K. Potato Starch Dispersions: Structure and
phological, thermal and rheological properties of starches Rheology, Ph.D. Thesis, Chalmers University of Technol-
from dierent botanical sources. Food Chem. 2003, 81, ogy: Gothenburg, Sweden, 1992.
219231. 35. Zobel, H.F. Gelatinization of starch and mechanical
15. Watson, S.A. Corn and sorghum starches: Production. In properties of starch pastes. In Starch, Chemistry and
Starch, Chemistry and Technology, 2nd Ed.; Whistler, R.L., Technology, 2nd Ed.; Whistler, R.L., BeMiller, J.N.,
BeMiller, J.N., Paschall, E.F., Eds.; Academic Press: New Paschall, E.F., Eds.; Academic Press: New York, 1984;
York, 1984; 417468. 285309.
16. Ranhotra, G.S. Wheat: Contribution to world food supply 36. Keetels, C.J.A.M.; van Vliet, T.; Luyten, H. Eect of
and human nutrition. Wheat: Production, Properties and retrogradation on the structure and mechanics of concen-
Quality; Blackie Academic & Professional: Glasgow, 1994; trated starch gels. In Food Macromolecules and Colloids;
1224. Dickinson, E., Lorient, D., Eds.; The Royal Society of
17. Oleson, B.T. World wheat production, utilization and Chemistry: Cambridge, 1995; 472479.
trade. Wheat: Production, Properties and Quality; Blackie 37. Hizukuri, S. Starch: Analytical aspects, carbohydrates in
Academic & Professional: Glasgow, 1994; 111. food. In Food Science and Technology 74; Eliasson, A.-C.,
18. Carioca, J.O.B.; Arora, H.L.; Pannir Selvam, V.; Tavares, Ed.; Marcel Dekker, Inc.: New York, 1996; 347429.
F.C.A.; Kennedy, J.F.; Knill, C.J. The industrial utilisation 38. Murphy, P. Starch. Handbook of Hydrocolloids; Phillips,
of starch and its derived products in Brazil. Starch/Starke G.O., Williams, P.A., Eds.; Woodhead Publishing
1996, 48, 322326. Limited: Cambridge, 2000; 4165.
19. Atwell, W.A. Wheat Flour; Eagan Press: St. Paul, 2001. 39. Berry, C.P.; DAppolonia, B.L.; Giles, K.A. The character-
20. Atwell, W.A.; Hood, L.F.; Lineback, D.R.; Varriano- ization of triticale starch and its comparison with starches
Marston, E.; Zobel, H.F. The terminology and method- of rye, durum, and HRS wheat. Cereal Chem. 1971, 48,
ology associated with basic starch phenomena. Cereal 415427.
Foods World 1988, 33, 306311. 40. Ring, S.G. Sti tests for designer starches. Chem. Br. 1995,
21. Blanshard, J.M.V. Starch granule structure and function: 31 (4), 303307.
A physicochemical approach. In Starch: Properties and 41. Zobel, H.F. Starch granule structure. In Developments in
Potential; Galliard, T., Ed.; John Wiley & Sons: Chichester, Carbohydrate Chemistry; Alexander, R.J., Zobel, H.F.,
1987; 1654. Eds., American Association of Cereal Chemists (AACC):
22. Cornell, H.J.; Hoveling, A.W. Wheat: Chemistry and Uti- St. Paul, 1992, 136.
lization; Technomic Publishing Co, Inc.: Lancaster, 1998. 42. Kuipers, N.J.M. GasSolid Hydroxyethylation of Potato
23. Coultate, T.P. Food: The Chemistry of Its Components, 4th Starch, Ph.D. thesis, Rijksuniversiteit, Groningen: The
Ed.; The Royal Society of Chemistry: Cambridge, 2002. Netherlands, 1995.
24. Cura, J.A.; Jansson, P.-E.; Krisman, C.R. Amylose is not 43. Guilbot, A.; Mercier, C. Starch. In The Polysaccharides;
strictly linear. Starch/Starke 1995, 47, 207209. Aspinall, G.O., Ed.; Academic Press, Inc.: Orlando, 1985;
25. Morris, V.J. In Gelation of Polysaccharides, Functional Vol. 3, 209282.
Properties of Food Macromolecules, 2nd Ed.; Hill, S.E., 44. Whistler, R.L.; Daniel, J.R. Molecular structure of starch.
Ledward, D.A., Mitchell, J.R., Eds.; Aspen Publishers In Starch, Chemistry and Technology, 2nd Ed.; Whistler,
Inc.: Gaithersburg, 1998; 143226. R.L., BeMiller, J.N., Paschall, E.F., Eds.; Academic Press:
26. Ring, S.G.; Noel, T.R.; Bull, V.J. The structure of the New York, 1984; 153182.
starch polysaccharides and their organisation in the starch 45. Manners, D.J. Recent developments in our understanding
granule. In Seed Storage Compounds: Biosynthesis, Inter- of amylopectin structure. Carbohydr. Polym. 1989, 11, 87
actions, and Manipulation; Shewry, P.R., Stobart, K., Eds.; 112.
Oxford University Press: Oxford, 1993; 2539. 46. Hew, C.L.; Unrau, A.M. Investigation of the starch com-
27. Takeda, Y.; Shitaozono, T.; Hizukuri, S. Structures of sub- ponents of a synthetic cereal species. J. Agric. Food Chem.
fractions of corn amylose. Carbohydr. Res. 1990, 199, 207 1970, 18, 657662.
214. 47. Shewry, P.R.; Bechtel, D.B. Morphology and chemistry of
28. Takeda, Y.; Tomooka, S.; Hizukuri, S. Structures of the rye grain. In Rye: Production, Chemistry, and Technol-
branched and linear molecules of rice amylose. Carbohydr. ogy, 2nd Ed.; Bushuk, W., Ed.; American Association of
Res. 1993, 246, 267272. Cereal Chemists (AACC): St. Paul, 2001; 69127.
29. Thomas, D.J.; Atwell, W.A. Starches; Eagan Press: St. 48. Juliano, B.O.; Villareal, C.P. Grain Quality Evaluation of
Paul, 1999. World Rices; International Rice Research Institute (IRRI):
30. Walstra, P. Physical Chemistry of Foods; Marcel Dekker, Manila, 1993.
Inc.: New York, 2003. 49. Manners, D.J. Structural analysis of starch components by
31. Yuryev, V.P.; Wasserman, L.A.; Andreev, N.R.; Tolsto- debranching enzymes. In New Approaches to Research on
guzov, V.B. Structural and thermodynamic features of low- Cereal Carbohydrates; Hill, D., Munck, L., Eds.; Elsevier
and high amylose starches. A review. In Starch and Starch Science Publishers B.V.: Amsterdam, 1985; 4554.
Containing Origins; Yuryev, V.P., Cesa`ro, A., Bergthaller, 50. Gidley, M.J. Starch structure/function relationships:
W.J., Eds.; Nova Science Publishers, Inc.: New York, 2002; Achievements and challenges. In Starch: Advances in
2355. Structure and Function; Barsby, T.L., Donald, A.M.,
32. Kitamura, S.; Hakozaki, K.; Kuge, T. Eects of molecular Frazier, P.J., Eds.; The Royal Society of Chemistry:
weight on the retrogradation of amylose. In Food Hy- Cambridge, 2001; 17.
51. Fitt, L.E.; Snyder, E.M. Photomicrographs of starches. In enzymes. In Carbohydrate Chemistry; Kennedy, J.F., Ed.;
Starch, Chemistry and Technology, 2nd Ed.; Whistler, R.L., Oxford University Press: Oxford, 1988; 342377.
BeMiller, J.N., Paschall, E.F., Eds.; Academic Press: New 70. Kennedy, J.F.; Cabral, J.M.S.; Sa-Correia, I.; White, C.A.
York, 1984; 675689. Starch biomass: A chemical feedstock for enzyme and
52. Karlsson, R.; Olered, R.; Eliasson, A.-C. Changes in starch fermentation processes. In Starch: Properties and Potential;
granule size distribution and starch gelatinisation proper- Galliard, T., Ed.; John Wiley & Sons: Chichester, 1987;
ties during development and maturation of wheat, barley 115148.
and rye. Starch/Starke 1983, 35, 335340. 71. Blanchard, P.H.; Katz, F.R. Starch hydrolysates. In Food
53. Hinrichs, W.; Buttner, G.; Steifa, M.; Betzel, C.; Zabel, V.; Polysaccharides and Their Applications; Stephen, A.M.,
Pfannemuller, B.; Saenger, W. An amylose antiparallel dou- Ed.; Marcel Dekker, Inc.: New York, 1995; 99122.
ble helix at atomic resolution. Science 1987, 238, 205208. 72. Reilly, P.J. Enzymic degradation of starch. In Starch
54. Gidley, M.J.; Bociek, S.M. C-13 cp/mas nmr-studies of Conversion Technology, Food Science and Technology 14;
amylose inclusion complexes, cyclodextrins, and the van Beynum, G.M.A., Roels, J.A., Eds.; Marcel Dekker,
amorphous phase of starch granulesrelationships be- Inc.: New York, 1985; 101142.
tween glycosidic linkage conformation and solid-state C-13 73. Gacesa, P.; Hubble, J. The enzymic treatment of waste
chemical-shifts. J. Am. Chem. Soc. 1988, 110, 38203829. materials. In Bioconversion of Waste Materials to Industrial
55. Pfannemuller, B. Inuence of chain length of short mono- Products; Martin, A.M., Ed.; Elsevier Applied Science:
disperse amyloses on the formation of A- and B-type X-ray London, 1991; 3962.
diraction patterns. Int. J. Biol. Macromol. 1987, 9, 105 74. Priest, F.G.; Stark, J.R. Starch-hydrolyzing enzymes with
108. novel properties. In Biotechnology of Amylodextrin Oligo-
56. Gidley, M.J.; Bulpin, P.V. Crystallisation of malto-oligosaccharides; ACS Symposium Series 458; Friedman, R.B.,
saccharides as models of the crystalline forms of starch: Ed.; American Chemical Society (ACS): Washington,
minimum chain-length requirement for the formation of 1991; 7285.
double helices. Carbohydr. Res. 1987, 161, 291300. 75. Frost, G.M.; Moss, D.A. Production of enzymes by fer-
57. Hizukuri, S.; Kaneko, T.; Takeda, Y. Measurement of the mentation. In Biotechnology; Kennedy, J.F., Ed.; VCH:
chain length of amylopectin and its relevance to the origin Weinheim, 1987; Vol. 7a, 65211.
of crystalline polymorphism of starch granules. Biochim. 76. Webb, E.C., Ed. Enzyme Nomenclature: Recommendations
Biophys. Acta 1983, 760, 188191. (1992) of the Nomenclature Committee of the International
58. Imberty, A.; Perez, S. A revisit to the 3-dimensional struc- Union of Biochemistry and Molecular Biology; Academic
ture of B-type starch. Biopolymers 1988, 27, 12051221. Press, Inc.: San Diego, 1992.
59. Zobel, H.F. Molecules to granules: A comprehensive 77. Kainuma, K. Starch oligosaccharides: Linear, branched,
starch review. Starch/Starke 1988, 40, 4450. and cyclic. In Starch, Chemistry and Technology, 2nd Ed.;
60. French, D. Organisation of starch granules. In Starch, Whistler, R.L., BeMiller, J.N., Paschall, E.F., Eds.; Aca-
Chemistry and Technology, 2nd Ed.; Whistler, R.L., BeMil- demic Press: New York, 1984; 125152.
ler, J.N., Paschall, E.F., Eds.; Academic Press: New York, 78. Venkatasubramanian, K.; Keim, C.R. Starch and energy:
1984; 183247. Technology and economics of fuel alcohol. In Starch
61. Jenkins, P.J.; Donald, A.M. Gelatinisation of starch: A Conversion Technology, Food Science and Technology 14;
combined SAXS/WAXS/DSC and SANS study. Carbo- van Beynum, G.M.A., Roels, J.A., Eds.; Marcel Dekker,
hydr. Res. 1998, 308, 133147. Inc.: New York, 1985; 143173.
62. Donald, A.M.; Perry, P.A.; Waigh, T.A. The impact of 79. Dasinger, B.L.; Fenton, D.M.; Nelson, R.P.; Roberts,
internal granule structure on processing and properties. In F.F.; Truesdell, S.J. Enzymic and microbial processes in the
Starch: Advances in Structure and Function; Barsby, T.L., conversion of carbohydrates derived from starch. In Starch
Donald, A.M., Frazier, P.J., Eds.; The Royal Society of Conversion Technology, Food Science and Technology 14;
Chemistry: Cambridge, 2001; 4552. van Beynum, G.M.A., Roels, J.A., Eds.; Marcel Dekker,
63. Jane, J.-L.; Robyt, J.F. Structure studies of amylose-V Inc.: New York, 1985; 237262.
complexes and retro-graded amylose by action of alpha 80. Otey, F.H.; Doane, W.M. Chemicals from starch. In
amylases, and a new method for preparing amylodextrins. Starch, Chemistry and Technology, 2nd Ed.; Whistler, R.L.,
Carbohydr. Res. 1984, 132, 105118. BeMiller, J.N., Paschall, E.F., Eds.; Academic Press: New
64. Gidley, M.J.; Bulpin, P.V. Aggregation of amylose in York, 1984; 389416.
aqueous systemsThe eect of chain-length on phase- 81. Kieboom, A.P.G.; van Bekkum, H. Chemical conversion of
behaviour and aggregation kinetics. Macromolecules 1989, starch-based glucose syrups. In Starch Conversion Tech-
22, 341346. nology, Food Science and Technology 14; van Beynum,
65. Dengate, H.N. Swelling, pasting and gelling of wheat G.M.A., Roels, J.A., Eds.; Marcel Dekker, Inc.: New York,
starch. In Advances in Cereal Science and Technology; 1985; 7399.
Pomeranz, Y., Ed.; American Association of Cereal 82. Krutos kova, A.; Uher, M. Natural and Synthetic Sweet
Chemists (AACC): St. Paul, 1984; Vol. VI, 4982. Substances; Ellis Horwood: Chichester, 1992.
66. Gudmundsson, M.; Eliasson, A.-C. Thermal and viscous 83. Kim, C.H.; Lee, G.M.; Abidin, Z.; Han, M.H.; Rhee, S.K.
properties of rye starch extracted from dierent varieties. Immobilisation of Zymomonas mobilis and amyloglucosi-
Cereal Chem. 1991, 68, 172177. dase for ethanol-production from sago starch. Enzyme
67. Hebeda, R.E.; Teague, W.M. Starch hydrolysing enzymes. Microb. Technol. 1988, 10 (7), 426430.
In Developments in Carbohydrate Chemistry; Alexander, 84. Cole, G.E.; McCabe, P.C.; Inlow, D.; Gelfand, D.H.; Ben-
R.J., Zobel, H.F., Eds.; American Association of Cereal Bassat, A.; Innis, M.A. Stable expression of Aspergillus
Chemists (AACC): St. Paul, 1992; 6585. awamori glucoamylase in distillers yeast. Biotechnology
68. Robyt, J.R. Enzymes in the hydrolysis and synthesis of 1988, 6 (4), 417421.
starch. In Starch, Chemistry and Technology, 2nd Ed.; 85. Inlow, D.; McRae, J.; Ben-Bassat, A. Fermentation of corn
Whistler, R.L., BeMiller, J.N., Paschall, E.F., Eds.; Aca- starch to ethanol with genetically engineered yeast.
demic Press: New York, 1984; 87123. Biotechnol. Bioeng. 1988, 32 (2), 227234.
69. White, C.A.; Kennedy, J.F. The carbohydrate-directed 86. Kumagai, M.H.; Sverlow, G.G.; della-Cioppa, G.; Grill,
L.K. Conversion of starch to ethanol in a recombinant 103. Klis, J.B. Ingredients round-table. Food Technol. 1984, 38
Saccharomyces cerivisiae strain expressing rice a-amylase (11), 9294.
from a novel Pichia pastoris alcohol oxidase promoter. 104. Howling, D. Introduction: Glucose syrupsPast, present
Biotechnology 1993, 11 (5), 606610. and future. In Glucose Syrups: Science and Technology;
87. Morris, C.E. Maltodextrins. Food Eng. 1984, 56 (7), 48. Dziedzic, S.Z., Kearsley, M.W., Eds.; Elsevier Applied
88. Kennedy, J.F.; Knill, C.J.; Taylor, D.W. Maltodextrins. In Science Publishers Ltd: London, 1984; 17.
Handbook of Starch Hydrolysis Products and their Deriv- 105. Lloyd, N.E.; Nelson, W.J. Glucose- and fructose-contain-
atives; Kearsley, M.W., Dziedzic, S.Z., Eds.; Blackie Aca- ing sweeteners from starch. In Starch, Chemistry and
demic & Professional: Glasgow, 1995; 6582. Technology, 2nd Ed.; Whistler, R.L., BeMiller, J.N.,
89. Frye, A.M.; Setser, C.S. Bulking agents and fat substitutes. Paschall, E.F., Eds.; Academic Press: New York, 1984;
In Low-Calorie Foods Handbook, Food Science and Tech- 611660.
nology 56; Altschul, A.M., Ed.; Marcel Dekker, Inc.: New 106. Fullbrook, P.D. The enzymic production of glucose syrups.
York, 1993; 211251. In Glucose Syrups: Science and Technology; Dziedzic, S.Z.,
90. Birch, G.G.; Azudin, M.N.; Grigor, J.M. Solution proper- Kearsley, M.W., Eds.; Elsevier Applied Science Publishers
ties and composition of dextrins. In Biotechnology of Ltd: London, 1984; 65115.
Amylodextrin Oligosaccharides; ACS Symposium Series 107. Dziedzic, S.Z.; Kearsley, M.W. Physico-chemical prop-
458; Friedman, R.B., Ed.; American Chemical Society erties of glucose syrups. In Glucose Syrups: Science and
(ACS): Washington, 1991; 261272. Technology; Dziedzic, S.Z., Kearsley, M.W., Eds.; Else-
91. Ring, S.G.; Whittam, M.A. Linear dextrins. In Biotechnol- vier Applied Science Publishers Ltd: London, 1984; 137
ogy of Amylodextrin Oligosaccharides; ACS Symposium 168.
Series 458; Friedman, R.B., Ed.; American Chemical 108. Olson, H.S. Enzymatic production of glucose syrups. In
Society (ACS): Washington, 1991; 273293. Handbook of Starch Hydrolysis Products and their Deriv-
92. Jasutiene, I.; Kolodzeiskis, V. Eects of various factors on atives; Kearsley, M.W., Dziedzic, S.Z., Eds.; Blackie
the process of hydrolysis of starch to maltodextrins. In Academic & Professional: Glasgow, 1995; 2664.
Starch and Starch Containing Origins; Yuryev, V.P., Ce- 109. Kearsley, M.W.; Dziedzic, S.Z. Physical and chemical
sa`ro, A., Bergthaller, W.J., Eds.; Nova Science Publishers, properties of glucose syrups. In Handbook of Starch Hy-
Inc.: New York, 2002; 211216. drolysis Products and their Derivatives; Kearsley, M.W.,
93. Ben-Bassat, A.A.; Grushka, E. High-performance liquid- Dziedzic, S.Z., Eds.; Blackie Academic & Professional:
chromatography of monosaccharides and oligosaccha- Glasgow, 1995; 129154.
rides. J. Liq. Chromatogr. 1991, 14, 10511111. 110. McDonald, M. Use of glucose syrups in the food industry.
94. Koizumi, K.; Kubota, Y.; Tanimoto, T.; Okada, Y. High- In Glucose Syrups: Science and Technology; Dziedzic, S.Z.,
performance anion-exchange chromatography of homo- Kearsley, M.W., Eds.; Elsevier Applied Science Publishers
geneous D -gluco-oligosaccharides and D -gluco-poly- Ltd: London, 1984; 247263.
saccharides (polymerisation degree R50) with pulsed 111. Jackson, E.B. Use of glucose syrups in the food industry. In
amperometric detection. J. Chromatogr. 1989, 464, 365 Handbook of Starch Hydrolysis Products and their Deriv-
373. atives; Kearsley, M.W., Dziedzic, S.Z., Eds.; Blackie
95. Hernandez, L.M.; Ballou, L.; Ballou, C.L. Separation of Academic & Professional: Glasgow, 1995; 245268.
yeast asparagines-linked oligosaccharides by high-per- 112. Wilms, J. Enzymatic production of maltose syrups. In
formance anion-exchange chromatography. Carbohydr. Starch and Starch Containing Origins; Yuryev, V.P., Ce-
Res. 1990, 203, 111. sa`ro, A., Bergthaller, W.J., Eds.; Nova Science Publishers,
96. Ammeraal, R.N.; Delgado, G.A.; Tenbarge, F.L.; Fried- Inc.: New York, 2002; 199209.
man, R.B. High-performance anion-exchange chromatog- 113. van Tilburg, R. Enzymic isomerisation of corn starch-
raphy with pulsed amperometric detection of linear and based glucose syrups. In Starch Conversion Technology,
branched glucose oligosaccharides. Carbohydr. Res. 1991, Food Science and Technology 14; van Beynum, G.M.A.,
215, 179192. Roels, J.A., Eds.; Marcel Dekker, Inc.: New York, 1985;
97. Cabalda, V.M.B.; Kennedy, J.F.; Jumel, K. Analysis of 175236.
amylodextrins. In Biotechnology of Amylodextrin Oligo- 114. Kobayashi, S. Cyclodextrin producing enzyme (CGTase).
saccharides; ACS Symposium Series 458; Friedman, R.B., In Enzymes for Carbohydrate Engineering, Progress in
Ed.; American Chemical Society (ACS): Washington, Biotechnology; Park, K.-H., Robyt, J.F., Choi, Y.-D.,
1991; 140170. Eds.; Elsevier Science B.V.: Amsterdam, 1996; Vol. 12,
98. Heyraud, A.; Rinaudo, M. Use of multidetection for 2341.
chromatographic characterization of dextrins and starch. 115. Li, S.; Purdy, W.C. Cyclodextrins and their applications in
In Biotechnology of Amylodextrin Oligosaccharides; ACS analytical chemistry. Chem. Rev. 1992, 92, 14571470.
Symposium Series 458; Friedman, R.B., Ed.; American 116. Friedman, R.B.; Hedges, A.R. Recent advances in the use
Chemical Society (ACS): Washington, 1991; 171188. of cyclodextrins in food systems. Frontiers in Carbohydrate
99. Altschul, A.M. Low-calorie foodsA scientic status sum- Research1: Food Applications; Elsevier Applied Science:
mary by the Institute of Food Technologists expert panel London, 1989; 7482.
on food safety and nutrition. Food Technol. 1989, 43 (4), 117. Kalinkevich, A.N.; Chemeris, I.I.; Pavlenko, P.A.; Pilipen-
113125. ko, V.V.; Rogulsky, Y.V.; Bugai, A.N.; Danilchenko, S.N.;
100. Kaper, F.S.; Gruppen, H. Replace oil and fat with potato- Sukhodub, L.F. Mass spectrometry study of cyclodextrins
based ingredient. Food Technol. 1987, 41 (3), 112113. and their interaction with biologically active molecules. In
101. Alexander, R.J. Carbohydrates used as fat replacers. In Starch and Starch Containing Origins; Yuryev, V.P., Ce-
Developments in Carbohydrate Chemistry; Alexander, R.J., sa`ro, A., Bergthaller, W.J., Eds.; Nova Science Publishers,
Zobel, H.F., Eds.; American Association of Cereal Inc.: New York, 2002; 154162.
Chemists (AACC): St. Paul, 1992; 343370. 118. Rutenberg, M.W.; Solarek, D. Starch derivatives: Produc-
102. Pszczola, D.E. Carbohydrate-based ingredient performs tion and uses. In Starch, Chemistry and Technology, 2nd
like fat for use in a variety of food applications. Food Ed.; Whistler, R.L., BeMiller, J.N., Paschall, E.F., Eds.;
Technol. 1991, 45 (8), 262. Academic Press: New York, 1984; 311388.
119. Fleche, G. Chemical modication and degradation of properties of starch triesters. Ind. Eng. Chem. 1942, 34,
starch. In Starch Conversion Technology, Food Science and 12071209.
Technology 14; van Beynurn, G.M.A., Roels, J.A., Eds.; 136. Wol, I.A.; Olds, D.W.; Hilbert, G.E. Triesters of corn
Marcel Dekker, Inc.: New York, 1985; 7399. starch, amylose and amylopectin: Properties. Ind. Eng.
120. Mentzer, M.J. Starch in the paper industry. In Starch, Chem. 1951, 43, 911914.
Chemistry and Technology, 2nd Ed.; Whistler, R.L., BeMil- 137. Wol, I.A.; Olds, D.W.; Hilbert, G.E. Mixed esters of
ler, J.N., Paschall, E.F., Eds.; Academic Press: New York, amylose. Ind. Eng. Chem. 1957, 48, 12471248.
1984; 543574. 138. Tessler, M.M.; Billmers, R.L. Preparation of starch esters.
121. Kennedy, H.M.; Fischer, A.C. Starch and dextrins in J. Environ. Polym. Degrad. 1996, 4, 8589.
prepared adhesives. In Starch, Chemistry and Technology, 139. Khalil, M.I.; Hashem, A.; Hebeish, A. Preparation and
2nd Ed.; Whistler, R.L., BeMiller, J.N., Paschall, E.F., characterisation of starch acetate. Starch/Starke 1995, 47,
Eds.; Academic Press: New York, 1984; 593610. 394398.
122. Kirby, K.W. Non-food uses of starch. In Developments 140. Knill, C.J.; Rahman, S.; Kennedy, J.F. New carbohydrate
in Carbohydrate Chemistry; Alexander, R.J., Zobel, H.F., polymer derivatives from renewable bioresources targeted
Eds.; American Association of Cereal Chemists (AACC): for industrial application. In Cellulosic Pulps, Fibres and
St. Paul, 1992; 371386. Materials; Kennedy, J.F., Phillips, G.O., Williams, P.A.,
123. Colonna, P.; Buleon, A.; Mercier, C. Physically modied Lonnberg, B., Eds.; Woodhead Publishing Limited: Cam-
starches. In Starch: Properties and Potential; Galliard, T. bridge, 2000; 319326.
Ed.; John Wiley & Sons: Chichester, 1987; 79114. 141. Kesler, C.C.; Hjermstad, E.T. Hydroxyethyl- and hydroxy-
124. Chiu, C.W.; Schiermeyer, E.; Thomas, D.J.; Shah, M.B. propylstarch. In Methods in Carbohydrate Chemistry IV:
Thermally inhibited starches and ours and process for their Starch; Whistler, R.L., Ed.; Academic Press: New York,
production, US Patent 5,725,676. 1998. 1964; 304306.
125. Imeson, A., Ed. Thickening and Gelling Agents for Food; 142. Kennedy, J.F. Chemically reactive derivatives of polysac-
Chapman & Hall: London, 1997 114. charides. Adv. Carbohydr. Chem. Biochem. 1974, 29, 305
126. Rohwer, R.G.; Klem, R.E. Acid-modied starch: Produc- 405.
tion and uses. In Starch, Chemistry and Technology, 2nd 143. Schoch, T.J. Swelling power and solubility of granular
Ed.; Whistler, R.L., BeMiller, J.N., Paschall, E.F., Eds.; starches. In Methods in Carbohydrate Chemistry IV:
Academic Press: New York, 1984; 529541. Starch; Whistler, R.L., Ed.; Academic Press: New York,
127. Autio, K.; Poutanen, K. Gelling properties of acid- 1964; 106108.
modied starches. In Food Hydrocolloids: Structures, 144. Kartha, K.P.R.; Srivastava, H. Reaction of epichlorohy-
Properties, and Functions; Nishinari, K., Doi, E., Eds.; drin with carbohydrate polymers. II. Starch reaction
Plenum Press: New York, 1993; 161165. mechanism and physicochemical properties of modied
128. Wurzburg, O.B. Modied starches. In Food Polysaccha- starch. Starch/Starke 1985, 37, 297306.
rides and Their Applications; Stephen, A.M., Ed.; Marcel 145. Wing, R.E.; Maiti, S.; Doane, W.M. Eectiveness of jet-
Dekker, Inc.: New York, 1995; 6798. cooked pearl cornstarch as a controlled release matrix.
129. Hullinger, C.H. Hypochlorite-oxidized starch. In Methods Starch/Starke 1987, 39, 422425.
in Carbohydrate Chemistry IV: Starch; Whistler, R.L., Ed.; 146. Trimnell, D.; Shasha, B.S. Autoencapsulation: A new
Academic Press: New York, 1964; 313315. method for entrapping pesticides within starch. J. Control.
130. Mehltretter, C.L. Dialdehyde starch. In Methods in Release 1988, 7, 2531.
Carbohydrate Chemistry IV: Starch; Whistler, R.L., Ed.; 147. Heller, J.; Pangburn, S.H.; Roskos, K.V. Development of
Academic Press: New York, 1964; 316317. enzymatically degradable protective coating for use in
131. Yalpani, M. Polysaccharides: Syntheses, Modications and triggered drug delivery systems: Derivatised starch hydro-
Structure/Property Relations, Studies in Organic Chemistry gels. Biomaterials 1990, 11, 345350.
36; Elsevier Science Publishers B.V.: Amsterdam, 1988. 148. Levy, M.C.; Andry, M.C. Microcapsules prepared through
132. Mullen, J.W.; Pacsu, E. Starch studies: Possible indus- interfacial crosslinking of starch derivatives. Int. J. Pharm.
trial utilisation of starch esters. Ind. Eng. Chem. 1943, 35, 1990, 62, 2735.
381384. 149. Dumitriu, S.; Vidal, P.F.; Chornet, E. Hydrogels based on
133. Whistler, R.L. Preparation and properties of starch esters. polysaccharides. In Polysaccharides in Medicinal Applica-
Adv. Carbohydr. Chem. 1945, 1, 279307. tions; Dumitriu, S., Ed.; Marcel Dekker, Inc.: New York,
134. Wurzburg, O.B. Acetylation. In Methods in Carbohydrate 1996; 125241.
Chemistry IV: Starch; Whistler, R.L., Ed.; Academic Press: 150. Flowerdrew, D. Food Additives: What Every Manager
New York, 1964; 286288. Needs to Know About the Law; Chandos Publishing
135. Mullen, J.W.; Pacsu, E. Starch studies: Preparation and Limited: Oxford, 1999.
26
StructureProperty Relationship in Chitosans
Kjell M. Varum and Olav Smidsrd
Norwegian University of Science and Technology (NTNU), Trondheim, Norway
I. GENERAL INTRODUCTION and 60 mol% of the deacetylated form. Since the amino
groups are very electronegative and can take up a proton
According to some marine biologist [1] chitin in all its
and become positively charged, and since the N-acetylated
forms, in a vast number of dierent species in the marine
amino groups are hydrophobic, chitosans can be regarded
and terrestrial environment, competes with cellulose in
as a family of reactive amphiphilic polymers with very
being the most abundant organic substance on our planet.
dierent chemical, physical, and biological properties. This
As a raw material for commercial exploitation it is, how-
mere fact, which so far has not been fully recognized, will
ever, almost invisible compared to cellulose from which
make it necessary to optimize the type of chitosan needed
commercial products are produced in the scale of 100200
for a given application to obtain the desired functionality.
million metric tons per year compared to less than 10,000
In the food, pharmaceutical, and biomedical elds the
metric tons for chitin [2]. There could be several reasons for
requirement for commercially available chitosans with
this enormous dierence: availability of sources, lack of
well-characterized and reproducible chemical and physical
proper harvesting techniques and logistics, and raw mate-
properties is absolute in order to trigger the development of
rial separated geographically from the marked and the
new products and to obtain approval for their usage. It is
developing technologies. Or it could be some inherent
hoped that the present review of the structureproperty
problems with the functionality of chitinous products
relationship of chitosans based on our present knowledge,
compared to cellulose due to their chemical dierences,
obtained in part by some 15 years of research in our
cellulose being very inert and chitosan, as shall be seen,
laboratory, may have an eect in increasing the interest
very reactive and an active ingredient in many applications.
for chitosans and thereby help to enhance the utilization of
In some instances the use of chitosans produced from
the whole family of chitosans. Since the structureproperty
chitin would be the best choice from just a functional point
relationship is in focus in the present review, the literature
of view, e.g., in several potential applications in the cellu-
cited and discussed deals mainly with the eect of the
lose industry, for removing humus from drinking water
degree of acetylation and the molecular weight on the
and occulation of wastewater, and as a lm-forming
physical and biological properties, and a large fraction of
material, but when the tonnage required and costs are
the literature has been omitted from among the increasing
compared with synthetic polycation alternatives, chitosan
number of papers published on chitosan.
is not the choice due to its unavailability and too high cost.
However, when environmental concerns are in focus, and
when waste products, e.g., humus removed from drinking A. Sources
water and proteins from food industry wastewater, are
collected for use as fertilizer or as animal feed, chitosan Chitin occurs mainly as a structural polysaccharide in the
could clearly compete with the synthetic polycations. outer skeleton of animals belonging to the phylum Ar-
The present authors believe that the chemical dier- thropoda (animals with an outer skeleton). Chitin is also
ences between cellulose and chitosans so far may have hin- found in, e.g., algae and the cell walls of certain fungi.
dered their bulk uses. In the simple chemical sense, Industrial sources of chitin are crustacean shells, where
chitosans are the 2-deoxy 2-amino derivative of cellulose. chitin comprises about one-third of the dry weight of the
In chitin the amino groups are highly acetylated, but chi- shells, depending on the species, and where it occurs in
tosan, the water-soluble polymer, contains between 100 close association with proteins, calcium carbonate, pig-
625
626 Varum and Smidsrd
ments, and lipids [3]. Chitosan is much less abundant in 2. Production of Chitosan
nature than chitin, and has so far only been found in the cell Chitin is used as raw material for all current commercial
walls of certain fungi. production of chitosan. However, it is also possible to
Chitin is raw material for all current commercial isolate chitosan from certain fungi [46] and to prepare
production of chitosan and the monosaccharide glucosa- chitosans with dierent fractions of acetylated units, FA, by
mine, with an estimated annual production (year 2000) of reacetylation of highly deacetylated chitosan [7].
10,000 metric tons [2]. The industrial applications of Deacetylation of chitin can in principle be performed
chitosan and the use of glucosamine form the basis of the by hydrolysis under acidic or alkaline conditions. The use
exploitation of crustacean shells, which represent a poten- of acidic conditions for deacetylation will result in severe
tial waste problem for the suppliers of shrimps, crabs, and degradation of the polysaccharide (see Sec. V.C.1 in this
other crustaceans. chapter). The acetamido group of chitin, however, is rela-
tively resistant to alkaline hydrolysis, although much less
resistant than the glycosidic linkage in chitin, as only limit-
B. Production
ed degradation occurs during the drastic alkaline condi-
1. Isolation of Chitin tions required for the production of chitosan from chitin.
The extraction of chitin from crustacean shells is shown Chitosans can be prepared from chitin by two dierent
schematically in Fig. 1. The shells are treated with dilute methods, i.e., homogeneous [8,9] and heterogeneous de-
acid to extract the calcium carbonate (CaCO3), which has a acetylation procedures, which are limited to the production
very low solubility in water. However, by lowering the pH of fully water soluble chitosans (at acidic pH values) with a
to about 1, the carbonate ions react with the protons to relatively low FA, i.e., less than 0.20.3. Attempts to
form bicarbonate (HCO prepare more highly acetylated chitosans using the heter-
3 ) and carbonic acid (H2CO3),
which is liberated from the solution as carbon dioxide ogeneous deacetylation process has been described, and it
(CO2). Thus, by keeping the pH low the calcium carbonate was found that the fractions were composed of an acid-
is eectively removed from the shells. In the second step, insoluble chitinlike fraction and an acid-soluble chitosan
the shells are treated with alkali at elevated temperatures to fraction [10,11]. Such heterogeneously deacetylated chito-
remove proteins. Proteins are generally more soluble at sans with a high FA could be obtained only by careful
higher pH values, and after washing the solubilized pro- control of the time of deacetylation and subsequent extrac-
teins, the chitin is dried and milled. Some producers include tion of the product by dilute acid. Recently, a new heter-
extraction with an organic solvent to remove pigments and ogeneous method to prepare more highly acetylated
fat, depending on the source of the chitin and the isolation chitosans with full acid solubility has been reported [12].
procedure.
II. CHITOSAN CHEMISTRY

A. Composition
Chitin is a linear polymer of (1!4)-linked 2-acetamido-2-
deoxy-h-D-glucopyranose (GlcNAc; A unit) which is in-
soluble in aqueous solvents. Chitin isolated from dierent
sources may be slightly de-N-acetylated, which may be the
result of the isolation procedure (see Fig. 1). Chitin shares
many structural similarities with cellulose, as, e.g., the
conformation of the monomers and the diequatorial gly-
cosidic linkages. Chitosans can be considered as a family of
linear binary copolymers of (1!4)-linked A units and 2-
amino-2-deoxy-h-D-glucopyranose (GlcN; D unit) (Fig. 2),
and not a uniquely dened compound; as it refers to
polysaccharides having dierent composition of A and D
units. It has been proposed to dene chitin and chitosan
based on their solubility in aqueous acetic acid, i.e., chito-
san as soluble and chitin as insoluble in 0.1 M acetic acid
[3]. Commercially produced chitosans are normally pre-
pared by alkaline deacetylation of chitin to FA values below
0.20.3.
A number of methods have been published and used
for determining the degree of N-acetylation of chitosans,
Figure 1 Procedure for isolation of chitin from crustacean such as IR, UV, gel permeation chromatography, colloid
shells (schematically). The order of the demineralization and titration, elemental analysis, dye adsorption, and acid
the deproteinization step may be reversed. base titration methods [3]. Some of the methods have been
StructureProperty Relationship in Chitosans 627
Figure 2 Chemical structure of chitin (a) and chitosan (b).
reported to be only partially quantitative. An enzymatic proton NMR spectrum of a chitosan is shown, which can
method based on complete hydrolysis of chitosan for deter- be used for determination of FA and obtaining sequential
mining the fraction of N-acetylated units, FA, has been information. In order to obtain quantitative data from
proposed [13] in which quantication of GlcNAc and GlcN such a spectrum, it is necessary that the observed resonan-
was done either by colorimetric or HPLC analysis. Also, a ces represent the relative amounts of the protons involved
similar method based on acid hydrolysis of the N-acetyl (see Ref. [16] for details). The characterization of chitosans
groups and detection of acetic acid by HPLC analysis and as given in Fig. 3 is, however, limited to samples that are
UV spectrometry has been reported [14]. This method is soluble in an acidic aqueous solvent, which limits the
advantageous in that both chitosan and chitin (soluble and analysis to chitosans with FA values lower than about
insoluble material) can be analyzed. Proton NMR spec- 0.7. However, CP-MAS NMR spectroscopy can be used
troscopy is an accurate method for determining the chem- to determine the chemical composition of samples contain-
ical composition of chitosans [15,16]. In Fig. 3, a 600-MHz ing water-insoluble fractions [11,17,18].
Figure 3 1H NMR spectrum (600 MHz) of solutions of a depolymerized chitosan with FA = 0.46 (5 mg/ml) in D2O at pD 3 and
90jC. The resonance at 0 ppm is the internal reference TSP [sodium-(trimethylsilyl)-propionate-d4]. Other assignments are given
in the Figure.
B. Sequence to polymers containing more than 60% acetylated residues,

1 giving very diverse physical properties. As shall be dis-
H and 13C NMR spectroscopy have been used to deter- cussed, Nature probably synthesizes chitosan via chitin,
mine the sequences of the monomers along the chain in which is deacetylated by enzymes capable of modifying the
chitosans. A limited depolymerization of the chitosans was chitin chain (deacetylases). Depending on the mode of
necessary in order to obtain spectra with sucient resolu- action of these enzymes, chitosans with a random (Ber-
tion to resolve the diad frequencies FAA and FAD, which noullian) distribution of the A and D units or a blockwise
were assigned as indicated in Fig. 3 [16]. 13C NMR distribution of the two units may be synthesized. Dierent
spectroscopy was used for more detailed sequential char- deacetylases of fungal origin have been isolated and char-
acterization of chitosans, including all four diad frequen- acterized, and for some, their mode of action has been
cies and some triad frequencies [19]. From these NMR data investigated [24].
and comparison with sequences based on random (Ber-
noullian) statistics, it was concluded that the units in the
chitosan chain were randomly distributed in water-soluble, A. Biological Function of Chitin and Chitosans
partially N-acetylated chitosans prepared from chitin by
alkaline de-N-acetylation [16,19]. Chitin functions as a structural polysaccharide in the outer
Characterization of the compositional distribution of skeleton of animals with an outer skeleton, where it is
chitosans has been attempted. Chitinlike particles could be cross-linked with proteins providing strength to the shells.
isolated from heterogeneously deacetylated chitosans, In the fungal cell wall, the chitin probably has a similar
which can be explained when the swelling of the chitin function as in the shells, where together with other poly-
particles is the rate-limiting step [11]. The compositional saccharides (e.g., h-glucans) it gives strength to the cell
distribution of chitosans that were fully soluble at acidic wall. It is not clear whether chitosans in the fungal cell walls
pH values has been characterized as a function of the have a dierent function from the chitin. However, it may
molecular weight and FA, and it was found that composi- be that changing the composition of one of its cell wall
tional distribution that was revealed upon degradation and components may be advantageous to the fungus, e.g., with
subsequent fractionation by increasing the pH to 7.5 was respect to fungi that attack plants and where the plants
consistent with what is expected from theoretical random recognition of the attacking fungus (or lack of such) may
degradation of chitosans with a random (Bernoullian) allow the fungus to invade plant cells before the plant has
distribution of the units [20]. mobilized its defense, such as chitinases, and enforcement
Nitrous acid degradation was used to degrade heter- of the plant cell wall by lignin formation [25].
ogeneously deacetylated chitosans to produce fully N- The presence of chitin-degrading enzymes in humans
acetylated oligosaccharides with a 2,5-anhydro-D-mannose [26,27] also raises questions related to the function of these
unit at the new reducing end [21], and it was found that the enzymes, and if their occurrence could be related to defense
weight fractions of the oligomers were in reasonable agree- mechanisms against fungi containing chitin/chitosan in the
ment with theoretical results from Bernoullian statistics. cell wall.
However, the solubility of the fully N-acetylated oligomers
decreases with increasing chain length, as recently noted B. Biosynthesis
[22]. Others have claimed that heterogeneously deacety-
lated chitosans contain blocks of acetylated units [23]. 1. Biosynthesis in Fungi
It is generally accepted that chitosans prepared by It is beyond the scope of this review to cover all aspects of
homogeneous deacetylation of chitin have a random dis- chitin biosynthesis, and only some of the classical work on
tribution of acetylated and deacetylated units along and the biosynthesis of chitin in fungi will be reviewed. In the
among the chains. However, for chitosans prepared by fungus Mucor rouxii, chitin synthetase synthesizes chitin by
heterogeneous deacetylation of chitin the same distribution the polymerization of UDPGlcNAc, and it was revealed
is more controversial. It is clear that such heterogeneously that chitin deacetylase operated in tandem with chitin
deacetylated chitosans (with FA larger than 0.2) can be synthetase by hydrolyzing the N-acetamido groups on the
separated into an acid-soluble chitosan fraction and an growing chitin chains [28]. This mechanism of biosynthesis
acid-insoluble chitinlike fraction [10,11], and that this of chitosan can be understood in view of the very limited
heterogeneous mixture of two molecular populations has enzymatic deacetylations (using deacetylase from M. rouxii
been misidentied as a block copolymer. There is a need for and Colletotricum lindemuthianum) that have been
more sophisticated methods in order to fully characterize obtained with chitin as substrate [29].
the distribution of acetylated and deacetylated units among
and along the chains, which may in the future reveal more 2. Chitin Deacetylases
detailed information on the ne structure of chitosans. It would be attractive to develop an enzymatic method as
an alternative method to the current chemical production
III. BIOSYNTHESIS of chitosan, where concentrated lye is used to deacetylate
chitin. However, so far the enzymatic production of chito-
A special feature of chitosans as dened here is that the san is possible only by fermentation of fungi and subse-
chemical composition ranges from pure polyglucosamine quent isolation of the chitosan from the cell wall [46].
Table 1 Selected Characteristics of Two Chitin Deacetylases from M. rouxii and C.

lindemuthianum
Origin of deacetylase
M. rouxii C. lindemuthianum
Localization Periplasm [30] Culture media [31]

Optimum pH value 4.5 [30] 11.5 [31]
Acetate inhibition Yes [30] No [31]
Mode of action Multiple attack [32,33] Multiple chain [34]
Hurdles to overcome in order to develop an enzymatic mer molecules according to the number of molecules
process for production of chitosan from chitin include the having a specic molecular weight) and the weight average
insolubility and crystallinity of the substrate. A recent (which weighs the polymer molecules according to the
review on chitin deacetylases has been published [24], and weight of molecules having a specic molecular weight).
here it will only be focused on some selected properties of In a population of molecules where Ni is the number of
two dierent fungal deacetylases, as given in Table 1. As molecules and wi the weight of molecules having a specic
can be seen from the table, the two chitin deacetylases from molecular weight Mi, these two averages are dened as:
M. rouxii and C. lindemuthianum are quite dierent, and P
especially there is a striking dierence in the pH optimum i Ni Mi
Mn P 1
of 4.5 of the M. rouxii deacetylase to 11.5 of the C. i Ni
P P
lindemuthianum deacetylase. As discussed previously (see i wi Mi Ni M2i
Sec. I.B.2), the hydrolysis of the acetamido group may Mw P Pi 2
i wi i Ni M i
occur by two dierent mechanisms, i.e., acid or alkaline
hydrolysis, and it can be speculated that the two deacety- In a polydisperse molecule population, the relation Mw >
lases from M. rouxii and C. lindemuthianum may oper- Mn is always valid, while for a monodisperse molecule
ate according to acid-catalyzed and alkaline-catalyzed population we have Mw = Mn. A polydispersity index, PI
hydrolysis, respectively, based on their dierent properties (= Mw/Mn), near 2 is characteristic of a linear polymer that
(Table 1) and especially the dierence in their pH optima. has been subjected to random depolymerisation [35]. A PI
Interestingly, these two enzymes also seem to dier in their of less than 2 suggests that some fractionation has occurred
mode of action, as the M. rouxii deacetylase is able to during the production process. Precipitation, solubiliza-
combine with the enzyme and deacetylate more than one tion, ltration, washing, or other separating procedures
acetyl group before it dissociates from the substrate (mul- may have caused loss of the high-molecular-weight or the
tiple attack) to produce a block copolymer, whereas the C. low-molecular-weight tail of the distribution. A PI of more
lindemuthianum deacetylase operates according to a multi- than 2.0 indicates a wider distribution, suggesting a mixing
ple-chain mechanism; that is, after combining with the of products of dierent molecular weights to obtain a
substrate the enzyme will remove only one acetyl group sample of a certain average molecular weight, or that a
before it dissociates from the substrate. nonrandom degradation of the polymer has occurred
during the production process or in the raw material.
IV. POLYMER PROPERTIES 2. Molecular Weight Distribution

A. Molecular Weight and Molecular Weight Size exclusion chromatography (SEC), which separates the
Distribution polymer molecules according to their size, combined with
detectors capable of determining the concentration and
1. Molecular Weight Averages preferably the molecular weight of the (monodisperse)
Polysaccharides are in general polydisperse with respect to fractions is a convenient method to determine the molec-
molecular weight, and polysaccharides resemble more the ular weight and the molecular weight distribution (MWD)
synthetic polymers than other biopolymers (proteins and of polymers. Otty et al. [36] reported on semipreparative
nucleic acids) with respect to this property. This may be and analytical use of GPC to fractionate chitosans. Typi-
because polysaccharides are not coded for in the DNA of cally, fractions with relatively low PI values (1.21.5) were
the organism but are synthesized by polymerase enzymes, obtained by fractionating a normally polydisperse sample
and/or that during extraction a substantial depolymeriza- with Mw = 270,000 using an SEC column (Sepharose CL-
tion of the polymer may take place. Thus, the molecular 4B and Sepharose CL-6B). Reversible interactions between
weight of a chitosan sample is an average over the whole chitosans and dierent column packings were found to
distribution of molecular weights. strongly inuence the log molecular weightelution volume
There are several methods of averaging; the two most relationships, showing that care must be taken when using
common are the number average (which weighs the poly- SEC to determine MWD without a molecular weight
Figure 4 SEC-MALLS elution proles (RI detector) and plots of log molecular weight vs. volume (calibration curve) for a
chitosan with FA = 0.10 (chitosan A) and a chitosan with FA = 0.50 (chitosan B). Columns: TSK 6000 + 5000 TWXL serially
connected. Mobile phase: 0.2 M ammonium acetate, pH 4.5. (From Ref. [38].)
detector, as has previously been noted [37]. Also, it is electrostatic repulsion between the charged groups on the
crucial that the fractionation range of the GPC column polymer chain, as demonstrated in Fig. 5 [39].
(i.e., the pore size distribution) is such that the column is Much work has been done by several research groups
able to separate the full molecular weight distribution of to elucidate the eect of the N-acetyl group on the confor-
the sample, which is a challenge for high-molecular-weight mation of the chitosans in solution (Ref. [39]and references
samples. Fig. 4 shows the elution proles [refractive index therein). The ionic strength dependence of the intrinsic
(RI) detector] and plots of log molecular weight vs. elution viscosity of the type given in Fig. 5 (chitosan with FA = 0.6)
volume for two chitosans of widely dierent FA values [38]. was performed for three series of chitosans with FA = 0,
Rather broad MWDs (see Sec. IV.A.1) were determined for 0.15, and 0.6, and dierent molecular weights were used to
both samples, with PI of 3.2 and 2.4 of chitosan A and obtain a parameter, B, reecting the inherent stiness of
chitosan B, respectively. the chain backbone [40]. The slope, S, of the salt tolerance
curves in Fig. 5 is determined by extrapolation to the
condition of an intrinsic viscosity of one (in dL/g) at an
B. Chain Conformation ionic strength 0.1 (M), and termed B. The B values
Polysaccharides are in general sti molecules, and chito- obtained in this way by Anthonsen et al. [39] were 0.002,
sans are no exception. This stiness leads to highly ex- 0.10, and 0.09 for chitosans having FA values 0.6, 0.15, and
tended chains. In addition, there is also the added extension 0, respectively. Similar values have also been obtained by
of polyelectrolytes due to ionic-strength-dependent elec- Terbojevich et al. [41].
trostatic repulsion between the charged groups on the
polymer chain.
Polysaccharides are in general quite sti molecules
compared to most synthetic water-soluble polymers. Ex-
cept for inulin and the (1!6)-linked pyranosidic polymers
such as dextran, rigid six-member sugar rings and restricted
rotation around glycosidic bonds provide the high chain-
stiness characteristic of polysaccharides. There is little
doubt that both the acetylated and deacetylated units in
chitosans exist in the 4C1 ring conformation, which means
that the chitosan molecule, independent of chemical com-
position, consists entirely of diequatorial glycosidic link-
ages. When the geometry of the glycosidic linkage restricts
rotation in diequatorially linked polymers, as in cellulose
and chitosans, the stiness leads to highly extended chains
and high intrinsic viscosity in solution. In chitosan, in its Figure 5 Intrinsic viscosities of a chitosan with FA of 0.6 as
protonated form, an added ionic-strength-dependent ex- a function of ionic strength. Similar curves were obtained for
tension is a result of its polyelectrolyte properties with two other chitosans. (From Ref. [39].)
Figure 6 Relationship between the Kuhn length and the B parameter.
The obtained B values are plotted in Fig. 6 together intrinsic viscosities compared to the chitosan with FA = 0.
with data given by Smidsrd and Christensen [42], where Similarly, for combinations of chain lengths and ionic
the B values are correlated to the Kuhn statistical segment strength lying below the curve, chitosans with FA = 0 will
length at h conditions. It is seen that the two chitosans with have the highest intrinsic viscosities. Such complex behav-
the lowest FA have B values close to that of cellulose ior suggests that when hydrodynamic data of chitosans are
derivatives, but the lower value of B for FA = 0.6 suggests compared both chemical composition, molecular weight,
that these molecules are stier than any of the other single- and ionic strength must be considered before comparison
stranded molecules given in the gure. Alternatively, be- are made at the molecular level of chain conformation and
cause the charge density of this sample is only 40% of the the shape and hydration of the hydrodynamic particle.
fully de-N-acetylated chitosan, the eective charge density A number of articles have been published to establish
(after Manning condensation) could be slightly less than the relationship between the intrinsic viscosity and the
for the other two samples and cause the lower B value. molecular weight of chitosans in the form of a Mark
Smidsrd and Haug, however, found B to be independent HouwinkKuhnSakurada (MHKS) equation [36,39,41,
of the stoichiometric charge density for pectins and poly- 4351]
acrylic acids in a broader range of charge densities than in
the present chitosan samples. However, no chain molecules g KMav
with the charged groups situated at position 2 of the sugar
ring were included in the study behind Fig. 6, and it is
possible that the empirical correlation between B values
and the stiness of the polysaccharide could be dierent in
such cases.
The fact that the salt tolerance is quite dierent for
chitosans with FA = 0 and FA = 0.6 causes the intrinsic
viscosity to vary dierently with the molecular weight with
increasing ionic strengths [39]. A double logarithmic plot of
the intrinsic viscosities against molecular weight for the
two samples at dierent ionic strengths gave curves cross-
ing each other. In Fig. 7 are given the number average de-
gree of polymerization, DPn, at which [g] is equal for
chitosans with FA = 0.6 and 0 vs. the ionic strength. It is
seen that the higher the ionic strength, the lower the DPn at
which the intrinsic viscosity of the chitosan with FA = 0.6
exceeds that for the chitosan with FA = 0. The gure
illustrates that for combinations of chain lengths and ionic Figure 7 Number average degree of polymerization (DPn)
strength lying above the curve in Fig. 7, the chitosan with at which the [g]I is equal for chitosans with FA = 0.6 and FA
the highest degree of acetylation ( FA = 0.6) has the higher = 0 vs. ionic strength. (From Ref. [39].)
where Mv is the viscosity average of the molecular weight,

and K and a are constants for a given polymersolvent
system. For nondrained Gaussian chains (the equivalent
sphere model of Flory) the exponent, a, equals 0.5. Values
of a higher than 0.5 are obtained for sti and expanded
molecules due to partial free drainage, and deviation from
Gaussian segment distribution due to inherently sti chain
backbone, or chain expansion due to solvent or polyelec-
trolyte eects. Theories based on the wormlike chain model
are often used for treatment of non-Gaussian coils [52].
Values of a and K obtained by Anthonsen et al. [39] by
viscometry, and Mn determinations by osmometry, are
given in Table 2. Here it is seen that the exponent a is
larger for the chitosan with FA = 0.6 than for the chitosans
with FA = 0 and 0.15 for all ionic strengths, suggesting
higher chain extension or more freely drained hydrody- Figure 8 Comparison of intrinsic viscosity vs. the weight
namic particles for this sample compared to the two others. average molecular weight of chitosans as reported in the
literature. ., FA = 0.15; I = 0.12 M [36]; o, FA = 0.52;
Wang et al. [53] obtained (solvent: 0.2 M HAc + 0.1 M
I = 0.12 M [36], z, FA = 0.11 and 0.48; I = 0.2 M) [43], q,
NaAc) similar results from light-scattering measurements
0.02 < FA < 0.21; 0.2 M < I < 0.3 M) [44]; n, FA = 0.250.30;
and viscometry I = 0.12 M [45]; 5, 0.03 < FA < 0.53; I = 0.12 M) [46];
logK 1:6800 7:25FA w , FA = 0.3; I = 0.12 M [47]. (From Ref. [46].)
a 0:805 1:01FA
using chitosans with FA ranging from 0 to 0.3. 0.3 M (Fig. 8), and obtained with some scattering a quite
Both Anthonsen et al. [39] and Wang et al. [53] good correlation to a straight line in a loglog plot of all the
proposed that the increase of the exponent a with FA could data, giving the following MHKS equation:
be caused by hydrogen bond formation between the N-
acetyl group and the C(6)OH of the neighboring unit. They g ml=g 8:43 103 Mw0:93
also considered that the bulky N-acetyl group would
restrict the rotation of sugar residues around the glycosidic All these ionic strengths were high enough to screen most
bond. Statistical mechanical calculations of chain exten- electrostatic repulsions and are therefore comparable, but
sion of chitosan with dierent FA values carried out by the lack of dependency of the intrinsic viscosity on FA is
Stokke [54] could reproduce the same trend of increasing apparently in disagreement with the results discussed ear-
extension with increasing FA provided that formation of lier of Anthonsen et al. [39] and Wang et al. [53]. In some of
hydrogen bonds with neighboring sugar units were allowed the works, the radius of gyration was determined, and these
for in the calculation. The calculation was done without sets of data were found to follow the equation
allowing for any competition from water molecules in the
RG;z 7:5 102 M0:55
w
hydrogen bond formation. Mazeau et al. [55] also found
that the average chain dimensions of chitosans increased The two scaling coecients (0.92 and 0.55) suggest, accord-
monotonously with FA by conformational analysis. ing to Berth and Dautzenberg [46], that all the samples of
Berth and Dautzenberg [46] compared Mw[g] data chitosan behave consistently with a model of a nearly free-
from several works [36,41,4345,47] with FA ranging from draining, wormlike chain (extended coil), and that dier-
0.11 to 0.52 and ionic strength ranging from 0.12 M to ences between samples were less than often had been
Table 2 Values of a and K from the MHKS Equations at Dierent Ionic Strengths
FA = 0.6 FA = 0.15 FA = 0
a a
l (M) a K R a K R a K Ra
lb 0.95 6.22e0.5 0.999 0.62 2.09e0.3 0.979 0.47 1.11e0.2 0.996
1.007 1.06 1.81e0.5 0.999 0.66 1.75e0.3 0.981 0.52 8.36e0.3 0.997
0.107 1.06 2.18e0.5 0.983 0.78 5.85e0.4 0.978 0.58 5.59e0.3 0.995
0.0131 1.29 2.31e0.6 0.998 0.79 9.69e0.4 0.960 0.64 5.20e0.3 0.998
0.005b 1.37 1.14e0.6 0.998 0.81 1.01e0.3 0.956 0.66 5.44e0.3 0.998
a
Correlation coecient from linear regression analysis.
b
Extrapolated values.
claimed by the dierent authors. As discussed earlier here parameter, which is linked to the charge of the D units. The
(Fig. 7) the hydrodynamic properties of chitosans are quite ionic strength of the solvent is also an important parameter
complex, and the eect of the bulky hydrophobic acetyl (salting-out eects). Furthermore, the content of ions in the
group and the hydrophilic, charged amino group seems to solvent that specically interact with chitosans (e.g., Cu
aect chain extension and hydration in such a way that the and multivalent negative ions such as molybdate) may also
macroscopic solution properties are relatively little depen- limit the solubility of chitosans.
dent on FA at moderately high ionic strengths in the range All chitosans are soluble at pH values below 6 but
of intrinsic viscosity/molecular weights commonly in use. solubility decreases as pH increases. In the pH range from 6
A complication in establishing the solution properties to 8, commercial chitosans will precipitate upon increase of
of chitosans with classical techniques is their tendency to the pH. However, the solubility increases with increasing
undergo a concentration-dependent aggregation in solu- FA, as shown in Fig. 9 [20]. Chitosans of medium molecular
tions, as discussed by Anthonsen et al. [56], and aggrega- weight with FA around 0.5 are neutral soluble; that is, they
tion has also been observed by Amiji [57]. Anthonsen et al. will not precipitate upon increasing the pH from below 6 to
were unable to remove the aggregates by ultraltration and neutral. Such solubility dierences may profoundly aect
ultracentrifugation, but later work by Otty et al. [36] and the accessibility of chitosans to enzymes as well as biolog-
Berth and Dautzenberg [46] indicated that such aggregates ical eects in the physiological pH range.
constituted small problems in determining molecular The charge density of chitosan, i.e., the degree of
weights and molecular weight distributions by size exclu- protonization of amino groups, is determined both by the
sion chromatography methods where the aggregates could chemical composition of the chitosan ( FA), the molecular
be separated from the molecular disperse chitosan prior to weight, and external variables such as pH and ionic
measurements of molecular weights, as also recently strength. Values of the dissociation constant (pKa) for
reported by Fredheim and Christensen [38]. In summary, chitosan range from 6.2 to 7, depending on the type of
it is recommended to perform a collaborative study (a chitosan and conditions of measurement [6063]. The
round-robin study) among established laboratories for titration behavior of chitosans has been studied by three
comparative [g]Mw determinations on the same set of dierent methods: colloid titration, 1H NMR spectrosco-
chitosan samples. Until then, the commercial use and the py, and electrophoretic light scattering (ELS). The chem-
regulatory aspects connected to biomedical applications ical shift of the H-2 resonance of the D units relative to an
must rely on carefully obtained [g]-M relationships set up internal standard can be monitored as a function of in-
with validated instrument in commercial Good Laboratory creasing pH in proton NMR studies, as shown in Fig. 10.
Practice laboratories. This chemical shift is directly inuenced by the charge of
the nitrogen atom bound to carbon 2 in a deacetylated unit.
In experiments, as shown in Fig. 10, chitosans with FA 0.01
V. PHYSICAL PROPERTIES
A. Ion Binding
It is well known that chitosan may form complexes with
certain metal ions, particularly transition metal and post-
transition ions [3]. In certain applications, it is important
with knowledge on the (selective) binding of metal ions to
chitosans. However, most studies of ion binding to chito-
san have been aimed at determining whether chitosan will
bind to a given ion, while only a few studies have involved
determination of the selectivity of binding of dierent ions
to chitosans. Recently, Rhazi et al. [58] determined the
selectivity of mixtures of the ions Cu > Hg > Zn > Cd >
Ni > Co = Ca, using potentiometric and spectrometric
methods. Vold et al. [59] reported the selectivity of dierent
chitosans in binary mixtures of Cu2+, Zn2+, Cd2+, and
Ni2+, showing that the uncharged amino group of chito-
sans ( FA of 0.01 and 0.49) could bind Cu in large excess of
the other metal ions, while low selectivity coecients were
found between the other ions that were studied.
B. Solubility and Charge Density

The solubility at acidic pH values and insolubility at basic
pH values is a characteristic property of commercial chi- Figure 9 Solubility vs. pH curves of chitosans. E FA =
tosans. Generally, three essential parameters determine the 0.01, x FA = 0.17, 5 FA = 0.37, n FA = 0.60. (From Ref.
solubility of chitosans in water. The pH is the most obvious [20].)
linkages. This is probably due to the combination of the

decrease in the rate when a positively charged amino group
is present close to the glycosidic linkage to be hydrolyzed,
and the substrate-assisted mechanism by which a glycosidic
linkage following an A unit is cleaved.
The activation energies for acid hydrolysis of the DD
glycosidic linkage were determined to be f155 kJ mol1,
which was much higher than the activation energies for
acid hydrolysis of the AA and AD glycosidic linkages of
f132 kJ mol1 [64]. This very large dierence in the rate
of hydrolysis of the glycosidic linkages implies that a fully
de-N-acetylated chitosan should be chosen for applica-
tions where a high stability of the chitosan is required at
low pH values, i.e., where depolymerization may occur by
Figure 10 Titration curves of chitosans monitored by 1H acid hydrolysis.
NMR. The chemical shift dierences between H-2 of D units Extended acid hydrolysis of chitosans with varying FA
and the internal reference sodium-(trimethylsilyl)-propio- using concentrated hydrochloric acid (to avoid de-N-acet-
nate-d4 (TSP) as a function of pH for chitosan with FA = ylation) was also recently used to prepare oligosaccharides
0.01 (.), FA = 0.13 (o), and FA = 0.49 (z). Solid lines of varying chain length composed of consecutive D units
indicate logistic regression. (From Ref. [63].) with an acetylated unit at the reducing end, in agreement
with what was expected from the large dierences in the
specicity of the hydrolysis of the glycosidic linkages [65].
and 0.13 will precipitate upon increase of the pH to The stability of chitosan hydrochloride powders with
approximately 6.5, making the titration curves incomplete. dierent chemical compositions has been studied at 60, 80,
However, the chitosan with FA 0.49 was soluble over the 105, and 120jC [66]. It was concluded that the dominant
entire pH interval studied and a complete titration curve degradation mechanism in the solid state of these chitosan
was recorded. It is apparent from Fig. 10 that all chitosans salts is by acid hydrolysis.
showed the same titration behavior. The same type of
logistic regression as above made it possible to extrapolate 2. Stability as a Function of pH
beyond the measured points and determine the pKa values.
A convenient way to characterize chitosan degradation is
All three chitosans were found to have the same pKa values
by the change in the reciprocal of the intrinsic viscosity with
of 6.7, only slightly higher than that derived from ELS
time, from which a degradation rate constant can be
study, while others have reported dierent pKa values of
calculated [64]. Chitosans of medium molecular weight
chitosans with dierent FA [62].
C. Chemical Stability
For many applications of chitosans it is important to be
aware of the factors that determine and limit the stability of
chitosans, both in the solid state and in solution, and the
chemical reactions responsible for the degradation. The
glycosidic linkages are susceptible to both acid and alkaline
degradation and oxidation by free radicals. If a certain
application requires that the viscosity of the solution is
maintained for extended time periods, it is better to use a
high concentration of a medium- or low-viscosity (molec-
ular weight) product instead of a low concentration of a
high-viscosity product. This is merely because a cleavage in
a long chain would decrease the viscosity to a much larger
extent than a cleavage in a short chain.
1. Acid-Catalyzed Degradation
The acid-catalyzed degradation rates of chitosans have
been found to be dependent on FA (Fig. 11), and the initial
degradation rate constant was found to increase in direct
proportion to FA [64]. Acid hydrolysis was found to be Figure 11 Degradation rate constants (k) as a function of
highly specic to cleavage of AA and AD glycosidic FA of the chitosans, determined by the viscosity assay at a
linkages, which was found to be hydrolyzed with about chitosan concentration of 1.5 mg/ml in 0.4 M HCl at 60jC.
three orders of magnitude higher rate than DD and DA (From Ref. [64].)
with FA around 0.5 are neutral soluble (see Sec. V.B), and seems therefore that autoclaving can be used to sterilize
the stability of this chitosan was determined as a function chitosan solutions without severe depolymerization.
of pH. Fig. 12 shows the relative degradation rate of a
chitosan ( FA = 0.55, [g] = 794 mL/g) at dierent pH
values, an ionic strength of 0.1 M and a temperature of D. Enzymatic Degradation
60jC. The degradation is at a minimum between pH 3 and
11 and increases at both lower and higher pH. However, The abundance of chitin in nature is reected in the
the degradation around neutral pH values is very slow, and abundance of chitin-degrading enzymes, which are found,
reliable estimates of the degradation rates are dicult, e.g., in arthopods when their rigid exoskeleton need peri-
particularly because traces of impurities can be expected odic replacement as a result of the growth process, in
to inuence the degradation when occurring according to animals feeding on arthropods that need to degrade the
the oxidativereductive depolymerization mechanism [67]. outer skeleton in order to digest the content of their prey,
The decreased stability at pH values less than 3 is due to and in microorganisms utilizing chitin as an energy source.
acid hydrolysis, and the relative increase in the degra- Chitosan-degrading enzymes are less abundant, reecting
dation at low pH will be proportional to FA [64]. The that chitosan has so far only been found naturally in certain
reaction responsible for the degradation at pH 10 and fungi. Lysozyme may also, in addition to its natural
above could be both OHcatalyzed hydrolysis and the substrate (the glycosidic linkage of certain bacterial cell
oxidativereductive degradation (ORD) reaction. The rate walls peptidoglycans), hydrolyze chitin and chitosans
of both reactions will be expected to increase with increas- [69,70].
ing pH. However, chitin and chitosans are relatively stable A variety of assays have been used to determine the
toward alkaline degradation, as the rather severe condi- activity of chitinases and chitosanases. One may make use
tions used for deacetylation of chitin (1520 M NaOH, of chitin oligomers where an organic group has been O-
100120jC) can be performed without severe degradation glycosidically linked to the reducing end and where the
of the polysaccharide. release of the group is accompanied by a strong increase in
the uorescence. This is a convenient but articial sub-
strate [71]. Other assays are based on the detection of the
3. Sterilization of Chitosan Solutions
new (reducing) ends introduced by the cleavage of the gly-
The sterilization of chitosan solutions may be performed cosidic linkages by the enzyme, or use of dye-labeled sub-
by autoclaving (typically 120jC for 20 min), which may strates [72].
lead to some depolymerization, depending on the pH and Both viscometry and NMR spectrometry were used to
the chemical composition of the chitosan. Autoclaving of investigate the degradation rates and the specicity of
chitosan solutions ( FA of 0.01, 0.35, and 0.60) at pH 4.5 at lysozyme-catalyzed degradation of chitosans. Using vis-
120jC for 20 min does not reduce the intrinsic viscosity of cometry and chitosans with widely dierent FA and known
the chitosan with FA = 0.01, while the intrinsic viscosity of Bernoullian distribution of monomers, the initial lysozyme
the other two chitosans was only moderately reduced, from degradation rates were determined, suggesting a very
760 to 550 mL/g for the chitosan with FA of 0.35, and from strong increase in the rate with FA, i.e., proportionally to
820 to 600 mL/g for the chitosan with FA of 0.60 [68]. It FA in about the fourth power [73]. Similar dependency was
obtained at dierent pH values, at dierent ionic strengths,
and for both hen egg white lysozyme (HEWL) and human
milk lysozyme [73,74]. When human blood serum was
added to chitosan solutions, the same strong degradation
rate dependence on FA was found, suggesting that lyso-
zyme, or another chitinase with the same specicity, is
responsible for the chitosan degradation reactions in serum
[75]. By a combination of the degradation rate studies and
NMR determination of the identities (A or D units) of the
new reducing and nonreducing end groups, it was sug-
gested that a minimum of four acetylated units had to be
contained in the lysozyme binding site to obtain maximum
initial degradation rates [76], as illustrated schematically in
Fig. 13. These studies suggest that it could be possible to
tailor chitosans with a predetermined degradation rate for
use in, e.g., the human body. Moreover, NMR studies of
endoenzymatic degradation of chitosans oer the possibil-
ity for determination of the specicities of enzymes with
Figure 12 Relative degradation rates of a chitosan with FA respect to cleavage of the four diererent glycosidic link-
= 0.55 ([g] = 794 mL/g at pH 4.5 and I = 0.1 M) as a function ages (AA, AD, DA, and DD) in chitosans as deter-
of pH. The degradation rates were obtained at 60j and I = 0.1 mined by the identity of the new reducing and nonreducing
M by the viscosity assay as described. (From Ref. [64].) ends, and in some cases also the variation in the identity of
VI. TECHNICAL PROPERTIES

A. Flocculation
As mentioned in the Introduction, chitosans represent a
promising alternative to synthetic polycations for applica-
tions as occulants (see, e.g., Refs. [81,82]). Water treat-
ment oers many possibilities, ranging from humic acid
removal from drinking water [83] to treatment of diverse
wastewaters or sludge dewatering. Suspended solids, dyes,
heavy metals, pesticides, and other toxic compounds may
all be eciently removed by chitosan.
However, the structurefunction relationship of chi-
tosans has rarely been addressed in occulation studies. As
within other application areas, a commercial chitosan with
FA 00.2 has been used in most studies, and the occulation
eciency of the chitosan has been shown to increase with
decreasing FA and pH, and thereby increasing charge
density. Consequently, electrostatic interactions have been
implicated to play a dominant role in interactions of
chitosans with negatively charged materials. However, by
Figure 13 Schematic illustration of the specicity of changing the chemical composition of the chitosan and
hydrolysis of lysozyme towards the 16 dierent tetrade
environmental conditions, the properties of chitosans may
sequences in chitosan. The tetrade sequences are positioned
be altered considerably. By systematically studying the
in the active site of lysozyme in such a way that the tetrades are
hydrolyzed at the arrow (between subsite 1 (D) and +1 (E)).
eects of these properties on occulation, the relative
importance of dierent interactions may be identied,
giving an opportunity to develop an optimal occulant
for a given application.
the nearest neighbors to the new reducing and nonreducing Flocculation of bacterial suspensions has been studied
ends [76]. as a model system [84,85], where occulation was moni-
The binding of partially N-acetylated oligomers to tored by residual turbidity measurements. Experiments
lysozyme has been studied [77]. By studying the binding with Eschericia coli and ve dierent chitosans varying in
of low-molecular-weight chitosans with a low FA to lyso- FA revealed that the chitosans with high FA occulated at
zyme, it was found that such an A unit predominantly lower concentrations compared to those with low FA [84].
surrounded by D units would bind preferentially in subsite The relationship between FA and occulation eciency is
C (-2) with a dissociation constant of 0.11 mM, dependent clearly illustrated in Fig. 14. A parameter referred to as the
on pH and ionic strength and without depolymerization of critical concentration, x75, was determined from the non-
the chitosan chain [78,79]. These ndings are in agreement linear regression of the experimental data, expressing the
with previous use of a chitosan with a low fraction of N- chitosan concentration needed to obtain 75% occulation.
acetylated units as an inhibitor to lysozyme [73]. Recently, Chitosans with high FA were clearly better occulants in
it was demonstrated that immobilized lysozyme could be our study. The increase in FA resulted in a rather dramatic
utilized to fractionate chitosan chains without acetyl decrease of x75 concentrations, in some cases by a factor of
groups from those containing acetyl groups [80]. 10 or more. Similar relationships between FA and x75 were
Figure 14 Chitosan concentration at 75% occulation (x75) of E. coli as a function of FA after 24 hours of sedimentation at pH
6.8 (a) and 5 (b). (From Ref. [84].)
obtained at pH 5, where all chitosans are completely relationships related to chitosans ability to enhance the
soluble and fully charged, and at pH 6.8, where low adsorption of poorly absorbable drugs will be discussed
acetylated chitosans precipitate and the charge density is [9294]. In vitro studies of the eect of the chemical
lowered to <50% (Fig. 14). An increase in FA resulted in composition of the chitosan ( FA) and molecular weight
dramatic decrease of necessary concentration; for instance, of the uptake of mannitol, as a model of a poorly adsorb-
x75 for chitosan FA 0.62 was about 10 times lower than that able drug, using monolayers of human epithelial Caco-2
for FA 0.01. Very similar relationships between FA and x75 cells have been reported [92]. The most eective adsorption
were obtained at pH 5 and 6.8. enhancers were chitosans with a low FA with low and high
In addition, pH is an important factor inuencing both molecular weight, as seen in Fig. 15a. A chitosan with FA of
solubility and charge density of chitosan. Therefore, it is 0.35 and relatively high molecular weight was found to
expected, and often found, that occulation eciency
largely depends on pH [8688]. However, no such depen-
dency was observed in the bacterial occulation study [84].
The low acetylated chitosans had similar x75 concentra-
tions in the range of pH 4.07.5. A further increase in pH
resulted in dramatic increase in critical concentration,
probably caused by so-called sweep occulation with ex-
cess insoluble chitosans.
B. Gelling
So far no simple ionic and nontoxic cross-linking agent has
been found that gives reproducible chitosan gels at low
concentrations, such as calcium ions for gelling of algi-
nates. However, aqueous chitosan gels cross-linked with
molybdate polyoxyanions have been reported, resulting in
transparent, thermo-irreversible gels that are able to swell
several times their original size in aqueous solutions,
depending on the ionic strength [89]. The gelling kinetics
and the nal elasticity of these gels were inuenced by the
FA of the chitosan, and it was suggested that interchain
interactions between acetylated units were responsible for
these eects [90], as was also suggested for a completely
dierent gelling system, where chitosans of varying FA were
cross-linked with glutardialdehyde [91].
VII. BIOMEDICAL PROPERTIES

Perhaps the most promising applications of chitin and
chitosans are related to their use in biotechnological,
biomedical, and pharmaceutical applications. Chitin and
chitosans for such applications need to be pure, and their
chemical compositions, chain lengths, and chain length
distributions need to be specied. Chitin and chitosans are
biodegradable, and their biodegradation should be con-
trollable in the human body as, e.g., by controlling the
crystallinity of chitin, which will inuence its rate of
degradation, or by controlling the chemical composition
( FA) of chitosan and thereby its lysozyme degradation rate
(see Sec. V.D). When used, e.g., in tissue engineering or for
wound healing, the biocompatibility of the polysaccharides
is also important. Figure 15 Eect of chitosans of varying FA on (a) the
permeability of mannitol in Caco-2 cell monolayer (b) intra-
A. Enhanced Adsorption of Drugs cellular dehydrogenase activity. n FA = 0.01; Mn = 31 000,
. FA = 0.01; Mn = 170 000, w FA = 0.15; Mn = 4700, z FA
Chitosan properties such as bioadhesivity, biodegradabil- = 0.15; Mn = 190 000, D FA = 0.35; Mn = 12 000, x FA =
ity, and low toxicity are important in drug delivery appli- 0.35; Mn = 170 000, 5 FA = 0.49; Mn = 22 000, o FA=0.49;
cations, and some recent studies on structurefunction Mn = 98 000. (From Ref. [92].)
have properties that were especially advantageous. How-

ever, the chitosans were also shown to display a clear dose-
dependent toxicity, although at concentrations well below
those needed to improve mannitol transport, with lowest
toxicity of the chitosans with the highest FA values and
little eect of the molecular weight (Fig. 15b). By choosing
the chitosan with FA of 0.35, eective adsorption with a low
toxicity could be achieved.
The mechanisms of the adsorption enhancement of
two chitosans with FA of 0.01 and 0.35 were found to be
similar, and it was concluded that the enhancement of ad-
sorption occurred through the paracellular pathway [93].
The inuence of the mucus layer on the adsorption
enhancement of chitosans was investigated by using a
mucin-producing intestinal goblet cell line in addition to
an in situ perfusion model of rat ileum, showing reduced
chitosan adsorption enhancement for poorly adsorbed
drugs [94]. However, as noted by the authors, it may be
possible to overcome this eect by specic formulations of
chitosan and the drug that increase the local concentration
of both chitosan and drug, as shown by, e.g., an eective
chitosan nasal delivery system [95].
B. Gene Delivery
Gene transfer carriers of nonviral origin have recently
attracted much attention as a method to deliver plasmid Figure 16 The eect of FA on (a) the stability of complexes
DNA to the nucleus of cells for gene therapy. Polycations between DNA and chitosan as tested in the agarose gel
including synthetic polycations [such as polyethyleneimine retardation assay at increasing charge ratios and (b) in vitro
(PEI)], cationic lipids, polylysines, and, recently, chitosans transfection eciency in 293 cells at increasing charge ratios.
Arrows indicate (1) loading position, (2) open circle, and (3)
have been successfully used as eective condensation
supercoiled form of pDNA. C(15;190) is a chitosan with FA
agents for the negatively charged plasmid DNA in gene
= 0.15 and a molecular weight of 190 000, while C(49;98) is a
delivery. Concerns about the toxicity and lack of biode- chitosan with FA = 0.49 and a molecular weight of 98 000.
gradability of synthetic polycations, which are rather ef- n C(15;190), . C(49;98). (From Ref. [98].)
fective in compacting DNA, have initiated search for
eective and nontoxic biodegradable gene delivery sys-
tems. Mumper et al. [96] reported on the use of chitosan
for gene delivery, which was followed up by several studies
(see Ref. [97] and references therein). Recently, the ability be bioadhesive. Chitosans also form complexes with poly-
of chitosans with varying FA to form physically stable anions such as DNA, alginate, xanthan, mucin, etc. and
DNA complexes were reported, and it was found that only these binding properties have been studied in relation to
chitosans with a FA lower than 0.35 form stable complexes dierent potential drug delivery systems [97,100102].
with DNA [98], as shown in Fig. 16a. The ability to form Several advantages can be achieved by adhering drug
stable complexes was correlated to the in vitro transfection carrying systems to mucosal surfaces such as a prolonged
eciency in 293 cells (Fig. 16b). The eect of the chain residence time at the site of drug action, and establishment
length and chain length distribution of low-FA chitosans of a steeper drug concentration gradient near the mucosal
has also recently been reported, and it has been found that surface for enhancing drug absorption [103]. Some work
fully de-N-acetylated monodisperse oligomers with DP 6, investigating the use of chitosan for coating of alginate
8, 10,12, or 14 are inecient as gene delivery systems [99]. microcapsules for enhancing the bioadhesive properties of
However, a chitosan with a number average degree of the capsules will be discussed here [104106].
polymerization (DPn) of 24 has been found to be as ecient The binding of chitosans to alginate beads was studied
as high-molecular-weight chitosans in forming physically quantitatively by using radioactive-labeled fractions of
stable DNA complexes [99], suggesting that low molecular chitosans [104]. By incubation of calcium alginate gel beads
weight chitosans may potentially be used for gene transfer. in solutions of chitosans an amount of absorbed chitosan
corresponding to 2 Ag/mm2 bead surface, and a maximum
C. Capsule Formation weight ratio of chitosan to alginate in microcapsule of 0.4
could be achieved at long residence times. The binding of
Because most cells and living tissues have negatively chitosan increased markedly by reducing the number av-
changed surfaces, chitosan, as a polycation, is known to erage molecular weight of chitosan below 2000 and by
increasing the porosity of the alginate gel. The binding of REFERENCES

chitosan was found to increase with decreasing FA and with
increasing pH in the range from pH 4 to 6. 1. New developments in marine biotechnology. Proceedings
of the 4th International Marine Biotechnology Confer-
The stability of alginatechitosan capsules at physio-
ence, Sorrento, Paestum, Oranto and Pugnochiuso, Italy;
logical conditions (saline) was found to depend strongly on Kluwer Academic Publishers, 1998.
the amount of chitosan bound to the capsules, [105]. 2. Sandford, P.A. Commercial sources of chitin & chitosan
Capsules with very high mechanical strength were obtained and their utilization. In Advances in Chitin Science Volume
by soaking calcium alginate microcapsules with diameters VI; Varum, K.M., Domard, A., Smidsrd, O., Eds.;
around 300 Am in solutions of chitosans with a number (2002). Department of Biotechnology: Trondheim, Nor-
way, 2002; 35 pp.
average molecular weight of 1500. When such capsules
3. Roberts, G.A.F. Chitin Chemistry; Macmillan Press:
were treated with calcium sequestering agents, the gel core Houndmills, UK, 1992.
did not dissolve indicating the presence of chitosan 4. McGahren, W.J.; Perkinson, G.A.; Growich, J.A.; Leese,
throughout the capsule matrix. The permeability of these R.A.; Ellestad, G.A. Chitosan by fermentation. Process
capsules could be modied by varying both the molecular Biochem. 1984, 19, 88.
weight and the degree of acetylation of the chitosans. 5. Shimahara, K.; Takiguchi, Y.; Kobayashi, T.; Uda, K.;
The alginatechitosan microcapsules were studied fur- Sannan, T. Screening of mucoraceae strains suitable for
chitosan products. In: Chitin and Chitosan; Skjak-Brk,
ther by Gaserd et al. [106] as a bioadhesive drug delivery G., Anthonsen, T., Sandford, P., Eds.; Elsevier: London,
system and compared with uncoated calcium alginate gel 1989; 171 pp.
beads. An in vitro tissue adhesion assay was used to 6. Rane, K.D.; Hoover, D.G. Production of chitosan by
quantitatively determine adhesion of beads to dierent fungi. Food Biotechnol. 1993, 7, 11.
tissues. The addition of chitosans to the gel beads increased 7. Kurita, K.; Koyama, Y.; Nishimura, S.-I.; Kamiya, M.
the adhesive properties considerably both to pig esophagus Facile preparation of water-soluble chitin from chitosan.
Chem. Lett. 1989, 9, 1597.
and stomach mucosa surfaces. The dierence in adhesive
8. Sannan, T.; Kurita, K.; Iwakura, Y. Studies on chitin: 1.
properties between the coated and the uncoated micro- Solubility change by alkaline treatment and lm casting.
particles was found to be considerably larger for the Makromol. Chem. 1975, 176, 1191.
stomach mucosa, most probably because of the presence 9. Sannan, T.; Kurita, K.; Iwakura, Y. Studies on chitin: 2.
of negatively charged mucins on this surface. These chito- Eect of deacetylation on solubility. Makromol. Chem.
san-coated gel beads clearly represent a potential drug 1976, 177, 3589.
10. Varum, K.M.; Anthonsen, M.W.; Grasdalen, H.; Smids-
delivery system.
rd, O. Chemical composition and sequences in chitosans
determined by high-eld proton and carbon n.m.r.-
spectroscopyRelation to solubility. In Advances in
VIII. CONCLUDING REMARKS Chitin and Chitosan; Brine, C.J., Sandford, P.A., Zikakis,
J.P., Eds.; London: Elsevier, 1992; 127 pp.
Compared to the established commercial applications of 11. Otty, M.H.; Varum, K.M.; Smidsrd, O. Compositional
other marine polysaccharides, such as alginate, carra- heterogeneity of heterogeneously deacetylated chitosans.
geenan, and agar, the commercial use of chitosan is still Carbohydr. Polym. 1996, 29, 17.
12. PCT Patent Application No. PCT/GB02/03471.
in its infancy despite an enormous number of suggestions
13. Nanjo, F.; Katsumi, R.; Sakai, K. Enzymatic method for
for applications in published papers. The present authors determination of the degree of deacetylation of chitosans.
believe that the potential for use of chitosan, especially in Anal. Biochem. 1991, 193, 164.
biomedical applications, is at least comparable with that of 14. Niola, F.; Basora, N.; Chornet, E.; Vidal, P.F. A rapid
other polysaccharides of marine origin. However, due to method for the determination of the degree of N-
the diverse physical and biological properties of chitosans acetylation of chitinchitosan samples by acid hydrolysis
and HPLC. Carbohydr. Res. 1993, 238, 1.
and its potent interaction with biological tissues, only a
15. Hirano, S.; Yamaguchi, R. N-Acetylchitosan gel: A
careful and systematic search for optimum performance polyhydrate of chitin. Biopolymers 1976, 15, 1685.
in each potential and the simultaneous developments of 16. Varum, K.M.; Anthonsen, M.; Grasdalen, H.; Smidsrd,
the correct regulatory requirements for approval of usage, O. Determination of the degree of N-acetylation and the
can develop the eld to its potential. Any food usage in distribution of N-acetyl groups in partially N-deacetylated
the West will also require approval by FDA or its Euro- chitins (chitosans) by high-eld n.m.r. spectroscopy.
pean counterpart. Carbohydr. Res. 1991, 211, 17.
17. Saito, H.; Tabeta, R.; Hirano, S. A high resolution 13C
The technical usage of chitosans has been hampered by nmr study of chitin, chitosan and N-acetyl chitosans by
high costs and lack of availability of large quantities. In this cross polarization/magic angle spinning (C/MS) nmr spec-
application area, one has a catch-22 situation where low troscopy. Conformational behaviour and gelation mech-
costs require large-scale production and large-scale pro- anism. Chitin and Chitosan, Sapporo, Japan, 1982; 7176.
duction require a large market. With more research, how- 18. Raymond, L.; Morin, F.G.; Marchessault, R.H. Degree of
ever, one could foresee that chitosans with superior deacetylation of chitosan using conductometric titration
and solid-state NMR. Carbohydr. Res. 1992, 246, 311.
properties to the synthetic analogues will be produced,
19. Varum, K.M.; Anthonsen, M.; Grasdalen, H.; Smidsrd,
and this together with increased environmental concerns O. 13C-N.M.R. studies of the acetylation sequences in
may lead to bulk uses of the renewable and biodegradable partially N-deacetylated chitins (chitosans). Carbohydr.
chitosan molecule. Res. 1991, 217, 19.
20. Varum, K.M.; Otty, M.H.; Smidsrd, O. Water-solubil- 37. Domard, A.; Rinaudo, M. Gel permeation chromatog-
ity of partially N-acetylated chitosans as a function of pH: raphy of cationic polymer on cationic porous silica gels.
Eect of chemical composition and depolymerization. Polym. Commun. 1984, 25, 55.
Carbohydr. Polym. 1994, 25, 65. 38. Fredheim, G.E.; Christensen, B.E. Polyelectrolyte com-
21. Sashiwa, H.; Saitmoto, H.; Shigemasa, Y.; Ogawa, R.; plexes: Interactions between lignosulfonate and chitosan.
Tokura, S. Distribution of the acetamide group in Biomacromolecules 2003, 4, 232.
partially deacetylated chitins. Carbohydr. Polym. 1991, 39. Anthonsen, M.W.; Varum, K.M.; Smidsrd, O. Solution
16, 291. properties of chitosans: conformation and chain stiness
22. Tmmeraas, K.; Varum, K.M.; Christensen, B.E.; Smids- of chitosans with dierent degrees of N-acetylation.
rd, O. Preparation and characterisation of oligosacchar- Carbohydr. Polym. 1993, 22, 193.
ides produced by nitrous acid depolymerisation of 40. Smidsrd, O.; Haug, A. Estimation of the relative stiness
chitosans depolymerisation of chitosans. Carbohydr. of the molecular chain in polyelectrolytes from measure-
Res. 2001, 333, 137. ments of viscosity of dierent ionic strengths. Biopol-
23. Aiba, S. Studies on chitosan: 3. Evidence for the pres- ymers 1971, 10, 1213.
ence of random and block copolymer structures in par- 41. Terbojevich, M.; Cosani, A.; Conio, G.; Marsano, E.;
tially N-acetylated chitosans. Int. J. Biol. Macromol. 1991, Bianchi, E. Chitosan-chain rigidity and mesophase
13, 4044. formation. Carbohydr. Res. 1991, 209, 251.
24. Tsigos, I.; Martinou, A.; Kafetzopoulos, D.; Bouriotis, V. 42. Smidsrd, O.; Christensen, B.E. Molecular structure
Chitin deacetylases; new, versatile tools in biotechnology. and physical behaviour of seaweed colloids as compared
Trends Biotechnol. 2000, 18, 305. to microbial polysaccharides. In Seaweed Resources in
25. Vander, P.; Varum, K.M.; Domard, A.; El Gueddari, Europe: Uses and Potential; Guiry, M.D., Blunden, G.
N.E.; Moerschbacher, B.M. Comparison of the ability of Eds.; John Wiley and Sons, 1991; 185 pp.
partially N-acetylated chitosans and chitooligosaccharides 43. Errington, N.; Harding, S.E.; Varum, K.M.; Illum, L.
to elicit resistance reactions in wheat leaves. Plant Physiol. Hydrodynamic characterization of chitosans varying in
1998, 118, 1353. degree of acetylation. Int. J. Biol. Macromol. 1993, 15,
26. Overdijk, B.; Van Steijn, G.J. Human serum contains a 113.
chitinase: identication of an enzyme formerly described 44. Rinaudo, M.; Milas, M.; Dung, P.L. Characterization of
as 4-methylumbelliferyl-tetra-N-acetylchitotetraoside hy- chitosan. Inuence of ionic strength and degree of
drolase (MU-TACT hydrolase). Glycobiology 1994, 4, acetylation on chain expansion. Int. J. Biol. Macromol.
797. 1993, 15, 281.
27. Escott, G.M.; Adams, D.J. Chitinase activity in human 45. Berth, G.; Dautzenberg, H.; Peter, M.G. Physico-chem-
serum and leukocytes. Infect. Immun. 1995, 63, 4770. ical characterization of chitosans varying in degree of
28. Davis, L.L.; Bartnicki-Garcia, S. Chitosan synthesis by acetylation. Carbohydr. Polym. 1998, 36, 205.
the tandem action of chitin synthetase and chitin 46. Berth, G.; Dautzenberg, H. The degree of acetylation of
deacetylase from Mucor ruoxii. Biochemistry 1984, 23, chitosans and its eect on the chain conformation in
1065. aqueous solution. Carbohydr. Polym. 2002, 47, 39.
29. Tsigos, I.; Martinou, A.; Varum, K.M.; Kafetzopoulos, D.; 47. Colfen, H.; Berth, G.; Dautzenberg, H. Hydrodynamic
Christodoulidou, A.; Tzanodaskalaki, M.; Bouriotis, V. characterisation of chitosans in aqueous solution. Carbo-
Enzymatic deacetylation of chitin employing chitin deace- hydr. Polym. 2001, 45, 373.
tylases. In: Chitin World; Karnicki, Z.S., Brzeski, M.M., 48. Beri, R.G.; Walker, J.; Reese, E.T.; Rollings, J.E.
Bykowski, P.J., Wojtasz-Pajak, A., Eds.; Wirtschaftsverlag Characterisation of chitosans via coupled size-exclusion
NW: Gdynia, Poland, 1994; 98 pp. chromatography and multi-angle laser light scattering
30. Kafetzopoulos, D.; Martinou, A.; Bouriotis, V. Biocon- technique. Carbohydr. Res. 1993, 11.
version of chitin to chitosan: Purication and character- 49. Wu, C.; Zhou, S.Q.; Wang, W. A dynamic laser light
ization of chitin deacetylase from Mucor rouxii. Proc. scattering study of chitosan in aqueous solution. Biopol-
Natl. Acad. Sci. U.S.A. 1993, 90, 2564. ymers 1995, 35, 385.
31. Tokuyasu, K.; Ohnishi-Kameyama, M.; Hayashi, K. 50. Berth, G.; Dautzenberg, H. Solution behaviour of some
Purication and characterization of extracellular chitin selected polysaccharides studied preferentially by static
deacetylase from Colletotrichum lindemuthianum. Biosci. light scattering. Recent Res. Dev. Macromol. Res. 1998,
Biotechnol. Biochem. 1996, 60, 1598. 3, 225248.
32. Martinou, A.; Bouriotis, V.; Stokke, B.T.; Varum, K.M. 51. Berth, G.; Colfen, H.; Dautzenberg, H. Physicochemical
Mode of action of chitin deacetylases from Mucor rouxii and chemical characterisation of chitosan in dilute
on partially N-acetylated chitosans. Carbohydr. Res. aqueous solution. Progr. Colloid Polym. Sci. 2001, 2882,
1998, 311, 71. 1.
33. Tsigos, I.; Zydowicz, N.; Martinou, A.; Domard, A.; 52. Yamakawa, H. Modern Theory of Polymer Solutions;
Bouriotis, V. Mode of action of chitin deacetylase from Harper and Row: New York, 1971.
Mucor rouxii on N-acetylchitooligosaccharides. Eur. J. 53. Wang, W.; Bo, S.L.; Qin, W. Determination of the Mark
Biochem. 1999, 261, 698. Houwink equation for chitosans with dierent degrees of
34. Tokuyasu, K.; Mitsutomi, M.; Yamaguchi, I.; Hayashi, deacetylation. Int. J. Biol. Macromol. 1991, 13, 281.
K.; Mori, Y. Recognition o chitooligosaccharides and 54. Anthonsen, M.W. Chitosan, chemical structure and phys-
their N-acetyl groups by putative subsites of chitin ical properties. Thesis, Norwegian Institute of Technol-
deacetylase from a deuteromycete, Colletotrichum linde- ogy, Trondheim, Norway, 1994.
muthianum. Biochem. 2000, 39, 8837. 55. Mazeau, K.; Perez, S.; Rinaudo, M. Predicted inuence of
35. Tanford, C. Physical Chemistry of Macromolecules; John N-acetyl group content on the conformational extension
Wiley and Sons: New York, 1961. of chitin and chitosan chains. J. Carbohydr. Chem. 2000,
36. Otty, M.H.; Varum, K.M.; Christensen, B.E.; Smidsrd, 19, 1269.
A. Preparative and analytical size-exclusion chromatog- 56. Anthonsen, M.W.; Varum, K.M.; Hermansson, A.M.;
raphy of chitosans. Carbohydr. Polym. 1996, 31, 253. Smidsrd, O.; Brant, D.A. Aggregates in acidic solutions
of chitosans detected by static light scattering. Carbohydr. 78. Kristiansen, A.; Varum, K.M.; Grasdalen, H. Compet-
Polym. 1994, 25, 13. itive binding of highly de-N-acetylated chitosans and
57. Amiji, M.N. Pyrene uorescence study of chitosan self- N,NV-diacetylchitobiose to lysozyme from chicken egg
association in aqueous solution. Carbohydr. Res. 1995, white studied by 1H NMR spectroscopy. Carbohydr. Res.
26, 211. 1996, 289, 143.
58. Rhazi, M.; Desbrieres, J.; Rinaudo, M.; Vottero, P.; 79. Kristiansen, A.; Varum, K.M.; Grasdalen, H. The
Alagui, A. Contribution to the study of the complexation interactions between highly de-N-acetylated chitosans
of copper by chitosan and oligomers. Polymer 2002, 43, and lysozyme from chicken egg white studied by 1H-
1267. NMR spectroscopy. Eur. J. Biochem. 1998, 251, 335.
59. Vold, I.M.N.; Varum, M.; Guibal, E.; Smidsrd, O. 80. Sasaki, C.; Kristiansen, A.; Fukamizo, T.; Varum, K.M.
Binding of ions to chitosan-selectivity studies. Carbohydr. Biospecic fractionation of chitosan. Biomacromolecules
Polym. 2003, 54, 471. 2003, 6, 1686.
60. Domard, A. pH and CD measurements on a fully 81. No, H.K.; Meyers, S.P. Application of chitosan for
deacetylated chitosan: application to copper(II)polymer treatment of wastewaters. Rev. Environ. Contam. Tox-
interactions. Int. J. Biol. Macromol. 1987, 9, 98. icol. 2000, 163, 1.
61. Anthonsen, M.W.; Smidsrd, O. Hydrogen ion titration 82. Meyer, H.; Butte, W.; Schlaak, M. Chitosan in wastewater
of chitosan with varying degrees of N-acetylation by treatment. In Advances in Chitin Science; Peter, M.G.,
monitoring induced 1H-NMR chemical shifts. Carbohydr. Domard, A., Muzzarelli, R.A.A., Eds.; Universitat Pots-
Polym. 1995, 26, 303. dam: Potsdam, Germany, 2000; Vol. IV, 153 pp.
62. Sorlier, P.; Denuziere, A.; Viton, C.; Domard, A. Relation 83. Eikebrokk, B. Coagulation-direct ltration of soft, low
between the degree of acetylation and the electrostatic alkalinity humic waters. Water Sci. Technol. 1999, 40, 55.
properties of chitin and chitosan. Biomacromolecules 84. Strand, S.P.; Vandvik, M.S.; Varum, K.M.; stgaard, K.
2001, 2, 765. Screening of chitosans and conditions for bacterial
63. Strand, S.P.; Tmmeraas, K.; Varum, K.M.; stgaard, occulation. Biomacromolecules 2001, 2, 126.
K.P. Electrophoretic light scattering studies of chitosans 85. Strand, S.; Varum, K.M.; stgaard, K. Interactions
with dierent degrees of N-acetylation. Biomacromole- between chitosans and bacterial suspensions: adsorption
cules 2001, 2, 1310. and occulation. Colloids Surf., B Biointerfaces 2003, 27,
64. Varum, K.M.; Otty, M.H.; Smidsrd, O. Acid hydrolysis 71.
of chitosans. Carbohydr. Polym. 2001, 46, 89. 86. Agerkvist, I. Characterization of and occulation in E. coli
65. Varum, K.M.; Grasdalen, H. Oligosaccharides prepared cell disintegrates. Ph.D. thesis, Department of Biochemis-
by extensive depolymerization of partially N-acetylated try and Biotechnology, Royal Institute of Technology:
chitosans in concentrated acid. Manuscript in prepara- Stockholm, Sweden, 1992.
tion. 87. Ashmore, M.; Hearn, J. Flocculation of model latex
66. Holme, H.K.; Foros, H.; Pettersen, H.; Dornish, M.; particles by chitosans of varying degrees of acetylation.
Smidsrd, O. Thermal depolymerization of chitosan Langmuir 2000, 16, 4906.
chloride. Carbohydr. Polym. 2001, 46, 287. 88. Ashmore, M.; Hearn, J.; Karpowitz, F. Flocculation of
67. Smidsrd, O.; Haug, A.; Larsen, B. Oxidativereductive latex particles of varying surface charge densities by
depolymerization: a note on the comparison of degrada- chitosans. Langmuir 2001, 17, 1069.
tion of dierent polymers by viscosity measurements. 89. Draget, K.I.; Varum, K.M.; Moen, E.; Gynnild, H.;
Carbohydr. Res. 1967, 5, 482. Smidsrd, O. Chitosan cross-linked with Mo(VI) poly-
68. Varum, K.M. unpublished results. oxyanions: a new gelling system. Biomaterials 1992, 13,
69. Berger, L.R.; Weiser, R.S. The glucosaminidase activity of 635.
egg-white lysozyme. Biochim. Biophys. Acta 1957, 26, 90. Draget, K.I.; Varum, K.M.; Smidsrd, O. Chitosan
517. crosslinked with Mo (VI) polyoxyanions: Eect of
70. Amano, K.I.; Ito, E. The action of lysozyme on partially chemical composition. In Advances in Chitin and Chitosan,
deacetylated chitin. Eur. J. Biochem. 1978, 85, 97. Proceedings International Conference on Chitin and
71. McCreath, K.J.; Gooday, G.W. A rapid and sensitive Chitosan; Brine, C.S., Sandford, P.A., Zikakis, J.P.,
microassay for determination of chitinolytic activity. J. Eds.; Elsevier Applied Science: London, 1991; 604 pp.
Microbiol. Methods 1992, 14, 229. 91. Draget, K.I. Associating phenomena in highly acetylated
72. Wirth, S.J.; Wolf, G.A. Dye-labelled substrates for the chitosan gels. Polym. Gels Netw. 1996, 4, 143.
assay and detection of chitinase and lysozyme activity. J. 92. Schipper, N.G.M.; Varum, K.M.; Artursson, P. Chitosans
Microbiol. Methods 1990, 12, 197. as absorption enhancers for poorly absorbable drugs. 1:
73. Nordtveit, R.J.; Varum, K.M.; Smidsrd, O. Degradation Inuence of molecular weight and degree of acetylation
of fully water-soluble, partially N-acetylated chitosans on drug transport across human intestinal epithelial
with lysozyme. Carbohydr. Polym. 1994, 23, 253. (Caco-2) cells. Pharm. Res. 1996, 13, 1686.
74. Nordtveit, R.J.; Varum, K.M.; Smidsrd, O. Degradation 93. Schipper, N.G.M.; Olsson, S.; Hoogstraate, J.A.; deBoer,
of partially N-acetylated chitosans with hen egg white and A.G.; Varum, K.M.; Artursson, P. Chitosans as absorp-
human lysozyme. Carbohydr. Polym. 1996, 29, 163. tion enhancers for poorly absorbable drugs 2: Mecha-
75. Varum, K.M.; Myhr, M.; Hjerde, R.J.N.; Smidsrd, O. In nism of absorption enhancement. Pharm. Res. 1997, 14,
vitro degradation rates of partially N-acetylated chitosans 923.
in human serum. Carbohydr. Res. 1997, 299, 99. 94. Schipper, N.G.M.; Varum, K.M.; Stenberg, P.; Ocklind,
76. Varum, K.M.; Holme, H.K.; Izume, M.; Stokke, B.T.; G.; Lennernas, H.; Artursson, P. Chitosans as absorption
Smidsrd, O. Determination of enzymatic hydrolysis enhancers of poorly absorbable drugs 3: Inuence of
specicity of partially N-acetylated chitosans. Biochim. mucus on absorption enhancement. Eur. J. Pharm. Sci.
Biophys. Acta 1996, 1291, 5. 1999, 8, 335.
77. Fukamizo, T.; Ikeda, Y.; Ohkawa, T.; Goto, S. 1H-NMR 95. Soane, R.J.; Hinchclie, M.; Davis, S.S.; Illum, L.
study on the chitotrisaccharide binding to hen egg white Clearance characteristics of chitosan based formulations
lysozyme. Eur. J. Biochem. 1992, 210, 351. in the sheep nasal cavity. Int. J. Pharm. 2001, 217, 183.
96. Mumper, R.J.; Wang, J.; Claspell, J.M.; Rolland, A.P. 101. Felt, O.; Buri, P.; Gurny, R. Chitosan: A unique poly-
Novel polymeric condensing carriers for gene delivery. saccharide for drug delivery. Drug Dev. Ind. Pharm. 1998,
Proc. Int. Symp. Control. Release Bioact. Mater. 1995, 22, 24, 979.
178. 102. Davis, S.S. Drug delivery systems. Interdiscip. Sci. Rev.
97. Borchard, G. Chitosans for gene delivery. Adv. Drug 2000, 25, 175.
Deliv. Rev. 2001, 52, 145. 103. Lehr, C.-M. From sticky stu to sweet receptorsAchieve-
98. Koping-Hoggard, M.; Tubulekas, I.; Guan, H.; Edwards, ments, limits and novel approaches to bioadhesion. Eur. J.
K.; Nilsson, M.; Varum, K.M.; Artursson, P. Chitosan as Drug Metab. Pharmacokinet. 1996, 21, 139.
a nonviral gene delivery system. Structure-property 104. Gaserd, O.; Smidsrd, O.; Skjak-Brk, G. Microcap-
relationships and characteristics compared with polyethy- sules of alginatechitosan: I. A quantitative study of the
lenimine in vitro and after lung administration in vivo. interaction between alginate and chitosan. Biomaterials
Gene Ther. 2001, 8, 1108. 1998, 19, 1815.
99. Koping-Hoggard, M.; Melnikova, Y.S.; Varum, K.M.; 105. Gaserd, O.; Sannes, A.; Skjak-Brk, G. Microcapsules
Lindman, B.; Artursson, P. Relationship between the of alginatechitosan: II. A study of capsule stability and
physical shape and the eciency of oligomeric chitosan as permeability. Biomaterials 1999, 20, 773783.
a gene delivery system in vitro and in vivo. J. Gene Med. 106. Gaserd, O.; Jolie, I.G.; Hampson, F.C.; Dettmar, P.W.;
2003, 5, 130. Skjak-Brk, G. The enhancement of the bioadhesive
100. Langer, R. Drug delivery and targeting. Nature 1998, 392, properties of calcium alginate gel beads by coating with
510. chitosan. Int. J. Pharm. 1998, 175, 237.
27
Chitosan as a Delivery System for the Transmucosal
Administration of Drugs
Lisbeth Illum
IDentity, Nottingham, United Kingdom
Stanley (Bob) S. Davis

University of Nottingham, Nottingham, United Kingdom
I. INTRODUCTION from the intestine into the main blood circulation (rst-
pass eect).
Therapeutic materials such as drugs and vaccines can be Many commercially available drugs are of low molec-
introduced to the body by many dierent routes of admin- ular weight (less than 300 Da) and have physicochemical
istration, although conventionally, over many years, par- properties (water solubility, distribution coecient) that
enteral and oral delivery have been the routes of choice cause them to be well absorbed across mucosal surfaces.
[1,2]. The route of preference will be dependent on many The amount of drug available in the blood circulation is
factors; these include required speed of onset of action and termed its bioavailability (expressed as a percentage of
bioavailability, the dose (potency) and physicochemical the total amount of drug administered) with reference to a
properties of the therapeutic substance, patient conve- standard (often the drug injected intravenously). Com-
nience, and therapeutic indication. Agents used in acute pounds which have sucient lipid solubility will be trans-
situations are often given by injection directly into the ported transcellularly across the mucosal membrane by a
blood stream, into a muscle, or into the subcutaneous process of passive diusion (Fig. 1). Some important drug
tissue [3]. Some drugs can be delivered across the skin using molecules do not have physicochemical properties that
transdermal patches if they have the required physico- allow them to be well transported across mucosal mem-
chemical properties to include a reasonable high lipophili- branes. Good examples are peptide and protein drugs, such
city and potency. With regard to patient convenience and as calcitonin, insulin, and leutinizing hormonereleasing
compliance with dosage regimens, noninvasive, noninject- hormone (LHRH). These molecules are large (e.g., >1000
able products are normally selected. Da) and are polar in nature with low distribution coe-
The oral route, where a drug substance is absorbed cients. More conventional, lower molecular weight drugs
across the mucosa of the small and large intestines into the with polar characteristics also show impaired transport
general blood circulation, is preferred. Other transmucosal across mucosal barriers (e.g., atenolol). The limited quan-
routes (nasal, pulmonary, buccal, sublingual, rectal, and tities that are absorbed will exploit a paracellular pathway
vaginal) can be employed for the delivery of therapeutic and pass between the cells of the membrane (Fig. 1). Many
materials directly into the systemic circulation. These of these challenging molecules also exhibit instability,
alternative mucosal routes may have advantages when especially when exposed to the harsh pH and enzyme
rapid onset of action is required or when the gastrointes- conditions found in the gastrointestinal tract.
tinal tract does not provide appropriate conditions for Some molecules are able to exploit natural pathways
good drug absorption (i.e., instability of the drug mole- because they have structures similar to peptides. The ACE
cule), metabolism of the drug in the gut wall during inhibitors such as captopril, L-DOPA, and gabapentin are
absorption, or metabolism in the liver when the drug passes good examples. These drugs are absorbed from the gut by a
643
644 Illum and Davis
Figure 1 Transcellular and paracellular routes of drug transport across mucosal membranes.
combination of paracellular transport and a carrier-medi- suggests that the NH2 and COOH termini of this protein
ated process, the latter being restricted to the upper regions are situated in the cytoplasma of the cell with two extra-
of the small intestine (absorption window). Since absorp- cellular loops projecting into the paracellular space be-
tion is following the same route as the breakdown products tween the cells [6]. The extracellular loops may interact
of digestion, the absorption of these drugs can be greatly with loops originating from occludin from the neighboring
inuenced by the co-administration of food. cell to promote interaction and sealing of the paracellular
space. The cytoplasmic COOH terminal domain of occlu-
din interacts with other tight junction proteins such as the
II. PARACELLULAR TRANSPORT scaolding proteins Z0-1, Z0-2, and Z0-3, also present in
AND TIGHT JUNCTIONS the cytoplasm. The third transmembrane protein, JAM, is
dierent structurally to the occludin and claudin and is
The reversible opening of tight junctions between cells
immunoglobulin-like in form.
represents an important strategy in increasing the trans-
Recent evidence has conrmed the notion that occlu-
mucosal transport of polar molecules. Much is now known
din is a functional component of the paracellular pathway
about junctional complexes, including the tight junctions
[8]. Treatment of cultured renal epithelial (A6) cells with
that are present between the cells of the mucosal mem-
peptides to the two extracellular occludin protein loops
branes and the manner in which small polar molecules can
(1 and 2) indicated that loop 2 had a direct role in forming
be transported across these junctions. Excellent reviews on
the morphology of junctional complexes and the intracel-
lular signaling processes that control junction integrity can
be found elsewhere [47]. A brief summary covering the
salient aspects relevant to this chapter is provided here.
The structural components of the tight junction and
the possible modulating factors are shown in Fig. 2. The
epithelial cells on the apical surface are closely connected
by intercellular junctions, the specialized sites and struc-
tural components of which are commonly known as the
junctional complex. Each junctional complex is composed
of three regions. One is the zonola occludens (ZO) (near the
apical surface) that forms a tight band around the upper
part of the cell and is normally known as the tight junction
(TJ). Under the ZO is the zonola adherens (ZA) and in the
lower again is the macula adherens (MA). These complexes
create a regulatable semipermeable diusion barrier be-
tween cells. The tight junction comprises a series of integral
membrane proteins. These interact with components of the
cytoskeleton through other proteins. The key membrane
components that participate in the extracellular cellcell
contacts between adjacent epithelial cells are the trans-
membrane proteins occludin and claudin and the junction- Figure 2 Schematic representation of the tight junction
al adhesion molecule (JAM). The topology of the occludin between cells. Zonola occludens (ZO) region. (From Ref. 9.)
Chitosan for Transmucosal Administration of Drugs 645
the paracellular permeability barrier. Cells treated with a and controlled fashion using selected agents to enable the
peptide to the extracellular loop 2 but not loop 1 of oc- uptake of many poorly absorbed drugs such as proteins
cludin increased membrane permeability (reduced trans- and peptides at local sites along the GI tract without
epithelial resistance and increased the paracellular ux) of associated adverse events.
molecules up to a size of 40 kDa. This occurred by reducing
the amount of occludin at the tight junction without
obviously aecting cell viability, morphology, or other III. ABSORPTION PROMOTERS
tight junction protein levels.
By means of electrophysiological analysis, it has been Previously, many research groups have sought ways in
shown that the size of the largest molecule that can which it would be possible to promote or enhance the
penetrate the ZO varies with the epithelial tissues of the transmucosal uptake of drugs that display low bioavail-
body. Generally, the permeation of tight junctions is ability when delivered to a mucosal surface. The litera-
limited for molecules with a radius larger than 3.6 A and ture contains reviews dealing with key routes such as the
the tight junctions are impermeable to molecules with a gastrointestinal tract and the nasal cavity [1216]. A
radius larger than 15 A [4]. wide variety of materials have been examined in terms
The ZO is closely associated with the ZA complex. The of their absorption-enhancing ability (Table 1). These
ZA complex holds cells close together but does not form a materials include compounds such as nonionic surfac-
tight barrier. The ZA is made up of transmembrane tants (e.g., laureth-9), alkylglycosides, bile salts, and bile
proteins known as cadherins. Both the ZO and ZA struc- salt derivatives such as sodium glycocholate, sodium
tures act to anchor cytoskeletal components. A dense band taurodihydrofusidate, phospholipids such as dideca-
of actin and myosin laments circumscribes the cell at the noyl-L-a-phosphatidylcholine, cyclodextrins such as di-
level of the ZA. Fine laments of actin extend into the tight methyl-h-cyclodextrin, and cationic polymers such as
junction and bind directly to Z0-1. Hence it is believed that chitosan and polyarginines.
eects on the cytoskeleton can regulate the tight junction
and, in turn, paracellular permeability.
Multiple regulatory pathways control the structure Table 1 Materials Used for the Enhanced Absorption
and function of tight junctions [9]. Much of the functional of Drugs
control of tight junction structure components seems to
occur through mechanisms involving phosphorylation and Surfactants
dephosphorylation. Protein kinase (PKC) inhibitors in- Alkylglycoside surfactants
crease paracellular permeability. The modulation of tight Polyoxyethylene 9-lauryl ether (Laureth 9) (L9)
Polysorbate 80
junction functions by G-protein-coupled events has been
Quillaja saponin
reviewed recently by Hopkins et al. [10]. Proteins act as
Bile salts (BS) and derivatives
messengers that control cellcell contact between scaold
Sodium glycocholate
proteins and the actin cytoskeleton. The barrier junction of Sodium tauro- 24,25,dihydrofusidate (STDHF)
intercellular tight junctions is regulated dynamically by Phospholipids
interactions between the scaolding proteins, the extracel- Didecanoyl-L-alpha phosphatidyl choline (DDPC)
lular matrix, and the actin cytoskeleton. Substances that Lysophosphatidylcholine (LPC)
perturb the actin cytoskeleton are likely to disrupt the tight Lysophosphatidylglycerol (LPG)
junction through eects on actin originating on the peri- Fatty acids and salts
junctional ring that projects onto the cytoplasmic surface Oleic acid
of the tight junction [6]. Sodium caprate (NaCAP)
Phosphorylation of the proteins located in the tight Caprylic acid derivatives
junction apparently regulates the formation of the junction Chelating agents
itself and also possibly the regulation of the tight junction. EDTA
Disruption of the interactions between the molecules di- Cyclodextrins (CD)
rectly involved in TJ structure would have a direct eect on Dimethyl h-cyclodextrin
paracellular transport. Microbial toxins are known to aect Cationic polymers
the cytoskeleton through aects on actin polymerization, Chitosan (CHI)
probably through the modication of a protein kinase C Modied chitosans
pathway [11]. Pharmaceutical excipients such as fatty acids, Poly-L-arginines
cyclodextrins, and chitosan can also inuence paracellular Aminated gelatine
transport, but the mechanism has yet to be fully under- Lipids and miscellaneous systems
Palmitoyl-DL-carnitine (AC)
stood. Such materials probably trigger a cascade of signal-
Glycyrrhetinic acid
ing events. Cytokines, chelating agents, growth factors, and
1-[2-(Decylthio)ethyl]azacyclopentane-2-one (HPE-101)
high concentrations of physiological nutrients can also Starch microspheres
provide enhanced paracellular permeability. PEGylated polymers
Daugherty and Mrsny [9] have suggested that it should
be possible to manipulate the tight junction in a dynamic Source: Ref. 27.
646 Illum and Davis
key areas of the intestine (so far as absorption is concerned)

can greatly aect the conditions. Clearly, for an absorption
promoter to work, it needs to be presented to the mucosal
surface, together with the therapeutic agent, at a concen-
tration that will provide improved absorption but not lead
to adverse reactions and side eects. Unfortunately, the
eectiveness of many absorption-promoting or -enhancing
agents can be related directly to their damaging eects
on membranes (Fig. 3).
In our research, we have sought to identify novel
materials that will provide enhanced transport of mole-
cules across mucosal surfaces in a safe and reliable manner.
The essential characteristics of such a material can be listed
(Table 2) [27]. We have examined in great detail lysophos-
pholipids (especially lysophosphatidyl glycerol), starch
microspheres (and their admixture with enhancers), and a
range of cationic materials to include polysaccharides and
discovered that the last group especially was able to
enhance transmucosal delivery of drugs without any de-
Figure 3 Schematic diagram of the relation between tectable damage to the membrane.
absorption enhancement (as measured by bioavailability) It was argued that these cationic materials should
and membrane damage (see Table 1 for abbreviations). provide a close contact with the nasal mucosa due to an
interaction between the negatively charged sialic acid
residues on the mucin and epithelial cell membrane and
the positive charges on the cationic polymers. Therefore a
Many of the compounds listed are polyvalent in nature decreased clearance of administered formulations from the
and, as a result, can have more than one mechanism of nasal cavity and an increased absorption were expected.
action, i.e., enhancing drug absorption. Some may aect The studies were initially focused on two cationic polymers,
the overlying mucus layer, while others can remove com- namely, DEAE-dextran and chitosan. Studies were per-
ponents from cell membranes or, in general, cause irrevers- formed in rats and in sheep with simple solutions of the
ible damage by stripping o the membrane. The eects seen enhancer systems and insulin as a model drug [28]. In the
are often dose-dependent. Some compounds can aect rat model, it was shown that both positively charged
both transcellular and paracellular pathways, once again polysaccharides were able to signicantly enhance the
in a dose-dependent fashion. The bile salts (and derivatives nasal absorption of insulin. However, when the sheep
thereof ), selected because of their endogenous nature, have model was used, chitosan was far superior to DEAE-
been studied in detail by many research groups [1719] as dextran in terms of the absorption-enhancing eect. It
have fatty acid materials (e.g., sodium caprate) [18] and was suggested that a reason for this dierence between
phospho- (and lyso-) phospholipids [2022]. Cell culture the two polymers was that DEAE-dextran is less densely
models (Caco-2), epithelial sheets (Ussing chamber meth- charged than chitosan and that DEAE-dextran is a
odology), animal models, and human studies have docu- branched polysaccharide as opposed to chitosan which
mented the use of these materials through clinical has a linear structure. This could result in some of the
evaluation. However, despite a large amount of detailed charges being hidden in the DEAE-dextran network and
research, there are few products that have been approved therefore being unavailable for interaction with the mucosa
by regulatory authorities that employ surfactants as ab-
sorption promoters. This is due to the fact that such
materials can be damaging in chronic use as well as irritant.
Attempts to develop nasal formulations of insulin using Table 2 Properties of an Ideal Absorption Enhancer
nonionic surfactants, bile salts, and phospholipids as
enhancers provide interesting case histories [19,20,23]. a) Pharmacologically inert at concentration used.
Nonsurfactant materials such as cyclodextrins and chelat- b) Nonirritating, nontoxic, and nonallergenic.
ing agents (the latter acting directly on tight junctions by c) Any eect on mucosa should be completely reversible.
the sequestering of calcium) have also been investigated d) Should be potent (i.e., requires small quantities to be
[24,25]. Often, exciting data obtained in animal models used).
e) Compatible with drugs.
have not translated well to clinical studies in man [26].
f) Should be able to remain in contact with mucosa long
When considering the use of these enhancer materials
enough to achieve maximal eect.
in the gastrointestinal tract for improved drug absorption, g) No taste or oensive odor.
there can be important dierences between in vitro studies h) Readily available and inexpensive.
involving isolated animal tissues and the in vivo situation
where dilution of the formulation and rapid transit through Source: Ref. 27.
[29,30], which, combined with the lower charge density, The early uses of chitosan as an excipient in pharma-
could result in a weaker bioadhesive eect. Moreover, such ceutical formulations have recently been reviewed by Illum
a dierence between the two polysaccharides would be [2], Dodane and Vilivalam [35], and Felt et al. [36]. The
more pronounced in the conscious sheep model as opposed positively charged nature of chitosan provides interesting
to the anaesthetized rat model where the mucociliary properties with respect to interaction with oppositely
clearance mechanism is impaired [31]. Consequently, it charged materials, especially those of high molecular
was concluded that chitosan (and derivatives thereof ) weight [29,30]. Hence it was appreciated by us and others
might be a material that fullled all of the requirements that chitosan would interact with mucus and mucosal
for a safe and reliable absorption promoter as listed in surfaces (through an interaction with charged sugar groups
Table 2, and we embarked upon an extensive investigation such as sialic acid) and therefore have possible use as a
on the use of chitosan as a delivery system for the trans- bioadhesive material [37,38]. Indeed, this aspect has been
mucosal administration of challenging drugs. exploited recently for the development of gastroretentive
bioadhesive microsphere formulations [39]. Other reports
on the use of chitosan as a bioadhesive agent have been
IV. CHITOSAN provided by us for nasal delivery as well as others for a
variety of mucosal routes [2,4042]. The interaction be-
Chitosan is a polysaccharide comprising copolymers of n- tween positively charged chitosan and negatively charged
acetyl glucosamine and glucosamine. It exists in small DNA has also been exploited for the improved delivery of
amounts naturally in certain fungi but is normally derived plasmid DNA [4345]. Under appropriate mixing condi-
from chitin by alkaline deacetylation. The chain length of tions, small positively charged nanoparticles (50100 nm in
the chitosan polymer can be controlled by acid hydrolysis size) can be obtained. These particles can be taken up by
or enzymatic degradation. Chitin is the second most target cells leading to gene expression. Indeed, the use of
abundant polysaccharide material after cellulose [2]. It chitosan nanoparticles for gene vaccines has been an area
makes up the exoskeleton of insects and crustacea as well of special attention [46,47].
as plant cell walls. Most of the chitosans available com- Our work on the bioadhesive properties of chitosan led
mercially are derived from crustacean sources. It is impor- to the important discovery that this polymer was not only
tant to appreciate that chitosans can be provided in bioadhesive (and could be used to alter intestinal transit of
dierent molecular weights (and molecular weight distri- formulations and nasocilliary clearance), but also had
butions), degrees of deacetylation, and salt forms (e.g., profound absorption-promoting eects when given nasally
acetate, hydrochloride, and glutamate). Chitosan salts are to a rat or sheep model together with highly polar mole-
positively charged at acidic pH due to protonization of the cules such as hormones (insulin, calcitonin, LHRH-ana-
NH2 groups. The physicochemical characteristics of a logs) and morphine-6-glucuronide [4850] (Fig. 4).
given chitosan will also depend on how it was produced
from the native chitin material. As a consequence, chito-
sans that appear nominally to be the same can have A. Mode of Action of Chitosans
dierent physical/chemical properties, especially regarding
their solubility in acidic solutions, but also in terms of their We realized that the dramatic increase in the bioavailability
ability to enhance the transport of drugs across mucosal of the drugs when administered together with chitosan and
surfaces. These considerations have been well discussed by the generally pulsatile shape of the pharmacokinetic
Roberts [32] who has urged workers in the eld to adopt a prole (drug concentration vs. time) could not be explained
more detailed method for describing chitosan samples used simply by a prolonged period of residence of an adminis-
for dierent aspects of research. In our work, we have used tered formulation at the absorption site. It has now been
chitosan materials obtained from Pronova Biomedical in generally accepted that the mechanism of action of chito-
Norway. These chitosans are produced by the total deacet- san in enhancing the transport of drugs across mucosal
ylation of chitin followed by reacetylation to a required membranes is due to a combination of bioadhesion
given specication and display good solubilities in acid (delayed clearance of the formulation from the site of
solutions (or as salt forms in water) even at high degrees of absorption) and the transient opening of the tight junctions
reacetylation (75%) [33]. between the cells of the mucosal membrane [51].
Chitosan has been of interest to workers in the phar- Dodane et al. [52] studied the eect of chitosan on
maceutical eld for decades. It is unusual in being a Caco-2 cell monolayers and found that chitosan caused a
positively charged polysaccharide with a good safety pro- reversible, time- and dose-dependent decrease in trans-
le, having been approved in certain countries (Italy, Nor- epithelial electrical resistance (TEER) associated with an
way, and Japan) for many years as a food additive. It is also increase in paracellular ux of the membrane-impermeant
used as a dietary supplement in putative agents for the tracer, mannitol. These studies conrmed the ndings of
reduction of fat absorption. Two pharmaceutical products Artursson et al. [53]. The involvement of the tight junctions
containing chitosan as an excipient have now been marin the increased permeation was visualized by confocal
keted, namely, an oral controlled release hydrogel-based scanning microscopy and showed that epithelial cells
system (SQZ GelTM, Macromed) and a skin adhesive gel (Caco-2) treated with chitosan expressed a thickened pat-
for wound healing (AquatrixTM II, Hydromer) [34]. tern of occludin at the cell periphery, ZO-1 decreased in
648 Illum and Davis
tight junctions [55]. Ranaldi et al. [56] have lately compared

the eect of chitosan and other polycations (polyethylene-
imines, poly-L-lysines of dierent molecular weights) on
the integrity of tight junctions and of the actin cytoskeleton
using the Caco-2 cell line. All polymers induced a reversible
increase in tight junction permeability which was concen-
tration-dependent and also energy-dependent. The authors
attempted to extrapolate their results to the oral adminis-
tration of chitosan as a weight reduction agent, but their
arguments were not convincing.
Chan et al. [57] have recently proposed an alternative
explanation for the mode of action of chitosans on cell
membranes. They showed in vitro, using a variety of
techniques, that chitosan induced perturbation of mem-
brane bilayers formed from phospholipids. The same slight
eect on the membrane was similarly described by Dodane
et al. [52] who also reported an increase in cellular activity
(in terms of appearance of large intracellular vacuoles)
after application of chitosan to the cell monolayer. How-
ever, the relevance of these in vitro studies to the in vivo
situation was not investigated by the authors.
V. NASAL DELIVERY
A. The Nasal Mucosa
The nose has four major functions, namely, those of
olfaction, mechanical and enzymatic defense, supply of
air to the lungs, and conditioning of the air before it reaches
the lungs. Of these, especially the defense system has an
impact on the nasal absorption of drugs due to the tight
junctions between the epithelial cells of the nasal mem-
brane, the mucociliary clearance mechanism, and the
possible enzymatic degradation of the drugs. Lipophilic
drugs are generally well absorbed in the nasal cavity with
bioavailabilities up to 100%, whereas small polar mole-
cules generally have a bioavailability of less than 10% and
peptides less than 1%.
The anterior part of the nasal cavity is lined with
Figure 4 Nasal administration of insulin to sheep. Eect of squamous epithelium that gradually changes further back
added chitosan. Insulin dose 20 IU/kg, chitosan concen- to pseudostratied columnar epithelium which constitutes
tration 0.5%. (A) Plasma insulin; (B) plasma glucose. the respiratory epithelium. This latter epithelium consists
of a layer of elongated columnar cells on the surface and
below is 24 layers of basal cells on top of the basal
membrane. The respiratory epithelium is covered with cilia
intensity in some areas, there was a slight shortening of and most of the cells also with a layer of 24 Am long cilia.
laments, and actin aggregates appeared specically at Cilia are ne hairlike structures which can move in a
cellcell boundaries [52]. These results conrmed the coordinated way to help the mucus ow across the epithe-
results of Schipper et al. [54] who stained Z0-1 protein lial surface. The mucus layer that covers the epithelium
and found a distinctive disruption of Z0-1 in the same cell consists of a sol layer, a low viscosity periciliary uid,
model after application of chitosan in the same cell mono- which surround the cilia, and a more viscous gel layer
layer model. The transmembrane protein occludin has been forming a layer on top of the sol layer and covering the tips
found to display a disrupted pattern after incubation with of the cilia.
chitosan derivatives [55]. Chitosan may also aect ion Clearly, for a polar molecule of reasonable molecular
transport through an interaction with the cell surface. Such weight, the membrane permeability can be considered the
interactions may well lead to activation of intracellular most important factor limiting the nasal membrane pene-
events that involve the participation of secondary messen- tration. Drugs can cross the epithelial cell membrane either
gers and the protein kinases that are known to modulate by the transcellular route exploiting simple concentration-
dependent passive diusion, receptor-mediated transport, poly-L-arginine showed no toxic eect on the membrane in
or vesicular transport mechanisms. This is the case for a similar way to chitosan.
lipophilic drugs which are readily absorbed and trans- The eect of chitosan molecular weight and chitosan
ported directly to the systemic circulation due to the high concentration on the nasal absorption of peptides has been
vascularity of the nasal mucosa. Polar molecules will studied in rats and in sheep [61]. Hence it was shown in the
generally pass the membrane via the tight junctions. As rat model that when insulin was applied nasally in solution
described above, the paracellular route will only allow mol- with chitosan fractions of molecular weights from about
ecule up to a certain size (less than 15 A) to pass through the 450 to 10 kDa, the absorption of insulin, expressed in terms
junction unless absorption enhancers opening these junc- of lowering of the blood level, was highly dependent on the
tions are employed. molecular weight of the chitosan. The absorption of insulin
Another contributing factor to the low transport of increased with an increase in molecular weight until about
polar molecules, which is especially of importance for 200 kDa after which the lowering in blood glucose was not
peptides, is the enzymatic degradation taking place in the signicantly dierent (Fig. 5). In terms of chitosan solution
nasal cavity or during passage across the epithelial barrier. concentration, it was found that at and above a concen-
The nasal cavity and the epithelial membrane contain both tration of about 0.5%, there was no increase in eect of
exopeptidases and endopeptidases that can attack N and C chitosan on promotion of nasal insulin absorption in rats
termini and internal peptide bonds. and sheep (Fig. 6). It should be noted that the blood glucose
The mucociliary clearance mechanism provides mam- lowering eect of the chitosaninsulin formulation was
mals with a very ecient defense system against inhaled highest in the rat model.
bacteria, particles, and irritants by transporting these on Chitosan can be formulated either as a simple solution
the surface of the mucus posteriorly in the nasal cavity and formulation, as spray-dried powder formulations, or as
down the throat. Hence pharmaceutical formulation ad- microsphere or nanoparticle formulations. It has been
ministered to the nasal cavity will also be transported away shown in most cases that the eects of chitosan powders
from the absorption site by the same mechanism. It has and chitosan microspheres are superior in providing en-
been shown that the half-life of clearance from the human hancement of the nasal absorption of polar drugs as
nose is in the order of 15 min [58]. This mucociliary compared to chitosan solution formulations. This is in part
clearance of drug formulations can be controlled to some due to the longer residence time of these formulations in the
degree by the use of bioadhesive materials such as chitosan. nasal cavity [58]. Hence the chitosaninsulin solution
formulation was optimized in the sheep model by produc-
B. Chitosan ing a nasal chitosan powder formulation which resulted in
an increase in bioavailability of nearly vefold as compared
About a decade ago, we were the rst to demonstrate that to the chitosan solution formulation [62]. Bioavailabilities
chitosan could enhance the nasal absorption of polar small
molecular weight drugs as well as peptides and proteins
[48]. Thus when insulin, in a simple chitosan solution
formulation, was administered nasally to sheep, the plasma
glucose levels fell to 43% of the control level within 90 min
compared to a control solution of insulin which only caused
a fall in blood glucose level to 83% of control levels. The
corresponding plasma insulin levels resulted in a sevenfold
increase in Cmax as compared to the control insulin solution
(Fig. 4). A later Phase 1 clinical trial showed a bioavail-
ability for the nasal chitosan solution formulation of about
10% vs. a subcutaneous injection [51].
Subsequently, other researchers have studied dierent
chitosans for the nasal delivery of drugs. Tengamnuay et al.
[59] investigated the eect of using dierent chitosans
(either initially in the form of free amines or as chitosan
salts with molecular weights ranging from 800 to 1860
kDa) on the nasal absorption in rats of (D-Arg2)-kyoto-
phin, a neutral dipeptide with morphine-like activity. The
degree of deacetylation was from >70% to 97%. Signi-
cant absorption of the peptide was found for all types of
chitosan studied especially at lower pH values. Natsume
et al. [60] studied various cationic enhancers for the en-
hancement of nasal absorption of dextran 4000. They
found that in the rat model, chitosan was an excellent Figure 5 Nasal administration of insulin to rats. Eect of
enhancer, but that poly-L-arginine and sodium dodecyl the molecular weight of chitosan. Insulin dose 4 IU/kg,
sulfate (SDS) were more powerful. However, of these, only chitosan concentration 0.5%.
650 Illum and Davis
[63]. All the investigated pharmacokinetic parameters

(Cmax, Tmax, AUC over 2h) were similar during and
outside migraine attacks. The product was well tolerated.
As a polar drug, morphine is not readily absorbed via
the nose when administered in simple formulations (bio-
availability of about 10% in humans) [64]. It has been
demonstrated by recently published Phase 1 clinical trial
data that a nasal chitosanmorphine solution formulation
can give rapid absorption of the drug with the peak plasma
concentration within 10 min and absorption increased
about sixfold to provide a bioavailability of 60% vs. an
intravenous injection [3]. An impressive increase in the
absorption of morphine with the addition of chitosan to
the nasal formulation was especially notable in this
study. Importantly, a much lower degree of metabolism
of morphine to its two most common metabolites (mor-
phine-6-glucuronide and morphine-3-glucuronide) was
found after nasal administration as compared to oral
administration of morphine. Indeed, the metabolic prole
of the nasal chitosanmorphine formulation was similar to
Figure 6 Nasal administration of insulin. Eect of chitosan
concentration in the rat and sheep. that obtained after intravenous administration. Further
optimized chitosanmorphine formulations have pro-
vided bioavailabilities in the region of 80% in later dose
ranging studies [27]. These initial studies have now resulted
in a pilot Phase 2 evaluation and the development of a
of 11.6%, 25.6%, and 36.6%, relative to subcutaneous product capable of oering patients rapid and ecient pain
injection, were likewise obtained in sheep for a goserelin relief by a noninjectable route [65]. This novel nasal
chitosan solution formulation, a dry blend of chitosan and morphine product containing chitosan as an absorption
goserelin, and for goserelin freeze-dried with chitosan promoter is expected to reach the market within the next
cross-linked microspheres, respectively [50]. few years.
A nasal calcitonin product, consisting of a simple
solution of calcitonin and benzalkonium chloride (BKC), C. Chitosan Derivatives
has been commercially available from Novartis in the last
56 years. Although it has been suggested by Novartis that The chitosan derivative, trimethylchitosan (TMC), has
at the concentration used the cationic surfactant BKC acts also been evaluated for the enhancement of nasal delivery
both as a preservative and as an absorption enhancer, the of polar drugs [66,67]. However, such chitosan derivatives
bioavailability of the product is only about 13%. Various that are soluble at basic pH values are more interesting for
companies are presently attempting to formulate an im- the purposes of oral delivery (see below) since the pH in the
proved product with a higher bioavailability and a lower nasal cavity (pH 56) is suciently low for the nonderiv-
absorption variability. Soane [61] reported the results from atized chitosan to stay in solution. Most of the studies
a pilot study in 5 volunteers where salmon calcitonin was performed with this chitosan derivative used either Caco-2
given nasally with and without chitosan as an absorption cells or in vivo animal models. When insulin was adminis-
promoter. The results showed that the chitosan signi- tered nasally to rats, in combination with chitosan or TMC
cantly increased the nasal absorption as compared to a (61.2% quaternization), as solution formulations at acid
nasal control. Studies to optimize the novel chitosan and basic pH values, it was shown that at pH 4.4, chitosan
calcitonin formulation are now underway in Phase 1 was superior in enhancing the absorption of insulin from
clinical trials. the nasal cavity as compared to TMC [67]. However, not
A chitosan-based liquid formulation has been devel- surprisingly, at pH 7.4, chitosan failed to show a signicant
oped for the polar antimigraine compound alniditan. This eect, whereas TMC showed a similar eect to that at pH
formulation has been evaluated both in Phase 1 and Phase 4.4. This, of course, was due to chitosan being insoluble at
2 studies. Phase 1 studies showed that chitosan enhanced pH 7.4! In a later paper, Hamman et al. [66] investigated the
the nasal absorption of alniditan to provide a bioavailabil- importance of the degree of quaternization of the amine
ity of about 60%. This result conrmed earlier results from groups in TMC for its absorption-enhancing eect on
sheep studies. In a Phase 2 study, a chitosan solution mannitol in the rat nose. They tested TMC with 12%,
formulation was administered to migraineurs at 2 dose 22%, 36%, 48%, and 59% degrees of quaternization
levels during and outside migraine attacks. An early rise in (TMC12, TMC22, TMC36, TMC48, and TMC59, respec-
plasma concentration and the amount of drug in the tively) and found that the absorption-enhancing eect
circulation were found to relate to headache improvement increased with increasing quaternization until a quaterni-
zation of 48% was reached. There was no signicant respectively. However, due to a high variability, there was
dierence between the eect of TMC48 and TMC59. no signicant dierence between the results obtained. In
the sheep model, the chitosaninsulin solution formulation
D. Chitosan Microparticles was found to give signicantly lower blood glucose levels
(53.0% as compared to 73.3% and 72.6%) and a signi-
Chitosan microparticles (or the combination of chitosan cantly higher Cmax (179.1 AIU/ml compared to 66.9 and
and other polymers) can be produced by processes of emul- 106.2 AIU/ml) for plasma insulin than the nanoparticle
sication, precipitation, complex coacervation, solvent formulations. Furthermore, the nasal chitosaninsulin
evaporation, or a combination of these. The microparticles powder formulation was found in all respects to be superior
are either unstabilized or stabilized by cross-linking or by to all other formulations.
complexation with oppositely charged (macro)molecules. Lim et al. [73] compared the absorption-enhancing
The drug is either encapsulated during the production eects of hyaluronanchitosan complex microparticles
process or sorbed into/onto the particles after production. with those of hyaluronan and chitosan microparticles
The prepared particles are normally freeze-dried [62,68]. loaded with gentamicin after nasal administration to
One of the more interesting concepts for the ecient rabbits as dry powder formulations. It was found that the
production of chitosan nanoparticles is the ability of microparticles containing chitosan gave the highest bio-
chitosan to gel in contact with the negatively charged availabilities, 31% and 42% for chitosan and hyaluronan
tripolyphosphate ions by ionotropic gelation that facili- chitosan complex microparticles, respectively. Unfortu-
tates instantaneous nanoparticle formation. The nanopar- nately, no comparison was made to a formulation com-
ticles are formed via inter and intramolecular linkages prising chitosangentamicin in solution. The size of the
created between the cationic amino groups of chitosan microparticles was given as 1930 Am.
and the tripolyphosphate anions [69]. It has been claimed
by some authors that using this method, high nanoparticle
yields and high drug loadings could be achieved, for VI. ORAL DELIVERY
example, with insulin [70,71].
The reasoning behind the use of chitosan nanopar- One of the current challenges in drug delivery is the
ticles for nasal drug delivery rather than a chitosan solution discovery of novel methods for the improved oral delivery
formulation appears to be the fact that certain bioadhesive of polar molecules such as peptides and proteins (or
nanoparticles have been shown to remain in the nasal polysaccharides in the form of low molecular weight
cavity of man for extended periods of time [72]. The mean heparin). Not surprisingly, such compounds are poorly
half-life of residence time of cationic nanoparticles (not absorbed across the mucosal surface in the gastrointesti-
chitosan-based) was found to be 2.3 F 1.7 hr in man. For nal tract due to severe degradation by enzymes present
the case of chitosan, Soane et al. [58] reported a half-life of within the intestines, their hydrophilicity, and the size of
residence of 45 min for chitosan solution and a half-life of the molecule. Interestingly, it was shown by Drewe et al.
residence time of 90 min for chitosan microspheres (2550 [74] that the somatostatin analog, octreotide, a stable
Am in diameter) in man. A simple saline solution (control) polypeptide, only achieved a bioavailability of 0.6% in
had a half-life of 15 min. man, and when administered with a surfactant absorption
It has been suggested in various scientic papers that enhancer, the absorption increased to 3.3%. Desmopres-
chitosan nanoparticles (300400 nm in diameter), prepared sin, which is marketed in the United States as an oral
by the ionic gelation method and loaded with insulin, were formulation, has a bioavailability in the order of 0.2%.
able to enhance the nasal absorption of insulin in the rabbit Sinko et al. [75] evaluated the oral absorption of calcitonin
model to a greater extent than a simple chitosaninsulin in a dog model and found a bioavailability of about 0.5%
solution formulation (results expressed by the reduction of when administered without enhancer systems and about
glucose blood levels) [70,71]. However, surprisingly, these 1.25% when administered in delivery systems containing
authors also showed that the chitosaninsulin solution either fatty acids or bile salts. In a Phase 1 clinical trial,
formulation induced only a minor decrease in plasma such a fatty acid enhancer system (caprylic acid derivative)
glucose levels in their rabbit model. A recent study in two was found to provide a bioavailability of orally adminis-
animal models, the rat and the sheep, has showed conclu- tered salmon calcitonin of 0.51.4% [76]. Similarly, it was
sively that chitosan nanoparticles (similar to those used in found that insulin administered orally in dogs gave neg-
the Fernandez-Urrusuno studies) were not able to provide ligible levels of insulin in the blood, but when adminis-
an improved absorption-enhancing eect as compared to tered insulin conjugated to a lipophilic (alkyl)/hydrophilic
that of chitosan in solution or in a powder form [62]. In the (PEG) polymer, the bioavailability was estimated to 8%
rat model, the chitosan solution formulation was shown to [77]. In man, no specic bioavailability study was per-
decrease the blood glucose levels to 40.1% of basal levels, formed, but the bioavailability was estimated to reach
whereas the two nanoparticle formulations only lowered about 5% in a dose ranging study. A study in monkeys
these levels to 59.7% and 52.9% of basal levels. The showed a bioavailability of 2.1% when parathyroid hor-
bioavailabilities of the various formulations, based on the mone (PTH) was delivered orally in combination with N-
pharmacodynamic data, were 47.9%, 37.7%, and 36.1%, (8-(2-hydroxy-4-methoxy)benzoyl)amino caprylic acid (4-
652 Illum and Davis
MOAC), whereas without the enhancer system, the up- It should also be mentioned that a range of publica-
take was negligible [78]. tions has appeared in the literature concerned with the use
Hence an oral delivery system that would enable the of chitosan nanoparticles for oral peptide absorption [83].
safe and therapeutic eect of peptides and possibly proteins These chitosan nanoparticles and other nanoparticle sys-
would be of great benet and interest to the pharmaceu- tems have all been tested in animal models where they have
tical industry. showed great promise for improvement of peptide absorp-
tion. However, it is at present unclear whether these
promising results would also translate into similar results
A. Chitosan in man. Studies in man with polystyrene particles have
shown that uptake of particles did not take place in the
It has been shown by our own group that chitosan can human gastrointestinal tract to a signicant extent [8486].
enhance the absorption of peptide drugs such as calcitonin Considerable work has been performed on the pro-
after oral administration to the rat. Others, such as Luessen duction of microparticle systems for improved oral delivery
et al. [79] in Leiden, have reported bioavailabilities of 5.1% of peptides and proteins in the same way as described for
for buserelin in rats when the drug was administered orally nasal delivery systems [87]. The particles were produced
as a solution formulation in combination with chitosan at either by chitosan alone, chitosan in combination with
pH 5.5. However, a disadvantage of chitosan as an oral polymer counterions, or microparticles coated with chito-
absorption promoter is its relative low pKa (f6.5) of the san. The main reason for using such microparticles for oral
preferred chitosan which has a degree of deacetylation of delivery is the potential of these bioadhesive particles to
more than 80%. This means that at pH values of about 6.5 reside longer in the small intestines due to mucoadhesion,
or higher, the protonation of chitosan decreases rapidly to protect the encapsulated drug against degradation in the
and the material becomes insoluble and precipitates out of stomach, and to promote absorption of the drugs in the
solution. Hence at the pHs of the distal small intestines and gastrointestinal tract. Aydin and Akbuga [88] prepared
distal parts of the colon, it is likely that chitosan will not chitosan beads by dropping a solution of chitosan contain-
have its full absorption-promoting eect. Moreover, it ing salmon calcitonin into tripolyphosphate solution. The
could prove dicult to formulate solid dosage forms encapsulation ecacy was 5459%. In vitro, the drug was
containing chitosan where the chitosan will dissolve rap- relased over nearly 30 days. Kawashima et al. [89] pro-
idly after the dosage form has disintegrated because the duced PLGA microparticles coated with chitosan and
dissolution of chitosan is relatively slow even in acid media. containing elcatonin (a calcitonin derivative). The chito-
In an attempt to overcome these problems, various san-coated particles were found to reduce signicantly the
derivatives of chitosan have been produced by introducing blood calcium levels in rats after intragastric administra-
into the molecule groups that are protonated at acid, tion as compared to an elcatonin solution and noncoated
neutral, and basic pH values (e.g., trimethyl chitosan) PLGA microparticles. Takishima et al. [90] similarly coat-
[67] or protonated at values higher than pH 6 such as ed ethylcellulose microparticles containing the antibiotic
carboxymethyl chitosan [80]. cephradine with chitosan and studied the intestinal trans-
The inuence of mucus on the absorption enhance- port of these particles in the rat after intraduodenal ad-
ment of chitosan has been studied by Schipper et al. [81] ministration. The chitosan-coated microparticles were
using an intestinal cell line (HT29) with and without mucus found to be retained for a longer period of time in the
layers and in an in situ perfusion model of the rat ileum. intestine (8 hr) than the noncoated microparticles.
The discharge of mucus from goblet cells was considered to
be a limiting factor that could inhibit the binding of
chitosan to the epithelial cells and hence decrease the B. Chitosan Derivatives
absorption-enhancing eect. The authors suggested that
increasing the local concentrations of chitosan and the The use of trimethylated and carboxymethylated chitosans
drug could overcome the problem, but no studies were as enhancers for the intestinal absorption of hydrophilic
conducted in intact animals to verify this proposal. drugs has been investigated in detail by Junginger et al.
Probably the most important problem in the use of [13,9196] especially for oral delivery of polar drugs. They
chitosan and chitosan derivatives for the improved drug have described such materials as safe permeation
absorption in the intestine is the need to deliver the drug enhancers that are nonabsorbable and therefore not
and the polymer concurrently in a soluble form to the mu- expected to show any systemic toxicity. However, a
cosal surface and thereby the problem of employing a detailed toxicological evaluation has yet to be provided.
preferred solid dosage form. Unfortunately, the dissolu- They chose chitosan derivatives to overcome the solubility
tion of chitosan (and its derivatives) can be slow. Recently, problems exhibited by chitosan at neutral and basic pH
attempts have been made to develop peptide products that values, especially when considering the conditions (pH,
are retained at critical sites in the intestine and which would ionic strength) found in certain regions of the gastrointes-
allow the drug and chitosan enhancer to be delivered at an tinal tract. Various in vitro and in vivo investigations have
appropriate rate for optimal eect. Rapidly swelling been described by these authors to include studies in a large
hydrogel formulations for this purpose comprising grafted animal modelthe pig [96]. However, as yet, no pivotal
chitosan have been described [82]. studies in man have been reported.
Comparative studies between chitosan and trimethyl to provide a bioavailability of 14% and 25% using a 5% or
chitosans (TMC) of dierent degrees of quaternization a 10% TMC concentration, respectively. For the highest
have been conducted using intestinal epithelial cells in TMC concentration, this represented a 14.8-fold increase
culture (Caco-2) [95,97]. Transepithelial electrical resist- as compared to a simple octreotide solution. Unfortunate-
ance (TEER) and the permeability of a polar marker (14C- ly, the paper did not indicate as to how it would be possible
mannitol) were investigated at pH values of 6.2 and 7.4. to provide a solid dose formulation for human use con-
Both chitosan materials aected membrane permeability at taining sucient TMC to provide a 10% concentration at
the lower pH value with chitosan being superior to chitosan the absorption site.
derivatives; however, at the higher pH value, the unmod- Less extensive reports on the use of mono-N-carboxy-
ied chitosan was insoluble, and as to be expected, only the methyl chitosan (MCC) as an intestinal permeation en-
TMC derivatives were eective. TMC materials with a high hancer have also been presented [80,103]. This material was
charge density were observed to be optimal. exploited by Thanou et al. [80] for the delivery of anionic
In a series of similar papers, Kotze et al. have described species that would interact unfavorably with cationic chi-
experiments wherein the eect of trimethylchitosan (TMC) tosan and TMCs. Transport studies using intestinal cell
on membrane permeability has been evaluated in the Caco- lines have shown that two dierent grades of MCC were
2 model of intestinal epithelial cells. The results have shown able to increase the permeation of low molecular weight
that TMC is able to open the tight junctions of intestinal heparin. Initial tests in the rat model indicated that MCC
epithelial cells to allow for paracellular transport of hy- could provide therapeutic levels of the anticoagulant.
drophilic molecules. It was concluded that the charge The delivery of chitosan-based formulations orally has
density of TMC, as determined by the degree of quaterni- been considered by Dorkoosh et al. [104] who have exam-
zation, is an important factor determining its potential use ined an oral delivery system based on a superporous
as an absorption enhancer across intestinal epithelia, espe- hydrogel (SPH) and composite polymers. The system was
cially in neutral and basic environments and could be made of two components: a conveyor system made of SPH
important for the better delivery of challenging hydrophilic and a core that contained octreotide. The core was inserted
molecules such as peptides and proteins [97101]. into the conveyor system (core inside) or attached to the
Cell-based toxicological investigations demonstrated surface of the conveyor system (core outside). Four dier-
that the TMC material was similar to chitosan and apparent oral formulations were investigated in pigs to include
ently opened paracellular pathways and thereby allowed a core outside system containing trimethyl chitosan. All
enhanced transport of markers such as uorescent dextran formulations were placed in enteric-coated gelatin cap-
and a dye, Texas red [93]. No staining of the nuclei was sules. The highest bioavailability (16.1F3.3%) was
found. Consequently, it was concluded that TMCs were achieved by the addition of trimethyl chitosan chloride as
safe and did not perturb cell membranes. Studies were also an extra absorption enhancer.
performed to ascertain the cellular mechanisms responsible Berkop-Schnurch et al. [105107] have studied inten-
for alterations in tight junction morphology [102]. As with sively the production and the in vivo characteristics of
chitosan itself, the eect of the TMCs was reversible. derivatives of chitosan such as chitosancysteine, chito-
Immunohistological techniques were employed to show santhiglycolic acid, and chitosanEDTA based on prin-
that the occludin pattern around the cell was disrupted ciples of increased enzymatic inhibition or tight junction
but was normalized after the removal of the polymer. opening of the copolymers for improvement of oral peptide
Cytoskeletal F-actin and intracellular calcium levels were absorption. No in vivo studies have yet been published, as
also measured. While calcium levels were unaltered, signif- far as we are aware.
icant rearrangement of F-actin, present at the subapical
level, was observed. While these results were similar to those
reported previously for chitosan, they were dierent to VII. BUCCAL DELIVERY
those for other known absorption enhancers such as sodi- A. The Buccal Mucosa
um caprate, which can also modify tight junction integrity.
The peptide drug, buserelin, was employed by Thanou The morphology of the oral mucosal membrane and the
et al. [94] as a model peptide compound in cell culture and various routes of transport across the membrane have been
for in vivo studies in a rat model. A dramatic increase in described by various authors and shall only be summarized
bioavailability was found following the direct administra- here [108110]. The mucosal lining of the oral cavity
tion of the drug and TMC into the duodenum of the rat at a represents an important topical route for delivery of drugs.
controlled pH of 7.2. Not surprisingly, at this pH, unmod- It is especially important for more lipophilic drugs but
ied chitosan was shown to be ineective. However, it could also be of relevance to polar drugs, such as small
should be borne in mind that in man, the upper regions of peptides, if the formulation contains an excipient that can
the intestine are acidic and a simple chitosan system could promote transport across the epithelium. The oral mucosa
well be just as eective. A similar study was recently has greater permeability and perfusion than the skin, and
published on the intestinal absorption of octreotide in pigs the oral cavity provides an environment almost free from
using N-trimethyl chitosan chloride as the absorption the acidity and protease activity present elsewhere in the
enhancer [96]. The formulations, all solutions, were applied gastrointestinal tract. The mucosa is well vascularized and
directly into the jejunum. The TMC formulation was able the veins drain directly into the jugular vein, so that drugs
654 Illum and Davis
penetrating the epithelium will pass straight into the sys- high bioavailabilities (e.g., 47% for nitroglycerin, 46%
temic circulation avoiding rst-pass metabolism. The ac- for ecainide, and 69.8% for indomethacin). However, in
cessibility of the oral cavity makes application of the drug order to increase the permeability for hydrophilic mole-
easy and acceptable to the patient. Any taste problems can cules and especially for peptides, it is necessary to include
often be overcome by formulation approaches. permeation enhancers in the formulations. Permeation
The structure of the oral mucosa is very similar to that enhancers that have been studied for this purpose include
of the skin in that the oral mucosa is covered with stratied bile salts, surfactants, fatty acids, chelators, cyclodextrins,
squamous epithelium. In part, the oral cavity, such as the and chitosan [109,115]. Studies performed in vivo in rats,
gums and the palate, is keratinized. This keratin layer rabbits, dogs, and humans showed very variable results
resembles the epidermis covering the skin. The mucosa [109]. For peptides such as insulin, generally little or no
lining the oor of the mouth and the buccal regions is drug could be detected in the blood stream after buccal
nonkeratinized (lining epithelium) and the epithelium is administration with or without traditional absorption
similar to that covering the esophagus or uterine cervix. enhancers. However, recently, Morishita et al. [116]
The expression buccal mucosa refers anatomically to the found a pharmacological availability (based on glucose
lining of the inside of the cheeks. levels) in rats of 15.9% after administration of insulin in a
The rate and degree of transport of drugs across the F-127 gel system with fatty acids as absorption enhancers.
oral mucosa are very dependent on lipophilicity and mo- Smaller peptides such as buserelin and LHRH showed
lecular weight of the molecule. Lipophilic drugs are some absorption especially when administered with ab-
thought to be transported across the epithelium by the sorption enhancers (bile salts), i.e., 12.7% for buserelin in
transcellular route, mainly by a mechanism of concentra- pigs. A human study delivering glucagon-like peptide in a
tion-dependent diusion. More hydrophilic small mole- buccal bioadhesive tablet with a bile salt showed a
cules and peptides are believed to cross the membrane by relative bioavailability of 47% to SC [117]. However,
the paracellular route. However, it has been suggested that long-term eects of mucosal irritation from these enhanc-
the main route of transport for most drugs across the oral er systems have to be considered carefully. In some
epithelium is paracellular [111] and hence that the perme- instances, the use of bioadhesive formulations (e.g., ad-
ability is dependent on the nature of the intercellular hesive patches) as compared to simple solutions was
material (arising from the membrane coating granules) sucient to increase the buccal transport of certain
being similar to the case for the epidermis of the skin. peptides (e.g., TRH and oxytocin).
However, it has also been shown that the major intercellu- Chitosan has been utilized in buccal delivery systems
lar barrier is lipid in nature (ceramides, glycosylceramides), either as a bioadhesive material or as an absorption
and hence these materials will prevent the transport of enhancer. For example, Remunan-Lopez et al. [118] have
more hydrophilic molecules [112]. described the preparation of buccal bilayered devices com-
prising a drug-containing mucoadhesive layer and a drug-
B. Buccal Delivery Systems free backing layer. The mucoadhesive layer was composed
of a mixture of drug and chitosan, with or without an
Traditionally, drugs have been delivered across the muco- anionic cross-linking polymer (polycarbophil, sodium al-
sal barrier in simple aqueous solutions or conventional ginate, and gellan gum), and the backing layer was made of
sublingual or buccal tablets. A major limitation to obtain- ethylcellulose. Using nifedipine and propranolol hydro-
ing good absorption by use of these simple formulations is chloride as slightly and highly water-soluble model drugs,
the lack of retention at the site of absorption due to respectively, it was demonstrated that these new devices
continuous dilution by salivary ow. Thus in later years, show promising potential for use in controlled delivery of
the use of bioadhesive materials for the creation of sticky drugs to the oral cavity. In vitro release data showed that
dosage forms has been utilized for buccal administration. the release of drug could be controlled by the counterion
By forming an adhesive interaction between the delivery used with the chitosan and that release times of more than 6
system and the oral mucosa, the residence time of the drug hr could be obtained. No in vivo data were given. Senel et
at the absorption site is extended which should allow al. [40] produced two types of buccal bioadhesive systems
therapeutic drug plasma levels to be maintained over based on high molecular weight chitosan, namely, cross-
extended periods especially if the delivery system contains linked chitosan lms and chitosan gels both containing
a controlled release entity. A great variety of bioadhesive chlorhexidine. The lms were cross-linked with tripoly-
polymers such as gelatin, agarose, hyaluronic acid, phosphate and showed in vitro release of 12 hr similar to
carbomers, polyacrylic acidhydroxypropyl methylcellu- that for the gels.
lose, as well as chitosan have been utilized in various Portero et al. [119] have investigated the potential of
formulations such as tablets, patches, tapes, lms, pow- chitosan to enhance buccal peptide and protein absorption,
ders, and microsphere delivery systems [110]. Claims have using a model of the buccal epithelium (cultured TR146
been made that such systems can stay at the site of cells). Permeability studies were performed with 3H-man-
application for periods of 6 hr [113,114]. nitol and uorescein isothiocyanate-labeled dextrans (FD)
Albeit normally slowly, small lipophilic drugs have with various MW (4.419.5 kDa) to determine the enhanc-
been shown to penetrate the oral mucosa (compared to ing eect of chitosan glutamate (dierent salts with dier-
absorption from the intestines) and to provide reasonably ent molecular weights). An enhancing eect was found for
chitosan concentrations of 20 Ag/mL and higher, correlat- bodies were induced at a level similar to those induced by
ing with a decrease in TEER values. the injection formulation. Moreover, it was found that the
Chitosan has been shown to have a signicant enhanc- guinea pigs vaccinated nasally with the chitosanCRM197
ing eect on the permeation of drugs across the buccal formulation were well protected against the disease after
mucosa without any toxic eect on the membrane [109]. In challenging with diphtheria [46]. Recently, these results
these studies, various drugs (hydrocortisone, TGF-ha have been conrmed clinically in a three-way Phase 1 study
large peptide) were incorporated into 2% chitosan gels in 25 adults whom had all previously been vaccinated
produced from a high molecular weight chitosan (MW: against diphtheria (>5 years ago) [122]. The nasal chitosan
1,400,000 Da, 80% degree of deacetylation) and drug powder formulation was shown to be superior in terms of
transport across excised porcine buccal mucosa evaluated IgA levels and induced neutralizing antibody levels as
and compared to a control. The results showed enhance- compared to the nasal diphtheria control formulation
ment of permeability of up to sixfold for these hydrophilic and in terms of IgA levels as compared to the conventional
drugs. Kremer et al. [120] investigated the eect of molec- injectable vaccine. Both Th1 and Th2 responses were
ular weight (2701400 kDa) and degree of deacetylation induced after nasal administration. The results indicate
(7397%) on the permeability-enhancing ability of dier- that the nasal CRM197 chitosan vaccine was eective,
ent chitosans on the movement of hydrocortisone across protective, and also induced both humoral and cellular
excised porcine buccal mucosa. In the same work, chitosan immunity against diphtheria toxoid [123].
gels were compared to carbomer gels. Chitosan and Likewise, it has also been shown that chitosan en-
carbomer showed a similar drug ux in the rst 3 hr after hances the immunoresponse obtained from inuenza vac-
which the chitosan system showed signicantly higher cine when given nasally to mice [46]. Hence IgG levels
uxes. There was no signicant dierence between the similar to those obtained after parenteral administration as
eects of the dierent chitosans except that the highest well as signicant IgA levels were found both in the nasal
molecular weight chitosan (MW 1400 kDa) provided a uid and in the lungs. The responses from the nasal
higher concentration of drug in the supercial layers of the chitosan formulations were found to be superior to those
mucosa than did the other chitosans. obtained from a simple nasal antigen formulation. Recently,
these encouraging results have been conrmed in a Phase 1
clinical study in 60 volunteers. The HI values were in-
VIII. VACCINES creased more than fourfold relative to predose HI values
A. Chitosan and HI values of 40 and higher were found. These results
are indicative of protective levels of antibodies according to
The chitosan concept has also been exploited for the the CPMP requirements. HI levels were similar for the
delivery of antigens for nasal vaccination against various nasal inuenza vaccine containing chitosan and the con-
respiratory diseases by Illum et al. [46]. Signicant and ventional intramuscular inuenza vaccine [46].
improved immunoresponses have been obtained for per- Westerink et al. [124] examined in mice the eect of
tussis, diphtheria, and inuenza in a range of animal administering tetanus toxoid (TT) nasally in combination
models (and in man for some antigens). It is likely that it with a thermal gelation agent Pluronic F127 and either
is the absorption-promoting ability of chitosan (opening of lysophosphatidylcholine (LPC) or chitosan. It was shown
tight junctions) that is promoting the contact of the antigen that the immune response, in terms of IgG and IgA levels in
with intraepithelial and submucosal lymphocytes. The lung and nasal washes, was superior for the formulation
mucoadhesive properties of chitosan may also aid the containing chitosan as compared to Pluronic F127 alone or
contact between the antigen (especially if in particulate in combination with LPC. There was no comparison to a
form) and the nose-associated lymphoid tissue (NALT) formulation comprising TT and chitosan in solution, and
and the specic M-like cells in the NALT. This has been hence it is not possible to evaluate whether a simple
reviewed recently elsewhere [47]. Some of the results chitosan formulation would be as eective as the F127/
obtained for two of the antigens tested are discussed below. chitosan formulation.
A formaldehyde stabilized recombinant diphtheria
toxoid (CRM197) was administered nasally in dierent B. Chitosan Microparticles
doses (from 5 to 20 Ag) in an initial study in mice [121]. The
formulations were solutions with and without chitosan. The use of chitosan for mucosal vaccination has been
IgG serum titers similar to those obtained for an intraperi- reviewed by van der Lubben et al. [125]. These authors
toneal (IP) injection of the antigen were obtained. High pointed out that chitosan easily forms microparticles and
IgA levels were found for all nasal chitosan formulations in nanoparticles which encapsulate large amounts of antigens
the nasal wash samples, whereas no response was seen for such as ovalbumin, diphtheria toxoid, or tetanus toxoid. It
the IP formulation. Similar encouraging results had previ- has been shown that ovalbumin-loaded chitosan micro-
ously been obtained in both primed and naive guinea pigs, particles are taken up by the Peyers patches of the gut-
with a chitosan powder formulation found to be superior to associated lymphoid tissue (GALT). Additionally, after
the chitosan solution formulation [46]. Further studies in co-administering chitosan with antigens in nasal vaccina-
primed guinea pigs showed that in the presence of chitosan tion studies, a strong enhancement of both mucosal and
in the nasal formulation, antidiphtheria-neutralizing anti- systemic immune responses can be observed. They con-
656 Illum and Davis
cluded that chitosan particles, powders, and solutions are It can be concluded that the use of chitosan as micro-
promising candidates for mucosal vaccine delivery. particles for mucosal delivery of vaccine has shown inter-
Calvo et al. [126] produced chitosan nanoparticles esting results that will need to be substantiated in human
containing nonionic surfactants in the form of diblock clinical studies.
copolymers of ethylene oxide and propylene oxide (Plur-
onics) and tripolyphosphate. The particles (2001000 nm)
were loaded with bovine serum albumin. These nanopar- IX. CONCLUSION
ticles had an entrapment eciency of 80% and released
the model antigen for up to 1 week in vitro dependent on the The oral route, where a drug substance is transported across
composition and loading degree of the nanoparticles. The the mucosa of the small and large intestines into the general
same authors later encapsulated tetanus toxoid in the same blood circulation, is generally the route of preference for
type of nanoparticles and found similar long release times delivery of drugs. Other transmucosal routes of delivery
[127]. The same authors have also recently investigated the (nasal, buccal) can also be employed for the delivery of
role of a chitosan coating on a 125I-tetanus toxoid-loaded therapeutic materials directly into the systemic circulation,
PLGA nanoparticle system in terms of transport across the especially when a rapid onset of action is required or when
nasal mucosa [128]. It was found in a rat model that after the gastrointestinal tract does not provide appropriate
nasal administration, the chitosan coating provided an conditions for good drug absorption. However, polar drugs
enhanced transport of toxoid into the blood stream as normally show poor absorption since they cannot pass
compared to noncoated PLGA nanoparticles. This sup- through cells and have to travel through cell junctions by
ported previous results for PLA-PEG nanoparticles loaded paracellular transport. Such transport can be enhanced by
with the same radiolabeled antigen [129]. In the most recent chitosan. Consequently, for some time, chitosan has been of
study from the same group, various types of nanoparticles interest as a delivery system and as an enhancer system for
(PLA-PEG NP, chitosan-coated PLGA NP, and chitosan the improved mucosal delivery of drugs and has been
NP) were tested. The loading capacity for proteins (to investigated for nasal, buccal, and oral delivery of drugs
include tetanus toxoid), the stability of the protein, the and vaccines. It is unusual in being a positively charged
ability to transport the antigen across the nasal and intes- polysaccharide with a good safety prole. It is now marketed
tinal mucosae, and the resultant immune responses were all as an excipient in two pharmaceutical products.
measured [130]. Surprisingly, the authors found that PLA- Work on the bioadhesive properties of chitosan led to
PEG particles were superior to PLA particles in trans- the important discovery that this polymer was not only
porting tetanus toxoid into the blood stream after nasal bioadhesive (and could be used to alter intestinal transit of
and intestinal application. It was likewise found that chi- formulations and nasocilliary clearance), but also had
tosan had the same eect on PLGA nanoparticles after profound absorption-promoting eects when administered
nasal administration. Finally, it was found that chitosan to a mucosal membrane such as that found in the nasal
nanoparticles compared to free antigen were superior in cavity. The present review has described how the use of
inducing an immune response in mice (in terms of serum chitosan has progressed especially for the nasal delivery of
IgG levels) after nasal administration. small polar molecules and peptides and that very promising
A number of papers have been published by van der clinical data have been obtained. It is expected that in the
Lubben et al. [131133], which have described the prepa- very near future, nasal products containing chitosan as an
ration of chitosan microparticles and the good incorpora- excipient/absorption enhancer will appear on the market.
tion of ovalbumin and diphtheria toxoid (DT). The In the rst instance, they will provide improved systems for
microparticles (<10 Am) were made by the precipitation/ pain management and vaccination. Systems for treatment
coacervation method. Release studies showed that the DT of osteoporosis, migraine, diseases of the brain, and erectile
remained entrapped for more than 3 months [131]. In vivo dysfunction are in development. Oral formulations con-
studies, using confocal laser scanning microscopy and taining chitosan or chitosan derivatives as absorption
immunohistochemistry, showed that the particles were promoters are also in development. However, these will
taken up by Peyers patches after intragastrical feeding to most likely take longer to reach the market.
mice [132]. Finally, the ability of the chitosan micropar-
ticles to enhance both systemic and local immune response
against DT after oral and nasal administration in mice was REFERENCES
investigated [133]. For both nasal and oral administration, 1. Davis, S. S. Delivery of peptide and non-peptide drugs
it was found that the chitosan microparticles signicantly through the respiratory tract. PSTT 1999, 2, 450456.
improved the immune response in terms of IgG and IgA 2. Illum, L. Chitosan and its use as a pharmaceutical
titers. Furthermore, DT neutralizing antibody levels were excipient. Pharm. Res. 1998, 15, 13261331.
also improved after oral administration for the micropar- 3. Illum, L.; Watts, P.; Fisher, A.N.; Hinchclie, M.;
ticle formulation. However, there was no comparison with Norbury, H.; Jabbal-Gill, I.; Nankervis, R.; Davis, S.S.
Intranasal delivery of morphine. J. Pharmacol. Exp. Ther.
a DTchitosan solution formulation for nasal administra- 2002, 301, 110.
tion, and it cannot be judged whether the chitosan nano- 4. Madara, J.L.; Dharmsathaphorn, K. Occludin junction
particles would be superior to this formulation for vaccine structure relationship in a cultured epithelial monolayer. J.
delivery and inducing an immune response. Cell Biol. 1985, 101, 21242133.
5. Balda, M.S.; Matter, K. Tight junctions. J. Cell. Sci. 1998, 24. Merkus, F.W.H.M.; Verhoef, J.C.; Marttin, E.; Romeijn,
111, 541547. S.G.; Van der Kuy, P.H.M.; Hermens, W.A.J.J.; Schipper,
6. Denker, B.M.; Nigam, S.K. Molecular structure and as- N.G.M. Cyclodextrins in nasal drug delivery. Adv. Drug
sembly of the tight junctions. Am. J. Physiol. 1998, 274, Deliv. Rev. 1999, 36, 4157.
F1F9. 25. Matsubara, K.; Abe, K.; Irie, T.; Uekama, K. Improve-
7. Madara, J.L. Modulation of tight junctional permeability. ment of nasal bioavailability of luteinizing hormone-
Adv. Drug Deliv. Rev. 2000, 41, 251253. releasing hormone agonist, buserelin, by cyclodextrin
8. Wong, V.; Gumbiner, B.M. A synthetic peptide corre- derivative in rats. J. Pharm. Sci. 1995, 84, 12951300.
sponding to the extracellular domain of occluding perturbs 26. Merkus, F.W.H.M.; Verhoef, J.C.; Romeijn, S.G.; Schip-
the tight junction permeability barrier. J. Cell Biol. 1997, per, N.G. Interspecies dierences in the nasal absorption of
136, 399409. insulin. Pharm. Res. 1991, 8, 1343.
9. Daugherty, A.L.; Mrsny, R.J. Regulation of the intestinal 27. Davis, S.S.; Illum, L. Absorption enhancers for nasal drug
epithelial paracellular barrier. PSTT 1999, 2, 281287. delivery. Clin. Pharmacokinet. 2003, 42, 11071128.
10. Hopkins, A.M.; Li, D.; Mrsny, R.J.; Walsh, S.V.; Nusrat, 28. Critchley, H. Intranasal drug delivery. Ph.D. Thesis, Not-
A. Modulation of tight junction function by G protein- tingham University, UK, 1989.
coupled events. Adv. Drug Deliv. Rev. 2000, 41, 329340. 29. Harding, S.E.; Davis, S.S.; Deacon, M.P.; Fiebrig, I.
11. Fasano, A.; Fiorentini, C.; Donelli, G.; Uzzau, S.; Kaper, Biopolymer mucoadhesives. Biotechnol. Genet. Eng. Rev.
J.B.; Margaretten, K.; Ding, X.; Guandalini, S.; Comstock, 1999, 16, 4186.
L.; Goldblum, S.E. Zonola occludens toxin modulates tight 30. Fiebrig, I.; Varum, K.M.; Harding, S.E.; Davis, S.S.;
junctions through protein kinase C-dependent actin re- Stokke, B.T. Colloidal gold and colloidal gold labelled with
organization, in vitro. J. Clin. Invest. 1995, 96, 710720. germ agglutinin as molecular probes for identication in
12. Verhoef, J.C.; Bodde, H.E.; de Boer, A.G.; Bouwstra, J.A.; mucin/chitosan complexes. Carbohydr. Polym. 1997, 33,
Junginger, H.E. Transport of peptide and protein drugs 9199.
across biological membranes. Eur. J. Drug Metab. 31. Mayor, S.H.; Illum, L. Investigation of the eect of
Pharmacokinet. 1990, 15, 8393. anaesthesia on nasal absorption of insulin in rats. Int. J.
13. Thanou, M.; Verhoef, J.C.; Junginger, H.E. Chitosan and Pharm. 1997, 149, 123129.
its derivatives as intestinal absorption enhancers. Adv. 32. Roberts, G.A.F. Chitin Chemistry; The MacMillan Press
Drug Deliv. Rev. 2001, 50, S91S101. Ltd: London, 1992.
14. Behl, C.R.; Pimplaskar, H.K.; Sileno, A.P.; Xia, W.J.; 33. Errington, N.; Harding, S.E.; Varum, K.M.; Illum, L.
Gries, W.J.; deMeireles, J.C.; Romeo, V.D. Optimisation Hydrodynamic characterisation of chitosan varying in
of systemic nasal drug delivery with pharmaceutical molecular weight and degree of deacetylation. Int. J. Biol.
excipients. Adv. Drug Deliv. Rev. 1998, 29, 117134. Macromol. 1993, 15, 11231127.
15. Kompella, U.B.; Lee, V.H.L. Delivery systems for pene- 34. Gupta, P.; Vermani, K.; Garg, S. Hydrogels from
tration enhancement of peptides and protein drugs: controlled release to pH responsive drug delivery. Drug
design considerations. Adv. Drug Deliv. Rev. 2001, 46, Discov. Today 2002, 7, 569579.
211245. 35. Dodane, V.; Vilivalam, V.D. Pharmaceutical applications
16. Smith, P.L.; Wall, D.A.; Gochoco, C.H.; Wilson, G. Oral of chitosan. PSTT 1998, 1, 246253.
absorption of peptides and proteins. Adv. Drug Deliv. Rev. 36. Felt, O.; Buri, P.; Gurni, R. Chitosan: A unique
1992, 8, 253290. polysaccharide for drug delivery. Drug Dev. Ind. Pharm.
17. Deurloo, M.J.; Hermens, W.A.; Romeyn, S.G.; Verhoef, 1998, 24, 979993.
J.C.; Merkus, F.W. Absorption enhancement of intra- 37. Lehr, C.M.; Bouwstra, J.A.; Schacht, E.H.; Junginger,
nasally administered insulin by sodium taurodihydrofusi- H.E. In vitro evaluation of mucoadhesive properties of
date (STDHF) in rabbits and rats. Pharm. Res. 1989, 6, chitosan and some other natural polymers. Int. J. Pharm.
853856. 1992, 78, 4348.
18. Yamamoto, A.; Morita, T.; Hashida, M.; Sezaki, H. Eects 38. Illum, L. Bioadhesive formulations for peptide delivery. In
of absorption promoters on the nasal absorption of drugs Bioadhesion in Drug DeliveryIssues in Fundamentals,
with various molecular weights. Int. J. Pharm. 1993, 93, 91 Novel Approaches and Development; Mathiowitz, E., Lehr,
99. C.M., Chickering, D., Eds.; Marcel Dekker: New York,
19. Lalej-Bennis, D.; Boillot, J.; Bardin, C.; Zirinis, P.; Coste, 1998; 507539.
A.; Escudier, E.; Chast, F.; Peynegre, R.; Slama, G.; Selam, 39. He, P.; Davis, S.S.; Illum, L. Sustained release chitosan
J.-L. Six month administration of gelied intranasal insulin microspheres prepared by novel spray drying methods. J.
in 16 type 1 diabetic patients under multiple injections: Microencapsul. 1999, 16, 343355.
ecacy vs. subcutaneous injections and local tolerance. 40. Senel, S.; Kremer, M.J.; Kas, S.; Wertz, P.W.; Hincal,
Diabetes Metab. 2001, 27, 372377. A.A.; Squier, C.A. Enhancing eect of chitosan on peptide
20. Drejer, K.; Vaag, A.; Bech, K.; Hansen, P.; Sorensen, A.R.; drug delivery across buccal mucosa. Biomaterials 2000, 21,
Mygind, N. Intranasal administration of insulin with 20672071.
phospholipid as absorption enhancer: Pharmacokinetics 41. Ramdas, M.; Dileep, K.J.; Anitha, Y.; Paul, W.; Sharma,
in normal subjects. Diabet. Med. 1992, 9, 335340. C.P. Alginate encapsulated bioadhesive chitosan micro-
21. Dua, R.; Duncan, M.; Hossein, Z.; Thomas, E.N. The spheres for intestinal drug delivery. J. Biomater. Appl.
inuence of the enhancer dimyristoylphosphatidylglycerol 1999, 13, 290296.
and formulation factors on the nasal absorption of salmon 42. Bugamelli, F.; Raggi, M.A.; Orienti, I.; Zecchi, V.
calcitonin. Drug Deliv. 1998, 5, 127134. Controlled insulin release from chitosan microparticles.
22. Richardson, J.L.; Farraj, N.F.; Illum, L. Enhanced vaginal Arch. Pharm. 1998, 331, 133138.
absorption of insulin in sheep using lysophosphatidylcho- 43. Iqbal, M.; Lin, W.; Jabbal-Gill, I.; Davis, S.S.; Steward,
line and a bioadhesive microsphere delivery system. Int. J. M.W.; Illum, L. Nasal delivery of chitosan-DNA plasmid
Pharm. 1992, 88, 319325. expressing epitopes of respiratory syncytical virus (RSV)
23. Hinchclie, M.; Illum, L. Intranasal insulin delivery and induces protective CTL responses in BALB/c mice. Vac-
therapy. Adv. Drug Deliv. Rev. 1999, 35, 199234. cine. in press.
658 Illum and Davis
44. Roy, K.; Mao, H.-Q.; Huang, S.K.; Leong, K.W. Oral gene study in two animal models between simple chitosan
delivery with chitosan-DNA nanoparticles generates im- formulations and chitosan nanoparticles. Pharm. Res.
munologic protection in murine peanut allergy model. Nat. 2002, 19, 9981008.
Med. 1999, 5, 387391. 63. Roon, K.I.; Soons, P.A.; Uitendaal, M.P.; De Beukelaar,
45. Koping-Hoggaard, M.; Ocklind, G.; Guan, H.; Jansson, F.; Ferrari, M.D. Pharmacokinetic prole of alniditan
B.; Artursson, P. Chitosan and PEI-pDNA polyplexes: In nasal spray during and outside migraine attacks. J. Clin.
vivo gene expression after tracheal, nasal and oral Pharmacol. 1999, 47, 285290.
administration to mice. Proc. Int. Symp. Control. Release 64. Behl, C. Experiences and the potential of nasal drug
Bioact. Mater. 1999, 26, 795796. delivery. In Symposium Notes, Management Forum Meet-
46. Illum, L.; Jabbal-Gill, I.; Hinchclie, M.; Fisher, A.N.; ing on Nasal Drug delivery; London, March 2324, 2000.
Davis, S.S. Chitosan as a novel nasal system for vaccines. 65. Wilcock, A.; Pavis, H.; Edgecombe, J.; Carr, D.; Man-
Adv. Drug Deliv. Rev. 2001, 51, 8196. derson, C.; Church, A. Nasal morphine for the treatment of
47. Davis, S.S. Nasal vaccines. Adv. Drug Deliv. Rev. 2001, 51, breakthrough pain in cancer patients. J. Pain Symptom
2442. Manage. 2003, 24, 598602.
48. Illum, L.; Farraj, N.F.; Davis, S.S. Chitosan as a novel 66. Hamman, J.H.; Stander, M.; Kotze, A.F. Eect of the
nasal delivery system for peptides drugs. Pharm. Res. 1994, degree of quaternisation of N-trimethyl chitosan chloride
11, 11861189. on absorption enhancement: in vivo evaluation in rat nasal
49. Illum, L.; Davis, S.S.; Pawula, M.; Fisher, A.N.; Barrett, epithelia. Int. J. Pharm. 2002, 232, 235242.
D.A.; Farraj, N.F.; Shaw, P.N. Nasal administration of 67. Kotze, A.F.; Thanou, M.; Verhoef, J.C.; Junginger, H.E.
morphine-6-glucuronide in sheepPharmacokinetic Chitosan and N-trimethyl chitosan chloride as absorption
study. Biopharm. Drug Dispos. 1996, 17, 717724. enhancers for nasal and rectal delivery of insulin. Proc. Int.
50. Illum, L.; Watts, P.; Fisher, A.N.; Jabbal-Gill, I.; Davis, Symp. Control. Release Bioact. Mater. 1998, 25, 479480.
SS. Novel chitosan-based delivery systems for the nasal 68. Kas, H.S. Chitosan: properties, preparation and applica-
administration of a LHRH-analogue. STP Pharma Sci. tion to microparticulate systems. J. Microencapsul. 1997,
2000, 10, 8994. 14, 689711.
51. Illum, L. Nasal drug deliveryPossibilities, problems and 69. Janes, K.A.; Fresneau, M.P.; Marazuela, A.; Fabra, A.;
solutions. J. Control. Release 2003, 87, 187198. Alonso, M.J. Chitosan nanoparticles as delivery systems
52. Dodane, V.; Khan, M.A.; Merwin, J.R. Eect of chitosan for doxorubicin. J. Control. Release 2001, 73, 255267.
on epithelial permeability and structure. Int. J. Pharm. 70. Fernandez-Urrusuno, R.; Calvo, P.; Remunan-Lopez, C.;
1999, 182, 2132. Vila-Jato, J.L.; Alonso, M.J. Enhancement of nasal
53. Artursson, P.; Lindmark, T.; Davis, S.S.; Illum, L. Eect absorption of insulin using chitosan nanoparticles. Pharm.
of chitosan on the permeability of monolayers of in- Res. 1999, 16, 15761581.
testinal epithelial cells (Caco-2). Pharm. Res. 1994, 11, 71. Fernandez-Urrusuno, R.; Romani, D.; Calvo, P.; Vila-
13581361. Jato, J.L.; Alonso, M.J. Development of a freeze dried
54. Schipper, N.G.M.; Olsson, S.; Hoogstraate, J.A.; de Boer, formulation of insulin loaded chitosan nanoparticles
A.G.; Varum, K.M.; Artursson, P. Chitosan as absorption intended for nasal administration. STP Pharm. Sci. 1999,
enhancers for poorly absorbed drugs 2: Mechanisms of 9, 429436.
absorption enhancement. Pharm. Res. 1997, 14, 923929. 72. Kravtzo, R.; Fisher, A.; de Miguel, I.; Perkins, A.; Major,
55. Stenson, W.F.; Easom, R.A.; Riehl, T.E.; Turk, J. M.; Betbeder, D.; Etienne, A. Nasal residence time
Regulation of paracellular permeability in Caco-2 cell evaluation of cationic Biovectork in human volunteers.
monolayers by protein kinase C. Am. J. Physiol. 1993, 265, Proc. Int. Symp. Control. Release Bioact. Mater. 1998, 25,
G955G962. 818819.
56. Ranaldi, G.; Marigliano, I.; Vespignani, I.; Perozzi, G.; 73. Lim, S.T.; Forbes, B.; Berry, D.J.; Martin, G.P.; Brown,
Sambuy, Y. The eect of chitosan and other polycations on M.B. In vivo evaluation of novel hyaluronan/chitosan
tight junction permeability in the human intestinal Caco-2 microparticulate delivery systems for the nasal delivery of
cell line(1). J. Nutr. Biochem. 2002, 13, 157167. gentamicin in rabbits. Int. J. Pharm. 2002, 231, 7382.
57. Chan, V.; Hai-Quan, M.; Leong, K.W. Chitosan-induced 74. Drewe, J.; Fricker, G.; Vonderscher, J.; Beglinger, C.
perturbation of dipalmitoyl-sn-glycero-3-phosphocholine Intestinal absorption of octreotide: absorption enhance-
membrane bilayer. Langmuir 2001, 17, 37493756. ment by polyoxyethyle-24-cholesterol ether. Br. J. Phar-
58. Soane, R.J.; Frier, M.; Perkins, A.; Jones, N.S.; Davis, S.S.; macol. 1993, 108, 298303.
Illum, L. Evaluation of the clearance characteristics of 75. Sinko, P.J.; Lee, Y.-H.; Makhey, V.; Leesman, G.D.;
bioadhesive systems in humans. Int. J. Pharm. 1999, 178, Sutyak, J.P.; Yu, H.; Perry, B.; Smith, C.L.; Hu, P.;
5565. Wagner, E.J.; Falzone, L.M.; McWhorter, L.T.; Gilligan,
59. Tengamnuay, P.; Sahamethapat, A.; Sailasuta, A.; Mitra, J.P.; Stern, W. Biopharmaceutical approaches for devel-
A.K. Chitosans as nasal absorption enhancers of peptides: oping and assessing oral peptide delivery strategies and
comparison between free amine chitosans and soluble salts. systems: In vitro permeability and in vivo oral absorption
Int. J. Pharm. 2000, 197, 5367. of salmon calcitonin (sCT). Pharm. Res. 1999, 16, 527533.
60. Natsume, H.; Iwata, S.; Ohtake, K.; Miyamoto, M.; 76. Buclin, T.; Cosma Rochat, M.; Burckhardt, P.; Azria, M.;
Yamaguchi, M.; Hosoya, K.; Kobayashi, D.; Morimoto, Attinger, M. Bioavailability and biological ecacy of a new
Y. Screening of cationic compounds as an absorption oral formulation of salmon calcitonin in healthy volun-
enhancer for nasal drug delivery. Int. J. Pharm. 1999, 185, teers. J. Bone Miner. Res. 2002, 17, 14781485.
112. 77. Still, J.G. Development of oral insulin: Progress and
61. Soane, R.J. Ph.D. Thesis. In Bioadhesive polymers as intra- current status. Diabetes Metab. Res. Rev. 2002, 18, S29
nasal drug delivery systems for peptide and protein drugs; S37.
University of Nottingham: UK, 1999. 78. Leone-Bay, A.; Sato, M.; Paton, D.; Hunt, A.H.; Sarubbi,
62. Dyer, M.; Hinchclie, M.; Watts, P.; Castile, J.; Jabbal- D.; Carozza, M.; Chou, J.; McDonough, J.; Baughman,
Gill, I.; Nankervis, R.; Illum, L. Nasal delivery of insulin R.A. Oral delivery of biologically active parathyroid
using novel chitosan based formulations: A comparative hormone. Pharm. Res. 2001, 18, 964970.
79. Luessen, H.L.; de Leeuw, B.J.; Langemeyer, M.W.E.; de 94. Thanou, M.; Florea, B.I.; Langemeyer, M.W.E.; Verhoef,
Boer, A.G.; Coos, J.; Junginger, H.E. Mucoadhesive J.C.; Junginger, H.E. N-trimethylated chitosan chloride
polymers in peroral peptide drug delivery. VI. Carbomer (TMC) improves the intestinal permeation of the peptide
and chitosan improve the intestinal absorption of the pep- drug buserelin in vitro (Caco-2 cells) and in vivo (rats).
tide drug buserelin in vivo. Pharm. Res. 1996, 13, 16681672. Pharm. Res. 2000, 17, 2731.
80. Thanou, M.; Nihot, M.T.; Jansen, M.; Verhoef, J.C.; 95. Thanou, M.; Kotze, A.F.; Scharringhausen, T.; Luessen,
Junginger, H.E. Mono-N-carboxymethyl chitosan (MCC), H.L.; de Boer, A.G.; Verhoef, J.C.; Junginger, H.E. Eect
a polyampholytic chitosan derivative, enhances the in- of degree of quaternization of N-trimethyl chitosan
testinal absorption of low molecular weight heparin across chloride for enhanced transport of hydrophilic compounds
intestinal epithelia in vitro and in vivo. J. Pharm. Sci. 2001, across intestinal Caco-2 cell monolayers. J. Control.
90, 3846. Release 2000, 64, 1525.
81. Schipper, N.G.; Varum, K.M.; Stenberg, P.; Ocklind, G.; 96. Thanou, M.; Verhoef, J.C.; Verheijden, J.H.M.; Junginger,
Lennernas, H.; Artursson, P. Chitosans as absorption H. Intestinal absorption of octreotide using trimethyl chito-
enhancers of poorly absorbable drugs. 3: Inuence of san chloride studies in pigs. Pharm. Res. 2001, 18, 823828.
mucus on absorption enhancement. Eur. J. Pharm. Sci. 97. Kotze, A.F.; Thanou, M.M.; Luessen, H.L.; de Boer, B.G.;
1999, 8, 335343. Verhoef, J.C.; Junginger, H.E. Eect of the degree of
82. Homan, A.S.; Matsura, J.E.; Wu, X.; Gombotz, W.R. quaternization of N-trimethyl chitosan chloride on the
Release of cytokine receptors from physical hydrogels of permeability of intestinal epithelial cells (Caco-2). Eur. J.
pluronic polyethers grafted to chitosan backbones. Proc. Pharm. Biopharm. 1999, 47, 269274.
Int. Symp. Control. Release Bioact. Mater. 1997, 24, 126 98. Kotze, A.F.; Luessen, H.L.; de Leeuw, B.J.; de Boer, B.G.;
127. Verhoef, J.C.; Junginger, H.E. N-trimethyl chitosan chlo-
83. Pan, Y.; Li, Y.; Zhao, H.; Zheng, J.; Xu, H.; Wei, G.; Hao, ride as a potential absorption enhancer across mucosal
J.; Cui, F. Bioadhesive polysaccharide in protein delivery surfaces: in vitro evaluation in intestinal epithelial cells
system: Chitosan nanoparticles improve the intestinal (Caco-2). Pharm. Res. 1997, 14, 11971202.
absorption of insulin in vivo. Int. J. Pharm. 2002, 249, 99. Kotze, A.F.; Luessen, H.L.; de Leeuw, B.J.; de Boer, A.G.;
139147. Verhoef, J.C.; Junginger, H.E. Comparison of the eect of
84. Lavelle, E.C.; Shari, S.; Thomas, N.W.; Holland, J.; dierent chitosan salts and N-trimethyl chitosan chloride
Davis, S.S. The importance of gastrointestinal uptake of on the permeability of intestinal epithelial cells (Caco-2). J.
particles in the design of oral delivery systems. Adv. Drug Control. Release 1998, 51, 3546.
Deliv. Rev. 1995, 18, 522. 100. Kotze, A.F.; Luessen, H.L.; de Boer, A.G.; Verhoef, J.C.;
85. Rinck, P.A.; Smevik, O.; Nilsen, G.; Klepp, O.; Onsrud, Junginger, H.E. Chitosan for enhanced intestinal perme-
M.; Oksendal, A.; Borseth, A. Oral magnetic particles in ability: prospects for derivatives soluble in neutral and
MR imaging of the abdomen and pelvis. Radiology 1991, basic environments. Eur. J. Pharm. Sci. 1999, 7, 145151.
178, 775779. 101. Kotze, A.F.; Thanou, M.M.; Luebetaen, H.L.; de Boer,
86. Lonnemark, M.; Hemmingsson, A.; Erichson, A.; Gun- A.G.; Verhoef, J.C.; Junginger, H.E. Enhancement of
dersen, H.G.; Bach-Gansmo, T. Oral superparamagnetic paracellular drug transport with highly quaternized N-
particles for magnetic resonance imaging. Eect in plain trimethyl chitosan chloride in neutral environments: in
and viscous aqueous suspensions. Acta Radiol. 1990, 31, vitro evaluation in intestinal epithelial cells (Caco-2). J.
303307. Pharm. Sci. 1999, 88, 253257.
87. Takeuchi, H.; Yamamoto, H.; Kawashima, Y. Mucoadhe- 102. Thanou, M.; Verhoef, J.C.; de Bont, H.J.G.M.; Nagel-
sive nanoparticulate systems for peptide drug delivery. kerke, J.F.; Junginger, H.E. Eects of N-trimethyl chitosan
Adv. Drug Deliv. Rev. 2001, 47, 3954. chloride (TMC) on cytoskeletal F-actin and the tight junc-
88. Aydin, Z.; Akbuga, J. Chitosan beads for the delivery of tions transmembrane protein occluding of intestinal Caco-
salmon calcitonin: Preparation and release characteristics. 2 cells. In Chitosan derivatives in drug delivery, Ph.D. The-
Int. J. Pharm. 1996, 131, 101103. sis, Leiden University, Leiden, The Netherlands, 2000; 91
89. Kawashima, Y.; Yamamoto, H.; Takeuchi, H.; Kuno, Y. 108.
Mucoadhesive DL-lactide/glycolide copolymer nano- 103. Muzzarelli, R.A.A.; Tanfani, F.; Emmanueli, M.; Mariotti,
spheres coated with chitosan to improve oral delivery of S. N-(carboxymethylidene)chitosans and N-(carboxy-
elcatonin. Pharm. Dev. Technol. 2000, 5, 7785. methyl)-chitosans: novel chelating polyampholytes ob-
90. Takishima, J.; Onishi, H.; Machida, Y. Prolonged intesti- tained from chitosan glyoxylate. Carbohydr. Res. 1982,
nal absorption of cephradine with chitosan-coated ethyl- 107, 199214.
cellulose microparticles in rats. Biol. Pharm. Bull. 2002, 25, 104. Dorkoosh, F.A.; Verhoef, J.C.; Verheijden, J.H.; Raee-
14981502. Tehrani, M.; Borchard, G.; Junginger, H.E. Peroral
91. Junginger, H.E.; Verhoef, J.C. Macromolecules as safe absorption of octreotide in pigs formulated in delivery
penetration enhancers for hydrophilic drugsA ction? systems on the basis of superporous hydrogel polymers.
PSTT 1998, 1, 370376. Pharm. Res. 2002, 19, 15321536.
92. Junginger, H.E.; Thanou, M.; Lueen, H.L.; Kotze, A.F.; 105. Bernkop-Schnurch, A. Chitosan and its derivatives:
Verhoef, J.C. Safe Mucosal Penetration Enhancers: A Potential excipients for peroral peptide delivery systems.
Fiction? Polymers as Absorption Enhancers for Trans- Int. J. Pharm. 2000, 194, 113.
mucosal Drug Delivery. In Controlled Drug Del. Designing 106. Bernkop-Schnurch, A.; Humenberger, C.; Valenta, C.
Technology for the Future; Park, K., Mrsny, R.J., Eds.; Basic studies on bioadhesive delivery systems for peptide
ACS Symposium Series 752, ACS: Washington, 2000; 25 and protein drugs. Int. J. Pharm. 1998, 165, 217225.
35. 107. Bernkop-Schnurch, A.; Walker, G. Multifunctional ma-
93. Thanou, M.; Verhoef, J.C.; Romeijn, S.G.; Nagelkerke, trices for oral peptide delivery. Crit. Rev. Ther. Drug Carr.
J.F.; Merkus, F.W.H.M.; Junginger, H.E. Eects of N- Syst. 2001, 18, 459501.
trimethyl chitosan chloride, a novel absorption enhancer, 108. Ho, N.F.H.; Barsuhn, C.L.; Burton, P.S.; Merkle, H.P.
on Caco-2 intestinal epithelia and the cilia beat frequency Mechanistic insights to buccal delivery of proteinaceous
of chicken embryo trachea. Int. J. Pharm. 1999, 185, 7382. substances. Adv. Drug Deliv. Rev. 1992, 8, 197235.
660 Illum and Davis
109. Senel, S.; Hincal, A.A. Drug permeation enhancement via Protective diphtheria immunity by unilateral prime-boost
buccal route: possibilities and limitations. J. Control. intranasal immunisation associated with restricted ipsi-
Release 2001, 72, 133144. lateral mucosal secretory IgA. J. Infect. Immun. 2003, 71,
110. Senel, S.; Kremer, M.; Nagy, K.; Squier, C. Delivery of 726732.
bioactive peptides and proteins across oral (buccal) 123. McNeela, E.A.; Jabbal-Gill, I.; Illum, L.; Pizza, M.; Rap-
mucosa. Curr. Pharm. Biotechnol. 2001, 2, 175186. puoli, R.; Podda, A.; Lewis, D.J.M.; Mills, K.H.G. Intra-
111. Squier, C.A.; Lesch, C.A. Penetration pathways of dierent nasal vaccination with genetically detoxied diphtheria
compounds through epidermis and oral epithelia. J. Oral toxin (CRM197) induces antigen-specic T cell response
Pathol. 1988, 17, 512516. in human volunteers: correlation between toxin neutraliz-
112. Squier, C.A.; Hall, B.K. Enhanced penetration of nitro- ing antibody and Th2 response. J. Infect. Immun. submitted
sonornicotine across oral mucosa in the presence of for publication.
ethanol. J. Oral Pathol. 1986, 15, 276279. 124. Westerink, M.A.; Smithson, S.L.; Srivastava, N.; Blonder,
113. Allemann, E.; Leroux, J.-C.; Gurny, R. Polymeric nano- J.; Coeshott, C.; Rosenthal, G.J. ProJuvant TM (Pluronic
and microparticles for the oral delivery of peptides and F127/chitosan) enhances the immune response to intra-
peptidomimetics. Adv. Drug Deliv. Rev. 1998, 34, 171 nasally administered tetanus toxoid. Vaccine 2002, 20, 711
189. 723.
114. Ponchel, G.; Irache, J.-M. Specic and non-specic bio- 125. van der Lubben, I.M.; Verhoef, J.C.; Borchard, G.;
adhesive particulate systems for oral delivery to the gastro- Junginger, H.E. Chitosan for mucosal vaccination. Adv.
intestinal tract. Adv. Drug Deliv. Rev. 1998, 34, 191219. Drug Deliv. Rev. 2001, 52, 139144.
115. Veuillez, F.; Kalia, Y.N.; Jacques, Y.; Deshusses, J.; Buri, 126. Calvo, P.; Remunan-Lopez, C.; Vila-Jato, J.L.; Alonso,
P. Factors and strategies for improving buccal absorption M.J. Chitosan and chitosan/ethylene oxide-propylene
of peptides. Eur. J. Pharm. Biopharm. 2001, 51, 93109. oxide block copolymer nanoparticles as novel carriers for
116. Morishita, M.; Barichello, J.M.; Takayama, K.; Chiba, Y.; proteins and vaccines. Pharm. Res. 1997, 14, 14311436.
Tokiwa, S.; Nagai, T. Pluronic F-127 gels incorporating 127. Calvo, P.; Remunan-Lopez, C.; Vila-Jato, J.L.; Alonso,
highly puried unsaturated fatty acids for buccal delivery M.J. Novel hydrophilic chitosan-polyethylene oxide nano-
of insulin. Int. J. Pharm. 2001, 212, 289293. particles as protein carriers. J. Appl. Polym. Sci. 1997, 63,
117. Gutniak, M.K.; Larsson, H.; Heiber, S.J.; Juneskans, O.T.; 125132.
Holst, J.J.; Ahren, B. Potential therapeutic levels of 128. Tobio, M.; Calvo, P.; Vila Jato, J.L.; Alonso, M.J. Chito-
glucagons-like peptide I achieved in humans by a buccal san coated PLGA nanoparticles: new vehicle for increasing
tablet. Diabetes Care 1996, 19, 843848. protein nasal absorption. Millennial World Congress of
118. Remunan-Lopez, C.; Portero, A.; Vila-Jato, J.L.; Alonso, Pharmaceutical Sciences, 2000; 69.
M.J. Design and evaluation of chitosan/ethylcellulose 129. Tobio, M.; Gref, R.; Sanchez, A.; Ianger, R.; Alonso, M.J.
mucoadhesive bilayered devices for buccal delivery. J. Stealth PLA-PEG nanoparticles as protein carriers for
Control. Rel. 1998, 55, 143152. nasal administration. Pharm. Res. 1998, 15, 270275.
119. Portero, A.; Remunan-Lopez, C.; Nielsen, H.M. The 130. Vila, A.; Sanchez, A.; Tobio, M.; Calvo, P.; Alonso, M.J.
potential of chitosan in enhancing peptide and protein Design of biodegradable particles for protein delivery. J.
absorption across the TR1146 cell culture modelAn in Control. Release 2002, 78, 1524.
vitro model of the buccal epithelium. Pharm. Res. 2002, 19, 131. van der Lubben, I.M.; Verhoef, J.C.; van Aelst, A.C.;
169174. Borchard, G.; Junginger, H.E. Chitosan microparticles for
120. Kremer, M.J.; Senel, S.; Wertz, P.W.; Hill, J.R.; Squier, oral vaccination: preparation, characterisation and pre-
C.A. Permeabilizing eect of dierent chitosans on drug liminary in vivo uptake studies in murine Peyers patches.
delivery across buccal mucosa. Proc. Int. Symp. Control. Biomaterials 2001, 22, 687694.
Release Bioact. Mater. 2001, 28, 315316. 132. van der Lubben, I.M.; Konings, F.A.J.; Borchard, G.;
121. McNeela, E.A.; OConnan, D.; Jabbal-Gill, I.; Illum, L.; Verhoef, J.C.; Junginger, H.E. In vivo uptake of chitosan
Davis, S.S.; Pizza, M.; Rappuoli, R.; Mills, K.H.G. A microparticles by murine Peyers patches: Visualization
mucosally delivered vaccine against diphtheria: Formula- studies using confocal laser scanning microscopy and
tion of cross reacting material (CRM 197) of diphtheria immunohistochemistry. J. Drug Target. 2001, 9, 3947.
toxin with chitosan enhances local and systemic antibody 133. van der Lubben, I.M.; Kersten, G.; Fretz, M.M.; Beuery,
and Th2 responses following nasal delivery. Vaccine 2000, C.; Verhoef, J.C.; Junginger, H.E. Chitosan microparticles
19, 11881198. for mucosal vaccination against diphtheria: oral and nasal
122. Mills, K.H.G.; Cosgove, C.; McNeela, E.A.; Sexton, A.; ecacy studies in mice. In Mucosal vaccine delivery with
Giemza, R.; Jabbal-Gill, I.; Church, A.; Lin, W.; Illum, L.; chitosan derived formulations. Ph.D. Thesis, Leiden Uni-
Podda, A.; Rappuoli, R.; Grin, G.E.; Lewis, D.J.M. versity, Leiden: The Netherlands, 2002; 6782.
28
Pharmaceutical Applications of Chitosan and Derivatives
M. Thanou
Cardi University, Cardi, United Kingdom
H. E. Junginger
Leiden University, Leiden, The Netherlands
I. INTRODUCTION Chitin is found in the exoskeleton of crustaceans, crab

and shrimp shells, insects, and fungal mycelia. The main
Chitin is a naturally occurring polysaccharide and its pro- sources of chitin are the shell wastes of shrimps, lobsters,
duction in biomass of up to 1012 tons/year makes it one and crabs, and chitin production is associated with food
of the most abundant polysaccharides on Earth [1]. Even industries such as shrimp canning. The isolation process
though the discovery of chitin and chitosan dates back includes: (1) treatment with 35% (wt/vol) aqueous NaOH
to the 19th century, it has been only in the last three dec- at 8090jC for a few hours to remove protein materials;
ades that this material has attracted the attention of bio- and (2) treatment with 35% (wt/vol) aqueous HCl to
medical scientists. remove inorganic materials (i.e., calcium carbonate). The
Chitin was discovered in 1811 by Braconnot [2] and resulting chitin is deacetylated by 40% sodium hydroxide
was initially termed fungine because it was discovered in at 120jC for 13 hr. This treatment produces 70% deacet-
mushrooms. It was later in 1823 that Odier gave the name ylated chitin [4].
chitin to the same material discovered now in the elytrum of In the last two decades, most forms of chitin, chito-
the cock chafer beetle based on the Greek term chitos, san, and their derivatives have found a number of phar-
meaning coat. maceutical or biomedical applications [58]. As a result, a
However, it was in 1859 that Rouget discovered chi- current search in scientic references databases for chito-
tosan after treating chitin with hot and concentrated alkali san applications in pharmaceutics can yield up to 1000
(KOH), proposing the term modied chitin. In a similar original studies where the abovementioned applications
work in 1894, Hoppe-Seyler, who ignored the work of have been investigated.
Rouget, named this polymer chitosan [2]. Even if chitosan has been an over-the-counter (OTC)
Nowadays, this popular term can lead to confusion if food supplement (mainly for weight loss) in the last years, it
someone considers that it represents a number of dierent is only this year that this polymer received limited approval
structural copolymers with dierent molecular weights from the Food and Drug Administration (FDA) as a
(Mw) and degrees of acetylation (DAs). In 1999, the newer hemostatic material in dressings for wounds.
term chitinosans was proposed by Rege et al. [3] to embrace With the recent advances in tissue engineering, drug
the spectrum of acetylated poly(N-glucosamines) although delivery, and gene delivery, chitin/chitosan and their deriv-
the name chitosans is strongly xed in the scientic nomen- atives will present a novel and useful class of biomaterials
clature and sometimes used to cover chitins, chitosans, and suitable for a number of applications. These molecules are
their derivatives. very versatile and small changes in their molecular struc-
Chitin and chitosan are presented in Fig. 1. Chitosan ture can bring large changes in their interactions with
is the deacetylated form of chitin, a (1!4)2-amino2- components of biological tissues or drugs. This, in combi-
deoxy-h-D-glucan. However, the term usually refers to nation with their inherent biocompatibility and signicant
a range of polysaccharides that dier in their degree of absence of cytotoxicity, makes these polymers (and in some
N-deacetylation (4098%) and average molecular weight cases oligomers) excellent candidates for applications in the
(Mw). biomedical eld.
661
662 Thanou and Junginger
Figure 1 Chemical structures of both chitin and chitosan.
This review aims to present the biological properties [17,18]. However, toxicity of chitosan might depend on
of chitosan, covering the number and types of pharmaceu- DA, Mw, purity, salt type, and the route of administration
tical applications that have been presented over the past [19,20].
years. In this aspect, we will present pharmaceutical appli- Intravenous administration of chitosan oligomers at
cations where chitin, chitosan, and their derivatives are 4.5 mg/kg body weight is well tolerated and no abnormal
investigated. Finally, we will highlight the elds that we physiological symptoms were observed after 11 days of
believe will emerge in the new era. treatment in mice. However, an increase in lysozyme
activity in the blood was observed to return to normal
values 5 days after treatment [21]. Lysozyme can hydrolyze
II. CHITINOUS MATERIALS AND chitosan in animals but its hydrolytic activity is minimal
PHYSICOCHEMICAL AND BIOLOGICAL when the DA is below 30% [22].
PROPERTIES The abovementioned properties suggest that chitosan
is a chemically versatile biomaterial that, when controlled
Chitosans physicochemical properties are strongly related
in silico, can lead to controllable biological eects in vitro,
with its biological properties. The ability of associating
which can guide the production of the desired bioactivity
with water and (poly)electrolytes plays an important role in
in vivo.
the formation of solutions or colloidal systems (hydrogels).
The main chemical characteristics that inuence its state
and its biological properties are DA and Mw [911]. III. CHITOSAN AS BIOMATERIAL IN WOUND
Chitosan is a weak base with a pKa value of 6.5 [2] HEALING
depending on the DA and Mw [12]. When the DA is below
25% chitosan is soluble at pH values between 2 and 6. N-acetylglucosamine is common structural unit in chitin
When DA is close to 50%, the polymer becomes soluble at and hyaluronic acid, a material necessary in wound repair.
any pH. At DA values over 60%, the polymer becomes It is therefore likely for the chitinous material to promote
completely insoluble in water and at pH above 6 due to the tissue regeneration and repair. The rst ndings on this
dierent hydrogen bonds between amide hydroxyl and application were reported in 1978 [23]. Up to now, there is a
ether groups [2]. Chitosan can form salts with inorganic substantial number of studies suggesting the benecial
and organic acids, which all give solutions of low pH value eects of chitinous materials on dierent types of wounds.
when dissolved in water. At such pH values, hydrogen This is probably the reason why the rst FDA approval of
bonding dissociates due to the protonation of amine chitosan is on this type of application.
groups. The most commonly used forms of chitosan salts Chitosan has been used as a wound-healing accelera-
in pharmaceutics are the glutamate and chloride salts [13]. tor in veterinary medicine. In the cellular level, it promotes
For polymeric chitosan, increases in Mw result in a the functions of inammatory cells such as polymorpho-
decrease of solubility. This is again due to the hydrogen nuclear leukocytes (PMNs), macrophages, and broblasts,
bonding within the polymeric chains [14]. On the contrary, resulting in an increase in granulation and organization
oligomeric chitosan with a degree of polymerization below [24]. Ueno et al. [25] have studied chitosan on experimental
8 is soluble at any pH and DA [2]. open skin wounds on the dorsal side of beagle dogs [25].
When derivatization on the functional groups of chi- Chitosan bers were applied for 15 days, and the process of
tosan (NH2 and CH2OH) occurs, more parameters are wound healing was evaluated histologically and immuno-
added to inuence solubility properties. The type of the histochemically. On day 3 postwounding, the chitosan-
substituent and the degree of substitution can inuence a treated wounds showed histologically strong inltration of
number of physicochemical and biological properties. PMN cells and an increase in eusion, whereas granulation
Chitosans biocompatibility was shown several times was more pronounced on days 9 and 15, accompanied by
in scientic reports, and there is evidence that it is biode- an increase of the production of type III collagen. The last
gradable [15,16]. It has been shown that it has low toxicity ndings were veried by the increase of osteopontin, a
in rabbits with LD50 of 16 g/day/kg (orally administered) ligand for the avh3 integrin found in PMNs [26]. It was also
Applications of Chitosan and Derivatives 663
observed that chitosan activates macrophage and IL-1 [35]. The reported antimicrobial properties of chitosans
production [24]. Similar observations were previously re- [36] appear to favor wound-healing applications [37,38].
ported for oligomeric chitosan where an in vitro stimu- These properties open the route to new applications of
latory eect on both macrophage nitric oxide (NO) chitinous materials in tissue regeneration and repair, and
production and chemotaxis was demonstrated. The macro- their potential use as coatings in biomedical devices by
phage NO secretion was attributed to the N-acetylgluco- functioning as inhibitors of biolm formation.
samine unit of the chitosan molecule rather than to the
glucosamine residue [27].
The benecial eect of chitin on wounds was presented
recently in the news. In Vietnam, a 3-year program of IV. CHITINOUS MATERIALS IN DRUG
clinical investigations showed that VinachitinR (wound DELIVERY
dressing) was highly successful in the treatment of burns Chitosans characteristics as biomaterial and polymer were
and scalds (http://vietnamnews.vnagency.com.vn/2002- employed in the eld of pharmaceutics and drug delivery.
09/14/Columns/). In the United States, the FDA approved Either as controlled-release matrix or functional biomate-
the application of chitosan bandage as a useful material to rial, chitosans have been investigated in a number of
stop bleeding in battleelds (http://www.hemcon.com// therapeutic delivery systems.
press.html). This limited approval was based on preclinical
studies in swine animals with liver injuries where chitosan
A. Controlled-Release Dosage Forms
was found to signicantly improve hemostasis [28]. The
hemostatic eect of chitosan has been previously presented Physicochemical properties of chitinous materials allow for
in a number of studies. Rao and Sharma [29] reported that interactions with a number of pharmaceutical excipients
in coagulation and hemagglutination tests, it was shown and drugs to give dosage forms with controlled-release
that the hemostatic mechanism of chitosan appeared to be characteristics. Such dosage forms may vary from tradi-
independent of the classical coagulation cascade, whereas tional tablets and granules to sophisticated particulate
interaction between the cell membrane of erythrocytes and formulations including sponges and gels. These delivery
chitosan took place [29]. systems should control the rate of administration at the site
In another study, microcrystalline chitosan (MCCh) of absorption and prolong the duration of the therapeutic
sealant installation (via an arterial sheath) at the comple- eects. These materials are designed for noninvasive drug
tion of catheterization improved hemostasis in dogs, and delivery, or as implantable and injectable systems. Sub-
signicant reductions in manual compression time of the stantial emphasis has been given in the design and charac-
artery after withdrawal of both the sheath introducer and terization of microspheres for specic therapeutic delivery.
the catheter were documented. Comparative results were Table 1 is a summary of the dosage forms in which chitosan
found in rats, wherein a created wound in the aorta could materials have been used.
be sealed relatively quickly and easily [30].
When chitosan (80% deacetylated, 20% DA) lm B. Dosage Forms for Peroral Administration
coating (10 nm) was exposed to serum or plasma, a weak
transient activation of the complement system and no Although chitosan has been mostly used as a diluent in
activation of the intrinsic pathway were observed. How- tablet manufacturing, it has been also proposed as a binder,
ever, upon further acetylation of the surface amino groups, lubricant, or potential disintegrating agent [7]. Sawayanagi
the polysaccharide turned into a strong activator of the et al. [39] used chitosan for the production of tablets with
alternative pathway of complement [10]. In the same study, sustained-release properties. The rate of release was related
it was presented that chitosan did not activate the intrinsic to the amount of chitosan used and a zero-order release
pathway of coagulation, which is in agreement with previ- prole was obtained [39].
ously reported data [29,31]. Chitosan granules containing indomethacin were
Chitosan has been suggested as a biomaterial in the found to maintain a plateau level in beagle dogs when
wound healing of articular cartilage [32]. When chitosan administered orally, in contrast to conventional indometh-
solution was injected into the rat knee articular cavity, it acin capsules that quickly reached the maximum level. The
provoked intra-articular brous tissue formation and authors suggested that this was due to the slow rate of
chondrocyte proliferation, and reduced the decrease in release and the longer residence time in the stomach [40]. It
epiphyseal cartilage thickness with time [32]. It is proposed is possible that when chitosan is in the stomach, it swells
that chitosan, being able to form insoluble ionic complexes and forms gels (low pH) that aggregate in the small
with negatively charged glucosaminoglucans (GAGs), can intestines (higher pH), entrapping the drug and forming
immobilize chondroitin sulphate molecules within the sustained-release particulates.
hydrogel, which can mimic the GAG-rich extracellular For the design of nifedipine prolonged-release dosage
matrix (ECM) of articular chondrocytes [33,34]. forms, a chitosan matrix was prepared where the drug was
However, a chitosan derivative (N,N-dicarboxymethyl embedded, forming microgranules or microbeads. A com-
chitosan) associated with bone morphogenetic protein parison between the two dosage forms showed that the
(BMP)-7 was also found to facilitate the repair of articular release rate of nifedipine from chitosan matrix was slower
cartilage lesions, probably due to a synergistic mechanism for beads than for granules. However, the amount and
Table 1 Application of Chitosan Materials in Conventional Dosage Forms

Dosage form Technological characteristics Routes of delivery
Tablets Directly compressed diluent Oral, buccal

Disintegrating agent Oral
Controlled release Oral, buccal
Capsules Film coating Colon
Granules, beads Matrix Oral
Particles, colloids Cross-linking Oral, implantables,
injectables, nasal, ocular
Film coating Liposomes, devices, capsules Oral, implants
Microspheres Cross-linking, interfacial Oral, implantables,
acylation, precipitation, injectables, nasal, ocular
spray drying, ionic gelation,
solvent evaporation
percentage of drug released were much higher in acidic after peroral administration. As shown from the few
medium than in phosphate buer [41]. abovementioned examples, dierent types of dosage forms
Henriksen et al. [42] suggested the technique of wet were designed and evaluated for controlled-release prop-
granulation to prepare chitosan oral formulations. The erties. It is expected that if the material gains full approval
authors compared chitosan malate and chitosan base on with a generally-regarded-as-safe (GRAS) status, such
their release characteristics of model drugs. They found that dosage forms will appear in the market.
theophylline granules containing from 25% to 75% chi-
tosan malate showed an increasing sustained-release eect C. Dosage Forms for Local Administration
when the chitosan malate salt content was increased [42]. in the Oral Cavity
The wet and dry states of chitosan blends with the-
ophylline were examined regarding their drug release rate The mucoadhesive properties of chitosan make it an
[43]. It was found that the lower the initial viscosity of the attractive material for the local delivery of drugs in the
polymer/drug blend in the wet state, the faster is the release oral cavity [46]. It has been shown in a number of studies
of theophylline. The swelling ability of the polymer greatly that chitosan can be easily formulated to provide pro-
inuences the release kinetics of the tablets prepared by longed delivery of antimicrobial or antifungal agents for
polymerdrug dry blending. Both types of tablets were periodontal diseases and mucosal lesions [47,48]. For this
rapidly disintegrated in acidic medium. The authors sug- purpose, chitosan was used alone or in combination with
gested the addition of alginate in the mixture to control other polymers (in lms, tablets, or microspheres) as
disintegration and release in the stomach [43]. dosage forms for the delivery of antimicrobial agents in
Chitosan has also been used for the formation of the oral cavity.
hollow gastroretentive microspheres [44]. The oating Combination of chitosan with alginate yielded bioad-
microcapsules containing melatonin were prepared by hesive tablets for intraoral drug delivery of ketoprofen [49].
ionic interaction of chitosan and a negatively charged The adhesion force of chitosan/alginate tablets was com-
surfactant, sodium dioctyl sulfosuccinate. These micro- parable to that of AftachR. After sublingual administra-
capsules showed near zero-order kinetics in simulated tion in rabbits, ketoprofen was rapidly absorbed without
gastric uid accompanied by greatly retarded release last- an initial sharp peak, with plasma concentration showing a
ing for several hours. sustained release 3 hr after administration.
To develop a stomach-specic drug delivery system Incorporation of anionic polymers such as alginates in
able to increase the ecacy of tetracycline against Helico- chitosan-consisting formulations promotes controlled-re-
bacter pylori, chitosan microspheres were prepared [45]. lease characteristics probably due to the formation of
Microspheres with a spherical shape and an average interpenetrating networks.
diameter of 2.03.0 Am were formed. When the drug was Remunan-Lopez et al. [50] prepared chitosan bilayer
added to the polymer solution before cross-linking, only a devices (mucoadhesive and backing layer) with or without
small drug amount (8% wt/wt) was optimally incorpo- the incorporation of an anionic cross-linking polymer
rated in the nal microsphere formulation. However, (polycarbophil, sodium alginate, and gellan gum). It was
when the drug was incubated with the preformed micro- shown that the uncross-linked chitosan-containing devices
spheres, a maximum amount of 69% (wt/wt) could be absorbed a large quantity of water, gelled, and then eroded,
loaded. The drug was stable for up to 12 hr even under allowing drug release. The lms showed a sustained drug
highly acidic condition. release. Tablets displayed controlled swelling and drug
Chitosans pH-dependent solubility has been em- release. These tablets were produced by in situ cross-linking
ployed in the design of formulations for delivery of drugs the chitosan with polycarbophil.
Cross-linking of chitosan was used to prepare erodible was associated with chitosan in a complex 1:1 ratio based
controlled-release systems [51]. Samples of cross-linked on charge and was formed between ammonium ions on the
palmitoyl glycol chitosan (GCP) with dierent hydropho- chitosan and SO3 groups on heparin.
bicities were prepared by freeze drying an aqueous disper- Chitosan (26% DA) microspheres containing mitox-
sion of the polymer with three dierent model drugs antrone were prepared for intramuscular implantation
varying in size and hydrophilicity. Hydration and erosion [59]. These microspheres were especially designed to slowly
were governed by the hydrophobicity of the gel and the release the drug. They were prepared using glutaraldehyde
presence of the amphiphilic additives. All gels were found for the cross-linking of chitosan in a mixture of apolar
to be mucoadhesive. solvents with a surfactant. Further processes included
The authors further used this system to deliver a model hardening of the spherical particles. Drug release was
hydrophobic drug, denbufylline, in the oral cavity of found to be eectively controlled by the extent of cross-
rabbits [52]. Denbufylline was detected 0.5 hr after dosing linking and only about 25% of the incorporated drug was
with the GCP formulation and delivery was sustained for at released over 36 days. It was shown that the microspheres
least 5 hr after dosing. were well tolerated by the living tissues after implantation
In some cases, chitosan was used as tablets composed in the skeletal muscles of rats. The authors did not observe
by microspheres [53] or lms [54] for the local delivery of signicant biodegradation of the material over a period of 3
chlorhexidine. These formulations improved dissolution of months. This can be explained by the fact that the highly
chlorhexidine, diminished adhesion of Candida albicans, deacetylated chitin used in this study is unlikely to be
and gave a prolonged release of the drug in the oral cavity. degraded by enzymes (lysozyme). Nevertheless, the later
Chitosan was investigated in lm dosage forms for delivery ndings of the negative eects of glutaraldehyde on cell
into the periodontal pocket. The emulsication/casting/ viability hindered the further application of glutaraldehyde
evaporation technique was used to incorporate poly(D,L- cross-linked microspheres.
lactide-coglycolide) (PLGA) micromatrices of ipriavone A thermosensitive solution (at 4jC) that gels at 37jC
in a chitosan lm [55]. In vitro release experiments demon- was prepared after addition of glycerophosphate in chito-
strated that such micromatricial lm systems can release san [60]. This system was shown to sustain the release of
the drug over a period of 20 days. macromolecules over a period of several hours to a few
With the aim of improving bone regeneration, Park et days. However, the release of small Mw drugs was com-
al. [56] developed chitosan sponges containing platelet- pleted in 24 hr. In order to improve the release of such
derived growth factor-BB (PDGF-BB). These sponges drugs, the authors incorporated liposomes that could delay
were formed when chitosan solution was freeze-dried, the release [61]. It was observed that the gelation rate and
cross-linked, and freeze-dried again. It was shown that gel strength increased upon liposome addition. An in vitro
release rate of the growth factor could be controlled. These release of a model compound that lasted for 2 weeks
chitosan sponges demonstrated marked increase in new was achieved.
bone formation and rapid calcication. Histomorphomet- The delivery of cytokines to the central nervous system
ric analysis conrmed that PDGF-BB-loaded chitosan is a challenge in the development of therapies for chronic
sponge signicantly induced new bone formation. In this degenerative diseases. To deliver hematopoietic cytokine
study, the physicochemical (hydrogel and formation of a colony-stimulating factor 1 (CSF-1) to the central nervous
porous matrix) and biological properties (bioadhesion and system, Maysinger et al. [62] investigated chitosan as
biocompatibility) were combined in a dosage form to encapsulating material of rhCSF and broblast-like LM-
enhance periodontal bone regeneration. 10 cells that produce CSF-1. These microcapsules were
implanted in mutant mice that lacked systemically bioac-
tive CSF-1, following cerebral cortex ischemic lesion either
V. TECHNOLOGIES FOR INJECTABLE in the lesion or into the peritoneum. The best level of cell
CHITOSAN DRUG DELIVERY SYSTEMS viability after encapsulation was achieved with highly
viscous chitosan. It was also found that rhCSF incorpo-
Chitosan has been suggested to be biocompatible and not rated into chitosan biodegradable spheres could be re-
to elicit adverse eects when administered by injections [57] leased and retain its biological activity. Additionally,
either intravenously (i.v.), intraperitoneally (i.p.), or as microencapsulated LM-10 cells that produce CSF-1 could
implant. Regarding toxicity, chitosan type has been pre- survive and constitutively release CSF-1 in alginatechito-
sented to be important when injected [19,20]. However, a san spheres for dierent lengths of time depending on the
number of studies were presented, where scientists used physical properties of the chitosan used [62].
chitosan in combination with other technologies to create Chitosan glycerophosphate salts were used for injec-
biocompatible dosage forms for prolonged release of drugs. tion in vivo for encapsulation of chondrocytes and bone
Gallo and Hassan [58] reported in 1988 the prepara- protein [63]. The solution was injected subcutaneously at
tion of chitosan magnetic microspheres to use the advan- the dorsal part of rats. The presence of dierentiating
tage of physical localization of drug release. The authors cartilage and bone tissue from rat explants indicated that
used chitosan, with the aim to bind onto anionic glycos- gels could deliver bone protein, leading to de novo cartilage
aminoglycan receptors on the surface of capillary endothe- and bone formation in an ectopic site. In the same study,
lial cells. As an in vitro GAG, heparin was used. Heparin chondrocytes were cultured with the thermosensitive gel
and injected subcutaneously in mice. After 3 weeks, the administration. Whereas toxicity is a concern, still it is
implants were removed and analysis showed several areas not clear which types of chitosans can be administered and
of chondrocytes secreting a matrix characteristic of normal by which route. From a technological point of view, this
cartilage, suggesting that chitosan/polyol thermosensitive polysaccharide can serve as a platform for the controlled
gels present a suitable material in drug delivery using delivery of drugs in the systemic circulation or internal
implants. To minimize doxorubicin-related cardiotoxicity, organs. It is important that clearance, metabolism, and
Mitra et al. [64] prepared chitosan nanoparticles contain- chronic toxicity should be studied extensively before fur-
ing dextrandoxorubicin conjugate. The size of these nano- ther development of chitosan injectable dosage forms. In
particles was found to be 100-nm diameter, which favored this aspect, modied chitosans could be developed with
the enhanced permeability and retention (EPR) eect minimal toxicity although degradation and elimination
observed in solid tumors. Using this eect, it is possible would be still issues.
to achieve passive tumor targeting. Results from studies in
mice bearing implanted macrophages tumor cells suggested
that encapsulation of the conjugate in nanoparticles not
only reduced the side eects but also improved its thera- VI. CHITOSAN AS A FUNCTIONAL MATERIAL
peutic ecacy in the treatment of solid tumors as indicated IN DRUG DELIVERY SYSTEMS
by the prolonged survival time and the decrease of tumor
As mentioned, chitosan attracted interest in biomedical
size [64].
applications in just the last three decades. This coincides
In the eld of cancer treatment using polymer drug
with signicant progress in biotechnology and the begin-
conjugates, chitosan has been investigated in the form of
ning of the genomics era. Novel therapeutics have been
water-soluble derivatives that allowed the formation of
developed and a number of novel drug delivery systems
such conjugates. Initially, chitosan was modied chemical-
have been proposed. Such therapeutics could fall into the
ly to give N-succinyl-chitosan, which was shown to have
category of macromolecules. The majority of these are
low toxicity and biodegradability [65]. Conjugates of this
either susceptible to degradation in body uids, or charac-
derivative with mitomycin [66] showed good pharmacoki-
terized by poor permeability across biological membranes.
netic prole in rats and an inhibition of the growth of B16
In most cases, both challenges occur, and the material in
melanoma tumor after i.p. administration. Results from
question must both protect the macromolecule and facili-
biodistribution studies using uorescein isothiocyanate-
tate its passage across membranes. Chitosan and its mod-
labeled succinylated chitosan showed that in tumor-bear-
ication have been investigated in most of the modern drug
ing mice, the polymerdrug conjugate was eliminated
delivery systems (Table 2). It has been found that these
faster compared to normal mice. Distribution of the deriv-
materials can facilitate the delivery of therapeutic peptides,
ative in the tumor reached 10% of the dose per gram at 48
proteins, and nucleic acids.
hr after i.v. injection [67]. To increase specic tumor uptake
and targeting, the authors introduced the lactosaminated
N-succinyl chitosan that bears lactose groups, which is able A. Mucoadhesion, Permeation Enhancement, and
to interact with the asialoglycoprotein receptor of the liver Enzyme Inhibition Properties for the Delivery
[68]. Conjugates of the lactose-bearing chitosan derivative of Hydrophilic Macromolecular Drugs
with mitomycin were investigated for liver targeting in the
early metastatic phase of liver cancer. It was observed Macromolecular drugs such as peptides and proteins are
though that the targeting moiety did not improve the eect characterized by susceptibility to enzymatic degradation
of highly succinylated chitosan mitomycin conjugate [69]. and poor permeation across membranes. In the last dec-
The abovementioned examples show the importance ade, the delivery of peptides and proteins across mucosal
of well-characterized chitosan materials for parenteral membranes appeared challenging for a number of scien-
Table 2 Application of Chitosan Materials in Drug Delivery Systems

Delivery system Characteristics Application
Polyelectrolyte complex Counterion plasmid DNA Nucleic acid delivery
DNA vaccine Oral mucosal vaccination
Microencapsulated cells rhCSF-1 Nerve growth
Solutions, gels Peptide protein delivery, Oral, nasal
Microparticles vaccination
Nanoparticles Peptide protein delivery Vaccination, delivery to the
ocular surface
Gels Implants, coatings Tissue engineering
Micelles Chemical modication Delivery of lipophilic agents
tists. The delivery material to be used should ideally: (1) be labelled dextran (Mw 4400 Da) in vitro in Caco-2 cells.
mucoadhesive to increase the residence time at the site of No eect on the permeability of the monolayer could be
absorption; (2) protect the peptidic drug from degrada- observed, indicating that at this pH value, chitosan is not
tion; and (3) increase its permeation across mucosal or eective as permeation enhancer [76].
cell membranes. Two chitosan salts (hydrochloride and glutamate)
The bioadhesive properties of chitosan were described were studied for their ability to enhance the transport of
14
by Lehr et al. [70], demonstrating, in studies using pig C mannitol across Caco-2 cell monolayers at two pH
intestinal mucosa, that chitosan in swollen state is an values, 6.2 and 7.4. At low pH value, both chitosans
excellent mucoadhesive suitable for repeated adhesion. showed a pronounced eect on the permeability of the
The authors also described that chitosan underwent min- marker, leading to 25-fold (glutamate salt) and 36-fold
imal swelling in articial intestinal uid due to its poor (hydrochloride salt) enhancement. At pH 7.4 though,
aqueous solubility at neutral pH values, proposing that both chitosans failed to increase the permeability due to
substitution of the free NH2 groups with short alkyl solubility problems. The authors concluded that there was
chains should change the solubility and therefore the a need for chitosan derivatives with increased solubility for
mucoadhesion prole. use as absorption enhancers in more basic environments
In 1994, protonated soluble chitosan was found to such as large intestines or colon [77].
enhance the paracellular permeability [71] of markers Chitosan and chitosan salts lack the advantage of
across Caco-2 intestinal cell monolayers, indicating a good solubility at neutral pH values. Chitosan aggregates
promising solution to oral peptide and protein delivery. in solutions at pH values above 6.5, and only protonated
The inuence of chitosans DA and Mw was also chitosan (i.e., in its uncoiled conguration) can trigger the
investigated on the permeability of Caco-2 cell intestinal opening of the tight junctions, thereby facilitating the
monolayers. Schipper et al. [72] investigated the eect of paracellular transport of hydrophilic compounds. This
chitosan solutions at pH 5.5 on the permeability of the property implies that chitosan can be eective as an
nonabsorbable paracellular marker 14C mannitol and in- absorption enhancer only in a limited area of the intestinal
tracellular dehydrogenase activity. It was found that chi- lumen where the pH values are close to its pKa. For this
tosans with a high degree of deacetylation were eective as reason, chitosan and its salts may not be a suitable carrier
permeation enhancers at low and high molecular weights for peptide drug delivery to specic sites of the intestine,
but showed a clear dose-dependent toxicity, whereas chi- for instance the jejunum or ileum. To overcome this
tosans of low degree of deactylation were eective at high problem, the chitosan derivative N,N,N-trimethyl chitosan
molecular weight only, showing low toxicity [72]. chloride (TMC) has been synthesized and characterized
Chitosans ability to bind to epithelial Caco-2 cell (Fig. 2). This partly quaternized chitosan shows higher
monolayers was further investigated. It was found that aqueous solubility than chitosan in a much broader pH
low and high Mw chitosans bound tightly to the epithelium, and concentration range. The reason for this improved
inducing a redistribution of F-actin and the tight junctions solubility is the substitution of the primary amine with
protein ZO-1. No intracellular uptake of chitosan was methyl groups and the prevention of hydrogen bond
observed. It was also shown that these eects were medi- formation between the amine and the hydroxylic groups
ated through chitosans cationic charges because addition of the chitosan backbone.
of the highly anionic heparin in the study solution inhibited TMC was further investigated for permeation-en-
permeation-enhancing eects [73]. hancing properties and toxicity using the Caco-2 cells as
Dodane et al. [74] investigated the eect of chitosan a model for intestinal epithelium. Initially, a trimethylated
(degree of deacetylation 80%) solutions at pH 6.06.5 chitosan having degree of trimethylation 12% (dimethyl-
on the structure and function of Caco-2 cell monolayers. ation 80%) was tested. The eect of this partially quater-
Using a series of uorescent microscopy techniques, the nized TMC on the permeability of intestinal epithelial cells
authors were able to show that chitosan had a transient was studied. This polymer (1.52.5% wt/vol, pH 6.7)
eect on the tight junctions permeability and that viability caused large increases in the transport rate of 14C mannitol
of the cells was not aected. However, it was also shown (3260 fold), uorescently labeled dextran 4400 (167373
that chitosan treatment slightly perturbed the plasma fold), and buserelin (2873 fold). Confocal laser scanning
membrane, yet this eect was shown to be reversible [74]. microscopy (CLSM) conrmed that N-trimethyl chitosan
Chitosan gels were rst tested in vivo for their ability chloride opens the tight junctions of intestinal epithelial
to increase the intestinal absorption by Luessen et al. [75]. cells to allow increased transport of hydrophilic com-
The enhancement in absorption of the peptide analog, pounds through the paracellular transport pathway. No
buserelin, using chitosan solution at pH 6.7, was studied intracellular transport of the uorescent marker could be
after intraduodenal administration in rats. Chitosan was observed [78].
found to substantially increase the bioavailability of the Chitosan HCl and TMCs of dierent degrees of
peptide (f5.1%) in comparison to control (no polymer) or trimethylation were tested for enhancing the permeability
Carbopol 934P (polyacrylate) formulations. of the radiolabelled marker 14C mannitol in Caco-2 intes-
Borchard et al. [76] investigated chitosan glutamate tinal epithelia at neutral pH values (pH z 7.2). Chitosan
solutions at pH 7.4 for their eect in increasing the para- HCl failed to increase the permeation of these monolayers
cellular permeability of 14C mannitol and uorescently and so did TMC with a degree of methylation of 12.8%.
Figure 2 Chemical structure of trimethyl chitosan. Degrees of trimethylation and dimethylation vary.
However, TMC with a degree of trimethylation of 60% with or without TMC60 (pH = 7.2) were compared with
increased signicantly the 14C mannitol permeability chitosan dispersions at neutral pH values after intraduo-
across Caco-2 intestinal monolayers, indicating that a denal administration in rats. A remarkable increase in
threshold value at the charge density of the polymer is buserelin serum concentrations was observed after coad-
necessary to trigger the opening of the tight junctions at ministration of the peptide with TMC60, whereas buserelin
neutral pH values [79]. alone was poorly absorbed. In the presence of TMC60,
TMC polymers were investigated to see if they pro- buserelin was rapidly absorbed from the intestines having
voke cell membrane damage on Caco-2 cell monolayers Tmax at 40 min, whereas chitosan dispersion (at pH 7.2)
while enhancing the transport of hydrophilic macromole- showed a slight increase in buserelin absorption compared
cules. Using cell membrane-impermeable uorescent to the control, but it did not manage to increase buserelin
probes and CLSM, it was visualized that TMC polymers concentrations to the levels achieved with TMC60. The
widen the paracellular pathways without cell membrane absolute bioavailability of buserelin after coadministration
damage [80]. From microscopy visualization studies, it with TMC60 was 13.0% [81]. Similar to the buserelin
appears that the mechanism of opening the tight junctions studies, octreotide absorption after intrajejunal adminis-
is similar to that of protonated chitosan. Because of the tration was substantially increased, resulting in peptide
absence of signicant cytotoxicity, TMC polymers (partic- bioavailability of 16% [82]. In pigs, octreotide oral bio-
ularly with high degree of trimethylation) appear to be safe availability increased to 25%, indicating that substantial
absorption enhancers for improved transmucosal delivery intestinal absorption of peptide analogs can take place
of peptide drugs. Highly quaternized TMC polymers were when the therapeutic is coadministered with the trimethyl-
studied for their ability to increase the paracellular perme- ated material.
ation of peptide drugs across intestinal monolayers in vitro. These ndings verify the hypothesis that TMC, being a
Caco-2 cell monolayers were used as an intestinal epithe- soluble polymer, is ecient in increasing the absorption of
lium model to investigate the ability of two TMC polymers hydrophilic compounds at neutral pH values similar to the
with degrees of substitution of 40% and 60% to increase values found at the jejunum of the small intestines. From
the paracellular transport of the peptide drug buserelin at mucoadhesion studies using pig intestinal mucosa, it was
neutral pH value. Both TMCs managed to signicantly found that TMC at pH 7 showed similar adhesion strength
increase the transport of buserelin compared to the control. as chitosan of pH 6 (unpublished data). Therefore, it is
However, the eect of TMC60 was more prominent. unlikely for this eect of permeation enhancement to be
TMC60 increased the transported amount of the peptide attributed to dierences in mucoadhesion. From in vivo
up to 6% of the apically applied buserelin [81]. The eect of studies in rats and pigs, it is clear that the chitosan and the
TMC60 concentration on the transport of hydrophilic quaternized modication are ecient absorption
compounds was investigated using the somatostatin ana- enhancers of such peptide analog drugs.
log, octreotide, at neutral pH values (7.4). In the presence In the abovementioned studies, peptide analogs were
of TMC60, octreotide permeation showed a TMC concen- used. Such analogs are designed to resist degradation and
tration-dependent increase, indicating a specic interaction appear rather stable against the proteolytic activity of the
of the cationic polymer with components of the tight gastrointestinal tract enzymes. However, a number of
junctions, nonsaturated within the concentration range peptides and proteins (e.g., insulin) can rapidly degrade
used (0.251.5% wt/vol) [82]. in the hostile environment of the intestinal lumen where the
The eects of TMC60 polymers were studied in vivo in secretion of serine proteases is abundant.
rats, using peptide drugs that nd application in clinical Bernkop-Schnurch et al. introduced the idea of cova-
use, administered mainly subcutaneously and/or nasally. lent attachment of enzyme inhibitors as and/or complexing
Buserelin (pI = 6.8) and octreotide (pI = 8.2) peptide agents at the 2-position of chitosan to create polymers that
analogs were used for this purpose. Buserelin formulations exhibited inhibitory properties. Chitosan was covalently
bound with ethylenediaminetetraacetic acid (EDTA) to rats and sheep. The mechanism of action of chitosan was
prepare a material with combined mucoadhesive and en- suggested to be a combination of bioadhesion and a
zyme-inhibitory properties [83]. The inhibitory eect of this transient widening of the tight junctions in the membrane.
polymer conjugate was evaluated by using leucine enkeph- Aspden et al. [91]. investigated chitosan salts of dierent
alin (Leu enkephalin) as a model drug cleaved by amino- Mw and degree of deacetylation for their ability to enhance
peptidase N. The chitosanEDTA conjugate was found to nasal absorption of insulin. All formulations produced
be able to bind zinc, an essential cofactor for aminopepti- signicant decreases in blood glucose levels. Additionally,
dase N, and enzyme activity could be completely inhibited it was shown using the rat nasal perfusion method that
under the use of 1.0% chitosanEDTA conjugate. The chitosan salts produced minimal membrane and cellular
novel polymer conjugate was more bioadhesive than un- damage when compared to Leureth-9 solution [91].
modied chitosan and is easily hydratable in water and Chitosans eect in nasal delivery is primarily attrib-
basic aqueous solutions exhibiting quick swelling proper- uted to its mucoadhesive properties that can prolong
ties. Antipain, a trypsin inhibitor, was covalently attached residence time in the nasal cavity. In a study performed
to chitosan [84]. In contrast to the unmodied polymer, with human volunteers, it was shown that chitosan delivery
chitosanantipain conjugates exhibited a signicant inhib- systems can reduce the rate of clearance from the nasal
itory eect toward enzymatic activity of trypsin. Based on a cavity, thereby increasing the contact time of the delivery
chitosanantipain conjugate, a drug delivery system was system with the nasal mucosa [92].
generated using insulin as model drug. Tablets containing This property has been employed in the development
the polymer conjugate demonstrated, after incubation with of a chitosan nasal delivery system of a small polar drug
trypsin, minor proteolysis of matrix-embedded insulin such as morphine. In a recent phase I study, chitosan
compared to tablets lacking the polymer conjugate. morphine nasal formulations have been tested in healthy
In an attempt to improve the inhibitory property of volunteers in comparison with a slow i.v. infusion of
chitosan conjugates, the authors introduced the multicon- morphine to achieve rapid and ecient pain relief. The
jugation approach [85]. The serine protease inhibitors results showed that the nasal formulation was rapidly
antipain, chymostatin, and elastatinal were covalently absorbed with a Tmax of 15 min or less, and a bioavailability
linked to chitosan (poly-1,2-h-D-glucosamine). Thereafter, of nearly 60%. The shape of the plasma prole for nasal
the complexing agent EDTA was bound to the remaining delivery of the chitosanmorphine formulation was found
primary amino groups of the polymer. The inhibitory eect similar to the one obtained for the slow i.v. administration
of the resulting polymer conjugate toward trypsin, chymo- of morphine. Furthermore, the metabolite prole obtained
trypsin, elastase, carboxypeptidase, carboxypeptidase B, after the nasal administration of the chitosanmorphine
and aminopeptidase N, as well as its mucoadhesive prop- nasal formulation was identical to the one obtained for
erties, were evaluated in vitro. It was found that the novel morphine administered by the intravenous route, indicat-
polymer conjugate exhibited adhesive force lower than that ing that nasal delivery of morphine using chitosan can be a
of unmodied chitosan. However, the polymer conjugate good alternative to i.v. injections [93].
showed a strong inhibitory activity toward all tested serine Chitosan powder or solution formulation has been
proteases. Due to its additional high binding anity shown to be very eective in delivering peptides such as
toward bivalent metal ions, it also inhibited the Zn2+- leuprolide (1300 Da), salmon calcitonin (3500 Da), and
dependent exopeptidases carboxypeptidase A and B, and parathyroid hormone (PTH) (4000 Da) across the nasal
aminopeptidase N. membrane, and nasal bioavailabilities of around 20% have
These chitosan conjugates have been presented as been obtained in clinical trials, indicating that such peptide
potential multifunctional polymers in oral peptide and delivery systems soon will be in the market [94]. In a
protein delivery [86,87]. number of studies, it has been shown that microspheres
Regarding oral delivery of macromolecules, chitosan (nanoparticles of powders) provide improved absorption
has been used in combination with liposomes. Multilamel- compared to solution [94].
lar liposomes coated with chitosan were found to be highly Insulinchitosan nanoparticles were prepared by the
adhesive in the rat intestines. The same system containing ionotropic gelation of chitosan glutamate and tripolyphos-
insulin was administered to normal rats. The blood glucose phate and by simple complexation of insulin. These nano-
level of rats was found to be signicantly decreased after particles were compared with chitosan solution and
administration of the chitosan-coated liposomes contain- chitosan powder for their eciency to increase insulin
ing insulin. The lowered glucose level was maintained for nasal absorption in anesthesized rats and/or in conscious
more than 12 hr after administration of the liposomal sheep. Insulinchitosan nanoparticle formulations pro-
insulin, which suggested mucoadhesion of the CS-coated duced a pharmacological response in the two animal
liposomes in the intestinal tract of the rats [88,89]. models, although in both cases the response in terms of
As an alternative to the oral route, the nasal mucosa lowering the blood glucose levels was less than that of the
can provide a route of delivery where the proteolytic nasal insulinchitosan solution formulation. The insulin
barrier appears to be of substantially lower signicance. chitosan solution formulation was found to be signicant-
Illum et al. [90] described that chitosan solutions at ly more eective than the complex and nanoparticle for-
0.5% (wt/vol) concentration were highly eective at in- mulations. However, in the sheep model, the most eective
creasing the absorption of insulin across nasal mucosa in chitosan formulation for nasal insulin absorption was a
chitosan powder delivery system with a bioavailability of Mw and DA. A chitosan sample with a lower Mw and a
17.0% as compared to 1.3% and 3.6% for the chitosan higher DA would be a more susceptible substrate and
nanoparticles and chitosan solution formulations, respec- would be a more suitable candidate for use in colon drug
tively [95]. delivery [101]. Dosage forms using chitosan for colonic
Chitosan was examined on airway epithelial cells for targeting have been reported in the last decade. Tozaki et
protein release, relative bioadhesive properties, and induc- al. [102] reported chitosan capsules for colonic delivery of
tion of cytokine release from respiratory epithelium. Chi- insulin. In vitro drug release experiments from chitosan
tosan microparticles containing bovine serum albumin capsules containing carboxyuorescein showed that the
(BSA) were prepared by spray drying. Chitosan micro- probe was not released in articial gastric juice (pH 1) or
particles were found to adhere onto polarized Calu-3 cells in articial intestinal juice (pH 7). However, the release of
(used as a model for nasal epithelia) and enhanced protein the probe was markedly increased in the presence of rat
transport across the cells. A mixture of chitosan micro- cecal contents. In agreement with the release proles, a
particles with lysophosphatidylcholine increased protein marked absorption of insulin and a corresponding de-
transport further. Microparticles applied apically induced crease in plasma glucose levels were observed following
the basolateral release of IL-6 and IL-8 from polarized the oral administration of these capsules in combination
Calu-3 cells. However, release of other cytokines, such as with sodium glycocholate, a permeation enhancer. The
IL-1h, tumor necrosis factor (TNF)-a, granulocytemac- hypoglycemic eect started from 8 hr after the adminis-
rophage colony-stimulating factor (GM-CSF), and trans- tration of chitosan capsules when the capsules entered the
forming growth factor (TGF)-h, were not aected by an colon, as evaluated by the transit time experiments with
apical exposure to polymer formulations [96]. chitosan capsules.
The mucoadhesion, biodegradability (lysozyme), and The capsules were further developed and tested for the
physicochemical (viscoelasticity) properties of chitosan colon-specic delivery of R68070, a new thromboxane
appear to be valuable in ocular drug delivery [7,97]. synthase inhibitor [103], and 5-aminosalicylic acid (5-
Chitosans gels displayed excellent tolerance after admin- ASA) [104] as anti-inammatory drugs for the therapy of
istration on the corneal surface and it was shown that ulcerative colitis.
corneal residence time increased by threefold compared to Chitosan microcores entrapped within acrylic micro-
commercial collyrium [98]. spheres were presented as colon-specic delivery system for
The potential of chitosan nanoparticles as a vehicle for the delivery of sodium diclofenac [105]. The chitosan
the improvement of the delivery of cyclosporin A to the entrapped the drug and then microencapsulated into
ocular mucosa for the treatment of local diseases was Eudragit L-100 and Eudragit S-100 using an oil-in-oil
investigated. In vivo experiments showed that, following solvent evaporation method. It was found that chitosan
topical instillation of nanoparticles to rabbits, it was Mw inuenced the release of the drug from the microcores.
possible to achieve therapeutic concentrations in external Furthermore, by coating the microcores with Eudragit,
ocular tissues (i.e., cornea and conjunctiva) during at least perfect pH-dependent release proles were attained. No
48 hr while maintaining negligible or undetectable cyclo- release was observed at acidic pH values; however, when
sporin A levels in the inner ocular structures blood and reaching the Eudragit pH solubility, a continuous release
plasma. These levels were signicantly higher than those for a variable time (812 hr) was achieved [105].
obtained following instillation of a chitosan solution con- Following the same rationale of drug pH-dependent
taining the peptide drug and an aqueous cyclosporin A release, chitosan powder was dispersed on aminoalkyl
suspension [99]. methacrylate copolymer RS (Eudragit) RS [106]. To obtain
As shown, chitosans have been intensively investigated the bifunctional releasing characteristics, such as time
for the delivery of macromolecular and hydrophilic thera- dependency and site specicity, capsules containing acet-
peutics. Based on mucoadhesive and permeation-enhanc- aminophen as active ingredient were coated by the chitosan
ing properties, chitosans have been used unmodied or as dispersed on hydrophobic polymer. It was noted that the
derivatives to provide a range of multifunctional materials release rate could be controlled by changing the thickness
for the delivery of peptides and proteins at mucosal sites of the layer. Enteric-coated chitosan-dispersed hydropho-
of absorption. bic polymer capsules reached the large intestines within 13
hr after oral administration and were degraded in the colon
B. Chitosan Delivery Systems for Colon Targeting of beagle dogs [106].
Proteolytic enzyme activity in the colon is signi-
It was recently suggested that chitosan is susceptible to cantly lower compared with that of the small intestines.
degradation by the microora available in the colon Colon targeting has been suggested for the delivery of
[100,101]. Colonic bacterial enzymes, such as glycosidases, macromolecular drugs. For this multiparticulate system
are responsible for the hydrolysis of disaccharides, oligo- of chitosan, hydrogel beads have been investigated for
saccharides and polysaccharides. These are produced colon-specic delivery [107]. The hydrogel bead was
by the anaerobic bacteria in the human colon, of which formed by polyelectrolyte complexation of chitosan with
Bacteroides and Bidobacteria are the predominant spe- its counterion, tripolyphosphate. Results showed that
cies. The susceptibility of chitosan to degradation by the hydrogel beads were degraded by rat cecal and
rat bacterial enzymes was found dependent on both its colonic enzymes, resulting in a marked acceleration in
the release of the uorescently labeled BSA, a model studies in mice demonstrated that uorescently labeled
protein [107]. chitosan microparticles are taken up by the epithelium of
murine Peyers patches, an essential step in oral vaccina-
C. Mucosal Vaccination Using Chitosan Solutions tion [111].
and Microparticulate Forms Chitosan microparticles were further tested for their
potential to enhance both the systemic and local immune
Mucosal vaccination presents the advantage of inducing responses against DT after oral and nasal administration in
the production of local antibodies at the sites where mice. Compared to intragastric feeding with DT in solu-
pathogens enter the body. For this application, vaccines tion, a strong enhancement of the systemic and local
are coadministered with penetration enhancers and adju- immune responses against DT was found in mice fed with
vants, or encapsulated in particles. In the last years, two DT-loaded chitosan microparticles. The immune reaction
major sites for vaccination have been investigated: the was found to be strongly dependent on the DT dose
nasal and oral mucosae. Emphasis was given on the associated with the microparticles. Signicant systemic
preparation of particles that could be loaded eciently humoral immune responses were also found after nasal
with the vaccine. vaccination with DT associated with chitosan micropar-
A number of particles have been prepared to deliver ticles [112].
vaccines. Chitosan cross-linked microspheres were tested Nasally administered vaccines can induce both muco-
for loading with BSA and diphtheria toxoid (DT). A burst sal and systemic immune responses. The nasal passages are
release eect was observed from these microspheres, which rich in lymphoid tissues and the target site for nasally
was overcome by coating of the particles with paran oil or administered vaccines is the nasal associated lymphoid
a dierent polymer. Immunogenicity studies on Wistar rats tissue (NALT) situated mainly in the pharynx. Nasal
using DT-loaded chitosan spheres showed that the anti- vaccine administration using chitosan has attracted a lot
body titers were fairly constant over a 5-month period, of attention and the results of the rst clinical study have
although very low compared to DT given on alum as been recently presented [5,94,113].
control [108]. Bacon et al. [114] found that chitosan in phosphate-
Chitosan and chitosan ethylene oxidepropylene ox- buered saline (PBS) substantially enhanced the local and
ide (PEOPPO) block copolymer nanoparticles were pre- systemic immune responses against inuenza virus in mice
pared by a very mild ionic cross-linking technique using [114]. Similar results were obtained for cross-reacting
tripolyphosphate and were tested for their eciency of materials of diphtheria toxin (CRM197) in mice [115] and
loading and release of BSA, tetanus, and diphtheria tox- guinea pigs [116].
oids. BSA tetanus and DT were highly associated with Mills et al. [113] recently reported on the results of the
chitosan nanoparticles partly due to electrostatic interac- phase I study of subunit intranasal vaccine. Using geneti-
tions between the carboxyl groups of the protein and the cally inactivated CRM197 in chitosan powder, they found
amine groups of chitosan. PEOPPO also interacted elec- that a single nasal immunization was well tolerated and
trostatically with chitosan, thus competing with the pro- boosted antitoxin-neutralizing activity in healthy volun-
teins for association with nanoparticles. The proteins were teers. This activity was substantially higher compared with
released from the nanoparticles at a constant rate. The CRM197 alone in solution and exceeded the accepted
tetanus vaccine was released in the active form for 15 days protective levels. Surprisingly, although unilateral intrana-
in aqueous solution [109]. The advantages and applications sal immunization induced systemic immune response, a
of such ionically cross-linked nanoparticles have been nasal antitoxin secretory immunoglobulin A response (in
recently reviewed [110]. mucosal secretions) was seen only after the second immu-
The ecacy of orally administered vaccines (antigens) nization and only in the vaccinated nostril. The authors
is low due to their susceptibility to degradation and low noted that the results were very promising and, if repeated
uptake by the gut associated lymphoid tissue (GALT). in a larger study, an intranasal diphtheria vaccine based
Chitosan microparticles were prepared by a precipitation/ on CRM197 chitosan could be rapidly licensed for human
coacervation method to deliver antigens to Peyers patches use [113].
of the lower ileum. These microparticles were smaller than Chitosan is a bioadhesive material, which is able to
10 Am, a size suitable for uptake by M-cells of Peyers increase the residence time of formulations in the nasal
patches. CLSM visualization studies showed that the mod- cavity. This, in combination with the fact that it can open
el antigen, ovalbumin, was entrapped within the chitosan transiently the tight junctions of mucosal epithelia, is the
microparticles. Using eld emission scanning electron mi- main factor of chitosans function in enhanced antigen
croscopy, it was demonstrated that chitosan microparticles mucosal uptake, which the described nasal vaccine delivery
had porous structure, which facilitated the entrapment of systems have shown [5].
the model antigen ovalbumin. Loading studies of the
chitosan microparticles with the model compound, oval- D. DNA and Oligonucleotide Delivery Using
bumin, resulted in loading capacities of about 40%. Sub- Chitosan as Complexing Agent
sequent release studies showed only a very low release of
ovalbumin within 4 hr and most of the ovalbumin (about Cationic polymers have been used to condense and deliver
90%) remained entrapped in the microparticles. In vivo DNA both in vitro and in vivo. Several cationic polymers
that gave increased transfection eciencies have been plasmid DNA from nuclease degradation as shown by gel
investigated. They form polyelectrolyte complexes with electrophoresis. It was also found that the transfection
plasmid DNA, in which the DNA becomes better protected eciency of chitosanDNA nanoparticles was cell type-
against degradation by nucleases. They show structural dependent as in the case for most transfection agents. In the
variability and versatility, including the possibility of sur- same study, transferrin (ligand for transferrin receptor) or
face modication with specic targeting ligands for gene KNOB protein (C-terminal globular domain of the ber
expression mediated through specic receptors [117]. Nat- protein on adenovirus capsid) was conjugated to the nano-
ural polymers biocompatibility, low immunogenicity, and particle surface to investigate the eect of receptor-medi-
minimal cytotoxicity can render them a good alternative to ated endocytosis on transfection eciency. The transferrin
viral or lipid-mediated transfection [118,119]. conjugation only yielded a maximum of fourfold increase
Chitosan polymers and depolymerized chitosans have in their transfection eciency in HEK293 cells and HeLa
been investigated for their ability to condense and deliver cells, whereas KNOB-conjugated nanoparticles (contain-
plasmid DNA in COS-1 cell cultures [120]. It was found ing DNA) could improve gene expression level in HeLa
that chitosans having a molecular weight lower than 105 Da cells by 130-fold. To improve stability and attribute stealth
were forming small complexes between 200 and 100 nm. characteristics, polyethylene glycol (PEG) was conjugated
These complexes showed variable stability in the presence on the surface of chitosan nanoparticles. This allowed
of 10% serum. The molecular weight of chitosan proved to lyophilization without aggregation, and without loss of
have limited inuence on plasmid expression in vitro, bioactivity for at least 1 month in storage. The clearance of
whereas in vivo experiments using complexes of chito- the PEGylated nanoparticles in mice following intravenous
sanDNA and an endosomolytic peptide led to expression administration was slower than unmodied nanoparticles
of the plasmid in the small intestines of rabbits [120]. at 15 min, and with higher depositions in the kidney and
Nanospheres of chitosanDNA were prepared by self- liver [124].
induced complex coacervation method to yield particles in Chitosan was hydrophobically modied with deoxy-
the size range of 200750 nm. These nanospheres were ef- cholic acid to form self-aggregates in aqueous media. The
cient vectors in the Luciferase HEK293 (human embry- size of self-aggregates varied (130300 nm) in diameter, and
onic kidney) cell system. However, their transfection their structures were found to depend strongly on the
eciency varied when tested in dierent cell lines. Inter- molecular weight of chitosan ranging from 5 to 200 kDa.
estingly, addition of transferrin or chloroquine on the The complex formation with DNA had strong dependency
surface of nanospheres did not increase transfection e- on the size and structure of chitosan self-aggregates and
ciency [121]. signicantly inuenced the transfection eciency of COS-1
Chitosan and lactosylated chitosan vectors were pre- cells [125].
pared and investigated for their transfection eciencies in Chitosans of dierent Mw (31190 kDa) and DA (1
vitro. In this study, the transfection eciency of chitosan in 49%) were investigated for their transfection in HEK293
HeLa cells in the presence of 10% serum was found cells. The optimum chitosan was found to induce compa-
comparable to that of polyethyleneimine (PEI). Lactosy- rable transfection to PEI on HEK293 cells. However, in
lated chitosan was tested as a vector targeted for cells contrast to PEI, chitosan was nontoxic at escalating doses.
expressing a galactose-specic membrane lectin (BNL These chitosanDNA complexes were administered intra-
CL.2 and Hep-G2 cells). It was shown that these vectors tracheally in Balb/c mice. Transfection eciency was
were poorly ecient in transfecting these cell lines. Again, found elevated compared to naked DNA; however, it was
the presence of chloroquine did not improve transfection lower compared to PEI [126].
eciency [122]. It has been proposed that chitosan/plasmid complexes
Galactosylated chitosangraftdextranDNA com- condense to form large aggregates (58 Am) that adhere to
plexes were prepared. Galactose groups were chemically the cell surface. Subsequently, the complexes are endocy-
bound to chitosan for liver specicity and dextran was tosed [127]. This adsorptive endocytosis was found to be
grafted for increasing the complex stability in water. It was partly mediated by clathrin-mediated process and it is
shown that this system could eciently transfect Chang initiated by nonspecic interactions between chitosan
liver cells expressing asialoglycoprotein receptor that indi- nanoparticles and cell membranes [128]. Chitosan was
cates a specic interaction of the galactose ligands on shown to destabilize the lipid bilayer. The eect was found
chitosan with this receptor [123]. dependent on the Mw and pH [129]. This can explain the
Mao et al. [124] prepared chitosanDNA nanopar- mechanism of chitosanDNA complex uptake by cells and
ticles using a complex coacervation process. A number of the reason why optimization of the complexes by means of
parameters related to the preparation process were inves- chitosans Mw is important [130,131].
tigated. It was found that an amino group-to-phosphate It was previously reported that trimethylated chitosan
group ratio (N/P ratio) between 3 and 8 was the most (80% degree of quaternization) polymers, bearing anten-
ecient, and the size of particles was optimized to 100250 nary galactose residues through a 6-O-linked carboxy-
nm. The surface charge of these particles was slightly methyl group, served as DNA carrier. The complexes
positive with a zeta potential of +12 to +18 mV at pH were tested for specic targeting to Hep-G2 cells and
lower than 6.0, and became nearly neutral at pH 7.2. These expression of h-galactosidase activity. The complexes e-
nanoparticles could partially protect the encapsulated ciently transfected the Hep-G2 cells and the transfection
eciency was signicantly inhibited in the presence of an tic potential. In this strategy, chitosan appeared to be a
inhibitor, indicating that the conjugates were specically valuable material for the delivery of such vaccines.
internalized via the galactose receptor present on the
cellular surface of Hep-G2 cells [132].
In our group, we prepared trimethyl chitosan oligo- VII. SUMMARY AND PERSPECTIVES FOR
mers (TMOs) and investigated them for transfection e-
FUTURE CHITOSAN TECHNOLOGIES
ciency in vitro. We chose oligomeric chitosan because of
the potential for faster cell metabolism (clearance out of In this report, we presented the most important pharma-
the cell) after transfection, and we introduced trimethyla- ceutical applications that appeared in the literature in the
tion to improve the complexing ability (positive charge two last decades. During this period, signicant progress
independent of the pH). The complexes were found to be was made in the physicochemical characterization of
more ecient in transfecting COS-1 cells compared with chitinous materials. It is now possible to have well-charac-
unmodied chitosan oligomers and transfection eciency terized materials and, in most cases, to choose the right
was unaltered in the presence of serum [133]. Later, it was chitosan for the right application. The de novo synthesis of
shown that TMO, but not oligomeric, chitosan was able to chitosan at dierent distinct Mw values will provide clear
protect DNA against nuclease degradation, indicating that information on their structure and their relationship
permanent positive charge is necessary for complex for- with bioactivity.
mation [134]. A landmark chitosan application in gene However, in our opinion, the immunological and
delivery was reported in 1999. Roy et al. [135] used chi- toxicological prole is not yet claried. The eect of
tosan to form nanoparticle complexes with plasmid con- dierent types of chitosans on cytokine release, tissue
taining the dominant peanut allergen gene (pCMVArah2) compatibility, and chronic toxicity could give information
to generate immunologic protection in a murine model on safety aspects and further approval of such materials
of peanut allergy after oral administration. Compared from regulatory authorities.
with nonimmunized mice or mice treated with naked We have given emphasis on the already approved
DNA, mice immunized with nanoparticles showed a sub- chitosan applications in wound healing and we presented
stantial reduction in allergen-induced anaphylaxis associ- the injectable forms of this biomaterial related to drug
ated with reduced levels of IgE, plasma histamine, and delivery. In this aspect, chitosans potential for cell encap-
vascular leakage. sulation for the production of therapeutic bioactives
The notion of using chitosan for DNA vaccination has (implants) shows promise for future applications [62,139].
been well accepted and, in the last few years, a number of Chitosan can form membranes around cells that allow the
studies have been presented. In the eld of oral immuniza- passage of molecules. Its biocompatibility and controllable
tion, chitosan has been complexed with plasmid DNA biodegradability are additional advantages that could pro-
encoding for house dust mite allergen and administered mote such application.
orally in mice. This administration successfully primed the We mainly focused on chitosan and some of the
murine immune response [136]. In the eld of nasal vacci- derivatives that found pharmaceutical application mainly
nation, a plasmid DNA encoding for the CTL epitope of in drug delivery. It is worth mentioning that grafted
respiratory syncytial virus (RSV; pathogen of the lower chitosan such as PEG chitosan [140], or branched chitosan
respiratory tract and responsible for severe illness) was such as dendrimerized chitosan sialic acid hybrid [141] may
administered nasally in the form of polyplexes with chito- present suitable nanostructures for drug and DNA deliv-
san in BALB/c mice. Following RSV challenge of chito- ery. Currently, a nasal morphine and a vaccine delivery
sanDNA immunized mice, a substantial reduction in the system show promise in clinical trials. The rst results
virus load was observed in the lungs with immunized mice from preclinical evaluation of chitosanDNA vaccines
compared with that in the control group [137]. Similar to for mucosal immunization predict that soon, these systems
the abovementioned study, a u vaccine using chitosan is will also enter phase I trials. The development of a number
being currently developed [5]. of dierent chitosan modications will provide a library
Oligonucleotides have been found to be associated of polyglucosamines that will lead to more pharmaceuti-
with chitosan. Using the ionotropic gelation technique cal applications.
(tripolyphosphate), stable and spherical nanoparticles
could be obtained [110]. Recently, it was proposed that
chitosan complexation with phosphorothioate oligonucle-
REFERENCES
otide gave stable complexes that protected the DNA from
nuclease degradation [138]. 1. Jeuniaux, C.; Vossfoucart, M.F. Chitin biomass and pro-
It appears that the biocompatible, biodegradable, and duction in the marine-environment. Biochem. Syst. Ecol.
mainly cationic characteristics of chitosan suggest that this 1991, 19, 347.
material is suitable for complexation with nucleic acids. 2. Domard, A.; Domard, M. Chitosan: StructureProperties
Relationship and Biomedical Applications; Dumitriu S.,
Further uptake from the cells by endocytosis can yield the Ed.; Marcel Dekker: New York, 2001; 187 pp.
therapeutic nucleic acid. In the beginning of the millenni- 3. Rege, P.R.; Shukla, D.J.; Block, L.H. Chitinosans as
um, a novel therapeutic approach emerged. DNA mucosal tableting excipients for modied release delivery systems.
vaccination attracted scientic interest due to its therapeu- Int. J. Pharm. 1999, 181, 49.
4. Kumar, M. A review of chitin and chitosan applications. 27. Peluso, G.; Petillo, O.; Ranieri, M.; Santin, M.; Am-
React. Funct. Polym. 2000, 46, 1. brosio, L.; Calabro, D.; Avallone, B.; Balsamo, G.
5. Illum, L.; Jabbal-Gill, I.; Hinchclie, M.; Fisher, A.N.; Chitosan-mediated stimulation of macrophage function.
Davis, S.S. Chitosan as a novel nasal delivery system for Biomaterials 1994, 15, 1215.
vaccines. Adv. Drug Deliv. Rev. 2001, 51, 81. 28. Pusateri, A.E.; McCarthy, S.J.G.; K.W.; Harris, R.A.;
6. Dodane, V.; Vilivalam, V.D. Pharmaceutical applications Cardenas, L.; McManus, A.T.; Goodwin, C.W., Jr. Eect
of chitosan. Pharm. Sci. Technol. Today 1998, 1, 246. of a chitosan-based hemostatic dressing on blood loss and
7. Felt, O.; Buri, P.; Gurny, R. Chitosan: a unique polysac- survival in a model of severe venous hemorrhage and
charide for drug delivery. Drug Dev. Ind. Pharm. 1998, hepatic injury in swine. J. Trauma 2003, 54, 177.
24, 979. 29. Rao, S.B.; Sharma, C.P. Use of chitosan as a biomaterial:
8. Singla, A.K.; Chawla, M. Chitosan: some pharmaceutical studies on its safety and hemostatic potential. J. Biomed.
and biological aspectsan update. J. Pharm. Pharmacol. Mater. Res. 1997, 34, 21.
2001, 53, 1047. 30. Hoekstra, A.; Struszczyk, H.; Kivekas, O. Percutaneous
9. Sabnis, S.; Block, L.H. Chitosan as an enabling excipient microcrystalline chitosan application for sealing arterial
for drug delivery systems: I. Molecular modications. Int. puncture sites. Biomaterials 1998, 19, 1467.
J. Biol. Macromol. 2000, 27, 181. 31. Malette, W.G.; Quigley, H.J.; Gaines, R.D.; Johnson,
10. Benesch, J.; Tengvall, P. Blood protein adsorption onto N.D.; Rainer, W.G. Chitosan a new hemostatic. Ann.
chitosan. Biomaterials 2002, 23, 2561. Thorac. Surg. 1983, 36, 55.
11. Chatelet, C.; Damour, O.; Domard, A. Inuence of the 32. Lu, J.X.; Prudhommeaux, F.; Meunier, A.; Sedel, L.;
degree of acetylation on some biological properties of Guillemin, G. Eects of chitosan on rat knee cartilages.
chitosan lms. Biomaterials 2001, 22, 261. Biomaterials 1999, 20, 1937.
12. Ottoy, M.H.; Varum, K.M.; Smidsrod, O. Compositional 33. Suh, J.-K.F.; Matthew, H.W. Application of chitosan-
heterogeneity of heterogeneously deacetylated chitosans. based polysaccharide biomaterials in cartilage tissue
Carbohydr. Polym. 1996, 29, 17. engineering: a review. Biomaterials 2000, 21, 2589.
13. Illum, L. Chitosan and its use as a pharmaceutical 34. Sechriest, V.F.; Miao, Y.J.; Niyibizi, C.; Westerhausen-
excipient. Pharm. Res. 1998, 15, 1326. Larson, A.; Matthew, H.W.; Evans, C.H.; Fu, F.H.; Suh,
14. Anthonsen, M.W.; Varum, K.M.; Smidsrod, O. Solution J.K. GAG-augmented polysaccharide hydrogel: a novel
properties of chitosansconformation and chain stiness biocompatible and biodegradable material to support
of chitosans with dierent degrees of N-acetylation. chondrogenesis. J. Biomed. Mater. Res. 2000, 49, 534.
Carbohydr. Polym. 1993, 22, 193. 35. Mattioli-Belmonte, M.; Gigante, A.; Muzzarelli, R.A.;
15. Onishi, H.; Machida, Y. Biodegradation and distribution Politano, R.; De Benedittis, A.; Speechia, N.; Bua, A.;
of water-soluble chitosan in mice. Biomaterials 1999, 20, Biagini, G.; Greco, F. N,N-dicarboxymethyl chitosan as
175. delivery agent for bone morphogenetic protein in the
16. Chandy, T.; Sharma, C.P. Chitosanas a biomaterial. repair of articular cartilage. Med. Biol. Eng. Comput.
Biomater. Artif. Cells Artif. Organs 1990, 18, 1. 1999, 37, 130.
17. Arai, K.; Kinumaki, T.; Fujita, T. Toxicity of chitosan. 36. No, H.K.; Park, N.Y.; Lee, S.H.; Meyers, S.P. Anti-
Bull. Tokai Reg. Fish Res. Lab. 1968, 43, 89. bacterial activity of chitosans and chitosan oligomers with
18. Kim, S.K.; Park, P.J.; Yang, H.P.; Han, S.S. Subacute dierent molecular weights. Int. J. Food Microbiol. 2002,
toxicity of chitosan oligosaccharide in SpragueDawley 74, 65.
rats. Arzneimittelforschung 2001, 51, 769. 37. Mi, F.L.; Shyu, S.S.; Wu, Y.B.; Lee, S.T.; Shyong, J.Y.;
19. CarrenoGomez, B.; Duncan, R. Evaluation of the bio- Huang, R.N. Fabrication and characterization of a
logical properties of soluble chitosan and chitosan micro- sponge-like asymmetric chitosan membrane as a wound
spheres. Int. J. Pharm. 1997, 148, 231. dressing. Biomaterials 2001, 22, 165.
20. Tanaka, Y.; Tanioka, S.; Tanaka, M.; Tanigawa, T.; 38. Muzzarelli, R.; Tarsi, R.; Filippini, O.; Giovanetti, E.;
Kitamura, Y.; Minami, S.; Okamoto, Y.; Miyashita, M.; Biagini, G.; Varaldo, P.E. Antimicrobial properties of N-
Nanno, M. Eects of chitin and chitosan particles on carboxybutyl chitosan. Antimicrob. Agents Chemother.
BALB/c mice by oral and parenteral administration. 1990, 34, 2019.
Biomaterials 1997, 18, 591. 39. Sawayanagi, Y.; Nambu, N.; Nagai, T. Use of chitosan
21. Miyai, K.; Kanno, T.; Ischikawa, E. Progress in Clinical for sustained-release preparations of water-soluble drugs.
Biochemistry; Hirano, S., Inui, H., Iwata, M., Yamanaka, Chem. Pharm. Bull. (Tokyo) 1982, 30, 4213.
K., Tanaka, H., Toda, T., Eds.; London, 1992. 40. Miyazaki, S.; Yamaguchi, H.; Yokouchi, C.; Takada, M.;
22. Muzzarelli, R.A. Human enzymatic activities related to Hou, W.M. Sustained release of indomethacin from
the therapeutic administration of chitin derivatives. Cell. chitosan granules in beagle dogs. J. Pharm. Pharmacol.
Mol. Life Sci. 1997, 53, 131. 1988, 40, 642.
23. Balassa, L.L.; Prudden, J.F. Application of chitin and 41. Chandy, T.; Sharma, C.P. Chitosan beads and granules
chitosan in wound healing acceleration. Conference on for oral sustained delivery of nifedipine: in vitro studies.
Chitin and Chitosan, 1978. Biomaterials 1992, 13, 949.
24. Ueno, H.; Mori, T.; Fujinaga, T. Topical formulations 42. Henriksen, I.; Skaugrud, O.; Karlsen, J. Use of chitosan
and wound healing applications of chitosan. Adv. Drug and chitosan malate as an excipient in wet granulation of
Deliv. Rev. 2001, 52, 105. 3 water-soluble drugs. Int. J. Pharm. 1993, 98, 181.
25. Ueno, H.; Yamada, H.; Tanaka, I.; Kaba, N.; Matsuura, 43. Mi, F.L.; Her, N.L.; Kuan, C.Y.; Wong, T.B.; Shyu, S.S.
M.; Okumura, M.; Kadosawa, T.; Fujinaga, T. Accelerat- Chitosan tablets for controlled release of theophylline:
ing eects of chitosan for healing at early phase of eect of polymerdrug wet or dry blending and anionic
experimental open wound in dogs. Biomaterials 1999, 20, cationic interpolymer complex. J. Appl. Polym. Sci. 1997,
1407. 66, 2495.
26. Ueno, H.; Murakami, M.; Okumura, M.; Kadosawa, T.; 44. El-Gibaly, I. Development and in vitro evaluation of
Uede, T.; Fujinaga, T. Chitosan accelerates the produc- novel oating chitosan microcapsules for oral use:
tion of osteopontin from polymorphonuclear leukocytes. comparison with non-oating chitosan microspheres.
Biomaterials 2001, 22, 1667. Int. J. Pharm. 2002, 249, 7.
45. Hejazi, R.; Amiji, M. Stomach-specic anti-H. pylori hematopoietic cytokine colony-stimulating factor 1 is also
therapy: I. Preparation and characterization of tetracy- a growth factor in the CNS: II. Microencapsulated CSF-1
line-loaded chitosan microspheres. Int. J. Pharm. 2002, and LM-10 cells as delivery systems. Exp. Neurol. 1996,
235, 87. 141, 47.
46. Needleman, I.G.; Smales, F.C. In vitro assessment of 63. Chenite, A.; Chaput, C.; Wang, D.; Combes, C.;
bioadhesion for periodontal and buccal drug delivery. Buschmann, M.D.; Hoemann, C.D.; Leroux, J.C.; Atkin-
Biomaterials 1995, 16, 617. son, B.L.; Binette, F.; Selmani, A. Novel injectable
47. Choi, B.K.; Kim, K.Y.; Yoo, Y.J.; Oh, S.J.; Choi, J.H.; neutral solutions of chitosan form biodegradable gels in
Kim, C.Y. In vitro antimicrobial activity of a chitooligo- situ. Biomaterials 2000, 21, 2155.
saccharide mixture against Actinobacillus actinomycetem- 64. Mitra, S.; Gaur, U.; Ghosh, P.C.; Maitra, A.N. Tumour
comitans and Streptococcus mutans. Int. J. Antimicrob. targeted delivery of encapsulated dextrandoxorubicin
Agents 2001, 18, 553. conjugate using chitosan nanoparticles as carrier. J.
48. Ikinci, G.; Senel, S.; Akincibay, H.; Kas, S.; Ercis, S.; Control. Release 2001, 74, 317.
Wilson, C.G.; Hincal, A.A. Eect of chitosan on a 65. Song, Y.H.; Onishi, H.; Nagai, T. Conjugate of mitomy-
periodontal pathogen Porphyromonas gingivalis. Int. J. cin-C with N-succinyl-chitosanin-vitro drug-release
Pharm. 2002, 235, 121. properties toxicity and antitumor-activity. Int. J. Pharm.
49. Miyazaki, S.; Nakayama, A.; Oda, M.; Takada, M.; 1993, 98, 121.
Attwood, D. Chitosan and sodium alginate based 66. Song, Y.; Onishi, H.; Nagai, T. Pharmacokinetic charac-
bioadhesive tablets for intraoral drug-delivery. Biol. teristics and antitumor activity of the N-succinylchito-
Pharmacol. Bull. 1994, 17, 745. sanmitomycin C conjugate and the carboxymethyl
50. Remunan-Lopez, C.; Portero, A.; Vila-Jato, J.L.; Alonso, chitinmitomycin C conjugate. Biol. Pharm. Bull. 1993,
M.J. Design and evaluation of chitosan/ethylcellulose 16, 48.
mucoadhesive bilayered devices for buccal drug delivery. 67. Kato, Y.; Onishi, H.; Machida, Y. Evaluation of N-
J. Control. Release 1998, 55, 143. succinyl-chitosan as a systemic long-circulating polymer.
51. Martin, L.; Wilson, C.G.; Koosha, F.; Tetley, L.; Gray, Biomaterials 2000, 21, 1579.
A.I.; Senel, S.; Uchegbu, I.F. The release of model 68. Kato, Y.; Onishi, H.; Machida, Y. Lactosaminated and
macromolecules may be controlled by the hydrophobicity intact N-succinyl-chitosans as drug carriers in liver meta-
of palmitoyl glycol chitosan hydrogels. J. Control. Release stasis. Int. J. Pharm. 2001, 226, 93.
2002, 80, 87. 69. Kato, Y.; Onishi, H.; Machida, Y. Ecacy of lactosami-
52. Martin, L.; Wilson, C.G.; Koosha, F.; Uchegbu, I.F. nated and intact N-succinylchitosanmitomycin C con-
Sustained buccal delivery of the hydrophobic drug jugates against M5076 liver metastatic cancer. J. Pharm.
denbufylline using physically cross-linked palmitoyl glycol Pharmacol. 2002, 54, 529.
chitosan hydrogels. Eur. J. Pharm. Biopharm. 2003, 55, 35. 70. Lehr, C.-M.; Bouwstra, J.A.; Schacht, E.H.; Junginger,
53. Giunchedi, P.; Juliano, C.; Gavini, E.; Cossu, M.; H.E. In vitro evaluation of mucoadhesive properties of
Sorrenti, M. Formulation and in vivo evaluation of chitosan and some other natural polymers. Int. J. Pharm.
chlorhexidine buccal tablets prepared using drug-loaded 1992, 78, 43.
chitosan microspheres. Eur. J. Pharm. Biopharm. 2002, 71. Artursson, P.; Lindmark, T.; Davis, S.S.; Illum, L. Eect
53, 233. of chitosan on the permeability of monolayers of
54. Senel, S.; Ikinci, G.; Kas, S.; Youse-Rad, A.; Sargon, intestinal epithelial cells (Caco-2). Pharm. Res. 1994, 11,
M.F.; Hincal, A.A. Chitosan lms and hydrogels of 1358.
chlorhexidine gluconate for oral mucosal delivery. Int. J. 72. Schipper, N.G.M.; Varum, K.M.; Artursson, P. Chitosans
Pharm. 2000, 193, 197. as absorption enhancers for poorly absorbable drugs: 1.
55. Perugini, P.; Genta, I.; Conti, B.; Modena, T.; Pavanetto, Inuence of molecular weight and degree of acetylation
F. Periodontal delivery of ipriavone: new chitosan/ on drug transport across human intestinal epithelial
PLGA lm delivery system for a lipophilic drug. Int. J. (Caco-2) cells. Pharm. Res. 1996, 13, 1686.
Pharm. 2003, 252, 1. 73. Schipper, N.G.; Olsson, S.; Hoogstraate, J.A.; deBoer,
56. Park, Y.J.; Lee, Y.M.; Park, S.N.; Sheen, S.Y.; Chung, A.G.; Varum, K.M.; Artursson, P. Chitosans as absorp-
C.P.; Lee, S.J. Platelet-derived growth factor releasing tion enhancers for poorly absorbable drugs: 2. Mechanism
chitosan sponge for periodontal bone regeneration. of absorption enhancement. Pharm. Res. 1997, 14, 923.
Biomaterials 2000, 21, 153. 74. Dodane, V.; Khan, M.A.; Merwin, J.R. Eect of chitosan
57. Hirano, S.; Noishiki, Y. The blood compatibility of on epithelial permeability and structure. Int. J. Pharm.
chitosan and N-acylchitosans. J. Biomed. Mater. Res. 1999, 182, 21.
1985, 19, 413. 75. Luessen, H.L.; de Leeuw, B.J.; Langemeyer, M.W.; de
58. Gallo, J.M.; Hassan, E.E. Receptor-mediated magnetic Boer, A.B.; Verhoef, J.C.; Junginger, H.E. Mucoadhesive
carriers: basis for targeting. Pharm. Res. 1988, 5, 300. polymers in peroral peptide drug delivery: VI. Carbomer
59. Jameela, S.R.; Jayakrishnan, A. Glutaraldehyde cross- and chitosan improve the intestinal absorption of the
linked chitosan microspheres as a long acting biodegrad- peptide drug buserelin in vivo. Pharm. Res. 1996, 13,
able drug delivery vehicle: studies on the in vitro release of 1668.
mitoxantrone and in vivo degradation of microspheres in 76. Borchard, G.; Luessen, H.L.; de Boer, A.G.; Verhoef,
rat muscle. Biomaterials 1995, 16, 769. J.C.; Lehr, C.-M.; Junginger, H.E. The potential of
60. Ruel-Gariepy, E.; Chenite, A.; Chaput, C.; Guirguis, S.; mucoadhesive polymers in enhancing intestinal peptide
Leroux, J. Characterization of thermosensitive chitosan drug absorption: 3. Eects of chitosanglutamate and
gels for the sustained delivery of drugs. Int. J. Pharm. carbomer on epithelial tight junctions in vitro. J. Control.
2000, 203, 89. Release 1996, 39, 131.
61. Ruel-Gariepy, E.; Leclair, G.; Hildgen, P.; Gupta, A.; 77. Kotze, A.F.; Luessen, H.L.; de Leeuw, B.J.; de Boer,
Leroux, J.C. Thermosensitive chitosan-based hydrogel A.G.; Verhoef, J.C.; Junginger, H.E. Comparison of the
containing liposomes for the delivery of hydrophilic eect of dierent chitosan salts and N-trimethyl chitosan
molecules. J. Control. Release 2002, 82, 373. chloride on the permeability of intestinal epithelial cells
62. Maysinger, D.; Berezovskaya, O.; Fedoro, S. The (Caco-2). J. Control. Release 1998, 51, 35.
78. Kotze, A.F.; Luessen, H.L.; deLeeuw, B.J.; deBoer, B.G.; 94. Illum, L. Nasal drug deliverypossibilities, problems and
Verhoef, J.C.; Junginger, H.E. N-trimethyl chitosan solutions. J. Control. Release 2003, 87, 187.
chloride as a potential absorption enhancer across 95. Dyer, A.M.; Hinchclie, M.; Watts, P.; Castile, J.; Jabbal-
mucosal surfaces: in vitro evaluation in intestinal epithe- Gill, I.; Nankervis, R.; Smith, A.; Illum, L. Nasal delivery
lial cells (Caco-2). Pharm. Res. 1997, 14, 1197. of insulin using novel chitosan based formulations: a
79. Kotze, A.F.; Thanou, M.M.; Luebetaen, H.L.; de Boer, comparative study in two animal models between simple
A.G.; Verhoef, J.C.; Junginger, H.E. Enhancement of chitosan formulations and chitosan nanoparticles. Pharm.
paracellular drug transport with highly quaternized N- Res. 2002, 19, 998.
trimethyl chitosan chloride in neutral environments: in 96. Witschi, C.; Mrsny, R.J. In vitro evaluation of micro-
vitro evaluation in intestinal epithelial cells (Caco-2). J. particles and polymer gels for use as nasal platforms for
Pharm. Sci. 1999, 88, 253. protein delivery. Pharm. Res. 1999, 16, 382.
80. Thanou, M.M.; Verhoef, J.C.; Romeijn, S.G.; Nagel- 97. Henriksen, I.; Green, K.L.; Smart, J.D.; Smistad, G.;
kerke, J.F.; Merkus, F.W.; Junginger, H.E. Eects of N- Karlsen, J. Bioadhesion of hydrated chitosans: an in vitro
trimethyl chitosan chloride, a novel absorption enhancer, and in vivo study. Int. J. Pharm. 1996, 145, 231.
on Caco-2 intestinal epithelia and the ciliary beat 98. Felt, O.; Furrer, P.; Mayer, J.M.; Plazonnet, B.; Buri, P.;
frequency of chicken embryo trachea. Int. J. Pharm. Gurny, R. Topical use of chitosan in ophthalmology:
1999, 185, 73. tolerance assessment and evaluation of precorneal reten-
81. Thanou, M.; Florea, B.I.; Langemeyer, M.W.; Verhoef, tion. Int. J. Pharm. 1999, 180, 185.
J.C.; Junginger, H.E. N-trimethylated chitosan chloride 99. De Campos, A.M.; Sanchez, A.; Alonso, M.J. Chitosan
(TMC) improves the intestinal permeation of the peptide nanoparticles: a new vehicle for the improvement of the
drug buserelin in vitro (Caco-2 cells) and in vivo (rats). delivery of drugs to the ocular surface. Application to
Pharm. Res. 2000, 17, 27. cyclosporin A. Int. J. Pharm. 2001, 224, 159.
82. Thanou, M.; Verhoef, J.C.; Marbach, P.; Junginger, H.E. 100. Hejazi, R.; Amiji, M. Chitosan-based gastrointestinal
Intestinal absorption of octreotide: N-trimethyl chitosan delivery systems. J. Control. Release 2003, 89, 151.
chloride (TMC) ameliorates the permeability and absorp- 101. Zhang, H.; Neau, S.H. In vitro degradation of chitosan by
tion properties of the somatostatin analogue in vitro and bacterial enzymes from rat cecal and colonic contents.
in vivo. J. Pharm. Sci. 2000, 89, 951. Biomaterials 2002, 23, 2761.
83. Bernkop-Schnurch, A.; Paikl, C.; Valenta, C. Novel 102. Tozaki, H.; Komoike, J.; Tada, C.; Maruyama, T.;
bioadhesive chitosanEDTA conjugate protects leucine Terabe, A.; Suzuki, T.; Yamamoto, A.; Muranishi, S.
enkephalin from degradation by aminopeptidase N. Chitosan capsules for colon-specic drug delivery: im-
Pharm. Res. 1997, 14, 917. provement of insulin absorption from the rat colon. J.
84. BernkopSchnurch, A.; Bratengeyer, I.; Valenta, C. Devel- Pharm. Sci. 1997, 86, 1016.
opment and in vitro evaluation of a drug delivery system 103. Tozaki, H.; Fujita, T.; Odoriba, T.; Terabe, A.; Suzuki,
protecting from trypsinic degradation. Int. J. Pharm. T.; Tanaka, C.; Okabe, S.; Muranishi, S.; Yamamoto, A.
1997, 157, 17. Colon-specic delivery of R68070, a new thromboxane
85. Bernkop-Schnurch, A.; Scerbe-Saiko, A. Synthesis and in synthase inhibitor, using chitosan capsules: therapeutic
vitro evaluation of chitosanEDTAprotease-inhibitor eects against 2,4,6-trinitrobenzene sulfonic acid-induced
conjugates which might be useful in oral delivery of ulcerative colitis in rats. Life Sci. 1999, 64, 1155.
peptides and proteins. Pharm. Res. 1998, 15, 263. 104. Tozaki, H.; Odoriba, T.; Okada, N.; Fujita, T.; Terabe,
86. Bernkop-Schnurch, A.; Walker, G. Multifunctional ma- A.; Suzuki, T.; Okabe, S.; Muranishi, S.; Yamamoto, A.
trices for oral peptide delivery. Crit. Rev. Ther. Drug Chitosan capsules for colon-specic drug delivery: en-
Carr. Syst. 2001, 18, 459. hanced localization of 5-aminosalicylic acid in the large
87. Bernkop-Schnurch, A.; Kast, C.E. Chemically modied intestine accelerates healing of TNBS-induced colitis in
chitosans as enzyme inhibitors. Adv. Drug Deliv. Rev. rats. J. Control. Release 2002, 82, 51.
2001, 52, 127. 105. Lorenzo-Lamosa, M.L.; Remunan-Lopez, C.; Vila-Jato,
88. Takeuchi, H.; Yamamoto, H.; Niwa, T.; Hino, T.; J.L.; Alonso, M.J. Design of microencapsulated chitosan
Kawashima, Y. Mucoadhesion of polymer-coated lip- microspheres for colonic drug delivery. J. Control.
osomes to rat intestine in vitro. Chem. Pharm. Bull. Release 1998, 52, 109.
(Tokyo) 1994, 42, 1954. 106. Shimono, N.; Takatori, T.; Ueda, M.; Mori, M.; Higashi,
89. Takeuchi, H.; Yamamoto, H.; Niwa, T.; Hino, T.; Y.; Nakamura, Y. Chitosan dispersed system for colon-
Kawashima, Y. Enteral absorption of insulin in rats from specic drug delivery. Int. J. Pharm. 2002, 245, 45.
mucoadhesive chitosan-coated liposomes. Pharm. Res. 107. Zhang, H.; Alsarra, I.A.; Neau, S.H. An in vitro
1996, 13, 896. evaluation of a chitosan-containing multiparticulate
90. Illum, L.; Farraj, N.F.; Davis, S.S. Chitosan as a novel system for macromolecule delivery to the colon. Int. J.
nasal delivery system for peptide drugs. Pharm. Res. 1994, Pharm. 2002, 239, 197.
11, 1186. 108. Jameela, S.R.; Misra, A.; Jayakrishnan, A. Cross-linked
91. Aspden, T.J.; Illum, L.; Skaugrud, O. Chitosan as a nasal chitosan microspheres as carriers for prolonged delivery
delivery system: evaluation of insulin absorption enhance- of macromolecular drugs. J. Biomater. Sci. Polym. Ed.
ment and eect on nasal membrane integrity using rat 1994, 6, 621.
models. Eur. J. Pharm. Sci. 1996, 4, 23. 109. Calvo, P.; Remunan-Lopez, C.; Vila-Jato, J.L.; Alonso,
92. Soane, R.J.; Frier, M.; Perkins, A.C.; Jones, N.S.; Davis, M.J. Chitosan and chitosan/ethylene oxidepropylene
S.S.; Illum, L. Evaluation of the clearance characteristics oxide block copolymer nanoparticles as novel carriers
of bioadhesive systems in humans. Int. J. Pharm. 1999, for proteins and vaccines. Pharm. Res. 1997, 14, 1431.
178, 55. 110. Janes, K.A.; Calvo, P.; Alonso, M.J. Polysaccharide
93. Illum, L.; Watts, P.; Fisher, A.N.; Hinchclie, M.; colloidal particles as delivery systems for macromolecules.
Norbury, H.; Jabbal-Gill, I.; Nankervis, R.; Davis, S.S. Adv. Drug Deliv. Rev. 2001, 42, 83.
Intranasal delivery of morphine. J. Pharmacol. Exp. Ther. 111. van der Lubben, I.M.; Verhoef, J.C.; van Aelst, A.C.;
2002, 301, 391. Borchard, G.; Junginger, H.E. Chitosan microparticles for
oral vaccination: preparation, characterization and pre- characteristics of size-controlled self-aggregates of deox-
liminary in vivo uptake studies in murine Peyers patches. ycholic acid-modied chitosan and their application as a
Biomaterials 2001, 22, 687. DNA delivery carrier. Bioconjug. Chem. 2001, 12, 932.
112. van der Lubben, I.M.; Kersten, G.; Fretz, M.M.; Beuvery, 126. Koping-Hoggard, M.; Tubulekas, I.; Guan, H.; Edwards,
C.; Coos Verhoef, J.; Junginger, H.E. Chitosan micro- K.; Nilsson, M.; Varum, K.M.; Artursson, P. Chitosan as
particles for mucosal vaccination against diphtheria: oral a nonviral gene delivery system. Structureproperty
and nasal ecacy studies in mice. Vaccine 2003, 21, 1400. relationships and characteristics compared with polyethy-
113. Mills, K.H.; Cosgrove, C.; McNeela, E.A.; Sexton, A.; lenimine in vitro and after lung administration in vivo.
Giemza, R.; Jabbal-Gill, I.; Church, A.; Lin, W.; Illum, Gene Ther. 2001, 8, 1108.
L.; Podda, A.; Rappuoli, R.; Pizza, M.; Grin, G.E.; 127. Ishii, T.; Okahata, Y.; Sato, T. Mechanism of cell
Lewis, D.J. Protective levels of diphtheria-neutralizing transfection with plasmid/chitosan complexes. Biochim.
antibody induced in healthy volunteers by unilateral Biophys. Acta Biomembr. 2001, 1514, 51.
primingboosting intranasal immunization associated 128. Huang, M.; Ma, Z.S.; Khor, E.; Lim, L.Y. Uptake of
with restricted ipsilateral mucosal secretory immunoglo- FITCchitosan nanoparticles by a549 cells. Pharm. Res.
bulin a. Infect. Immun. 2003, 71, 726. 2002, 19, 1488.
114. Bacon, A.; Makin, J.; Sizer, P.J.; Jabbal-Gill, I.; 129. Fang, N.; Chan, V.; Mao, H.Q.; Leong, K.W. Interactions
Hinchclie, M.; Illum, L.; Chateld, S.; Roberts, M. of phospholipid bilayer with chitosan: eect of molecular
Carbohydrate biopolymers enhance antibody responses weight and pH. Biomacromolecules 2001, 2, 1161.
to mucosally delivered vaccine antigens. Infect. Immun. 130. Koping-Hoggard, M.; Melnikova, Y.S.; Varum, K.M.;
2000, 68, 5764. Lindman, B.; Artursson, P. Relationship between the
115. Jabbal-Gill, I.; Fisher, A.N.; Rappuoli, R.; Davis, S.S.; physical shape and the eciency of oligomeric chitosan as
Illum, L. Stimulation of mucosal and systemic antibody a gene delivery system in vitro and in vivo. J. Gene Med.
responses against Bordetella pertussis lamentous haemag- 2003, 5, 130.
glutinin and recombinant pertussis toxin after nasal admin- 131. Lee, M.; Nah, J.W.; Kwon, Y.; Koh, J.J.; Ko, K.S.; Kim,
istration with chitosan in mice. Vaccine 1998, 16, 2039. S.W. Water-soluble and low molecular weight chitosan-
116. McNeela, E.A.; OConnor, D.; Jabbal-Gill, I.; Illum, L.; based plasmid DNA delivery. Pharm. Res. 2001, 18, 427.
Davis, S.S.; Pizza, M.; Peppoloni, S.; Rappuoli, R.; Mills, 132. Murata, J.; Ohya, Y.; Ouchi, T. Design of quaternary
K.H. A mucosal vaccine against diphtheria: formulation chitosan conjugate having antennary galactose residues as
of cross reacting material (CRM(197)) of diphtheria toxin a gene delivery tool. Carbohydr. Polym. 1997, 32, 105.
with chitosan enhances local and systemic antibody and 133. Thanou, M.; Florea, B.I.; Geldof, M.; Junginger, H.E.;
Th2 responses following nasal delivery. Vaccine 2000, 19, Borchard, G. Quaternized chitosan oligomers as novel
1188. gene delivery vectors in epithelial cell lines. Biomaterials
117. Merdan, T.; Kopecek, J.; Kissel, T. Prospects for cationic 2002, 23, 153.
polymers in gene and oligonucleotide therapy against 134. Florea, B.I.; Ravenstijn, P.G.M.; Junginger, H.E.; Borch-
cancer. Adv. Drug Deliv. Rev. 2002, 54, 715. ard, G. N-trimethylated oligomeric chitosan (TMO)
118. Liu, W.G.; De Yao, K. Chitosan and its derivativesa protects plasmid DNA from DNase I degradation and
promising non-viral vector for gene transfection. J. promotes transfection eciency in vitro. STP Pharma Sci.
Control. Release 2002, 83, 1. 2002, 12, 243.
119. Borchard, G. Chitosans for gene delivery. Adv. Drug 135. Roy, K.; Mao, H.Q.; Huang, S.K.; Leong, K.W. Oral
Deliv. Rev. 2001, 52, 145. gene delivery with chitosanDNA nanoparticles generates
120. MacLaughlin, F.C.; Mumper, R.J.; Wang, J.; Tagliaferri, immunologic protection in a murine model of peanut
J.M.; Gill, I.; Hinchclie, M.; Rolland, A.P. Chitosan and allergy. Nat. Med. 1999, 5, 387.
depolymerized chitosan oligomers as condensing carriers 136. Chew, J.L.; Wolfowicz, C.B.; Mao, H.-Q.; Leong, K.W.;
for in vivo plasmid delivery. J. Control. Release 1998, 56, Chua, K.Y. Chitosan nanoparticles containing plasmid
259. DNA encoding house dust mite allergen, Der p 1 for oral
121. Leong, K.W.; Mao, H.Q.; Truong-Le, V.L.; Roy, K.; vaccination in mice. Vaccine 2003, 21, 2720.
Walsh, S.M.; August, J.T. DNApolycation nanospheres 137. Iqbal, M.; Lin, W.; Jabbal-Gill, I.; Davis, S.S.; Steward,
as non-viral gene delivery vehicles. J. Control. Release M.W.; Illum, L. Nasal delivery of chitosanDNA plasmid
1998, 53, 183. expressing epitopes of respiratory syncytial virus (RSV)
122. Erbacher, P.; Zou, S.M.; Bettinger, T.; Stean, A.M.; induces protective CTL responses in BALB/c mice.
Remy, J.S. Chitosan-based vector/DNA complexes for Vaccine 2003, 21, 1478.
gene delivery: biophysical characteristics and transfection 138. Ferreiroa, M.G.; Crooke, R.M.; Tillman, L.; Hardee, G.;
ability. Pharm. Res. 1998, 15, 1332. Bodmeier, R. Stability of polycationic complexes of an
123. Park, Y.K.; Park, Y.H.; Shin, B.A.; Choi, E.S.; Park, antisense oligonucleotide in rat small intestine homoge-
Y.R.; Akaike, T.; Cho, C.S. Galactosylated chitosan nates. Eur. J. Pharm. Biopharm. 2003, 55, 19.
graftdextran as hepatocyte-targeting DNA carrier. J. 139. Zielinski, B.A.; Aebischer, P. Chitosan as a matrix for
Control. Release 2000, 69, 97. mammalian cell encapsulation. Biomaterials 1994, 15,
124. Mao, H.Q.; Roy, K.; Troung-Le, V.L.; Janes, K.A.; Lin, 1049.
K.Y.; Wang, Y.; August, J.T.; Leong, K.W. Chitosan 140. Ohya, Y.; Cai, R.; Nishizawa, H.; Hara, K.; Ouchi, T.
DNA nanoparticles as gene carriers: synthesis, character- Preparation of PEG-grafted chitosan nanoparticles as
ization and transfection eciency. J. Control. Release peptide drug carriers. STP Pharma Sci. 2000, 10, 77.
2001, 70, 399. 141. Sashiwa, H.; Shigemasa, Y.; Roy, R. Highly convergent
125. Kim, Y.H.; Gihm, S.H.; Park, C.R.; Lee, K.Y.; Kim, synthesis of dendrimerized chitosansialic acid hybrid.
T.W.; Kwon, I.C.; Chung, H.; Jeong, S.Y. Structural Macromolecules 2001, 34, 3211.
29
Macromolecular Complexes of Chitosan
Naoji Kubota and Kei Shimoda
Oita University, Oita, Japan
I. INTRODUCTION B. Structure and Properties of Chitosan
A. Macromolecular Complexes Chitin, poly[h(1!4)-2-acetoamido-2-deoxy-D-glucopyra-

nose], is one of the most abundant natural polysaccharides
Macromolecular complexes are molecular aggregates of and is present in crustacea, insects, fungi, and yeasts. The
two or more complementary polymers, which have denite total annual global estimates of accessible chitin amount
composition and characteristics and arise from intermo- to 1 109 tons [2]. It is obtained primarily as a by-product
lecular interactions, such as Coulomb forces, hydrogen- of the seafood industry. Deacetylation of chitin by alkali
bonding forces, charge-transfer forces, and van der Waals readily aords chitosan (Ch), poly[h(1!4)-2-amino-2-de-
forces. The complexes can be divided into the following oxy-D-glucopyranose]. Ch is also found in various fungi.
four classes according to the nature of their interactions [1]: However, the molecular structure of Ch is believed to be a
polyelectrolyte complexes, hydrogen-bonding complexes, copolymer of N-acetyl-glucosamine and glucosamine; usu-
charge-transfer complexes, and stereocomplexes. ally the glucosamine content is more than 90%. It is
It is rare that these intermolecular interactions take known that 50% N-deacetylated chitin (50% N-acetylated
place singly, and hydrophobic interactions are also signif- Ch) is water soluble [3]. Kubota and Eguchi showed that
icant factors in aqueous media. Although these secondary the water solubility of half N-acetylated Ch increased
binding forces, among which Coulomb forces are stron- with decreasing the molecular weight, as shown in Fig. 1
gest, are weaker than primary binding forces, they play [4]. In addition, the solubility of half N-acetylated Ch
very important roles in the biological systems (e.g., molec- with the molecular weight lower than 10,000 is rather high
ular recognition, accumulation and interpretation of ge- even in aqueous dimethylacetamide and aqueous dimethyl
netic information, and formation of higher-order structure sulfoxide (DMSO) [5].
of proteins). Because Ch has an amino group in the repeating unit,
Biological systems comprise dierent kinds of macro- it aords ammonium group in aqueous acidic media.
molecules, such as nucleic acids, enzymes, proteins, and Owing to its cationic nature, Ch spontaneously forms
polysaccharides, and most of them have denite structure water-insoluble complexes with anionic polyelectrolytes.
and specic functions. Antigenantibody reactions and Therefore, Ch has been used mainly as a occulant for the
enzymic reactions, for example, are very specic. In these treatment of wastewater. However, it has recently been
cases, intermacromolecular interactions play important used in biomedical and pharmaceutical elds because of its
roles. Many bioreactions proceed via formation of macro- favorable properties of good biocompatibility, low toxici-
molecular complexes in the beginning of the reaction, and ty, and biodegradability.
the concerted interactions are included. As a simplied The intrinsic viscosity [g] of Ch depends on the pH and
model of these bioreactions, it is rational to investigate the the ionic strength, as shown in Fig. 2 [6]. If the pH of the
macromolecular complexes. The majority of researches on solution is increased, the intermolecular and intramolecu-
macromolecular complexes have been directed toward a lar electrostatic repulsions between cationic charges are
better understanding of biological systems and of structure reduced. This allows the Ch chains to come closer and thus
and properties of functional units. However, macromolec- lowers the hydrodynamic volume of the Ch molecules. This
ular complexes have recently found their way into practical eect may enhance the interchain and intrachain hydrogen
applications, in particular, as biomaterials. bonding. Similarly, as the ionic strength increases, [g]
679
680 Kubota and Shimoda
decreases due to the shielding eect of the counterions.

Although Colfen et al. suggested that the hydrodynamic
behavior of Ch was consistent with wormlike chain model
[7], the Ch molecule is rather sti. The exibility of some
polysaccharides has been investigated in terms of a sti-
ness parameter B (i.e., the dependence of the intrinsic
viscosity on the ionic strength). The B values for a variety
of polymers are compiled in Table 1. The B value of Ch was
estimated as 0.08 [8]. Terbojevich et al. obtained B values
from 0.043 to 0.091 for Ch with degrees of acetylation
ranging from 52.2% to 12.1% [9]. These values are essen-
tially the same as those for carboxymethyl cellulose and
hyaluronic acid, greater than DNA, and less than poly-
acrylate. These solution properties of Ch greatly aect the
formation of macromolecular complexes.
II. FORMATION OF MACROMOLECULAR

COMPLEXES OF CHITOSAN
The formation of macromolecular complexes in solution,
depending on the intensity of polymerpolymer and poly-
mersolvent interactions, leads to the separation of com-
plexes as a solid or liquid phase. Because Ch is a cationic
polyelectrolyte, most studies on the macromolecular com-
plexes of Ch are concerned with polyelectrolyte complexes
Figure 1 Dependence of water solubility of half N-acety- (PECs). However, only a few articles dealt with the poly-
lated Ch on the molecular weight. (From Ref. 4.) ionic interaction between polysaccharides, until Kikuchi
Figure 2 Eects of pH and ionic strength on intrinsic viscosity [g] of Ch solution: x, 0.050 mol/L; E
z, 0.075 mol/L; E, 0.100 mol/
L; n, 0.200 mol/L; ., 0.300 mol/L; z, 0.500 mol/L NaCl. (From Ref. 6.)
Macromolecular Complexes of Chitosan 681
Table 1 Stiness Parameter, B, for Ch and Some Polymers range interactions such as hydrophobic interactions and
hydrogen bonds.
Degree of acetylation
Polymer (%) B
A. Thermodynamics and Stoichiometry
Ch 12.1 0.091
20.1 0.060
of Complex Formation
42.1 0.061 The complexation reaction of macromolecules is signi-
52.2 0.043 cantly dierent from that of low-molecular weight mole-
Polyphosphate 0.44 cules due to the polymer eects by which a relatively stable
Polyacrylate 0.23 complex occurs, although the force of each bound pair is
Amylose xanthate 0.22
small. This implies that the apparent equilibrium constant
Carboxymethylamylose 0.20
of the polymerpolymer complex is extremely large and the
Carboxymethylcellulose 0.065
reaction is apparently irreversible. The free-energy change
Hyaluronic acid 0.07
Sodium pectinate 0.044 DGo for macromolecular complexation is a function of the
DNA 0.006 degree of polymerization, and the equilibrium constant
abruptly increases at the critical chain length [13]. Perez-
Source: Refs. 9 and 8. Reproduced by permission of Routledge, Inc., Gramatges et al. revealed that complexation of Ch with
part of The Taylor & Francis Group. poly(acrylic acid) (PAA) proceeded cooperatively and the
larger degree of conversion, h, was obtained for the higher-
molecular weight Ch [14]. The relationship between the
stability constant, K, and h (Fig. 3) indicates that this
rst reported the PEC containing Ch as a component [10]. complex consists of a long sequence of bound pairs of
Several reviews on PEC have been published so far [11,12]. repeating units. The slope of this curve shows the inuence
The formation of PEC is governed not only by the nature of of neighboring functional groups on their reactivity.
the individual polyelectrolyte components, such as charac- Kanbayashi and Arai determined thermodynamic
teristics of polyions (strong or weak), position of ionic sites, parameters for PEC formation in the methyl glycol Ch
charge density, molecular weight, exibility, functional (MGC)carboxymethyl cellulose (CMC) system [15]. The
group structure, hydrophilicity and hydrophobicity, and formation of PEC gained a large negative DGo, but the
stereoregularity, but also by the reaction conditions, such factors were dependent on the charge density of CMC. The
as pH, ionic strength, polymer concentration, mixing ratio, PEC formation using CMC with low charge density
and temperature. This, therefore, may lead to a diversity of showed a marked exothermic tendency and was classied
physical and chemical properties of the complexes. PECs as an enthalpy-dominating reaction. On the other hand, the
arise due to interactions between oppositely charged poly- reaction using CMC with high charge density showed a
mers and can be additionally stabilized through short- smaller exothermic tendency and gained large positive
Figure 3 Linear relationship between the stability constant, K, and the degree of conversion h for ChPAA complexes: D, Mv =
8.5 104; o, Mv = 1.1 105; 5, Mv = 2.3 105. (From Ref. 14.) Copyright 1996 Springer-Verlag.
entropy, indicating an entropy-dominating type. They also was observed. As shown in Fig. 4, pKa of Ch increased as
investigated the dependence of thermodynamic parameters the addition of the polyanion increased, suggesting that the
on ionic strength using the MGCpartially hydrolyzed charged carboxylate group induced the ionization of the
poly(acrylamide) system [16]. The degree of complexation amino group of Ch [22]. Therefore, the supernatant pH
did not change much with the ionic strength (0.0010.2), increased at pH 6 as the yield of the complex increased:
and a gain of a large negative DGo was accompanied by the
PEC formation. However, DHo became a large positive NH2 OOC H ! NH
3 OOC
value with increasing ionic strength. Therefore, the higher
ionic strength suggested the entropy-dominating-type PEC The supernatant pH decreased at pH 3 as the yield of
formation reaction. A much higher ionic strength aects complex increased:
PEC formation; for example, when Ch and n-carrageenan
NH
3 HOOC ! NH3 OOC H
(Car) were mixed in the presence of 5.7% NaCl at pH 2.8
on a boiling water bath, such a phase separation did not However, changes in the supernatant pH by complexation
occur, but a viscous and macroscopically homogeneous was dependent on the type of polyanions [23]. For ChCar,
PEC mixture was obtained [17]. The presence of Na+ and Chalginate (Alg), and Chpectin (Pec) complexes, no
Cl reduces the electrostatic attraction between oppositely signicant pH change occurred at the initial pH = 3. At
charged polyelectrolytes. This mixture gelled as its temper- pH 4.5, there was a slight increase in pH as a result of
ature or ionic strength decreased. complexation of Ch with Alg and Pec, and a more signif-
In general, the composition of the reaction mixture, Z, icant increase with Car. At pH 5.4, there was an increase in
is unity at the equivalence point, suggesting a 1:1 stoichi- pH analogous to that at pH 6 for the ChPAA system.
ometry for the complex, no matter what order of mixing is However, unlike PAA, a maximum pH change was main-
chosen. However, h varied with Z in the case of Ch tained in the wide mixing ratio from 0.2 to 0.4. Dierences
polygalacturonate (PGal) complex [18]. It fell from unity in polyanion conformation are responsible for the dier-
to 0.8 as Z increased from 0.2 to 0.5 and then rose again up ences in pH changes between ChPAA and ChCar, Ch
to 0.85 for Z = 1. If the complexation reaction is termi- Alg, ChPec complex formation. For example, structural
nated before a 1:1 stoichiometry of charge neutralization is similarity between Ch and Car, Alg, and Pec or exibility of
reached, water-soluble complexes are possible. In addition, PAA is possible.
when the PEC involves two polyelectrolytes of dierent Takahashi et al. observed the dierence in basic
molecular size, soluble nonstoichiometric PEC can be properties between ChAlg and ChPAA complexes [24].
formed. In such a complex, the larger polymer chain In the ChAlg system, the insoluble complexes were
behaves as a host macromolecule to the shorter one, and formed at a constant unit molecular ratio of 1:1.3 (Ch:Alg)
the host polymer should be a strong polyelectrolyte. For at pH 3.74.7. However, the unit molecular ratio of the
weak polyelectrolytes, not all of the repeating units need to ChPAA system was greatly aected by pH, showing a
have a 1:1 stoichiometry. change from 1:2.4 to 1:1.7 (Ch:PAA) with an increase in
pH from 3.7 to 4.7. They concluded that this was due to
B. Complexes with Polysaccharides the higher exibility of the polymer chain of PAA than
that of Alg.
The interaction between oppositely charged polyelectro- On the other hand, owing to the ease of interfacial
lytes involving weak polyacid and/or weak polybase is PEC formation, the tness of the structures of the back-
aected by the solution pH because of the change in the bones of Ch and some polyanions was estimated as fol-
degree of dissociation. In this sense, it is of great interest to lows [25]:
examine the properties of complexes involving weak poly-
electrolytes under various pH conditions. When Ch, a heparin Hep > Alg > carboxymethyl
weak polybase, is reacted with a weak polyacid, the insol- chitin CMChitin > PAA
uble complex formation occurs only in the narrow pH
range. Arguelles-Monal et al. reported the complex forma- Nakajima and Shinoda showed that the backbone chain
tion reaction of the ChCMC system [19]. At pH 3.6, the conformations of component polymers together with the
PEC was rich in CMC, whereas at pH 4.8 the excess kind and location of ionizable groups were important
component was Ch. At pH 4, the yield of PEC increased factors to discuss the formation and structure of PEC
with the addition of one polyelectrolyte solution to the [26]. They used glycol Ch (GC), which is water soluble at
other, and the maximum yield corresponded to the ratio all pHs, as a polycation and hyaluronic acid (HA), chon-
[CMC]/[Ch] = 1. Beyond this point, the yield remained droitin sulfate (CS), Hep, and sulfated cellulose (SCS) as
constant, and the composition of the PEC obtained showed polyanions. In the GCHA and GCCS systems, the
the stoichiometry. Similar results were obtained in the case experimental curves of complex composition, R, to pH
using PAA, a synthetic polymer, as a polyacid [20,21]. The crossed the theoretical curves at R = 0.5 (Fig. 5 shows the
mixing ratio Ch/(Ch + PAA) for maximum insoluble GCHA system as an example.) In both complexes, the
complex formation, Rmax, increased with increasing solu- neutral complex appears only at R = 0.5. The positive and
tion pH. Interestingly, at the initial pH = 6, the superna- negative charges may remain in the regions R < 0.5 and
tant pH of the reaction mixture increased as the complex R > 0.5, respectively. This result shows that the dominant
was formed. At the initial pH=3, the opposite behavior factor in these cases is the pyranose structure itself rather
Figure 4 Degree of ionization for amino groups of Ch in the presence of xanthan. The mole ratio of carboxyl groups to amino
groups: ., 0.30; E, 0.60; n, 1.19. (From Ref. 22.)
than the kind and location of the ionizable groups on the pH region. They suggested that one of the possible struc-
pyranose ring. In the case of the GCHep system, the tures having these compositions was the ladder form
discrepancy between theoretical and experimental curves sandwiching a Hep molecule between two GC molecules.
was rather small, and complex formation seemed to pro- Moreover, the experimental curve of the GCSCS system
ceed almost stoichiometrically. However, the complex was remarkably dierent from the theoretical curve, as
composition was GC/(GC + Hep) = 0.65 at the lower shown in Fig. 6. However, the complex composition at the
Figure 5 Composition of complex plotted against pH for GCHA system. (From Ref. 26.) Copyright 1976, reprinted with
permission from Elsevier Science.
Figure 6 Composition of complex plotted against pH for GCSCS system. (From Ref. 26.) Copyright 1976, reprinted with
permission from Elsevier Science.
lower pH region was the same value as that of the GCHep curves appeared at pH 4.5 at the higher substitution degree
system. In this case, it is thought that the GCSCS system is of N-propionyl, N-hexanoyl, and N-myristoyl. The higher
similar to the GCHep system in structure, but negative the acyl group, the stronger the eect, due to the insolu-
charges are always conserved. The reason for such dier- bility of the complexes and the hydrophobic interactions of
ence may be the location of the OSO 3 groups on SCS and N-fatty acyl groups. It is dicult to form a ladderlike
Hep molecules. Similarly, the theoretical and experimental structure in this system owing to bulky acyl groups.
curves of the mixing ratio intersect at 0.46, 0.52, and 0.53, Therefore, some of the free groups that are not involved
for the GCCMC, GCAlg, and GCPGal complexes, in the PEC formation may exist inside the complexes.
respectively [27]. On the contrary, in the case of the GC
dextran sulfate (DS) complex, the stoichiometric compo-
C. Complexes with Proteins
sition occurs at R = 0.62 at pH < 3.5. It was concluded
that the GCDS complex had a dierent structure from the Remunan-Lopez and Bodmeier investigated optimal con-
other three [28]. ditions for the complexation between Ch and type B
In the case of n-Car, K+ ions induce helix conforma- gelatin (Gel) [31]. All of the optimal preparation condi-
tion and promote helixhelix aggregation. The decisive tions were essentially coincident with those for Chpoly-
factor of the ChCar complex formation seems to be saccharide complexes. In addition, complexation was
whether n-Car is in a helixhelix aggregated state [29]. found to depend on the molecular weight of Ch and Gel;
The interaction between Ch and n-, L-, and E-Car in their higher-molecular weight Ch resulted in higher amounts of
coil or nonaggregating helical conformation normally complex formed, whereas higher-molecular weight Gel
resulted in the formation of PEC with stoichiometric solubilized the complexes.
charge ratios of unity. Nevertheless, the formation of When a protein reacts with an oppositely charged
PEC was signicantly aected by the fraction of n-Car in polyelectrolyte to form PEC, a conformational change
K+-induced helical conformation. If the n-Car exists in the occurs. Park et al. reported the conformational change of
helixhelix aggregated state then the interaction with Ch a-keratose (Ker) by complexing with Ch at pH 5.2 [32].
produces PEC with a charge ratio below unity, thereby The a-helical structure in Ker was transformed into a
providing a means of complexes with a surplus of negative random structure by complexing with Ch at Rmax, whereas
charge. In this case, Ch molecules act as binding elements the h-sheet was not aected. It seemed that helix-
between helixhelix aggregated n-Car. favoring amino acid residues, aspartic and glutamic acids,
Partially N-acylated Ch samples were used to examine participated in forming electrostatic linkages. As shown in
the eect of the N-acyl groups on the complex formation Fig. 7, the lower the molecular weight of Ch, the higher
with CS [30]. The Rmax values became larger with increas- the destruction of the a-helix in Ker. low-molecular
ing substitution degree of N-acyl groups but were not weight Ch seems to cause easier or complete complexa-
dependent on the kind of N-acyl group. Broad turbidity tion reaction.
Figure 7 Eect of molecular weight of Ch on the secondary structure of keratose at Rmax: (A) Mv = 5.91 105; (B) Mv =
4.83 105; (C) Mv = 1.91 105; (D) Mv = 1.08 105; (E) Mv = 0.31 105; n, a-helix; m, h-sheet; n, random structure.
(From Ref. 32.)
Taravel and Domard investigated the interaction be- of triple helices. The presence of an infrared (IR) absorp-
tween Ch and bovine atelocollagen (Col) in detail [33,34]. tion band at 1600 cm1, the shift of the amide I band, and
When a solution of ChHCl was added to a Col solution of the insolubility at pH 5.65.8 of this complex suggested the
pH 7.8, a pure PEC was formed. Interestingly, the aggre- formation of a hydrogen-bonding complex (HBC). HBC is
gation of the triple helices of Col was not inuenced by this often found in polymer blends, such as Chpoly(viny
interaction. During the formation of PEC, Col behaves like alcohol) (PVA) [36] and N-acetylated ChPVA systems
dispersions of encapsulated microgels. Accordingly, only [37].
some of the negative charges of Col can participate in the Hydrophobic interactions are also important in the
PEC. The weight proportion of Ch in these complexes was complexes with proteins. a-, h-,and n-caseins (Cas), which
14.2% and 10.2% for low-molecular weight and high- are phosphoproteins in milk, are precipitated by Ch [38].
molecular weight Col, respectively. These values are much NaCl up to 1 mol/L was ineective to prevent interaction
lower than that of the theoretical value (28.5%). This between Ch and Cas, and a nonionic detergent, Tween 20,
limitation is attributed to a competition between the for- up to 2% was unable to prevent the ChCas interactions.
mation of PEC and Col triple helices. Moreover, they However, the complex could be dissolved in a mixture of
attempted to prepare a 1:1 complex by two dierent increased NaCl and Tween 20. In this case, hydrophobic
methods [35]. The rst method was to form the complex and electrostatic interactions participate in the association
at a temperature higher than the denaturation temperature and coagulation of Cas with Ch. The fraction of hydro-
of Col to avoid gelation of Col. Denaturation allowed the phobic surface on a Ch molecule was estimated as
formation of a pure PEC with much higher Ch/Col ratio 51.5% at pH 6.3, and it increased slightly to 52.4% on
than the values obtained at lower temperature. However, a increasing the ionization degree to 100% [39]. Therefore,
theoretical complex was not formed, as denaturation was an increase in the ionic strength would reinforce the
not entirely completed. The presence of a large excess of Ch hydrophobic interactions between Ch and hydrophobic
was the second method. The solution of ChHCl was added residues of Cas. The complexation with Ch also imparts to
to a Col solution of pH 3.6. The solutions thus prepared faba bean legumin (FBL), a seed storage protein, the
could contain a great excess of Ch up to 1200%. By insolubility at the isoelectric point, pI = 5.2, and higher
creasing the pH of these mixture solutions to pH 5.8, the pH [40]. Only at pH > 7.0 did the ChFBL complex
complexation between the two polymers was achieved. A precipitate from the solution due to the insolubility of Ch,
large excess of Ch could destabilize the gelation of Col, and and the complex was stable even at high ionic strength as
Col did not precipitate in the intermolecular associations 1 M NaCl. This result points to a substantial contribution
of noncoulombic interactions to the ChFBL complex. DNA. For example, DNA in the complex could be
The increase in negative deviations of limited viscosity digested with a mixture of DNase I and phosphodiester-
numbers from additive values with increasing NaCl con- ase, and cytosine residues in the DNA (denatured DNA)
centration may be evidence for a substantial role of hydro- could be deaminated by treatment with sodium bisulfate.
phobic interactions. In addition, carcinogenic heterocyclic amines showed ad-
sorption to DNA and RNA in the complex. On the
D. Complexes with DNA contrary, it was reported that DNA complexed with Ch,
at all charge ratios, resulted in a signicant decrease in
As expected, Ch interacts very securely with DNA in degradation by DNase II; the lower the molecular weight
solution and causes DNA to be precipitated from solution. of Ch, the higher the inhibition eect [45].
This complexation is very similar to the Chpolyphosphate N-Dodecylated Ch (Ch12) also reacted with DNA to
(PP) [41], GCmetaphosphate (MP) [42], and MGCMP form a PEC [46]. Although DNA in the ChDNA complex
[42] systems. Phosphoric acid is a slightly stronger acid was not suciently protected when it was exposed to
than carboxylic acids. Therefore, ChDNA complex is DNase, DNA in the Ch12DNA remained intact due to
stable in 0.1 mol/L HCl, but unstable in 0.1 mol/L NaOH. the protection from nuclease oered by Ch12. In addition,
Kendra and Hadwiger showed that the Ch must be 7 or complexation with Ch12 enhanced the thermal stability of
more sugar units in length both to optimally induce plant DNA. However, the complex was dissociated by the addi-
genes and to inhibit fungal growth [43]. This length re- tion of microions; the ability of Mg2+ to break the PEC
quirement suggests that a series of positive charges match was greater than that of Na+ and K+. This is related to the
up with phosphate negative charges in the grooves of the dierent anity of ions to DNA; Mg2+ has a much higher
DNA helix in the B form. anity to DNA compared with Na+ and K+ [47].
Hayatsu et al. added Ch solution (pH 5) to the solu- After complexation between Ch and DNA, phase
tion of DNA, RNA, and homopolynucleotides (pH 7.2) separation occurs to yield coacervates that represent the
to form insoluble complexes [44]. The double strandedness aggregated colloidal complexes. The ChDNA particles
of DNA was retained in the complex because the PEC had a negative surface charge when the complexes were
preparation process is mild enough not to inict any made at N/P ratio below 2, and became positively charged at
damage on DNA. In this system, DNA molecule was N/P ratio above 2 through neutral at N/P = 2, as shown in
accessible to enzymes and reagents having an anity to Fig. 8 [48]. The pH of the solution at 55.8 and a temperature
Figure 8 N/P ratio dependence of zeta potential of ChDNA complex. Measurements were performed with 20 mg of DNA in
1 mL of 0.15 mol/L NaCl. (From Ref. 48.)
Figure 9 Eect of pH on zeta potential of ChDNA nanoparticles. (From Ref. 49.) Copyright 2001, reprinted with permission
from Elsevier Science.
of the solution above 50jC resulted in ChDNA nano- E. Ternary Complexes

particles [49]. The size of ChDNA nanoparticles was
optimized to 150250 nm with a narrow distribution at N/ Kikuchi and his coworkers reported complexes consisting
P ratio between 3 and 8 and Ch concentration of 100 Ag/mL. of three polyelectrolyte components, strong polybase
The zeta potential of the ChDNA nanoparticles was +22 weak polybasestrong polyacid system and strong poly-
to +18 mV at pH < 6.5, and decreased dramatically to 20 basestrong polyacidweak polyacid system.
mV at pH 88.5, as shown in Fig. 9. At pH 7.07.4, the In the MGCGCpoly(vinyl sulfate) (PVS) system,
nanoparticles appeared to be electrostatically neutral and the molar ratio, S(PVS)/N(MGC+GC), to form insoluble
may oer an eective protection to the encapsulated DNA complexes decreased with increasing solution pH [50,51].
from nuclease degradation. Experimental conditions and results of elemental analyses
Table 2 Reaction Conditions and Elemental Analyses of MGCGCPVS Complexes

Reacting conditions
Molar ratio Molar ratio

in mixture Sulfur Nitrogen in PEC
Samplea [H+] (S/N) (%) (%) (S/N)
1-A 7% HCl 2.00 6.70 2.63 1.11
1-B 4% HCl 1.60 7.02 2.98 1.03
1-C 1% HCl 1.20 8.15 3.13 1.14
1-D pH 2.0 1.00 8.12 3.18 1.12
1-E pH 6.5 0.80 8.10 3.04 1.16
1-F pH 13.0 0.70 6.82 2.88 1.03
2-A 7% HCl 2.00 6.83 2.08 1.43
2-B 4% HCl 1.60 6.93 2.75 1.10
2-C 1% HCl 1.20 7.68 3.10 1.08
2-D pH 2.0 1.00 8.17 3.22 1.11
2-E pH 6.5 0.50 6.51 3.19 0.89
2-F pH 13.0 0.50 8.19 3.13 1.14
a
Series 1: PVS solution was added dropwise to MGC + GC solution. Series 2: MGC + GC solution was added dropwise to PVS solution.
Source: Ref. 51. Copyright n 1988 Wiley. Reprinted by permission of John Wiley & Sons, Inc.
for each PEC prepared are given in Table 2. The compo- ization of acrylic acid (AA) and sodium 4-styrene sulfonate
sitions of sulfur and nitrogen are almost the same. Because (NaSS), respectively, in the presence of Ch as a template at
coagulation did not occur at pH > 6.5 in the GCPVS pH 44.5 [56]. It was concluded that the template polymer-
system [52,53], only MGC was expected to react with PVS ization technique appeared advantageous only for the
in the solution of pH > 6.5. However, in IR spectra, the synthesis of the ChPAA complex. The molecular weight
absorption band at 1540 cm1 assigned to NH+ 3 in GC of PAA obtained increased with increasing molecular
was present in the PEC except for the PEC prepared at pH weight of Ch, as shown in Table 3 [57].
13.0, and the absorption band assigned to NH2 that However, the structure and conformation of Ch mol-
should appear at 1600 cm1 was absent. This is due to ecule, in which NH+ 3 groups are separated from each
the strong induction eect by charged PVS as described other by a sequence of four atoms and oriented towards
above, because the absorption band at 1230 cm1 assigned opposite directions in space, are disadvantages for the
to OSO 3 in PVS was present in each PEC. coordination of AA and NaSS molecules. Therefore, the
The MGCpoly(L-glutamate) (PLG)PVS system is eect of Ch as a template seems to be quite poor in
more convenient for investigating the composition of the comparison with that of vinyl polymer such as polyallyl-
complexes by IR spectrometry, as each component in this amine. At least the rst step of this template polymeriza-
system has a dierent reactive group [54]. The intensity of tion is not an ionic preadsorption of monomer molecules
the IR absorption based on the COO group increased onto Ch template, but the formation of a growing oligomer
with increasing pH from 2.0 to 5.0, and was constant at pH in the solution, which complexes with the template only
6.013.0. These results inferred that PLG was absent in the beyond a critical chain length. In other words, an oligomer
PEC at pH < 2, increased with increasing pH from 2.0 to formation seems more favored than the polymerization of
5.0, and was constant at pH > 6.0. The intensities of the monomer molecules adsorbed onto Ch molecules.
absorption band around 1240 cm1 assigned to the OSO 3 ChPAA complex nanoparticles were also prepared
groups of PVS for the PEC prepared at pH < 2.0 were by template polymerization of AA in Ch solution [57]. The
medium, and those for the PEC prepared at 2.0 < pH < diameter of the nanoparticles was 200300 nm, and the
5.0 were weak. The absorption band around 1740 cm1 surface of the nanoparticles had positive charges. At pH
assigned to the COOH group of PLG was present in the 7.4, PAA was highly extended while Ch was insoluble,
PEC prepared at pH 4.0 and 5.0. These results were resulting in the phase separation of nanoparticles where
identical with those of the elemental analyses. This kind Ch was coated on the nanoparticles. These nanoparticles
of PEC was also prepared in the MGCcarboxymethyl seem to form a sort of coreshell structure.
dextran (CMD)PVS system under various pH conditions
and in a dierent order of mixing, and similar results were
obtained [55]. The PECs obtained are classied into three III. PROPERTIES OF MACROMOLECULAR
groups by means of elemental analysis, IR spectroscopy, COMPLEXES OF CHITOSAN
and solubilities. The rst is the group of PECs prepared at
pH < 3, which are composed of MGC and PVS connected The properties of macromolecular complexes depend not
by ionic bond with each other. The second is the group of only on the component polymers but also preparation
PECs formed at pH > 6.5, which are made up of MGC, conditions as mentioned above. The stoichiometric com-
CMD, and PVS linked by ionic bond with one another. The plexes are generally insoluble and make hydrogel.
last is the group of PECs prepared at intermediate pH,
which also constitute MGC, CMD, and PVS, but have a A. Swelling Properties
more complex three-dimensional network structure.
When weak polyelectrolytes are involved in the PEC, the
F. Complexes by Template Polymerization swelling ratio of the PEC depends on the environmental
pH. Figure 10 depicts the swelling behavior of the ChCar
Water-insoluble PECs of ChPAA and Chpoly(styrene complex gel in the various pH media [17]. Similarly, ChDS
sulfonate) (PSS) can be obtained from the radical polymer- gel swelled at pH 1012, and took the maximum volume at
Table 3 Relationship of the Molecular Weight of Ch and PAA
Molecular weight Polymerization Molecular weight Yield of Yield of

of Ch time of PAA PAA nanoparticles
Mw (h) Mn (%) (%)
40,000 2 367 85 70.0
80,000 2 1087 83 68.6
100,000 2 2138 72 62.3
200,000 2 4526 78 63.1
300,000 2 8026 71 60.0
Source: Ref. 57. Copyright 2002, reprinted with permission from Elsevier Science.
Figure 10 Equilibrium swelling ratio of ChCar complex gel: o, HClaq.; 5, NaOHaq.; D, KOHaq. (From Ref. 17.) Copyright n
1993 Wiley. Reprinted by permission of John Wiley & Sons, Inc.
pH 10.5 [58]. These are weak basestrong acid-type PECs. group. Furthermore, if the complex gel has net negative
In acidic and neutral solutions, amino groups remain charge, anions including OH will be excluded from the
positively charged, and the electrostatic bond between complex gel. Then the internal pH is lower than the

NH+ 3 and OSO3 groups remains, resulting in no swell- external pH, and the complex gel does not swell even at
ing. If the complex is immersed in an alkaline solution, the pH 9 [58]. At pH > 10.5, the equilibrium swelling ratio
amino group is deionized, although the sulfate group holds decreased with increasing pH, probably due to the increase
the negative charge. Therefore, the electrostatic bond in ionic strength of the external solution with increasing
between the two functional groups disappears, as shown pH. However, in a KOH solution, the equilibrium swelling
in Fig. 11. Generally, swelling of ionic gels is explained by ratio of the gel was smaller than that in the NaOH solution,
the dierence in osmotic pressures due to ionic solutes in because K+ has stronger anity than Na+ to sulfate
the gel and in the ambient solution. Na+ attracted by the groups (Fig. 10). Sakiyama et al. also prepared ChDS
negatively charged sulfate group increases the osmotic complex gel containing more amino groups than sulfate
pressure of the gel. Thus, the swelling of the gel occurs. groups [60]. In this case, part of the protonated amino
According to this mechanism, the complex gel is expected groups remained free from electrostatic interaction with
to swell at pH above 6.3, pKa of Ch [59]. However, the gel sulfate groups. When the molar ratio of amino group to
did not swell even at pH 9. This discrepancy suggests the sulfate group was higher than unity, the equilibrium vol-
induction eect of the ionized sulfate group on the amino ume at pH 10.5 was low.
Figure 11 Schematic representation of swelling mechanism of ChCar complex gel.

A PEC gel of the weak baseweak acid system was also enced not only by the diusion of mobile ions but also by
made from Ch and xanthan (Xan), which has a carboxyl other factors such as the charge state of Ch in the complex
group [61,62]. When the complex is placed in acidic or basic gel [64].
media, dissociation of the intermolecular bonds takes place Dumitriu et al. discussed the inuence of the acetyla-
according to: tion degree and molecular weight of Ch as well as pH on the
swelling behavior of the ChXan complex [65]. The com-
COOH H3 N p COO H3 N plexes derived from Ch with acetylation degrees of 10%
and 28% swelled with very similar proles, whereas the
! COO H2 N
complex from Ch having the acetylation degree of 34%
The swelling ratio of the ChXan complex was greater showed a distinctly lower swelling degree. This could be
than that of the ChCar complex, and this PEC is explained considering that the lower the degree of acetyla-
considered one of the amphoteric gels. As expected, in tion, the more ionic linkages occurred, resulting in a denser
addition to the pH range 1012, the gel swelled at pH < gel that has the potential to absorb increased amounts of
1.5 and dissolved in the pH range 0.21.0 in the course of water. On the other hand, the molecular weight of Ch had a
swelling. With the increase in concentration of NaCl, the strong inuence on the water absorption; the lower the
equilibrium swelling ratio decreased, and dissolution of molecular weight, the higher the swelling degree. Yao et al.
the gel was depressed. Furthermore, the pH values at the prepared the PEC membrane consisting of Ch and Pec
maximal equilibrium swelling in alkaline and acidic sol- using formic acid as the casting medium, and obtained a
utions were shifted to the neutral side in the presence of similar swelling behavior [66]. With decreasing acetylation
NaCl. Unlike the neutral gel, the pH-sensitive swelling degree of Ch, the swelling degree of the complex increased
rate of the ChXan complex gel can be described by the in acidic and alkaline regions. When the complex was
collective diusion of the gel network, which is based on prepared with Pec of higher methoxylation degree, the
the diusion of a polymer network, but it is not controlled complex swelled largely over the whole pH range. With
by the cooperative diusion of the network, which reects increasing preparation pH, the swelling degree of the
the concentration uctuation of the network in the gel, complex increased in the acidic range and decreased in
because the swelling rate of an ionic gel was inuenced not the alkaline region.
only by the diusion of network but also by the diusion Arguelles-Monal et al. reported the dependence of
of mobile ions and the dissociation of functional groups swelling of the ChCMC complex membrane on pH [67].
on the polyelectrolytes [63]. As a result, the swelling rate They found that the swelling of the membrane in water
of the ChXan complex gel placed in a solution of pH 10 exhibited a characteristic pattern; the membranes
after previous equilibration at pH 11 was dominated by adsorbed water until a maximum swelling was achieved,
the diusion rate of such mobile ions as Na+ in the gel after which the membranes slowly shrank to an equilibrium
rather than by cooperative diusion of the network, value. The maximum swelling value, time, and rate in-
whereas the swelling rate of the gel placed in the solution creased as the preparation pH increased from 4.2 to 5.7,
of pH 10 after previous equilibration at pH 7 was inu- indicating that the PEC prepared at pH 5.7 had more free
Figure 12 Temperature dependence of apparent diusion coecients for ChCMC complexes: o, PEC prepared at pH 4.46; F,
PEC prepared at pH 5.51. (From Ref. 68.) Copyright Society of Chemical Industry, reproduced with permission. Permission is
granted by John Wiley & Sons, Ltd. on behalf of the SCI.
ionic groups than that formed at pH 4.2. The shrinkage

observed could be accounted for by the formation of new
electrostatic bonds. PEC formation and its consequent
precipitation occur very rapidly and, as a result, the
polymer chains of the PEC become trapped in arrange-
ments that are not the equilibrium ones. By removing salt
from the complex in water, the electrostatic attraction is
restored and results in the formation of new bonds [61].
Polymer chains in PEC having electrostatic interactions
with another polymer chain experience a restriction in
mobility due to ionic cross-linking. However, the segmen-
tal mobility of the polyelectrolyte chains in the swollen
state may be sucient to allow adequate spatial rearrange-
ments of the reacting groups.
The sorption of water vapor by the same PEC mem-
branes was not a Fickian process [68]; the PEC exhibited a
linear dependence of water uptake on the time during the
rst 120 min. This is a characteristic feature that occurs
when the diusion rate and the mobility of the penetrant
are much higher than the segmental mobility of the poly-
mer. It ceased at the moment when the diusion fronts
advancing from the opposite surfaces met at the center of
the membrane. After that, the swelling of membranes
obeyed second-order kinetics. The pseudo-zero-order rate
constants were almost the same, but the pseudo-second-
order rate constant of the PEC prepared at pH 4.46 was
almost ve times as large as that of the PEC prepared at pH
5.51. From an Arrhenius plot of the diusion coecients,
the apparent activation energies were estimated as 30.3 kJ/
mol for pH 5.51 and 47.0 kJ/mol for pH 4.46. The higher
activation energy for the PEC prepared at pH 4.46 was Figure 13 Degree of swelling of ChCMC and ChPLG
expected from the more cross-linked structure resulting complex lms in water: o, ChCMC; ., ChPLG. (From
from the intermacromolecular bonding as compared with Ref. 70.)
the PEC prepared at pH 5.51. In addition, the abrupt
changes in the slope for both PECs suggest that these PECs
possibly experience some sort of conformational transition prepared by casting an aqueous formic acid solution, and
at 60jC (Fig. 12). similar results were obtained [70] (Fig. 13).
Ichikawa et al. reported the water-sorption behavior The ChPAA complexes also swell at pH 11 and
of the ChCMC complex obtained by changing the mixing dissolve at pH < 2. To make the ChPAA complex
ratio [69]. PEC lms were prepared by casting clear so- insoluble at acidic pH, Lee et al. prepared an interpene-
lutions of Ch and CMC in aqueous formic acid. The degree trating polymer network (IPN) hydrogel by UV irradiation
of swelling in water was signicantly aected by the ratio of to the mixture of AA and Ch [71]. In this way, they could
the amounts of the amino group and the carboxyl group, as avoid the precipitation problem by mixing two oppositely
shown in Fig. 13; the larger the dierence in the amount of charged polymers. Ch:PAA = 50:50 complex showed the
the two groups, the larger the degree of swelling. The highest swelling ratio in an acidic solution, whereas
addition of electrolytes had little inuence on the swelling Ch:PAA = 18:83 complex had the highest swelling ratio
of the neutral PEC lms, whereas in the case of Ch-rich and in a basic solution.
CMC-rich lms, the degree of swelling decreased remark-
ably with the addition of electrolytes. Moreover, the CMC- B. Solubility
rich complex lms immersed in aqueous solution contain-
ing Ca2+ ion exhibited a volume collapse. This is due to the Stoichiometric PEC of strong polybasestrong polyacid is,
existence of a specic eect of Ca2+ on the carboxylate generally, insoluble in water and common organic solvents,
groups in PEC, possibly by the formation of Ca2+ com- but soluble in specic ternary solvent mixtures consisting
plexes. This eect increases with increasing pH of PEC of salts, water, and water-compatible organic solvents.
formation, since at higher pH a greater amount of carbox- This phenomenon is explained by the cooperative eect
ylate groups exists due to the lesser complexation of the that microions weaken the electrostatic interactions and
PEC formation reaction. Repeated soaking of the lm in organic solvents weaken the hydrophobic interactions
water removed the water-soluble components from the between macromolecules. Phase diagrams for the MGC
lms, suggesting that electrostatic bonds were formed. PLGPVS system in the ternary solvent of NaBr/acetone/
Clear PEC lms consisting of Ch and PLG were also water are shown in Fig. 14 [54]. Phase diagrams were not
drawback. Soil lamentous fungi grew on PEC ber

consisting of Ch and gellan gum (GG), and some of the
fungi, especially Aspergillus oryzae, Penicillium caseico-
lum, and Penicillium citrinum, degraded the PEC bers
into CO2 [74]. The productive secretions of hydrolytic
enzymes, such as polysaccharide lyase, from the microor-
ganism during their growth caused degradation of the
PEC. The total CO2 production and biodegradation indi-
cate the biodegradability of the PEC. On the contrary,
because the interactions between Ch and CS are very
strong, the production of disaccharides from the ChCS
complex with condroitinase at pH 7 and 7.5 occurred to a
lesser extent as compared with that from CS alone [75].
This result suggests that Ch protects CS from depolymer-
ization at the physiological pH.
Ch in the interaction with Col aects the hydrolysis of
Col by inhibition of the recognition process or by direct
interaction with the enzyme. Taravel and Domard de-
Figure 14 Phase diagram of MGCPLGPVS complexes in scribed the stability of the two kinds of ChCol complexes
ternary solvent of NaBr/Acetone/H2O at 30jC: o, 7% HCl; (PEC and HBC) toward collagenase, as well as their
D, pH 6; 5, pH 8; q, pH 11; ., pH 13. (From Ref. 54.) mechanical properties [76]. As for the enzymatic results
Copyright n 1985 Wiley. Reprinted by permission of John obtained for the PEC, the behavior was quite similar to that
Wiley & Sons, Inc. observed with pure Col, although Ch stabilized the triple
helix and thus protection toward the proteolytic enzyme
could be expected. On the other hand, when HBC was
formed, increase of the content of Ch in the HBC improved
obtained for the complexes prepared at pH 2.0, 4.0, and the stability of the complex. This kind of interaction
5.0. The complexes prepared in 7%, 4%, and 1% HCl certainly resists the recognition of the Col structure by
solutions were partially soluble. The complexes prepared at collagenase because of denaturation of the triple helices,
pH 6.0, 8.0, 11.0, and 13.0 were soluble. These results which is less sensitive to collagenase. This kind of complex
suggest that the PEC prepared at pH 2.05.0 are almost could be of great interest for biomedical applications (e.g.,
neutral and include hydrogen bonding. Similar results were long-lifetime biomaterials). However, the presence of Ch
obtained in the MGCCMDPVS system [55]. did not show a hardening of the material, but a softening.
On the other hand, nonstoichiometric PEC can be For example, tensile strength is lower, the Youngs mod-
soluble in water and waterorganic solvent mixtures. The ulus is less than half the values for the polymers alone, and
behavior of the ChDS complex solutions was strongly strain at break is higher.
dependent on both the nature and the concentration of the
microions added [72]. The dependence of the relative
turbidity of the PEC solution on the concentration of NaCl IV. APPLICATION OF MACROMOLECULAR
had a maximum at 0.9 mol/L. Moreover, the cations COMPLEXES OF CHITOSAN
showed the following order of potency in terms of their
ability to increase the solubility: K+ > Na+ > Li+. This Weak polyelectrolyte-containing complexes may aord
tendency seems to be related to dierences in the anity of particularly interesting materials for biomedical uses from
these counterions to sulfate groups of DS. The solubility of the viewpoint of structural resemblance to various macro-
PEC in organic solvents could be increased by adding water molecular complexes in the living systems. The important
and by increasing the temperature. The highest solubility of factors for biomedical materials are practical character-
the PEC was observed in aqueous DMSO (water:DMSO istics, medical functionality, and biocompatibility.
= 30:70). In addition, the nature of microions present in
the solutions remarkably
p
inuenced the mean square iner- A. Antithrombogenic Materials
+
tia radius, R2, of the complex [73].
pThe change of Na for
+ 2
Li caused a drastic increase of R . Evidently, chaotropic It is possible that the ionic group on Ch may exhibit a
properties and high dissociation constants of lithium salts biological activity in contact with blood. However, whole
prevent intramolecular association and promote electro- blood is slightly basic and the chance for protonation of the
lytic swelling of PEC particles. amino group of Ch is rather small. Perhaps this is why the
polycationic character of the Ch surface is not decisive with
C. Biological Stability respect to its activity on blood clotting. Kikuchi and Noda
prepared a series of the PECs containing Ch as a compo-
The complex consisting of natural polymers is easily nent and investigated their antithrombogenic properties.
degraded in biological media. Depending on the circum- They reacted a dilute acetic acid solution of Ch with the
stances, this can be considered as either an advantage or a aqueous solution of Hep, which is known for its high
antithrombus activity, to form an insoluble precipitate exhibited a maximum on the PEC of N/S=0.11 [72]. It
[77,78]. The compositional ratio of Ch to Hep (N/S) in seems that the high anticoagulant activity of the PEC is
the complex was changed from 0.34 to 1.18 by changing the underlain by optimum density and distribution of sulfate
mixing ratio. A blood-clotting test was performed on groups on the PEC.
sample tablets of N/S = 1.06 and 0.57 by measuring, Kikuchi et al. also reported antithrombogenic prop-
gravimetrically, the amount of thrombus formed. A rm erties of Chhigh-molecular weight CMD [82], Chlow-
clot was not formed for 10 days (N/S = 1.06) and 20 days molecular weight CMD [83], and ChCMC complexes
(N/S = 0.57). Some of plasma proteins are active in [84,85]. The amount of thrombus on each complex is
detaching of Hep from the complex. Fibrinogen and smaller than that on the glass. The mechanism for the
transferrin are known to have high anity to Hep; there- suppression of clotting suggests the possibility that neg-
fore, it is also possible that these proteins may win with Ch ative charges such as the carboxyl group may exist on
in the competition for the antithrombus. the surface of the complexes because the physiological
DS was developed as an alternative of Hep and shows pH is 7.4.
antithrombus properties. Kikuchi and Fukuda prepared Biocompatibility largely depends on the surface prop-
ChDS complexes at various pHs and mixing ratios erties. Surface grafting on polymeric materials is an impor-
[79,80]. The mole ratios of N/S in the complexes were tant method for changing surface properties. One
estimated to be 0.792.54. The complex prepared at pH approach to develop antithrombogenic materials is surface
6.5 (lower N/S ratio) suppressed the clotting of blood heparinization. Uragami et al. prepared anticlotting active
considerably rather than that prepared in 1% HCl (higher PEC membranes from quaternized Ch and Hep [86].
N/S ratio). However, their antithrombogenic activity was Quaternized Ch membrane was previously cross-linked
not higher than ChHep complex. They also discussed the with ethylene glycol diglycidyl ether and then immersed
dependence of the clot-inhibiting property on the molecu- in Hep solution. No thrombus was macroscopically ob-
lar weight of DS [81]. The complex consisting of Ch and served on the ChHep membranes in vivo. Aiba et al.
lower-molecular weight DS (6000) revealed more clot immobilized antithrombogenic PEC on the surface of a
inhibiting than higher-molecular weight DS (640,000). It styrenebutadienestyrene block copolymer lm [87]. By
seems that the antithrombus properties may be due to the irradiation of UV light, partially N-acetylated Ch was
leaking of DS from the surface. In addition, it was found covalently immobilized onto the lm using the photosen-
that the PECs prepared in the mixing order of DS solution sitive methyl 4-azidobenzoimidate, which was previously
to Ch solution are more clot inhibiting than those prepared attached to the amino group of Ch, and then it was treated
in the reverse mixing order, as shown in Fig. 15. When the with Hep. It was revealed that Hep immobilized on the lm
N/S ratio was further decreased, the anticoagulant activity surface was removed immediately but not completely. This
method is considered to be an eective means of preparing
biomaterials that are used in contact with blood.
Clot formation is also inuenced by the proper hydro-
phobicity and electronegativity on the surface of the mate-
rials. It was found that there was some correlation between
the wettability of several Ch derivatives and their throm-
boresistance by coating glass surfaces [88]. The complex
with partially cross-linked PAA (Carbopol 934) was a
stable system that did not dissociate easily and had much
better wettability than a siliconized glass surface, but the
corresponding clotting-time ratio was not higher than that
for the surface of Ch coating alone. On the contrary, the
surface complexation with PVS could improve anti-
thrombogenic activity of Ch. Zou et al. prepared the
ChPVS complex by soaking Ch lm in PVS solution at
pH 3.5 and obtained better antithrombogenic activity than
did Ch alone [89]. With the increase of the S/N ratio of the
surface element, antithrombogenic activity was increased.
This is due to the increase in the concentration of OSO 3
on the surface.
B. Controlled-Release Formulation
Figure 15 Percentage of clot formed on ChDS complexes Controlled release of drugs has been benecial to attain
compared with that on glass after a lapse of 20 min. Storage their slow, and thereby prolonged, release. The dried
time of blood is 7 days: ., Ch was added to DS in 2% HCl complexes of ChPec and Chacacia (Aca) systems were
solution; o, DS was added to Ch in 2% HCl; E, glass. used as tablet materials for chlorpromazine [90]. Unfortu-
(From Ref. 81.) Copyright n 1978 Wiley. Reprinted by nately, these dried complexes were not superior to any of
permission of John Wiley & Sons, Inc. the individual polymers in retarding drug release because
the preformed and completely dried complexes showed complex of a 2:1 (+:) charge ratio was most stable. In
poor swelling degree. On the other hand, the physical vitro transfection of Cos-1 cells, the highest level of expres-
mixtures of Ch and either Pec or Aca resulted in tablets sion in the absence of serum, was obtained using the
with retarding drug release, suggesting that interpolymer complex made with 102-kDa Ch, but was approximately
complexes were formed during the dissolution process. 250-fold lower than that observed with Lipofectaminek.
Takayama et al. investigated drug-release phenomena of The ChDNA complex containing a pH-sensitive endo-
tablets consisting of the physical mixture of Ch and HA somolytic peptide enhanced the levels of expression four-
[91]. The release rate of Brilliant Blue from the ChHA fold. Furthermore, expression of this complex in rabbits
mixture (3:7) tablet was greatly decreased, suggesting a after administration in the upper small intestine and colon
possible PEC formation between Ch and HA in the tablet was observed.
following water penetration into the tablet. Sato et al. pointed out that pH condition was very
Silk peptide as a model drug was loaded into ChPAA important to achieve high transfection eciency; the
nanoparticles prepared by template polymerization of AA optimal pH was 6.9 [95]. At pH below 7, the ChDNA
in Ch solution [57]. The release of silk peptide from the complexes are positively charged, as shown in Fig. 9, and
nanoparticles depended greatly on the pH. At pH 4.5, there can bind with the negatively charged cells. Accordingly,
was very limited swelling, and silk peptide in the nano- Ch may be available to deliver gene into tumor cells in
particles could not be released easily. At pH 7.4, the nano- vivo, because the extracellular pH of tumor sometimes is
particles were swollen to a great extent, resulting in a fairly around 67, which is lower than that of normal tissue.
fast release of silk peptide. At pH < 4, however, the They also concluded that the transfection level was highest
nanoparticles dissolved quickly, and silk peptide release when the Ch molecular weight was 40 kDa, N/P ratio was
rate was very fast. 3, and transfection medium contained 10% serum [93].
In addition to swelling, shrinking of the swollen gel The promotion of gene expression in the presence of serum
also exhibits a promotion eect on the release of drugs suggests that Ch might possess an ecient in vivo gene
loaded into the gel by the squeezing eect [60]. The transfer capability.
promotion eect of the ChDS gel on the release of high- Transfection ability of luciferase plasmid complexed
molecular weight dextran (Dex) was pH dependent; the with Ch was cell type dependent [48]. The complex e-
ChDS gel released Dex more rapidly at pH 8 than at pH 2 ciently transfected HeLa cells, independently of the pres-
because the degree of shrinking was greater at pH 8. ence of 10% serum, while the complex was inecient at
However, the release rates at both pHs became closer for transfecting BNL CL.2 or HepG2 cells. In addition, the
low-molecular weight Dex because the PEC network is transfection eciency of ChDNA nanoparticles was
rather loose for low-molecular weight Dex. investigated by using several cell lines [49]; higher gene
expression levels were found in HEK293 and IB-3-1 cells
C. Gene Carriers as compared with those in 9HTEo and HeLa cells (Fig.16),
which diered from the result obtained above. Further-
Ch is useful as a nonviral vector for gene delivery, which more, conjugation of KNOB, which is the C-terminal
has several advantages such as low cost, noninfective, globular domain of the ber protein on adenovirus capsid,
absence of immunogenicity, good compliance, and the resulted in 130-fold increase in the transfection eciency
possibility of repeated clinical administration. To achieve in HeLa cells, whereas only severalfold in HEK293 cells.
ecient gene delivery via encapsulation of DNA into a Ch can be a useful oral gene carrier because of its
polycation, two basic requirements must be met: DNA mucoadhesive property [96]. Oral administration of the
protection from nuclease attack and eective dissociation ChDNA nanoparticles containing a dominant peanut
of DNA from its complex [46]. allergen-gene to mice produced secretory IgA and serum
Mumper et al. rst described the possibility of Ch IgG2a and showed a substantial reduction in allergen-
DNA complexes as carrier for gene delivery [92]. In this induced anaphylaxis [97].
case, Ch can compact DNA and mask its negative charges, However, ChDNA nanoparticles were prone to ag-
and the cationic complexes obtained in the large N/P ratio gregation when stored at room temperature or at 4jC over
are uptaken by cells through the electrostatic interaction a couple of days. It was revealed that PEG conjugation
because the cell surface is negatively charged. After uptake, eliminated aggregation of the nanoparticles in solution and
ChDNA complexes are endocytosed and possibly re- kept them aggregation free even after lyophilization [49]. In
leased from endosomes due to swelling and rupture of addition, the dried particles were easily resuspended in
endosome [93]. The ChDNA complexes have high pro- saline or PBS, even after storage over 1 month, and the
ton-accumulation ability, and such buering eect of the PEGylation did not aect the transfection potency for at
complexes may perturb the endosomal membrane and least 1 month in storage. When injected intravenously, the
release the complexes from endosomes as the pH of endo- clearance of the PEGylated nanoparticles was slightly
somes decline. They also showed that parameters such as slower than that of the unmodied nanoparticles.
Ch molecular weight, DNA concentration, and charge DNA can be coated on the preformed cationic nano-
ratio inuenced the stability of the ChDNA complexes particles consisting of Ch and CMC [98]. The obtained
[94]. The complexes made with higher-molecular weight Ch DNA-coated nanoparticles were stable and applied topi-
were more stable to salt and serum challenge, and the cally to the skin of shaved mice, and resulted in detectable
Figure 16 Transfection of HEK293, IB-3-1, 9HTEo, and HeLa cells with ChDNA nanoparticles containing pRE-luciferase
plasmid. (From Ref. 49.) Copyright 2001, reprinted with permission from Elsevier Science.
and quantiable levels of luciferase expression in skin D. Membrane Application

after 24 h.
Gene therapy by delivering genes to target cells is an The best-known application of PEC is in the preparation of
interesting topic. Murata et al. reported the enhanced membranes. It has been stressed that the main expectation
cellular recognition ability when using quaternary Ch of PEC membranes is the possibility to control the rate and
containing pendant galactose residues [99]. They pre- selectivity of uxes for solutes by changing the chemical
pared an N,N,N-trimethylchitosangalactose conjugate and physical properties of the membrane induced by
(gTMCh) and complexed the resulting gTMCh with changes in local conditions such as pH.
plasmid DNA to achieve an ecient gene delivery via If PEC contains a strong polyelectrolyte such as PVS,
receptor-mediated endocytosis. The gTMChDNA com- its ionic bond is maintained at acidic and neutral pHs. The
plex could interact with APA lectin, which is known to GCPVS membrane was prepared at pH 3 and the possi-
bind specically to galactose or the N-acetylgalactos- bility of a permeability control was investigated [102]. The
amine unit, and increase h-galactosidase activity in GCPVS membrane obtained was quite sturdy and resem-
HepG2 cells. bled cellophane. The permeability of solute through the
To prepare a hepatocyte-targeting DNA carrier, Park membrane can be estimated by the equation derived from
et al. coupled Ch with lactobionic acid bearing galactose, Ficks law of diusion [103]. Fig. 17 shows that the GC
and the resulting galactosylated Ch (gCh) was further PVS membrane can control the permeabilities of KCl,
grafted with hydrophilic Dex (gChDex) to enhance the urea, and sucrose on pH. Each value of the permeability
stability in water, then complexed with DNA (gChDex decreases with molecular weight in the order of KCl > urea
DNA) [100]. They also prepared gChPEGDNA com- > sucrose. In the IR spectra of the GCPVS membranes
plexes in a similar manner [101]. The gChDexDNA and treated with the buer solutions of pH 3.07.0, the absorp-
gChPEGDNA complexes were more stable than the gCh tion band at 1530 cm1 assigned to the NH+ 3 group of
DNA complex, suggesting that the Dex and PEG chains on GC was present in all membranes, whereas the band at
the complex surfaces were ecient in preventing particle 1600 cm1 assigned to the NH2 group was absent, indi-
aggregation. The gChPEGDNA complexes were stable cating that the dissociation of the membrane did not occur
against DNase I in the blood plasma during intravenous at pH 3.07.0. An increase in the permeability is not
injection of the complexes. These complexes were eciently attributed to the breaking of ionic bonds. These investiga-
transfected into Chan liver and HepG2 cells having asia- tions suggest a practicability of permeation control by the
loglycoprotein receptor, whereas CT-26 and HeLa cells GCPVS membrane.
without asialoglycoprotein receptor were not transfected For separation of azeotropic mixtures, pervaporation
with the complexes, suggesting that the gChDexDNA and is an eective process. The PEC membranes prepared from
gChPEGDNA complexes transfected the cells through clear solution of Ch and PAA in aqueous acetic acid had
the asialoglycoprotein receptors. very excellent selectivity and permeability for the separa-
because methanol and MTBE are larger than water and

ethanol molecules.
Although PEC formation has been recognized as a
very useful method to make highly water-selective mem-
branes, PEC is usually a compact precipitate that is insol-
uble. Therefore, it is dicult to fabricate the PEC
membrane from the precipitate. Even if PEC membrane
can be formed from the solution, the microions yielded
cannot be removed easily from the membrane. Then the
ChPAA complex membrane was prepared by bringing a
Ch membrane into contact with a PAA solution [106]. The
membrane can be regulated by varying the molecular
weight of PAA, temperature, and reaction time. low-mo-
lecular weight PAA diused into Ch, while high-molecular
weight PAA remained only near the surface. As the mo-
lecular weight of PAA increased, the water selectivity of the
membrane in water/ethanol pervaporation was highly
improved, despite the small quantity of PAA. Owing to
the thin PEC layer, the permeation ux of the membrane
was higher as compared with the ChPAA complex mem-
brane formed by the conventional solution mixing method.
This method can be applied to the improvement of Alg
membrane. An Alg membrane was dipped into the Ch
solution containing CaCl2 to prepare the reverse osmosis
(RO) membrane for separation of an anionic surfactant
[107]. Because the complexation reaction between oppo-
sitely charged polyelectrolytes is very fast and cationic Ch
is too large to diuse into the Alg membrane, the Alg
membrane was coated with the ChAlg complex. The
double-layered structure obtained was very stable, main-
taining constant membrane performance with operating
time, because the PEC can serve as a protective layer for the
interior CaAlg from washing out of Ca2+ ions.
Figure 17 Eect of pH on permeability of solutes through The ChPLG complex could be layered on the quartz
GCPVS membrane at 30jC: o, 0.005 mol/L KCl; D, 0.01 crystal microbalance (QCM) by dipping the QCM in Ch
mol/L urea; 5, 0.02 mol/L sucrose. (From Ref. 102.) and PLG solutions alternately (20 layers = 10 bilayers)
[108]. The ultrathin PEC of the positively charged Ch
alternating with the negatively charged PLG was formed
tion of ethanol and water; PEC membranes always have on the QCM surface. The aqueous solutions with high
highly preferential permeability to water [104]. At high solubility readily caused not only adsorption but also
water concentration, the complex structure was loosened desorption referred to as the polymer peeling o. However,
by the adsorption of water, which increased the perme- the addition of organic solvents, such as acetonitrile and
ability and decreased the separation factor. On the other acetone, into aqueous polymer solutions reduced the de-
hand, the strength of complex structure increased at higher sorption of polymer and promoted the adsorption of
ethanol concentration, which signicantly reduced the greater quantities of the PEC, indicating that the adsorp-
permeability and notably increased the separation factor. tion was driven by the poor solubility of component
Preparation of the PEC membrane having higher charge polyelectrolytes. Similarly, ChCh sulfate (ChS) was ap-
density is a way to enhance the pervaporation perform- plied to the preparation of micrometer-size hollow shells by
ance. The ChPAA complex membranes prepared in means of a layer-by-layer technique, coating the monodis-
aqueous formic acid by blending both polymer solutions perse polymer particles with eight layers of ChChS com-
in dierent ratios exhibited a good pervaporation perfor- plex [109].
mance for dehydrating ethanol at 80jC [105]. The swelling If an aqueous solution of a polyanion is placed care-
ratio decreased with increasing PAA content, and then the fully on a surface of the Ch solution, an instantaneous
permeation ux decreased and the water content in per- formation of lm separating the two liquids can be
meate increased. Moreover, in the case of the separation of observed [25]. PEC ber could be spanned from interfa-
methyl t-butyl ether (MTBE) and methanol, the perme- cial membranes between Ch and GG just as in the case of
ation ux increased with increasing methanol content in interfacial polycondensation [110], because the reaction
the feed. However, the ux decreased to a lesser extent between polyelectrolytes with opposite charges is very
than in the case of waterethanol mixtures, possibly fast. The PEC bers of the ChPLG [111] and ChPAA
[112] systems were spanned in the same way. However, Ch/TPP beads stayed intact in articial intestinal uids but
the swelling and solubility of these bers are within the disintegrated easily in articial gastric uids. Then Bod-
limit aforementioned. meier et al. developed an oral, multiple-unit dosage form,
which may spread out uniformly in the gastrointestinal
E. Encapsulation of Drugs tract, ingeniously using its dissolution property [114]. Ethyl
cellulose microspheres containing propranolol were dis-
Because capsules possess interfacial membranes, encapsu- persed into aqueous acetic acid solutions of Ch. The
lation is a kind of membrane application. The most im- dispersion was dropped into a TPP solution to form Ch/
portant aspect of the encapsulation process is the control of TPP complex beads. As expected, the Ch/TPP beads
the porosity and mechanical strength of the capsule mem- released the microparticles in 0.1 mol/L HCl. This results
brane. The encapsulation process involves the forming of in more reproducible drug absorption and reduces local
capsules by adding drops of a solution of either a cationic irritation compared to single-unit dosage forms.
or an anionic polymer to an oppositely charged polymer Lin and Lin devised emulsion-phase separation pro-
solution. Therefore, the selection of counterions for com- cedure to prepare Ch/TPP beads [115]. Ch solutions of
plex formation determines the characteristics of the capsu- dierent acetic acid concentrations containing methanol
les. For example, ionic cross-linking of polyelectrolytes and theophylline were added to liquid paran to form a
with low-molecular weight counterions results in gel for- water/oil (W/O) emulsion, and various amounts of powder
mation, such as Ch gels formed by PP. or solution of TPP were added to the system at dierent
stages during the process. Thereafter, ethyl acetate was
1. Ch/Polyanion Capsules added to cause phase separation. The formation of Ch/TPP
Shiraishi et al. examined the eect of the molecular weight beads depended on the adding sequence and the form
of Ch on the release rate of indomethacin from Ch/tripo- (powder and solution) of TPP in the encapsulation process.
lyphosphate (TPP) complex gel beads [113]. Indomethacin As a result, TPP could not be added before emulsication,
was dispersed in a solution of various Ch hydrolysates (M whereas, by adding after the emulsication, both forms of
= 83,000 7600) in a dilute acetic acid. These suspensions TPP could give the Ch/TPP beads. The release rate of
were dropped through a syringe nozzle into a TPP solution theophylline from the Ch/TPP beads slowed with an
of pH 6, then Ch/TPP beads were formed instantaneously. increase of TPP. In addition, the slower release rate was
The amount of released drug was linear with the square observed for the beads made by the higher acetic acid
root of time, and the apparent release rate constant of concentration. This suggests that higher acetic acid con-
indomethacin from the Ch/TPP beads decreased with centration enables complete protonation of the amino
increasing molecular weight of Ch, as shown in Fig. 18. group of Ch to improve the interaction with TPP. This
The intermolecular and intramolecular linkages increase method was applied to the Ch/CMC capsules [116].
with increasing molecular weight, accompanied by the NaCMC powder was added to liquid paran before
decrease of porosity and increase of tortuosity. However, emulsication in this case. The higher the acetic acid
Figure 18 Correlation between molecular weight of Ch and release rate constant of indomethacin from Ch/TPP beads.
Copyright 1993, reprinted with permission from Elsevier Science.
concentration, the smaller the particle size, resulting in of Ap-5FU was faster than that of the Schi s base bond.
faster release of theophylline. Furthermore, the capsules The PEC membrane on the Ch gel acted as an eective
prepared using ascorbic acid were larger than those pre- barrier to release of 5-FU. When the APA lectin was added
pared using acetic or citric acids. However, capsules made to the suspension of Ch/CM-NAPGA, a drastic change in
from acetic acid resulted in a slower release of theophylline turbidity of the suspension was observed. On the other
as compared with those prepared by citric or ascorbic hand, the aggregation of the Ch/CM-NAPGA microcap-
acids, probably because the surface topography of capsules sule was dissociated by the addition of excessive lactose.
prepared with citric or ascorbic acids was more porous in These results suggest that saccharide chain on the surface
structure. Naturally, the greater the CMC concentration, of the Ch gel can be specically recognized by lectinlike
the larger the capsule size. Excess CMC in the Ch/CMC receptors of cells. The Ch/CM-NAPGA microcapsules
capsules might be responsible for such larger capsule size containing 5-FU indeed exhibited higher growth-inhibi-
and slower-release behavior. tory eect against hepatoma cells.
Xie et al. entrapped rotundine in PEC capsules in a
two-step process: rst, gelation of a Ch acetate solution
containing rotundine by dropping into an alcoholic alka- 2. Polyanion/Ch Capsules
line solution, and next, complexation of the Ch surface Sulfoethyl cellulose (SEC)/Ch capsules were prepared by
with a solution of carboxymethyl glucomannan (CMGM) dropping SEC solution into Ch solution [120]. The me-
[117]. An increase of the substitution degree of CMGM chanical stability increased with increasing SEC concen-
from 0.26 to 0.40 led to an increase of drug-release time. It tration, and showed a maximum value when the Ch
seemed that complexing with CMGM of a higher substi- concentration was increased. Because the PEC membrane
tution degree resulted in a denser cross-linking network is formed in a fast reaction on the surface of the droplet, the
and reected the increase in the drug-release time. The core remains in a liquid state in this system. On the
release-sustaining eect increased as the concentration of contrary, when the NaAlg solution was introduced drop-
CMGM increased from 0.5 to 2.0%. However, the release wise into Ch solution containing CaCl2, Alg/Ch gel beads
of rotundine in an articial gastric uid was faster than in with the solid core were obtained [121]. Ca2+ is supposed to
an articial intestinal uid, because the swelling degree of penetrate Alg droplets more readily than Ch molecule. The
the Ch/CMGM capsule at pH 1.0 was greater than that at inner core of CaAlg is coated with an interphasic ChAlg
pH 7.0. membrane, which is in turn surrounded by a layer of Ch.
Ch/Alg complex capsules were prepared by dropping MacKnight et al. extruded the Alg solution into a coagu-
Ch solution into Alg solution containing low-molecular lation bath of CaCl2, and then reacted with a Ch solution of
weight cross-linker, genipin, for Ch [118]. In consideration pH 6.5. The exterior of the capsules was further reacted
of the kinetic aspect, the reaction rate of Ch with Alg is very with Alg. They examined the parameters believed to
quick, but that of cross-linking is slow. In consideration of strongly aect Alg/Ch capsule formation: the molecular
the diusion aspect, Alg is restricted to penetrate through weight of Ch, the distance of reactive groups from the main
outer skin layer into the inner core, while genipin can Ch chain, and the type of reactive group [122]. As a result, a
penetrate Ch droplets more readily than Alg. As a result, key factor in membrane formation was found to be the
Alg was accumulated on the surface. The Ch/Alg capsules molecular weight of Ch. Capsules prepared with the inter-
showed the higher swelling degree at pH < 2.0. In addition, mediate-molecular weight Ch (M = 1.6 105 to 3.3 105)
the release rate of indomethacin from the Ch/Alg capsules were strong and had exible membranes. Increased dura-
increases with decreasing pH of gelling solution and de- bility of capsules appeared to be due to the thickness of the
creasing concentration of Alg. The eects of both the PEC membrane; the lower-molecular weight Ch presum-
formation of thicker ChAlg skin layer and higher degrees ably diused more easily into the Alg gel matrix. The
of cross-linked inner core lead to the decrease in drug- permeability studies for Alg/Ch capsules showed that the
release rate. lower the molecular weight of protein, the faster it diused
Microcapsules having saccharide moieties on their into the capsules. For the capsules prepared with a Ch of M
surfaces are expected to show targetability to specic = 2.4 105, for example, the molecular weight cuto was
organs or cells because receptors on the cell membrane approximately 200,000.
have specicity to some sugar moieties. The Ch/6-O-car- Polk et al. extruded Alg solution containing bovine
boxymethyl-N-acetyl-a-1,4-polygalactosamine (CM- serum albumin (BSA) into CaCl2 solution containing Ch
NAPGA) capsules were expected to have targetability to [123]. They showed that capsule strength and exibility
the hepatocyte [119]. Ch gel containing 1-[N-(5-amino- were aected by pH and CaCl2 concentration as well as the
pentyl)carbamoyl]-5-uorouracil (Ap-5FU) was obtained molecular weight of Ch; the most durable Alg/Ch capsule
by the W/O emulsion method in toluene and cross-linked was obtained at pH 5.5 and in the CaCl2 concentration
with glutaraldehyde. When the Ch gel was cross-linked, range 1.51.8% (Table 4). At the same time, swelling was
Ap-5FU was simultaneously immobilized by Schis base an important factor in the release of BSA. Higher swelling,
formation. The Ch gel obtained was resuspended in CM- for example, suggests a reduced AlgCh ionic interaction
NAPGA solution. In physiological saline media, only free and increases membrane permeability. The swelling degree
5-FU was released from the Ch/CM-NAPGA microcap- was aected by Ch molecular weight, Ch concentration,
sule. This is because the hydrolytic rate of carbamoyl bond and pH. Capsules prepared with higher-molecular weight
Table 4 Strength and Flexibility of Alg/Ch Capsules tion (f0.25% w/v) was suitable for BSA retention during
Prepared acid incubation. BSA retention during microcapsule prep-
aration was not signicantly altered by pH, whereas BSA
Durability retention during acid incubation decreased signicantly
with decreasing the pH. Furthermore, in microcapsules
Condition Strengtha Flexibilityb
dried 10% by weight with acetone, BSA retention was over
pH 6.5 +++ +++ 80% during 24-h acid incubation, while only 20% BSA
6.0 ++ ++ retention in non-acetone-dried microcapsules.
5.5 +++ ++++ However, the ChAlg system does not form stable
5.0 ++ ++ membranes under physiological conditions [125]. As
4.5 ++ +++ Dainty et al. reported, phosphate may disrupt the CaAlg
4.0 ++ ++ gel at pH > 5.5 because the anity of phosphate to Ca2+ is
3.5 + + higher than that of Alg [126]. Murata et al. attempted to
3.0 + + control the degradation of Alg/Ch capsules by further
adding HA [127]. An aqueous solution of Alg and HA
CaCl2 Concentration 2.0 ++ ++
containing drug was dropped into CaCl2 solution (pH 4.7)
(wt.%) 1.8 +++ +++
1.6 +++ +++
containing Ch. As a result, the lower the molecular weight
1.5 +++ +++ of Alg, the higher the addition eect of HA on decreasing
1.4 ++ ++ the disintegration rate of the Alg/Ch capsules in the
1.2 ++ ++ phosphate buer (pH 6.8). Accordingly, the delayed disin-
1.0 ++ ++ tegration reected slow drug release from these capsules.
The rate of diclofenac and urbiprofen release from the
a
Strength: + weak, ++++ strong. Alg/Ch capsule with HA was slower than that from the
b
Flexibility: + fragile, ++++ very exible. Alg/Ch capsule without HA. The change of drug-release
Source: Refs. 122 and 123. Copyright n 1994 Wiley. Reprinted by pattern appeared to be attributable to the complex forma-
permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons,
tion between HA and Ch. They also used CS to control the
Inc.
disintegration of the Alg/Ch capsule [128]. The CS-rein-
forced Alg/Ch capsule swelled slowly and disintegration
was hardly observed.
Ch delayed the release of BSA signicantly, and the In the Alg/Ch system, however, a burst eect at the
capsules prepared with the 0.2% Ch released the BSA onset of drug-release measurement is occasionally ob-
faster than those prepared with the 0.1% Ch. The low- served, resulting in a sudden rise in the amount of drug
pH conditions strengthened the capsule membrane by released [129]. This burst release of drugs must be a serious
enhancing the interaction between Alg and Ch. Conse- problem in drug delivery systems. Also, when PEC mem-
quently, the release rate at low pH was reduced greatly, brane capsules are prepared by dropping an anionic poly-
whereas that at high pH was little reduced. Hwang et al. saccharide solution into a cationic Ch solution, the capsule
obtained the same tendency for the pH dependence of wall is very thin, 34 Am in the wet state and 0.3 Am in the
diusion of BSA and g-globulin [6]. They emphasized the dry state, because the interfacial membrane that is formed
eect of the hydrodynamic volume of the Ch molecule on immediately around the droplet acts as a barrier and
the diusion properties. The low release at low pH and high prevents cross-linking polymers from producing additional
release at high pH are benecial to the oral delivery of PEC layers. Therefore, PEC capsules with much thicker
protein drugs in the intestines. The acidic condition of the capsule membranes are needed for the controlled-release
stomach should aid the capsules in retaining the bioactive system of drugs, in particular, of low-molecular weight
material, whereas the more neutral environment of the drugs. Tomida et al. developed a novel method for the
intestines should promote release. preparation of thicker PEC membrane capsules containing
Vandenberg et al. asserted that under optimal encap- theophylline [130]. In the rst step, theophylline and wheat
sulation conditions, Alg/Ch microcapsules should fulll starch (used as an excipient) were dispersed in the aqueous
the following criteria as oral delivery system for proteins; solution comprising KCl and pullulan (used as an agent to
protein is encapsulated with an eciency of >95%, of modulate the viscosity), and this dispersion was dropped
which 80% is retained within the microcapsules during a into Car solution (pH 9) containing Ch powder. A capsule
24 h incubation at pH 1.5 (gastric), and following transfer membrane containing Ch powder was formed instantly
into a pH 7.5 buer solution (intestinal), over 90% of the around each droplet due to the ionotropic gelation of Car
remaining protein is released within 120 min [124]. Then, by K+. In the second step, these capsules were incubated in
they obtained the following results. Higher Alg concentra- an aqueous solution (pH 2.1) containing citric acid and
tion (>2.5% w/v) and lower CaCl2 concentration (0.05% KCl. During this treatment, Ch powder that was uniformly
w/v) were suitable for BSA retention during microcapsule embedded in the coating gel membrane dissolved and
preparation and acid incubation. On the other hand, higher cross-linked with Car in the gel membrane to form a
Ch concentration was suitable for BSA retention during CarCh complex membrane. The capsule core was uni-
microcapsule preparation, but appropriate Ch concentra- formly coated with a dense PEC layer about 300 Am thick.
As the Ch concentration increased, the capsule diameter for enzyme immobilization. PEC hydrogels have excellent
increased and, hence, the drug-release rate decreased, as porous structure that facilitates diusion of both the sub-
shown in Fig. 19. strates and the products of an enzymatic reaction. Yama-
Kawashima et al. also developed a simple technique to guchi et al. immobilized glucoamylase by coprecipitation
coat theophylline granules with a thick PEC of Ch and TPP with Ch and some polyanions: ChDS, ChPVS, and Ch
[131]. The theophylline granules containing TPP were Hep systems [133]. At pH 4.5, the glucoamylase solution
stirred in a dilute HCl solution of Ch. During the process, was added to each of the polyanion solutions. The enzyme
TPP in the granule dissolved and moved to the surface. On was precipitated almost completely after the centrifuga-
the surface, TPP reacts with Ch, resulting in the formation tion. Glucoamylase activities of the precipitate for maltose
of an insoluble PEC membrane. The theophylline release were 100%, 84%, and 46% in the ChDS, ChPVS, and
from the granules coated with the ChTPP complex fol- ChHep systems, respectively, and 76% in the ChDS
lowed zero-order kinetics after an induction period [132]. system for soluble starch. Moreover, about 80% of the
The induction period depended on the swelling behavior of enzymatic activity was retained in the ChDS system even
the granules, and the degree of swelling was aected by the after repeated use (six times).
coating lm thickness of the granules and the pH of the The CaAlg gel is essentially inapplicable to enzyme
dissolution medium. Then the drug-release rate depended immobilization because its porosity is so large that enzymes
on the lm thickness and pH of the dissolution medium. leak. Kokufuta et al. immobilized h-amylase in the CaAlg
When the pH is less than 4, the drug-release rate was gel stabilized by the PEC consisting of MGC and PVS
enhanced and the induction period was shortened, as [134]. The immobilization was made by dropping aqueous
compared with that at pH > 6. This pH dependence of Alg and PVS solution containing the enzyme into a CaCl2
drug release is essentially the same as the Ch/TPP beads solution containing MGC. The loose network structure of
described above. the CaAlg gel enabled diusion of MGC into the gel phase.
The PEC-stabilized CaAlg seemed to have a compact
network structure cross-linked with PVS and MGC and,
F. Immobilization of Enzymes consequently, could be used for preventing the leakage of
enzymes from the gel support. When the unstabilized
Although Ch itself has been used as a support for immo- CaAlg gel was used, about 70% of the enzyme leaked out
bilization of enzymes, PEC of Ch can be successfully used from the gel for the rst 20 h. In contrast, when the PEC-
Figure 19 Release proles of theophylline from Car/Ch capsules prepared with varying Ch concentrations: o, 0%; ., 0.10%;
D, 0.25%; E, 0.50%; 5, 1.00%; n, 1.50%. (From Ref. 130.)
stabilized CaAlg gel was employed, the amount of the the protease activity was observed in the case of the
released enzyme was less than 20% during a period of combined xylanaseprotease immobilization. Due to this
more than 400 h and retained 40% of the activity in its synergy, the protease activity was increased up to 185%
native form. of the free enzyme.
The advantage of hydrogels, such as PEC, for enzyme
immobilization is their hydrophilic microenvironment fa- G. Immobilization of Cells
vorable for enzymatic activity. Dumitriu et al. used the
Xan/Ch capsule for immobilization of lipase [135]. The The immobilization of whole cells is advantageous com-
aqueous Xan solution containing enzymes was introduced pared to immobilized enzymes, where the reaction requires
into the Ch solution of pH 6.5. The initial rate of formation a multistep enzyme system. However, the immobilization
of oleic acid from olive oil increased with increasing initial of whole cells is suitable only for the cells that show a very
enzyme concentration in O/W emulsion. After 22 days at high catalytic activity. When the cells are encapsulated, the
4jC, the immobilized lipase had lost less than 2% of its capsule membrane can protect cells from shear and me-
initial activity. In lipase reactions, solubilization of the chanical impact and allow for compartmentalization that
substrate requires the use of organic solvents. The Xan/ leads to higher cell density. Such high-density-cell systems
Ch capsule, when immersed in isooctane, appeared as rigid may simulate natural tissue conditions.
spheres, but the immobilized enzyme fairly maintained its The eects of organic and inorganic compounds used
catalytic activity, lower than in the O/W emulsion, as as nutrients do not need to be considered so long as a PEC
shown in Fig. 20. consisting of strong polyacid and strong polybase is used
Dumitriu et al. also adopted the Xan/Ch capsule to as support for the immobilization of cells. Kokufuta et al.
immobilize xylanase [136]. The immobilized xylanase did immobilized gram-negative Escherichia coli cells in the
not migrate out of the complex after 10 washing cycles PEC consisting of MGC and PVS [137]. Ch is the only
with acetate buer. The immobilized enzymes were ther- successful occulant in both minimal and complex media
mally stable, temperatures of 8595jC were possible, and for the occulation of E. coli [138]. Therefore, MGC is
showed 6070% higher activity than free enzymes. The also eective to aggregate E. coli cells. When PVS was
ChXan hydrogel may provide a favorable microenviron- added to the suspension (pH 68) containing the cells
ment for enzymatic activity. They used the same complex aggregated with an excess of MGC, an amorphous com-
capsule for the immobilization of two enzymes (xylanase plex resulted from complexing of PVS with the excess of
and protease) as a binary system [65]. The yields of the MGC. No remarkable dierence was observed in the
enzyme were 8598%, which was far superior to the other glucose-consumption proles between free and immobi-
immobilization methods. Although the activity of prote- lized cells. Although the glucose-oxidizing activity for free
ase did not increase, a synergistic eect of the xylanase on cells was inhibited completely at pH 10 due to alkaline
Figure 20 Lipase-catalyzed hydrolysis of olive oil in isooctane at 34jC. Olive oil concentration: 34.5% w/v. Evolution of oleic
acid formation as a function of initial lipase concentration: o, 0.35% w/v; 5, 0.70% w/v; w , 1.0% w/v; , 1.5% w/v. (From
Ref. 135.) Copyright Kluwer: reprinted with kind permission of Kluwer Academic Publishers.
denaturation of E. coli cells, the immobilized cells had an

activity even at pH 10. This seems to be an advantage of
the cell entrapment, which arises from the protection of
cells with PEC support.
However, the mixing of polycation and polyanion
solution in the presence of microorganisms leads to amor-
phous aggregates of PEC. Chu et al. reported a novel
method to shape PEC beads [139]. They rst prepared clear
PEC solution of Ch and Xan containing Corynebacterium
glutamicum cells in the presence of concentrated NaCl
(5.7%), then dropped the suspension into distilled water to
form PEC gel beads. The fumarase activity of the immobi-
lized cells was about ve times higher than that of intact cells,
and the higher the Ch/Xan ratio, the higher the fumarase
activity. Furthermore, about 90% of the initial fumarase
activity was retained for 240 h in a column experiment.
Klein et al. immobilized E. coli cells in the Ch/PP beads
for industrial application to produce 6-APA [140]. The
result demonstrated the success in obtaining immobilized
cell preparations with very high activity. In this case, the
penicillin-G solution was brought to nearly complete con-
version (>98%) and the beads were reused after separa-
tion of the solution. However, cells in the Ch/PP beads
were found to grow at a reduced rate, about one-third that
of the control, and the cells in the Ch/Alg capsules were
able to achieve growth rates comparable to those of the free
cells [8]. The explanation for these dierences is that the
Figure 21 Changes in nitrogen concentrations during
Ch/PP complex probably has a tighter network and growth aerobic cultures of singly immobilized N. europaea cells (a)
is limited by diusion of materials to the cell. Both com- and coimmobilized N. europaea plus P. denitiricans cells (b):
plexes could withstand compression forces of up to 2000 g o, NH4+-N; ., NO2-N. For the singly immobilized cells,
and could undergo a 90% deformation without rupture. 375 beads containing 3.1 mg dry wt. of N. europaea were
This fact makes the system suitable for industrial applica- cultured under the same conditions as was used for the
tions, because of the resistance to mechanical or handling coimmobilized cells, except the medium was free from
stresses, which may be imposed in several separations or ethanol as a hydrogen donor. (From Ref. 142.)
fermentation processes. Furthermore, in the case of Ch/
Alg capsules, addition of CaCl2 to Ch and glucose to Alg
increased capsule strength, the capsules became strong ations. Li used a similar method for increasing the stability
enough to require up to 100 N during uniaxial compression of CaAlg beads including Saccharomyces cerevisiae (yeast)
to burst a capsule [141]. cells adopting the TPPCh complex as a surface coating
However, the use of CaAlg gel has been limited due to [143]. The mixture solution of Alg and TPP containing S.
its instability upon contact with various anions, such as cerevisiae was extruded into CaCl2 solution containing Ch.
phosphate, citrate, and lactate. Specically, the lack of The Alg beads coated with PEC maintained their rigidity
resistance to the phosphate ion is a serious problem as during ethanol production and were chemically stable in
mentioned above. Kokufuta et al. used CaAlg gel that was phosphate buer. No dierence in ethanol production was
reinforced with a PEC consisting of MGC and PVS [142]. found between the uncoated CaAlg and the CaAlg coated
Nitrosomonas europaea and Paracoccus denitricans were with PEC of TPPCh.
suspended in aqueous solution containing Alg and PVS. Similar to the Alg/Ch system, the CMC/Ch capsules
The cell suspension was then added dropwise into CaCl2 also show lower mechanical strength and low swelling
solution including MGC. The coimmobilized cells were degree. In order to improve these defects, complexation
aerobically cultured on a medium containing 3 mmol/L of between Ch and CMC was carried out in the presence of
phosphate ions, using (NH4)2SO4 as a substrate. The PEC- Al2(SO4)3 [144]. The AlCMC/Ch capsules obtained higher
stabilized CaAlg gel was resistant to phosphate ions, and mechanical strength, which was 710 times higher than that
no breakage of the capsules was observed during the of the CMC/Ch complexes, and higher permeability to
cultivation. As shown in Fig. 21, ammonia was consumed substrate based on larger swelling capacity, as compared
without forming nitrite, indicating that the N. europaea and with the CMC/Ch capsules. Then the AlCMC/Ch gel could
P. denitricans cells coimmobilized into the PEC make it be used as a support for yeast cell immobilization (1 109
possible for nitrication and denitrication to occur simul- cells/mL) and gave high ethanol productivity.
taneously. This result could be used to remove nitrogen Pandya and Knorr immobilized plant cells of carrot
from wastewater instead of conventional two-stage oper- (Daucus carota) and celery (Apium graveolens) in the PEC
capsules consisting of Ch and Alg or Car [145]. The lier by these PECs than by the other tissue culture materi-
formation of capsules was done by extruding either Car als. The increased alkaline phosphatase activity, which is
or Alg solution into either KCl or CaCl2 solution contain- an initial dierentiation marker for osteoblast, on the Ch
ing either acid-soluble Ch or water-soluble Ch salt. Plant CMChitin was higher than that of the ChPChitin, sug-
cells were more viable in the capsules containing water- gesting that the dierentiation inducement ability of the
soluble Ch salt than in the capsules containing acid-soluble PEC with carboxymethyl groups was stronger than that of
Ch, and more viable in the Car/Ch capsules than in the Alg/ PEC with phosphate groups. These results suggest that
Ch capsules. From the dierence in the release of dye, it PEC can control cell functions, such as proliferation and
was concluded that the matrix of Car/Ch could possibly dierentiation, without the loss of cell functions in vitro.
have larger pore size than that of Alg/Ch. This type of However, the control of cell functions in association with
capsule can be used for continuous production of bio- PEC is still not clear.
chemicals from plant cells and microorganisms. Proteoglycans and ECM polysaccharides termed
Tay et al. presented the applicability of PEC of the Ch GAGs are major components of the hematopoietic matrix.
Alg system to encapsulate and germinate secondary embry- Within the ECM, GAGs are immobilized by combinations
oids of Brassca napus in the two methods: Ch/Alg and Alg/ of covalent, ionic, and hydrogen-bonding interactions. The
Ch capsules [146]. As a result, the Ch/Alg capsules have ChGAG complexes, containing Hep, HA, and CS (CSA,
desirable toughness, but none of the secondary embryoids condroitin-4-sulfate, CSB, dermatan sulfate, and CSC,
survived. The high acidity of the Ch matrix (citric acid, pH condroitin-6-sulfate) as GAG, were coated on tissue cul-
2.5) and permeabilizing eect of Ch on cell membranes, ture plates [151]. Then, human umbilical cord blood
which increases the permeability of the embryoid and CD34+ cells were seeded onto the PEC surfaces, and their
causes leakage of solutes from the tissue, probably proliferation and retention of CD34 expression were fol-
accounts for this nonsurvival. On the contrary, germina- lowed. The ability of GAG species to bind and enhance the
tion frequency of 100% was achieved in the Alg/Ch activities of some hematopoietic growth factors is recog-
capsules. However, the Ch outer layer with positive charge nized. The ChCSA showed an inhibitory eect on cell
makes the coat soft in the presence of water. The structural proliferation and survival, resulting in complete cell death
stability of the coating could be enhanced by neutralizing by the end of week 2. All other surfaces produced a 25- to
excess positive charge on Ch with alkali. Khor et al. also 40-fold increase in total cells after 6 weeks. Only the Ch
prepared articial seeds by dropping the Alg solution Hep and ChCSB surfaces consistently exhibited signi-
containing orchid seeds and protocorms, which have an cant expansion of the CD34+ population. All other PEC
inner Alg matrix that simulates the endosperm of many surfaces exhibited declines in CD34 expression, with neg-
plant seeds and an outer seed coat of Ch that is hard ligible CD34+ percentages remaining after 4 weeks. In
enough for protection and easy handling [147]. contrast, the ChHep and ChCSB surfaces exhibited
CD34+ fractions as high as 90% after 4 weeks. However,
H. Tissue Culture these eects may partly be mediated by desorbed GAGs,
since most of GAG leached into solution over the course of
In general, when cells are cultured on tissue culture plates the culture. Sustained high Hep levels had toxic eects,
in vitro, they grow to form a monolayer on the surface while CSB exhibited more rapid early proliferation of
and are dedierentiated. However, PEC of Ch can control CD34+ cells.
such cell functions as proliferation, morphology, and
dierentiation.
For example, human periodontal ligament broblast ACKNOWLEDGMENTS
formed three-dimensional cell aggregates on the Ch We would like to acknowledge Dr. Yasuo Kikuchi, who
CMChitin complex, and the cells were spread and well has retired, for his important comments.
proliferated on the Chsulfated chitin (SChitin) complex
[148,149]. The formation of cell aggregates was caused by
the inhibition of bronectin adsorption, which is known to REFERENCES
promote the attachment and spreading of cells. CMChitin
and SChitin are similar in structure to glycosaminoglycans 1. Tsuchida, E.; Abe, K. Interactions Between Macromole-
(GAGs), which are an integral part of proteoglycans that cules in Solution and Intermacromolecular Complexes;
Springer-Verlag: Berlin, 1982; 1418.
are major components of extracellular matrix (ECM), and 2. Tracy, M.V. Rev. Pure Appl. Chem. 1957, 7, 1.
cells seem to recognize the PEC as a component of ECM. In 3. Sannan, T.; Kurita, K.; Iwakura, Y. Makromol. Chem.
addition, the Chphosphated chitin (PChitin) and the Ch 1976, 177, 3589.
CMChitin complexes exhibited a high adhesion of osteo- 4. Kubota, N.; Eguchi, Y. Polym. J. 1997, 29, 123.
blast as well as a low adsorption of bronectin [150]. These 5. Kubota, N.; Tatsumoto, N.; Sano, T.; Toya, K. Carbo-
PECs induced aggregates of the osteoblast and inhibited hydr. Res. 2000, 324, 268.
6. Hwang, C.; Rha, C.K.; Sinskey, A.J. In Chitin in Nature and
the osteoblast proliferation on the early culture days. The Technology; Muzzarelli, R.A.A., Jeuniaux, C., Gooday,
osteoblast on the ChCMChitin formed smaller aggregates G.W., Eds.; Plenum: New York, 1986; 389396.
than those on the ChPChitin. Furthermore, the osteoblast 7. Colfen, H.; Berth, G.; Dautzenberg, H. Carbohydr.
dierentiation, the mineralization stage, was induced ear- Polym. 2001, 45, 373.
8. Rha, C.K.; Rodriguez-Sanchez, D.; Kienzle-Sterzer, C. In 37. Kubota, N.; Konaka, G.; Eguchi, Y. SenI Gakkaishi
Biotechnology of Marine Polysaccharides; Colwell, R.R., 1997, 54, 212.
Pariser, E.R., Sinskey, A.J., Eds.; Hemisphere: Washing- 38. Ausar, S.F.; Bianco, I.D.; Badini, R.G.; Castagna, L.F.;
ton, DC, 1985; 283311. Modesti, N.M.; Landa, C.A.; Beltramo, D.M. J. Dairy
9. Terbojevich, M.; Casani, A.; Scandola, M.; Fornasa, A. In Sci. 2001, 84, 361.
Chitin in Nature and Technology; Muzzarelli, R.A.A., 39. Plashchina, I.G.; Mrachkovskaya, T.A.; Danilenko, A.N.;
Jeuniaux, C., Gooday, G.W., Eds.; Plenum: New York, Kozhevnikov, G.O.; Starodubrovskaya, N.Y.; Braudo,
1986; 349351. E.E.; Schwenke, K.D. Spec. Publ.R. Soc. Chem. 2001,
10. Kikuchi, Y. Nippon Kagaku Kaishi 1973, 1973, 2436. 258, 293.
11. Bixler, H.J.; Michaels, A.S. In Encyclopedia of Polymer 40. Braudo, E.E.; Plashchina, I.G.; Schwenke, K.D. Nahrung
Science and Technology; Bikales, N.M., Mark, H.F., 2001, 45, 382.
Gaylord, N.G., Eds.; John Wiley & Sons: New York, 1969; 41. Frossard, E.; Tekely, P.; Morel, J.L. Fertil. Res. 1994, 37,
Vol. 10, 765. 151.
12. Tsuchida, E.; Abe, K. Interactions Between Macromole- 42. Kubota, N.; Kikuchi, Y. Makromol. Chem. 1992, 193, 559.
cules in Solution and Intermacromolecular Complexes; 43. Kendra, D.F.; Hadwiger, L.A. Exp. Mycol. 1984, 8, 276.
Springer-Verlag: Berlin, 1982; 1847. 44. Hayatsu, H.; Kubo, T.; Tanaka, Y.; Negishi, K. Chem.
13. Tsuchida, E.; Abe, K. Interactions Between Macromole- Pharm. Bull. 1997, 45, 1363.
cules in Solution and Intermacromolecular Complexes; 45. Richardson, S.C.W.; Kolbe, H.V.J.; Duncan, R. Int. J.
Springer-Verlag: Berlin, 1982; 83. Pharm. 1999, 178, 231.
14. Perez-Gramatges, A.; Arguelles-Monal, W.; Peniche- 46. Liu, W.G.; Yao, K.D.; Liu, Q.G. J. Appl. Polym. Sci.
Covas, C. Thermodynamics of complex formation of 2001, 82, 3391.
polyacrylic acid with poly(N-vinyl-2-pyrrolidone) and 47. Izumrudov, V.A.; Zhiryakova, M.V. Macromol. Chem.
Chitosan. Polym. Bull. 1996, 37, 127. Phys. 1999, 200, 2533.
15. Kanbayashi, S.; Arai, T. Kobunshi Ronbunshu 1994, 51, 48. Erbacher, R.; Zau, S.; Bettinger, T.; Stean, A.M.; Remy,
115. J.S. Pharm. Res. 1998, 15, 1332.
16. Kanbayashi, S.; Arai, T. Kobunshi Ronbunshu 1994, 51, 49. Mao, H.-Q.; Roy, K.; Troung-Le, V.L.; Janes, K.A.; Lin,
323. K.Y.; Wang, Y.; August, J.T.; Leong, K.W. Chitosan
17. Sakiyama, T.; Chu, C.H.; Fujii, T.; Yano, T. Preparation DNA nanoparticles as gene carriers: synthesis, character-
of a polyelectrolyte complex gel from Chitosan and n- ization and transfection eciency. J. Control. Release
carrageenan and its pH-sensitive swelling. J. Appl. Polym. 2001, 70, 399.
Sci. 1993, 50, 2021. 50. Kikuchi, Y.; Kubota, N.; Tanaka, H. Nippon Kagaku
18. Arguelles-Monal, W.; Cabrera, G.; Peniche, C.; Rinaudo, Kaishi 1986, 1986, 706.
M. Polymer 2000, 41, 2373. 51. Kikuchi, Y.; Kubota, N.; Mitsuishi, H. Structures,
19. Arguelles-Monal, W.; Garciga, M.; Peniche-Covas, C. properties, and alkali metal ion transport membrane of
Polym. Bull. 1990, 23, 307. polyelectrolyte complexes consisting of methyl glycol
20. Chavasit, V.; Kienzle-Sterzer, C.; Torres, J.A. Polym. Bull. Chitosan, glycol Chitosan, and poly(vinyl sulfate). J.
1988, 19, 223. Appl. Polym. Sci. 1988, 35, 259.
21. Chavasit, V.; Torres, J.A. Biotechnol. Prog. 1990, 6, 2. 52. Kikuchi, Y.; Kubota, N. Bull. Chem. Soc. Jpn. 1985, 58,
22. Ikeda, S.; Kumagai, H.; Sakiyama, T.; Chu, C.-H.; 2121.
Nakamura, K. Biosci. Biotechnol. Biochem. 1995, 59, 1422. 53. Kikuchi, Y.; Kubota, N. Bull. Chem. Soc. Jpn. 1987, 60,
23. Mireles, C.; Martino, M.; Bouzas, J.; Torres, J.A. 375.
Advances in Chitin Chitosan; Elsevier: Amsterdam, 1992; 54. Kikuchi, Y.; Kubota, N. Structure and properties of
506515. polyelectrolyte complexes consisting of three materials:
24. Takahashi, T.; Takayama, K.; Machida, Y.; Nagai, T. polysaccharide, polypeptide, and synthetic macromole-
Int. J. Pharm. 1990, 61, 35. cule. J. Polym. Sci., Polym. Lett. Ed. 1985, 23, 537.
25. Dutkiewicz, J.; Tuora, M.; Judkiewicz, L.; Ciszewski, R. 55. Kikuchi, Y.; Kubota, N. Nippon Kagaku Kaishi 1985,
Advances in Chitin Chitosan; Elsevier: Amsterdam, 1992; 1985, 1465.
496505. 56. Cerrai, P.; Guerra, G.D.; Tricoli, M.; Maltinti, S.;
26. Nakajima, A.; Shinoda, K. Complex formation between Barbani, N.; Petarca, L. Macromol. Chem. Phys. 1996,
oppositely charged polysaccharides. J. Colloid Interface 197, 3567.
Sci. 1976, 55, 126. 57. Hu, Y.; Jiang, X.; Ding, Y.; Ge, H.; Yuan, Y.; Yang, C.
27. Srinivasan, R.; Kamalam, R. Biopolymers 1982, 21, 251. Synthesis and characterization of Chitosan-poly(acrylic
28. Srinivasan, R.; Kamalam, R. Biopolymers 1982, 21, 265. acid) nanoparticles. Biomaterials 2002, 23, 3193.
29. Hugerth, A.; Caram-Lelham, N.; Sundelof, L.-O. Carbo- 58. Sakiyama, T.; Takata, H.; Kikuchi, M.; Nakanishi, K. J.
hydr. Polym. 1997, 34, 149. Appl. Polym. Sci. 1999, 73, 2227.
30. Hirano, S.; Mizutani, C.; Yamaguchi, R.; Miura, O. 59. Muzzarelli, R.A.A. Chitin; Pergamon Press: Oxford, 1977;
Biopolymers 1978, 17, 805. 103 pp.
31. Remunan-Lopez, C.; Bodmeier, R. Int. J. Polym. 1996, 60. Sakiyama, T.; Takata, H.; Toga, T.; Nakanishi, K. J.
135, 63. Appl. Polym. Sci. 2001, 81, 667.
32. Park, W.H.; Lee, K.Y.; Ha, W.S. Macromol. Chem. Phys. 61. Chu, C.-H.; Sakiyama, T.; Fujii, T.; Yano, T. Food
1996, 197, 2175. Hydrocolloids, Structures, Properties, and Functions;
33. Taravel, M.N.; Domard, A. Biomaterials 1993, 14, Plenum: New York, 1993; 247250.
930. 62. Chu, C.-H.; Sakiyama, T.; Yano, T. Biosci. Biotechnol.
34. Taravel, M.N.; Domard, A. Macromol. Rep. 1994, A31, Biochem. 1995, 59, 717.
1237. 63. Kumagai, H.; Chu, C.-H.; Sakiyama, T.; Ikeda, S.;
35. Taravel, M.N.; Domard, A. Biomaterials 1995, 16, 865. Nakamura, K. Biosci. Biotechnol. Biochem. 1996, 60, 1623.
36. Miya, M.; Iwamoto, R.; Mima, S. J. Polym. Sci., Polym. 64. Chu, C.-H.; Kumagai, H.; Sakiyama, T.; Ikeda, S.;
Phys. Ed. 1984, 22, 1149. Nakamura, K. Biosci. Biotechnol. Biochem. 1996, 60, 1627.
65. Dumitriu, S.; Magny, P.; Montane, D.; Vidal, P.F.; 99. Murata, J.; Ohya, Y.; Ouchi, T. Carbohydr. Polym. 1996,
Chornet, E. J. Bioact. Compat. Polym. 1994, 9, 184. 29, 69.
66. Yao, K.D.; Tu, H.; Cheng, F.; Zhang, J.W.; Liu, J. 100. Park, Y.K.; Park, Y.H.; Shin, B.A.; Choi, E.S.; Park, Y.R.;
Angew. Makromol. Chem. 1997, 245, 63. Akaike, T.; Cho, C.S. J. Control. Release 2000, 69, 97.
67. Arguelles-Monal, W.; Hechavarr a, O.L.; Rodr guez, L.; 101. Park, I.K.; Kim, T.H.; Park, Y.H.; Shin, B.A.; Choi, E.S.;
Peniche, C. Polym. Bull. 1993, 31, 471. Chowdhury, E.H.; Akaike, T.; Cho, C.S. J. Control.
68. Peniche-Covas, C.; Arguelles-Monal, W.; Roman, J.S. Release 2001, 76, 349.
Sorption and desorption of water vapor by membranes of 102. Kikuchi, Y.; Kubota, N. Bull. Chem. Soc. Jpn. 1988, 61,
the polyelectrolyte complex of Chitosan and carboxy- 2943.
methyl cellulose. Polym. Int. 1995, 38, 45. 103. Kubota, N.; Ohga, K.; Moriguchi, M. J. Appl. Polym. Sci.
69. Ichikawa, T.; Yoshikawa, E.; Kubota, H.; Nakayama, H.; 1991, 42, 495.
Nakajima, T. Kobunshi Ronbunshu 1990, 47, 709. 104. Shieh, J.-J.; Huang, R.Y.M. J. Membr. Sci. 1997, 127, 185.
70. Ichikawa, T.; Araki, C.; Nakajima, T. Kobunshi Ron- 105. Nam, S.Y.; Lee, Y.M. J. Membr. Sci. 1997, 135, 161.
bunshu 1991, 48, 789. 106. Iwatsubo, T.; Kusumocahyo, S.P.; Shinbo, T. J. Appl.
71. Lee, J.W.; Kim, S.Y.; Kim, S.S.; Lee, Y.M.; Lee, K.H.; Polym. Sci. 2002, 86, 265.
Kim, S.J. J. Appl. Polym. Sci. 1999, 73, 113. 107. Yeom, C.K.; Kim, C.U.; Kim, B.S.; Kim, K.J.; Lee, J.M.
72. Gamzazade, A.I.; Nasibov, S.M. Carbohydr. Polym. J. Membr. Sci. 1998, 143, 207.
2002, 50, 339. 108. Tachaboonyakiat, W.; Serizawa, T.; Endo, T.; Akashi, M.
73. Gamzazade, A.I.; Nasibov, S.M. Carbohydr. Polym. Polym. J. 2000, 32, 481.
2002, 50, 345. 109. Berth, G.; Voigt, A.; Dautzenberg, H.; Donath, E.;
74. Ohkawa, K.; Yamada, M.; Nishida, A.; Nishi, N.; Mohwald, H. Biomacromolecules 2002, 3, 579.
Yamamoto, H. J. Polym. Environ. 2000, 8, 59. 110. Yamamoto, H.; Senoo, Y. Macromol. Chem. Phys. 2000,
75. Denuziere, A.; Taverna, M.; Ferrier, D.; Domard, A. 201, 84.
Electrophoresis 1997, 18, 745. 111. Ohkawa, K.; Takahashi, Y.; Yamada, M.; Yamamoto, H.
76. Taravel, M.N.; Domard, A. Biomaterials 1996, 17, 451. Macromol. Mater. Eng. 2001, 286, 168.
77. Kikuchi, Y. Makromol. Chem. 1974, 175, 2209. 112. Ohkawa, K.; Ando, M.; Shirakabe, Y.; Takahashi, Y.;
78. Kikuchi, Y.; Noda, A. J. Appl. Polym. Sci. 1976, 20, 2561. Yamada, M.; Shirai, H.; Yamamoto, H. Textile Res. J.
79. Kikuchi, Y.; Fukuda, H. Makromol. Chem. 1974, 175, 2002, 72, 120.
3593. 113. Shiraishi, S.; Imai, T.; Otagiri, M. Controlled release of
80. Fukuda, H.; Kikuchi, Y. Makromol. Chem. 1977, 178, indomethacin by Chitosanpolyelectrolyte complex: opti-
2895. mization and in vivo/in vitro evaluation. J. Control.
81. Fukuda, H.; Kikuchi, Y. In vitro clot formation of the Release 1993, 25, 217.
polyelectrolyte of sodium dextran sulfate with Chitosan. 114. Bodmeier, R.; Chen, H.; Paeratakul, O. Pharm. Res. 1989,
J. Biomed. Mater. Res. 1978, 12, 531. 6, 413.
82. Fukuda, H.; Kikuchi, Y. Bull. Chem. Soc. Jpn. 1978, 51, 115. Lin, S.Y.; Lin, P.C. STP Pharma. Sci. 1992, 2, 500.
1142. 116. Lin, S.Y.; Lin, P.C. Chem. Pharm. Bull. 1992, 40, 2491.
83. Kikuchi, Y.; Takebayashi, T. Bull. Chem. Soc. Jpn. 1982, 117. Xie, S.S.; Liu, X.J.; Zhang, Y.X.; Zhang, J.L.; Zhang,
55, 2307. D.Y.; Wang, Y.L.; Li, D.; Ni, D. J. Macromol. Sci., Pure
84. Fukuda, H.; Kikuchi, Y. Makromol. Chem. 1979, 180, Appl. Chem. 1992, A29, 31.
1631. 118. Mi, F.-L.; Sung, H.-W.; Shyu, S.-S. Carbohydr. Polym.
85. Fukuda, H. Bull. Chem. Soc. Jpn. 1980, 53, 837. 2002, 48, 61.
86. Uragami, T.; Mori, H.; Noishiki, Y. Jinko Zoki 1988, 17, 119. Ohya, Y.; Takei, T.; Kobayashi, H.; Ouchi, T. J.
511. Microencapsul. 1993, 10, 1.
87. Aiba, S.; Minoura, N.; Taguchi, K.; Fujiwara, Y. 120. Rose, T.; Neumann, B.; Thielking, H.; Koch, W.; Vorlop,
Biomaterials 1987, 8, 481. K.-D. Chem. Eng. Technol. 2000, 23, 769.
88. Dutkiewicz, J.; Szosland, L.; Kucharska, M.; Judkiewicz, 121. Lee, O.-S.; Ha, B.-J.; Park, S.-N.; Lee, Y.-S. Macromol.
L.; Ciszewski, R. J. Bioact. Compat. Polym. 1990, 5, 293. Chem. Phys. 1997, 198, 2971.
89. Zou, H.; Li, X.-Y.; Xu, G.-F. Polymers and Biomaterials, 122. McKnight, C.A.; Ku, A.; Goosen, M.F.A.; Sun, D.;
Int. Symp. Proc. Elsevier: Amsterdam, 1991; Vol. 3, 465 Penney, C. J. Bioact. Compat. Polym. 1988, 3, 334.
470. 123. Polk, A.; Amsden, B.; De Yao, K.; Peng, T.; Goosen,
90. Meshali, M.M.; Gabr, K.E. Int. J. Pharm. 1993, 89, 177. M.F.A. Controlled release of albumin from Chitosan-
91. Takayama, K.; Hirata, M.; Machida, Y.; Masada, T.; alginate microcapsules. J. Pharm. Sci. 1994, 83, 178.
Sannan, T.; Nagai, T. Chem. Pharm. Bull. 1990, 38, 1993. 124. Vandenberg, G.W.; Drolet, C.; Scott, S.L.; de la Noue, J.
92. Mumper, R.J.; Wang, J.; Claspell, J.M.; Rolland, A.P. J. Control. Release 2001, 77, 297.
Proc. Int. Symp. Control. Rel. Bioact. Mater. 1995, 22, 125. Wandrey, C.; Grigorescu, G.; Hunkeler, D. Progr. Colloid
178. Polym. Sci. 2002, 119, 84.
93. Ishii, T.; Okahata, Y.; Sato, T. Biochim. Biophys. Acta 126. Dainty, A.L.; Gouldin, K.H.; Robinson, P.K.; Simpkins,
2001, 1514, 51. I.; Travan, M.D. Biotechnol. Bioeng. 1986, 28, 210.
94. MacLaughlin, F.C.; Mumper, R.J.; Wang, J.; Tagliaferri, 127. Murata, Y.; Miyamoto, E.; Kawashima, S. Drug Delivery
J.M.; Gill, I.; Hinchclie, M.; Rolland, A.P. J. Control. Syst. 1994, 9, 357.
Release 1998, 56, 259. 128. Murata, Y.; Miyamoto, E.; Kawashima, S. J. Control.
95. Sato, T.; Ishii, T.; Okahata, Y. Biomaterials 2001, 22, Release 1996, 38, 101.
2075. 129. Filipovic-Grcic, J.; Maysinger, D.; Zorc, B.; Jalsenjak, I.
96. Fiebrig, I.; Varum, K.M.; Harding, S.E.; Davis, S.S.; Int. J. Pharm. 1995, 116, 39.
Stokke, B.T. Carbohydr. Polym. 1999, 33, 91. 130. Tomida, H.; Nakamura, C.; Kiryu, S. Chem. Pharm. Bull.
97. Roy, K.; Mao, H.-Q.; Huang, S.-K.; Leong, K.W. Nat. 1994, 42, 979.
Med. 1999, 5, 387. 131. Kawashima, Y.; Handa, T.; Kasai, A.; Takenaka, H.; Lin,
98. Cui, Z.; Mumper, R.J. J. Control. Release 2001, 75, 409. S.Y.; Ando, Y. J. Pharm. Sci. 1985, 74, 264.
132. Kawashima, Y.; Handa, T.; Kasai, A.; Takenaka, H.; Lin, 141. Daly, M.M.; Knorr, D. Biotechnol. Prog. 1988, 4, 76.
S.Y. Chem. Pharm. Bull. 1985, 33, 2469. 142. Kokufuta, E.; Yukishige, M.; Nakamura, I. J. Ferment.
133. Yamaguchi, R.; Arai, Y.; Hirano, S.; Ito, T. Agric. Biol. Technol. 1987, 65, 659.
Chem. 1978, 42, 1297. 143. Li, X. Biotechnol. Appl. Biochem. 1996, 23, 269.
134. Kokufuta, E.; Shimizu, N.; Tanaka, H.; Nakamura, I. 144. Long, D.D.; Luyen, D.V. J. Macromol. Sci., Pure Appl.
Biotechnol. Bioeng. 1988, 32, 756. Chem. 1996, A33, 1875.
135. Dumitriu, S.; Chornet, E.; Vidal, P.F.; Moresoli, C. 145. Pandya, Y.; Knorr, D. Process Biochem. 1991, 26, 75.
Polyionic hydrogels as support for immobilization of 146. Tay, L.F.; Khoh, L.K.; Loh, C.S.; Khor, E. Biotechnol.
lipase. Biotechnol. Tech. 1995, 9, 833. Bioeng. 1993, 42, 449.
136. Dumitriu, S.; Chornet, E. Biotechnol. Prog. 1997, 13, 539. 147. Khor, E.; Ng, W.-F.; Loh, C.-S. Biotechnol. Bioeng. 1998,
137. Kokufuta, E.; Matsumoto, W.; Nakamura, I. J. Appl. 59, 635.
Polym. Sci. 1982, 27, 2503. 148. Hamano, T.; Suzuki, H.; Teramoto, A.; Iizuka, E.; Abe,
138. Hughes, J.; Ramsden, D.K.; Symes, K.C. Biotechnol. K. J. Macromol. Sci., Pure Appl. Chem. 1998, A35, 439.
Tech. 1990, 4, 55. 149. Hamano, T.; Teramoto, A.; Iizuka, E.; Abe, K. J. Biomed.
139. Chu, C.H.; Kumagai, H.; Nakamura, K. J. Appl. Polym. Mater. Res. 1998, 41, 257.
Sci. 1996, 60, 1041. 150. Hamano, T.; Chiba, D.; Nakatsuka, K.; Nagahata, M.;
140. Klein, J.; Heckenberg, U.; Kressdorf, B.; Mueller, R.; Teramoto, A.; Kondo, Y.; Hachimori, A.; Abe, K. Polym.
Vorlop, K.D.; Wagner, F. Proc. 3rd European Congress Adv. Technol. 2002, 13, 46.
on Biotechnology; Verlag Chemie: Weinheim, 1984, Vol. 1, 151. Madihally, S.V.; Flake, A.W.; Matthew, H.W.T. Stem
267272. Cells 1999, 17, 295.
30
Polysialic Acid: Structure and Properties
Tadeusz Janas
University of Colorado, Boulder, Colorado, U.S.A.
Teresa Janas
University of Colorado, Boulder, Colorado, U.S.A. and University of Zielona, Gora, Poland
I. INTRODUCTION II. STRUCTURE OF POLYSIALIC ACID

Polysialic acids (polySia) form a structurally unique group A. Sialic Acids Monomers that Form
of linear carbohydrate chains with a degree of polymeri- Polysialic Acid Chain
zation (DP) up to 200 sialyl residues. PolySia chains are
covalently attached to membrane glycoconjugates (glyco- Sialic acids (Sia) comprise a family of about 40 derivatives
proteins or glycolipids) on cells that range in evolutionary of the nine-carbon sugar neuraminic acid (Neu) (for review
diversity from bacteria to human brains. In this paper the see Ref. 1). The carboxyl group at position 1 confers a neg-
following topics are reviewed: biophysical and biochemical ative charge on the molecule under physiological condi-
studies on the structure of polySia chains including NMR, tions and characterizes it as a strong organic acid (pK 2.2).
optical activity, atomic force microscopy, electrophoresis, Sia usually occur as monosialyl residues joined through a
and chromatography; dynamic properties of polySia glycosidic linkage to oligosaccharides, glycoproteins, and
chains including their interactions with membranes, anti- gangliosides [2]. In rarer cases, sialic acid monomers form
bodies, endoneuraminidases, and other proteins; topology polysialic acids (polySia) chains, which are a structurally
and energetics of biosynthesis, transmembrane transloca- diverse family of linear carbohydrate chains that consist of
tion and expression of polySia chains in bacterial cells; the N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic
structure and properties of polySia in eukaryotic cells acid (Neu5Gc), or a deaminated form of Sia 3-deoxy-D-
including structure of polysialylated neural cell adhesion glycero-D-galacto-2-nonulosonic acid (KDN) (for review
molecule (N-CAM) in vertebrates, eect of polySia charge see Ref. 3). KDN formally is a 5-deamino-, 5-hydroxy-
and hydration on molecular interaction and intercellular neuraminic acid [2]. Thus only three members of the Sia
space, developmental changes in the length of polySia family form polySia chains. The structure of these sialic
chains attached to N-CAM, role of polySia chains in neural acids is shown in Fig. 1. The amino group of Neu can be
development and in activity-induced synaptic plasticity, acetylated (forming Neu5Ac) or glycolylated (forming
polySia chains in membrane ion channels, in animal eggs, Neu5Gc). Neu5Gc diers from Neu5Ac only by an addi-
in human tumor cells, and in invertebrates; polySia in ther- tional oxygen atom in the N-acyl moiety. In the case of
apeutics including molecular engineering of polySia chains KDN there is an additional hydroxyl group present instead
and polySia chains in drug delivery and drug selection. of the amino group at carbon 5 of Neu.
707
708 Janas and Janas
Figure 1 The structure of sialic acids that form polysialic

acid chains. Neu5Ac: N-acetylneuraminic acid; Neu5Gc: N-
glycolylneuraminic acid; KDN: 3-deoxy-D-glycero-D-galacto-
2-nonulosonic acid (deamino-, 5-hydroxy-neuraminic acid).
These sialic acids are derivatives of neuraminic acid (Neu).
B. Intersialyl Linkages in Polysialic Acids

The polySia glycotope has been shown to exhibit structural
diversity both in the sialic acid components and in the
intersialyl linkages (for review see Ref. 4). The DP of
polySia chain ranges from 8 to ca. 200 Sia residues. The
chains with DP from 2 to 7 are named oligosialic acids
(oligoSia). Figure 2 shows the intersialyl linkages of oligo-
Sia and polySia. Both the reducing and nonreducing ends
of the chain are indicated. Sialic acid monomers in bacterial
capsular polySia chains are joined internally through a2,8-,
a2,9-, or alternating a2,8-/a2,9-ketosidic linkage between
Neu5Ac. The intersialyl linkages in eukaryotic cells are
usually a2,8-ketosidic linkage between Neu5Ac, a2,8-keto-
sidic linkage between KDN, and a2,8-ketosidic linkage
between Neu5Gc, although other kinds of linkages were
also reported (for review see Ref. 3).
Figure 2 The most frequent intersialyl linkages in polysialic

acids chains. A: a2,8-ketosidic linkage between Neu5Ac; B:
a2,9-ketosidic linkage between Neu5Ac; C: a2,8-ketosidic
linkage between Neu5Gc; D: a2,8-ketosidic linkage between
KDN. The reducing and nonreducing ends of the chain are
indicated.
Polysialic Acid: Structure and Properties 709
C. NMR Studies of PolySia Chains The solution conformations of N. meningitides B

polySia were analyzed by 2-D NOE NMR at 500 MHz,
The dierences in the orientation of the a2,8Neu5Ac and molecular modeling of oligoSia conformers, and complete
a2,9Neu5Ac polySia chains and in the segmental motion relaxation matrix analysis [9]. The analysis suggested that
of the polysaccharides were determined from 1H and 13C polySia adopted helical structures, with three to four Sia
NMR studies [5]. Polysaccharides were extracted from residues forming one turn of the helix with a pitch of 0.9
bacterial capsules of Neisseria meningitides serogroup B 1.1 nm. The conformational properties of colominic acid
(intersialyl linkages a2,8-) and non-O-acetylated and (Neu5Ac)5 were evaluated by NMR studies at 600
serogroup C (intersialyl linkages a2,9-). It was found that MHz, in conjugation with potential energy calculations
the side-chain of Neu5Ac in non-O-acetylated serogroup [10]. These calculations were used to estimate the energet-
C polySia chain adopts a conformation such that H-7 and ically favorable conformers and to describe the wide range
H-8 are approximately antiperiplanar. In serogroup B of helices polySia can adopt. Similar helical parameters for
polySia H-7 and H-8 were in gauche conformation. Mo- a2,8Neu5Ac polysaccharide and poly(A) were proposed
lecular mechanics calculations were used to probe the as the basis for their cross-reactivity to a monoclonal
conformational preferences of both B and C polySia antibody. The structure of (Neu5Ac)9 helix was presented
chains, and the results were in agreement with those based with a pitch of 0.55 nm. This study was extended by
on 1H NMR data. From the 13C NMR spin-lattice relax- comparison of the conformation of the polySia epitope
ation times, the molecular correlation times have been with its reduced and N-acyl derivatives [11]. The critical
calculated. The C polySia was characterized by internal or importance of the carboxylate group to the stability of the
segmental motion in the C-7 to C-9 side chain of the extended (Neu5Ac)9 helical epitope was ascertained from
monomer, in contrast the B polySia had little or no such NMR studies and potential energy calculations on the
movement and tumbled as a rigid species with internal carboxyl reduced polySia. These studies indicated that the
rotation of only the pendant C-9 group. extended helix was not stabilized in the reduced polymer.
The chain lengths of oligoSia puried from depolym- Studies on N-acyl derivatives showed that the increasing
erized polySia were analyzed using 1H NMR data [6]. size of these substituents did not disrupt the extended
PolySia were extracted from bacterial capsules of Esche- helical conformer, indicating that the bulky N-acyl groups
richia coli K1 (colominic acid), E. coli K92, and N. menin- protruded outward from the helix. Sensitive pulse sequen-
gitides serogroup B. A comparison of the intensities of the ces were developed in order to determine geometry-depen-
H-3e signals for the nonreducing, internal, and reducing dent 1H and 13C chemical shift anisotropy terms of
residues of the oligoSia gave ratios of 1:5:1 and 1:8:1 for Neu5Ac and its a2,8 homopolymer (colominic acid) in
heptaSia (Neu5Ac)7 and undecaSia (Neu5Ac)11, respec- solution [12]. Large changes in the chemical shift anisot-
tively. Both one-dimensional and two-dimensional 1H and ropy values were observed and the most pronounced
13
C NMR experiments were carried out on the triSia dierences were at C5, C6, and C7, which might be
(Neu5Ac)3 and colominic acid [7]. In addition to the attributed to conformational changes around C6/C7 and
assignment of the signals in the spectra, these studies C7/C8 bonds, and possible changes in hydrogen bonding
demonstrated that both linkages of (Neu5Ac)3 diered in interactions. 1H NMR chemical shifts for poly(Neu5Ac)
conformation from each other and from the inner linkages chain (colominic acid) obtained at 300 K in D2O are
of colominic acid. The data indicated that these conforma- presented in Table 1. (for details see Ref. 11).
tional dierences extended to both terminal disaccharides
of oligosaccharides larger then (Neu5Ac)5. It also provided D. Optical Activity Studies of PolySia Chains
an explanation for the conformational epitope of N. men-
ingitides B polySia: because the two terminal disaccharides Solution structure determination is greatly facilitated both
of (Neu5Ac)10 dier in conformation to its inner residues, by NMR spectroscopy and circular dichroism (CD) [13].
the immunologically functional part of (Neu5Ac)10 resides The common property that these biopolymers possess is
in its inner six residues. Thus this number of residues was chirality. Solution properties of Neu5Ac homopolymers,
consistent with the maximum size of an antibody site. The colominic acid, were reported [14], including a study of the
1
H NMR spectrum of polySia extracted from the capsule process of intramolecular lactonization of polySia. The
of N. meningitides B bacterial cells was completely assigned polarimetric pH-jump experiments showed that at pH
by two-dimensional homonuclear (COSY and HOHAHA) lower than 5 the chiroptical properties of colominic acid
and heteronuclear (1H, 13C) NMR experiments [8]. exhibit characteristic time dependence traceable to a pro-
1
Table 1 H NMR Chemical Shifts (y) for Poly(Neu5Ac) Chain (Colominic Acid) Obtained at 300 K in D2O
Proton H-3a Me H-3e H-4 H-6 H-5 H-7 H-8 H-9
y, ppm 1.74 2.07 2.67 3.60 3.63 3.82 3.90 4.10 4.19
Meprotons of methyl group within the acetyl group linked to carbon 5.
710 Janas and Janas
gressive increase in lactonic ring formation along the L-CA, 3 for H-CA and M-CA, and was estimated to be 2
chains. These experiments demonstrated that lactonization for KDN-gp, H-PSGP, and L-PSGP.
was catalyzed by protons and followed simple second- OligoSia (DP = 26), long-chain polySia (colominic
order rate kinetics (irreversible) during the early stages of acid, DP > 50), and short-chain polySia (obtained by mild
the process. This kinetics could be tentatively interpreted acid hydrolysis of colominic acid, DP = 417) were inves-
by assuming that at pH values 4.15 and 2.79 colominic acid tigated by CD spectroscopy and viscometry at dierent
is protonated at 50% and 100%, respectively. temperatures and salt concentrations to elucidate confor-
Equilibrium dialysis and CD methods were used to mational transitions and the degree of exibility of the
investigate the Ca2+ binding properties of three dierent polySia glycotope [16]. The eect of colominic acid N-de-
types of both oligoSia and polySia chains: oligo/poly acylation on these properties was also studied. N-Deace-
(Neu5Ac), oligo/poly(Neu5Gc), and oligo/poly(KDN) tylated colominic acid (i.e., a homopolymer of neuraminic
[15]. The apparent binding constants (Ka) and the number acid) was prepared by treatment with 10 N NaOH at 80jC
of binding sites (n) were estimated. PolyNeu5Ac was sep- for 3 hr. PolySia chain has two chromophoric groups: N-
arated from colominic acid using Sephacryl S-200 chroma- acyl and carboxyl, both of which exhibit n!P* electron
tography column in the form of high molecular weight transition in the region 205225 nm. The dichroic absorp-
(H-CA) of DPc24, and medium molecular weight (M-CA) tion of N-acetyl groups generally occurred at a lower
of DPc15. OligoNeu5Ac of DPc5 was prepared from wavelength than in the case of carboxyl groups. Therefore
colominic acid by treatment with 0.01 N HCl at 80jC for 10 in the case of N-deacylated colominic acid, the CD spectra
min and then chromatographed on the same type Sephacryl resulted only from the optical properties of the carboxyl
S-200 column. Poly(Neu5Gc) and oligo(Neu5Gc) were group. The spectrum of N-deacylated colominic acid
obtained from high-molecular weight polysialoglycopro- showed a negative dichroic band, which represented the
teins (H-PSGP) and low-molecular weight polysialoglyco- chiral environment of the carboxyl group. For colominic
proteins (L-PSGP) puried from unfertilized and fertilized acid, the carboxyl band overlapped with the N-acetyl band.
eggs, respectively, of rainbow trout. Oligo/poly(KDN) From the CD measurements of polySia with dierent DP,
were prepared by periodate Smith degradation of KDN it was concluded that the spectra vary signicantly from
glycoprotein (KDN-gp) which was isolated and puried the monosaccharide to trisaccharide, and this variation
from the ovarian uid of rainbow trout. The CD spectrum diminished when the DP was bigger than 9. This conclusion
of M-CA exhibited a weak negative band at ca. 225 nm and was consistent with previously described results of NMR
a stronger positive band at a lower level at ca. 205 nm. The experiments [11] suggesting that more than nine Neu5Ac
addition of calcium ions to the colominic acid solution did units are needed for a cooperative binding of polySia
not cause any change of the negative band, but resulted in a antigen to the antibody.
decrease of the lower wavelength positive band. Similar
experiments were performed for Mg2+ and Na+ ions. The E. Atomic Force Microscopy of PolySia Chains
results indicated that colominic acid had higher binding
anities for calcium ions than for Mg2+ and Na+ ions. Atomic force microscopy has become a new method to
KDN-gp, H-PSGP, and L-PSGP all had positive CD bands visualize supramolecular structures under conditions close
at 213 nm, and the intensities of these bands were reduced to their native state (for review see Ref. 17). In the imaging
upon binding to calcium ions. Table 2 shows the values mode the probe is scanned across the sample keeping a
of apparent binding constant, Ka, for binding of Ca2+ to constant force, amplitude, or frequency shift. This tech-
dierent polySia chains. Ka values were calculated from nique was used to image polySia chains [18] in the form of:
measured Ca2+-induced changes in molar ellipticity of . oligomers consisting of 6, 9, 12, 15, or 18 residues of
appropriated solution using CD method. The number of
a2,8-linked Neu5Ac, which were puried by high-
sialic acid residues required to bind one Ca2+ ion was 5 for
performance liquid chromatography (HPLC) from
colominic acid
. polysialyl glycopeptides of embryonic brain neural
cell adhesion molecule (N-CAM), in which the
Table 2 Apparent Binding Constant, Ka, for Binding of polySia is formed by a2,8-linked Neu5Ac
Ca2+ to Dierent PolySia Chains The experiments revealed the presence of lamentous
CA H-PSGP KDN-gp L-PSGP structures in samples of oligomers of 12 or more sialyl
residues, whereas shorter oligomers did not display these
Ka, M1 10.9103 2.98103 2.89103 1.00103 structures. Individual laments had a thickness of ca. 1 nm
and tended to occur as lament bundles. As the DP of
Ka values were calculated from measured Ca2+-induced changes in
oligoSia increased, an extensive branching of the lament
molar ellipticity of appropriated solution using circular dichroism
method. bundles into networks was observed. Similar network was
CAcolominic acid, H-PSGPhigh-molecular weight polysialogly- observed for a sample containing puried polysialylated
coproteins, L-PSGPlow-molecular weight polysialoglycoproteins, glycopeptides of N-CAM. The network was degraded by
KDN-gppoly(KDN) glycoprotein. sialidase, thus conrming that it consisted of sialic acid.
Source: From Ref. 15. When the sample containing the same glycopeptides, but
without attached polySia, was examined, lament bundles

were not observed. It was suggested that the potential of
polySia chains to associate into bundle networks could
result in associative interactions between cells, as a novel
mechanism by which N-CAM and other cell membrane
components may mediate their interactions with other
molecules and cells.
F. Capillary Electrophoresis of PolySia Chains

A direct and rapid method for the separation of oligoSia
and polySia consisting of a2,8-linked Neu5Ac with various
DP was presented using capillary electrophoresis (CE) [19,
20]. It was also demonstrated that CE was a useful tool for
kinetic studies of the degradation of polySia and lactonized
polySia by neuraminidase. Comparative structural analysis Figure 3 Relationship between the electrophoretic mobility
of oligoSia and polySia chains from diverse sources was and degree of polymerization of oligoNeu5Ac in the presence
carried out using high-performance capillary electropho- of poly(ethylene glycol) at concentration 10% and applied
resis (HPCE) [21]. Controlled acid hydrolysates of three voltage 10 kV. (From Ref. 22.)
dierent series of a2,8-linked polySia chains were analyzed:
. (!8Neu5Aca2!)n chains from colominic acid
. rainbow trout egg polysialoglycoprotein (PSGP) tion of oligoNeu5Ac for short and long oligomers could be
containing (!8Neu5Gca2!)n chains explained on the basis of the formation of helical structure
. rainbow trout egg KDN-rich glycoprotein (KDN- in the case of longer oligoNeu5Ac. Similar experiments
gp) containing (!8KDNa2!)n chains. were also performed for hyaluronic acid chains [22] with
similar results.
These three dierent types of a2,8-linked oligoSia Lactonization and hydrolysis of oligoNeu5Ac dimers,
having the same degree of polymerization could be sepa- trimers, and tetramers were studied by capillary electro-
rated by HPCE. In addition, the HPCE technique showed phoresis [23]. The reaction temperature was the key factor
that the lactonization of a2!5-Oglycolyl-linked oligo/poly- that determined whether these two reactions would pro-
Neu5Gc chains, i.e., (!5-Oglycolyl-Neu5Gca2!)n chains, ceed simultaneously. At higher temperatures (60jC) lacto-
prepared from sea urchin egg jelly sialic acid-rich glyco- nization and hydrolysis of oligoNeu5Ac were competitive
protein (polySia-gp) did not take place as readily as in under acidic conditions (0.1 N acetic acid). At lower
(!8Neu5Aca2!)n chains from colominic acid. temperatures (<10jC), only lactonization could proceed
High-resolution analysis of oligoSia and polySia under the acidic conditions. The experiments showed that
chains was performed by capillary electrophoresis using initial lactonization took place in the internal (middle)
various types of capillaries and buer additives [22]. The position of the oligomers. The a2,8-linked glycosidic bonds
best resolution in separating individual chain-lengths was in oligoSia were protected from hydrolysis if the bonds
achieved using a capillary coated with phenylmethylsili- were in the lactone ring, especially in the dimer. This could
cone and a buer solution containing a neutral polymer, explain why the dimer was the major product in the
poly(ethylene glycol) as an additive. It was found that hydrolysis of oligoSia and polySia under acidic conditions.
smaller oligoSia composed of less than ve Neu5Ac resi-
dues migrated in the reverse order of their molecular
G. Electrochemical Detector of PolySia Chains
masses on the electrophoregrams. However, oligoSia
chains larger than pentamer migrated in the order of their An electrochemical method for the determination of poly-
molecular masses. PolySia chains having up to 100 Neu5Ac Sia chains has been developed, by using a potentiometric
residues were clearly distinguishable. Figure 3 shows the thick-lm sensor [24]. The biosensor was coated with
relationship between the electrophoretic mobility and depoly(vinyl acetate)-polyethylene copolymer membranes
gree of polymerization of oligoNeu5Ac in the presence of containing immobilized sialidase (N-acetylneuraminidase).
poly(ethylene glycol) at concentration 10% and applied These membranes with immobilized sialidase were placed
voltage 10 kV (for details see Ref. 22). The mobility of each on top of a H+-selective poly(vinyl chloride)poly(vinyl
oligoSia was calculated according to the equation: acetate) indicator membrane. The enzymatic cleavage of
l l=tE 1 the terminal Sia residue from polySia (colominic acid)
chain led to the release of sialic acid monomers in the form
2 1 1
where l (cm V sec ) is the mobility; l (cm) is the length of a a-ketocarboxylic acid. A related local pH change in the
from the injection port to the detector; t (sec) is the sialidase-containing membrane was generated, which was
migration time; E (V cm1) is the applied electric eld. measured by the H+-selective indicator membrane. The
As the smaller molecules can pass through the matrix more soluble sialidase from Clostridium perfringens had its pH
easily than the larger ones, the dierent behavior in migra- optimum in the pH range 3.54.5 when immobilized on
712 Janas and Janas
group in the rigid components of polySia chains was

relatively broad and became visible as a separate signal
below the sharp signal. After sonication of polySia chains
with lipid dispersion, sevenfold increase of the amount of
polySia exible segments and twofold decrease of the
amount of rigid segments were observed in comparison
with the case of sonicated polySia chains without lipo-
somes. Similar phenomenon was reported in the case of
sonication of the hyaluronic acid chains with phosphati-
dylcholine [27]. Both the presence of net negative charges
on the surface of phosphatidylocholine liposomes (after
modication of the liposomes with phosphatidic acid) and
net positive charges (after modication of the liposomes
with octadecylamine) resulted in the decrease of the
amount of exible segments and the increase of the
amount of rigid segments within polySia chains in com-
parison with the case of nonmodied phosphatidylcholine
Figure 4 Relationship between biosensor potential and pH liposomes [26].
1
of the buer solution for colominic acid concentration 10 H NMR technique was also used for studies of the
mg/mL. (From Ref. 24.) eect of polySia chains on the dynamics of phosphatidyl-
choline liposomes [28]. Upon addition of the paramagnetic
ion Pr3+ separate signals of the protons of the lipid choline
the biosensor. The relationship between the biosensor group could be observed from the inner and outer layer of
potential and the pH of the buer solution for colominic the bilayer lipid membrane. The presence of polySia chains
acid concentration 10 mg/mL is presented in Fig. 4 (for resulted in an increase of adsorption of praseodymium ions
details see Ref. 24). The values of the potential in the to the external surface of phosphatidylcholine/octadecyl-
optimum range of pH were ca. 10 mV. Similar experiments amine vesicles. A decrease of half-width of proton signal
were also performed for other (not polySia) sialic acid- both from lipid choline group and CH2 groups, and CH3
containing polysaccharides: mucine and sialyl-lactose. terminal group of the lipid alkyl chain was also detected
for these liposomes indicating an increase of the rate of
H. High-Performance Anion-Exchange segmental movement of these groups in the presence of
Chromatography of PolySia Chains polySia chains. Figure 5 shows the relationship between
the half-width (Dm1/2) of 1H NMR spectra maximum
High-performance anion-exchange chromatography corresponding to N-methyl choline signals of phosphati-
(HPAEC) with pulsed electrochemical detection was ap- dylcholine from the inner monolayer of vesicles, and the
plied for analysis of oligoSia and polySia chains for DP up concentration of polySia. A strong sharpening of the spec-
to 70 [25]. However, the limitations in sensitivity and tral peaks was observed indicating an increase of the
selectivity of the pulse electrochemical detector became
signicant when the material contained a large portion of
protein or the material was available only in small amount.
Therefore a new method was developed for more sensitive
DP analysis of polySia chains [25].
III. DYNAMICS OF POLYSIALIC ACID

A. Interactions of PolySia Chains
with Membranes
The eect of phospolipid liposomes on the conformational
mobility of polySia chains (colominic acid) was studied
using 1H NMR technique [26]. In liposomal suspension,
typical 1H NMR spectra of polySia were obtained with the
assignments of the signal from protons based on the data
in Ref. 11. The sharp signal at 2.08 of the three-methyl
protons of the acyl group was well separated from other
signals as a sharp singlet. The calculated spinspin relax- Figure 5 Relationship between the half-width (Dr1/2) of 1H
ation times from the half-width of this signal were pro- NMR spectra maximum corresponding to N-methyl choline
portional to the conformational mobility of the exible signals of phosphatidylcholine from the inner monolayer of
segments of the polySia chains. The signal from the acyl vesicles, and the concentration of polySia. (From Ref. 28.)
segmental movement of lipid molecules within this mono- phosphodiester linkage. The phospholipid might play a
layer. Cyclic voltammetry, uorescence spectroscopy, and role in linking the capsule to the outer bacterial membrane.
electron microscopy studies on the eect of polysialic acid The readily water-soluble K1-antigen incorporated spon-
on phospholipid model membranes were performed [29 taneously via its lipid anchor group into the lipid vesicle
32]. PolySia chains aected the electrochemical properties membrane as proven by immunouorescence using a uo-
of bilayer lipid membranes, changed the morphology of rescein-labeled monoclonal antibody anti-K1-IgG
lipid vesicles, and modulated the uorescence response of a mAb735 and the enzyme-linked immunoadsorbant assay
voltage-sensitive dye, Oxonol-V, in lipid vesicles. Similar (ELISA) procedure. Without the lipid molecule no binding
results were observed in the case of neuraminidase treat- of polySia to the lipid vesicles was observed. This was
ment of the porcine intestinal brush-border membrane shown by using colominic acid, which consisted of polySia
vesicles [33,34]. Two uorescence dyes, pyrene and 1,6- chains (of shorter length in comparison to K1 antigen as
diphenyl-1,3,5-hexatriene (DPH), were used to study the they were derived from the larger polySia chains by acid-
lipid uidity in the membranes [33]. After treatment with catalyzed hydrolysis [37]) with free reducing end, instead of
neuraminidase a shift of thermal transition temperature, an puried K1-antigen. An additional proof for the lipid
increase in the uorescence-quenching rate for Tl+ and a anchor insertion was obtained from experiments using
decrease in the uorescence lifetime were observed. The pH-treated K1-antigen. Because the phoshodiester bond
results indicated that the lipid organization of the mem- by which the lipid is linked to the polySia chain is acid labile
branes was susceptible to neuraminidase treatment and [38], puried K1-antigen was treated at 60jC for 1 hr at pH
that the membrane uidity increased by desialylation 5. This treatment resulted in complete loss of incorporation
through neuraminidase treatment. Three uorescent dyes, of K1-antigen into liposomes. The incorporated K1-anti-
DPH, 1-anilino-8-naphthalene sulfonate (ANS), and 3,3V- gen was also treated with polySia-cleaving enzyme endo-
dipropyl-2,2V-thiadicarbocyanine iodide (DiS-C3(5)), were neuraminidase (Endo-N) [39]. Endo-N was added to the
used to study the surface charge density and the potential of K1-antigen-bearing lipid vesicles before or after the assay
the membranes [34]. The results of quenching experiments for the antibody binding. In the rst set of experiments
using DPH-labeled membranes and cationic and anionic Endo-N was added before addition of the antibody, and no
quenchers suggested an increase of the membrane surface antibody binding could be detected. Therefore the chain
density of negative charges by desialylation through neur- length of the vesicle-associated polySia was reduced by the
aminidase treatment. These results were supported by a enzyme action to less than 10 Neu5Ac groups, which is the
decrease of ANS-binding anity of the membranes after critical chain length for binding of the mAb735 antibody.
desialylation. The Kd value of the ANSmembrane com- In the second set of experiments, the Endo-N was added
plex increased after neuraminidase treatment from 36.0 to after the antibody was allowed to bind to the K1-antigen-
54.9 AM. The degree of valinomycin-induced uorescence bearing lipid vesicles. A complete loss of the membrane-
change of the membrane-potential-sensitive probe DiS- bound uorescence was observed indicating that Endo-N
C3(5) in membrane vesicles was reduced by desialylation was able to cleave the K1-antigen polySia even when the
suggesting that penetration of the dye molecules toward the antibody was bound. Because the K1-antigen was an
vesicle interior was facilitated by this treatment. The mem- amphiphilic polymer, it formed micelles in water [38]. Thus
brane potentials estimated from the valinomycin-induced the driving force for the insertion of the lipid residue into
changes in DiS-C3(5) were from 25 to 29.7 mV in lipid vesicles was the gain in free energy resulting from the
control membranes, and from 40 to 48.8 mV in neur- transition of the micellar system to a system in which the
aminidase-treated membranes. lipid residues were located in the hydrophobic interior of
The Langmuir monolayer technique was used to in- the bilayer lipid membrane.
vestigate the inuence of polySia chains on phosphatidyl-
choline/octadecylamine lipid lms [35]. Isotherms, limiting B. Binding of PolySia Chains to Antibodies
molecular areas, and collapse pressure were measured for
various concentrations of polySia in the subphase. The The antibodies recognizing oligoSia or polySia glycotope
isothermal isobars showed deviations, both negative and were classied into three groups, based on the immuno-
positive, from the additivity rule. The specic molecular specicity for oligo/polySia chain length and the involve-
areas of the lipid molecules were calculated showing the ment of the nonreducing terminus in antibody recognition
modulation of the value of these areas in the presence of [4]. Group I antibodies (Table 3) recognized chains of a2,8-
polySia. Interaction of polySia chains with the lipid head- linked sialic acid monomers with DP 8 or greater. These
groups resulted also in the changes of limiting molecular antibodies could recognize the helical structure formed by
areas, collapse pressure, and the Gibbs free energy of Sia monomers within the internal region of polySia chain.
mixing of the monolayer components. The nonreducing terminal residues did not participate in
The insertion of puried K-antigen from E. coli K1 these antigens recognition. Group II antibodies recognized
into giant liposomal membrane was investigated [36]. The both oligoSia with DP = 37 and longer polySia chains.
K1-antigen consisted of a large hydrophilic parta homo- These antibodies could recognize the distal part of oligo/
polymer of ca. 200 a-2,8-linked Neu5Ac residues, which is polySia chain, including the nonreducing terminal mono-
connected at its potential reducing end to a hydrophobic mer. Group II antibodies recognized oligoSia chains with
part1,2-diacylglycerol (phosphatidic acid)through a DP = 24, but do not bind polySia. These antibodies
714 Janas and Janas
Table 3 Immunospecicity for Oligo/PolySia Chain Length interface between two media of dierent refractive index
of Oligo/PolySia Antibodies (for review see Ref. 44). Under conditions of total internal
reection of a plane polarized light, an evanescent wave
Group I Group II Group III penetrates into the medium of lower refractive index,
antibodies antibodies antibodies causing free electrons in the metal layer to oscillate, thus
DiSia, DP = 2 + generating the surface plasmon waves. This phenomenon
OligoSia, DP = 37 + + or occurs at one specic angle of the incident light and can be
PolySia, DP z 8 + + monitored in the reected light as a reduction in the light
intensity at this angle. After binding of the substrate to the
Source: From Ref. 4. surface, this angle changes. The dierence between the
angles is indicative of the amount of substrate bound to the
surface. The rate at which this change occurs depends on
recognized specic conformations of these short-chain the kinetics of the interaction. The antipolySia antibody
oligomers. A subgroup within group III could be dened was immobilized on the gold surface and was in contact
for antibodies that recognized only dimers of sialic acid. with the buer, whereas the polySia chains of dened chain
The antigen-binding fragment from IgG murine length were dissolved in the buer. The interaction be-
monoclonal antibodies (designated mAb735) with specic- tween these two biomolecules resulted in the changes in the
ity for long polySia chains (i.e., belonging to group I local refractive index at the surface, thus changing the
antigens) has been prepared and crystallized [40]. This angle of reected light. The obtained response curves
antibody recognized a specic helical conformation of (sensorgrams) were used to evaluate the kinetics of the
polySia chain and a minimum of 10 residues was required interaction and the steady-state binding constants. Lower
to form a helical epitope. The binding site deduced from responses were observed for polySia samples, in compar-
X-ray diraction analysis displayed a shape and distribu- ison with protein samples, because of the lower refractive
tion of charges that was complementary to that of the indexes typical for carbohydrates. Both the association
predicted conformation of the oligosaccharide helical epi- rates (kon) and the antibody binding anity dramatically
tope with six Sia residues per turn and a pitch of 3.6 nm. increased with the increase of polySia chain length
Thermodynamics of polySia binding to the mAb735 anti- (Table 5, for details see Ref. 43). A subnanomolar disso-
body was determined using titration microcalorimetry for ciation constant (KDc0.85 109 M) was obtained for
polySia ranging in length from 9 to 15 residues (Table 4, for the binding of the long (DPc200) polySia chains isolated
details see Ref. 40). The binding of these ligands (KA = 4 from group B N. meningitides. Colominic acid (DPc100)
104 M1) was ca. 10 times weaker than for long chain (DP from E. coli K1 (KDc6.8 109 M) and shorter polymers
= 41, KA = 3 105 M1). There was only little chain (DPc50, 31, and 16) isolated from hydrolyzed colominic
length dependence for any of the thermodynamic functions acid exhibited progressively lower association rate con-
in the range of DP from 9 to 15. The thermodynamic and stants and weaker anities. The polysialylated glycopep-
X-ray crystallographic data were consistent with a binding tides from human embryonal brain material exhibited high
site that accommodated at least eight sialic acid residues. anities (KD c5 109 M) indicating the presence of long
A method for screening and characterizing antipolySia chain epitopes on the glycopeptide. The inuence of pH,
antibodies using lipid-conjugated polySia was developed temperature, ionic environment, and polyamines was also
[41]. Homopolymers of a2,8-linked Neu5Ac, Neu5Gc and determined. Both divalents (Ca2+, Mg2+, Mn2+; concen-
KDN were conjugated with dipalmitoyl-phosphatidylcho- tration up to 25 mM) and a polyamine (spermine) in-
line by reductive amination to form neopolysialoglyco- creased the anity of polySia to the antibody.
lipids. The lipidation of polySia chains facilitated their Several monoclonal antibodies directed to polysaccha-
noncovalent immobilization on the solid surface thus ride antigens were tested for their cross-reactivity with
permitting to immunodetect the polySia antigen rapidly polynucleotides and DNA [45,46]. With the antibodies
and with high sensitivity. Using a series of these immobi- mAb735 and IgMNOV no binding to polysaccharides other
lized neopolysialoglycolipids with dened DP, the mini-
mum chain length was determined which was required
for binding of new anti-polyNeu5Ac and anti-KDN anti- Table 4 Thermodynamic Parameters for the Binding of
bodies. A determination of the minimum polySia chain a2,8-linked PolySia to mAb735 Antibody
length that is recognized by these antipolySia antibodies
was important as the length of polySia chains on the neural DP of
cell adhesion molecule (N-CAM) changed markedly dur- polySia K DGo DHo
ing vertebrate development. The change in DP was found chain (104 M1) (kJ M1) (kJ M1)
to be inversely correlated with the strength of the N-CAM-
9 3.9 26.2 45.4
mediated homophilic binding of neural cells [42].
11 4.8 26.7 42.6
The interaction of polySia chains with the antipolySia 14 5.9 27.2 38.7
antibody mAb735 was evaluated by surface plasmon 15 4.4 26.5 41.6
resonance [43]. Surface plasmon resonance is an optical
phenomenon occurring at a metal (e.g., fold) surface at the Source: From Ref. 40.
Table 5 Association Rate (kon) and the Antibody Binding Constant (KD) for the Binding of Poly(Neu5Ac) to the
Antibody mAb735
kon (103 M1 sec1) KD (109 M)
Poly(Neu5Ac) from hydrolyzed colominic acid, DP=16 Not determined 8260

Poly(Neu5Ac) from hydrolyzed colominic acid, DP=31 36 92.5
Poly(Neu5Ac) from hydrolyzed colominic acid, DPc50 290 24.9
Poly(Neu5Ac) from colominic acid, DPc100 1300 6.8
Poly(Neu5Ac) from Neisseria meningitides group B, 2000 0.85
DPc200
than poly(a2,8-Neu5Ac) was observed even with such IV. POLYSIALIC ACID IN BACTERIAL CELLS
closely related compound as a2,8-/a2,9-poly(Neu5Ac).
However, IgMNOV showed marked cross-reactivity with a A. Occurrence, Structure, and Function
variety of polynucleotides and DNA, which could result of PolySia in Bacterial Cells
from a nonspecic reactivity of this antibody with nega-
PolySia chains, attached to the outer face of the outer
tively charged polyanionic chains. The specicity of the
membrane through a phospholipid, form a capsule in some
antibody mAb735 proved to be unique among 11 other
bacterial strains (for review see Refs. 3,50,51). The a,2,8-
monoclonal antibodies directed against various bacterial
linked polySia capsule of neuropathogenic N. meningitides
polysaccharides; these antibodies reacted at least with one
serogroup B was found to be immunologically and struc-
kind of polynucleotide.
turally identical to the capsule of neuroinvasive E. coli K1
strains (Table 6). These capsular polysaccharides were
C. Binding of PolySia Chains to Melittin reported to be neuroinvasive determinants associated with
The interaction of polySia chains (colominic acid) with neonatal meningitides in humans [52,53]. Structural anal-
melittin was studied using CD measurements [47]. Melittin ysis showed DP of at least 200 Sia residues for capsular
is a 26-residue peptide, which adopts an amphipathic a- polySia chains on E. coli K1 cells grown at pH 7.0 [54]. The
helical conformation in the presence of biological mem- sialic acid monomers in the polySia capsules of E. coli K92
branes, micelles, and surfactants, and can self-associate and E. coli strain Bos-12 were reported to be joined through
into a tetrameric complex. The CD spectra of melittin and alternating a2,8- and a2,9-ketosidic linkages [55]. In N.
its analogues were measured for dierent concentration meningitides serogroup C sialic acid monomers in polySia
(mg/ml) ratio of colominic acid and the peptide. Melittin chain were connected through the a2,9- linkage [56]. It was
was induced into an a-helical conformation upon increas- shown that O-acetylated sialic acid residues in the poly
ing this ratio to 1 with no further changes upon increasing (Neu5Ac) chains of some substrains of E. coli K1 (desig-
the ratio beyond 1. No helix formation was observed for nated OAc+) could decrease the pathogenicity and in-
melittin in the presence of sialic acid monomers. Similar crease the immunogenicity of these bacteria [57]. These
conformational changes were also observed for several
melittin analogues, some of them revealing a signicant
h-structure content.
Table 6 Occurrence and Structure of Polysialic Acid in
Bacterial Cells
D. Binding of PolySia Chains to a
Mutant Bacteriophage Bacterial strain Structure
Endo- and exoglycosidases have proven to be useful Escherichia coli K1 (a2,8-Neu5Ac)n;

reagents for structural analysis of glycoconjugates [48]. 7-O-Ac-; 9-O-Ac-
One such enzyme is an endo-N-acetylneuraminidase Escherichia coli K92 Alternating
(Endo-N) associated with bacteriophages that specically (a2,8-Neu5Ac)n
recognize the polysialic acid capsule of E. coli K1 as a and (a2,8-Neu5Ac)n
receptor and subsequently degrade it. These bacteriophage linkages
mutants that could bind to polySia chains but lost the Moraxella nonliquefaciens (a2,8-Neu5Ac)n
catalytic activity (do not cleave polySia) were tested wheth- Neisseria meningitides (a2,8-Neu5Ac)n
serogroup B
er they could recognize and bind to bacterial and eukary-
Neisseria meningitides (a2,9-Neu5Ac)n
otic polySia [49]. A minimum binding requirement was
serogroup C
determined to be 10 sialyl monomers in the polySia chains
Pasteurella (a2,8-Neu5Ac)n
using oligo/polySia of dened length prepared from colo- haemolytica A2
minic acid.
716 Janas and Janas
OAc+ substrains could either be xed or could undergo a membrane-spanning protein containing six transmem-
reversible form variation between OAc+ and OAc at a brane hydrophobic helices whereas KpsT is a peripheral
characteristic frequency. inner membrane protein (localized at the cytoplasmic side
The expression of polySia capsule in N. meningitides of the membrane).
serogroups B and C, and E. coli K1 strains appeared to Region 1 of ca. 1012 kb encodes for six proteins that
facilitate bacterial invasion and colonization of the have been implicated in polySia assembly and export to the
meninges in neonates; it might represent an elaborate cell surface. The proposed functions for the gene products
survival mechanism that evolved to trick the human im- of region 1 are as follows:
mune system by masking the O-antigen moieties of lipo- . KpsFarabinose-5-phosphate isomerase, re-
polysaccharides (reviewed in Ref. 50). This polyanionic
quired for KDO biosynthesis
capsule also provides a barrier against phagocytosis. . KpsEinitiase responsible for starting polySia
Poly(a2,8-Neu5Ac) is poorly immunogenic because these
chain synthesis on undecaprenyl phosphate
chains are structurally identical to the poly(Neu5Ac) epi- . KpsDpolySia transport in the periplasm
tope on the embryonic form of N-CAM in vertebrates. . KpsUCMPKDO synthetase
Poly(a2,9-Neu5Ac) has opposite immunogenic properties . KpsCcoupling of polySia biosynthesis and
as its structure diers from the polySia chains attached to
transport
N-CAM. The polySia capsule in E. coli K1 cells is a . KpsSpolySia chain elongation
receptor for polySia-specic bacteriophages that require
expression of this capsule for infectivity [50,58]. Bacterial cells with mutations in region 1 and region 3 genes
generally accumulated intracellular polySia chains, which
B. Genetic Organization of the KPS could be observed by transmission electron microscopy.
Gene Cluster in E. coli K1
C. Biosynthesis of PolySia Chains in
The KPS gene cluster in E. coli K1 is divided into three E. coli K1 Cells
functional regions (for review see Refs. 51,5961). The K1-
specic central region 2 of ca. 56 kb encodes six proteins The major steps in Neu5Ac synthesis, Neu5Ac activation
that direct the synthesis of Neu5Ac, activation of Neu5Ac, to form CMP-Neu5Ac, and poly(Neu5Ac) synthesis are
and polymerization (Fig. 6). Region 2 genes are unique for presented as reactions 15:
each E. coli serotype synthesizing a chemically and struc- ManNAc PEP X Neu5Ac Pi 4:1
turally distinct capsular polysaccharide. The proposed
functions for the gene products of region 2 are as follows: Neu5Ac CTP X CMP Neu5Ac PPi 4:2
. CMP Neu5Ac P C55 X Neu5Ac P
NeuSpolysialyltransferase 4:3
. NeuEpolymerization/transport coupling C55 CMP
. NeuCN-acetylmannosamine synthesis nCMP Neu5Ac P C55 X
. NeuACMP-Neu5Ac synthetase
. a2; 8 Neu5Acn P C55 4:4
NeuBNeu5Ac synthase
. NeuDacyltransferase n 1P C55
Region 3 of 1.6 kb encodes two proteins, which are essential a2; 8 Neu5Acn P C55
for the transport of polySia chains across the inner bacte- acceptor X a2; 8 Neu5Acn 4:5
rial membrane from the cytoplasmic side to the periplasmic acceptor P C55
side of the membrane. The proposed functions for the gene
products of region 3 are as follows: where PC55 is undecaprenyl monophosphate, PEP is
. phosphoenolpyruvate.
KpsMtransmembrane transporter of polySia
Reaction 1 is catalyzed by a soluble enzyme Neu5Ac
chains
. synthase (NeuB), reaction 2 is catalyzed by a soluble
KpsTATP binding and ATP hydrolysis
enzyme CMP-Neu5Ac synthetase (NeuA), reactions 35
KpsM and KpsT belong to the ATP-binding cassette are catalyzed by enzymes of the inner membrane-bound
(ABC) superfamily of membrane transporters. KpsM is a polysialyltransferase complex. The extent to which
Figure 6 Genetic organization of the kps gene cluster in E. coli K1. The boxes show the location of each gene. The arrows
indicate the directions of transcription for each region.
Neu5Ac monomers are polymerized on P-C55 is not chloro-2-methoxyacridine uorescence. These results indi-
known, but similar oligoSia-P-C55 intermediates have been cated that the polyST activity was modulated both by DlH
reported for polySia synthesis in N. meningitides serogroup and ATP.
B [62]. It was shown [51] that Sia residues might be fully
polymerized on P-C55 by the sequential addition of mono- E. Translocation of PolySia Chains Across the
mers (DP=ca. 200). Such a preassembled polysialyl-P-C55 Inner Membrane of E. coli K1 Cells
might be translocated across the inner membrane. PolySia
chain elongation occurred by tail growth, i.e., the addition Two proteins within the membraneassociate polysialyl-
of sialic acid monomers to the nonreducing terminus of the transferase complex in E. coli K1, KpsT, and KpsM are
growing chain in which the activated linkage in CMP- involved in the transmembrane translocation of polySia
Neu5Ac was used for its own addition [54]. Identication chains: KpsT (which hydrolyses ATP) and KpsM (which
of a presumed undecaprenol/dolichol recognition sequence forms a transmembrane channel). An in vivo system was
in the deduced protein sequence of NeuE but not NeuS [54] developed which used spheroplasts prepared from E. coli
suggested that NeuE might be an initiase responsible for K1 cells that are unable to degrade sialic acid (nanA4
starting polySia chain synthesis, whereas NeuS might mutation) in order to determine the molecular mechanism
catalyze chain polymerization. of the translocation of sialyl polymers across the inner
bacterial membrane [66,67]. PolySia chains that were trans-
D. Topology and Energy-Dependence of the located across the inner bacterial membrane were depoly-
Polysialyltransferase Complex Activity merized by endo-N-acylneuraminidase (Endo-N). In
in E. Coli K1 Cells contrast, those chains remaining inside vesicles were not
sensitive to Endo-N. Synthesis and translocation were
The membrane-associated polysialyltransferase complex followed kinetically after pulse-labeling the spheroplasts
(polyST) in E. coli K1 catalyses the synthesis, transmem- with 14C-Neu5Ac. The theory of compartmental analysis
brane translocation, and assembly of polySia chains. was applied for the determination of polySia distribution
Sealed membrane vesicles of dened orientation were used: between dierent compartments. The eect of valinomy-
right-side-out vesicles (ROV) which have the same topol- cinas a modulator of transmembrane electrical potential
ogy as intact cells, and inside-out vesicles (IOV) which have gradient (Dw)and CCCPas a modulator of proton
opposite orientation, in order to determine the transmem- transmembrane electrochemical potential gradient (DlH)
brane organization of the polysialyltransferase [6365]. on translocation rateswas tested. It was shown that both
The eect of vesicle permeabilization and vesicle inversion DlH and Dw were actively involved in the transmembrane
on the activity of the polyST was studied. It was found that translocation of polySia; DlH was found to be essential for
there was no polyST activity in ROV above the back- the translocation. In addition, the results indicated that
ground level whereas IOV prepared by French press polySia chains are translocated across the inner membrane
showed over vefold increase in polyST activity. In addi- while being membrane bound. A mechanism of the expres-
tion, there was a substantial increase in polyST activity in sion of polySia capsule in E. coli K1 cells was presented
ROV after inversion by sonication or French press, or by which included Dw-dependent insertion of polySia chains
permeabilization with toluene or Triton X-100. These into the KpsM channel within the inner membrane.
experiments showed that the functional domain of the
polysialyltransferase was located on the cytosolic face of
the inner membrane. The eect of partial trypsin treatment
on inactivation of the sialyltransferase activity in sealed
vesicles was assessed both before and after vesicles inver-
sion. There was only a slight decrease in polyST activity
when ROV were treated with trypsin, and then inverted,
but more than 90% of the polyST activity was lost when
ROV are inverted before trypsinolysis. These results con-
rmed the localization of the polyST at the cytoplasmic
side of the membrane. The eect of energy-rich com-
pounds, ATPase inhibitors, and uncouplers of the proton
electrochemical potential gradient (DlH) was studied in
IOV in order to determine the energy requirement for the
polyST complex activity. Both NADH and lactate oxida-
tion, and ATP hydrolysis caused an increase in polyST
activity whereas the nonhydrolyzable ATP analog, AMP-
PNP, reduced this activity. The treatment of KpsT protein
in IOV by a photoanity probe, 8-azido-ATP, reduced Figure 7 The transmembrane potential, Vm, as a function of
markedly the polyST activity with partial restoration after the polySia chain length (number of Sia monomers, DP).
NADH addition. The changes were correlated with the Calculated for the ratio of permeability coecients for
changes in DlH measured by quenching of 9-amino-6- positive and negative ions equal to 100. (From Ref. 68.)
718 Janas and Janas
legionaminic acid. The chain length of this polymer was

4075 sugar residues joined by a2,4 linkage. The binding of
this lipopolysaccharide-specic monoclonal antibody
mAb2625 to isolated poly(legionaminic acid) was studied
using surface-plasmon-resonance biomolecular interaction
analysis and saturation-transfer-dierence NMR spectros-
copy [71]. It was shown that mAb2625 antibody bound
directly to the N-methylated units of the polymer.
V. POLYSIALIC ACID IN EUKARYOTIC CELLS

A. PolySia Attached to the N-CAM
Protein in Vertebrates
1. Structure of Polysialylated N-CAM
Figure 8 The reversal curves showing the relationship PolySia chains, unlike most polysaccharides found on the
between the time, t, and the polySia chain length (number cell surface, in the vertebrate embryo appear to be conned
of Sia monomers, DP) for the polySia ux J=0. Calculated to the N-CAM protein (integral membrane protein), in
for the membrane conductance equal to 108 V1 cm2. particular the fth Ig domain (for review see Refs. 72, 73).
(From Ref. 69.)
Figure 10 shows the proposed structure of polySia attached
to N-CAM via typical N-linked glycosylation (in trans
Golgi compartment) through the expression of sialyl trans-
ferases (PST and STX) that are each dierentially regulated
An analysis, based on the GoldmanHodgkinKatz during development. Polysialylated N-CAM represents a
equation, of transmembrane potential changes resulting less-adhesive form of N-CAM, thus facilitating the synaptic
from transmembrane translocation of polySia was per- reorganization during brain development, and synaptic
formed [68]. The relationships between the transmembrane plasticity in mature neural cell. The mechanism of polySia
electric potential and the polySia chain length (up to 200 action probably involves steric hindrance created by its
monomers), the inner/outer concentration ratio of polySia, negative electric multicharge and large hydration volume.
the cation/anion permeability ratio and the temperature PolySia chains were also found in extraneural cells and
were discussed. It was found that the maximal membrane tissues, e.g., human kidney, heart, muscle, pancreas, lung,
potential changes, up to 118 mV, were developed for a thyroid, T-lymphocytes, and sodium membrane channels
permeability ratio greater than 1. The increase of the for sodium ions (for review see Ref. 2).
polySia chain length resulted in the decrease of this eect
(Fig. 7, for details see Ref. 68). 2. Eect of PolySia Charge and Hydration on
The analysis of transmembrane translocation of poly- Molecular Interaction and Intercellular Space
Sia chains was performed using membrane electrical equiv-
alent circuit [69]. The dependence of polySia transmem- Using a neuroblastoma/sensory neuron cell hybrid, it was
found that the removal of polySia chains with a neur-
brane ux on time, polySia chain length, membrane
electrical capacitance, membrane electrical conductance, aminidase (Endo-N) augmented cellcell aggregation me-
and applied membrane potential was discussed. It was diated by the L1 cell adhesion molecule as well as cell
found that the changes in polySia ux were up to 88%
after 1 msec. The decrease of membrane conductance and
the increase of polySia chain length resulted in the dimi-
nution of this eect. Inversion of polySia ux direction was
observed as a result of external potential changes. Reversal
curves, representing the values of considered parameters
for zero-ux, were presented (Fig. 8, for details see Ref. 69).
F. PolySia Chains in Bacterial

Lipopolysaccharides
The 1H- and 13C NMR spectroscopic studies were used to
elucidate the structure of the O-specic chain of Gram-
negative bacterium Legionella pneumophila serogroup 1
lipopolysaccharide [70]. It was found to be a homopolymer
of a sialic acid derivative: 5-acetamidino-7-acetamido-8-O- Figure 9 Chemical structure of legionaminic acid (5-
acetyl-3,5,7,9-tetradeoxy-nonulosonic acid, termed legio- acetamidino-7-acetamido-8-O-acetyl-3,5,7,9-tetradeoxy-non-
naminic acid. Figure 9 shows the chemical structure of ulosonic acid).
Figure 10 Proposed structure of polySia attached to N-CAM. (Based on information in Refs. 72, 73, 80, 82)
attachment to a variety of tissue culture substrates [74]. In adhesion receptors as diverse as an immunoglobin cell
studies of embryonic spinal cord axon bundling, the adhesion molecule, a cadherin, and an integrin, and did not
pronounced defasciculation produced by the removal of require N-CAM functional domains other than those
polySia was explained by an increase in cellsubstrate minimally required for polysialylation. These ndings
interaction [74]. Using F11 neuron/neuroblastoma hybrid indicated that the regulation of cell adhesion was based
cells, the measured density and size of polySia indicated solely on polySia biophysical properties.
that a substantial fraction of the space between two
opposed cell surface membranes could be sterically inu-
enced by the presence of polySia chains [75]. The distance 3. Developmental Changes in the Length of
between opposed cell membranes upon enzymatic removal PolySia Chains Attached to N-CAM
of polySia, using Endo-N, decreased by 1015 nm and The rst proof that internally linked Neu5Ac residues were
thus would be expected to have a substantial eect on attached to N-CAM was provided by Finne [77] by using
receptor-mediated interactions. Manipulations of ionic mass fragmentography. The reported polySia chain length
strength indicated that the steric properties of polySia from 8 to 12 was underestimated because of the lability of
directly eected embryonic membrane adhesion [42]. the intersialyl linkage to mildly acidic condition [37,78].
Two mechanisms were considered by which steric eects Similar underestimation of polySia chain length was pre-
could inuence the membranes interactions: rstin viously considered in the case of bacterial colominic acid
which polySia impedes trans interactions between recep- because the polySia chains were isolated from an E. coli
tors on opposing cells, and the secondthat involve a K-235 culture ltrate of pH below 5.0 (discussed in Ref. 2).
change in cis interactions between receptors on the same Although the bacterial capsular polySia chains were found
cell. In the trans mechanism, the hydrated polySia at- to have ca. 200 sialyl monomers [54], the polySia chain
tached to N-CAM serves as an impediment to intermem- length in eukaryotic cell can reach only ca. 5055 sialyl
brane contact, and therefore decreases the eciency of residues [7981]. PolySia-containing glycoproteins con-
encounter between complimentary receptors on opposing sisting of extended chains of at least 55 sialyl residues were
cells. In the cis mechanism, polySia eects cellcell inter- expressed on human neuroblastoma cells [79]. High-pres-
actions via interference with clustering of N-CAM pro- sure liquid chromatography method was applied to analyze
teins or by association of N-CAM with other receptor on the DP of polySia chains expressed on embryonic and adult
the same cell membrane [42, 72]. It was shown [76] that chicken brain N-CAM [80]. The DP of polySia in embry-
regulation of cell adhesion by polySia occurred with onic chicken brain was 4050 (average DP was ca. 45)
720 Janas and Janas
throughout developmental stages from 5 to 21 days after myotubes [88]. PolySia have also been found to play a
fertilization. The data indicated that: permissive role in axon path-nding, reducing the fascicu-
.
lative interactions between axons both in the peripheral
Only one of the three antennae is polysialylated.
.
and central nervous system.
One of the remaining two antennae is either
Polysialylated N-CAM could negatively regulate my-
disialylated or oligosialylated with low DP.
.
elin formation [89]. During development, polysialylated N-
The third antenna is not sialylated but terminated
CAM was rst expressed on all growing bers; then, axonal
by unsubstituted Gal residue.
expression was downregulated and myelin deposition oc-
This polysialylation prole was in contrast to that of the curred only on polySiaN-CAMnegative axons. As axo-
adult chicken brain, which showed polySia (attached to N- nal electrical activity strongly induced myelination, the
CAM) with polydispersity ranging from DP 15 to 35. The data indicated that myelination is tightly controlled by
occurrence of disialic acid (diSia) residues was found as the both positive (electrical activity) and negative (polysia-
most frequent form of lower oligoSia throughout all lylated N-CAM) regulatory signals. While polysialylated
developmental stages including adult. N-CAM is abundantly expressed in the embryonic brain, in
N-glycosylation pattern of polysialylated N-CAM adults polySia level is decreased and persists mainly in
from the brains of newborn calves was analyzed using brain regions, which preserves a potential for morpholog-
matrix-assisted laser desorption/ionization mass spectros- ical and physiological plasticity. Spatial learning increased
copy [82]. PolySia chains were found to be exclusively the number of polySia-positive neurons in the hippocam-
linked to the fth (Asn 439) and sixth (Asn 468) N-glyco- pus, an area involved in learning and memory.
sylation sites in the fth immunoglobulin-like domain of
N-CAM. The DP of polySia chains was found, using anion
exchange chromatography, to be at least 30. The core 5. Role of PolySia in Activity-Induced
structures of polysialylated glycans included hybrid, di-, Synaptic Plasticity
tri-, and small amount of tetraantennary carbohydrates, A number of studies have shown that polysialylated N-
which were all fucosylated at the innermost N-acetylglu- CAM has an important role in synaptic plasticity, both in
cosamine. In the case of triantennary glycans, the 2,6 the brain and at the neuromuscular junction, and is re-
arm was preferred in polysialylation. In addition, high level quired for synaptic activity in some regions of hippocam-
of sulfated groups was found on polysialylated structures. pus junction (for review see Refs. 90, 91) and for circadian
The structure of sialic acid chains was analyzed in clock function. The possible mechanism of long-term
mammalian (mouse) adult brain using two sensitive chem- potentiation included modulation of synaptic AMPA re-
ical methods [83]. The preexistence of a2,8-linked diSia and ceptor channel open time by polySia chains [92]. Long-term
oligoSia structures with up to seven Sia monomers was potentiation was impaired in hippocampal mossy ber
shown to occur on a large number of brain glycoproteins, system [93]. In addition, enzymatic removal of polySia by
including N-CAM. This nding was conrmed using sev- Endo-N also impaired long-term potentiation in the hip-
eral antibodies. Immunospecicities of these antibodies pocampus [94]. Washout of the enzyme resulted in reex-
were determined to be diSia and oligoSia residues with pression of polySia immunoreactivity, which correlated,
dened chain lengths. Thus a surprising diversity was with a complete recovery of long-term potentiation. This
found in the number and molecular masses of proteins reexpression is blocked by a blocker of sodium-dependent
distinct from N-CAM that were covalently modied by action potentials, TTX. Thus expression of polysialylated
these short sialyl glycotopes. N-CAM at the synapse could be modulated by neuronal
and synaptic activity. Taken together, these reports showed
that both N-CAM knockout and enzymatic removal of
4. Role of PolySia in Neural Development polySia resulted in similar electrophysiological phenotype.
In the vertebrate nervous system, polySia chains have been The role of polySia chains was examined during the
found to have an important role in cell migration, axonal formation of oculomotor synapses on chick ciliary neurons
guidance, axon branching, axon myelination, synapse [95]. Prior to synaptogenesis, polySia was abundantly and
formation, and functional plasticity of the system (for uniformly expressed on the surface of the ciliary neuron
review see Refs. 2,8486). PolySia has been identied to body. At the time synaptic bonds started to form, polySia
be a factor in axophilic (along axon bers) and cooperative was lost from the point of synaptic contact. Thereafter,
cell migration, but not in gliophilic or radial migration polySia was progressively lost from the neuron surface. It
(along glial bers). PolySia chains have been found to be was suggested [95] that the synapse eliminated polySia
expressed on the surface of myotubes during the secondary chains as part of its normal development, and that the loss
myogenesis. In this process the newly formed secondary of polySia from the site of axontarget interaction might
myotubes detached from the primary myotube in order to serve to stabilize structures formed during synaptogenesis.
generate dierent muscle bers. An expression of poly- The translocation of polysialylated N-CAM to the
sialylated N-CAM occurred at the time that these cell surface of cultured cortical neurons was examined [96].
clusters break apart [87]. In contrast, the increase in Endo-N was used to remove preexisting polySia chains
expression of N-CAM, although typically observed during from the plasma membrane and the reexpression of the
myogenesis, was not essential to myoblast fusion to form polymer was monitored by immunocytochemistry, and
68% of polySia immunostaining was restored within 1 hr. fractional open time as a function of the applied membrane
This recovery was almost completely inhibited by TTX, potential:
suggesting that spontaneous electrical neuron activity was
required for mobilization of polysialylated N-CAM to the f 1=f1 expzm FV Vm =RTg 2
cell surface of neurons. K + -induced depolarization where f is the channel fractional open time; V (mV) is
allowed the recovery of polySia surface immunostaining the applied membrane potential; Vm (mV) is the potential
in the presence of TTX and this eect required the presence at which the channel was opened half of the time; zm is
of extracellular Ca2+. It was also suggested that polysia- the eective valences of the apparent gating charges;
lylated N-CAM can be translocated to the cell surface via F is Faraday constant; R is the gas constant; T (K) is
regulated exocytosis. temperature.
The brain suprachismatic nuclei (SCN), which express After neuraminidase treatment a large depolarizing
polysialylated N-CAM, are the principal site for the gen- shift in the average potential required for channel activa-
eration and entrainment of mammalian circadian rhythms. tion was observed. In addition, desialidated channels
The daily locomotor rhythm was examined in mice with a showed an increase in the frequency of reversible transi-
mutation, which in adults results in the deletion of the N- tions to subconductance states. Table 7 shows the sodium
CAM isoform that carried polySia in the brain [97]. The channel parameters obtained from single-channel measure-
circadian eects of polySia depletion in the SCN region ments before and after neuraminidase treatment (for
were also examined using intra-SCN microinjection of details see Ref. 101). It was suggested that sialic acid
Endo-N. The results (e.g., shortening of the free-running residues played a signicant role in the function of sodium
periods for both mutant and Endo-N microinjection) channels, possibly through their inuence on the local
indicate critical roles for polysialylated N-CAM in the electric eld and/or conformational stability of the channel
physiology and development of the mammalian SCN protein. Eleven monoclonal antibodies were identied that
circadian clock. recognized eel electroplax sodium channel. One monoclo-
nal antibody, 3G4, was found to bind to an epitope
involving terminal polySia [102]. Except for this antibody,
B. PolySia Attached to the Membrane the epitopes recognized by other antibodies were resistant
Ion Channels to deglycosylation of the channel.
The sodium channel a subunit in the adult rat brain
The voltage-sensitive Na+-channel from Electrophorus was the second mammalian glycoprotein (the rst was N-
electricus electroplax is a single large peptide of 208 kDa CAM) to be shown to contain a2,8Neu5Ac polySia chains
with an estimated 85 kDa of carbohydrate including ca. [103]. Immunoprecipitation using a polySia-specic mono-
40 kDa or 113 residues of sialic acid attached [98]. Char- clonal antibody mAb735 precipitated not only N-CAM
acterization of carbohydrate substituents on this mem- isoforms carrying polySia chains, but also the a subunit of
brane channel was performed using several glycosidases the sodium channel. Specicity of this immunoreactivity
[99]. The results indicated the presence of two classes of was conrmed by showing that pretreatment of the channel
polysaccharides: neutral, high mannose or hybrid polysac- with endoneuraminidase specic for a2,8-linked polySia of
charides, and acidic, complex polysaccharides with a core- eight or more residues abolished immunoreactivity.
structure terminating in a linear homopolymer of sialosyl Analysis of sialidase sensitivity of Shaker-type K+
monomers in a2,8-linkages. The immunoreactivity of the channels from mammalian brain suggested that they con-
electroplax sodium channel with several antibodies raised tain 120220 sialic acid monomers per functional protein
against bacterial polySia chains was tested [100]. It was molecule [104,105]. To examine the role of sialidation in
found that the electroplax sodium channel bound these Shaker-type K+ channels function, Chinese hamster ovary
antibodies with high anity and the sodium channel was cell lines decient in glycosylation were transfected with rat
the only membrane protein that had attached this antigen. brain K+-channel cDNA [106]. The channel was function-
As the antibodies tested required a minimum of 10 consec-
utive sialic acid residues, these results suggested the pres-
ence of a long polySia chain or multiple oligomers of 10 or
Table 7 Sodium Channel Parameters Obtained from Single-
more residues potentially extending 1030 nm from the cell Channel Measurements of Planar Lipid Bilayers Before and
surface into the extracellular environment. It was suggested After Neuraminidase Treatment
that polySia chains might serve to maintain a solute reser-
voir, creating an anionic cloud over the channel molecule, Vm, mV zm
which in turn might shield the channel protein from extra-
cellular proteins. Channel before 71 1.02
Puried electroplax sodium channels were treated with neuraminidase treatment
neuraminidase to remove sialic acid residues and then Channel after 40 0.66
neuraminidase treatment
examined for functional changes in planar lipid bilayers
[101]. To analyze the voltage-dependent gating properties Vmthe potential at which the channel was opened half of the time;
of sodium channels in the lipid bilayers, steady-state acti- zmthe eective valences of the apparent gating charges.
vation curves were obtained by measuring the channel Source: From Ref. 101.
722 Janas and Janas
ally expressed in all cell lines, but the voltage dependence of containing only poly(Neu5Ac) or poly(Neu5Gc) chains,
activation was shifted to more positive values. It was also and one hybrid polySia structure that contained both
found that the activation kinetics was slower in the mutant Neu5Ac and Neu5Gc monomers in the same chain was
cell lines compared with control. After treatment of control also discovered. A glycosyltransferase that catalyzed the
cells with sialidase, a similar positive shift in the voltage transfer of deaminated neuraminic acid (KDN) from
dependence of activation was also recorded. These experi- CMP-KDN to the nonreducing termini of oligo/polySia
ments suggested that the over 100 monomers of sialic acid chains of PSGP was found in the ovary of rainbow trout
attached to this channel could modify the channel function, [109]. Incorporation of the KDN residues into the oligo/
possibly by inuencing the local electric eld detected by its polySia chains prevented their further elongation, resulting
voltage sensor. in capping of the chain.
Chemical and 1H NMR spectroscopic studies revealed
C. PolySia in Animal Eggs the presence in the jelly coat of sea urchin eggs polysia-
lylated glycoproteins, designated polySia-gp [110]. The
A glycoprotein containing deaminated neuraminic acid structure of the polySia chains was characterized as (-5-
(KDN) was isolated from the vitalline envelope of the Oglycolyl-Neu5Gca2-)n, where n ranged from 4 to more than
unfertilized eggs of rainbow trout [107]. This glycoprotein 40 Neu5Gc residues. The poly(Neu5Gc) chains were linked
was designated as KDN-glycoprotein because it to core oligosaccharides that were O-glycosidically joined
contained only KDN and no other kind of sialic acid to threonine residues on a core polypeptide. In two dier-
monomers. Further studies were performed to determine ent sea urchin species studied, the polypeptide contained
the structures of the polySia chains on Salmonidae sh egg ca. 17 or 25 O-linked polySia chains.
polysialoglycoproteins (PSGPs) isolated from the eggs of Sulfated oligoSia chains in the O-linked glycan of the
eight dierent sh species from three genera [108]. Using sea urchin egg receptor for sperm were identied [111]. It
chemical, immunochemical, enzymatic, and 1H NMR was found that these oligosaccharides had the structure:
methods, a wide range of diversity in polySia structures (SO4)-9Neu5Gca2(-5-Oglycolyl-Neu5Gca2-)n. The sulfate
was found including dierences in the relative proportion unit was located on the hydroxyl group of C9 of the
of monomeric units (KDN, Neu5Gc, Neu5Ac), the pres- nonreducing terminus of the chain, the oligoSia chain
ence of hybrid structures, and the extent of O-acylation (O- contained one sulfate group per two to four Sia residues.
acetyl substitution at C4, C7, or C9, or O-lactyl substitution It was concluded that this sulfated polySia chain was a
at C9) in the PSGPs. Two homopolymers were found component of a GalNAc-containing chain that was O-
Figure 11 Chemical structure of the polySia chains attached to glycoproteins found in sea urchin eggs: Asea urchin jelly coat;
Bsea urchin egg receptor for sperm. (From Ref. 112.)
linked to the polypeptide chain of the sea urchin egg release the polysialyllactose chains from the ceramide
receptor for sperm. It was suggested that the sulfate moiety. The polyacrylamide gel electrophoresis of these
residues in the receptor might function in the sperm chains revealed that 6080 sialyl units were transferred to
binding process via interaction with bindin, the sea urchin the disialyllactose ganglioside moiety. It was suggested that
sperm adhesive protein. polysialylated ganglisides could have potentially important
Figure 11 shows the chemical structure of the polySia biological and pharmacological properties.
chains attached to glycoproteins found in sea urchin eggs Antiviral activity of sulfated colominic acids against
(for details see Ref. 112). human immunodeciency virus type 1 (HIV-1) was exam-
ined [117]. Sulfated colominic acids were prepared by
D. PolySia in Human Tumor Cells treatment of colominic acid with chlorosulfuric acid in
pyridine at 70jC for 1 hr or with sulfur trioxide trimethyl-
Poly(Neu5Ac) chains were found in several human tumors amine complex in N,N-dimethylformamide at 25jC for 20
(for review see Ref. 2). The rst human tumors, which were hr and then raised to 50jC for 5 hr. Sulfated colominic
demonstrated to express a2,8-polysialylated N-CAM, acids with dierent sulte content were obtained. Figure 12
were neuroblastomas [79,113] and nephroblastomas shows the chemical structure of the sulfated polySia chains.
(Wilms tumor) [114]. The chain length of polySia chains The polysaccharides, containing 612% sulfur, blocked the
in neuroblastomas cell reached 55 Sia residues [79]. Subse- expression of HIV-1 antigen. It was also found that sul-
quent studies revealed a2,8-polySia chains on a variety of fated colominic acid had much more potent anti-HIV-1
human tumors including breast cancer, congenital meso- activity than nonsulfated colominic acids. It was suggested
blastic nephroma, head and neck tumors, leukemias, lung that sulfated colominic acids might represent a new class of
carcinoid tumors, malignant melanomas, medullary thy- anti-HIV-1 agents and good candidates for therapeutic use
roid carcinomas, medulloblastomas, multiple myelomas, against HIV-1 infection.
pheochromocytomas, pituitary adenomas, small-cell lung It was demonstrated that the toxic eect of scrapie
cancers, T-cell malignant lymphomas; a2,9-polySia chains prion protein (PrPSc) could be blocked by sulfated colo-
were found in ovarian teratocarcinoma. The possible func- minic acid [118]. PrPSc-induced apoptosis of rat cortical
tions of polysialylation in tumor cells include the enhance- neurons in vitro and sulfated polySia acted neuroprotec-
ment of the metastatic potential (simulating migration) and tively when preincubated with the neurons or PrPSc. The
tumor cell motility on substrates, and the enhancement of HIV-1 coat protein gp120 also induced the neural cell death
detachment and/or penetration of the bloodbrain barrier and this eect was abolished by sulfated polySia when
for colonization of the central nervous system [2]. preincubated with the cells or gp120 protein. It was sug-
Polysialylated proteins were identied in rat basophilic gested that sulfated colominic acid might be a promising
leukemia cells and human breast cancer cells [115]. The compound for the treatment of dementia caused by PrPSc
presence of a2,8-polySia on these cells was conrmed by the and HIV-1 infections.
elimination of antipolySia antibody staining after treat- Neuron cell surface expression of polySia was revers-
ment with a2,8-polySia-specic Endo-N. The 180260- ibly inhibited by N-butanoylmannosamine (ManBut)
kDa proteins were neither the neural cell adhesion molecule [119]. NT2 neurons were treated with ManBut and polySia
nor the sodium channel a subunit as conrmed using expression was visualized by immunouorescence micros-
immunouorescence microscopy and Northern analysis. copy with an antipolySia monoclonal antibody. Inhibition
Thus new polysialylated proteins were discovered in mam- occurred through a metabolic mechanism in which Man-
malian cells as N-CAM and sodium channels were the only
known polysialylated proteins in mammalian cells. It was
suggested that a2,8-polysialylation might be widespread
especially in cancer cells.
VI. POLYSIALIC ACID IN THERAPEUTICS

A. Molecular Engineering of PolySia Chains
It was demonstrated that the bacteria E. coli K1 polysia-
lyltransferase complex (polyST) could selectively polysia-
lylate several structurally related eukaryotic glycosphin-
golipids, when added as exogenous sialyl acceptor [116].
While bacteria do not synthesize glycosphingolipids, the
results showed that the polyST exposed on the cytoplasmic
surface of IOV prepared from the bacteria cells could be
used as a synthetic reagent to construct a class of structur-
ally dened poly(Neu5Ac)-glycosphingolipids. PolySia
chains of variable length were created by using endoneur- Figure 12 Chemical structure of the sulfated polySia chains
aminidase (Endo-N). Ceramide glycanase was applied to (sulfated colominic acid). (From Ref. 117.)
724 Janas and Janas
But was converted to unnatural sialic acid derivatives that Escherichia coli K1 and K92. Carbohydr. Res. 1986, 156,
eectively acted as chain terminators during cellular poly- 123.
7. Michon, F.; Brisson, J.-R.; Jennings, H.J. Conformational
Sia biosynthesis. It was suggested that the transient dis-
dierences between linear a(2!8)-linked homosialooligo-
ruption of polySia expression by ManBut might be used to saccharides and the epitope of group B meningococcal
study the roles of polySia in synapse formation, tumor polysaccharide. Biochemistry 1987, 26, 8399.
metastasis, organ development, and modulating cell adhe- 8. Yamasaki, R. 2D NMR analysis of group B capsular
sion. The application of ManBut as an antimetastatic agent polysaccharides of N. meningitides: complete assignment of
1
was also suggested. H-NMR spectrum of B polysaccharide of strain 6275.
Biochem. Biophys. Res. Commun. 1988, 154, 159.
9. Yamasaki, R.; Bacon, B. Three-dimensional structural
B. PolySia Chains in Drug Delivery analysis of the group B polysaccharide of Neisseria menin-
and Drug Selection gitidis 6275 by two-dimensional NMR: the polysaccharide
is suggested to exist in helical conformations in solution.
PolySia chains exhibited long half-lives in the blood circu- Biochemistry 1991, 30, 851.
lation after intravenous injection, thus the polysaccharides 10. Brisson, J.-R.; Baumann, H.; Imberty, A.; Peres, S.;
were proposed as carriers of short-lived drugs and small Jennings, H.J. Helical epitope of the group B meningo-
peptides [120]. Shorter-chain polySia increased the circu- coccal a(2-8)-linked sialic acid polysaccharide. Biochemis-
try 1992, 31, 4996.
latory half-lives of proteins; therefore these polymers could 11. Baumann, H.; Brisson, J.-R.; Michon, F.; Pon, R.;
serve as an alternative to the nonbiodegradable monome- Jennings, H.J. Comparison of the conformation of the
thoxypoly(ethylene glycol). Covalent coupling of colominic epitope of a(2!8) polysialic acid with its reduced and N-
acid to enzymes: catalase and asparaginase, increased these acyl derivatives. Biochemistry 1993, 32, 4007.
enzymes stability in the presence of proteolytic enzymes or 12. Batta, G.; Gervay, J. Solution-phase 13C and 1H chemical
blood plasma. Comparative studies in vivo with intact and shift anisotropy of sialic acid and its homopolymer
(colominic acid) from cross-correlated relaxation. J. Am.
polysialylated asparaginase showed that polysialylation Chem. Soc. 1995, 117, 368.
increased over twofold the half-life of the enzyme. 13. McReynolds, K.D.; Gervay-Hague, J. Examining the
PolySia chains (colominic acid) were found as a new secondary structures of unnatural peptides and carbohy-
chiral selector for capillary electrophoresis of basic drugs drate-based compounds utilizing circular dichroism. Tet-
[121]. Using a low concentrated phosphate buer contain- rahedron: Asymmetry 2000, 11, 337.
ing polySia, and polybre/colominic acid double-coated 14. Crescenzi, V.; Dentini, M.; Coviello, T. Solution and
gelling properties of polysaccharide polyelectrolytes. Bio-
capillary allowed excellent separation of the enantiomers
phys. Chem. 1991, 41, 61.
of basic drugs having the amino/imino group adjacent to 15. Shimoda, Y.; Kitajima, K.; Inoue, S.; Inoue, Y. Calcium
the chiral carbon atom. As the selective binding of the ion binding of three dierent types of oligo/polysialic acids
carboxyl group with the amino/imino or hydroxyl group as studied by equilibrium dialysis and circular dichroic
through hydrogen bonding is widely observed in nature methods. Biochemistry 1994, 33, 1202.
(e.g., in the DNA double-helix formation), a similar mech- 16. Bystricky, S.; Pavliak, V.; Szu, S.C. Characterization of
anism could be expected for the intermolecular binding colominic acid by circular dichroism and viscosity analysis.
Biophys. Chem. 1997, 63, 147.
between the amino/imino groups of the basic drugs and the 17. Tromas, C.; Garcia, R. Interaction forces with carbohy-
carboxyl group of sialic acid within polySia chain. Longer drates measured by atomic force microscopy. Top. Curr.
polySia chains gave better enantioseparation, whereas the Chem. 2002, 218, 115.
sialic acid monomers did not give any enantioseparation. 18. Toikka, J.; Aalto, J.; Hayrinen, J.; Pelliniemi, L.J.; Finne, J.
The polysialic acid units of the neural cell adhesion
molecule N-CAM form lament bundle networks. J. Biol.
REFERENCES Chem. 1998, 273, 28557.
19. Ogawa, T.; Terabayashi, T.; Kawanishi, Y. Analysis of
1. Traving, C.; Schauer, R. Structure, function and metabo- oligo- and poly-N-acetylneuraminic acids and their lac-
lism of sialic acids. Cell. Mol. Life Sci. 1998, 54, 1330. tones by capillary electrophoresis. J. Chromatogr. A 1996,
2. Troy, F.A. Sialobiology and the polysialic acid glycotope. 741, 295.
Occurrence, structure, function, synthesis, and glycopa- 20. Kakehi, K.; Hirose, A.; Tamai, T.; Taga, A.; Honda, S.
thology. In Biology of the Sialic Acids; Resenberg, A., Ed.; Analysis of N-acetylneuraminic acid oligomers by high-
New York and London: Plenum Press, 1995; 95 pp. performance capillary electrophoresis. Anal. Sci. 1996, 12,
3. Schauer, R. Origin and biological role of the great chemical 171.
diversity of natural sialic acids. Trends Glycosci. Glyco- 21. Cheng, M.-C.; Lin, S.-L.; Wu, S.H.; Inoue, S.; Inoue, Y.
technol. 1997, 9, 315. High-performance capillary electrophoretic characteriza-
4. Sato, C.; Kitajima, K. Glycobiology of di- and oligosialyl tion of dierent types of oligo- and polysialic acid chains.
glycotopes. Trends Glycosci. Glycotechnol. 1999, 11, 371. Anal. Biochem. 1998, 260, 154.
5. Lindon, J.C.; Vinter, J.G.; Lifely, M.R.; Moreno, C. 22. Kakehi, K.; Kinoshita, M.; Hayase, S.; Oda, Y. Capillary
Conformational and dynamic dierences between N. electrophoresis of N-acetylneuraminic acid polymers and
meningitides serogroup B and C polysaccharides, using hyaluronic acid: correlation between migration order re-
NMR spectroscopy and molecular mechanics calculations. versal and biological functions. Anal. Chem. 1999, 71,
Carbohydr. Res. 1984, 133, 59. 1592.
6. Lifely, M.R.; Nowicka, U.T.; Moreno, C. Analysis of the 23. Yu, Y.-P.; Cheng, M.-C.; Lin, H.-R.; Lin, C.-H.; Wu, S.-H.
chain length of oligomers and polymers of sialic acid Acid-catalyzed hydrolysis and lactonization of a2,8-linked
isolated from Neisseria meningitidis group B and C and oligosialic acids. J. Org. Chem. 2001, 66, 5248.
24. Aubeck, R.; Eppelsheim, C.; Brauchle, C.; Hampp, N. C.; Kratzin, H.D.; Klebert, S.; Vaesen, M.; Bitter-
Potentiometric thick-lm sensor for the determination of Suermann, D.; Rose, D.R.; Young, N.M.; Bundle, D.R.
the tumour marker bound sialic acid. Analyst 1993, 118, Evidence for the extended helical nature of polysaccharide
1389. epitopes. The 2.8 A resolution structure and thermody-
25. Lin, S.-L.; Inoue, Y.; Inoue, S. Evaluation of high- namics of ligand binding of an antigen binding fragment
performance anion-exchange chromatography with pulsed specic for a-(2!8)-polysialic acid. Biochemistry 1995, 34,
electrochemical and uorometric detection for extensive 6737.
application to the analysis of homologous series of oligo- 41. Sato, C.; Kitajima, K.; Inoue, S.; Seki, T.; Troy, F.A.;
and polysialic acids in bioactive molecules. Glycobiology Inoue, Y. Characterization of the antigenic specicity of
1999, 9, 807. four dierent anti-a(2!8-linked polysialic acid) antibodies
26. Timoszyk, A.; Janas, T. Wplcyw fosfolipidow na zmiane using lipid-conjugated oligo/polysialic acids. J. Biol. Chem.
ruchliwos ci lcancucha kwasu polisjalowego. Curr. Top.D 1995, 270, 18923.
Biophys. 2001, 24, 97. 42. Yang, P.; Major, D.; Rutishauser, U. Role of charge and
27. Ghosh, P.; Hutadilok, N.; Adam, N.; Lentini, A. Inter- hydration in eects of polysialic acid on molecular inter-
actions of hyaluronan (hyaluronic-acid) with phospholi- actions on and between cell membranes. J. Biol. Chem.
pids as determined by gel-permeation chromatography, 1994, 269, 23039.
multi-angle laser-light-scattering photometry and H-1- 43. Hayrinen, J.; Haseley, S.; Talaga, P.; Muhlenho, M.;
NMR spectroscopy. Int. J. Biol. Macromol. 1994, 16, 237. Finne, J.; Vliegenthart, J.F.G. High anity binding of long
28. Janas, T.; Krajinski, H.; Timoszyk, A.; Janas, T. Trans- chain polysialic acid to antibody, and modulation by
location of polysialic acid across model membranes: kinetic divalent cations and polyamines. Mol. Immunol. 2002, 39,
analysis and dynamic studies. Acta Biochim. Pol. 2001, 48, 399.
163. 44. Haseley, S.R.; Kamerling, J.P.; Vliegenthart, J.F.G. Un-
29. Janas, T.; Janas, T. Inuence of polysialic acid on the ravelling carbohydrate interactions with biosensors using
uorescence response of Oxonol-V in lipid vesicles. Cell. surface plasmin resonance (SPR) detection. Top. Curr.
Mol. Biol. Lett. 1997, 2, 256. Chem. 2002, 218, 93.
30. Janas, T.; Nowotarski, K.; Janas, T. Cyclic voltammetry 45. Kabat, E.A.; Nickerson, K.G.; Liao, J.; Grossbard, L.;
and uorescence spectroscopy studies on the eect of Osserman, E.F.; Glickman, E.; Chess, L.; Robbins, J.B.;
polysialic acid on phospholipid model membranes. Cell. Schneerson, R.; Yang, Y. A human monoclonal macro-
Mol. Biol. Lett. 1999, 4, 277. globulin with specicity for a(2!8)-linked poly-N-acetyl
31. Janas, T.; Krajinski, H.; Janas, T. Translocation of neuraminic acid, the capsular polysaccharide of group B
polysialic acid across liposomal membranes: energy barrier meningococci and Escherichia coli K1, which crossreacts
model and electron microscopy studies. Cell. Mol. Biol. with polynucleotides and with denaturated DNA. J. Exp.
Lett. 1999, 4, 278. Med. 1986, 164, 642.
32. Janas, T.; Janas, T. The eect of polysialic acid on the 46. Husmann, M.; Roth, J.; Kabat, E.A.; Weisgerber, C.;
uorescence response of Oxonol-V in lipid vesicles. Folia Frosch, M.; Bitter-Suermann, D. Immunohistochemical
Histochem. Cytobiol. 1999, 37, 29c. localization of polysialic acid in tissue sections: dieren-
33. Ohyashiki, T.; Taka, M.; Mohri, T. Eect of neuramini- tial binding to polynucleotides and DNA of a murine IgG
dase treatment on the lipid uidity of the intestinal brush- and a human IgM monoclonal antibody. J. Histochem.
border membranes. Biochim. Biophys. Acta 1987, 905, 57. Cytochem. 1990, 38, 209.
34. Ohyashiki, T.; Taka, M.; Mohri, T. Changes in the 47. Perez-Paya, E.; Houghten, R.A.; Blondelle, S.E. The role
membrane surface charge density and/or membrane of amphipathicity in the folding, self-association and
potential of the porcine intestinal brush-border membrane biological activity of multiple subunit small proteins. J.
vesicles induced by treatment with neuraminidase. J. Biol. Chem. 1995, 270, 1048.
Biochem. 1989, 106, 584. 48. Hallenbeck, P.C.; Vimr, E.R.; Yu, F.; Bassler, B.; Troy,
35. Nowotarski, K.; Janas, T.; Gruszecki, W.I.; Janas, T. F.A. Purication and properties of a bacteriophage-
Badania oddzialcywan polimerow kwasu sjalowego z induced endo-N-acetylneuraminidase specic for poly-a-
monowarstwami fosfolipidowymi. Curr. Top. Biophys. 2,8-sialosyl carbohydrate units. J. Biol. Chem. 1987, 262,
2001, 24, 85. 3553.
36. Decher, G.; Ringsdorf, H.; Venzmer, J.; Bitter-Suermann, 49. Aalto, J.; Pelkonen, S.; Kalimo, H.; Finne, J. Mutant bac-
D.; Weisgerber, C. Giant liposomes as model membranes teriophage with non-catalytic endosialidase binds to both
for immunological studies: spontaneous insertion of bacterial and eukaryotic polysialic acid and can be used as
puried K1-antigen (poly-a-2,8-NeuAc) of Escherichia probe for its detection. Glycoconj. J. 2001, 18, 751.
coli. Biochim. Biophys. Acta 1990, 1023, 357. 50. Troy, F.A. Polysialylation: from bacteria to brains.
37. Troy, F.A.; McCloskey, M.A. Role of a membranous Glycobiology 1992, 2, 5.
sialyltransferase complex in the synthesis of surface 51. Troy, F.A. Polysialic acid capsule synthesis and chain
polymers containing sialic acid in Escherichia coli: temper- translocation in neuroinvasive Escherichia coli K1: acti-
ature-induced alteration in the assembly process. J. Biol. vated intermediates and a possible bifunctional role for
Chem. 1979, 254, 7377. undecaprenol. In Polysialic Acid. From Microbes to Man;
38. Gotschlich, E.C.; Fraser, B.A.; Nishimura, O.; Robbins, Roth, J., Rutishauser, U., Troy, F.A., Eds.; Basel: Birk-
J.B.; Liu, T.-Y. Lipid on capsular polysaccharides of hauser Verlag, 1993; 95 pp.
Gram-negative bacteria. J. Biol. Chem. 1981, 256, 8915. 52. Robbins, J.B.; McCracke, G.H.; Gotschlich, E.C.; rskov,
39. Hallenbeck, P.C.; Vimr, E.R.; Yu, F.; Bassler, B.; Troy, F.; rskov, I.; Hanson, L.A. Escherichia coli K1 capsular
F.A. Purication and properties of a bacteriophage- polysaccharide associated with neonatal meningitis. N.
induced endo-N-acetylneuraminidase specic for poly-a- Engl. J. Med. 1974, 290, 1216.
2,8-sialosyl carbohydrate units. J. Biol. Chem. 1987, 262, 53. Troy, F.A. Chemistry and biosynthesis of selected bac-
3553. terial capsular polymers. Annu. Rev. Microbiol. 1979, 33,
40. Evans, S.V.; Sigurskjold, B.W.; Jennings, H.J.; Brisson, J.- 519.
R.; To, R.; Tse, W.C.; Altman, E.; Frosch, M.; Weisgerber, 54. Rohr, T.E.; Troy, F.A. Structure and biosynthesis of
726 Janas and Janas
surface polymers containing polysialic acid in Escherichia 73. Benson, D.L.; Schnapp, L.M.; Shapiro, L.; Huntley, G.W.
coli. J. Biol. Chem. 1980, 255, 2332. Making memories stick: cell-adhesion molecules in synap-
55. Egan, W.; Liu, T.-Y.; Dorow, D.; Cohen, J.S.; Robbins, tic plasticity. Trends Cell Biol. 2000, 10, 473.
J.D.; Gotschlich, E.C.; Robbins, John B. Structural studies 74. Acheson, A.; Sunshine, J.L.; Rutishauser, U. NCAM
on the sialic acid polysaccharide antigen of Escherichia coli polysialic acid can regulate both cellcell and cellsubstrate
strain Bos-12. Biochemistry 1977, 16, 3687. interactions. J. Cell Biol. 1991, 114, 143.
56. Bhattacherjee, A.K.; Jennings, H.J.; Kenny, C.P.; Martin, 75. Yang, P.; Yin, X.; Rutishauser, U. Intercellular space is
A.; Smith, I.C.P. Structural determination of the poly- aected by the polysialic acid content of NCAM. J. Cell
saccharide antigens of Neisseria meningitides serogroup Y, Biol. 1992, 116, 1487.
W-135, and BO. Can. J. Biochem. 1976, 54, 1. 76. Fujimoto, I.; Bruses, J.L.; Rutishauser, U. Regulation of
57. Higa, H.H.; Varki, A. Acetyl-coenzyme A: polysialic acid cell adhesion by polysialic acid. Eects on cadherin, immu-
O-acetyltransferase from K1-positive Escherichia coli. J. noglobin cell adhesion molecule, and integrin function and
Biol. Chem. 1988, 263, 8872. independence from neural cell adhesion molecule binding
58. Vimr, E.R.; McCoy, R.D.; Vollger, H.F.; Wilkinson, N.C.; or signaling activity. J. Biol. Chem. 2001, 276, 31745.
Troy, Frederic A. Use of prokaryotic-derived probes to 77. Finne, J. Occurrence of unique polysialosyl carbohydrate
identify poly(sialic acid) in neonatal neuronal membranes. units in glycoproteins of developing brain. J. Biol. Chem.
Proc. Natl. Acad. Sci. USA 1984, 81, 1971. 1982, 257, 11966.
59. Bliss, J.M.; Silver, R.P. Coating the surface: a model for 78. Manzi, A.E.; Higa, H.H.; Diaz, S.; Varki, A. Intra-
expression of capsular polysialic acid in Escherichia coli molecular self-cleavage of polysialic acid. J. Biol. Chem.
K1. Mol. Microbiol. 1996, 21, 221. 1994, 269, 23617.
60. Whiteld, C.; Roberts, I.S. Structure, assembly and 79. Livingston, B.D.; Jacobs, J.L.; Glick, M.C.; Troy, Frederic
regulation of expression of capsules in Escherichia coli. A. Extended polysialic acid chains (n>55) in glycoproteins
Mol. Microbiol. 1999, 31, 1307. from human neuroblastoma cells. J. Biol. Chem. 1988, 263,
61. Silver, R.P.; Prior, K.; Nsahlai, C.; Wright, L.F. ABC 9443.
transporters and the export of capsular polysaccharides 80. Inoue, S.; Lin, S.-L.; Inoue, Y. Chemical analysis of the
from Gram-negative bacteria. Res. Microbiol. 2001, 152, developmental pattern of polysialylation in chicken brain.
357. Expression of only an extended form of polysialyl chains
62. Masson, L.; Holbein, B.E. Physiology of sialic acid cap- during embryogenesis and the presence of disialyl residues
sular polysaccharides synthesis in serogroup B Neisseria in both embryonic and adult chicken brains. J. Biol. Chem.
meningitides. J. Bacteriol. 1983, 154, 728. 2000, 275, 29968.
63. Janas, T.; Troy, F.A. The Escherichia coli K1 poly-a-2,8- 81. Inoue, S.; Inoue, Y. Developmental prole of neural cell
sialosyl sialyltransferase is topologically oriented towards adhesion molecule glycoforms with a varying degree of
the cytoplasmic face of the inner membrane. Proceedings polymerization of polysialic acid chains. J. Biol. Chem.
of the Xth International Symposium on Glycoconjugates, 2001, 276, 31863.
Jerusalem, Israel, 1989; 207208. 82. von der Ohe, M.; Wheeler, S.F.; Wuhrer, M.; Harvey, D.J.;
64. Troy, F.A.; Janas, T.; Janas, T.; Merker, R.I. Topology of Liedtke, S.; Muhlenho, M.; Gerardy-Schahn, R.; Geyer,
the poly-a-2,8-sialyltransferase in E. coli K1 and energetics H.; Dwek, R.A.; Geyer, R.; Wing, D.R.; Schachner, M.
of polysialic acid chain translocation across the inner Localization and characterization of polysialic acid-con-
membrane. Glycoconj. J. 1990, 7, 383. taining N-linked glycans from bovine NCAM. Glycobiol-
65. Troy, F.A.; Janas, T.; Merker, R.I. Topology of the ogy 2002, 12, 47.
polysialyltransferase complex in the inner membrane of E. 83. Sato, C.; Fukuoka, H.; Ohta, K.; Matsuda, T.; Koshino,
coli K1. FASEB J. 1990, 4, A2246. R.; Kobayashi, K.; Troy, F.A.; Kitajima, K. Frequent
66. Troy, F.A.; Janas, T.; Janas, T.; Merker, R.I. Trans- occurrence of pre-existing a2!8-linked disialic and oligo-
membrane translocation of polysialic acid chains across the sialic acids with chain lengths up to 7 Sia residues in
inner membrane of neuroinvasive E. coli K1. Glycoconj. J. mammalian brain glycoproteins. J. Biol. Chem. 2000, 275,
1991, 8, 153. 15422.
67. Troy, F.A.; Janas, T.; Janas, T.; Merker, R.I. Vectorial 84. Kiss, J.Z.; Rougon, G. Cell biology of polysialic acid. Curr.
translocation of polysialic acid chains across the inner Opin. Neurobiol. 1997, 7, 640.
membrane of neuroinvasive E. coli K1. FASEB J. 1991, 5, 85. Muhlenho, M.; Eckhardt, M.; Gerardy-Schahn, R.
A1548. Polysialic acid: three-dimensional structure, biosynthesis
68. Janas, T.; Janas, T.; Krajinski, H. Membrane transport of and function. Curr. Opin. Struc. Biol. 1998, 8, 558.
polysialic acid chains: modulation of transmembrane 86. Bruses, J.L.; Rutishauser, U. Roles, regulation, and
potential. Eur. Biophys. J. 2000, 29, 507. mechanism of polysialic acid function during neural
69. Janas, T.; Krajinski, H.; Janas, T. Electromigration of development. Biochimie 2001, 83, 635.
polyion homopolymers across biomembranes: a biophys- 87. Fredette, B.; Rutishauser, U.; Landmesser, L. Regulation
ical model. Biophys. Chem. 2000, 87, 167. and activity-dependance of N-cadherin, NCAM isoforms,
70. Knirel, Y.A.; Rietschel, E.T.; Marre, R.; Zahringer, U. The and polysialic acid on chick myotubes during development.
structure of the O-specic chain of Legionella pneumophila J. Cell Biol. 1993, 123, 1867.
serogroup 1 lipopolysaccharide. Eur. J. Biochem. 1994, 88. Charlton, C.A.; Mohler, W.A.; Blau, H.M. Neural cell
221, 239. adhesion molecule (NCAM) and myoblast fusion. Dev.
71. Kooistra, O.; Herfurth, L.; Luneberg, E.; Frosch, M.; Biol. 2000, 221, 112.
Peters, T.; Zahringer, U. Epitope mapping of the O-chain 89. Charles, P.; Hernandez, M.P.; Stanko, B.; Aigrot, M.S.;
polysaccharide of Legionella pneumophila serogroup 1 Colin, C.; Roigon, G.; Zalc, B.; Lubetzki, C. Negative
lipopolysaccharide by saturation-transfer-dierence regulation of central nervous system myelination by
NMR spectroscopy. Eur. J. Biochem. 2002, 269, 573. polysialylated-neural cell adhesion molecule. Proc. Natl.
72. Rutishauser, U. Polysialic acid at the cell surface: Acad. Sci. USA 2000, 97, 7585.
biophysics in service of cell interactions and tissue 90. Martin, P.T. Glycobiology of the synapse. Glycobiology
plasticity. J. Cell. Biochem. 1998, 70, 304. 2002, 12, 1R.
91. Yamaguchi, Y. Glycobiology of the synapse: the role of glycoprotein: a novel deaminated neuraminic acid-rich
glycans in the formation, maturation, and modulation of glycoprotein isolated from vitelline envelope of rainbow
synapses. Biochim. Biophys. Acta 2002, 1573, 369. trout eggs. Biochem. Biophys. Res. Commun. 1988, 153,
92. Suppiramaniam, V.; Yilma, S.; Bowens, A.; Manivannan, 172.
K.; Bahr, B.; Dityatev, A. Colominic acid (polysialic acid) 108. Sato, C.; Kitajima, K.; Tazawa, I.; Inoue, Y.; Inoue, S.;
alters the channel properties of AMPA receptors recon- Troy, F.A. Structural diversity in the a2!8-linked poly-
stituted in lipid bilayers. Soc. Neurosci. Abs. 1999, 25, sialic acid chains in Salmonid sh egg glycoproteins
1489. occurrence of poly(Neu5Ac), poly(Neu5Gc), poly(-
93. Cremer, H.; Chazal, G.; Carleton, A.; Goridis, C.; Vincent, Neu5Ac, Neu5Gc), poly(KDN), and their partially acety-
J.-D.; Lledo, P.-M. Long-term but not short-term plasticity lated forms. J. Biol. Chem. 1993, 268, 23675.
at mossy ber synapses is impaired in neural cell adhesion 109. Angata, T.; Kitazume, S.; Terada, T.; Kitajima, K.; Inoue,
molecule-decient mice. Proc. Natl. Acad. Sci. USA 1998, S.; Troy, F.A.; Inoue, Y. Identication, characterization,
95, 13242. and developmental expression of a novel a2?8-KDN-
94. Muller, D.; Wang, C.; Skibo, G.; Toni, N.; Cremer, H.; transferase which terminates elongation of a2?8-linked
Calaora, V.; Rougon, G.; Kiss, J.Z. PSA-NCAM is oligo-polysialic acid chain synthesis in trout egg polysialo-
required for activity-induced synaptic plasticity. Neuron glycoproteins. Glycoconj. J. 1994, 11, 493.
1996, 17, 413. 110. Kitazume, S.; Kitajima, K.; Inoue, S.; Troy, F.A.; Cho, J.-
95. Bruses, J.L.; Chauvet, N.; Rubio, M.E.; Rutishauser, U. W.; Lennarz, W.J.; Inoue, Y. Identication of polysialic
Polysialic acid and the formation of oculomotor synapses acid-containing glycoprotein in the jelly coat of sea urchin
on chick ciliary neurons. J. Comp. Neurol. 2002, 446, eggsoccurrence of a novel type of polysialic acid struc-
244. ture. J. Biol. Chem. 1994, 269, 22712.
96. Kiss, J.Z.; Wang, C.; Olive, S.; Rougon, G.; Lang, J.; 111. Kitazume-Kawaguchi, S.; Inoue, S.; Inoue, Y.; Lennarz,
Baetens, D.; Harry, D.; Pralong, W.-F. Activity-dependent W.J. Identication of sulfated oligosialic acid units in the
mobilization of the adhesion molecule polysialic NCAM to O-linked glycan of the sea urchin egg receptor for sperm.
the cell surface of neurons and endocrine cells. EMBO J. Proc. Natl. Acad. Sci. USA 1997, 94, 3650.
1994, 13, 5284. 112. Kitazume-Kawaguchi, S. Polysialylated glycoproteins
97. Shen, H.; Watanabe, M.; Tomasiewicz, H.; Rutishauser, found in sea urchin eggs. Trends Glycosci. Glycotechnol.
U.; Magnuson, T.; Glass, J.D. Role of neural cell adhesion 1998, 10, 383.
molecule and polysialic acid in mouse circadian clock 113. Lipinski, M.; Hirsch, M.R.; Deagostini-Bazin, H.; Yama-
function. J. Neurosci. 1997, 17, 5221. da, O.; Tursz, T.; Goridis, C. Characterization of neural cell
98. Miller, J.A.; Agnew, W.S.; Levinson, S.R. Principal adhesion molecules (NCAM) expressed by Ewing and
glycopeptide of the tetrodotoxin/saxitoxin binding protein neuroblastoma cell lines. Int. J. Cancer 1987, 40, 81.
from Electrophorus electricus: isolation and partial chem- 114. Roth, J.; Zuber, C.; Wagner, P.; Taatjes, D.J.; Weigerber,
ical and physical characterization. Biochemistry 1983, 22, C.; Heitz, P.U.; Goridis, C.; Bitter-Suermann, D. Reex-
462. pression of poly(sialic acid) units of the neural cell adhesion
99. James, W.M.; Agnew, W.S. Multiple oligosaccharide molecule in Wilms tumor. Proc. Natl. Acad. Sci. USA 1988,
chains in the voltage-sensitive Na channel from Electro- 85, 2999.
phorus electricus: evidence for a2,8-linked polysialic acid. 115. Martersteck, C.M.; Kedersha, N.L.; Drapp, D.A.; Tsui,
Biochem. Biophys. Res. Commun. 1987, 148, 817. T.G.; Colley, K.J. Unique a2,8-polysialylated glycopro-
100. James, W.M.; Agnew, W.S. a(2!8)-P olysialic acid immu- teins in breast cancer and leukemia cells. Glycobiology
noreactivity in voltage-sensitive sodium channel of eel 1996, 6, 289.
electric organ. Proc. R. Soc. Lond. B 1989, 237, 233. 116. Cho, J.-W.; Troy, F.A. Polysialic acid engineering: syn-
101. Recio-Pinto, E.; Thornhill, W.B.; Duch, D.S.; Levinson, thesis of polysialylated neoglycosphingolipids by using the
S.R.; Urban, B.W. Neuraminidase treatment modies the polysialyltransferase from neuroinvasive Escherichia coli
function of electroplax sodium channels in planar lipid K1. Proc. Natl. Acad. Sci. USA 1994, 91, 11427.
bilayers. Neuron 1990, 5, 675. 117. Yang, D.-W.; Ohta, Y.; Yamaguchi, S.; Tsukada, Y.;
102. Ivey, S.; Thornhill, W.B.; Levison, S.R. Monoclonal anti- Haraguchi, Y.; Hoshino, H.; Amagai, H.; Kobayashi, I.
bodies raised against post-translational domains of the Sulfated colominic acid: an antiviral agent that inhibits the
electroplax sodium channel. J. Membr. Biol. 1991, 121, human immunodeciency virus type 1 in vitro. Antivir.
215. Res. 1996, 31, 95.
103. Zuber, C.; Lackie, P.M.; Catterall, W.A.; Roth, J. 118. Ushijima, H.; Perovic, S.; Leuck, J.; Rytik, P.G.; Muller,
Polysialic acid is associated with sodium channels and the W.E.G.; Schroder, H.C. Suppression of PrPSc- and HIV-1
neural cell adhesion molecule N-CAM in adult rat brain. J. gp120 induced neuronal cell death by sulfated colominic
Biol. Chem. 1992, 267, 9965. acid. J. Neurovirol. 1995, 5, 289.
104. Rehm, H. Enzymatic deglycosylation of the dendrotoxin- 119. Mahal, L.K.; Charter, N.W.; Angata, K.; Fukuda, M.;
binding protein. FEBS Lett. 1989, 247, 28. Koshland, D.E. Jr.; Bertozzi, C.R. A small-molecule
105. Scott, V.E.S.; Parcej, D.N.; Keen, J.N.; Findlay, J.B.C.; modulator of poly-a2,8-sialic acid expression on cultured
Dolly, J.O. a-Dendrotoxin acceptor from bovine brain is a neurons and tumor cells. Science 2001, 294, 380.
K+ channel protein. J. Biol. Chem. 1990, 265, 20094. 120. Gregoriadis, G.; Fernandes, A.; Mital, M.; McCormack, B.
106. Thornhill, W.B.; Wu, M.B.; Jiang, X.; Wu, X.; Morgan, Polysialic acids: potential in improving the stability and
P.T.; Margiotta, J.F. Expression of Kv1.1 delayed rectier pharmacokinetics of proteins and other therapeutics. Cell.
potassium channels in Lec mutant Chinese hamster ovary Mol. Life Sci. 2000, 57, 1964.
cell lines reveals a role for sialidation in channel function. J. 121. Du, Y.; Taga, A.; Suzuki, S.; Liu, W.; Honda, S. Colominic
Biol. Chem. 1996, 271, 19093. acid: a novel chiral selector for capillary electrophoresis of
107. Inoue, S.; Kanamori, A.; Kitajima, K.; Inoue, Y. KDN- basic drugs. J. Chromatogr. A 2002, 962, 221.
31
Brain Proteoglycans
Russell T. Matthews and Susan Hockfield
Yale University School of Medicine, New Haven, Connecticut, U.S.A.
I. INTRODUCTION features have generally led to the belief that GAGs dom-
inate the function of a glycoprotein even when other
In the vertebrate brain, two major classes of cells, glia and carbohydrate modications are present. Despite the rela-
neurons, are generated from a proliferative zone that tively simple primary structure of each GAG chain, being
surrounds the central ventricular space. The newly gener- composed of only two sugars, they are in fact tremendously
ated cells migrate from the proliferative zone to their adult heterogeneous molecules [13].
positions and extend processes. Neurons elaborate rela- A number of enzymatic modications give rise to
tively long and complex axonal and dendritic processes GAG chain heterogeneity. GAGs can be sulfated at many
that interconnect in highly stereotyped ways to generate the dierent positions. They can be subjected to epimeriza-
circuitry of the mature brain. Glial cells also extend pro- tion. Sulfation and epimerization alone can lead to an
cesses that enwrap neural and vascular elements. Once the extraordinary array of structurally unique GAG chains [1
complex circuitry of the central nervous system (CNS) has 3]. Furthermore, the length of dierent GAG chains can
achieved its mature structure and connections, it becomes vary signicantly. Although an understanding of the com-
stabilized and is maintained throughout adulthood. Pro- plexity of GAG chains is truly in its infancy and beyond
teoglycans, a group of glycoproteins that are invested the scope of this review, it is an important variable to keep
with covalently bound glycosaminoglycan (GAG) chains, in mind as we begin to dissect the diverse functions of
are one of the important classes of molecules in brain proteoglycans.
development and maturation. Although the role of proteo- Historically, proteoglycans have been dened by their
glycans in other tissues, such as cartilages, was established GAG chains, and the protein cores to which they are
long ago, an appreciation of their importance in the CNS attached have been largely ignored. However, the growing
has only been established relatively recently. identication of the protein cores of these molecules has
The GAG chains that dene proteoglycans are long, greatly enhanced our appreciation of the combinatorial
unbranched polysaccharides composed of repeating disac- importance of both the protein core and the attached
charide units. There are four major classes of GAG chains carbohydrates in dening their unique functions. Further-
found on proteoglycans: heparan sulfate (HS) has a back- more, there is a growing appreciation of the microhet-
bone structure of glucuronic acid or iduronic acid and erogeneity created by regulated glycosylation patterns,
N-acetylglucosamine; chondroitin sulfate (CS) is com- both between dierent proteoglycans and within a single
posed of glucuronic acid and N-acetylgalactosamine; der- proteoglycan species.
matan sulfate (DS) is similar to CS in structure, except that In the CNS, the expression of all four proteoglycans
glucuronic acid is epimerized to iduronic acid; and, nally, subtypes has been demonstrated. However, HS and CS
keratan sulfate (KS) is composed of galactose and proteoglycans (HSPG and CSPG) appear to be the pre-
N-acetylglucosamine. Important structural features of dominant species [3]. In general, the most prevalent HSPGs
GAGs dierentiate them from other carbohydrate mod- are found on the cell surface, whereas the most prevalent
ications. They are typically longer than other sugars and CSPGs are secreted and are components of the extracellu-
are highly negatively charged due to the presence of lar matrix (ECM) [3]. Although there are many exceptions
carboxyl and sulfate groups on many sugar residues. These to this rule, it is generally a useful distinction. Accordingly,
729
730 Matthews and Hockfield
this review will focus on the roles of these two subtypes of CNS, including cell surface proteoglycans such as synde-
proteoglycans in the CNS. cans and glypicans.
II. HSPGs
III. GLYPICANS
Specic ligands for HS GAGs have been identied in a
variety of systems, including a variety of growth factors Glypicans are a family of membrane-bound HSPGs that
and cell adhesion molecules that can directly aect cell are expressed in distinct temporal and spatial patterns in
proliferation, identity, and motility. In the CNS, HSPGs the CNS [14,15]. Six family members have been identied
are predominantly expressed on the cell surface either as [1621]. All glypicans are synthesized with an attachment
transmembrane proteoglycans, such as syndecans, or as signal for a GPI anchor at their C-terminus and, accord-
GPI-anchored proteoglycans, such as glypicans. A variety ingly, all are attached to the membrane via this anchor
of HS-binding molecules, including matrix ligands, and (Fig. 1). The predominant structural feature of glypicans is
other cell surface molecules, are involved in neural devel- a highly conserved and unique stretch of 14 cysteine
opment. Molecules that are known to be involved in CNS residues. This stretch of cysteine creates a structural motif
development that are modulated by HS GAGs include not found in any other protein family. HS GAG attach-
members of the broblast growth factor (FGF) family, ment sites are located toward the C-terminus of the protein,
heparin-binding factors such as heparin-binding growth- in close proximity to the cell membrane (Fig. 1). Glypican-1
associated molecule (HB-GAM; pleiotrophin), the neural and glypican-2 have additional GAG attachment sites
cell adhesion molecule (NCAM), midkine, slit-1 and slit-2, close to the N-terminus of the molecules. Glypican-1,
Wingless/Wnt, transforming growth factor-h (TGF-h), glypican-3, and glypican-4 have a conserved cleavage site
and Hedgehog family [413] (Table 1). near the cysteine-rich domain [17,22,23] and a similar site
Although there are a number of potential common in glypican-5 [24], but the functional implications of cleav-
ligands for the HS chains of HSPGs, the functional roles of age at this site are unknown. Based on sequence similari-
these molecules depend critically on their spatial and ties, the glypicans can be considered in two groups: one
temporal expression and the nature of the protein core to including glypican-1, glypican-2 (cerebroglycan), glypican-
which they are attached. In this review, we will focus on 4 (K-glypican), and glypican-6, and the other including
some of the more prominently expressed HSPGs in the glypican-3 (OCI-5, MXR7) and glypican-5.
Table 1 Cellular Origin and Ligands of Cell Surface HSPGs in the Central Nervous System
Approximate
Family Name Cellular origin Mw (kDa) Extracellular ligands
Glypicans Glypican-1 Neurons, glia 64 FGF-1, FGF-2, FGF-7,
laminin, Slit-1, Slit-2, VEGF
Glypican-2 Neurons 52 FGF-2, laminin,
(cerebroglycan, thrombospondin
M13)
Glypican-3 Unknown 70 FGF-2, IGF-II
(OCI-5)
Glypican-4 Neurons 60 FGF-2
(K-glypican)
Glypican-5 Neurons 65 Not determined
Glypican-6 Unknown 65 Not determined
Syndecans Syndecan-1 Glia 50 FGF-2, bronectin,
tenascin-C, HB-GAM/PTN,
midkine
Syndecan-2 Neurons 48 FGF-2
(broglycan)
Syndecan-3 Neurons 120 FGF-2, HB-GAM/PTN,
(N-syndecan, midkine, laminin
neuroglycan,
M7)
Syndecan-4 Glia 35 FGF-2
(amphiglycan,
ryudocan)
Brain Proteoglycans 731
Figure 1 Schematic representation of the structure of the glypican and syndecan families of cell surface HSPGs. Wavy line
represents attached HS chains. The springlike structure represents a GPI anchor in the glypicans. Tm, transmembrane domain;
C1, conserved intracellular domain in syndecans with a common amino acid sequence (. . .RM(K/R)KKDEGSY. . .); V, variable
intracellular domain; C2, a second conserved intracellular domain in syndecans (. . .EFYA).
IV. SYNDECANS the syndecan-1/3 and syndecan-2/4 subgroupings, syndec-

an-2 and syndecan-3 seem to exclusively carry HS GAG
Syndecans are type I transmembrane proteins that pre- chains, whereas syndecan-1 and syndecan-4 carry mixed
dominantly carry HS GAG chains (they can also carry CS CS/HS side chains [8]. Syndecans share a conserved cleav-
chains) [2527]. There are four members of this family: age site near the plasma membrane that enables these
syndecan-1 [28,29], syndecan-2 (broglycan) [30,31], syn- proteins to be shed from the cell surface, which may be
decan-3 (N-syndecan) [32,33], and syndecan-4 [34,35] (Fig. an important mechanism for regulating their functions
1, Table 1). All known mammalian syndecans are [33,4244].
expressed in the CNS. The cytoplasmic domains of syndec-
ans are highly conserved. All syndecans have a virtually
identical stretch of 10 amino acids adjacent to the trans- V. FUNCTIONS OF HSPGs IN THE CNS
membrane domain, termed C1, followed by a short vari-
able region, V, and, nally, a conserved C-terminal The role of HSPGs in FGF signaling is one of the most
domain, C2, with the amino acid sequence EFYA (Fig. 1) thoroughly characterized interactions. The biological ac-
[27]. The C2 region of syndecans has been shown to interact tivity of FGFs is believed to be dependent on their binding
with proteins containing type II PDZ domains, four of to cell surface HSPGs. The involvement of HSPGs in FGF
which have now been identied, including syntenin [36,37], signaling is dependent on interactions with their HS chains.
CASK/Lin-2 [38,39], synbindin [40], and synectin [41]. In the absence of HS, cells that express the high-anity
Binding to these proteins is believed to localize syndecans FGF receptors neither bind nor respond to their ligands
to subdomains of the plasma membrane and to the cyto- [8,11]. There are a number of mechanisms by which HSPG
skeletal signaling apparatus. The V-regions of syndecans aects FGF signaling. Interacting with HSPG sequesters
may mediate specic interactions of dierent syndecans, FGFs and protects them from proteolytic degradation
thereby imparting unique functions to each syndecan. [45,46]. Interactions with proteoglycans can increase the
Although the sequence similarity between syndecan local concentration of FGF, functionally enhancing the
family members is low, signicant sequence similarities anity of FGF for its receptors [45,47]. HS serves to
have been noted between syndecan-1 and syndecan-3 and oligomerize FGF (and other ligands) and thereby facilitate
between syndecan-2 and syndecan-4. The two groups also receptor dimerization and subsequent signaling [48]. The
dier in the location of potential GAG attachment sites. In large amount of soluble HSPG not attached to membranes
syndecan-1 and syndecan-3, these sites are clustered near in brain tissues parallels the shedding of HSPG observed in
the N-terminus and in the vicinity of the transmembrane cells in culture. Membrane shedding could provide a
domain. Syndecan-2 and syndecan-4 display additional mechanism to terminate activity that depends on plasma
GAG attachment sites closer to the N-terminus. Beyond membrane attachment [16,21,42], such as cellcell adhesion
or other functions mediated by cytoskeletal elements, or by mutants were identied based on cell patterning defects in
intracellular signaling. HSPGs can be considered to func- the larval eye and brain [59]. Dally mutants show a
tion as low-anity coreceptors for certain factors [49,50]. disruption of a division cycle that is triggered by an
In the CNS, FGF signaling plays a role in neuro- intercellular signal from photoreceptor axons arriving
genesis. Neurogenesis is the process of the generation and from the developing eye. There is strong evidence that
dierentiation of neurons from neural stem cells. In brief, these defects are mediated through the TGF-h/BMP-re-
in the mammalian CNS, stem cells reside in the ventricular lated protein, decapentaplegic. These observations indi-
zonethe deepest layer of the neural tube. Cells generated cate a role for glypicans in the regulation of cell division
from the stem cells in the ventricular zone migrate to and the development of particular cell types [60].
specic locations and dierentiate into one of three cell Syndecan-2 is expressed in the rodent nervous system
types, including neurons, astrocytes, and oligodendrocytes, and accumulates at synapses [39]. Detailed studies of
early in embryogenesis. The time course of the generation hippocampal neurons indicate that syndecan-2 localizes
of neurons precedes the generation of glial cells in each area specically to dendritic spines [61], which are prominent
of the developing CNS. Neurogenesis is regulated by a postsynaptic specializations at excitatory synapses. Inter-
number of growth factors, including FGF [5154]. estingly, CASK/Lin-2 binds syndecan-2 at neuronal syn-
Glypican-4 can modulate FGF signaling during neu- apses [39]. Furthermore, when a C-terminal fragment of
rogenesis. Glypican-4 is expressed in the ventricular zone, syndecan-2 is expressed in neuronal cultures, CASK/Lin-2
particularly in the most rostral part of the developing accumulates at synapses [39]. Further studies have indi-
brain, telencephalon, coincident with both FGF-2 expres- cated that this is likely a physiological interaction as they
sion and neurogenesis [55]. Cortical neural stem cells are colocalized during development [39,62]. In culture,
express FGF receptors [53] and FGF-2 can aect the fate overexpression of syndecan-2 leads to the precocious for-
of neuroprogenitor cells [52,54]. Cells that retain stem cell mation of mature spines, but does not lead to more spines
properties express glypican-4, but as neurons dierentiate, or more synapses [61], suggesting that syndecan-2 is in-
glypican-4 is rapidly turned o [55]. Furthermore, recom- volved in spine maturation [62]. Further studies have
binant glypican-4 can suppress the mitogenic eects of indicated that tyrosine phosphorylation is necessary for
FGF-2 on cortical precursor cells. Interestingly, glypican-4 syndecan-2 accumulation in spines and that syndecan-2
expression is maintained in the adult dentate gyrus [55], phophorylation is mediated by EphB2 [63].
one of the few brain regions where neural stem cells Like syndecan-2, syndecan-3 is expressed at high levels
replicate throughout life. in the nervous system. Early in development, syndecan-3 is
Glypican-1 is also expressed in the ventricular zone expressed in oligodendrocyte lineage precursor cells in the
and may also play a role in modulating FGF signaling. ventricular zone [64]. Syndecan-3 is turned o after the
Unlike glypican-4, expression of glypican-1 appears to be terminal division when these cells dierentiate into mature
present in the ventricular zone during both neurogenesis oligodendrocytes [64]. There is strong evidence that FGF-2
and gliogenesis [55], and is maintained in mature cells [56]. may play a role in oligodendrocyte dierentiation [6571].
During the phase of axonal elongation, glypican-1 interacts FGF is a ligand for syndecan-3, suggesting a role for
with Slit proteins, which play a role in axonal guidance [57]. syndecan-3 in the generation and dierentiation of oligo-
Glypican-1 and Slit proteins colocalize in the developing dendrocytes [64,66]. Syndecan-3 also localizes to developing
brain and bind with high anity to one another [57]. Slit-2 axon tracks, and neurite outgrowth in vitro on an HB-
and glypican-1 are coexpressed in reactive astrocytes fol- GAM matrix is blocked by soluble syndecan-3 or exoge-
lowing CNS injury in the adult and may have a role in nous heparan [72,73]. HB-GAM is also expressed in devel-
inhibiting nerve regeneration. oping axons and can promote neurite outgrowth [74]. It has
Glypican-2 is expressed exclusively in the developing been suggested that HB-GAM can cluster syndecan-3 into
nervous system. Expression peaks as neurons dierentiate, adhesion complexes that regulate the cytoskeleton and
migrate to their appropriate locations, and extend neurites, growth cone [75].
and then is rapidly turned o [20]. The regulation of glypi-
can-2 expression has been most elegantly demonstrated in
late stages of hippocampal development when most neurons VI. CSPGs
no longer express glypican-2, but newly generated granule
cells do. Interestingly, in granule cells in the adult, glypican-2 Although a number of ligands bind specically to HS
is excluded from the somatodendritic compartment and is chains, relatively few that bind CS chains have been found.
strongly polarized to the axon. Consistent with a role in CSPGs are major constituents of the CNS and their
axonal guidance, glypican-2 is expressed in axonal tracts and expression is developmentally regulated. CSPG expression
in growth cones coincident with active neurite growth [58]. peaks early in development at a time of robust neurite
Other glypicans, including glypican-3, glypican-5, and outgrowth. It follows then that the most clearly demon-
glypican-6, are also expressed in the mammalian CNS, but strated function for CS chains is in directing neurite
their functional roles are unclear. In Drosophila, mutations outgrowth (discussed in more detail below). However, the
at the dally (division abnormally delayed) locus, which role of CSPGs is likely to be much more varied and
contains a gene coding for Drosophila glypican, shed light complex. Here, we will focus on the major family of CSPGs
on the role of glypicans in CNS development [59]. Dally in the CNS: the lecticans and phosphacans.
Although many of the HSPGs found in the CNS are factors. HA expression in adults is markedly lower than
cell surface molecules, the most prevalent CSPGs in the during development [7779].
CNS are secreted ECM molecules [76]. The ECM of the
CNS is markedly dierent from other tissues. The brain
ECM contains none of the major forms of collagen and, at VII. LECTICANS
least in the adult, little bronectin or laminin. With the
exception of meningeal surfaces and blood vessels, there The lectican family includes four CSPGs (aggrecans, versi-
are no basal laminae. Among the constituents of brain cans, neurocans, and BEHAB/brevicans), which share
matrix are glycoproteins, the GAG hyaluronan (HA), and several conserved domains [80,81] (Fig. 2). All of the
an increasingly complex variety of proteoglycans. lecticans are expressed in the CNS and, interestingly,
An HA-based matrix occupies the extracellular space neurocans and BEHAB/brevicans are expressed only in
of brain at all stages of development, similar to the matrices the CNS [8290]. The high degree of structural similarity
found in cartilages and other tissues. HA is an extremely among lecticans is reected in the virtually identical orga-
large (up to 10 million daltons) polyanionic polymer con- nization of their genes [9194]. The intronexon bound-
sisting of repeating disaccharide units, constituting a GAG aries are highly conserved and coincide with the boundaries
chain; unlike all other GAGs, HA is not covalently bound between functionally related domains. It is likely that the
to protein. Given the importance of HA in forming the lectican family members evolved by gene duplication [95].
ECM of the CNS, we will pay particular attention to the The N-terminus of lecticans consists of immunoglob-
lecticans (hyalulectins), a family of HA-binding CSPGs ulin-binding and HA-binding tandem repeat domains
that includes aggrecans, versicans, neurocans, and brain- [80,81] (Fig. 2). Both domains are highly conserved among
enriched hyaluronan-binding protein (BEHAB)/brevicans. all family members. As predicted from their structure, all
As will be described below, the lecticans ability to bind HA lecticans can bind HA (Table 2). The C-terminus also
and other matrix and cell surface molecules places them in contains highly conserved domains including epidermal
a unique position as organizers of the ECM in the CNS. growth factor (EGF)-like repeats, a C-type lectin domain,
HA is present at the highest levels in the brain during and a complement regulatory protein (CRP) domain. All
the embryonic and early postnatal periods [7779]. In the lecticans also contain a central domain that is the site of
rat brain, HA peaks at 7 days after birth, and almost 90% carbohydrate attachment. The central domains, unlike the
of the HA present at this stage of development is water- N-termini and C-termini, are less conserved and are vari-
soluble [77]. HA is extremely hydroscopic and, therefore, able both in size and in the number of sites for carbohy-
can organize water molecules, forming a hydrated perme- drate attachment [80,81]. Although the N-terminus of all
able matrix through which cellular migration and process lecticans binds HA, the lectin domain of these molecules
elaboration occur, as well as permitting the diusion (or interacts with a common set of ligands, specically tenas-
ltration) of low-molecular weight solutes, such as growth cin-R and sulfatide (Table 2). These interactions with
Figure 2 Schematic representation of the domain structure of the lectican family of CSPGs. The black domain represents the
GAG attachment domain. Notice that the size and the number of attached CS chains (represented by wavy lines) change
signicantly between family members. In versicans, the a and h chains can be alternatively spliced out by creating a number of
dierent splice variants. HABD, hyaluronan-binding domain; EGF, epidermal growth factor-like domain; lectin, lectinlike
domain; CRP, complement-regulatory proteinlike domain.
Table 2 Cellular Origin and Ligands of Secreted CSPGs in the Central Nervous System
Approximate
Family Name Cellular origin Mw (kDa) Extracellular ligands
Lecticans Aggrecan Neurons 500+ Hyaluronan, sulfatides
(hyalectans) Versican Glia 400 Hyaluronan, tenascin-R,
(PG-M, HABP) sulfatides
Neurocan Neurons, glia 250 Hyaluronan, tenascin-R,
tenascin-C, FGF-2, NCAM,
NG-CAM/L1, TAG-1/axonin,
sulfatides
BEHAB, brevican Glia, neurons 150 Hyaluronan, tenascin-R,
sulfatides
RPTP~/h Phosphacan Glia, neurons 300 Tenascin-R, tenascin-C,
(DSD-1-PG) HB-GAM/PTN, amphoterin,
NCAM, NG-CAM/L1,
TAG-1/axonin, contactin
matrix proteins (at the N-terminus) and cell surface pro- structural dierences, phosphacan and neurocan appear to
teins (at the C-terminus) can position the lecticans to play overlapping roles in the developing CNS, which will
mediate cellmatrix interactions. be discussed together.
The number and types of carbohydrate moieties added
to individual lectican molecules vary considerably. Aggre-
can has around 100 GAG attachment sites, whereas
BEHAB/brevican has only three. Furthermore, BEHAB/ IX. FUNCTIONS OF CSPGs IN THE CNS
brevicans in the CNS exist both with and without attached Early in development, phosphacan and neurocan are com-
GAG chains, making them part-time proteoglycans [96]. mon in the CNS and may account for up to 50% of the
Aggrecan is heavily glycosylated with KS-GAG chains in
the cartilage appears to carry no KS-GAG chains but in the
CNS [89,97].
All lecticans are subjected to proteolytic cleavage.
Aggrecan is cleaved at multiple sites by a variety of
dierent metalloproteinases [98,99]. BEHAB/brevican
has cleavage sites very similar to those found in aggrecans
[96,100]. Specic cleavage sites have been identied in the
central domain of neurocan [101,102], and a variety of
versican cleavage products have also been identied [103].
VIII. PHOSPHACANS
Phosphacan is derived from alternative splicing of recep-
tor-type protein tyrosine phosphatase h (RPTPh; also
known as PTP~) [104,105]. There are three RPTP~/h splice
variants and at least two of them carry CS GAG chains
[106111] (Fig. 3). The largest RPTP~/h isoform is a
transmembrane protein composed of a carbonic anhydrase
domain, a bronectin type III repeat, and a novel cysteine-
free stretch where the GAG chains are likely to be attached.
Two intracellular tyrosine phosphatase domains follow the Figure 3 Schematic representation of the domain structure
single transmembrane domain. A smaller transmembrane of the three major RPTP~/h/phosphacan splice variants. The
form lacks the GAG attachment region, named dvRPTP~/ black domain represents the CS attachment domain. Notice
h. The third RPTP~/h splice form, called phosphacan that that short membrane-bound form dvRPTP~/h is
(DSD-1-PG in mouse), contains the entire extracellular invested with very few or no CS chains. Phosphacan lacks
domain but lacks the transmembrane domain and is a the entire transmembrane domain and is a secreted CSPG.
secreted proteoglycan [110] (Fig. 3). Phosphacan can carry CA, carbonic anhydrase-like domain; F, bronectin type III
dierent compliments of KS and CS [110]. Despite their domain; PTP, protein tyrosine phosphatase domain.
CSPG in the young brain [105]. Despite the fact that they trativerather than the proliferativephase of reactive
show essentially no sequence homology at the amino acid gliosis. A role for BEHAB/brevican in normal glial devel-
level, they interact with a similar set of ligands and appear opment and following injury has not yet been determined
to be functionally similar [105]. Although neurocan is and, to this date, no marked defects have been reported in a
largely neuronal in origin and phosphacan is produced BEHAB/brevican knockout mouse [127].
predominantly by glia, expression of each has been dem- An insight into possible functions of BEHAB/brevican
onstrated in both cell types. Neurocan and phosphacan are in development comes from studies of primary glial tumors
expressed at high levels in the developing CNS, and in the CNS. In gliomas, glial-derived cells exhibit aberrant
although neurocan drops to lower levels in adulthood, cell proliferation and motility. Gliomas are notoriously
phosphacan is maintained at relatively stable levels [102]. invasive and deadly CNS tumors. Consistent with
Neurocan and phosphacan interact with an overlapping set BEHAB/brevican upregulation when normal glial cells
of ligands including tenacin-C, NCAM, NG-CAM/L1, proliferate and migrate, BEHAB/brevican expression is
and TAG-1/axonin [87,112117] (Table 2). It is somewhat dramatically upregulated in surgical samples of human
surprising that two proteins that show little homology at glioma and in a rodent experimental glioma model [128
the amino acid level could interact with a common set of 131]. In experimental rat gliomas, overexpression of
diverse ligands and it is, therefore, possible that common BEHAB/brevican and of an N-terminal fragment of
carbohydrate modications mediate these interactions. BEHAB/brevican increases tumor invasion [132135].
Moreover, although carbohydrates do mediate some of Furthermore, invasion appears to require, in addition to
these interactions, unique carbohydrates on neurocan and increased expression, BEHAB/brevican cleavage by
phosphacan appear to mediate these interactions. For ADAMTS-4, a member of the ADAMTS family of pro-
example, although the binding of neurocan to NCAM teases [132]. This work has led to the suggestion that
and NG-CAM is dependent on CS chains, the interaction interruption of BEHAB/brevican expression and cleavage
of phosphacan with the same ligands is dependent on may present novel therapeutic targets for gliomas.
sialylated complex N-linked sugars [112,113,116,118]. This The most clearly demonstrated function for CSPGs in
example serves to highlight the complexity of these mole- the CNS is inhibition of axonal outgrowth. This has been
cules and the diculties in determining their functions. shown for essentially every CSPG discussed in this review.
Early work indicated that CSPGs were generally CSPGs localize to areas that act as barriers to axonal
inhibitory to cell movement and axonal outgrowth. Indeed, outgrowth in vivo [136141]. In vitro experiments have
initial studies in chick brain suggested that both neurocan also demonstrated the inhibitory eect on neurite out-
and phosphacan are potent inhibitors of axonal outgrowth growth [112,120,142146] and indicated that inhibition
[112,115]. However, this function does not t the expres- can be mediated both by the isolated core proteins
sion prole of neurocan and phosphacan, which are both [120,142] and by the isolated CS chains [147,148]. CSPGs
expressed at times and in places of substantial cell move- represent one of the major barriers to regeneration in the
ment and neurite outgrowth. It is more likely that neurocan adult CNS. Many CSPGs including, neurocans, BEHAB/
and phosphacan may instead function in a bidirectional brevicans, phosphacans, and versicans, are upregulated in
manner and play an important role in directinginstead of response to neural injury [149]. The upregulated CSPGs
inhibitingcell movement and processes extension. In- then contribute to the formation of a glial scar, a barrier to
deed, in vitro, both promote neurite outgrowth in the regenerating axons. Removal of the CS chains from these
presence of certain substrates [119121]. Interestingly, both proteoglycans can enhance regeneration [150152] and may
can also have the opposite eect on neurite outgrowth. present an important therapeutic target for CNS injury.
Although neurocan inhibits neurite outgrowth on sub- One of the most interesting, but poorly understood,
strate-bound TAG-1, phosphacan promotes outgrowth ECM structures is the perineuronal net that decorates the
on the same substrate [116]. Moreover, the same molecules surface of CNS neurons. Camillo Golgi in the late 19th
are expressed in the developing cortex, spinal cord, and century described this structure as . . .a delicate covering,
cerebellum before and during robust invasion by axons mainly reticular in structure, but also in the form of tiny
[105]. Knockout mice for phosphacan and neurocan do not tiled slices or scales or an interrupted envelop, which
exhibit any obvious change in neurite outgrowth [122,123], surrounds the cell body of all nerve cells and continues
leaving the function of these proteins as yet unresolved. along their protoplasmic extensions. . . [153]. Until quite
BEHAB/brevican expression peaks late in develop- recently, the existence of this structure in the CNS was
ment and is maintained at high levels into adulthood in the questioned [154]. The advent of better xation techniques
adult rat brain [86,124], where it is expressed by both and the use of antibodies against perineuronal net-specic
neurons and glia [125]. Importantly, BEHAB/brevican is components have conrmed the existence of this structure
expressed at high levels in the ventricular zone coincident [155]. However, the complete composition and function of
with early gliogenesis, suggesting a role for BEHAB/brevi- this delicate covering or perineuronal net described by
can in the proliferation and/or migration of glial cells [125]. Golgi remains elusive, and an understanding of this struc-
Consistent with such a role, BEHAB/brevican expression is ture is still in its infancy [156].
also upregulated following brain injury when glia are The perineuronal net is a neuron-specic ECM. Peri-
activated to proliferate and migrate [126]. The expression neuronal nets appear as a meshwork of molecules covering
of BEHAB/brevican correlates most closely with the inl- the surface of neurons as a sheetlike structure with many
small holes [153,157]. The holes in the net represent sites SUMMARY
of synaptic contact [158,159]. A variety of reagents reveal
perineuronal nets, including immunohistochemical stains Over the past 20 years, substantial progress has been made
that label polyanionic constituents such as GAG chains in identifying the core protein structure of many proteo-
[155], indirect histochemical methods for localizing HA glycans in the CNS. As the precise temporal and regional
[82], and lectins that bind N-acetylgalactosamine [160 specicity of CSPG expression has been revealed, we have
162]. In addition, monoclonal antibodies directed against begun to unravel the mystery of their functions. We have
CSPGs that reveal perineuronal nets have been generated reviewed here experiments that support critical roles for
from several laboratories [163169]. What has emerged proteoglycans at every stage of neural development, as well
from studies utilizing these antibodies is the observation as in the adult nervous system. Proteoglycans constitute an
that dierent neuronal subsets can be distinguished by the increasingly complex set of molecules, which present a
complement of CSPGs that their nets contain [165,170 remarkable degree of molecular diversity derived from
173]. The diverse CSPGs in perineuronal nets could regu- alternate splicing, regulated proteolytic processing, and
late the extracellular microenvironment surrounding each variations in glycosylation. New genetic models that delete
neuron in a cell type-specic way. or add specic proteoglycans, their proteases, and the
Lecticans are constituents of these perineuronal nets. glycotransferases responsible for GAG chain synthesis
Versicans [174,175], neurocans [101], BEHAB/brevicans hold the promise of increasing our understanding of this
[175], and phosphacans have all been localized to peri- complex class of molecules.
neuronal nets. However, to date, the most extensively
characterized molecule in the perineuronal net is the CSPG
recognized by the monoclonal antibody Cat-301 [159], REFERENCES
which was recently shown to be an aggrecan [132]. Cat- 1. Hascall, V.C.; Calabaro, A.; Midura, R.J.; Yanagishita,
301-labeled aggrecan is distributed over the surface of cell M. Isolation and characterization of proteoglycans.
bodies and the proximal dendrites of specic subsets of Methods Enzymol. 1994, 230, 390417.
neurons in several areas of the mammalian CNS, including 2. Lindahl, U.; Kusche-Gullberg, M.; Kjellen, L. Regulated
the visual thalamus and visual cortex of cats [167,176] and diversity of heparan sulfate. J. Biol. Chem. 1998, 273,
primates [171,177]. Cat-301 immunoreactivity is not 2497924982.
3. Margolis, R.U.; Aquino, D.A.; Klinger, M.M.; Ripellino,
detected until late in development and onset of immuno- J.A.; Margolis, R.K. Structure and localization of nervous
reactivity coincides with the consolidation of the mature set tissue proteoglycans. Ann. N.Y. Acad. Sci. 1986, 481, 46
of synaptic contacts [178,179]. The expression of Cat-301 54.
immunoreactivity is modulated by patterns of neuronal 4. Binari, R.C.; Staveley, B.E.; Johnson, W.A.; Godavarti,
activity during the early developmental period, in parallel R.; Sasisekharan, R.; Manoukian, A.S. Genetic evidence
with the requirement for activity for the normal maturation that heparin-like glycosaminoglycans are involved in
wingless signaling. Development 1997, 124, 26232632.
of anatomical and physiological properties of neurons 5. Cole, G.J.; Loewy, A.; Glaser, L. Neuronal cellcell
[178185]. Studies on the activity-dependent expression adhesion depends on interactions of N-CAM with
of Cat-301 raised the possibility that the Cat-301 CSPG heparin-like molecules. Nature 1986, 320, 445447.
may play a role in the stabilization of mature synapses and 6. Liang, Y.; Annan, R.S.; Carr, S.A.; Popp, S.; Mevissen,
may contribute to the reduced plasticity seen in the adult M.; Margolis, R.K.; Margolis, R.U. Mammalian homo-
CNS [179]. Recent work has shown that treatment of adult logues of the Drosophila slit protein are ligands of the
heparan sulfate proteoglycan glypican-1 in brain. J. Biol.
rat visual cortex with chondroitinase, which releases neu-
Chem. 1999, 274, 1788517892.
ronal surface-associated CSPGs, reopens the window of 7. Nurcombe, V.; Ford, M.D.; Wildschut, J.A.; Bartlett,
synaptic plasticity, conrming a role for CSPGs in synapse P.F. Developmental regulation of neural response to
stabilization [186]. FGF-1 and FGF-2 by heparan sulfate proteoglycan.
The perineuronal nets in the mammalian brain are Science 1993, 260, 103106.
extraordinarily diverse in molecular composition. Immu- 8. Rapraeger, A.C.; Krufka, A.; Olwin, B.B. Requirement of
nostaining with antibodies directed at dierent CSPGs heparan sulfate for bFGF-mediated broblast growth and
demonstrates nets on overlapping but distinct subsets of myoblast dierentiation. Science 1991, 252, 17051708.
9. Reichsman, F.; Smith, L.; Cumberledge, S. Glycosami-
neurons [187]. We have shown that each of three antibodies noglycans can modulate extracellular localization of the
to aggrecan Cat-301, Cat-315, and Cat-316 recognizes a wingless protein and promote signal transduction. J. Cell
dierent aggrecan glycoform and also decorates a distinct Biol. 1996, 135, 819827.
subset of perineuronal nets in the CNS [132]. The dierent 10. Ruppert, R.; Homann, E.; Sebald, W. Human bone
aggrecan glycoforms are synthesized by the neurons them- morphogenetic protein 2 contains a heparin-binding site
selves, giving each neuron precise control over the compo- which modies its biological activity. Eur. J. Biochem.
sition of its ensheating net [132]. The growing evidence that 1996, 237, 295302.
11. Yayon, A.; Klagsbrun, M.; Esko, J.D.; Leder, P.; Ornitz,
these nets play a role in the stabilization of synapses D.M. Cell surface, heparin-like molecules are required for
suggests that molecularly distinct nets may play distinct binding of basic broblast growth factor to its high
roles in stabilizing a unique set of synapses on specic anity receptor. Cell 1991, 64, 841848.
subpopulations of neurons. 12. Raulo, E.; Julkunen, I.; Merenmies, J.; Pihlaskari, R.;
Rauvala, H. Secretion and biological activities of heparin- 29. Saunders, S.; Jalkanen, M.; OFarrell, S.; Berneld, M.
binding growth-associated molecule. Neurite outgrowth- Molecular cloning of syndecan, an integral membrane
promoting and mitogenic actions of the recombinant and proteoglycan. J. Cell Biol. 1989, 108, 15471556.
tissue-derived protein. J. Biol. Chem. 1992, 267, 11408 30. David, G.; Bai, X.M.; Van der Schueren, B.; Marynen, P.;
11416. Cassiman, J.J.; Van den Berghe, H. Spatial and temporal
13. Merenmies, J.; Rauvala, H. Molecular cloning of the 18- changes in the expression of broglycan (syndecan-2)
kDa growth-associated protein of developing brain. J. during mouse embryonic development. Development
Biol. Chem. 1990, 265, 1672116724. 1993, 119, 841854.
14. David, G. Integral membrane heparan sulfate proteogly- 31. Marynen, P.; Zhang, J.; Cassiman, J.J.; Van den Berghe,
cans. FASEB J. 1993, 7, 10231030. H.; David, G. Partial primary structure of the 48- and 90-
15. Lander, A.D.; Stipp, C.S.; Ivins, J.K. The glypican family kilodalton core proteins of cell surface-associated heparan
of heparan sulfate proteoglycans: major cell-surface sulfate proteoglycans of lung broblasts. Prediction of an
proteoglycans of the developing nervous system. Perspect. integral membrane domain and evidence for multiple
Dev. Neurobiol. 1996, 3, 347358. distinct core proteins at the cell surface of human lung
16. David, G.; Lories, V.; Decock, B.; Marynen, P.; Cassi- broblasts. J. Biol. Chem. 1989, 264, 70177024.
man, J.J.; Van den Berghe, H. Molecular cloning of a 32. Carey, D.J.; Evans, D.M.; Stahl, R.C.; Asundi, V.K.;
phosphatidylinositol-anchored membrane heparan sulfate Conner, K.J.; Garbes, P.; Cizmeci-Smith, G. Molecular
proteoglycan from human lung broblasts. J. Cell Biol. cloning and characterization of N-syndecan, a novel
1990, 111, 31653176. transmembrane heparan sulfate proteoglycan. J. Cell Biol.
17. Filmus, J.; Shi, W.; Wong, Z.M.; Wong, M.J. Identica- 1992, 117, 191201.
tion of a new membrane-bound heparan sulphate 33. Carey, D.J.; Conner, K.; Asundi, V.K.; OMahony, D.J.;
proteoglycan. Biochem. J. 1995, 311, 561565. Stahl, R.C.; Showalter, L.; Cizmeci-Smith, G.; Hartman,
18. Paine-Saunders, S.; Viviano, B.L.; Saunders, S. GPC6, a J.; Rothblum, L.I. cDNA cloning, genomic organization,
novel member of the glypican gene family, encodes a and in vivo expression of rat N-syndecan. J. Biol. Chem.
product structurally related to GPC4 and is colocalized 1997, 272, 28732879.
with GPC5 on human chromosome 13. Genomics 1999, 34. Kojima, T.; Shworak, N.W.; Rosenberg, R.D. Molecular
57, 455458. cloning and expression of two distinct cDNA-encoding
19. Saunders, S.; Paine-Saunders, S.; Lander, A.D. Expres- heparan sulfate proteoglycan core proteins from a rat
sion of the cell surface proteoglycan glypican-5 is endothelial cell line. J. Biol. Chem. 1992, 267, 48704877.
developmentally regulated in kidney, limb, and brain. 35. David, G.; van der Schueren, B.; Marynen, P.; Cassiman,
Dev. Biol. 1997, 190, 7893. J.J.; van den Berghe, H. Molecular cloning of amphigly-
20. Stipp, C.S.; Litwack, E.D.; Lander, A.D. Cerebroglycan: can, a novel integral membrane heparan sulfate proteo-
an integral membrane heparan sulfate proteoglycan that is glycan expressed by epithelial and broblastic cells. J. Cell
unique to the developing nervous system and expressed Biol. 1992, 118, 961969.
specically during neuronal dierentiation. J. Cell Biol. 36. Asundi, V.K.; Carey, D.J. Self-association of N-syndecan
1994, 124, 149160. (syndecan-3) core protein is mediated by a novel
21. Watanabe, K.; Yamada, H.; Yamaguchi, Y. K-glypican: a structural motif in the transmembrane domain and
novel GPI-anchored heparan sulfate proteoglycan that is ectodomain anking region. J. Biol. Chem. 1995, 270,
highly expressed in developing brain and kidney. J. Cell 2640426410.
Biol. 1995, 130, 12071218. 37. Grootjans, J.J.; Zimmermann, P.; Reekmans, G.; Smets,
22. Liang, Y.; Haring, M.; Roughley, P.J.; Margolis, R.K.; A.; Degeest, G.; Durr, J.; David, G. Syntenin, a PDZ
Margolis, R.U. Glypican and biglycan in the nuclei of protein that binds syndecan cytoplasmic domains. Proc.
neurons and glioma cells: presence of functional nuclear Natl. Acad. Sci. U. S. A. 1997, 94, 1368313688.
localization signals and dynamic changes in glypican 38. Cohen, A.R.; Woods, D.F.; Marfatia, S.M.; Walther, Z.;
during the cell cycle. J. Cell Biol. 1997, 139, 851864. Chishti, A.H.; Anderson, J.M.; Wood, D.F. Human
23. Watanabe, E.; Maeda, N.; Matsui, F.; Kushima, Y.; Noda, CASK/LIN-2 binds syndecan-2 and protein 4.1 and
M.; Oohira, A. Neuroglycan C, a novel membrane- localizes to the basolateral membrane of epithelial cells.
spanning chondroitin sulfate proteoglycan that is restricted J. Cell Biol. 1998, 142, (erratum appears in J. Cell Biol.
to the brain. J. Biol. Chem. 1995, 270, 2687626882. 1998, 142 (4), following 1156 note: D.F. Wood (corrected
24. Veugelers, M.; Vermeesch, J.; Reekmans, G.; Steinfeld, R.; to D.F. Woods)). 129138.
Marynen, P.; David, G. Characterization of glypican-5 and 39. Hsueh, Y.P.; Yang, F.C.; Kharazia, V.; Naisbitt, S.;
chromosomal localization of human GPC5, a new member Cohen, A.R.; Weinberg, R.J.; Sheng, M. Direct inter-
of the glypican gene family. Genomics 1997, 40, 2430. action of CASK/LIN-2 and syndecan heparan sulfate
25. Berneld, M.; Kokenyesi, R.; Kato, M.; Hinkes, M.T.; proteoglycan and their overlapping distribution in neuro-
Spring, J.; Gallo, R.L.; Lose, E.J. Biology of the nal synapses. J. Cell Biol. 1998, 142, 139151.
syndecans: a family of transmembrane heparan sulfate 40. Ethell, I.M.; Hagihara, K.; Miura, Y.; Irie, F.; Yama-
proteoglycans. Annu. Rev. Cell Biol. 1992, 8, 365393. guchi, Y. Synbindin, a novel syndecan-2-binding protein
26. Carey, D.J. Syndecans: multifunctional cell-surface co- in neuronal dendritic spines. J. Cell Biol. 2000, 151, 5368.
receptors. Biochem. Biophys. Res. Commun. 1997, 240, 41. Gao, Y.; Li, M.; Chen, W.; Simons, M. Synectin, syndecan-
502506. 4 cytoplasmic domain binding PDZ protein, inhibits cell
27. Rapraeger, A.C.; Ott, V.L. Molecular interactions of the migration. J. Cell. Physiol. 2000, 184, 373379.
syndecan core proteins. Curr. Opin. Cell Biol. 1998, 10, 42. Elenius, K.; Maatta, A.; Salmivirta, M.; Jalkanen, M.
620628. Growth factors induce 3T3 cells to express bFGF-binding
28. Mali, M.; Jaakkola, P.; Arvilommi, A.M.; Jalkanen, M. syndecan. J. Biol. Chem. 1992, 267, 64356441.
Sequence of human syndecan indicates a novel gene 43. Kato, M.; Wang, H.; Kainulainen, V.; Fitzgerald, M.L.;
family of integral membrane proteoglycans. J. Biol. Ledbetter, S.; Ornitz, D.M.; Berneld, M. Physiological
Chem. 1990, 265, 68846889. degradation converts the soluble syndecan-1 ectodomain
from an inhibitor to a potent activator of FGF-2. Nat. 60. Perrimon, N.; Berneld, M. Specicities of heparan
Med. 1998, 4, 691697. sulphate proteoglycans in developmental processes. Cel-
44. Subramanian, S.V.; Fitzgerald, M.L.; Berneld, M. lular functions of proteoglycansan overview. Nature
Regulated shedding of syndecan-1 and -4 ectodomains 2000, 404, 725728.
by thrombin and growth factor receptor activation. J. 61. Ethell, I.M.; Yamaguchi, Y. Cell surface heparan sulfate
Biol. Chem. 1997, 272, 1471314720. proteoglycan syndecan-2 induces the maturation of
45. Vlodavsky, I.; Miao, H.Q.; Medalion, B.; Danagher, P.; dendritic spines in rat hippocampal neurons. J. Cell Biol.
Ron, D. Involvement of heparan sulfate and related 1999, 144, 575586.
molecules in sequestration and growth promoting activity 62. Hsueh, Y.P.; Sheng, M. Regulated expression and
of broblast growth factor. Cancer Metastasis Rev. 1996, subcellular localization of syndecan heparan sulfate
15, 177186. proteoglycans and the syndecan-binding protein CASK/
46. Saksela, O.; Rifkin, D.B. Release of basic broblast LIN-2 during rat brain development. J. Neurosci. 1999,
growth factorheparan sulfate complexes from endothe- 19, 74157425.
lial cells by plasminogen activator-mediated proteolytic 63. Ethell, I.M.; Irie, F.; Kalo, M.S.; Couchman, J.R.;
activity. J. Cell Biol. 1990, 110, 767775. Pasquale, E.B.; Yamaguchi, Y. EphB/syndecan-2 signal-
47. Rapraeger, A.C.; Guimond, S.; Krufka, A.; Olwin, B.B. ing in dendritic spine morphogenesis. Neuron 2001, 31,
Regulation by heparan sulfate in broblast growth factor 10011013.
signaling. Methods Enzymol. 1994, 245, 219240. 64. Winkler, S.; Stahl, R.C.; Carey, D.J.; Bansal, R.
48. Mason, I. Cell signalling. Do adhesion molecules signal Syndecan-3 and perlecan are dierentially expressed by
via FGF receptors? Curr. Biol. 1994, 4, 11581161. progenitors and mature oligodendrocytes and accumulate
49. Guimond, S.; Maccarana, M.; Olwin, B.B.; Lindahl, U.; in the extracellular matrix. J. Neurosci. Res. 2002, 69,
Rapraeger, A.C. Activating and inhibitory heparin 477487.
sequences for FGF-2 (basic FGF). Distinct requirements 65. McKinnon, R.D.; Matsui, T.; Dubois-Dalcq, M.; Aar-
for FGF-1, FGF-2, and FGF-4. J. Biol. Chem. 1993, 268, onson, S.A. FGF modulates the PDGF-driven pathway
2390623914. of oligodendrocyte development. Neuron 1990, 5, 603
50. Pye, D.A.; Vives, R.R.; Turnbull, J.E.; Hyde, P.; 614.
Gallagher, J.T. Heparan sulfate oligosaccharides require 66. Oh, L.Y.; Denninger, A.; Colvin, J.S.; Vyas, A.; Tole, S.;
6-O-sulfation for promotion of basic broblast growth Ornitz, D.M.; Bansal, R. Fibroblast growth factor
factor mitogenic activity. J. Biol. Chem. 1998, 273, 22936 receptor 3 signaling regulates the onset of oligodendrocyte
22942. terminal dierentiation. J. Neurosci. 2003, 23, 883894.
51. Vescovi, A.L.; Reynolds, B.A.; Fraser, D.D.; Weiss, S. 67. Gard, A.L.; Pfeier, S.E. Glial cell mitogens bFGF and
bFGF regulates the proliferative fate of unipotent (neuro- PDGF dierentially regulate development of O4+GalC-
nal) and bipotent (neuronal/astroglial) EGF-generated oligodendrocyte progenitors. Dev. Biol. 1993, 159, 618
CNS progenitor cells. Neuron 1993, 11, 951966. 630.
52. Vicario-Abejon, C.; Johe, K.C.; Hazel, T.G.; Collazo, D.; 68. Bansal, R.; Pfeier, S.E. Inhibition of protein and lipid
McKay, R.D.G. Functions of basic broblast growth sulfation in oligodendrocytes blocks biological responses
factor and neurotrophins in the dierentiation of hippo- to FGF-2 and retards cytoarchitectural maturation, but
campal neurons. Neuron 1995, 15, 105114. not developmental lineage progression. Dev. Biol. 1994,
53. Qian, X.; Davis, A.A.; Goderie, S.K.; Temple, S. FGF-2 162, 511524.
concentration regulates the generation of neurons and glia 69. Bansal, R.; Pfeier, S.E. Regulation of oligodendrocyte
from multipotent cortical stem cells. Neuron 1997, 18, 81 dierentiation by broblast growth factors. Adv. Exp.
93. Med. Biol. 1997, 429, 6977.
54. Ghosh, A.; Greenberg, M.E. Distinct roles for bFGF and 70. Bansal, R.; Kumar, M.; Murray, K.; Morrison, R.S.;
NT-3 in the regulation of cortical neurogenesis. Neuron Pfeier, S.E. Regulation of FGF receptors in the oligo-
1995, 15, 89103. dendrocyte lineage. Mol. Cell. Neurosci. 1996, 7, 263275.
55. Hagihara, K.; Watanabe, K.; Chun, J.; Yamaguchi, Y. 71. Osterhout, D.J.; Ebner, S.; Xu, J.; Ornitz, D.M.; Zazanis,
Glypican-4 is an FGF-2-binding heparan sulfate proteo- G.A.; McKinnon, R.D. Transplanted oligodendrocyte
glycan expressed in neural precursor cells. Dev. Dyn. progenitor cells expressing a dominant-negative FGF
2000, 219, 353367. receptor transgene fail to migrate in vivo. J. Neurosci.
56. Litwack, E.D.; Stipp, C.S.; Kumbasar, A.; Lander, A.D. 1997, 17, 91229132.
Neuronal expression of glypican, a cell-surface glycosyl- 72. Kinnunen, A.; Kinnunen, T.; Kaksonen, M.; Nolo, R.;
phosphatidylinositol-anchored heparan sulfate proteogly- Panula, P.; Rauvala, H. N-syndecan and HB-GAM
can, in the adult rat nervous system. J. Neurosci. 1994, 14, (heparin-binding growth-associated molecule) associate
37133724. with early axonal tracts in the rat brain. Eur. J. Neurosci.
57. Ronca, F.; Andersen, J.S.; Paech, V.; Margolis, R.U. 1998, 10, 635648.
Characterization of slit protein interactions with glypi- 73. Kinnunen, A.; Niemi, M.; Kinnunen, T.; Kaksonen, M.;
can-1. J. Biol. Chem. 2001, 276, 2914129147. Nolo, R.; Rauvala, H. Heparan sulphate and HB-GAM
58. Ivins, J.K.; Litwack, E.D.; Kumbasar, A.; Stipp, C.S.; (heparin-binding growth-associated molecule) in the
Lander, A.D. Cerebroglycan, a developmentally regulated development of the thalamocortical pathway of rat brain.
cell-surface heparan sulfate proteoglycan, is expressed on Eur. J. Neurosci. 1999, 11, 491502.
developing axons and growth cones. Dev. Biol. 1997, 184, 74. Mitsiadis, T.A.; Salmivirta, M.; Muramatsu, T.; Mur-
320332. amatsu, H.; Rauvala, H.; Lehtonen, E.; Jalkanen, M.;
59. Nakato, H.; Sugiura, M.; Jannuzi, A.; Oakes, R.; Kaluza, Thesle, I. Expression of the heparin-binding cytokines,
V.; Golden, C.; Selleck, S.B. The division abnormally midkine (MK) and HB-GAM (pleiotrophin) is associated
delayed (dally) gene: a putative integral membrane with epithelialmesenchymal interactions during fetal
proteoglycan required for cell division patterning during development and organogenesis. Development 1995,
postembryonic development of the nervous system in 121, 3751.
Drosophila. Development 1997, 124, 41134120. 75. Kinnunen, T.; Kaksonen, M.; Saarinen, J.; Kalkkinen, N.;
Peng, H.B.; Rauvala, H. CortactinSrc kinase signaling Beier, D.R.; Fassler, R. Structure and chromosomal
pathway is involved in N-syndecan-dependent neurite localization of the mouse neurocan gene. Genomics
outgrowth. J. Biol. Chem. 1998, 273, 1070210708. 1995, 28, 405410.
76. Margolis, R.K.; Margolis, R.U. Nervous tissue proteo- 94. Rauch, U.; Meyer, H.; Brakebusch, C.; Seidenbecher, C.;
glycans. EXS 1994, 70, 145177. Gundelnger, E.D.; Beier, D.R.; Fassler, R. Sequence and
77. Margolis, R.U.; Margolis, R.K. Distribution and metab- chromosomal localization of the mouse brevican gene.
olism of mucopolysaccharides and glycoproteins in Genomics 1997, 44, 1521.
neuronal perikarya, astrocytes, and oligodendroglia. 95. Iozzo, R.V. Matrix proteoglycans: from molecular design
Biochemistry 1974, 13, 28492852. to cellular function. Annu. Rev. Biochem. 1998, 67, 609
78. Jenkins, H.G.; Bachelard, H.S. Developmental and age- 652.
related changes in rat brain glycosaminoglycans. J. 96. Yamaguchi, Y. Brevican: a major proteoglycan in adult
Neurochem. 1988, 51, 16341640. brain. Perspect. Dev. Neurobiol. 1996, 3, 307317.
79. Oohira, A.; Matsui, F.; Matsuda, M.; Shoji, R. Devel- 97. Li, H.; Domowicz, M.; Hennig, A.; Schwartz, N.B. S103L
opmental change in the glycosaminoglycan composition reactive chondroitin sulfate proteoglycan (aggrecan)
of the rat brain. J. Neurochem. 1986, 47, 588593. mRNA expressed in developing chick brain and cartilage
80. Yamaguchi, Y. Lecticans: organizers of the brain extra- is encoded by a single gene. Brain Res. Mol. Brain Res.
cellular matrix. Cell. Mol. Life Sci. 2000, 57, 276289. 1996, 36, 309321.
81. Bandtlow, C.E.; Zimmermann, D.R. Proteoglycans in the 98. Sandy, J.D.; Neame, P.J.; Boynton, R.E.; Flannery, C.R.
developing brain: new conceptual insights for old pro- Catabolism of aggrecan in cartilage explants. Identica-
teins. Physiol. Rev. 2000, 80, 12671290. tion of a major cleavage site within the interglobular
82. Bignami, A.; Asher, R.; Perides, G.; Rahemtulla, F. The domain. J. Biol. Chem. 1991, 266, 86838685.
extracellular matrix of cerebral gray matter: Golgis 99. Sandy, J.D.; Flannery, C.R.; Neame, P.J.; Lohmander,
pericellular net and Nissls nervosen grau revisited. Int. L.S. The structure of aggrecan fragments in human
J. Dev. Neurosci. 1992, 10, 291299. synovial uid. Evidence for the involvement in osteo-
83. Engel, M.; Maurel, P.; Margolis, R.U.; Margolis, R.K. arthritis of a novel proteinase which cleaves the Glu 373
Chondroitin sulfate proteoglycans in the developing Ala 374 bond of the interglobular domain. J. Clin. Invest.
central nervous system: I. Cellular sites of synthesis of 1992, 89, 15121516.
neurocan and phosphacan. J. Comp. Neurol. 1996, 366, 100. Matthews, R.T.; Gary, S.C.; Zerillo, C.; Pratta, M.;
3443. Solomon, K.; Arner, E.C.; Hockeld, S. Brain-enriched
84. Jaworski, D.M.; Kelly, G.M.; Hockeld, S. BEHAB, a hyaluronan binding (BEHAB)/brevican cleavage in a
new member of the proteoglycan tandem repeat family of glioma cell line is mediated by a disintegrin and metal-
hyaluronan-binding proteins that is restricted to the brain. loproteinase with thrombospondin motifs (ADAMTS)
(erratum appears in J. Cell Biol. 1997, 137 (2), 521) J. Cell family member. J. Biol. Chem. 2000, 275, 2269522703.
Biol. 1994, 125 495509. 101. Matsui, F.; Nishizuka, M.; Yasuda, Y.; Aono, S.;
85. Margolis, R.K.; Margolis, R.U. Nervous tissue proteo- Watanabe, E.; Oohira, A. Occurrence of a N-terminal
glycans. Experientia 1993, 49, 429446. proteolytic fragment of neurocan, not a C-terminal half,
86. Milev, P.; Maurel, P.; Chiba, A.; Mevissen, M.; Popp, S.; in a perineuronal net in the adult rat cerebrum. Brain Res.
Yamaguchi, Y.; Margolis, R.K.; Margolis, R.U. Dier- 1998, 790, 4551.
ential regulation of expression of hyaluronan-binding 102. Meyer-Puttlitz, B.; Milev, P.; Junker, E.; Zimmer, I.;
proteoglycans in developing brain: aggrecan, versican, Margolis, R.U.; Margolis, R.K. Chondroitin sulfate and
neurocan, and brevican. Biochem. Biophys. Res. Com- chondroitin/keratan sulfate proteoglycans of nervous
mun. 1998, 247, 207212. tissue: developmental changes of neurocan and phospha-
87. Milev, P.; Fischer, D.; Haring, M.; Schulthess, T.; can. J. Neurochem. 1995, 65, 23272337.
Margolis, R.K.; Chiquet-Ehrismann, R.; Margolis, R.U. 103. Perides, G.; Asher, R.A.; Lark, M.W.; Lane, W.S.;
The brinogen-like globe of tenascin-C mediates its Robinson, R.A.; Bignami, A. Glial hyaluronate-binding
interactions with neurocan and phosphacan/protein-tyro- protein: a product of metalloproteinase digestion of
sine phosphatase-zeta/beta. J. Biol. Chem. 1997, 272, versican? Biochem. J. 1995, 312, 377384.
1550115509. 104. Grumet, M.; Friedlander, D.R.; Sakurai, T. Functions of
88. Schmalfeldt, M.; Dours-Zimmermann, M.T.; Winterhal- brain chondroitin sulfate proteoglycans during develop-
ter, K.H.; Zimmermann, D.R. Versican V2 is a major ments: interactions with adhesion molecules. Perspect.
extracellular matrix component of the mature bovine Dev. Neurobiol. 1996, 3, 319330.
brain. J. Biol. Chem. 1998, 273, 1575815764. 105. Margolis, R.K.; Rauch, U.; Maurel, P.; Margolis, R.U.
89. Schwartz, N.B.; Domowicz, M.; Krueger, R.C., Jr.; Li, Neurocan and phosphacan: two major nervous tissue-
H.; Mangoura, D. Brain aggrecan. Perspect. Dev. Neuro- specic chondroitin sulfate proteoglycans. Perspect. Dev.
biol. 1996, 3, 291306. Neurobiol. 1996, 3, 273290.
90. Yamada, H.; Watanabe, K.; Shimonaka, M.; Yamaguchi, 106. Barnea, G.; Grumet, M.; Milev, P.; Silvennoinen, O.;
Y. Molecular cloning of brevican, a novel brain proteo- Levy, J.B.; Sap, J.; Schlessinger, J. Receptor tyrosine
glycan of the aggrecan/versican family. J. Biol. Chem. phosphatase beta is expressed in the form of proteoglycan
1994, 269, 1011910126. and binds to the extracellular matrix protein tenascin. J.
91. Doege, K.J.; Garrison, K.; Coulter, S.N.; Yamada, Y. Biol. Chem. 1994, 269, 1434914352.
The structure of the rat aggrecan gene and preliminary 107. Barnea, G.; Grumet, M.; Sap, J.; Margolis, R.U.; Schles-
characterization of its promoter. J. Biol. Chem. 1994, 269, singer, J. Close similarity between receptor-linked tyrosine
2923229240. phosphatase and rat brain proteoglycan. Cell 1994, 76,
92. Naso, M.F.; Zimmermann, D.R.; Iozzo, R.V. Character- 205.
ization of the complete genomic structure of the human 108. Krueger, R.C., Jr.; Hennig, A.K.; Schwartz, N.B. Two
versican gene and functional analysis of its promoter. J. immunologically and developmentally distinct chondroi-
Biol. Chem. 1994, 269, 3299933008. tin sulfate proteoglycans in embryonic chick brain. J. Biol.
93. Rauch, U.; Grimpe, B.; Kulbe, G.; Arnold-Ammer, I.; Chem. 1992, 267, 1214912161.
109. Maeda, N.; Hamanaka, H.; Oohira, A.; Noda, M. Puri- ization of neurocan and phosphacan. J. Comp. Neurol.
cation, characterization and developmental expression of 1996, 366, 4454.
a brain-specic chondroitin sulfate proteoglycan, 6B4 122. Sakiyama, J.; Kurazono, S.; Mori, S.; Nakata, Y.;
proteoglycan/phosphacan. Neuroscience 1995, 67, 2335. Nakaya, N.; Oohira, A.; Zhou, X.H. Neurocan is
110. Maurel, P.; Rauch, U.; Flad, M.; Margolis, R.K.; dispensable for brain development. Cell Tissue Res.
Margolis, R.U. Phosphacan, a chondroitin sulfate pro- 2001, 306, 217229.
teoglycan of brain that interacts with neurons and neural 123. Harroch, S.; Palmeri, M.; Rosenbluth, J.; Custer, A.;
cell-adhesion molecules, is an extracellular variant of a Okigaki, M.; Shrager, P.; Blum, M.; Buxbaum, J.D.;
receptor-type protein tyrosine phosphatase. Proc. Natl. Schlessinger, J. No obvious abnormality in mice decient
Acad. Sci. U. S. A. 1994, 91, 25122516. in receptor protein tyrosine phosphatase beta. Mol. Cell.
111. Shitara, K.; Yamada, H.; Watanabe, K.; Shimonaka, M.; Biol. 2000, 20, 77067715.
Yamaguchi, Y. Brain-specic receptor-type protein-tyro- 124. Seidenbecher, C.I.; Gundelnger, E.D.; Bockers, T.M.;
sine phosphatase RPTP beta is a chondroitin sulfate Trotter, J.; Kreutz, M.R. Transcripts for secreted and
proteoglycan in vivo. J. Biol. Chem. 1994, 269, 20189 GPI-anchored brevican are dierentially distributed in rat
20193. brain. Eur. J. Neurosci. 1998, 10, 16211630.
112. Friedlander, D.R.; Milev, P.; Karthikeyan, L.; Margolis, 125. Jaworski, D.M.; Kelly, G.M.; Hockeld, S. The CNS-
R.K.; Margolis, R.U.; Grumet, M. The neuronal chon- specic hyaluronan-binding protein BEHAB is expressed
droitin sulfate proteoglycan neurocan binds to the neural in ventricular zones coincident with gliogenesis. J. Neuro-
cell adhesion molecules NG-CAM/L1/NILE and N- sci. 1995, 15, 13521362.
CAM, and inhibits neuronal adhesion and neurite out- 126. Jaworski, D.M.; Kelly, G.M.; Hockeld, S. Intracranial
growth. J. Cell Biol. 1994, 125, 669680. injury acutely induces the expression of the secreted
113. Grumet, M.; Milev, P.; Sakurai, T.; Karthikeyan, L.; isoform of the CNS-specic hyaluronan-binding protein
Bourdon, M.; Margolis, R.K.; Margolis, R.U. Interac- BEHAB/brevican. Exp. Neurol. 1999, 157, 327337.
tions with tenascin and dierential eects on cell adhesion 127. Brakebusch, C.; Seidenbecher, C.I.; Asztely, F.; Rauch,
of neurocan and phosphacan, two major chondroitin U.; Matthies, H.; Meyer, H.; Krug, M.; Bockers, T.M.;
sulfate proteoglycans of nervous tissue. J. Biol. Chem. Zhou, X.; Kreutz, M.R.; Montag, D.; Gundelnger, E.D.;
1994, 269, 1214212146. Fassler, R. Brevican-decient mice display impaired
114. Milev, P.; Chiba, A.; Haring, M.; Rauvala, H.; Schachner, hippocampal CA1 long-term potentiation but show no
M.; Ranscht, B.; Margolis, R.K.; Margolis, R.U. High obvious decits in learning and memory. Mol. Cell. Biol.
anity binding and overlapping localization of neurocan 2002, 22, 74177427.
and phosphacan/protein-tyrosine phosphatase-zeta/beta 128. Jaworski, D.M.; Kelly, G.M.; Piepmeier, J.M.; Hockeld,
with tenascin-R, amphoterin, and the heparin-binding S. BEHAB (brain enriched hyaluronan binding) is ex-
growth-associated molecule. J. Biol. Chem. 1998, 273, pressed in surgical samples of glioma and in intracranial
69987005. grafts of invasive glioma cell lines. Cancer Res. 1996, 56,
115. Milev, P.; Friedlander, D.R.; Sakurai, T.; Karthikeyan, 22932298.
L.; Flad, M.; Margolis, R.K.; Grumet, M.; Margolis, 129. Gary, S.C.; Kelly, G.M.; Hockeld, S. BEHAB/brevican:
R.U. Interactions of the chondroitin sulfate proteoglycan a brain-specic lectican implicated in gliomas and glial cell
phosphacan, the extracellular domain of a receptor-type motility. Curr. Opin. Neurobiol. 1998, 8, 576581.
protein tyrosine phosphatase, with neurons, glia, and 130. Gary, S.C.; Zerillo, C.A.; Chiang, V.L.; Gaw, J.U.; Gray,
neural cell adhesion molecules. J. Cell Biol. 1994, 127, G.; Hockeld, S. cDNA cloning, chromosomal localization,
17031715. and expression analysis of human BEHAB/brevican, a
116. Milev, P.; Maurel, P.; Haring, M.; Margolis, R.K.; brain specic proteoglycan regulated during cortical
Margolis, R.U. TAG-1/axonin-1 is a high-anity ligand development and in glioma. Gene 2000, 256, 139147.
of neurocan, phosphacan/protein-tyrosine phosphatase- 131. Gary, S.C.; Hockeld, S. BEHAB/brevican: an extra-
zeta/beta, and N-CAM. J. Biol. Chem. 1996, 271, 15716 cellular matrix component associated with invasive
15723. glioma. Clin. Neurosurg. 2000, 47, 7282.
117. Milev, P.; Monnerie, H.; Popp, S.; Margolis, R.K.; 132. Matthews, R.T.; Kelly, G.M.; Zerillo, C.A.; Gray, G.;
Margolis, R.U. The core protein of the chondroitin sul- Tiemeyer, M.; Hockeld, S. Aggrecan glycoforms con-
fate proteoglycan phosphacan is a high-anity ligand of tribute to the molecular heterogeneity of perineuronal
broblast growth factor-2 and potentiates its mitogenic nets. J. Neurosci. 2002, 22, 75367547.
activity. J. Biol. Chem. 1998, 273, 2143921442. 133. Nutt, C.L.; Zerillo, C.A.; Kelly, G.M.; Hockeld, S. Brain
118. Milev, P.; Meyer-Puttlitz, B.; Margolis, R.K.; Margolis, enriched hyaluronan binding (BEHAB)/brevican increases
R.U. Complex-type asparagine-linked oligosaccharides on aggressiveness of CNS-1 gliomas in Lewis rats. Cancer
phosphacan and protein-tyrosine phosphatase-zeta/beta Res. 2001, 61, 70567059.
mediate their binding to neural cell adhesion molecules 134. Nutt, C.L.; Matthews, R.T.; Hockeld, S. Glial tumor
and tenascin. J. Biol. Chem. 1995, 270, 2465024653. invasion: a role for the upregulation and cleavage of
119. Fukuda, T.; Kawano, H.; Ohyama, K.; Li, H.P.; Takeda, BEHAB/brevican. Neuroscientist 2001, 7, 113122.
Y.; Oohira, A.; Kawamura, K. Immunohistochemical 135. Zhang, H.; Kelly, G.; Zerillo, C.; Jaworski, D.M.;
localization of neurocan and L1 in the formation of Hockeld, S. Expression of a cleaved brain-specic
thalamocortical pathway of developing rats. J. Comp. extracellular matrix protein mediates glioma cell invasion
Neurol. 1997, 382, 141152. in vivo. J. Neurosci. 1998, 18, 23702376.
120. Maeda, N.; Noda, M. 6B4 proteoglycan/phosphacan is a 136. Landolt, R.M.; Vaughan, L.; Winterhalter, K.H.; Zim-
repulsive substratum but promotes morphological dier- mermann, D.R. Versican is selectively expressed in
entiation of cortical neurons. Development 1996, 122, embryonic tissues that act as barriers to neural crest cell
647658. migration and axon outgrowth. Development 1995, 121,
121. Meyer-Puttlitz, B.; Junker, E.; Margolis, R.U.; Margolis, 23032312.
R.K. Chondroitin sulfate proteoglycans in the developing 137. Oakley, R.A.; Tosney, K.W. Peanut agglutinin and
central nervous system: II. Immunocytochemical local- chondroitin-6-sulfate are molecular markers for tissues
that act as barriers to axon advance in the avian embryo. 157. Celio, M.R.; Blumcke, I. Perineuronal netsa specialized
Dev. Biol. 1991, 147, 187206. form of extracellular matrix in the adult nervous system.
138. Perris, R.; Krotoski, D.; Lallier, T.; Domingo, C.; Sorrell, Brain Res. Brain Res. Rev. 1994, 19, 128145.
J.M.; Bronner-Fraser, M. Spatial and temporal changes in 158. Hockeld, S.; McKay, R.D. A surface antigen expressed by
the distribution of proteoglycans during avian neural crest a subset of neurons in the vertebrate central nervous
development. Development 1991, 111, 583599. system. Proc. Natl. Acad. Sci. U. S. A. 1983, 80, 57585761.
139. Jhaveri, S. Midline glia of the tectum: a barrier for devel- 159. Zaremba, S.; Guimaraes, A.; Kalb, R.G.; Hockeld, S.
oping retinal axons. Perspect. Dev. Neurobiol. 1993, 1, Characterization of an activity-dependent, neuronal sur-
237543. face proteoglycan identied with monoclonal antibody
140. Snow, D.M.; Watanabe, M.; Letourneau, P.C.; Silver, J. Cat-301. Neuron 1989, 2, 12071219.
A chondroitin sulfate proteoglycan may inuence the 160. Schweizer, M.; Streit, W.J.; Muller, C.M. Postnatal devel-
direction of retinal ganglion cell outgrowth. Development opment and localization of an N-acetylgalactosamine
1991, 113, 14731485. containing glycoconjugate associated with nonpyramidal
141. Brittis, P.A.; Canning, D.R.; Silver, J. Chondroitin sulfate neurons in cat visual cortex. J. Comp. Neurol. 1993, 329,
as a regulator of neuronal patterning in the retina. Science 313327.
1992, 255, 733736. 161. Nakagawa, F.; Schulte, B.A.; Spicer, S.S. Selective
142. Dou, C.L.; Levine, J.M. Inhibition of neurite growth by cytochemical demonstration of glycoconjugate-containing
the NG2 chondroitin sulfate proteoglycan. J. Neurosci. terminal N-acetylgalactosamine on some brain neurons. J.
1994, 14, 76167628. Comp. Neurol. 1986, 243, 280290.
143. Niederost, B.P.; Zimmermann, D.R.; Schwab, M.E.; 162. Bruckner, G.; Brauer, K.; Hartig, W.; Wol, J.R.;
Bandtlow, C.E. Bovine CNS myelin contains neurite Rickmann, M.J.; Derouiche, A.; Delpech, B.; Girard, N.;
growth-inhibitory activity associated with chondroitin Oertel, W.H.; Reichenbach, A. Perineuronal nets provide a
sulfate proteoglycans. J. Neurosci. 1999, 19, 89798989. polyanionic, glia-associated form of microenvironment
144. Perris, R.; Johansson, S. Amphibian neural crest cell around certain neurons in many parts of the rat brain. Glia
migration on puried extracellular matrix components: a 1993, 8, 183200.
chondroitin sulfate proteoglycan inhibits locomotion on 163. Bertolotto, A.; Rocca, G.; Canavese, G.; Migheli, A.;
bronectin substrates. J. Cell Biol. 1987, 105, 25112521. Schier, D. Chondroitin sulfate proteoglycan surrounds a
145. Schmalfeldt, M.; Bandtlow, C.E.; Dours-Zimmermann, subset of human and rat CNS neurons. J. Neurosci. Res.
M.T.; Winterhalter, K.H.; Zimmermann, D.R. Brain 1991, 29, 225234.
derived versican V2 is a potent inhibitor of axonal growth. 164. Bertolotto, A.; Manzardo, E.; Guglielmone, R. Immuno-
J. Cell Sci. 2000, 113, 807816. histochemical mapping of perineuronal nets containing
146. Snow, D.M.; Lemmon, V.; Carrino, D.A.; Caplan, A.I.; chondroitin unsulfated proteoglycan in the rat central
Silver, J. Sulfated proteoglycans in astroglial barriers nervous system. Cell Tissue Res. 1996, 283, 283295.
inhibit neurite outgrowth in vitro. Exp. Neurol. 1990, 109, 165. Fujita, S.C.; Tada, Y.; Murakami, F.; Hayashi, M.;
111130. Matsumura, M. Glycosaminoglycan-related epitopes sur-
147. Carbonetto, S.; Gruver, M.M.; Turner, D.C. Nerve ber rounding dierent subsets of mammalian central neurons.
growth in culture on bronectin, collagen, and glycos- Neurosci. Res. 1989, 7, 117130.
aminoglycan substrates. J. Neurosci. 1983, 3, 23242335. 166. Guimaraes, A.; Zaremba, S.; Hockeld, S. Molecular and
148. Verna, J.M.; Fichard, A.; Saxod, R. Inuence of morphological changes in the cat lateral geniculate
glycosaminoglycans on neurite morphology and out- nucleus and visual cortex induced by visual deprivation
growth patterns in vitro. Int. J. Dev. Neurosci. 1989, 7, are revealed by monoclonal antibodies Cat-304 and Cat-
389399. 301. Neuronal subsets express multiple high-molecular-
149. Morgenstern, D.A.; Asher, R.A.; Fawcett, J.W. Chon- weight cell-surface glycoconjugates dened by monoclonal
droitin sulphate proteoglycans in the CNS injury antibodies Cat-301 and VC1.1. J. Neurosci. 1990, 10,
response. Prog. Brain Res. 2002, 137, 351359. 30143024.
150. Moon, L.D.; Asher, R.A.; Rhodes, K.E.; Fawcett, J.W. 167. Hockeld, S.; McKay, R.D.; Hendry, S.H.; Jones, E.G. A
Regeneration of CNS axons back to their target following surface antigen that identies ocular dominance columns
treatment of adult rat brain with chondroitinase ABC. in the visual cortex and laminar features of the lateral
Nat. Neurosci. 2001, 4, 465466. geniculate nucleus. Cold Spring Harbor Symp. Quant.
151. Bradbury, E.J.; Moon, L.D.; Popat, R.J.; King, V.R.; Biol. 1983, 48 (Pt 2), 877889.
Bennett, G.S.; Patel, P.N.; Fawcett, J.W.; McMahon, S.B. 168. Watanabe, E.; Fujita, S.C.; Murakami, F.; Hayashi, M.;
Chondroitinase ABC promotes functional recovery after Matsumura, M. A monoclonal antibody identies a novel
spinal cord injury. Nature 2002, 416, 636640. [comment] epitope surrounding a subpopulation of the mammalian
152. Yick, L.W.; Wu, W.; So, K.F.; Yip, H.K.; Shum, D.K. central neurons. Neuroscience 1989, 29, 645657.
Chondroitinase ABC promotes axonal regeneration of 169. Wintergerst, E.S.; Vogt Weisenhorn, D.M.; Rathjen,
Clarkes neurons after spinal cord injury. Neuroreport F.G.; Riederer, B.M.; Lambert, S.; Celio, M.R. Temporal
2000, 11, 10631067. and spatial appearance of the membrane cytoskeleton and
153. Celio, M.R.; Spreaco, R.; De Biasi, S.; Vitellaro- perineuronal nets in the rat neocortex. Neurosci. Lett.
Zuccarello, L. Perineuronal nets: past and present. Trends 1996, 209, 173176.
Neurosci. 1998, 21, 510515. 170. Arimatsu, Y.; Naegele, J.R.; Barnstable, C.J. Molecular
154. Peters, A.; Palay, S.L.; Webster, H.D. The Fine Structure markers of neuronal subpopulations in layers 4, 5, and 6
of the Nervous System: The Neurons and Supporting Cells; of cat primary visual cortex. J. Neurosci. 1987, 7, 1250
W.B. Saunders: Philadelphia, 1976. 1263.
155. Castejon, H.V. Histochemical demonstration of sulphated 171. Hockeld, S. Molecular dierences among neurons reveal
polysaccharides at the coat of nerve cells in the mouse an organization of human visual cortex. Neuroscience
central nervous system. Acta Histochem. 1970, 38, 5564. 1990, 34, 391401.
156. Celio, M.R. Evolution of the concept of extracellular 172. Sano, S.; Kudo, J.; Fujita, S.C. Dierent subsets of CNS
matrix in the brain. J. Hist. Neurosci. 1999, 8, 186190. neurons express dierent glycosaminoglycan epitopes on
large perineuronal proteoglycans. Brain Res. 1993, 630, acquisition of mature neuronal properties in the mamma-
6574. lian brain. Cold Spring Harbor Symp. Quant. Biol. 1990,
173. Zaremba, S.; Naegele, J.R.; Barnstable, C.J.; Hockeld, S. 55, 505514.
Neuronal subsets express multiple high-molecular-weight 180. Hockeld, S.; Zaremba, S. Characterization of an activity-
cell-surface glycoconjugates dened by monoclonal anti- dependent, neuronal surface proteoglycan identied with
bodies Cat-301 and VC1.1. J. Neurosci. 1990, 10, 2985 monoclonal antibody Cat-301. Vis. Neurosci. 1989, 3,
2995. 433443.
174. Bignami, A.; Perides, G.; Rahemtulla, F. Versican, a 181. Kalb, R.G.; Hockeld, S. Molecular evidence for early
hyaluronate-binding proteoglycan of embryonal precarti- activity-dependent development of hamster motor neu-
laginous mesenchyma, is mainly expressed postnatally in rons. J. Neurosci. 1988, 8, 23502360.
rat brain. J. Neurosci. Res. 1993, 34, 97106. 182. Kalb, R.G.; Hockeld, S. Induction of a neuronal
175. Hagihara, K.; Miura, R.; Kosaki, R.; Berglund, E.; proteoglycan by the NMDA receptor in the developing
Ranscht, B.; Yamaguchi, Y. Immunohistochemical evi- spinal cord. Science 1990, 250, 294296.
dence for the brevicantenascin-R interaction: colocaliza- 183. Kalb, R.G.; Hockeld, S. Large diameter primary aerent
tion in perineuronal nets suggests a physiological role for input is required for expression of the Cat-301 proteogly-
the interaction in the adult rat brain. J. Comp. Neurol. can on the surface of motor neurons. Neuroscience 1990,
1999, 410, 256264. 34, 391401.
176. Hockeld, S.; Garren, H.; Van Essen, D.C. Monoclonal 184. Sur, M.; Frost, D.O.; Hockeld, S. Expression of a
antibody Cat-301 identies Y-cells in the dorsal lateral surface-associated antigen on Y-cells in the cat lateral
geniculate nucleus of the cat. Vis. Neurosci. 1990, 5, 67 geniculate nucleus is regulated by visual experience. J.
81. Neurosci. 1988, 8, 874882.
177. Hockeld, S.; Deyoe, E.A. Antibody labeling of func- 185. Kalb, R.G.; Hockeld, S. Electrical activity in the
tional subdivisions in visual cortex: Cat-301 immuno- neuromuscular unit can inuence the molecular develop-
reactivity in striate and extrastriate cortex of the macaque ment of motor neurons. Dev. Biol. 1994, 162, 539548.
monkey. J. Comp. Neurol. 1990, 301, 575584. 186. Pizzorusso, T.; Medini, P.; Berardi, N.; Chierzi, S.;
178. Guimaraes, A.; Zaremba, S.; Hockeld, S. Molecular and Fawcett, J.W.; Maei, L. Reactivation of ocular domi-
morphological changes in the cat lateral geniculate nance plasticity in the adult visual cortex. Science 2002,
nucleus and visual cortex induced by visual deprivation 298, 12481251.
are revealed by monoclonal antibodies Cat-304 and Cat- 187. Lander, C.; Kind, P.; Maleski, M.; Hockeld, S. A family
301. J. Neurosci. 1990, 10, 30143024. of activity-dependent neuronal cell-surface chondroitin
179. Hockeld, S.; Kalb, R.G.; Zaremba, S.; Fryer, H. sulfate proteoglycans in cat visual cortex. J. Neurosci.
Expression of neural proteoglycans correlates with the 1997, 17, 19281939.
32
Crystal Structures of Glycolipids
Yutaka Abe
Process Development Research Center, Lion Corporation, Tokyo, Japan
Kazuaki Harata
Biological Information Research Center, National Institute of Advanced Industrial Science and Technology,
Ibaraki, Japan
I. INTRODUCTION ods, and researchers in a wide variety of elds report about

this type of bonding. X-ray structure analysis is one of the
Glycolipids are very important functional materials for most powerful methods to prove the presence of hydrogen-
daily life and human activity. The glycolipids play roles as bonding and to characterize its strength based on atomic
the structural holder of membrane proteins suspended in coordinates.
bilayer or bicontinuous cubic phases, and as the key code In this chapter, we focus on the characterization of the
of the intercellular communication or immune system. glycolipid crystal structure especially for the sugar moieties
The denition of glycolipids could be expanded to amphi- and hydrogen-bonding.
philes with sugar moieties as those with hydrophilic heads.
These materials are commonly used for industry and are
necessary for human life. In the area of consumer prod-
ucts, glycolipids are used as main ingredients in detergents II. HISTORICAL VIEW AND GENERAL ASPECTS
for the kitchen and emulsiers of foods and cosmetics OF GLYCOLIPIDS
where their excellent surface activity are advantageously
utilized. In the area of biochemical research, glycolipids Glycolipids show various kinds of biological activity.
are key compounds for dissolving and crystallizing mem- Oligosaccharides in the glycolipids or glycoproteins have
brane proteins. a great variety of combinations with many types of mono-
It is quite natural that organs produce amphiphiles saccharides, and operate as a marker of the type of cell, e.g.,
with a combination of sugar and lipid moieties for hydro- the antigen of the blood type [1] or tumor cell [2]. Another
philic and hydrophobic components, respectively. It is an important function of the glycolipids is its role as a material
important assignment for researchers focusing on glyco- for constructing the bilayer [3]. However, in this article, the
lipids to clarify the functional mechanism of sugar moie- description related to these functions is abridged because
ties in its molecular assembly from the physicochemical details of these functions have already been reported in
viewpoint. many articles and overviews.
We have been focusing on the crystal structures of On the other hand, synthesized glycolipids have been
glycolipids to understand the role of sugar moieties in the studied for more than 100 years. The alkyl glycoside was
molecular assembly. First, crystal is a phase of the molec- rst reported by Fisher in 1893 [4]. Recently, this material
ular assembly, and it is similar to the molecular arrange- has been used as a commodity detergent [5]. The main syn-
ment of the liquid crystal in solution. Second, we can thesized glycolipids are shown in Fig. 1.
quantitatively clarify the functions and eects of the sugar There are various reports on the physicochemical
moieties by analyzing the detailed crystal structure from properties of glycolipids. In 1911, Fischer et al. [6] reported
the coordinate. The most important function of the sugar that there are two melting temperatures of the alkyl
moieties is the formation of hydrogen-bonding. Nowa- glycoside, which was the observation of the thermotropic
days, we can analyze hydrogen-bonding by many meth- liquid crystal identied by Noller in 1938 [7]. These phe-
743
744 Abe and Harata
Figure 1 Principle synthesized glycolipids.

Crystal Structures of Glycolipids 745
The physicochemical properties of alkyl glycosides

have been studied since the rst report related to micelles
by Shinoda et al. [11]. The phase behavior of glycolipids
was dierent from that of the other types of amphiphiles.
Glycolipids with disaccharide or oligosaccharide as sugar
Figure 2 Alcoholethoxylate. moieties in aqueous solution show micelle phase, but that
with a monosaccaride shows a turbid solution or a phase
separation [18,19]. It is considered that the hydrophilicity
nomena were recognized as the molecular assembly of of the monosaccharide is not enough to form a micelle. One
glycolipids in the pioneering era. of the characteristic properties of glycolipids is that there is
A glycolipid shows a liquid crystal characteristic no ionic charge on the head group or the surface of the
because an ordered structure is maintained by the hydro- molecular assembly. There are reports about the easy
gen-bonding between the sugar moieties in both the thermo- aggregation of solid particles with glycolipids [20,21].
tropic and aqueous environments [8,9]. Many glycolipids Measurement by a surface force apparatus showed an
showing liquid crystal behavior are known and have been attractive force between the layers of glycolipid head
described in the comprehensive work by Jerey [10]. groups in the LangumuirBloget lm on mica [21]. This
The formation of a micelle, a type of molecular as- result suggests an interaction between the surface and an
sembly in aqueous solution, was rst reported for alkyl ordered sugar moiety by hydrogen-bonding. It is an im-
glycosides by Shinoda et al. in 1961 [11]. The alkyl glycoside portant characteristic for glycolipids that there is an inter-
shows a hexagonal and lamellar liquid crystal in concen- action between the sugar moieties.
trated aqueous solution. These characteristics of the glyco- Recently, new applications of glycolipids have been
lipid phase behaviors are more stable at high temperature developed in the eld of life sciences. In 1979, a glycolipid
than any other types of nonionic amphiphilesalcohol- was reported as the solubilizer of a membrane protein
ethoxylate shown in Fig. 2, which has only a hydroxyl because of the surface activity to dissolve a protein and
group. This behavior shows the strong interaction of the low denaturizing ability, which is a necessity to purify the
hydrogen-bonding between the sugar moieties [12]. proteins [22].
Some glycolipids have macro structures. Fourhop and Recently, structural biology, which explains the func-
Helfrich [13] reported that N-alkylgluconamide, as shown tion and activity of an enzyme from the viewpoint of the
in Fig. 1, forms a gel phase with a helical and brous molecular structure of a protein, has been playing an
structure observed by electron microscopy. It is considered increasingly important role in life science. X-ray structure
that this superstructure is caused by strong hydrogen- analysis is the key method in this strategy. Proteins are
bonding between the hydrogen atoms in the amide group categorized as membrane proteins and soluble proteins,
and oxygen atoms in the hydroxyl group of the sugar and the former accounts for 30% of all proteins and has a
moiety [14]. This helical and brous superstructure is only very important function in cells. Some structures of the
observed in chiral, and not in racemic molecule. membrane proteins were successfully solved by X-ray and
Another type of glycolipid that causes the helical their functional mechanism was claried, but obtaining an
brous superstructure, is N,NV-bis-(h-D-glucopyranosyl)al- adequately sized single crystal is still the greatest obstacle in
kandicarboxyamide, called a bolamphiphile (Fig. 1), and this strategy. There are few examples of the successful
reported by Shimizu [15]. The formation of this super- structural solution of the wild-type membrane proteins
structure is limited to the even number of the carbon atoms
in the acyl chain. The odd-numbered compound forms
plate crystals instead of the helical structure. Thus, these
glycolipids show characteristic superstructures in aqueous
condition.
There is a systematic research about glycolipids with
double alkyl chains, i.e., 1,3-di-O-dodecyl-2-O-glycosylgly-
cerols, as shown in Fig. 1. [16,17] Two of the three hydroxyl
groups in glycerin were modied with long alkyl chain
ether, and another was glycosidated with various degrees of
a sugar moiety. The melting points were dierent between
the derivatives of the maltooligosaccharides and cello-
oligosaccharides. The melting point of the former is de-
creased with the number of saccharides, but that of the
latter is increased. Because the chain gures by conforma-
tion analysis with molecular mechanics show helical and
extended conformations for the maltooligosaccharides and
cellooligosaccharides, respectively, the bulky sugar moiety
of the former causes a lower melting point than the Figure 3 Schematic description of the complex state of
compact moieties of the latter. membrane proteins and amphiphiles in a crystal.
746 Abe and Harata
Figure 5 Atomic geometry of hydrogen-bonding.
There are interesting results about the location of

glycolipid molecules in the crystal of a protein. Dissolved
membrane protein should be surrounded by the glycolipid
adjacent to the hydrophobic part of the protein like a
doughnut [27], and it should be crystallized by keeping
the complex schematically shown in Fig. 3. The locations of
the amphiphile molecules using the above hypothesis are
Figure 4 Solubility curve and Krat point of an amphi- claried by neutron diraction [2830]. In these results,
phile. there is a fused area between the doughnuts of the glyco-
lipid assembly with protein molecules. The same experi-
ment was carried out with dimethylamineoxyde, but the
fused area was not observed.
except for the mutants, and two of the structures have It is dicult to crystallize a membrane protein, but it
become the subject of Nobel prize-winning studies [23,24]. should be promoted by research progress in glycolipids.
Commercially available crystal screening kits show good hydrogen-bonding of sugar moieties in glycolipids and its
results for the crystallization of soluble proteins, but that of physicochemical properties will constitute a very important
the membrane proteins is still quite problematic. subject for the crystallization of a protein.
In the 1980s, the crystallization and x-ray analysis of a
membrane protein was successfully realized by Michel et al.
III. STRUCTURAL ANALYSIS OF GLYCOLIPID
[23] for subunits in the photosynthetic reaction center of
Rhodospeudomonas viridis, by using a surfactantdodecyl- CRYSTALS
amineoxide. Nowadays, the selection of the surfactant is A. Why Are the Crystal Structures
one of the most important techniques for the successful of Glycolipids Analyzed?
crystallization of the membrane protein. There are success-
ful examples of the dicult crystallization of the mem- As mentioned above, a glycolipid crystal is one of the
brane protein. phases of the molecular assembly itself. Is hydrogen-bond-
Bovine heart cytochrome c oxidase was successfully ing the essential force of interaction between the sugar
crystallized, and the crystal structure was solved by x-ray moieties and the formation of the assembly? This is an
[25]. Formation of the crystal was related to the structure of important starting point when we investigate the various
the sugar moieties and length of the alkyl chain [26]. This states of a glycolipid.
should be caused by the assembled structure of the protein The crystal phase is not only the state of the materials,
and glycolipid molecules. but also of the molecular assembly which can be analyzed
Table 1 Type and Strength of Hydrogen Bondings [39]
Property Very strong bonds Normal or weak bonds

Type of bonds FUUH: : : F XUUH: : : A
OUUH: : :O where A is an electronegative atom
O+UUH: : : O
Bond lengths Narrow range Broad range
H: : : A 1.2f1.5 A H: : : A 1.5f3.0 A
H: : : AcHUUX H: : : A > HUUX
Bond angles Strongly directional Weakly directional
HUUX: : :Ac180j HUUX: : : A c 160j F 20j
Bond energy >40 kJ/mol <20 kJ/mol
Reproduced by permission of Springer Verlagk.
Figure 6 Hydrogen-bonding linkage of the hydroxyl group.
as atomic coordinates that provide information about

molecules locations and is determined by the hydrogen-
bonding geometry. In addition, since most of the crystals of
amphiphiles show a lamella structure, which is similar to
the molecular assembly of thermotropic and lyotropic
liquid crystals, the crystal structures provide important
information for the investigation of the interaction and
packing state of the molecule. There is a report about
phospholipids which suggests a correlation between the
crystal structure and conformation of the molecule in
aqueous solution [31].
One of the utilities of the crystal structures of glyco-
lipids from a practical viewpoint is clarifying the mecha-
nism of the Krat point of surfactants. The Krat point,
shown in Fig. 4, is the critical point for a coexisting
monomer, micelle, and solid phase in an aqueous solution
of the surfactant. The solubility is indicated by this tem-
perature parameter. A low Krat point denotes high
solubility of the surfactant [32]. The solubility is a very
important subject for the practical use of surfactants
as light surface activity is observed under the Krat point
temperature.
Dissolution in water is a very important property in
the actual use of a glycolipid as a surfactant. There has been
a solubility problem of the glycolipid developed for a
surfactant. Alcoholethoxylates, shown in Fig. 2 as one of
Table 2 Energy in Sequential Hydrogen

Bonding of Water Cluster by Ab Initio Figure 7 Crystal structure of glucocylphytosphingosine
Calculation [41,42] reproduced from the coordinates of Ref. 73, with permission
from Elsevierk.
Bonding energy per
hydrogen-bonding [kJ/mol]
the nonionic surfactants, dissolve at 0jC, in contrast with
(H2O)n Cyclic Linear chain the low solubility of most of the glycolipids. A few glyco-
lipids dissolve at room temperature, but change to dicult
n=3 23.5 20.4 dissolution with slight modication of the surfactant struc-
n=4 39.7 25.7
ture. Octyl h-D-glucopyranoside [33], decyl, dodecyl h-D-
n=5 44.5 34.6
maltoside [34], and octanoyl-N-methylglucamide [35] are
n=6 45.0
dissolved in the commercial glycolipids, but octyl a-D-
748 Abe and Harata
tants, in spite of the long discussion about the association

between the Krat point and micellization or melting
temperature. There was no theory as to how the molecular
structure and the crystal structure and its physicochemical
properties are aected.
The crystal structures of glycolipids provide important
information when designing a surfactant molecule, because
the assumption of the molecular potential energy based on
the structural coordinate analysis claries the eect of the
hydrophilic and hydrophobic moieties on the static stabil-
ity of a crystal.
We next describe the crystal structures of glycolipids
especially regarding the function of the sugar moiety and
hydrogen-bonding.
Figure 8 Crystal structures of decyl a-D-glucopyranoside

reproduced from the coordinates of the structure of Ref. 74,
with permission from American Chemical Society.
glucopyranoside [36], dodecyl h-D-cellobioside [37], and

N-octylgluconamide [38] are dicult to dissolve and are
actually not usable as surfactants.
The solubility of a surfactant often causes trouble
during practical use, so the development of a surfactant
or products containing these materials can be a problem. In
these cases, there is an empirical hypothesis that a branched
alkyl chain reduces the Krat point because of the disor-
dering of the crystal structure. However, the structures of
the head groups have a signicant eect on changing the
Krat point, which will be described later. Figure 9 Complex crystal structure of octyl a-D-glucopy-
There was no clear description about the relation ranoside and octyl a-D-glucopyranoside reproduced with the
between the Krat points and crystal structures of surfac- coordinate of Ref. 80, with permission from Elsevierk.
hydroxyl group with a hydrogen atom, respectively, the

hydroxyl group possesses both functions of donor and
acceptor. This means that the hydroxyl groups have the
ability to form hydrogen-bonding linkages.
Typical hydrogen-bonding linkages are schematically
shown in Fig. 6. The bonding enthalpies of clusters of water
estimated by molecular orbital calculations of Pople listed
in Table 2 show that a longer hydrogen-bonding linkage
has a larger bonding energy [40,41]. It is considered that
continuous hydrogen-bonding linkages have stronger hy-
drogen-bonding than that of isolated bonding, because of
the increasing electrolytic polarization between the hydro-
gen and oxygen atoms in each hydroxyl group.
Figure 10 Crystal structure of gluconamide reproduced from

the coordinates of Ref. 38., with permission from Elsevierk.
B. Crystal Structures of Sugar and Hydrogen

Bonding
The crystal structures of a sugar have been analyzed for a
long time. The characteristic point of the crystal structures
is the hydrogen-bonding. There are various forces of
general hydrogen-bonding depending on the types of
donors and acceptors, which are listed on Table 1 [39].
The energy of hydrogen-bonding between the hy-
droxyl groups of a sugar is usually 20 kJ/mol, which is the
medium range for various types of hydrogen-bonding. The
shorter hydrogen-bonding has a stronger bonding energy.
The average geometry of the hydrogen-bonding between
hydroxyl groups in sugar molecules is shown in Fig. 5 [39]. Figure 11 Crystal structure of a bolaamphiphile of N-
Because the acceptor and donor of the hydrogen- alkyldigluconamide reproduced from the coordinates of Ref.
bonding are an oxygen atom in the hydroxyl group and a 85., with permission from Elsevierk.
750 Abe and Harata
Identication of the hydrogen-bondings in a crystal

structure is based on the atomic geometry, distance be-
tween the hydrogen atom in the donor hydroxyl group and
oxygen atom in the acceptor, and the hydrogen-bonding
angle between the hydrogen-bonding and the bonding
between the hydrogen and oxygen atoms in the donor
hydroxyl group. The accuracy of the position of the
hydrogen atoms determined by x-ray analysis depends on
the quality of the crystal and the intensity data of x-ray
reections because of the low atomic scattering factor of
hydrogen atom, but it is actually obtained by using a good
single crystal and reections in recent analyses. The bond-
ing length between these hydrogen and oxygen atoms in a
hydroxyl group is shorter than the actual distance between
these atomic nuclei, because the atomic position is deter-
mined as a maximum point of electron density, and that
point is shifted in the direction of the oxygen atomic
nucleus. The actual positions of the hydrogen or deuterium
atoms are determined by neutron scattering, which is not
the usual method used except for special cases because of
the necessity of nuclear reactors with diractometer for
single crystals and those machine times.
Figure 12 Categories of crystal structures of amphiphiles. Re- In the x-ray analysis, the hydrogen atom was deter-
produced by permission of the Royal Society of Chemistry [62]. mined at the distance 0.97 A from the oxygen atom, based
on the value of the neutron diraction results by taking into
account the angles between the hydrogen-bonding and
The cyclic linkage of hydrogen-bonding shows a bonding between the hydrogen and oxygen atoms in the
stronger bond than that of liner. In actual sugar crystals, hydroxyl group. The hydrogen-bondings are determined
the hydrogen-bonding energy, estimated by the molecular whether there is an oxygen atom for the acceptor that is
orbital calculations applying the coordinate of atoms in located in adequate geometry from the atomic position of
the cyclic hydrogen-bonding linkage in cyclodextrin crys- the hydrogen donor. Most of the hydrogen-bonding dis-
tal, accounted for the strong bonding and stable crystal- tance between the hydrogen and oxygen atoms are 1.82.0
linity [42]. A approximately, and it was reported that the hydrogen-
Table 3 Conditions of Single Crystal Formation for Glycolipids

Compounds Solvents Methods Ref.
Alkyl glycosides
Hexyl a-D-glycopyranoside Ethyl acetate [77]
Heptyl a-D-glycopyranoside monohydrate Water [77]
Octyl a-D-glycopyranoside Ethyl acetate zeolite KA [76]
Octyl a-D-glycopyranoside hemihydrate Methanol water [75]
Octyl a-D-glycopyranoside monohydrate Methanol water 2-propanol [75]
Decyl a-D-glycopyranoside Anhydrous methanol [74]
Glyconamides
N-(1-Octyl)-D-taloamide Water Slow cooling [84]
N-(n-Heptyl)-D-gluconamide Water [81]
N-(n-Octyl)-D-gluconamide Water [38]
N-(n-Decyl)-D-gluconamide Water [38]
N-(n-Dodecyl)-D-gluconamide Water [82]
N-(n-Octyl)-6-deoxy-D-gluconamide Ethyl acetate Slow evaporation [86]
N-Alkanoyl-N-glycamide
Nonanoyl-N-methylglucamide Acetone/methanol = 1/1 [87]
Bolaamphiphiles
N,NV-Bis(h-D-glucopyranosyl) Water Slow cooling and [88,89]
undecan -1,11-dicarboxyamide evaporation
Glycosylsphingolipid
Celebroside Ethanol Slow evaporation [73]
Table 4 Crystallographic Data for Crystals of Glycolipids
Cell constants
Molecular Space
Compounds weight group a [A] b [A] c [A] a [j] h [j] g [j] Z
Alkyl glycosides
Hexyl a-D-glycopyranoside [77] 264.32 P21 5.115(2) 7.589(2) 17.795(4) 96.11(3) 2
Heptyl a-D-glycopyranoside 296.36 C2 18.304(4) 4.971(2) 19.620(4) 92.18(2) 4
monohydrate [77]
Octyl a-D-glycopyranoside [76] 292.37 P21 5.140(2) 7.604(2) 19.939(4) 92.18(2) 2
Octyl a-D-glycopyranoside C2 15.190(5) 5.136(3) 19.944(7) 92.74(3) 4
Hemihydrate [75]
Octyl a-D-glycopyranoside C2 17.896(2) 5.154(1) 18.303(2) 90.30(1) 4
Monohydrate [75]
Decyl a-D-glycopyranoside [74] 320.4 P21 5.153(2) 7.626(4) 22.125(7) 90.95(4) 2
Glyconamides
N-(1-Octyl)-D-taloamide [84] P212121 9.140(2) 5.814(1) 62.57(1) 8
N-(n-Heptyl)-D-gluconamide [81] 293 P1 5.183(7) 16.18(2) 4.803(6) 96.1(1) 94.2(1) 99.0(1) 1
N-(n-Octyl)-D-gluconamide [38] 307 P21 5.252(1) 32.426(9) 4.805(1) 94.96(5) 2
N-(n-Decyl)-D-gluconamide [38] 335 P21 5.254(4) 35.97(1) 4.807(2) 94.81(3) 2
N-(n-Undecyl)-D-gluconamide [38] 362 P1 5.227(1) 19.628(9) 4.781(4) 93.23(2) 1
N-(n-Dodecyl)-D-gluconamide [38] 362 P21 5.31(2) 39.85(4) 4.82(2) 95.0(1) 2
N-(n-Octyl)-6-deoxy 291 P212121 5.452(1) 16.662(3) 36.897(5) 8
-D-gluconamide [86]
N-Alkanoyl-N-glycamide
Nonanoyl-N-methylglucamide [87] 335 P1 4.985(2) 5.603(2) 17.449(7) 85.4(3) 86.0(3) 76.2(3) 1
Bolaamphiphiles
N,NV-Bis(h-D-glucopyranosyl) 556.30 P21 6.220(1) 48.25(1) 4.922(2) 105.67(2) 2
undecan-1,11-dicarboxyamide [88,89]
Glycosylsphingolipid
Celebroside [73] 769.17 P21 11.202(5) 9.262(7) 46.46(3) 99.00(5) 4
bonding force is eective within the distance of about 3.0 between the hydrogen and oxygen atoms. Shorter hydro-
A [39]. gen-bonding distance means a strong bond.
Various crystal structures of sugars using X-ray and
neutron scattering have been reported, and these studies C. Crystal Structures of Lipids
were summarized in the general remarks by Jerey and
Sundaralingam [4349]. In their reports, the various hy- The rst report of x-ray powder diraction analysis of a
drogen bondings in sugar crystals are arranged and cate- lipid crystal was about the order of the saturated aliphatic
gorized by the strength and type of linkage. They reported carbonic acids, which have 1022 carbon atoms in acyl
that the strength of the hydrogen-bonding increased with chains, by Muller in 1923 [50]. The three-dimensional
the degree of linkage, because a longer bonding linkage structures with a single crystal of aliphatic acids and their
shows an average shorter hydrogen-bonding distance salts were reported for the potassium caprirate [51] and
Table 5 The cmc, 1% Krat Points, and Melting Temperatures of the Alkanoylglycoside [97]
Number of carbon cmc 1% Krat Points Melting temperature
Compounds atoms in an acyl chain [mM] [jC] [jC]
MeaGlc 10 1 27 70
MehGlc 10 1 40 8889
MeaGal 10 >80 136137
MeaMan 10 0.9 <0 a
a
Syrup.
752 Abe and Harata
The crystal structures of phospholipids, which are

the structural material of biomembranes, were systemati-
cally analyzed by Hitchcock et al. [57] and Pascher et al.
[58,59]. Those crystals show a bilayer structure like mem-
branes of cells or organs, and the conformation of the
hydrophilic moieties have a similar state as those in an
aqueous environment.
There are a few examples of the crystal structures of
anionic surfactants. Sodium dodecyl sulfate [60,61] and
aliphatic acid methyl ester sulfonate salts [62] used for
detergents were reported. These crystal structures of
Figure 13 Synthesis scheme of acylglycosides. amphiphiles have a lamellar structures with repeated layers
of molecules, which are divided to regions of hydrophobic
and hydrophilic layers.
For cationic surfactants, the crystal structures of an
lauric acid [52] by Vand et al. in 1949 and 1951, respective- alkyl ammonium salt, n-dodecylammoniumbromide by
ly. He categorized the packing of the alkyl chains as the Launden [63], and a dialkyl chains ammonium salt, di-
symmetrical type for various crystal structures of alkenes, octadecyldimethylammoniumbromide by Okuyama et al.
aliphatic alcohols, and aliphatic acids with long alkyl [64], were reported. Toda et al. [65] and Okuyama et al. [66
chains [53,54]. The crystals of the aliphatic acids with long 68] reported that alkyl ammonium salts and some aromatic
chains are known to have polymorphism such that those compounds form complex crystal structures. This is an
crystal systems transform into other systems at a critical important phenomenon to consider in the viewpoint of
temperature [55]. Goto [56] explained the stable state of the molecular interaction such that tetradecyl ammonium salts
crystal system in the polymorphism by the enthalpy value with a benzoic acid-like complex show a wormlike micelle
of the crystallize energy estimated by molecular mechanics. in aqueous solution [69].
Table 6 The Crystallization Condition of Methyl 6-O-Alkanoylglycosides [9094]

Concentration Temperature Crystal
Chain lengtha Solvents Ratio [wt.%] [jC] Methods description
10 Ether r.t. n.e. Plate
10 Ether r.t n.e. Plate
2 Acetone 10 4 Cooling hexagonal Plate
3 Acetone r.t n.e. Plate
4 Acetone/ether 1/3 r.t n.e. Plate
5 Syrup
7 Acetone/ether 1/9 5 4 Cooling Plate
10 Acetone/ether 5/95 r.t. n.e. Plate
12 Acetone r.t. n.e. Plate
14 Acetone/methanol 1/1 r.t. n.e. Plate
3 methanol 5 r.t. Cooling Plate
5 Methanol 10 r.t. Cooling Plate
6 Acetone/Methanol 4/1 r.t. n.e. Plate
8 Methanol r.t. n.e. Plate
a
No. of carbon atoms in acyl chain.
Ether: diethylether.
n.e.: natural evaporation.
Table 7 Crystallographic Data for the Methyl Alkanoylglycosides [9094]
Compounds MeaGlc MehGlc
Carbon number in acyl chains 10 12 3 4 6 7 8
Molecular formula C17H32O7 C19H36O7 C10H18O7 C11H24O7 C13H24O7 C14H26O7 C15H28O7
Molecular weight 348.4 376.5 250.2 264.3 292.3 306.4 320.4
Crystal system monoclinic monoclinic monoclinic monoclinic monoclinic monoclinic monoclinic
Space group P21 P21 P21 P21 P21 P21 P21
Z 2 2 2 2 2 2 2
a [A] 4.967(1) 4.968(1) 7.932(1) 7.842(1) 7.793(1) 7.906(2) 7.760(1)
b [A] 7.513(1) 7.503(1) 7.100(1) 7.389(1) 7.363(1) 7.459(1) 7.373(1)
c [A] 25.917(2) 28.443(1) 10.984(1) 11.341(5) 13.534(5) 14.218(3) 15.514(1)
Crystal Structures of Glycolipids
b [j] 92.92(1) 92.68(1) 101.106(1) 93.21(1) 99.99(2) 102.627(1) 102.91(1)

Cell volume [A3] 965.9(1) 1059.0(1) 607.1(1) 656.1(1) 764.8(3) 818.2(1) 865.2(1)
Dc 1.198 1.181 1.369 1.338 1.269 1.244 1.230
Molecule MehGlc MeaGal

Carbon number in acyl chains 9 10 11 12 13 2 3 4
Molecular formula C16H30O7 C17H32O7 C18H34O7 C19H36O7 C21H40O7 C9H20O7 C10H18O7 C11H20O7
Molecular weight 334.4 348.4 362.5 376.5 404.5 240.3 250.2 264.3
Crystal size 0.3 0.2 0.1 0.4 0.40.1 0.3 0.4 0.1 0.4 0.3 0.1 0.3 0.4 0.1 0.3 0.3 0.05 0.5 0.5 0.1 0.3 0.4 0.05
Crystal system monoclinic monoclinic monoclinic monoclinic monoclinic monoclinic monoclinic monoclinic
Space group P21 P21 P21 P21 P21 P21 P21 P21
Z 2 2 2 2 2 2 2 2
a [A] 7.922(1) 7.724(1) 7.923(5) 7.729(1) 7.713(1) 5.752(2) 5.767(1) 5.772(1)
b [A] 7.449(1) 7.351(1) 7.437(3) 7.346(1) 7.346(1) 8.082(2) 7.971(1) 8.038(1)
c [A] 15.815(1) 16.957(2) 17.217(1) 18.626(2) 20.473(1) 12.561(4) 13.462(1) 14.686(1)
b [j] 100.010(1) 94.81(1) 92.626(1) 91.51(1) 96.76(1) 94.72(1) 91.473(1) 94.32(1)
Cell volume [A3] 919.0(1) 959.4(2) 1013.5(1) 1056.2(1) 1151.9(1) 582.0(3) 618.6(1) 679.4(2)
Dc 1.208 1.206 1.188 1.184 1.166 1.348 1.343 1.292
Molecule MeaGal
Carbon number in acyl chains 5 6 7 8 9 10 11 12
Molecular formula C12H22O7 C13H24O7 C14H26O7 C15H28O7 C16H30O7 C17H32O7 C18H34O7 C19H36O7
Molecular weight 278.3 292.3 306.4 320.4 334.4 348.4 362.5 376.5
Crystal size 0.4 0.4 0.04 0.3 0.3 0.05 0.3 0.3 0.02 0.5 0.5 0.1 0.3 0.3 0.03 0.5 0.4 0.1 0.3 0.2 0.03 0.4 0.3 0.05
Crystal system monoclinic monoclinic monoclinic monoclinic monoclinic monoclinic monoclinic monoclinic
Space group P21 P21 P21 P21 P21 P21 P21 P21
Z 2 2 2 2 2 2 2 2
a [A] 5.770(1) 5.784(1) 5.775(1) 5.774(1) 5.768(1) 5.762(1) 5.778(1) 5.760(1)
b [A] 7.973(1) 8.024(1) 7.946(1) 8.013(1) 7.903(1) 8.003(1) 7.903(1) 7.986(1)
c [A] 15.451(1) 16.792(1) 17.883(1) 19.183(1) 20.113(1) 21.227(2) 22.326(1) 23.339(1)
b [j] 93.690(1) 95.40(1) 97.146(1) 98.50(1) 96.633(2) 93.93(1) 92.830(1) 90.21(1)
Cell volume [A3] 709.3(1) 775.8(1) 814.3(1) 877.8(1) 910.6(4) 976.6(1) 1018.3(1) 1073.6(1)
Dc 1.303 1.251 1.249 1.212 1.220 1.185 1.182 1.165
753
754 Abe and Harata
Figure 14 Single crystals of dodecanoyl MeaGal.
A serious problem in the x-ray structure analysis of

amphiphiles is the diculty in obtaining an adequate single
crystal size. The necessary crystal size is 0.20.3 mm for
each axis for the usual x-ray measurements using an in-
house x-ray generator. If synchrotron radiation is used, the
sizes of the crystals are smaller than those by conventional
measurement of the normal 3- to 18-kW x-ray generator.
The formation of adequate crystal size depends on the
crystallinity of the amphiphiles and the crystallization
conditions. The crystallization conditions include various
combinations of organic solvents, water, temperature,
and concentration.
Good eort and patience is needed, but successful
crystallization is possible. Water addition is eective for
crystallizing amphiphiles because it often produces a hy-
drate crystal. Slow cooling of a saturated solution is also
available. Recrystallizing solvents for surfactant synthesis Figure 15 Melting temperatures of MehGlc and MeaGal [94].
and purication are eective to obtain single crystals Reproduced by permission of the Royal Society of Chemistry.
because these are selected to grow crystals for eective
purication and ltration.
After the crystallization condition is determined, the D. Examples of the Crystal Structure of
successful solving of the structure to locate the hydrogen- Glycolipids
bonding depends on the quality of the crystal and good
diraction. High-quality diractions enable easy determi- As a natural biomaterial, the crystal structure of a sphingo-
nation of the structure. glycolipid, glucosylphytosphingosine, shown in Fig. 7, was
The strategy for structure determination and rene- reported by Abrahamsson and Pascher [73]. The crystal
ment is based on the packaged softwares [70,71]. The structure of this compound has a simple parallel bilayer
actual method to solve the crystal structure can be found structure without interdigitated alkyl chain, because of its
in some references [72]. double alkyl chainlike phospholipids. The hydrogen-
Figure 16 (a) Crystal structures of decanoyl MeaGlc reproduced from the coordinates of Ref. 92. (b) Crystal structures of
decanoyl MeaGal reproduced from the coordinates of Ref. 91. (c) Crystal structures of decanoyl MehGlc reproduced from the
coordinates of Ref. 90.
756 Abe and Harata
bonding network is observed between its hydroxyl groups and the crystal structures are caused by the layering of
and nitrogen atoms. There is no example of the crystal these bilayers.
structure of a natural glycolipid except for glucosylphytos- As other types of synthesized glycolipids, crystal
phingosine, because of the dicult crystallization of other structures of N-alkylgluconamide (Fig. 10) [38,8186]
natural glycolipids. and alkanoylglucamide [87] were solved. The object of
In 1976, a synthesized glycolipid, decyl a-D-gluco- the structure analysis was to clarify the mechanism of
pyranoside dehydrate, was crystallized and the structure liquid crystal formation or to compare the compounds
solved by Moews and Knox [74], which is shown in Fig. 8. that form either helix superstructures or plate crystal.
The crystal structure of heptyl and octyl a-D-glycopyrano- These crystal structures are characterized by the types of
side, which has a shorter alkyl chain than decyl a-D- sugar moieties. These glycoamide-type lipids show a
glucopyranoside, was reported for the anhydride, hemi- simple bilayer or monolayer. There is a dierence in
hydrate, and monohydrate crystals [7577]. Details of the packing between the monolayer- and bilayer-type crystals
hydrogen-bondings were discussed regarding their crystal caused by the hydrogen bond bridging over the layer for
structures. The hydrogen-bonding linkage and the pack- the former and only xed in a layer for the latter,
ing of sugar moieties are maintained in the dehydrate respectively. Most of the glycoamide-type compounds
crystals with two alkyl chain length, but there is a slightly
dierent hydrogen-bonding distance between the two
crystals. The crystal structures of similar compounds,
octyl thiomanoside [78] and octyl thioxyloside [79], were
also reported.
These crystals have a bilayer structure with an inter-
digitated alkyl chain. The alkyl chains in the glycolipids
crystal form an interdigitated structure to ll the space
caused by the bulky sugar moiety, in which the occupied
area per molecule in the layer is two times that of the
alkyl chain.
There is still no report for the crystal structure of octyl
h-D-glucopyranoside because of the diculty in producing
an adequate single crystal, but the structure of the complex
crystal of octyl a- and h-D-glucopyranosides was solved
(Fig. 9) [80]. Each monolayer in the bilayer of the crystal Figure 17 Occupied area of the sugar moieties and acyl
consists of a-anomers and h-anomers, respectively, chains in the crystals of methyl 6-O-alkanoylglycosides [94].
do not show an interdigitated crystal structure with an higher value of cmc Krat point. The values of cmc have a
alkyl chain because of the linear sugar moieties, but not slight dierence between the three derivatives, in contrast
with a bulky pyranose ring. with the large dierence in Krat point between those
The crystal structures of the bolaamphiphiles with derivatives. This means that the monomer dispersion of
sugar moieties at both ends of the hydrophobic chain were the glycolipids in water could not be related to the Krat
reported for the glycolic amide by Muller-Fahrnow et al. points. The Krat points are highest for the MeaGal and
[85], and for the glucosamide by Masuda and Shimizu lowest for MeaGlc, respectively. The methyl 6-O-dec-
[88,89], shown in Fig. 1. These crystals form a monolayer anoylglycosides reported by Gama and Kawaguchi [98]
structure. The crystal structure of glycolic amide is shown shows dierent melting temperatures between the dierent
in Fig. 11. types of sugar moieties, and the order of the melting
The crystal structures of the glycolipids consist of a temperature is the same as that of 1% Krat points. Thus,
monolayer or bilayer structure with a hydrophilic and this order of Krat points is related with the melting
hydrophobic layer like those of the other amphiphiles. temperature. The dierence in the Krat points with the
The bilayer and monolayer structures of the crystals are types of sugar moieties suggests the dierence between
called head-to-head and head-to-tail structures, respective- these crystal structures. The dierence in the structures
ly. These structures are categorized and schematically between the sugar moieties is slight, at rst glance.
shown in Fig. 12 [62]. These alkanoylglycosides were synthesized by the
The hydrogen-bondings between the hydroxyl groups acylation of sugar derivatives with acylchloride, which
of the sugar moieties are identied in all of the reported shows a major yield for primary alcohol derivative, and
crystal structures. Above the melting temperature, a lamel- puried by column chromatography. Highly regioselective
lar-type liquid crystal is formed because the layer structure synthesis methods for alkanoyl glycosides using special
is retained by the hydrogen-bonding. leaving groups have been recently developed [98]. The
Most of the alkyl chains are extended in the crystals enzyme reaction is eective for regioselectivity [99]. The
and is consistent with Vands criteria [53,54]. alkanoylglycosides are synthesized with good yield by
The methods and conditions to produce single crys- lipase from Candida antarctica as a catalyst reported by
tals and the crystallographic data for the glycolipids whose Bjorkling shown in Fig. 13.
crystal structures have been reported are listed in Tables 3 The systematic analyses of the crystal structures for
and 4, respectively. the methyl alkanoylglycosides have been reported for
various derivatives, as shown in Fig. 1 [9094]. The struc-
E. The Crystal Structures of Alkanoyl Glycosides tural dierence between the sugar moieties is the location
of the hydroxyl groups. Methyl a-galactoside is an a-
There is a systematic approach to crystal structure analysis anomer of the methyl glycoside and has an axial hydroxyl
of a series of glycolipidsthe methyl 6-O-alkanoylglyco- group at the fourth-ring carbon atom and other equatorial
sides as shown in Fig. 1 having three types of sugar moieties hydroxyl groups. The methyl glucosides all have equatorial
and various lengths of alkyl chains [9094]. The physico- hydroxyl groups. The structures of these glucoside deriva-
chemical properties of these glycolipids were measured for tives were analyzed for each of the a- and h-anomers. These
surface activity by Tamura et al. [95], and for the phase derivatives are expressed with symbols in Fig. 1.
diagrams by Ohbu and Fujiwara [18]. The derivatives with The systematic analysis of methyl alkanoylglycosides
various types of sugar moieties were synthesized. Di- was accomplished by determining the crystallization con-
alkanoyl trehalose shows immunoactivity as a code factor dition. The crystallization solvent was selected from the
(Fig. 1) [96]. aliphatic alcohols, acetone, dimethyl ether, and mixture of
The alkanoylglycosides have dierent melting temper- these solvents, and there is a specic condition for each type
atures and Krat points for various types of sugar moieties. of the sugar moieties with lengths of acyl chains. The
The cmc, 1% Krat points, and melting temperatures of conditions of the producing single crystals and the crystal-
methyl 6-O-decanoylglycosides are listed in Table 5 [97]. lographic data are listed in Tables 6 and 7, respectively.
The 1% Krat point is the solving temperature of 1 wt.% The microscopic photograph of a single crystal is
surfactant in aqueous solution and usually shows slightly shown in Fig. 14.
Table 8 Occupied Area of the Sugar Moieties and Tilt Angle of the Acyl Chain [9094]
Occupied areas in crystal
Number of carbon atoms Sugar moiety Acyl chain Tilt angle of acyl chain
Derivatives in acyl chain S [A2] R [A2] / [deg]
MeaGlc even 37.3 18.6 1

MehGlc odd 58.959.0 18.519.1 5051
even 56.757.9 18.119.3 4750
MeaGal odd 45.646.0 19.119.4 3134
even 46.046.4 19.119.5 3233
758 Abe and Harata
Figure 18 Thickness of hydrophobic region in crystal is dierent between acetyl and dodecanoyl, and acetyl and tetradecanoyl
for MeaGal and MehGlc, respectively [94]. Reproduced by permission of the Royal Society of Chemistry.
F. Relation Between the Crystal Structures and ages are schematically shown in Fig. 21, and are aected by
Physicochemical Properties of Methyl the type of sugar moieties. A disordered structure was
Alkanoylglycosides observed in the sugar moieties of MeaGlc shown in Fig.
22, and the linkage of the hydrogen-bondings is much
The melting temperatures of these crystals are shown in shorter than those of the other types of sugar moieties.
Fig. 15. The melting temperature is dierent between these The hydrogen-bonding linkage of the MeaGals is innite,
types of sugar moieties, as follows; in contrast with the nite linkage in the crystal of MehGlcs.
The hydrogen-bonding linkage of the MeaGals does not
MeaGal > MehGlc > MeaGlc include ring oxygen atom in pyranoside, but that of
This shows the order of the crystal thermal stability. MehGlcs ended at the oxygen atom. These dierences
Derivatives with the longer alkyl chain of MeaGal between the gure of hydrogen-bondings aect the bond-
have a lower melting temperature, but MehGlcs has a lower ing energy, as will be elaborated later.
melting temperature with a longer alkyl chain in the short- The molecular structures in the crystals are shown in
length range and higher melting point with a longer alkyl Fig. 23.
chain of more than 8 carbon atoms per chain. The methyl What is the important factor in determining these
alkanoylglycosides with odd and even numbers of acyl gures of hydrogen-bonding linkages? The linkage of
chains show a systematic dierence in these melting tem- MehGlc originates from the molecular structure of the
peratures. MeaGals with an even number of acyl chains sugar moieties of methyl h-D-glucopyranoside hemihy-
show a higher melting temperature than those with an odd drate. The crystal structure of methyl h-D-glucopyranoside
number, whereas MehGlcs show the opposite tendency hemihydrate reported by Jerey shows the hydrogen-
those with an odd number of chains have higher melting bonding linkage, O(4) U H(O4) : : : O(3) U H(O3) : : :
temperature than those with even number of chains. O(2) U H(O2) : : : O(5), similar to those of MehGlc (Fig.
The basic gure of the crystal structures of the methyl 24). However, the crystal structure of MeaGal has hydro-
alkanoylglycosides are the same as those of the alkylglyco-
sides with pyranoside sugar moieties (Fig. 16). The
arrangements of the alkyl chain are dierent between the
derivatives of each type of sugar moiety. The tilt angles of
the alkyl chains in the crystal are related to the occupied
area of the sugar moieties in the hydrophilic layer (Fig. 17).
The relation between the cross-sectional area of an
alkyl chain, S, the tilt angle of the acyl chain, /, and the
occupied area of a sugar moiety in the layer, R, are shown in
Fig. 17 and expressed as the following formula
S cos / 2 R
The larger tilt angle of the alkyl chain is shown by the
larger occupied area of the sugar moieties because the cross
section of an alkyl chain is slightly changed. As listed in
Table 8, MehGlc shows a larger tilt angle of the alkyl chain
with larger occupied area for sugar moieties, in contrast
with the smaller tilt angle with smaller area for MeaGlc.
The packing gure of the derivatives of the same sugar
moieties with a variety of acyl chain lengths are not aected
by the acyl chain length (Fig. 18). The space group showing
the molecular symmetry in a crystal is the same as only P21,
which is one of the symmetric types of monoclinic crystal
for all of the derivatives of the methyl alkanoylglycosides.
The relative location of the sugar moieties and hydrogen-
bonding length is slightly changed by the acyl chain length,
but the hydrogen-bonding linkage are maintained, and
only the thickness of the hydrophobic layer is changed by
the length.
The packing gure for the sugar moiety of the
methyl alkanoylglycosides is highly maintained by the
acyl-chain changing.
The three-dimensional structure of hydrogen-bond-
ings in crystals of MehGlc and MeaGal are shown in Figure 19 Hydrogen bondings of MehGlc in the crystal
Figs. 19 and 20, respectively. The hydrogen-bonding link- [91]. Reproduced with permission of Elsevierk.
760 Abe and Harata
Figure 20 Hydrogen bondings of MeaGal in the crystal [90]. Reproduced with permission of Elsevierk.
gen-bonding linkages dierent from that of methyl a-D- molecule or moieties in a crystal with static ratio. Some
galactopyranoside (Fig. 24). Methyl a-D-glucopyranoside may think that the crystals have a strict molecular arrange-
has a hydrogen-bonding linkage involving 6-hydroxyl ment order, but those molecules are actually in thermal
group which cannot form hydrogen-bonding for MeaGlc vibration, uctuation, and are arranged in various con-
because of the modication with the acyl groups (Fig. 24). formations in a crystal. It takes from a few to 12 sec to
Acetyl MeaGlc cannot be crystallized and shows syrup. measure the x-ray diraction, and a few hours to a few days
This suggests an unstable hydrogen-bonding network be- to complete all measurements for a crystal. Thus, the crystal
tween the sugar moieties form the disordered structure for structure by x-ray means the average structure for the
MeaGlc in crystal. Thus MehGlc maintains the hydrogen- measuring time. Actually, it could not be clearly identied
bonding linkage of methyl h-D-glucopyranoside without whether the disorder structure revealed the dynamic chang-
acyl chain, in contrast with the MeaGlc and MeaGal ing or static equilibrium of the molecular conformation.
which have these dierent linkages caused by modied 6- The disordered structures of the glycolipids are observed in
hydroxyl group with acyl group. Few hydrogen-bonding both sugar moieties and alkyl chains, and are suggested to
linkages in the crystal of MeaGlc suggest that the stable occur because of the thermal stability of the crystal. During
hydrogen-bonding linkage is prevented by the 6-O-acyl the crystal structure analysis by x-ray, the peaks of electron
group and leads to the lowest thermal stability of those density map show duplicated molecules or moieties of
crystals of the three derivatives. those. This is the disorder structure observed two confor-
Disordered structures of glycolipid molecules in crys- mations in a molecule.
tal are often observed. Disorder in crystallography means Those two conformations of MeaGlc are shown in
that more than two conformations are observed in a Figs. 22 and 23. A similar type of disorder structure of
Figure 21 Schematic illustration of the hydrogen-bonding linkage in crystals of MehGlc and MeaGal.
Figure 22 Two conformations in the disorder structure of MeaGlc.

7
Figure 23 The molecular structures of the alkanoylglycosides in crystal.

Figure 24 (a) Schematic illustration of the hydrogen-bonding linkage in crystals of methyl h-D-glucopyranoside hemihydrate.
(b) Schematic illustration of the hydrogen-bonding linkage in crystals of methyl a-D-galactopyranoside monohydrate. (c)
Schematic illustration of the hydrogen-bonding linkage in crystals of methyl a-D-glucopyranoside.
764 Abe and Harata
Figure 25 Atomic thermal motion of MehGlc and MeaGal.

values of the thermal parameters for three axes of the

ellipsoid plotted for each atomic numbering, as shown in
Fig. 25. The sugar moieties show a lower thermal vibration
at atoms with rigid hydrogen-bondings than the acyl chains
adjacent to the van der Waals potential.
The hydrogen-bonding distances of the methyl alka-
noylglycosides related to the length of acyl chains and their
average distances of those derivatives are shown in Figs.
26 and 27, respectively. The average hydrogen-bonding
distance of MeaGals is shorter than those of MehGlcs. The
hydrogen-bonding of H(O2) to O5 of MehGlcs is longer
than O3 to H(O2) and O4 to H(O3) in Fig. 27. These
suggest that a hydrogen-bonding including ring oxygen is
weaker than that consisting only of hydroxyl groups. The
hypothesis that the bonding energy is increased by the
linkage of it is also adopted.
The static potential and LennardJones potential for
the crystal packing of molecules are calculated with
CHARMm [100]. The hydrogen-bonding energy is treated
as the static potential of the sugar moieties. The static
interaction between the sugar moieties of MeaGal is a
lower energy value than that of MehGlc (Fig. 28). The
increasing energy of the hydrogen-bonding formed by this
linkage could not be reproduced by the bonding parame-
ters in molecular mechanics, but it is reproduced by the
stronger bonding energy values including shorter hydro-
gen-bonding distances with a longer linkage.
In conclusion, the hydrogen-bonding force between
the sugar moieties of MeaGal was stronger than that of
MehGlc.
Figure 26 Hydrogen-bonding distances of MehGlc and The tightness of the packing of the acyl chain was
MeaGal in crystals [94]. Reproduced by permission of the estimated by the cross-sectional area, density, thermal
Royal Society of Chemistry. parameter, and packing energy with molecular mechanics.
The acyl chains of MeaGal have larger thermal vibrations
or disorder structure than that of MeaGlc and MehGlc,
because the thermal parameter of the chains in MeaGal
MeaGlc with two conformations of pyranoside rings in a have larger values than those in MeaGlc and MehGlc. The
molecule has been observed for the crystal structure of cross-sectional area of the acyl chain in MeaGal is larger
octyl 1-S-h-xylopyranoside [79]. These crystals have very
weak diractions equivalent to cell axes: as=5a, bs=2b,
cs=c for the crystal of MeaGlc and as=a, bs=2b, cs=2c
for that of octyl 1-S-h-xylopyranoside. These diractions
mean that the crystals have larger cell axes, which are called
superlattice, with more than two dierent molecular con-
formations in the crystal. But those diractions for super-
lattice are very weak and are dicult to measure, and all of
the conformations in the crystal are dicult to solve. These
superlattices suggest having a large regular structure but it
is not clear for the detail structures.
Information about the atomic thermal vibration is
obtained via an x-ray structure analysis. Atomic vibration
is expressed as a thermal ellipsoid whose shape is similar to
an American football, as shown in Fig. 23, which means the
domain of atomic probability is usually more than 50%.
The larger ellipsoid denotes a larger atomic vibration and
the longer axes of the ellipsoid mean a larger vector of
vibration. The molecular structures of the glycolipids show
a lower thermal vibration of the sugar moieties than that of
the alkyl chains based on the observation of the thermal Figure 27 Average hydrogen-bonding distances with each
ellipsoid in the gures. This was expressed as average type of linkage for MehGlc and MeaGal in crystals [94].
766 Abe and Harata
acyl chains aected by the terminal contact of the chain

induces secondary eect to change the packing of the
hydrophobic moieties. Gradual change of the chain length
caused the systematic change of the structures for each odd
and even number of chains. The molecular structures of
methyl alkanoylglycosides with an odd number of chain in
crystal are shown in Fig. 29. The arrangement of the acyl
chain shows obvious dierence between the odd and even
number of chains. The packing category of the acyl chain of
MeaGal is deferent between odd and even chains, and the
thermal parameters of the even number of acyl chain is
larger than those of odd chain. The odd number of acyl
chains in MehGlcs shows disorder structure with two
conformations of the chains, and the derivatives of the
odd-numbered chain have lower thermal stability of crystal
than those of even derivatives.
The acyl chains all have trans conformation for vari-
ous lengths of the chains. Those chain shapes may be
directly observed in the gures at a glance, but it is obvious
that these are bent using the following analysis. X and Y
axes are dened as the same as a and b axes of the cell
lattice, respectively. The Z axis is dened as the perpendic-
ular of the ab plane with direction to the positive space of Z
coordinate. The XV and ZV axes are dened with principal
component analysis for the middle points of the CUC
bonding in an acyl chain schematically shown in Fig. 30.
The middle points of the CUC bondings are plotted for
dened XY, YZ, and XVZV planes, respectively, as shown in
Fig. 30. Figures drawn for the acyl chains show obviously
the bending of the acyl chains. A longer chain shows more
winding and complex gure. The acyl chains are bent at the
point of the connected region between the sugar and the
acyl chain moieties, in contrast with less-bent chain near
terminal. Thus, these windings of the chains are caused by
the distortion between the packing domains of the sugar
moieties and the acyl chains in the crystals. The larger tilt
angles of the acyl chains of MehGlcs and MeaGals are
shown for shorter chains in Fig. 31. The curvatures of the
Figure 28 The potential energies of a molecule in the crys- acyl chains systematically and gradually changed with the
tals of MehGlc and MeaGal [94]. Reproduced by permission number of carbon atoms in the chains. The directions of the
of the Royal Society of Chemistry. curve of the chains on the ZVXV plane and the curvature of
those on XY and YZ planes of MeaGals are dierent
between the odd and even number of chains shown in
Fig. 31. MehGlcs have dierent curvatures and tilt angles
than those in MeaGlc and MehGlc. The LennardJones between the derivatives with odd and even numbers of
potentials of a methylene group, CH2, in the acyl chain chains. These are caused by the symmetric dierence
calculated by molecular mechanics as listed in Table 9, between the odd and even chains shown in Fig. 32.
show that the packing of the acyl chain of MeaGal is In addition to the eect to the acyl chains on each
less than those of MehGlc because the former has a other, the terminal methyl group in the acyl chain aects
higher value than the latter. These results show a tighter the packing of the sugar moieties. Reduced hydrogen-
packing of the acyl chain in MeaGlc and MehGlc than that bonding energy of the MehGlc with an odd number of
in MeaGal. chain was shown by the result from molecular mechanics
The eect on the crystal structure and the melting calculation because the sugar moiety is pushed by the
temperature of the dierence between the odd and even terminal methyl group and the hydrogen-bonding is
numbers of carbon atoms in the acyl chain is found to be stretched. The obvious odd and even eect of the acyl
the symmetrical position of the terminal methyl group of chains of MehGlc was initiated and brought to an end.
the chain, which cause the dierent contact environment No observation of the stretching of the hydrogen-
between the molecules. As a result, the arrangement of the bonding for MeaGal was explained by the rather strong
Table 9 LennardJones Potential of a Methylene Group in Acyl Chain [94]

LennardJones potential
Number of carbon of a methylene group in
Derivatives atoms in acyl chain acyl chain [kJ/mol]
MeaGal even 5.99

odd 6.38
MehGlc even 6.67
odd conformer A 7.24
odd conformer B 7.14
hydrogen-bonding by the innite linkage and weak contact crystal, while weaker hydrogen-bonding like MehGlc has a
by the terminal methyl group. lower crystal stability, and MeaGlc, which does not have
an eective hydrogen-bonding, produces a more unstable
G. The Relations Between the Crystal Structure crystal. The order of the Krat point for the same acyl
and Physicochemical Properties chain length is as follows:
The characteristic properties of the glycolipids, the melting

temperature, and the Krat point are an indicator of the MeaGal > MehGlc > MeaGlc
solubility, and are aected by the slight dierence in the
structure of the sugar moieties. The origin of these phe- The dissolving behavior of the surfactants in an aque-
nomena is the crystal structure. The strength of the hydro- ous solution shows the Krat point phenomena, as previ-
gen-bonding aects the thermal stability of the crystal, and ously mentioned. The Krat point is the critical point of
the rigid crystal with a stronger hydrogen-bonding shows a the three phasessolid, monomolecular dispersion, and
lower aqueous solubility and surface activity. micelle in an aqueous solutionand the value is shifted
Examples of the crystal structures of the alkanoylgly- with the stability of these phases. The Krat point is
cosides show that the crystal with stronger hydrogen- reduced and the amphiphile becomes easy to dissolve in
bondings, like MeaGal, forms a highly thermally stable the case of the highly stable molecular diusion in
Figure 29 Two conformers of MehGlc with an odd-numbered acyl chain in crystal [94].
768 Abe and Harata
Figure 30 Dened Cartesian coordinates for analysis of bending acyl chains.
aqueous solution, low cmc value, which means that it is of crystals, a thermotropic liquid crystal, and liquid state
easy to form a micelle, or the low stability of the crystal. [8]. A crystal structure in the polymorphism of just below
When describing the glycolipids, its Krat point is the melting temperature is necessary to relate the struc-
increased with a more stabilized crystal with stronger ture and the melting temperature. However, the critical
hydrogen-bonding. The diusive stability of a single mole- crystal state of octyl a-D-glucopyranoside has yet to be
cule in an aqueous solution increased with the increasing identied. Thus it is dicult to discuss the relation
hydrophilicity by increasing the number of hydroxyl between the structure and the melting temperature for
groups or reducing the length of the hydrophobic chain. these compounds.
Its Krat point is considered to be reduced with the Methyl alkanoylglycosides only have a single crystal
increasing number of hydroxyl groups and increased with state through the thermal transformation, and show a crys-
the longer alkyl chain. However, the actual Krat point of tal structure in an aqueous solution, same as that of a single
the glycolipids is often higher with more hydroxyl groups, crystal produced in an organic solvent. Thus the crystal
which induce strong hydrogen-bonding. structures of alkanoyl glycosides relate between the struc-
The explanations for the solubility of the amphiphiles tures and the melting temperature or the Krat points.
to increase suddenly above the Krat point are: (1) a The Krat point mechanism of the glycolipids with
crystalline amphiphile in aqueous solution suddenly melts hydrogen-bonding was claried by the systematic ap-
at this point, and (2) the formed micelles promote dissolu- proach of this study about alkanoylglycosides.
tion. The hypothesis suggests that the Krat point is the
melting temperature of amphiphiles in an aqueous envi-
ronment, and melted amphiphiles become micelles [101]. IV. CONCLUSION
This hypothesis suggests that an amphiphile with lower
crystal melting point in an aqueous environment has a Glycolipids show dierent crystal structures between types
lower Krat point. Laughlin reported [32] that this rela- of sugar moieties, which induce the characteristic physico-
tionship of the melting temperature and Krat point is chemical properties, aqueous solubility, or thermal stabil-
simply consistent because nonamphiphilic organic com- ity related to the crystal.
pounds which do not have a cmc were also observed to Hydrogen bonding is the most important factor for
suddenly dissolve above the melting temperature. The these phenomena. It is relevant not only for scientic
common view of both hypotheses is the stability of the research, but also for industrial applications in that the
crystalline phase in an aqueous environment. geometry of the sugar structure is the determining factor of
The Krat point of glycolipids obviously shows a the characteristics for the physicochemical properties of the
dierence between the amphiphiles with a slight structural glycolipids. The hydrogen-bondings between hydroxyl
modication. There was no detailed study for the complex groups in a sugar moiety should have an important role
Krat point mechanism of glycolipids. in an aqueous environment. Naturally, hydration of the
Details of the crystal structures and the physicochem- hydroxyl group has been discussed, but the function of the
ical properties in an aqueous solution and a solid state of hydrogen-bonding to the assembly of glycolipid molecules
the alkyl glycosides have been studied. Those compounds in an aqueous condition is one of the most interesting
have characteristic properties in thermal transformation, points for researchers. At least, the hydrogen-bondings of
which shows a polymorphism and thermotropic liquid sugar bundle between the molecules maintain the assem-
crystals. Octyl a-D-glucopyranoside shows a complex bly in the crystal phase from the viewpoint of the crystal
phase transformation with rising temperature, three types structure analysis of glycolipids.
Figure 31 The plotting of the acyl chains of MeaGal for (i), (ii), and (iii), and those of MehGlc for (iv), (v), and (vi). The
numbers in the gures pointed at the curves are the number of carbon atoms in acyl chain for each derivative a and b show the
two conformers of the disorder structures [94]. Reproduced by permission of the Royal Society of Chemistry.
770 Abe and Harata
22. Baron, C.; Thompson, T.E. Biochim. Biophys. Acta 1975,

382, 276.
23. Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H.
Nature 1985, 318, 618.
24. Abrahams, J.P.; Leslie, A.G.W.; Lutter, R.; Walker, J.E.
Nature 1994, 370, 621.
25. Tsukihara, T.; Aoyama, H.; Yamashita, E.; Tomizaki T., ;
Yamaguchi, H.; Shinzawa-Itoh, K.; Nakashima, R.;
Yaono, R.; Yoshikawa, S. Science 1995, 269, 1069.
26. Yoshikawa, S. J. Struct. Biol. Sakabe Project 1998, 4 (3), 31.
27. Michel, H. Trends Biochem. Sci. 1983, 8, 56.
28. Pebay-Peyroula, E.; Gravito, R.M.; Rosenbusch, J.P.;
Zulauf, M.; Timmins, P.A. Structure 1995, 3, 1051.
Figure 32 The symmetric dierence between the odd- and 29. Timmins, P.A.; Pebay-Peyroula, E. In Neutron in Biology;
even-numbered acyl chains. Schoenborn, B.P., Knott, R.B., Eds.; Plenum Press: New
York, 1996; 267 pp.
30. Penel, S.; Pebay-Peyroula, E.; Rosenbusch, J.; Rummel,
G.; Schirmer, T.; Timmins, P.A. Biochimie 1998, 80, 543.
31. Hauser, H.; Pascher, I.; Sundell, S. Biochimie 1988, 27,
Selection of the sugar type is very important because it 9166.
determines the nature of physicochemical properties. This 32. Laughlin, R.G. The Aqueous Phase Behaviour of Surfac-
tants; Academic Press: London, 1995; 106117.
property can be changed by the sugar moiety with a slightly 33. Nilsson, F.; Soderman, O.; Johansson, I. Langmuir 1996,
dierent geometric structure. Chemistry in glycolipids will 12, 902.
be promoted with great progress as various types of 34. Warr, G.G.; Drummond, C.J.; Grieser, F.; Ninham,
oligosaccharides become available. B.W.; Evans, D.F. J. Phys. Chem. 1986, 90, 4581.
35. Hall, C.; Tiddy, G.J.T.; Pfannemuller, B. Liq. Cryst. 1991,
9, 527.
REFERENCES 36. Sakya, P.; Seddon, J.M.; Vill, V. Liq. Cryst. 1997, 23, 409.
37. Koeltzow, D.E.; Urfer, A.D. J. Am. Oil Chem. Soc. 1984,
1. Hakomori, S.; Strycharz, G.D. Biochemistry 1968, 7, 1279. 61, 1651.
2. Magnani, J.; Nilsson, B.; Brockhaus, M.; Zopf, D.; 38. Zabel, V.; Muller-Fahrnow, A.; Hilgenfeld, R.; Saenger,
Steplewski, Z.; Koprowski, H.; Ginsburg, V. J. Biol. W.; Pfannemuller, B.; Enkelmann, V.; Welte, W. Chem.
Chem. 1982, 257, 14365. Phys. Lipids 1986, 39, 313.
3. Quinn, P.J.; Williams, W.P. Biochim. Biophys. Acta 1983, 39. Jerey, G.A.; Saenger, W. Hydrogen Bonding in Biological
737, 223. Structure; Springer-Verlag: Berlin, Hydelberg, 1991; 18 pp.
4. Fischer, E. Ber 1893, 26, 2400. 40. Bene, J.D.; Pople, J.A. J. Chem. Phys. 1970, 52, 4854.
5. Hill, K., von Rybinski, W., Stoll, G., Eds.; Alkyl Poly- 41. Bene, J.D.; Pople, J.A. J. Chem. Phys. 1973, 58, 3605.
glycosides; VCH Verlagsgesellschaft: Weinheim, Ger- 42. Lesyng, B.; Saenger, W. Biochim. Biophys. Acta 1981,
many, 1997. 678, 408.
6. Fischer, E.; Helferich, B.; Leibig, J. Ann. Chem. 1911, 43. Jerey, G.A.; Sundaralingam, M. Adv. Carbohydr. Chem.
383, 68. Biochem. 1974, 30, 445.
7. Noller, C.R.; Rockwell, W.C. J. Am. Chem. Soc. 1938, 60, 44. Jerey, G.A.; Sundaralingam, M. Adv. Carbohydr. Chem.
2076. Biochem. 1975, 31, 347.
8. Goodby, J.W. Mol. Cryst. Liq. Cryst. 1984, 110, 205. 45. Jerey, G.A.; Sundaralingam, M. Adv. Carbohydr. Chem.
9. Dorset, D.L. Carbohydr. Res. 1990, 206, 193. Biochem. 1976, 32, 353.
10. Jerey, G.A. Acc. Chem. Res. 1986, 19, 168. 46. Jerey, G.A.; Sundaralingam, M. Adv. Carbohydr. Chem.
11. Shinoda, K.; Yamaguchi, T.; Hori, R. Bull. Chem. Soc. Biochem. 1977, 34, 345.
Jpn. 1961, 34, 237. 47. Jerey, G.A.; Sundaralingam, M. Adv. Carbohydr. Chem.
12. Plats, G.; Polike, J.; Thunig, C.; Hofmann, R.; Nickel, D.; Biochem. 1980, 37, 373.
Rybinski, W. Langmuir 1995, 11, 4250. 48. Jerey, G.A.; Sundaralingam, M. Adv. Carbohydr. Chem.
13. Fuhrhop, J.-H.; Helfrich, W. Chem. Rev. 1993, 93, 1565. Biochem. 1981, 38, 417.
14. Fuhrhop, J.-H.; Svenson, S.; Boettcher, C.; Rossler, E.; 49. Jerey, G.A.; Sundaralingam, M. Adv. Carbohydr. Chem.
Vieth, H.-M. J. Am. Chem. Soc. 1990, 112, 4307. Biochem. 1985, 43, 203.
15. Shimizu, T.; Masuda, M. J. Am. Chem. Soc. 1997, 119, 50. Muller, A. J. Chem. Soc. 1923, 123, 2043.
2812. 51. Vand, V.; Lomer, T.R.; Lang, A. Acta Cryst., 2, 214.
16. Hato, M.; Minamikawa, H. Langmuir 1998, 12, 1658. 52. Vand, V.; Morley, W.M.; Lomer, T.R. Acta Crystallogr.
17. Hato, M.; Minamikawa, H.; Tamada, K.; Baba, T.; 1951, 4, 324.
Tanabe, Y. Adv. Colloid Interface Sci. 1999, 80, 233. 53. Vand, V. Acta Cryst. 1953, 6, 797.
18. Ohbu, K.; Fujiwara, M. IMFORM 1995, 6, 1122. 54. Abrahamsson, S.; Dahlen, B.; Lofgren, H.; Pascher, I.
19. Nilsson, F.; Soderman, O.; Hansson, P.; Johansson, I. Prog. Chem. Fat Other Lipids 1978, 16, 125.
Langmuir 1998, 14, 4050. 55. von Sydow, E. Acta Chem. Scand. 1955, 9, 1685.
20. Waltermo, A. Dissertation; Laboratory for Chemical Sur- 56. Goto, M. Yukagaku 1987, 36, 909.
face Science, Department of Chemistry, Physical Chemis- 57. Hitchcock, P.B.; Mason, R.; Thomas, K.M.; Shipley,
try, Royal Institute of Techology, S-100 44 Stockholm G.G. J. Chem. Soc. Commun. 1974, 539.
Sweden. 58. Pearson, R.H.; Pascher, I. Nature 1979, 281, 499.
21. Waltermo, A.; Sjoberg, M.; Anhede, B.; Claesson, P.M. J. 59. Hauser, H.; Pascher, I.; Pearson, R.H.; Sundell, S.
Colloid Interface Sci. 1993, 156, 365. Biochim. Biophys. Acta 1981, 650, 21.
60. Sundell, S. Acta Chem. Scand. Ser. A 1986, 31, 799. 81. Muller-Fahrnow, A.; Hilgenfeld, R.; Hesse, H.; Saenger,
61. Coiro, V.M.; Mazza, F.; Pochetti, P. Acta Cryst. 1986, W.; Pfannemuller, B. Carbohydr. Res. 1988, 176, 165.
C42, 991. 82. Jerey, G.A.; Maluszynska, H. Carbohydr. Res. 1990,
62. Abe, Y.; Harata, K.; Fujiwara, M.; Ohbu, K. J. Chem. 207, 211.
Soc. Perkin Trans. 2 1999, p. 85. 83. Andre, C.; Luger, P.; Svenson, S.; Fuhrhop, J.-H.
63. Launden, B.-M. Acta Crystallogr. 1974, B30, 1756. Carbohydr. Res. 1992, 230, 31.
64. Okuyama, K.; Sobi, Y.; Iijima, N.; Hirabayashi, K.; 84. Andre, C.; Luger, P.; Svenson, S.; Fuhrhop, J.-H.
Kunitake, T.; Kajiyama, T. Bull. Chem. Soc. Jpn. 1988, Carbohydr. Res. 1993, 240, 47.
61, 1485. 85. Muller-Fahrnow, A.; Saenger, W.; Fritsch, D.; Schnieder,
65. Toda, F.; Tanaka, K.; Okada, T.; Bourne, S.A.; P.; Fuhrhop, J.-H. Carbohydr. Res. 1993, 242, 11.
Nassimbeni, L.R. Supramol. Chem. 1994, 3, 291. 86. Herbst, R.; Steiner, T.; Pfannemuller, B.; Saenger, W.
66. Noguchi, K.; Okuyama, K.; Vongpnimit, K. Mol. Cryst. Carbohydr. Res. 1995, 269, 29.
Liq. Cryst. 1996, 276, 185. 87. Muller-Fahrnow, A.; Zabel, V.; Steifa, M.; Hilgenfeld, R.
67. Kamitori, S.; Sumimoto, Y.; Vongbupnimit, K.; Noguchi, J. Chem. Soc., Chem. Commun. 1986; 1573.
K.; Okuyama, K. Mol. Cryst. Liq. Cryst. 1997, 300, 31. 88. Masuda, M.; Shimizu, T. Chem. Commun. 1996, 1057.
68. Okuyama, K.; Ishii, T.; Vongbupnimit, K.; Noguchi, K. 89. Masuda, M.; Shimizu, T. Carbohydr. Res. 1997, 302, 139.
Mol. Cryst. Liq. Cryst. 1998, 312 101. 90. Abe, Y.; Fujiwara, M.; Harata, K.; Ohbu, K. Carbohydr.
69. Sakaiguchi, Y.; Shikata, T.; Urakami, H.; Tamura, A.; Res. 1995, 269, 43.
Hirata, H. Colloid Polym. Sci. 1987, 265, 750. 91. Abe, Y.; Fujiwara, M.; Ohbu, K.; Harata, K. Carbohydr.
70. Hall, S.R.; Stewart, J.M. Xtal3.0 Reference Manual; Uni- Res. 1995, 275, 9.
versity of Western Australia: Maryland, Australia, 1990. 92. Abe, Y.; Harata, K.; Fujiwara, M.; Ohbu, K. Langmuir
71. Sheldrick, G.; Herbst-Irmer, R.; Clegg, B. SHELX 1996, 12, 636.
Workshop, Montreal ACA; July 23, 1995; 129. 93. Abe, Y.; Harata, K.; Fujiwara, M.; Ohbu, K. J. Chem.
72. Staut, G.H.; Jensen, L.H. X-ray Structure Determination; Soc., Perkin Trans. 2, 1998; 177.
John Wiley & Sons: New York, 1989. 94. Abe, Y.; Fujiwara, M.; Ohbu, K.; Harata, K. J. Chem.
73. Pascher, I.; Sundell, S. Chem. Phys. Lipids 1977, 20, 175. Soc., Perkin 2 2000; 341.
74. Moews, P.C.; Knox, J.R. J. Am. Chem. Soc. 1976, 98, 95. Tamura, T.; Shimizu, S.; Sasaki, Y.; Hirai, C. Yukagaku
6628. 1991, 40, 321.
75. Jerey, G.A.; Yeon, Y.; Abola, J. Carbohydr. Res. 1987, 96. Lederer, E. Chem. Phys. Lipids 1976, 16, 91.
169, 1. 97. Miyake, M., Personal communication, In preparation for
76. von Koningsveld, H.; Jansen, J.C.; Straathof, J.J. Acta publication.
Cryst. 1988, C44, 1054. 98. Gama, Y.; Kawaguchi, Y. Yukagaku 1993, 42, 685.
77. Adasch, V.; Homann, B.; Milius, W.; Platz, G.; Voss, G. 99. Bjorkling, F.; Godtfredsen, S.E.; Kirk, O. J. Chem. Soc.,
Carbohydr. Res. 1998, 314, 177. Chem. Commun. 1989; 934.
78. Carter, D.C.; Ruble, J.R.; Jerey, G.A. Carbohydr. Res. 100. Brooks, B.R.; Bruccoleri, R.E.; Olafson, B.D.; States,
1982, 102, 59. D.J.; Swaminathan, S.; Karpuls, M. J. Comp. Chem.
79. Bhattacharjee, S.; Jerey, G.A. Mol. Cryst. Liq. Cryst. 1983, 4, 187.
1983, 101, 247. 101. Tsujii, K.; Saito, N.; Takeuchi, T. J. Phys. Chem. 1980,
80. Jerey, G.A.; Yeon, Y. Carbohydr. Res. 1992, 237, 45. 84, 2287.
33
Synthetic and Natural Polysaccharides
with Anticoagulant Properties
Fuming Zhang, Patrick G. Yoder, and Robert J. Linhardt
University of Iowa, Iowa City, Iowa, U.S.A.
I. INTRODUCTION exploited as therapeutic agents [2]. Such compounds along

with heparin will be discussed within this chapter.
Anticoagulant polysaccharides have been of interest to the
medical profession since discovery of heparin by McLean
B. Anticoagulants and Antithrombotics
in 1916 [1]. Since that time considerable research has been
directed at improving anticoagulant and antithrombotic It is dicult to understand the action of anticoagulants and
properties of polysaccharides. Other compounds with antithrombotics without a basic understanding of the
structures similar to heparin have been studied as heparin hemostatic system, therefore a brief overview will be
substitutes. This chapter will discuss polysaccharides, provided here. When blood vessels are damaged, bleeding
both natural and synthetic, which have action on the occurs and the formation of a hemostatic plaque is initiat-
hemostatic system. ed. Platelets adhere to the perivascular collagen through
platelet surface glycoproteins and adhesion proteins [3].
A. Polysaccharides Platelet aggregation is also important to the hemostatic
system, but the physiology and biochemistry surrounding
Polysaccharides are important molecules that are often platelet function is beyond the scope of this chapter. Of
neglected in most reviews of bioactive biopolymers. Other most importance is thrombin formation in the blood
biopolymers such as proteins, DNA, and RNA have been coagulation system. Blood coagulation is based on an
highly publicized in both the scientic literature and the lay intrinsic homeostasis and represents a balance of procoag-
press. Polysaccharides have received little such promotion ulant and anticoagulant factors [3]. Thrombin (factor IIa)
even though they are widely distributed throughout nature formation is essential to coagulation as it catalyzes the
and have highly organized structure. These are important formation of an insoluble brin clot, which occurs late in
molecules involved throughout the body in signal trans- hemostasis [4]. Thrombin is formed through the sequential
duction and cell adhesion. Polysaccharides are also widely activation (Fig. 1) of coagulation factors normally circu-
distributed as constituents of foods found in most of our lating within plasma as zymogens (Table 1). Upon activa-
diets. These biodegradable molecules are also often utilized tion (i.e., factor II!factor IIa) a catalytic carboxy-terminal
as gellants, thickeners, lm formers, llers, and delivery domain is exposed and the activated coagulation factor
systems in pharmaceutical and cosmetic applications. functions as a serine protease. Activation of coagulation
Moreover, they are often derived from renewable sources occurs through two distinct pathways. The intrinsic path-
and therefore serve the above purposes in a relatively cost- way is initiated by collagen or contact with a charged
eective fashion. Although often used as adjuncts in surface. The extrinsic pathway begins with damage to
pharmaceutical applications, some polysaccharides are vessel walls, causing the release of tissue factor [3]. These
also used for their pharmacological action. The most two pathways converge with the activation of factor X
notable pharmacologically active polysaccharide is hepa- (factor X!factor Xa) and the cascade continues down a
rin, which is used medicinally as an anticoagulant. Others common pathway, resulting in thrombin (factor IIa) gen-
polysaccharides with pharmacological activity are also eration and eventually brin (clot) formation (Fig. 1) [4].
773
774 Zhang et al.
Figure 1 Blood coagulation cascade.
The current consensus is that coagulation is predominantly having a structure closely related to heparin, residing on
initiated through the extrinsic pathway [3]. the surface of endothelial cells, forms a complex with
Several anticoagulant systems regulate the procoagu- ATIII. This complex interacts with coagulation factors
lant pathways described above. The rst and most well IIa, IXa, Xa, and XIa rendering them inactive [3]. Heparin
known of these systems is the antithrombin III (ATIII) cofactor II (HCII) is another serpin capable of inhibiting
system. Antithrombin III is a circulating serine protease thrombin after binding heparan sulfate or the structurally
inhibitor (serpin) synthesized in the liver. Heparan sulfate, related polysaccharide, dermatan sulfate. Protein C and
thrombomodulin are the main players in another anti-
coagulation system. Activation of protein C occurs when
it is presented with thrombin and thrombomodulin (endo-
Table 1 Coagulation Factors thelial cofactor) on the endothelial cell surfaces. Active
protein C together with protein S, a cofactor, causes the
Pathway Activity
degradation of factors V and VIII. This system is believed
Intinsic to be a major anticoagulation pathway, based on data
Factor XII Contact factor from studies of acquired and inherited coagulation defects
Factor XI Serine protease [3]. Another anticoagulation system employs a protein
Factor IX Serine protease called tissue factor pathway inhibitor (TFPI) as its major
Factor VIII Cofactor player. This system is a major inhibitor of the extrinsic
pathway and studies suggest that its disruption is not
Extrinsic compatible with life. TFPI (a protein bound to the endo-
Tissue factor Cofactor thelium) is capable of forming quaternary complexes with
Factor VII Serine protease factors VIIa and Xa in the presence of calcium and phos-
pholipids [3]. This quaternary complexation renders these
Common factors inactive.
Factor X Serine protease New studies on hemostasis are reported daily and this
Factor V Cofactor
brief review does not do justice to the immense complexity
Prothrombin (factor II) Serine protease
of this system. The human body requires consistent circu-
Fibrinogen Structural protein
Factor XII Fibrin network
lation of nutrients and oxygen to its tissues while main-
taining a repair process capable of reversing the liquid
Anticoagulant Properties 775
nature of blood when needed. This feat is truly amazing 1 kg starting material followed by complex formation,
and to this day is unreplicated by man. fractionating precipitation, alkaline treatment, and bleach-
ing results in only 150 mg heparin [23]. Heparin obtained
from dierent tissues and dierent species dier structur-
II. NATURAL POLYSACCHARIDES WITH ally (Table 2) [11]. In addition, individual manufacturers
ANTICOAGULANT PROPERTIES use dierent methods of isolation and purication. A
multitude of dierent commercial heparin products exists
A. Heparin and Low Molecular Weight Heparins with some variations in their chemical and physiological
properties [23]. Other species of mammals as well as birds,
1. Heparin and Heparan Sulfate sh, and even invertebrates such as lobster and clams,
Background which do not have a blood coagulation system, also contain
heparin [22].
Heparin is derived from animal tissues and has been
used widely as a clinical anticoagulant since 1935. It was Structure
discovered in 1916 by Jay McLean, a second-year medical Heparin is a polydisperse, highly sulfated, linear poly-
student, working under the direction of physiologist saccharide consisting of repeating units of 1!4-linked
William Howell at Johns Hopkins University [5]. An pyranosyluronic acid and 2-amino-2-deoxyglucopyranose
understanding of heparins structure developed gradually. (glucosamine) residues [24,25]. The uronic acid typically
In 1928, Howell correctly identied one of the sugars in consists of 90% L-idopyranosyluronic acid (L-iduronic
heparin to be a uronic acid [6]. Early researchers showed acid) and 10% D-glucopyranosyluronic acid (D-glucuronic
that heparin also contained O-sulfo esters and N-sulfo- acid). Heparin has the highest negative charge density of
substituted glucosamine residues. By 1970, iduronic acid any known biological macromolecule. This is the result of
was demonstrated to be the major uronic acid component its high content of negatively charged sulfo and carboxyl
and a generalized structure of heparin could be drawn [7]. groups. Indeed, the average heparin disaccharide contains
It is only in the past 20 years that the molecular mech- 2.7 sulfo groups. The most common structure occurring in
anisms behind the anticoagulant/antithrombotic eects heparin is the trisulfated disaccharide (Fig. 2). However, a
were elucidated. With the discovery of the structure of number of structural variations of this disaccharide exist,
the ATIII pentasaccharide binding site, portions of hep- leading to the microheterogeneity of heparin. The amino
arins ne structure have been elucidated, and an improved group of the glucosamine residue can be substituted with
understanding of its conformation [810] and interaction an acetyl or sulfo group or unsubstituted. The 3- and 6-
with proteins established [1120]. A most important depositions of the glucosamine residues can be either substi-
velopment in recent years has been the growing awareness tuted with an O-sulfo group or unsubstituted. The uronic
of the ubiquitous distribution, structural diversity, and acid, which can be either L-iduronic or D-glucuronic
biological importance of heparan sulfate (HS). Formerly acid, may also contain a 2-O-sulfo group. Glycosamino-
an unwanted by-product of heparin manufacture, HS is glycan (GAG) heparin has a molecular weight range of
now recognized as a family of closely related yet distinct 540 kDa, with an average molecular weight of f15 kDa
polysaccharide species. In fact, heparin is now considered and an average negative charge of approx. 75. This
by many as just another member of in the HS family [21]. structural variability makes heparin extremely challenging
Heparin is the most commonly used clinical anticoag- molecule to characterize.
ulant. Over 33 metric tons of heparin is manufactured Heparan sulfate is structurally related to heparin but is
worldwide each year representing over 500 million doses much less substituted with sulfo groups than heparin and
[22]. For industrial-scale production, heparin is prepared has a more varied structure (or sequence). Like heparin,
by extraction from mammalian tissues that are rich in mast heparan sulfate is a repeating linear copolymer of uronic
cells (i.e., porcine intestine, bovine lung). The extraction of acid 1!4-linked to glucosamine (Fig. 2) [26]. While D-
Table 2 Heparins from Dierent Species and Tissues

Average number in one heparin chaina
N-acetylated N-sulfonated Trisulfated Disulfated
Species Tissue AT binding site AT binding site disaccharide disaccharide
Porcine Intestine 0.5 (0.30.7) 0.1 10 (1015) 1.2 (12)
Bovine Lung 0.3 0.3 14 1.0
Bovine Intestine 0.3 0.3 10 1.7
Ovine Intestine 0.7 0.4 11 1.4
Hen Intestine 0.3 0.2 6.7 1.7
Clam 0.5 0.4 5.0 1.9
a
The numbers shown in parentheses indicate a range of values typically observed.
776 Zhang et al.
Figure 2 Structure of heparin and heparan sulfate.
glucuronic acid predominates in heparan sulfate, it can the HS chain to protein, (2) generation of the polysaccha-
contain substantial amounts of L-iduronic acid. Heparan ride chain, and (3) enzymatic modication of the chain to
sulfates generally contain only f1 sulfo group per disac- yield the specic saccharide sequences and structural or-
charide, but individual heparan sulfates may have higher ganization that are responsible for protein binding [32].
content of O-sulfo groups. Heparan sulfate chains also Studies on heparin biosynthesis were performed in a
often contain domains of extended sequences having low or mastocytoma cell culture system with radiolabeled meta-
high sulfation [27]. While heparan sulfate contains all of the bolic precursors of heparin [34]. The core protein, sergly-
structural variations found in heparin (and vice versa), the cin, contains a high number of serine and glycine repeats
frequency of occurrence of the minor sequence variants is and is primarily synthesized in the rough endoplasmic
greater than in heparin, making heparan sulfates structure reticulum. The biosynthesis of the GAG chain predomi-
and sequence much more complex. Heparan sulfate chains nantly takes place in the Golgi apparatus. The rst step in
are also polydisperse, but are generally longer than heparin the pathway involves the attachment of a tetrasaccharide
chains, having average molecular weight (MWavg) of f30 fragment to a serine residue in the core protein [35]. The
kDa ranging from 5 to 50 kDa [28]. Heparan sulfate is sequence of this linkage-region tetrasaccharide is h-GlcAp-
biosynthesized, as a proteoglycan, through the same path- (1!3)-h-Galp-(1!3)-h-Galp-(1!4)-h-Xylp-(1!Ser.
way as heparin; however, unlike heparin, the heparan There are four dierent glycosyltransferases responsible
sulfate GAG chain remains connected to core protein. for the synthesis of the linkage region [36]. Onto this
The core protein of PG heparin and PG heparan sulfate neutral sugar linkage region the rst GlcNpAc residue or
are dierent. Heparan sulfate PG is ubiquitously distrib- N-acetylgalactosamine (GalNpAc, in the biosynthesis of
uted on cell surfaces and is a common component of the chondroitin sulfates) is added. This addition decides
extracellular matrix, whereas heparin is only found intra- whether the chain will be either a glucosaminoglycan
cellularly in certain granule-containing cells [29,30]. There (heparin and heparan sulfate) or a galactosaminoglycan
are two types of HS core proteins, the syndecans (an (chondroitin sulfate/dermatan sulfate). It has been sug-
integral membrane protein) and the glypicans (a GPI- gested that peptide sequence motifs close to the linkage-
anchored protein) [31,32]. Although structurally similar, region substituted serine residues act as a signal for the
heparin and heparan sulfate GAGs can often be structur- addition of a GlcNpAc residue, thus initiating heparin/
ally distinguished through their dierent sensitivity toward heparan sulfate formation [37,38].
a family of GAG-degrading, microbial enzymes, the hep- After the rst residue has been added, alternating
arin lyases [33]. transfer of GlcAp and GlcNpAc residues from their
corresponding UDP-sugar nucleotides to the nonreducing
Biosynthesis termini of growing chains forms the rest of the GAG chain.
The biosynthesis of heparin and heparan sulfate and One enzyme (present in humans in two isoforms) has both
the regulatory mechanisms resulting in the placement of GlcAp transferase and GlcNpAc transferase activities [39].
dierent saccharide sequences in their structure are only Approximately 300 sugar residues are added to the linear
partly understood (Fig. 3) [20]. Heparin and heparan polysaccharide chain before its synthesis terminates [34].
sulfate are synthesized by (1) formation of a region linking As the chain elongates it also undergoes other modication
Figure 3 Pathway of heparin proteoglycan synthesis and its degradation to peptidoglycans and glycosaminoglycans. Protein
synthesis takes place in the endoplasmic reticulum, linkage synthesis, chain elongation, and modication take place in the Golgi,
and proteolysis and glucuronidase digestion take place in the mast cell granules. Heparan sulfate is biosynthesized through a
similar pathway but with reduced modication of the GAG chain and little breakdown of the proteoglycan.
reactions [40]. Modication of the polymer is initiated by (ATIII). ATIII functions as the principal plasma inhibitor
N-deacetylation and N-sulfonation of the GlcNpAc resi- of most blood coagulation proteinases [45]. This serpin
dues by an N-deacetylase/N-sulfotransferase enzyme. Sub- forms tight, irreversible, equimolar complexes with its
sequent steps occur sequentially and either on or adjacent target enzymes by formation of an ester between an
to the N-sulfoglucosamine (GlcNpS)-containing residue. A arginine residue of its active center and the serine residue
C-5 epimerase then catalyzes transformation of some of the of the active center of the enzyme. This slow, time-,
D-glucuronic acid residues to L-iduronic acid residues [41]. temperature-, and pH-dependent process is accelerated
This is followed by O-sulfonation of the iduronic acid 2000-fold by heparin [46]. An essential component of the
residues at the C-2 position by an iduronosyl 2-O-sulfo- mechanism of heparins eect is the binding of a lysine-rich
transferase [42]. Studies have also shown that a very active region of ATIII to the highly specic ATIII-binding site in
glucuronosyl 2-O-sulfotransferase in mouse mastocytoma the heparin molecule (Fig. 4) [47]. This reversible, electro-
microsomal fractions is responsible for the O-sulfonation static interaction induces a conformational change in
of GlcAp residues at the C-2 position [43,44]. The 2-O- ATIII, which considerably reinforces its anticoagulant
sulfonation of the uronic acid is followed by the action of activity [23].
glucosamine 6-O-sulfotransferase, which transfers an O- Another anticoagulant activity mechanism of heparin
sulfo group to the C-6 position of GlcNpAc and GlcNpS is mediated through heparin cofactor II (HCII), which is
[41]. Finally a 3-O-sulfotransferase acts upon the polymer structurally similar to ATIII having a similar carboxyl
and modies certain GlcNpS6S residues [31]. The 3-O- terminal sequence but a distinctly dierent amino terminal
sulfonation is required for the anticoagulant activity of sequence [48]. The physiological role of HCII might be as
heparin, and the pentasaccharide sequence formed by the reserve of thrombin inhibitor when the plasma concentra-
3-O-sulfotransferase is the minimum structure required for tion of ATIII becomes abnormally low [49]. Unlike ATIII,
binding antithrombin III. HCII can inhibit thrombin but no other coagulation
proteases [46]. In addition to this unusual specicity, HCII
Anticoagulant Activity and Its Mechanism also can be potentiated by dermatan sulfate in addition to
The anticoagulant activity of heparin is primarily heparin and heparan sulfate [50,51]. The mechanism by
mediated through its binding and regulation of a plasma which HCII inhibits thrombin is similar to that proposed
serine proteinase inhibitor (serpin) antithrombin III for ATIII [46].
778 Zhang et al.
Figure 4 Mechanism of the rate-enhancing eect of heparin (H) on thrombin (T) inhibition by ATIII.
Other Biological Activities its charge using anion exchange chromatography. Strong
The interaction of heparin with various proteins that anion exchange (SAX) chromatography of heparin typi-
play important roles in the regulation of normal physio- cally involves the use of a salt gradient [61]. Heparin
logical processes as well as disease states has led to an fractions can be prepared with from two to three sulfo
interest in using heparin in roles outside its normal appli- groups per disaccharide repeating unit [62]. Anticoagulant
cation as an anticoagulant/antithrombotic agent. Ran- activities of fractionated heparin vary with degree of
domized trials to study the eectiveness of low molecular sulfonation (Table 3) [22] making this a useful technique
weight heparin (LMWH, MWavg f6000) as compared to separate or enrich specic heparin fraction based on
with unfractionated heparin (see Sec. II.A.2) (MWavg activities. Chemical sulfonation or desulfonation can also
f12,000) in treating venous thromboembolism in cancer used to prepare oversulfated or undersulfated heparins
patients led to a surprising observation: that treatment with with various activities (see Sec. III.A)
heparin may aect survival of patients with malignancy
[52,53]. Cancer patients who had been treated with LMWH 2. Low Molecular Weight Heparin
for their thrombosis had a slightly improved 3-month Background
survival as compared to cancer patients receiving unfrac-
tionated heparin. Heparin can potentially exert its activity Low molecular weight heparins (also referred to as low
at various stages in cancer progression and malignancy- molecular mass heparins (LMMHs)) are a group of hepa-
related processes. It can aect cell proliferation, interfere
with the adherence of cancer cells to vascular endothelium, Table 3 Heparin Activities as a Function of Polymer Chain
regulate the immune system, and have both inhibitory and Length and Degree of Sulfonation or Charge
stimulatory eects on angiogenesis [54]. There is recent
evidence showing that heparin treatment reduces tumor Anti IIa activity
metastasis in mice by inhibiting P-selectin-mediated inter- (U/mg)
actions of platelets with carcinoma cell-surface mucin
ATIII HCII
ligands [55].
Problems in the Medical Applications of Heparin Based on size
Tetrasaccharides (MW 1200) <1 <1
In clinical applications, heparin is administered intra- Hexasaccharides (MW 1900) <1 <1
venously (LMWH can be administered either intravenously Octasaccharides (MW 2400) <1 <1
or subcutaneously, improving its scope of therapeutic Decasaccharides (MW 2900) <1 <1
applications) during most extracorporeal procedures Dodecasaccharides (MW 3500) 5 2
(where blood is removed from the body and passed through Tetradecasaccharides (MW 4100) 13 16
a device), such as kidney dialysis and membrane oxygen- Hexadecasaccharides (MW 4700) 17 16
ation, used in heart bypass procedures [56]. The use of these Octadecasaccharides (MW 5300) 26 18
devices requires heparinization and can often lead to Eicosasaccharides (MW 5900) 24 14
hemorrhagic complications. Systemic heparinization is Heparin fraction (MW f4000) 6 23
also used in treatment of deep vein thrombosis and in a Heparin fraction (MW f5700) 26 44
variety of other surgical procedures [57]. Heparin-induced Heparin fraction (MW f14,500) 196 271
thrombocytopenia (HIT), a complex process that results in Heparin fraction (MW f16,900) 130 331
loss of platelets, is currently recognized as one of the most Heparin fraction (MW f25,300) 92 356
catastrophic complications of heparin treatment [58,59]. Unfractionated heparin 171 225
(MW f14,000)
Anticoagulant Activity of Fractionated Heparins
Heparin can be fractionated, on the basis of size, using Based on charge
gel permeation chromatography (GPC) [60]. Both low- Heparin fraction (very low charge) 17 90
Heparin fraction (low charge) 108 261
pressure and high-pressure GPC has been used to obtain
Heparin fraction (intermediate 125 336
heparin fractions of MWavg between 5000 and 40,000.
charge)
Anticoagulant activities of fractionated heparin with dif-
Heparin fraction (high charge) 227 468
ferent chain size are shown in Table 3 [22]. Being a sulfated Heparin fraction (very high charge) 540 448
polysaccharide, heparin can be fractionated on the basis of
rin-derived anticoagulant/antithrombotic agents. The in- with (1) suitable average molecular weight and low poly-
troduction of LMWHs primarily resulted from an im- dispersity, (2) antifactor Xa/antifactor IIa activity >1, (3)
proved understanding of the molecular basis of the structurally similar to a LMWH prepared through frac-
biochemistry associated with the coagulation cascade tionation and with few structural artifacts resulting from
[63]. The isolation of the serine protease inhibitor, anti- the depolymerization method used, (4) no residual toxic
thrombin III (ATIII), and the characterization of coagu- reagents, and 5) a cost-eective reproducible and scalable
lation factors (serine proteases), such as thrombin (factor process having a minimum number of process steps, little if
IIa) and factor Xa (inhibited by ATIII), were critical in any required purication, neither labor, reagent, nor cap-
driving the development of LMWHs [64,65]. Heparin ital intensive, and high yielding. While no current manu-
accelerates the inhibition of these coagulation factors by facturing process meets all of these goals, the currently used
ATIII, preventing the generation of a brin clot. In the processes have aorded a rst generation of clinically
coagulation cascade (Fig. 1), one factor activates the next useful LMWHs [63].
until prothrombin (factor II) is converted to thrombin Heparin can be oxidatively broken down using a
(factor IIa) by factor Xa. It is thrombin that acts on variety of oxygen-containing reagents including hydrogen
brinogen to form a brin clot. The very nature of this peroxide, hypochlorous acid, Cu+/O2, Fe2+/O2, or ioniz-
cascade suggested a therapeutic opportunity, to develop an ing g-irradiation (Fig. 5) [66]. Each of these methods relies
agent that was more specic than heparin (which acts at on the generation of oxygen radicals that are believed to act
many points in the cascade) that might provide more subtle by oxidizing sensitive saccharide residues within the hepa-
regulation of coagulation, reducing the major hemorrhagic rin polymer. Nonreducing sugars are essentially inert to
side eects associated with heparin. LMWHs were origi- aqueous hydrogen peroxide except in the presence of alkali
nally developed based on this rationale. or in the presence of a metal catalyst. Both of these
conditions lead to the generation of the hydroxyl radical,
Preparation of LMWH which will react with sugar residues and degrade them to 1-,
LMWHs are generally prepared through the con- 2-, and 3-carbon fragments without modifying the residues
trolled, partial, chemical, or enzymatic depolymerization on either side of the point of attack. The most susceptible
commercial heparin (Fig. 5 and Table 4) [22]. These residues appear to be those that are unsubstituted at
depolymerization methods were selected to give a product positions numbered 2 and 3 in the sugar ring. Studies of
Figure 5 Depolymerization of heparin to prepare LMWHs. The heparin chain in the center can undergo depolymerization by
each of the four processes shown. Heparin chain size is reduced (n > m), aording a LMWH.
780 Zhang et al.
Table 4 Properties of Commercially Available LMWHs

Trade name Manufacturer Preparation MWavga Potencyb
Natroparin calcium Sano/Choay EtOH fractionation 5,500 95

Fraxiparine
Fraxiparin
Fraxiparina
Seleparina
Fragmine Kabi/Pharmacia Nitrous acid degradation 3,400 60
Dalteparin sodium
Tedelparin
Fragmin
Low liquemine
Certiparin sodium Sandoz Isoamylnitrite degradation 6,300
Sandoparin
Parnaparin sodium Opocrin Peroxidative degradation 6,500 83
Fluxum
Minidalton
Ardeparin sodium Hepar/Wyeth-Ayerst Peroxidative degradation 6,200 62
Sandoparin
Enoxaparin sodium Rhone Poulenc/Aventis Benzylation/h-elimination 3,800 96
Clexane
Lovenox
Klexane
Tinzaparine sodium Novo/Leo/Dupont Enzymatic h-elimination 4,900 87
Logiparin
Reveparin sodium Knoll AG Nitrous acid degradation 4,000
Clivarin
Clivaparin
Std LMWH85/600 NIBSC Nitrous acid/GPC 100
Heparin 14,000 150
a
By GPC.
b
Relative anti-Xa potency against NIBSC standard LMWH.
the composition of disaccharides that result from oxidative dride. Partial deaminative cleavage is possible by
depolymerization of LMWHs suggest that nonsulfated controlling the process conditions (i.e., temperature, pH,
uronic acid residues in heparin are selectively oxidized to time) or by limiting the amount of nitrosation reagent
volatile acids, i.e., formic acid [67]. Under controlled [69,70]. The LMWH product formed using these controlled
conditions (i.e., temperature, pressure, time, oxidant), conditions is obtained in high yield and has the appropriate
LMWHs having appropriate molecular weights and activ- chemical and biological properties. Several LMWHs pre-
ity can be obtained in reasonable yields [68]. Of these pared through deaminative cleavage are currently used
oxidative methods, only hydrogen peroxide has been uti- clinically (Table 4).
lized to commercially prepare LMWHs (ardeparin sodium Enzymatic h-eliminative methods are used to com-
and panaparin sodium) for clinical use (Table 4). mercially prepare LMWHs. Heparin lyases eliminatively
In addition to oxygen radical processes, it is possible to depolymerize heparin aording unsaturated oligosaccha-
oxidatively depolymerize heparin through deamination ride products. Three major polysaccharide lyases (heparin
(Fig. 5). In these reactions, heparin is N-nitrosated, using lyases I, II, and III), isolated from Flavobacterium hepa-
either nitrous acid or another nitrosating reagent such as rinum, are capable of cleaving linkages present in heparin
isoamyl nitrite, at the amino group of its N-sulfoglucos- [71]. The extent of this reaction can be conveniently
amine residues [63]. The resulting unstable N-nitrososulfa- monitored by measuring the change in absorbance associ-
mide loses nitrogen and sulfate and generates a carbocation ated with the unsaturated uronate residue formed in each
at C-2 of the saccharide residue. Subsequent ring contrac- product molecule. Cleavage with heparin lyase I takes
tion of this residue and hydrolysis of the adjacent glycosidic place only at the 2-O-sulfoiduronic acid residue [72]. The
bond aords an LMWH. Each product chain resulting depolymerization is stopped by removing or inactivating
from this process contains an anhydromannose residue the enzyme. This method is used to prepare the clinically
(bearing a terminal aldehyde) at the reducing terminus. used LMWH product, tinzaparin sodium (Table 2).
This residue can subsequently be converted to anhydro- Chemical h-elimination can involve the direct treat-
mannitol using a reducing agent, such as sodium borohy- ment of heparin or its quaternary ammonium salt with
base [73,74]. Alternatively, the benzyl ester can be prepared enhancement of HCII activity [80]. DS is also able to
by treatment of the benzethonium salt of heparin with inhibit thrombin generation and thrombin-mediated am-
benzyl chloride and base with heating [75]. Under these plication reactions, but less eectively than heparin
conditions, chemical h-elimination takes place, aording [81,82]. Several DS preparations have been developed for
LMWH containing an unsaturated uronate residue in the prophylaxis of venous thromboembolism [23]. Compared
nonreducing end. Cleavage occurs specically at iduronic to heparin, native DS is a less active but safer antithrombin
acid without preference for the presence or absence of a drug by intravenous administration due to its low bleeding
2-O-sulfo group. Proper control of the process conditions potential, even at very high doses (up to 16 mg/kg). Low
aords the clinically used LMWH, enoxaparin sodium molecular weight DS has been developed (e.g., Desmin
(Table 4). 370) to improve bioavailability. Chemical modications
such as oversulfation and succinyl derivatization were
Medical Applications of LMWHs
examined to improve anticoagulant/antithrombotic activ-
Based on the method of the preparation, dierent ity and safety [83,84]. Dermatan disulfate (Intimatan),
structural modications are induced with the consequence prepared by chemical 6-O-sulfonation of dermatan sulfate,
that LMWHs may dier considerably in their chemical and shows substantial antithrombotic activity [85].
physical as well as in their biological and pharmacological
properties (Table 4). Although each LMWH is dierent, Chondroitin Sulfates
they all have several features in common such as being Chondroitin sulfates are families of structurally com-
polydisperse with MWavg f5000 and MW ranging from plex, sulfated, linear polysaccharides with repeating
2000 to 8000. In comparison with unfractionated heparin, (1!3,4-linked) disaccharide units of D-glucuronic acid
LMWHs have better dened chemical and biological prop- and N-acetyl-D-galactosamine, with the O-sulfo groups at
erties giving predictable pharmacological actions, sus- either the 4-position (chondroitin sulfate A) or 6-position
tained activity, improved bioavailability, and a better (chondroitin sulfate C) of the N-acetyl-D-galactosamine
therapeutic index. These can be administered subcutane- [86] (Fig. 6). This polysaccharide is part of the proteogly-
ously once a day due to their high bioavailability and longer cans found localized on cell surfaces and in the extracellu-
half-life [23]. Thus, LMWHs have been successfully intro- lar matrix and is important in cellcell communication [87].
duced as new eective and improved anticoagulant/antith- While chondroitin sulfates appear to be involved in main-
rombotic agents. taining hemostasis, chondroitin sulfates lack clinically
relevant levels of anticoagulant activity, presumably due
B. Nonheparin Glycosaminoglycans to the lack of iduronic acid and their low level of sulfation
[88]. Oversulfated chondroitin sulfates with two to three
Heparin has been the drug of choice in clinical, presurgical sulfate groups per disaccharide unit have been shown to
and postsurgical prophylaxis of thrombotic events. How- exhibit enhanced antithrombotic activity [89]. These chem-
ever, because of its side eects, such as bleeding and other ically prepared oversulfated chondroitin sulfates still con-
disadvantages, developing alternatives to heparin is an tain glucuronic acid residues, making them both
important research goal. Other polysaccharides and mod- structurally and conformationally dierent from the idur-
ied polysaccharides have been examined as potential onic acid residues found in heparin [90,91].
heparin analogs in drug development [76].
Acharan Sulfate
1. Animal Nonheparin Glycosaminoglycans Acharan sulfate is composed of a simple disaccharide
Nonheparin GAGs, originally side products of heparin repeating unit of 1!4-linked N-acetyl-D-glucosamine and
manufacture, have raised great interest as anticoagulant/ 2-O-sulfo-L-iduronic acid (Fig. 6) and is isolated from the
antithrombotic drugs in the past several years, especially giant African snail, Achatina fulica [92]. The natural func-
after the demonstration that the interaction with ATIII is tion of this molecule in snail, while still unclear, may be as
not essential to produce antithrombotic eects [77]. an antidesiccant, a metal chelator, an anti-infective, or a
locomotive (slime) agent. Its structure is related to heparin
Dermatan Sulfate and heparan sulfate but is distinctly dierent from all
Dermatan sulfate (DS, chondroitin sulfate B) is found known members of these classes of glycosaminoglycans.
in a variety of tissues in virtually all animals, and is a Because of its structural similarities to heparin, chemically
(1!3,4-linked) copolymer of N-acetyl-D-galactosamine, L- modied acharan sulfate was studied to understand the
iduronic acid, and D-glucuronic acid with O-sulfo groups chemical structure eecting its anticoagulant activity [93].
commonly found on the 4-position of the N-acetyl-D- After de-N-acetylation, acharan sulfate was N-sulfonated
galactosamine residues (Fig. 6). DS has been used clinically using either chlorosulfonic acidpyridine or sulfur triox-
as an antithrombotic agent [23]. Similar to HS, DS is a idetrimethylamine complex. The level of sulfonation in
relatively weak anticoagulant in vitro (70 times less than these products ranged from 22% to 24% (w/w), signi-
heparin) and, apart from the inhibition of the thrombin- cantly less than that of heparin at 36%. The MWavg of both
induced platelet aggregation, does not interact with plate- N-sulfoacharan sulfates were comparable with that of
lets [78,79]. DS has neither ATIII-mediated anti-Xa nor heparin. In vitro anticoagulant activity assays showed that
anti-IIa activity. Its anticoagulant eect is mainly based on N-sulfoacharan sulfate derivatives were moderately active
782 Zhang et al.
Figure 6 The structures of the major disaccharide units of chondroitin sulfate, dermatan sulfate, dermatan disulfate (Intimatan),
acharan sulfate, keratan sulfate and per-O-sulfonated hyaluronic acid (Ac is acetate and n is the degree of polymerization).
(corresponding from 20% to 40% of heparins activity) for A corneal cell culture system has been developed in which
the inhibition of thrombin, and neither product showed long-term KS biosynthesis is maintained. Progress has
any measurable antifactor Xa activity [93]. been made toward identication of the glycosyl- and
sulfotransferases responsible for KS biosynthesis. Evi-
Keratan Sulfate dence has also been presented supporting functional roles
Keratan sulfate (KS), the only GAG not to possess a of KS in cellular recognition of protein ligands, axonal
uronic acid residue, is composed of repeating disaccharide guidance, cell motility, and in embryo implantation. These
units of galactose and N-acetylglucosamine [94]. The glu- ndings have served to expand the concept of what KS is
cosamine and galactose residues are commonly 6-O-sulfo- and the potential roles it may play in the cellular biology of
nated (Fig. 6). The last 5 years have seen a marked increase diverse tissues [94]. No report has been found so far on KS
in research on KS and a concomitant increase in our as an anticoagulant agent.
understanding of the range of molecules that carry this
adaptable polysaccharide [94]. More than 15 KS-linked 2. Nonheparin Glycosaminoglycans from Plant
proteins have been identied and many of the genes Sources
encoding these have been cloned. KS-containing molecules
have been identied in numerous epithelial and neural Carrageenans
tissues in which KS expression responds to embryonic Besides animal GAGs, important natural sources for
development, physiological variations, and wound healing. sulfated polysaccharides are the marine ora and fauna,
i.e., algae and organisms like mussels and corals [23]. from three species of brown algae. The results indicate that
Carrageenans are obtained commercially by aqueous dierent structural features determine not only the antico-
extraction of certain species of red seaweeds in the Rho- agulant potency of the sulfated fucans but also the mech-
dophyceae class. They consist of sulfate esters of 1!3,4- anism by which they exert this activity. Thus, the branched
linked D-galactose and 3,6-anhydro-D-galactose copoly- fucans from brown algae are direct inhibitors of thrombin,
mers [95]. Carrageenans provide structural support for whereas the linear fucans from echinoderms require the
the spatial arrangement of plants and are widely used in presence of antithrombin or heparin cofactor II for inhi-
food, pharmaceutical, and industrial applications. They bition of thrombin, as reported for mammalian glycosa-
have been well known for a long time and are used minoglycans. The linear sulfated fucans from echinoderms
therapeutically as antipeptic and immunosuppressive have an anticoagulant action resembling that of mamma-
agents. Their anticoagulant activity, which was rst dem- lian dermatan sulfate and a modest action through anti-
onstrated by Elsner, is considered to be a side-eect [23]. In thrombin. A single dierence of one sulfo per
vitro, the carrageenans prolong a PTT and the PT, show tetrasaccharide repeating unit modies the anticoagulant
only a small eect in the HeptestR, and inhibit the amido- activity of the polysaccharide markedly. Possibly, the
lytic thrombin but not FXa activity [9698]. Recently, the spatial arrangements of sulfo groups in the repeating
antiviral and anticoagulant properties of the insoluble tetrasaccharide unit of the echinoderm fucan mimic the
polysaccharide fraction from carrageenans extracted from site in dermatan sulfate with high anity for heparin
cystocarpic and tetrasporic Stenogramme interrupta were cofactor II.
analyzed [98]. These analyses showed the polysaccharides To improve the activities of genuine sulfated fucans,
exhibit no or very slight anticoagulant activity at the chemical modications of a fucoidan from Fucus vesiculo-
concentrations assayed for antiviral activity, indicating a sus were conducted, indicating that oversulfation as well as
lack of correlation between antiviral and anticoagulant the introduction of alkylamino residues increase in vitro
properties of the polysaccharides studied in their work. and in vivo anticoagulant/antithrombotic activity [23].
Sulfated Fucans Sulfated Galactan
Since the description of high amounts of sulfated Farias and coworkers [108] reported an algal-sulfated
fucans in marine brown algae over 50 years ago, these D-galactan with repeating structure [!4-D-Galp-(1!3)-D-
polysaccharides have been widely tested for biological Galp-(1!)], but with a variable sulfation pattern (one-
activities in dierent mammalian systems [99,100]. These third of the total saccharide units are 2,3-di-O-sulfonated
fucans comprise a wide, continuous spectrum ranging from and another one-third contain only 2-O-sulfo groups), has
high-uronic-acid-, low-sulfate-containing polymers, which a potent anticoagulant activity (similar potency as unfrac-
are glucuronofucoglycans and fucoglucuronans, to the tionated heparin) due to enhanced inhibition of thrombin
relatively pure fucans, the so-called homofucans or fucoi- and factor Xa by ATIII and/or HCII. They also extended
dans [23]. Algal-sulfated fucans have anticoagulant activity the experiments to several sulfated polysaccharides from
as measured in several dierent assays and have also marine invertebrates with simple structures composed of a
venous antithrombotic activity [101,102]. Apart from the single, repeating structure. A 2-O- or 3-O-sulfonated
anticoagulant and antithrombotic activity, these polysac- L-galactan has a weak anticoagulant action when com-
charides are inhibitors of native and recombinant human pared with the potent action of the algal-sulfated D-gal-
immunodeciency virus reverse transcriptase activity in actan. Possibly, the addition of two sulfo esters to a single
vitro [103]. Furthermore, because of their interference with galactose residue has an amplifying eect on the antico-
molecular mechanisms of cell-to-cell recognition, algal- agulant action, which cannot be totally ascribed to the
sulfated fucans are potent blockers of a wide range of increased charge density of the polymer. These results
biological processes. Thus, algal-sulfated fucans are inhib- indicate that the wide diversity of polysaccharides from
itors of cell invasion by retroviruses such as human immu- marine alga and invertebrates is a useful tool to elucidate
nodeciency virus, herpes, cytomegalovirus, and African structure/anticoagulant activity relationships [108].
swine fever virus [104106].
The mechanisms by which the algal fucans exert their 3. Nonheparin Glycosaminoglycans from Microbial
anticoagulant action remain controversial. Mechanisms Sources
related to both antithrombin and heparin cofactor II Sulfated polysaccharides are rarely found as microbial
mediated activity have been described for algal-sulfated products. This may be due to the absence of a highly
fucans from dierent species [100,102]. Direct anticoagu- compartmentalized cellular structure found in eucaryotes
lant activity by an antithrombin-independent pathway has that is required for the biosynthesis of sulfated polysac-
also been reported for some preparations of algal fucans charides [109]. Heparin-like compounds have been pre-
[107]. In addition, some fucans have antifactor Xa activity, pared by chemical modication of capsular polysaccharide
whereas others do not. The structures of sulfated fucans from Escherichia coli K5. Capsular polysaccharide from E.
vary from species to species and must give rise to variation coli K5, with the basic structure [GlcA h (1!4) GlcNAc a
in the detailed mechanisms of anticoagulant action [99]. (1!4)]n, was chemically modied through N-deacetyla-
Pereira and coworkers [99] compared the anticoagu- tion, N-sulfonation, and O-sulfonation [110]. Depending
lant activity of the regular and repetitive fucans from on the reaction conditions, the products showed dierent
echinoderms with that of the more heterogeneous fucans proportions of components with high anity for ATIII.
784 Zhang et al.
Derivatives with high anity for ATIII and high anti-Xa Scientists have been sulfonating polysaccharides since
activity contained the trisulfated amino sugar GlcNSO3-3, the middle of the 19th century but only recently have
6-SO3. The 3-O-sulfo GlcN unit has been identied as a adequate methods been described [116]. Currently, several
marker component of the ATIIIbinding pentasaccharide hundred methods for sulfation of polysaccharides have
sequence in heparin (Fig. 2) suggesting that the modied been described in the literature. However, most sulfonation
bacterial polysaccharide interacts with ATIII and pro- reactions induce excessive destruction of the polysaccha-
motes its anticoagulant action in a manner similar to that ride or produce scarce. uneven sulfonation of the nal
of heparin [110]. Thus, homogeneous heparin-like sub- product [117]. Furthermore, such reactions result in poly-
stances with special anticoagulant properties might be saccharide sulfate mixtures, which must be separated [23].
produced by biotechnological methods [23]. Under these conditions, structureactivity relationships
Hyaluronic acid (HA) is a very high molecular weight (SAR), used to yield improved therapeutics, are very
GAG having no sulfo groups [22]. Bacterial HA is identical dicult to study.
to HA from animals and can substitute for animal HA Years of investing time, money, and other resources
previously used in eye or joint operations to replace the into the sulfonation of polysaccharides with the intent to
material lost during surgical manipulations [111]. Chemical produce the perfect anticoagulant have yielded mixed
O-sulfonation of this polysaccharide (Fig. 6) aords a results [118122]. This demonstrates the immense complex-
polysaccharide displaying anticoagulant activity [112]. ity of the bodys ability to recognize and metabolize such
Magnani and coworkers [113] synthesized seven dierently compounds. Sulfonation of dened oligosaccharides such
sulfonated HA derivatives (having a general formula as oligomannas might oer better antithrombotic agents
HyalSx, where x can be 1, 2, 2.5, 3, 3.5, 3.8, 4). Coagulation for the study of structureactivity relationships [123].
tests (whole-blood clotting time and thrombin time) The quest for the ultimate anticoagulant has associat-
showed that signicant prolongations were observed ed with it yet another level of diculty. Heparin-induced
from HyalS2.5 up to HyalS4, suggesting that the antico- thrombocytopenia, a major complication of heparin ther-
agulant activity increases by increasing the sulfonation apy in specic patients, is mediated through an immune
degree of HA. reaction. With the elucidation of the HIT mechanism at
hand, the ultimate modern anticoagulant must not
demonstrate cross-reactivity with heparin antibodies in
HIT patients [124].
III. MODIFIED AND SYNTHETIC
POLYSACCHARIDES WITH
ANTICOAGULANT PROPERTIES B. Dextran Sulfate
A. Synthetic Chemistry: Sulfonation Dextrans have a historical mainstay in medicine in which

they were used as plasma extenders in postsurgical and
As researchers began to look for analogs to build the medical thromboembolic prophylaxis. Dextran is of bac-
pharmacological anticoagulant arsenal, chemical sulfona- terial origin and therefore is unsulfated in nature. It is a
tion became an area of intense interest. The starting (!4)-h-D-Glc (1!3)-a-D -Glc (1! branched polymer
materials for such studies were nonsulfated polysaccha- [125]. The natural polysaccharide has a large MWavg
rides. It was believed that molecules comparable in struc- (f1000 kDa) but for medicinal purposes the MWavg is
ture to heparin upon sulfonation would produce reduced by partial degradation (10100 kDa). Dextrans
compounds with the ability to perform as anticoagulants eects are purely physical; the unsulfated polysaccharide
within the body. Many polysaccharides were targeted for has no direct eect on the coagulation system [23]. There-
sulfonation including agarose, amylose, arabinoglactan, fore, unsulfated dextran has low ecacy as an anticoagu-
cellulose, chitin, curdlan, dextran, guaran, hyaluronic acid, lant and also exhibits multiple side eects rendering its use
inulin, lentinan, laminarin, pectin, xanthan, xylan, and less than ideal.
others [23]. It was hypothesized sulfonation of such mate- Upon sulfonation, dextran is converted to dextran
rials could produce improved anticoagulants for either sulfate (Fig. 7), which was one of the rst polysaccharides
systemic administration or coating of implantable objects to be chemically sulfated. Dextran sulfate exhibits heparin-
[114]. Data indicate that specic desulfonation of heparin like activity but has a very narrow therapeutic indexan
and selective sulfonation of heparinoid molecules may de- undesirable characteristic for a therapeutic agent [121].
crease the thrombogenesis of implanted objects. This eect There is direct relationship between MWavg and toxicity,
is believed to be due to these molecules inability to aect with molecules less than 10 kDa showing little or no
platelet function, therefore decreasing platelet adhesion toxicity. This is a problem because dextran requires a
and aggregation [115]. These data are interesting because relatively high degree of sulfonation for maximum activity
the molecules, which cause the least eect on platelets, (f1.3) [120]. Although the relative anticoagulant activity
often provide little anticoagulation. Further studies on of dextran sulfate is low the molecule does exhibit lipopro-
molecular modications and their eect on platelet func- tein-lipase-releasing activity, a characteristic exploited as
tion, along with continued study of the anticoagulant an agent for the treatment of atherosclerosis [126]. Cur-
eects, may provide additional insights into the discovery rently, the use of dextran sulfate as an anticoagulant and
of new and more eective antithrombotic coatings. antilipemic has lost favor due to its narrow therapeutic
Figure 7 Structure of chitin, pentosan, and dextran sulfates. Representative saccharide units composing each of these polymers
are shown where n is the degree of polymerization.
index and the marketing of more eective alternatives. It expected [147]. It has also become evident this adverse
continues to be used within the laboratory in some diag- reaction is mediated through an immune complex mecha-
nostic assays [127] and has regained some popularity due to nism where the critical element for reactivity is a high
its ability to inhibit HIV binding to CD4+ T cells [128]. negative charge rather than specic polymer molecular
structure [148]. This would indicate this reaction could
C. Pentosan occur with other sulfated polysaccharides, making the
search for polysaccharide anticoagulants devoid of prop-
Pentosan is a compound extracted from beech tree bark erties capable of inducing HIT even more dicult.
and sulfonated to form a clinically useful anticoagulant
called pentosan polysulfate (PPS), often referred to as SP D. Chitosan Sulfate
54. It is a 1!4-linked xylopyranose with one lateral 4-O-
methyl-a-D-glucuronic acid branch (Fig. 7) [20]. PPS has Chitin, a 2-acetamido-2-deoxy-D-glucopyranose polymer
performed as an anticoagulant in Europe for well over 30 (Fig. 7), is the major component of the exoskeleton of
years and is often given subcutaneously twice daily [129]. It insects, crabs, and shrimp, and can also be found in cell
is thought that PPS functions as an anticoagulant by walls of bacteria and fungi. Like heparin, chitin and
various mechanisms, independent of ATIII [130]. First, chitosan, the products of N-deacetylation of chitin, have
PPS exhibits behavior consistent with inactivation of factor 1!4 linkages making them suitable targets for anticoagu-
VIII [131]; second, PPS appears to enhance brinolytic lant construction through chemical sulfonation. Further-
activity via activation of factor XII and kallikrein [132]. more, their linear structure and inherent presence of
PPS also exhibits weak antifactor Xa properties believed to acetamido and amino groups are even more attractive
be the result of hepatic trigylceride lipase release [133]. because such characteristics are challenging to introduce
Interestingly, PPS also has antiangiogenic activity due to chemically [118]. Likewise, the sulfonation of both of these
its ability to eectively block heparin-binding growth compounds yields products with anticoagulant properties.
factor (HBGF) both in vitro and in vivo [134]. Angiogen- Since chitin is highly crystalline its modication to chitosan
esis is important in rapid tissue growth and development as sulfate requires two-phase reactions [149]. The highest
observed in tumor progression and metastasis [135]. This activity is observed with O-sulfated N-acetylated chitosan
activity alone has led to considerable recent study of (>300 U/mg), followed by N,O-sulfonated chitosan (>200
pentosan polysulfate as an antitumor agent [136]. U/mg), both showing more activity than heparin [150]. N-
Another interesting characteristic of PPS is its ability Sulfo chitosan is devoid of activity, but interestingly, the
to be absorbed orally; although the bioavailability is low, addition of a carboxyl group at the 6 position aords a
signicant reduction in thrombus formation is observed product with one-fourth the activity of heparin [151]. This
[137]. A derivative of PPS called Bego 0391 was prepared activity is potentiated by further O-sulfonation [149]. Posi-
by Bene-Arzneimittel GmbH. This derivative is signicant- tion 6 [152] or amino group [118,153] carboxymethylation
ly more bioavailable relative to PPS, although its use potentiates the anticoagulant activity of either sulfonated
clinically has been limited [138]. Both oral and subcutane- chitin or chitosan and if the degree of sulfation is high these
ous administration of PPS yield increased plasminogen molecules function as well as heparin. A 6-O-sulfo group
activator activity within the plasma [139,140]. Multiple represents the main active site, although sulfonation at
studies suggest this increased brinolytic activity is due to multiple positions is benecial. The sulfamido group is not
endothelial tissue plasminogen activator (t-PA) release, not essential when a high degree of sulfonation is achieved
the factor XII pathway mentioned earlier [140142]. [154]. In addition to the degree of sulfonation, molecular
There are multiple reports that PPS induces thrombo- weight also inuences anticoagulant activity [118,150,153],
cytopenia when it is used therapeutically [143148]. Inter- with reasonably higher molecular weight molecules exhib-
estingly, PPS not only has the ability to induce iting greater activity [150].
thrombocytopenia, but exposure to PPS seems to sensitize Recently, chitosan has been extensively studied as a
patients to heparin. Subsequent exposure to heparin (and component of drug delivery. More specically, chitosans
to a lesser extent LMWHs) thrombocytopenia should be biocompatible properties [154] along with its ability to
786 Zhang et al.
complex biomolecules [155157] and its eects on trans- the above conditions plus the glycosidic branching play a
mucosal transit [158] led to its exploitation as a drug role in the activity of h-1,3-sulfoglucans not only in vitro
absorption enhancer. Chitosan polymers could potentially but also in vivo.
be used for delivery of a broad spectrum of therapeutics; Although h-1,3-sulfoglucans seem to be very compa-
DNA complexes with chitosan to form structures that have rable to heparin as antithrombotics in vivo the data from
been shown to increase transfection rates in vitro as well as coagulation assays indicate that these compounds have
in vivo [155], whereas peptide and protein absorption also lower activity [169,170]. This suggests the antithrombotic
increases using chitosan carriers [156,157]. Chitosans abil- activity observed is the result of additional mechanisms
ity to enhance absorption is believed to occur through beyond anticoagulation activity. One study suggests new
interaction with cellular membrane components and tight anticoagulants, such as h-1,3-sulfoglucans, whose eects
junctions. This interaction increases paracellular perme- are not adequately measured using current anticoagulant
ation of hydrophilic compounds [158]. These properties assays, and comparison of these agents to the gold stan-
show signicant enhancements in absorption of LMWH. dard (heparin) using PT and aPTT does not adequately
A 3% (w/v) low-viscosity mono-N-carboxymethyl show the agents antithrombotic potential [170].
chitosan (LMCC) signicantly increased the absorption Curdlan sulfate (CurS) serves as an anticoagulant
of LMWH across the intestinal membrane. Area under the through three dissimilar mechanisms: (1) the ability to
curve (AUC), a measure of total absorption of a drug, directly inhibit the thrombin-brinogen interaction; (2)
increased by 7 times, and the Cmax, a measure of the activation of plasma inhibitors (predominately HCII),
maximum concentration achieved in the plasma, increased which results in thrombin inhibition; and (3) complexation
by 5.4 times. Moreover, anti-Xa concentrations rose to a with brinogen [169]. CurS also activates the contact
level at or slightly above the therapeutic level [159]. Even system as indicated by its ability to stimulate kallikrein
though chitosan sulfate may not be the perfect anticoagu- generation [169]. This property of CurS is insignicant in
lant its use with LMWH may provide signicant improve- regard to anticoagulation in vivo but rather functions to
ments in oral administration over products that are break down brin. Exploitation of CurS as a probrinol-
currently available. ytic needs further investigation.
E. Curdlan F. Xylans
Curdlan is a natural polysaccharide with a linear h-1,3- Aside from pentosan, the original xylan to gain status as a
glucan structure. It is produced by Alcaligenes faecalis var. sulfonated polysaccharide with anticoagulant properties,
myxogenes, a bacterium normally found in the soil [160]. others have also been found to have such properties.
This naturally occurring carbohydrate is produced in large Xylans isolated from corncobs, oatspelts, and larchwood
amounts and used in the food industry; chemical sulfona- have been sulfated, resulting in potent anticoagulants more
tion results in h-1,3-glucans, which have been studied active than pentosan polysulfate [171]. Larchwood xylan
because of their biological properties. Curdlan possesses sulfate is the most active of these derivatives with an
immunostimulating properties in its natural form, and activity comparable to heparin [172]. This xylan seems to
chemically modied (sulfonation mainly) derivatives have function in a similar fashion as heparin, accelerating the
been shown to possess antiviral, anticoagulant, and anti- interaction between ATIII and factor IIa. Furthermore,
tumor properties [161164]. Some sulfonation products larchwood xylan sulfate seems to interact with thrombin,
also exhibit the ability to interact with growth factors directly inhibiting its eects on brinogen [171].
[165]. The chemical sulfonation of curdlan and other
polysaccharides with similar structure and the prod- G. Total Synthesis of Polysaccharide Sulfates
ucts of their chemical sulfonation have been extensively
studied due to the discrepancies between their in vitro and Only recently has complete synthesis of sulfated polysac-
in vivo activity. charides become an area of intense interest in anticoagu-
Data from the study of curdlan sulfates in coagulation lant development. The rst fully synthetic heparin
assays indicate the anticoagulant eect of these compounds oligosaccharide was produced in 1984 [173]. The synthe-
is dependent upon MW [166] and degree of sulfonation sized molecule consisted of a high-anity ATIII-binding
[167]. In addition to MW and degree of sulfonation, the pentasaccharide found naturally in heparin. It was quickly
substitution pattern of sulfo groups on the glucose mono- realized that this molecule had signicant advantages over
mer is also important in anticoagulant activity. Uniform the therapeutic agents available at that time. It also became
sulfo group distribution around glucose monomers and, as evident to researchers that for the rst time it may be
a consequence, more secondary hydroxyl-linked sulfo possible to produce heparin derivatives with very specic
groups improves the anticoagulant activity of h-1,3-sulfo activities [125]. Furthermore, use of homogeneous agents
glucans [168]. This indicates primary hydroxyl-linked sulfo in humans would be far easier to monitor, thereby reducing
groups at position 6 are not required for anticoagulant adverse eects and improving therapy [174]. Aside from the
eects [168]. Furthermore, this provides valuable informa- above, this synthesis provided extensive SAR data to the
tion about the need for primary hydroxyl-linked sulfo scientic community. It conrmed the reported structure
groups as a qualication for anticoagulant eects. In fact and activity described for the natural product, provided
complete explanation of ATIII SAR [175], and has allowed safer than heparin, generally resulting in less hemorrhagic
the production of unnatural derivatives [176]. complication, and have greater bioavailability and longer
The rst synthetic scheme resulting in an ATIII-bind- half-lives resulting in better dose control. In the future,
ing pentasaccharide was complicated with many steps and more subtle structural modications may be used to pre-
resulted in low yield of the product [173]. Subsequent pare heparin analogs that are entirely free from hemor-
schemes reported by multiple researchers decreased the rhagic side-eects.
number of steps, thereby increasing the yield [177179]. Over the last few decades heparin and heparan sulfate
The synthesis of this pentasaccharide ATIII-binding se- have been shown to interact with a number of biologically
quence resulted in the development of methods for the important proteins [20], thereby playing an essential role in
synthesis of larger sulfated polysaccharides modifying the the regulation of various physiological processes. Our
function of the molecule. understanding of these interactions at the molecular level
The extended synthetic targets were chosen based is important for the design of new, highly specic thera-
upon the structure, activity, and side eects of heparin. peutic agents. In addition, an understanding of the speci-
Mechanistic data from heparin studies suggest oligosac- city of heparin and heparan sulfate will be necessary to
charides with the ability to mimic heparin fully need two understand normal physiological and pathophysiological
distinct activities: the ability to bind to ATIII and thrombin processes (Fig. 9). These processes are particularly impor-
[180]. With the full elucidation of ATIII binding at hand, tant wherever cellcell interaction plays an important
further synthetic oligosaccharides were derived from exten- role, such as in developmental biology, cancer, wound
sions of the known ATIII-binding pentasaccharide (Fig. 2). healing, infectious diseases, inammatory processes, and
Thrombin binding is based on electrostatic interactions neurite outgrowth.
between the oligosaccharide and the protein [181]. There- Substantial research eort has focused on the prepa-
fore, introduction of the thrombin-binding domain signif- ration of improved heparins and heparin analogs that
icantly increases the anionic charge of the oligosaccharide might exhibit higher specicity and decreased side-eects.
and decreases the specicity of protein binding [182] (e.g., These heparin analogs or heparinoids include polysaccha-
more PF4 binding). This was the downfall of the initial rides from animals, plants, microorganisms, and inverte-
oligosaccharides that were generated [183]. Further rene- brates, synthetic derivatives of polysaccharides, and acidic
ment of the synthetic scheme was based on four consid- oligosaccharides and their small synthetic analogs. Many
erations: (1) a specic pentasaccharide sequence is required mammalian nonheparin glycosaminoglycans also exhibit
for ATIII binding [184,185]; (2) a thrombin-binding do- anticoagulant properties and present considerable thera-
main must consist of two to three disaccharides [186]; (3) a peutic potential. However, like heparin, they are heteroge-
17-saccharide chain is required for signicant thrombin neous mixtures and not much is known about their
inhibition [183]; and (4) the six to eight central saccharides mechanism of action. Several GAG mixtures are now
are not critical to either ATIII or thrombin interactions, clinically used as antithrombotic drugs; for example, dana-
therefore the charge of these residues can be altered paroid has been use as the usual alternative for anticoag-
without signicant loss in anticoagulant properties [183]. ulant treatment of HIT patients. Despite some successes
These ndings lead to the synthesis of a hexadecasaccha- in developing new heparinoid agents, substantial addi-
ride containing an ATIII-binding domain and a thrombin- tional eorts are needed to expand both the types of
binding domain separated by a neutral hexasaccharide molecules used as heparinoids and their dierent thera-
sequence possessing the ability of binding ATIII and peutic applications.
thrombin and devoid of PF4 binding (Fig. 8) [183,187]. There is now more interest in therapeutics prepared
Although these modications to the original pentasaccha- from nonmammalian sources to avoid the risk of contam-
ride have yielded products with improved properties and ination with pathogenic agents such as prions. Sulfated
activities the utility of theses derivatives in humans is still polysaccharides are widespread in the marine organisms,
under investigation. such as the carrageenans in red algae or the fucoidans
in brown algae, which show anticoagulant activity. Sub-
stantial research eort has been put in this large eld of
IV. CONCLUSIONS AND PERSPECTIVE potential heparinoids, but so far none of these has been
clinically tested. Heparin-like compounds with anticoagu-
Heparin has been the most commonly used clinical antico- lant activity were prepared by chemical modication of
agulant for more than 60 years. However, there are some bacteria polysaccharides. Thus, industrial-scale, homog-
side eects related to it, e.g., bleeding and heparin-induced eneous heparin-like substances with special anticoagu-
thrombocytopenia. Low molecular weight heparins were lant properties might be produced by biotechnological
developed as a new class of therapeutic agents called methods.
antithrombotics and oer several advantages over antico- Many other nonsulfated polysaccharides were used as
agulant heparin. These advantages include a decreased starting materials in an attempt to nd alternatives to
eect on platelets (reducing both their activation and natural anticoagulants. These unsulfated polysaccharides
aggregation) and increased specicity of action (presum- often had similar structure to heparin. Upon sulfonation, it
ably reducing their hemorrhagic side eects). The results of was found that many of these compounds were anticoagu-
clinical trials are promising suggesting that LMWHs are lants exhibiting considerable antithrombotic activity.
788
Figure 8 Structure of SR123781A.

Zhang et al.
4. Sixma, J.J.; Bouma, B.N. Physiology and biochemistry of

the haemostatic system. In The Thromboembolic Disor-
ders; van de Loo, J., Prentice, C.R.M., Beller, R.K., Eds.;
Schattauer: New York, 1983; 1 pp.
5. Linhardt, R.J. Heparin: An important drug enters its
seventh decade. Chem. Ind. 1991, 2, 45.
6. Howell, H.W. The purication of heparin and its chemical
and physiological reactions. Bull. Johns Hopkins Hosp.
1928, 42, 199.
7. Perlin, A.S.; Mazurek, M.; Jaques, L.B.; Kavanaugh,
L.W. A proton magnetic resonance spectral study of
heparin. L-Iduronic acid residues in commercial heparins.
Carbohydr. Res. 1968, 7, 369.
8. Lindahl, U.; Backstrom, G.; Thunberg, L.; Leder, I.G.
Figure 9 Involvement of heparin and heparan sulfate in Evidence for a 3-O-sulfated D-glucosamine residue in the
antithrombin-binding sequence of heparin. Proc. Natl.
important physiological processes.
Acad. Sci. USA 1980, 77, 6551.
9. Atha, D.H.; Lormeau, J.C.; Petitou, M.; Rosenberg,
R.D.; Choay, J. Contribution of monosaccharide residues
Nonetheless, only two such semisynthetic anticoagulants in heparin binding to antithrombin III. Biochemistry
1985, 24, 6723.
are currently in use: pentosan polysulfate and dextran 10. Linhardt, R.J.; Rice, K.G.; Kim, Y.S.; Lohse, D.L.;
sulfate. Although this search has not yielded considerably Wang, H.M.; Loganathan, D. Mapping and quantica-
better anticoagulants, it has yielded information about tion of the major oligosaccharide components of heparin.
structure and activity, which may ultimately play an im- Biochem. J. 1988, 254, 781.
portant role in the development of further clinically useful 11. Loganathan, D.; Wang, H.M.; Mallis, L.M.; Linhardt,
compounds. One concern about semisynthetic molecules is R.J. Structural variation in the antithrombin III binding
site region and its occurrence in heparin from dierent
that they contain unnatural structures that may be resistant sources. Biochemistry 1990, 29, 4362.
to metabolism, preventing clearance and resulting in long 12. Pervin, A.; Gallo, C.; Jandik, K.A.; Han, X.J.; Linhardt,
half-lives and toxicity. R.J. Preparation and structural characterization of large
Dependence on natural sources for pharmaceutical heparin-derived oligosaccharides. Glycobiology 1995, 5,
products has its downside. The desire to be free from the 83.
variability of nature has led to the development of synthetic 13. Gatti, G.; Casu, B.; Hamer, G.K.; Perlin, A.S. Studies on
the conformation of heparin by 1H and 13CNMR
heparin oligosaccharides with considerable anticoagulant
spectroscopy. Macromolecules 1979, 12, 1001.
activity. These compounds lack the polydispersity found in 14. Mulloy, B.; Forster, M.J.; Jones, C.; Davies, D.B. NMR
the unfractioned heparin, low molecular weight heparin, and molecular-modelling studies of the solution confor-
and the semisynthetic compounds developed to date. These mation of heparin. Biochem. J. 1993, 293, 849.
fully synthetic products have considerable consistency, a 15. Mikhailov, D.; Linhardt, R.J.; Mayo, K.H. NMR
characteristic the Food and Drug Administration and solution conformation of heparin-derived hexasaccharide.
other regulatory bodies admire. The most notable, fully Biochem. J. 1997, 328, 51.
16. Faham, S.; Hileman, R.E.; Fromm, J.R.; Linhardt, R.J.;
synthetic heparin mimetic is the ATIII-binding pentasac- Rees, D.C. Heparin structure and interactions with basic
charide found in heparin. The pentasaccharide was initially broblast growth factor. Science 1996, 271, 1116.
synthesized via a complicated and expensive scheme that 17. Hileman, R.E.; Fromm, J.R.; Weiler, J.M.; Linhardt, R.J.
may not be suitable for wide-scale, commercial exploita- Glycosaminoglycan-protein interaction: Denition of
tion. Far less complex synthetic procedures are under consensus sites in glycosaminoglycan binding proteins.
development of larger oligosaccharides with additional BioEssays 1998, 20, 156.
18. Conrad, H.E. Heparin-Binding Proteins; Academic Press:
activities. These studies have also yielded considerable data San Diego, CA, 1998; 1 pp.
about the cause of platelet activation in HIT patients, 19. Jackson, R.L.; Busch, S.J.; Cardin, A.D. Glycosamino-
possibly facilitating the synthesis of oligosaccharides de- glycans: Molecular properties, protein interactions, and
void of such activity. role in physiological processes. Pharmacol. Rev. 1991,
71, 481.
20. Ishan, C.; Linhardt, R.J. Heparinprotein interactions.
REFERENCES Angew. Chem. Int. Ed. Engl. 2002, 41, 390.
21. Casu, B.; Lindahl, U. Structure and biological interac-
1. McLean, J. The thromboplasticaction of cephalin. Am. J. tions of heparin and heparan sulfate. Adv. Carbohydr.
Physiol. 1916, 41, 250. Chem. Biochem. 2001, 57, 159.
2. El-Nokaly, M.A.; Soini, H.A. Preface. In Polysaccharide 22. Linhardt, R.J.; Toida, T. Heparin oligosaccharides: new
Applications; Cosmetics and Pharmaceuticals; EI-Nokaly, analoguesdevelopment and applications. In Carbohy-
M.A., Soini, H.A., Eds.; American Chemical Society: drates in Drug Design; Witczak, Z.J., Nieforth, K.A., Eds.;
Washington, DC, 1999; XIII pp. Marcel Dekker: NY, 1997; 277 pp.
3. Brenner, B. In Hemostasis and thrombosis: Physiological 23. Alban, S. Carbohydrates with anticoagulant and antith-
and pathological aspects, Thrombosis and Antithrombotic rombotic properties. In Carbohydrates in Drug Design;
Therapy; Lugassy, G., Ed.; Martin Dunitz, London, 2000; Witczak, Z.J., Nieforth, K.A., Eds.; Marcel Dekker: NY,
1 pp. 1997; 209 pp.
790 Zhang et al.
24. Casu, B. Structure and biological activity of heparin. Adv. heparan sulfate hexuronic acid 2-O-sulfation. J. Biol.
Carbohydr. Chem. Biochem. 1985, 43, 51. Chem. 1996, 271, 17711.
25. Comper, W.D. Heparin (and Related Polysaccharides); 45. Bjork, I.; Olson, S.T.; Shore, J.D. Molecular mechanisms
Gordon and Breach Science Publ., 1981; Vol. 7. of the accelerating eect of heparin on the reactions
26. Lane, D.A.; Lindahl, U. Heparin Chemical and Biological between antithrombin and clotting proteinases. In Hep-
Properties Clinical Applications; CRC Press: Boca Raton, arin, Chemical and Biological Properties; Lane, D.A.,
FL, 1989. Lindahl, U., Eds.; Edward Arnold: London, 1989; 229 pp.
27. Gallagher, J.T.; Turnbull, J.E.; Lyon, M. Heparan 46. Rosenberg, R.D.; Damus, P.S. The purication and
sulphate proteoglycans: Molecular organisation of mem- mechanism of action of human antithrombinheparin
brane-associated species and an approach to polysaccha- cofactor. J. Biol. Chem. 1973, 248, 6490.
ride sequence analysis. Adv. Exp. Med. Biol. 1992, 313, 47. Marcum, J.A.; Rosenberg, R.D. The biochemistry, cell
49. biology, and pathophysiology of anticoagulantly active
28. Grin, C.C.; Linhardt, R.J.; VanGorp, C.L.; Toida, T.; heparin-like moleculars of the vessel wall. In Heparin,
Hileman, R.E.; Schubert, R.L.; Brown, S.E. Isolation and Chemical and Biological Properties; Lane, D.A., Lindahl,
characterization of heparan sulfate from crude porcine U., Eds.; Edward Arnold: London, 1989; 275 pp.
intestinal mucosa peptidoglycan heparin. Carbohydr. Res. 48. Grith, M.J.; Noyes, C.M.; Church, F.C. Reactive site
1995, 276, 183. peptide structural similarity between heparin cofactor II
29. Berneld, M.; Kokeyesi, R.; Kato, M.; Hinkes, M.T.; and antithrombin III. J. Biol. Chem. 1985, 257, 2162.
Spring, J.; Gallo, R.L.; Lose, E.J. Biological of syndecans. 49. Tran, T.H.; Zbinden, B.; Lammle, B.; Duckert, F.
Annu. Rev. Cell. Biol. 1992, 8, 365. Methodology and clinical signicance of heparin cofactor
30. Lindahl, U.; Lindholt, K.; Spillmann, D.; Kjellen, L. II. Semin. Thromb. Hemost. 1985, 11, 342.
More to heparin than anticoagulation. Thromb. Res. 50. Tollefsen, D.M. Activation of heparin cofactor II by
1994, 75, 1. heparin dermatan sulfate. Nouv. Rev. Fr. Hematol. 1984,
31. Rosenberg, R.D.; Schworak, N.W.; Liu, J.; Schwartz, J.J.; 26, 233.
Zhang, L. Heparan sulfate proteoglycans of the cardio- 51. Linhardt, R.J.; Hileman, R.E. Dermatan sulfate as a
vascular system. Specic structures emerge but how is potential therapeutic agent. Gen. Pharmacol. 1995, 26,
synthesis regulated? J. Clin. Invest. 1997, 99, 2062. 443.
32. Berneld, M.; Gotte, M.; Park, P.W.; Reizes, O.; 52. Bijsterveld, N.R.; Hettiarachchi, R.J.K.; Peters, R.; Prins,
Fitzgerald, M.L.; Lincecum, J.; Zako, M. Functions of M.H.; Levi, M.; Buller, H.R. Low-molecular weight
cell surface heparan sulfate proteoglycans. Annu. Rev. heparins in venous and arterial thrombotic disease.
Biochem. 1999, 68, 729. Thromb. Haemost. 1999, 82 (Suppl. 2), 139.
33. Linhardt, R.J. Analysis of glycoconjugates. In Current 53. Hettiarachchi, R.J.; Smorenburg, S.M.; Ginsberg, J.;
Protocols in Molecular Biology; Varki, A., Ed.; Wiley Levine, M.; Prins, M.H.; Buller, H.R. Antithrombotic
Interscience: Winston-Salem, 1994; 17.13.1717.13.32. drugs in the primary medical management of intermittent
34. Lindahl, U.; Kjellen, L. The Biology of the Extracellular claudication: A meta-analysis. Thromb. Haemost. 1999,
Matrix: Proteoglycans; Academic Press: New York, 1987. 82, 947.
35. Salmivirta, M.; Lindholt, K.; Lindahl, U. Heparan 54. Smorenburg, S.M.; van Noorden, C.J.F. The complex
sulfate: A piece of information. FASEB J. 1996, 10, 1270. eects of heparins on cancer progression and metastasis in
36. Sugahara, K.; Kitagawa, H. Recent advances in the study experimental studies. Pharmacol. Rev. 2001, 53, 93.
of the biosynthesis and functions of sulfated glycosami- 55. Brister, S.J.; Buchanaw, M.R.; Grin, C.C.; VanGorp,
noglycans. Curr. Opin. Struct. Biol. 2000, 10, 518. C.L.; Linhardt, R.J. Dermatan Disulfate, an Inhibitor of
37. Zhang, L.; David, G.; Esko, J.D. Repetitive Ser-Gly Thrombin and Complement Activation. US Patent
sequences enhance heparan sulfate assembly in proteo- 5,922,690, 1999.
glycans. J. Biol. Chem. 1995, 270, 27127. 56. Borsig, L.; Wong, R.; Feramisco, J.; Nadeau, D.R.;
38. Fritz, T.A.; Gabb, M.M.; Wei, G.; Esko, J.D. Two N- Varki, N.M.; Varki, A. Heparin and cancer revisited:
acetylglucosaminyltransferases catalyze the biosynthesis Mechanistic connections involving platelets, P-selectin,
of heparan sulfate. J. Biol. Chem. 1994, 269, 28809. carcinoma mucins, and tumor metastasis. Proc. Natl.
39. McCormick, C.; Duncan, G.; Goutsos, K.T.; Tufaro, F. Acad. Sci. USA 2001, 98, 3352.
The putative tumor suppressors EXT1 and EXT2 form a 57. Langer, R.; Linhardt, R.J.; Hoberg, S.; Larsen, A.K.;
stable complex that accumulates in the Golgi apparatus Cooney, C.L.; Tapper, D.; Klein, M. An enzymatic
and catalyzes the synthesis of heparan sulfate. Proc. Natl. system for removing heparin in extracorporeal therapy.
Acad. Sci. USA 2000, 97, 668. Science 1982, 217, 261.
40. Lindholt, K.; Lindahl, U. Biosynthesis of heparin. The D- 58. Freedman, M.D. Pharmacodymamics, clinical indications
glucuronosyl- and N-acetyl-D-glucosaminyltransferase re- and adverse eects of heparin. J. Clin. Pharmacol. 1992,
actions and their relation to polymer modication. 32, 584.
Biochem. J. 1992, 287, 21. 59. Wallis, D.E.; Quintos, R.; Wehrmacher, W.H.; Messmore,
41. Lindahl, U.; Backstrom, G.; Malmstrom, A.; Fransson, H. Safety of warfarin anticoagulation in patients with
L.-A. Biosynthesis of L-iduronic acid in heparin: Epime- heparin-induced thrombocytopenia. Chest 1999, 116,
rization of D-glucuronic acid on the polymer level. 1333.
Biochem. Biophys. Res. Commun. 1972, 46, 985. 60. Walenga, J.M.; Bick, R.L. Heparin-induced thrombocy-
42. Jacobsson, I.; Lindahl, U. Biosynthesis of heparin. topenia, paradoxical thromboembolism, and other side
Concerted action of late polymer-modication reactions. eects of heparin therapy. Med. Clin. North Am. 1998,
J. Biol. Chem. 1980, 255, 5094. 82, 635.
43. Kusche, M.; Torri, G.; Casu, B.; Lindahl, U. Biosynthesis 61. Laurent, T.; Tengbland, A.; Thunberg, L.; Hook, M.;
of L-iduronic acid in heparin: Epimerization of D- Linhahl, U. The molecular-weight-dependence of the anti-
glucuronic acid on the polymer level. J. Biol. Chem. coagulant activity of heparin. Biochem. J. 1978, 175, 691.
1990, 265, 15403. 62. Linhardt, R.J.; Rice, K.G.; Kim, Y.S.; Lohse, D.L.;
44. Bai, X.; Esko, J.D. An animal cell mutant defective in Wang, H.M.; Loganathan, D. Mapping and quantica-
tion of the major oligosaccharide components of heparin. man, M.A. Inhibition of factor X and factor V activation
Biochem. J. 1988, 254, 781. by dermatan sulfate and a pentasaccharide with high
63. Kim, Y.S.; Linhardt, R.J. Structural features of heparin anity for antithrombin II in human plasma. Eur. J.
and their eect on heparin cofactor II mediated inhibition Biochem. 1990, 193, 485.
of thrombin. Thromb. Res. 1989, 53, 55. 83. Maarou, R.M.; Tapon-Bretaudiere, J.; Mardiguian, J.;
64. Gunay, N.S.; Linhardt, R.J. Production and chemical Sternberg, C.; Dautzenberg, M.D.; Fischer, A.M. Inu-
processing of low molecular weight heparins. Semin. ence of the oversulfation method and the degree of
Thromb. Haemost. 1999, 25, 5. sulfation on the anticoagulant properties of dermatan
65. Fareed, J.; Jeske, W.; Hoppensteadt, D.; Clarizio, R.; sulfate derivatives. Thromb. Res. 1990, 59 (4), 749758.
Walenga, J.M. Low-molecular-weight heparins: Pharma- 84. Saivin, S.; Caranobe, C.; Petitou, M.; Sie, P.; Lormeau,
cologic prole and product dierentiation. Am. J. J.C.; Level, M.; Crepon, B.; Houin, G.; Boneu, B.
Cardiol. 1998, 82, 3L10L. Antithrombotic activity, bleeding eect and pharmacody-
66. Rosenberg, R.D. Biochemistry and pharmacology of low namics of a succinyl derivative of dermatan sulphate in
molecular weight heparin. Sem. Hematol. 1997, 34, 2. rabbits. Br. J. Haematol. 1992, 80 (4), 509.
67. Linhardt, R.J. Chemical and enzymatic methods for the 85. Maruyama, T.; Toida, T.; Imanari, T.; Yu, G.; Linhardt,
depolymerization and modication of heparin. In Carbo- R.J. Conformational changes and anticoagulant activity
hydratesSynthetic Methods and Applications in Medici- of chondroitin sulfate following its O-sulfonation. Carbo-
nal Chemistry; Weinheim Ogura, H., Hasegawa, A., hydr, Res. 1998, 306, 35.
Suami, T., Eds.; Kodansha/VCH Publishers: Tokyo, 86. Bourin, M.C.; Ohlin, A.K.; Lane, D.; Steno, J.; Lindahl,
Japan, 1992; Vol. 20, 385 pp. U. Relationship between anticoagulant activities and
68. Linhardt, R.J.; Loganathan, D.; Al-Hakim, A. Oligosaccha- polyanionic properties of rabbit thrombomodulin. J. Biol.
ride mapping of low molecular weight heparins: Structure Chem. 1988, 263, 8044.
and activity dierences. J. Med. Chem. 1990, 33, 1639. 87. Sie, P.; Ofosu, F.; Fernadez, F.; Buchanan, M.R.; Petitou,
69. Fussi, F. Process for Obtaining Low Molecular Weight M.; Boneu, B. Br. J. Haematol. 1986, 64, 707.
Heparins. European Patent GB2, 002, 406B, 1982. 88. Bourin, M.C.; Lundgren, A.; Kerlund, E.; Lindahl, U.
70. Lormeau, J.-C.; Petitou, M.; Choay, J. Oligosaccha- Isolation and characterization of the glycosaminoglycan
rides Having Anti-Xa Activity and Pharmaceutical component of rabbit thrombomodulin proteoglycan. J.
Compositions Containing Them. US Patent #RE 35770, Biol. Chem. 1990, 265, 15424.
1998. 89. Qiu, G.; Toida, T.; Imanari, T. Structural diversity of
71. Lormeau, J.-C.; Petitou, M.; Choay, J. Method for Ob- dermatan sulphate in porcine dermis. Biol. Pharm. Bull.
taining Biologically Active Mucopolysaccharides of High 1997, 102, 721.
Purity, by Controlled Depolymerization of Heparin. US 90. Casu, B. Structural features and binding properties of
Patent #5019649, 1991. chondroitin sulfates, dermatan sulfate, and heparan
72. Lohse, D.L.; Linhardt, R.J. Purication and character- sulfate. Semin. Thromb. Haemost. 1991, 17S, 9.
ization of heparin lyases from Flavobacterium heparinum. 91. Kim, Y.S.; Jo, Y.T.; Chang, I.M.; Toida, T.E.; Park, Y.;
J. Biol. Chem. 1992, 267, 24347. Linhardt, R.J. A new glycosaminoglycan from the giant
73. Linhardt, R.J. Analysis of glycosaminoglycans with African snail Achatina fulica. J. Biol. Chem. 1996, 271,
polysaccharide lyases. In Current Protocols in Molecular 11750.
Biology, Analysis of Glycoconjugates; Varki, A., Ed.; Wiley 92. Wu, S.J.; Chun, M.W.; Shin, K.H.; Toida, T.; Park, Y.;
Interscience: Boston, MA, 1994; 17.13.1717.13.32. Linhardt, R.J.; Kim, Y.S. Chemical oversulfation and
74. Piani, S.; Tamagnone G.; Alpino, R.R.; Milani, M.R. anticoagulant activity of acharan sulfate. Thromb. Res.
Heparin Derivatives and Process for Their Preparation. 1998, 92, 273.
US Patent #5010063, 1991. 93. Greiling, H., Scott, J., eds. Keratan Sulfate Chemistry,
75. Lopez, L.L. Depolymerization Method of Heparin. US Biology, Chemical Pathology; T.J. Press: Great Britain,
Patent #4981955, 1991. 1989.
76. Maruyama, T.; Toida, T.; Imanari, T.; Yu, G.; Linhardt, 94. Funderburgh, J.L. Keratan sulfate: Structure, biosyn-
R.J. Conformational changes and anticoagulant activity thesis, and function. Glycobiology 2000, 10, 951.
of chondroitin sulfate following its O-sulfonation. Carbo- 95. Kennedy, J.F., White, C.A., Eds.; Bioactive Carbohy-
hydr. Res. 1998, 306, 35. drates; New York: Ellis Harwood, 1983.
77. Marcum, J.A.; Rosenberg, R.D. Anticoagulantly active 96. Kindness, G.; Long, W.F.; Williamson, F.B. Enhancement
heparan sulfate proteoglycan and the vascular endothe- of ATIII activity by carrageenans. Thromb. Res. 1979, 15,
lium. Semin. Thromb. Hemost. 1987, 13, 464. 49.
78. Cofrancesco, E.; Colombi, M.; Gianese, F.; Cortellaro, 97. Guven, K.V.; Ozsoy, Y.; Ulutin, O.N. Anticoagulant,
M. The eect of dermatan sulfate on in vitro human brinolytic and antiaggregant activity of carrageenans
plasma coagulation, platelet aggregation and beta TG/ and alginic acid. Bot. Mar. 1991, 34, 429.
PF4 release. Thromb. Res. 1990, 57, 405. 98. Caceres, P.J.; Carlucci, M.J.; Damonte, E.B.; Matsuhiro,
79. Hoppensteadt, D.; Walenga, J.; Fareed, J. Eect of B. Carrageenans from Chilean samples of Stenogramme
dermatan sulfate and heparan sulfate on platelet activity interrupta (Phyllophoraceae): Structural analysis and bio-
compared to heparin. Semin. Thromb. Hemost. 1991, 17 logical activity. Phytochemistry 2000, 53, 81.
(Suppl. 1), 60. 99. Pereira, M.S.; Mulloy, B.; Mourao, P.S. Structure and
80. Sie, P.; Dupouy, D.; Caranobe, C.; Petitou, M.; Boneu, B. anticoagulant activity of sulfated fucans. J. Biol. Chem.
Antithrombotic properties of dermatan sulfate hexadeca- 1999, 274, 7656.
saccharide fractionated by anity for heparin cofactor II. 100. Church, F.C.; Meade, J.B.; Treanor, R.E.; Whinna, H.C.
Blood 1993, 81, 1771. Antithrombin activity of fucoidan. The interaction of
81. Merton, R.E.; Thomas, D.P. Experimental studies on fucoidan with heparin cofactor II, antithrombin III, and
the relative ecacy of dermatan sulfate and heparin as thrombin. J. Biol. Chem. 1989, 264, 3618.
antithrombotic agents. Thromb. Haemost. 1987, 58, 839. 101. Nishino, T.; Aizu, Y.; Nagumo, T. The inuence of sulfate
82. Ofosa, F.A.; Choay, J.; Anvari, N.; Smith, L.M.; Blajch- content and molecular weight of a fucan sulfate from the
792 Zhang et al.
brown seaweed Ecklonia kurome on its antithrombin 119. Nishimura, S.-I.; Nishi, N.; Tokura, S.; Okiei, W.;
activity. Thromb. Res. 1991, 64, 723. Somorin, O. Inhibition of the hydrolytic activity of
102. Colliec, S.; Fisher, A.M.; Tapon-Bretaudiere, J.; Boisson, thrombin by chitin heparinoids. Carbohydr. Res. 1986,
C.; Durant, P.; Jozefonvicz, J. Anticoagulant properties of 156, 286.
a fuco dan fraction. Thromb. Res. 1991, 64, 143. 120. Nagasawa, D.; Harada, H.; Hayashi, S.; Misawa, T.
103. Baba, M.; Nakajima, M.; Schols, D.; Pauwels, R.; Sulfation of dextran with piperidine-N-sulfonic acid.
Balzarini, J.; de Clerq, E. Pentosan polysulfate, a sulfated Carbohydr. Res. 1972, 21, 420.
oligosaccharide, is a potent and selective anti-HIV agent 121. Ricketts, C.R. Dextran sulphateA synthetic analogue of
in vitro. Antiviral Res. 1988, 9, 335. heparin. Biochemistry 1952, 51, 129.
104. McClure, M.O.; Moore, J.P.; Blanc, D.F.; Scotting, P.; 122. Kamide, K.; Saito, M. Cellulose and cellulose derivatives:
Cook, G.M.; Keynes, R.J.; Weber, J.N.; Davies, D.; Recent advances in physical chemistry. Adv. Polym. Sci.
Weiss, R.A. Investigations into the mechanism by which 1987, 83, 1.
sulfated polysaccharides inhibit HIV infection in vitro. 123. Yu, G.; Gunay, N.S.; Linhardt, R.J.; Toida, T.; Fareed,
AIDS Res. Hum. Retroviruses 1992, 8, 19. J.; Hoppensleadt, D.A.; Shadid, H.; Ferro, V.; Li, C.;
105. Baba, M.; Snoeck, R.; Pauwels, R.; de Clercq, E. Sulfated Fewings, K.; Parermo, M.C.; Podger, D. Preparation,
polysaccharides are potent and selective inhibitors of composition, fractionation, structural characterization
various enveloped viruses, including herpes simplex virus, and anticoagulant activity of phosphosulfomannan PI-
cytomegalovirus, vesicular stomatitis virus, and human 88. Eur. J. Med. Chem. 2002, 37, 783.
immunodeciency virus. Antimicrob. Agents Chemother. 124. Greinacher, A.; Alban, S.; Dummel, V.; Franz, G.;
1988, 32, 1742. Mueller-Eckhardt, C. Characterization of the structural
106. Garc a-Villalon, D.; Gil-Fernandez, C. Antiviral activity requirements for a carbohydrate based anticoagulant with
of sulfated polysaccharides against African swine fever a reduced risk of inducing the immunological type of
virus. Antiviral Res. 1991, 15, 139. heparin-associated thrombocytopenia. Thromb. Haemost.
107. Grauel, V.; Kloareg, B.; Mabeau, S.; Durand, P.; 1995, 74, 866.
Jozefonvicz, J. New natural polysaccharides with potent 125. Linhardt, R.J.; Toida, T. Heparin oligosaccharides: New
antithrombic activity: Fucans from brown algae. Bio- analogues development and applications. In Carbohy-
materials 1989, 10, 363. drates in Drug Design; Witczak, Z.J., Nieforth, K.A., Eds.;
108. Farias, W.R.L.; Valente, A.-P.; Pereira, M.S.; Mourao, Marcel Dekker: New York, 1997; 277 pp.
P.A.S. Structure and anticoagulant activity of sulfated 126. The Merck Index, 9th Ed.; Windholz, M., Ed.; Merck
galactans. J. Biol. Chem. 2000, 275, 29299. Publishing Co.: Rahway, 1976; 2911 pp.
109. Razi, N.; Feyzi, E.; Bjork, I.; Naggi, A.; Casu, B.; 127. Wafenvoord, R.J.; Hendrix, H.H.; Kolde, H.J.; Hemker,
Lindahl, U. Structural and functional properties of H.C. Development of a rapid and sensitive chromogenic
heparin analogues obtained by chemical sulphation of heparin assay for clinical use. Haemostasis 1993, 23, 26.
Escherichia coli K5 capsular polysaccharide. Biochem. J. 128. Mitsuya, H.; Looney, D.J.; Duno, S.; Ueno, R.; Wong-
1995, 309, 465. Staal, F.; Broder, S. Dextran sulfate suppression of viruses
110. Casu, B.; Grazioli, G.; Razi, N.; Guerrini, M.; Naggi, A.; in the HIV family: Inhibition of virion binding to CD4+
Torri, G.; Oreste, P.; Tursi, F.; Zoppetti, G.; Lindahl, U. cells. Science 1988, 240, 646.
Heparin-like compounds prepared by chemical modica- 129. Bergqvist, D.; Efsing, H.O.; Hallbrook, T.; Lindblad, B.
tion of capsular polysaccharide from E. coli K5. Prevention of postoperative thromboembolic complica-
Carbohydr. Res. 1994, 263, 271. tions. Acta Chir. Scand. 1980, 146, 599.
111. Sutherland, I. A sticky business, microbial polysacchar- 130. Fischer, A.M.; Dautzenberg, M.D.; Aurousseau, M.H.;
ides: Current products and future trends. Microbiol. Beguin, S.; Goudemand, G.; Hemker, H.C. Comparison
today 2002, 29, 70. between the eect of pentosan polysulphate, heparin and
112. Gunay, N.S.; Linhardt, R.J. Heparinoids: Structure, antithrombin III injections in antithrombin III decient
biological activities and therapeutic applications. Planta patients. Thromb. Res. 1985, 37, 295.
Med. 1999, 65, 301. 131. Wagenvoord, R.; Hendrix, H.; Soria, C.; Hemker, H.C.
113. Magnani, A.; Albanese, A.; Lamponi, S.; Barbucci, R. Localization of the inhibitory site(s) of pentosan polysulfate
Blood-interaction performance of dierently sulphated in blood coagulation. Thromb. Haemost. 1988, 60, 220.
hyaluronic acids. Thromb. Res. 1996, 81, 3. 132. Wagenvoord, R.; Hendrix, H.; Soria, C.; Hemker, H.C.
114. Horton, D.; Usui, T. Sulfated carbohydrates obtained by Determination of the non-antithrombin II dependent
chemical modication of polysaccharides. In Carbohy- inhibitor sites of pentosan polysulfate in the blood
drate Sulfates; Schweiger, R.G., Ed.; American Chemical coagulation. First International Symposium on Pentosan
Society: Washington DC, 1978; 95 pp. Polysulfate, Paris, 1985, 5pp.
115. Baumann, H. The role of regioselectively sulfated and 133. Fischer, A.M.; Merton, R.E.; Marsh, N.A.; Williams, S.;
acetylated polysaccharide coatings of biomaterial for Ganey, P.J.; Barrow Cli, T.W.; Thomas, D.P. A com-
reducing platelet and plasma protein adhesion. Semin. parison of pentosan polysulfate and heparin: II. Eects of
Thromb. Hemost. 2001, 27, 445. subcutaneous injection. Thromb. Haemost. 1982, 47, 109.
116. Guiseley, D.B. Some Novel Methods and Results in the 134. Augmaier, G.; Lippman, M.E.; Wellstein, A. Inhibition
Sulfation of Polysaccharides. In Carbohydrate Sulfates; by pentosan polysulfate (PPS) of heparin-binding growth
Schweiger, R.G., Ed.; American Chemical Society: Wash- factors released from tumor cells and blockage by PPS of
ington DC, 1978; 144 pp. tumor growth in animals. J. Natl. Can. Inst. 1992, 84,
117. Whistler, R.L.; Spencer, W.W. Preparation and properties 1716.
of several polysaccharides. Arch. Biochem. Biophys. 1961, 135. Maciag, T.; Hoover, F.; Sternerman, M.; Weinstein, R.
95, 36. Serial propagation of human endothelial cells in vitro. J.
118. Muzzarelli, R.A.A.; Tanfani, F.; Emanuelli, M.; Pace, Cell. Biol. 1981, 91, 420.
D.P.; Chiurazzi, E.; Piani, M. Sulfated N-(carboxyme- 136. Swain, S.M.; Parker, B.; Wellstein, A.; Lippman, M.E.;
thyl)chitosan: Novel blood anticoagulants. Carbohydr. Steakley, C.; DeLap, R. Phase I trial of pentosan
Res. 1986, 126, 255. polysulfate. Invest. New Drugs 1995, 13, 55.
137. Giedrojc, J.; Klimiuk, M.; Radqiwon, P.; Kloczko, J.; immunologic protection in murine model of peanut
Bielawiec, M.; Breddin, H.K. Comparative study on the in allergy. Nat. Med. 1999, 5, 387.
vitro and in vivo activities of heparinoids derivative 156. Illum, L.; Farraj, N.R.; Davis, S.S. Chitosan as a novel
investigated on the animal model. J. Cardiovasc. Pharma- nasal delivery system for peptide drugs. Pharm. Res. 1994,
col. 1999, 34, 340. 11, 1186.
138. Klocking, H.P.; Hauptmann, J.; Richter, M. Probrino- 157. Thanou, M.; I orae, B.; Langermeyer, M.W.E.; Verhoef,
lytic and anticoagulant properties of the pentosan poly- J.C.; Junginger, H.E. N-Trimethylated chitosan chloride
sulphate derivative bego 0391. Pharmazie 1991, 46, 543. (TMC) improves the intestinal permeation of the peptide
139. Vinazzer, H.; Stermberger, A.; Haas, S.; Blumel, G. drug buserelin in vitro (Caco-2 cells) and in vivo (rats).
Inuence of heparin, of dierent heparin fractions and of Pharm. Res. 2000, 17, 27.
a low molecular weight heparin-like substance on the 158. Schipper, N.G.M.; Olsson, S.; Hoogstraate, J.A.; deBoer,
mechanism of brinolysis. Thromb. Res. 1982, 27, 341. A.G.; Varum, K.M.; Artursson, R. Chitosans as absorp-
140. Marsh, N.A.; Peyser, P.M.; Creighton, L.J.; Mahmound, tion enhancers for poorly absorbable drugs. Pharm. Res.
M.; Ganey, P.J. The eect of pentosan polysulphate 1997, 14, 923.
(SP54) on the brinolytic enzyme systemA human 159. Thanou, M.; Nihot, M.T.; Jansen, M.; Verhoef, J.C.;
volunteer and experimental animal study. Thromb. Hae- Junginger, H.E. Mono-N-carboxymethyl chitosan
most. 1985, 54, 833. (MCC), a polyampholytic chitosan derivative, enhances
141. Ganey, P.J.; Marsh, N.A. The eect of pentosan the intestinal absorption of low molecular weight heparin
polysulphate (SP54) on human brinolytic system. Folia across intestinal epithelia in vitro and in vivo. J. Pharm.
Haematol. 1986, 113, 262. Sci. 2001, 90, 38.
142. Klocking, H.P.; Markwardt, F. Release of plasminogen 160. Harada, T. The story of research into curdlan and the
activator by pentosan polysulphate. Thromb. Res. 1986, bacteria producing it. Trends Glycosci. Glycotechnol.
41, 739. 1992, 4, 309.
143. Vioux, J.F.; Roncato, M.; Hourdebaigt, P.; Aiach, M.; 161. Sasaki, T.; Abiko, N.; Sugino, Y.; Nitta, K. Dependence
Fiessinger, J.N. Heparin-induced thrombocytopenia and on chain length of antitumor activity of h-1,3-glucan from
pentosan polysulfate: Treatment with a low molecular Alcaligenes faecalis var. myxogenes, IFO 13140, and its
weight heparin despite in vitro platelet aggregation. acid degraded products. Cancer Res. 1978, 38, 379.
Thromb. Haemost. 1985, 55, 294. 162. Yoshida, T.; Hatanaka, K.; Uryu, T.; Kaneko, Y.; Suzuki,
144. Gouault-Heilmann, M.; Payen, D.; Contant, G.; Intrator, E.; Miyano, H.; Mimura, T.; Yoshida, O.; Yamamoto, N.
L.; Huet, Y.; Schaeer, A. Thrombocytopenia related to Synthesis and structural analysis of curdlan sulfate with
synthetic heparin analogue therapy. Thromb. Haemost. potent inhibitory eect in vitro and on AIDS virus
1985, 54, 557. infection. Macromolecules 1990, 23, 3717.
145. Follea, G.; Hamandjian, I.; Trzeciak, M.C.; Nedey, C.; 163. Whistler, R.L.; Bushway, A.A.; Singh, P.P. Nontoxic,
Streichenberger, R.; Dechavanne, M. Pentosan polysul- antitumor polysaccharides. Adv. Carbohydr. Chem. 1976,
phate associated thrombocytopenia. Thromb. Res. 1986, 32, 235.
42, 413. 164. Alban, S.; Franz, G. Anticoagulant activity of curdlan
146. Marand, J.; Herbert, J.; Bernat, A.; Defreyn, G.; sulfates in dependence on their molecular weight. Pure
Delebassee, D.; Savi, P.; Pinot, J.; Sampol, J. Exper- Carbohydr. Chem. 1994, 66, 2403.
imental and clinical pharmacology of pentosan polysul- 165. Paper, D.H.; Homan, R.; Alban, S.; Franz, G.; Blechen,
fate. Semin. Thromb. Hemost. 1991, 17 (Suppl. 2), 186. N.M. Eect of laminarin sulfates on transforming growth
147. Tardy-Poncet, B.; Tardy, B.; Brelac, F.; Reynaud, J.; factor a and basic broblast growth factor. Planta Med.
Mismetti, P.; Bertrand, J.C.; Guyotat, D. Pentosan 1992, 58, A585.
polysulfate-induced thrombocytopenia and thrombosis. 166. Alban, S. Synthese and physiologische testung neuartiger
Am. J. Hematol. 1994, 45, 252. heparinode; Dissertation, University of Regensburg, 1993.
148. Goad, K.E.; Horne, M.K. III; Gralnick, H.R. Pentosan- 167. Alban, S.; Krasus, J.; Franz, G. Synthesis of laminarin
induced thrombocytopenia: Support for an immune sulfates with anticoagulant activity. Arzneim.-Forsch./
complex mechanism. Br. J. Haematol. 1994, 88, 803. Drug Res. 1992, 42, 1005.
149. Vongchan, P.; Sajomsang, W.; Subyen, D.; Kongtawelert, 168. Alban, S.; Franz, G. Gas liquid chromatography-mass
P. Anticoagulant activity of a sulfated chitosan. Carbo- spectrometry analysis of anticoagulant active curdlan
hydr. Res. 2002, 337, 1239. sulfates. Semin. Thromb. Hemost. 1994, 20, 152.
150. Hirano, S.; Tanaka, Y.; Hasegawa, M.; Tobetto, K.; 169. Alban, S.; Franz, G. Characterization of the anticoagu-
Nishioka, A. Eect of sulfated derivatives of chitosan on lant action of a semisynthetic curdlan sulfate. Thromb.
some blood coagulant factors. Carbohydr. Res. 1985, 137, Res. 2000, 99, 377.
205. 170. Alban, S.; Jeske, W.; Welzel, D.; Franz, G.; Fareed, J.
151. Horton, D.; Just, E.K. Preparation from chitin of (1-4)-2- Anticoagulant and antithrombotic action of semisynthetic
amino-2-deoxy-h-D-glucopyranuronan and 2-sulfamino h-1,3-glucan sulfate. Thromb. Res. 1995, 78, 201.
analog having blood-anticoagulant properties. Carbo- 171. Campbell, A.; Nesheim, M.E.; Doctor, V.M. Mechanism
hydr. Res. 1973, 29, 173. of potentiation of antithrombin III ATIII inhibition by
152. Nichimura, S.I.; Nishi, N.; Tokura, S.; Okiei, W.; sulfated xylans. Thromb. Res. 1987, 47, 341.
Somorin, O. Inhibition of the hydrolytic activity of chitin 172. Doctor, V.M.; Lewis, D.; Coleman, M.; Kemp, M.T.;
heparinoids. Carbohydr. Res. 1986, 156, 286. Marbley, E.; Sauls, V. Anticoagulant properties of semi-
153. Muzzarelli, R.A.A.; Giacomelli, G. The blood anti- synthetic polysaccharide sulfates. Thromb. Res. 1991, 64,
coagulant activity of N-carboxymethylchitosan trisulfate. 413.
Carbohydr. Polym. 1987, 7, 87. 173. Sinan, P.; Jacquinet, J.-C.; Petitou, M.; Duchaussoy, P.;
154. Dodane, V.; Vilivalam, V.D. Pharmaceutical application Lederman, I.; Choay, J.; Torri, G. Total synthesis of a
of chitosan. Pharm. Sci. Technol. Today 1998, 1, 246. heparin pentasaccharide fragment having high anity for
155. Roy, K.; Mao, H.Q.; Huang, S.K.; Leong, K.W. Oral antithrombin III. Carbohydr. Res. 1984, 132, c5.
gene delivery with chitosan-DNA nanoparticles generates 174. Freedman, M.D. Pharmacodynamics, clinical indications
794 Zhang et al.
and adverse eects of heparin. J. Clin. Pharmacol. 1992, synthetic heparin analogues with tailor-made coagulation
32, 584. factor inhibitory activity. Nat. Struct. Biol. 1995, 2, 736.
175. Petitou, M.; Duchaussoy, P.; Lederman, I.; Choay, J.; 181. Stubbs, M.T.; Bode, W. The clot thickens: Clues provided
Sinan, P. Binding of heparin to antithrombin III: A by thrombin structure. Trends Biochem. Sci. 1995, 20, 23.
chemical proof of the critical role played by a 3-sulfated 2- 182. Casu, B. Structure and biological activity of heparin. Adv.
amino-2-deoxy-D-glucose residue. Carbohydr. Res. 1988, Carbohydr. Chem. Biochem. 1985, 43, 51.
179, 163. 183. Petitou, M.; Herault, J.-P.; Bernat, A.; Driguez, P.-A.;
176. van Boeckel, C.A.A.; Beetz, T.; van Aelst, F. Synthesis of Duchaussoy, P.; Lormeau, J.-C.; Herbert, J.-M. Synthesis
a potent antithrombin activating pentasaccharide: A new of thrombin-inhibiting heparin mimetics without side
heparin-like fragment containing two 3-O-sulfated glucos- eects. Nature 1999, 398, 417.
amines. Tetrahedron Lett. 1988, 29, 803. 184. Choay, J.; Petitou, M.; Lormeau, J.C.; Sinan, P.; Casu, B.;
177. Petitou, M.; Duchaussoy, P.; Lederman, I.; Choay, J.; Gatti, G. Structureactivity relationship in heparin: A
Jacquinet, J.-C.; Sinan, P.; Torri, G. Synthesis of heparin synthetic pentasaccharide with high anity for antith-
fragments: A methyl alpha-pentaoside with high anity rombin III and eliciting high anti-factor Xa activity.
for antithrombin III. Carbohydr. Res. 1987, 167, 67. Biochem. Biophys. Res. Commun. 1983, 116, 492.
178. van Boeckel, C.A.A.; Beetz, T.; Vos, J.N.; De Jong, 185. van Boeckel, C.A.A.; Petitou, M. The unique antithrom-
A.J.M.; van Aelst, F.; Van den Bosch, H.; Mertens, bin binding domain of heparin: A lead to new synthetic
J.M.R.; Van der Vlught, A. Synthesis of a pentasaccharide antithrombotics. Angew. Chem. Int. Ed. Engl. 1993, 32,
corresponding to the antithrombin III binding fragment 1671.
of heparin. J. Carbohydr. Chem. 1985, 4, 293. 186. Olson, S.T.; Halvorson, H.R.; Bjork, I. Quantitative
179. Ichikawa, Y.; Monden, R.; Kuzuhara, H. Synthesis of characterization of the thrombin-heparin interaction:
methyl glucoside derivatives of tri- and penta-saccharides Discrimination between specic and non-specic binding
related to the antithrombin III-binding sequence of models. J. Biol. Chem. 1991, 266, 6342.
heparin, employing allobiose as a key starting-material. 187. Herbert, J.M.; Herault, J.P.; Bernat, A.; Savi, P.;
Carbohydr. Res. 1988, 172, 37. Schaeer, P.; Driguez, P.A.; Duchaussoy, P.; Petitou,
180. Grootenhuis, P.D.; Westerduin, P.; Meuleman, D.; M. SR123781A, a synthetic heparin mimetic. Thromb.
Petitou, M.; van Boeckel, C.A.A. Rational design of Haemost. 2001, 85, 852.
34
Structural Elucidation of Heparan Sulfate-Like
Polysaccharides Using Miniaturized LC/MS *
Balagurunathan Kuberan, Miroslaw Lech, and Robert D. Rosenberg
Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A.
and Beth Israel Deaconess Medical Center, Harvard Medical School,
Boston, Massachusetts, U.S.A.
ABSTRACT the characterization of the biologically important anti-

thrombin III (ATIII)-binding pentasaccharide and its pre-
Heparan sulfate (HS), a cell surface-bound glycosaminogly- cursors, which dier from each other by sulfation pattern
can polysaccharide, has been implicated in numerous bio- and/or degree of sulfation. All of these pentasaccharides
logical functions. Heparan sulfate molecules are highly were well resolved and characterized by the LC/MS system
complex and diverse, yet deceivingly look simple and sim- using 34SO4 as a mass spectral probe. This newly developed
ilar, rendering structurefunction correlation tedious. Cur- methodology facilitates the purication and rapid charac-
rent chromatographic and mass spectrometric techniques terization of biologically signicant HS oligosaccharides,
have limitations in analyzing glycosaminoglycan samples and will thus expedite their synthesis. These ndings
that are in low abundance and large in size due to their should undoubtedly pave the way in deciphering multiple
highly acidic nature arising from the large number of sulfate functional arrangements, ascribed to many biological ac-
and carboxylate groups. A new methodology was developed tivities, which are predictably embedded in a single, large,
using capillary ion-paired reversed-phase C18 high-perfor- chaotic, yet well-organized, HS polysaccharide chain. De-
mance liquid chromatography (HPLC) directly coupled to velopment of newer techniques for HS oligosaccharide
electrospray ionization time-of-ight mass spectrometry analysis is greatly needed in the postgenome era as atten-
(ESI-TOF-MS) to address the above issues. Based on HS tion shifts to the functional implications of proteins and
disaccharide analysis, dibutylamine was found to be the best carbohydrates in general and HS in particular.
suited for HS analysis among many ion-pairing agents
investigated. Next, analysis of oligosaccharides derived
from heparosan, precursor for heparan sulfate, was under- KEYWORDS
taken to demonstrate its greater applicability in a more
complex structural analysis. The established chromato- Glycosaminoglycan; Heparin lyase Sulfotransferases;
graphic conditions enabled the characterization of hepar- PAPS; Capillary HPLC; ESI; MS; Reversed-phase col-
osan oligosaccharides of sizes up to tetracontasaccharide umns; Ion-pairing agents; Oligosaccharides; Antithrombin
with high resolution in a single run and were amenable to III; Mass spectral probes; Stable isotopes; Heparan sulfate;
negative ion electrospray MS in which sodium adduction Heparin; Polysaccharides.
and fragmentation were avoided. To date, these are the
largest nonsulfated HS precursor oligosaccharides to be ABBREVIATION
characterized by liquid chromatography/mass spectrometry
(LC/MS). Finally, the current methodology was applied to ESI TOF MS, Electrospray ionization time-of-ight mass
spectrometry; cHPLC capillary high performance liquid
chromatography; GlcN, glucosamine; Glca, glucuronic
* Reproduced with permission from American Chemical Society; acid; dp, degree of polymerization; FAB, fast atom bom-
J. Am. Chem. Soc., 124, 87078718, 2002. bardment; GAG, glycosaminoglycan; MALDI-TOF, ma-
795
796 Kuberan et al.
trix-assisted laser desorption/ionization time-of-ight; HS, During the past decade, advances made in MS have
Heparan Sulfate; ATIII, Antithrombin III; PAPS, 3-Phos- led to the development of soft ionization techniques,
phoadenosine-5- phosphosulfate; 3-OSTI, Heparan Sul- namely electrospray ionization (ESI) and matrix-assisted
fate 3-O sulfotransferase 1; 6-OSTI, Heparan Sulfate 6-O laser desorption/ionization (MALDI), which are be-
sulfotransferasel coming indispensable tools in HS analysis and replacing
FAB-MS analysis.
Although MALDI-MS is far more sensitive than FAB
I. INTRODUCTION ionization, sulfated HS oligosaccharides ionize poorly
when mixed directly with any of the matrixes commonly
Proteoglycans are dominant glycoconjugates located on the used for MALDI-TOF-MS. In order to overcome this
cell surface and in extracellular spaces and consist of a core problem, sulfated oligosaccharides are often mixed with a
protein with one or more glycosaminoglycan (GAG) side basic polypeptide to form a neutral complex that can be
chains covalently linked. Heparan sulfate (HS) belongs to ionized readily [6]. The method, originally developed by
the family of glycosaminoglycans. HS has been assigned in Rhomberg et al. [7], has been applied in conjunction with
the following physiological and pathological functions: capillary electrophoresis to investigate the mechanisms of
cellcell adhesion, cellmatrix adhesion, cell proliferation, heparinases. Sulfated HS oligosaccharides ionize readily
motility and dierentiation, lipoprotein metabolism, blood by negative ion ESI mass spectrometry and are detected as
coagulation, inammation, tissue regeneration, tumor pro- alkali or ammonium salts. The molecular mass distribu-
gression and invasion, and pathogenic infections caused by tion can be extracted from these spectra, and results have
bacteria, protozoa, and viruses. HS chains orchestrate these been reported for HS up to decasaccharide size [8], der-
functions through their specic interactions with a wide matan sulfate up to dodecasaccharide size [9], and chon-
array of proteins, ligands, receptors, and pathogens [13]. droitin sulfate up to tetradecasaccharide size [10]. ESI
HS is a highly acidic polysaccharide with repeating spectra of sulfated GAGs are very complex due to several
disaccharide units consisting of a glucosamine and hexu- factors: (1) samples from biological sources often contain a
ronic acid (ido- and/or gluco-). HS is biosynthesized in the mixture of dierent chain lengths; (2) each molecule is
Golgi by addition of nucleotide sugars to the reducing end detected with multiple charge states; (3) the sulfate/car-
of the growing polysaccharide chain, followed by subse- boxylate groups have a tendency to form stable adducts
quent modication by dierent enzymes in a concerted with such alkali earth metal cations as Na+, K+, and
fashion. The nascent chain may be epimerized at the C5 Ca2+ to varying degrees; and (4) the larger oligosacchar-
position and/or sulfated at the C2 position of uronic acid ides are relatively fragile and may fragment or lose sulfate
residues, and may be N-sulfated or O-sulfated and/or N- groups during ionization. Despite that all HS oligosac-
acetylated in glucosamine residues. Although core proteins charides produce abundant ions by negative ESI-MS, it is
have fairly homogeneous compositions, the lengths and often dicult to extract molecular mass information from
compositions of HS chains are highly variable. The struc- the spectra due to the presence of overlapping peaks. Thus,
tural diversity of HS poses signicant challenges in the eld removal of metal cations during sample preparation is
of structural and functional glycobiology. In most cases, mandatory to ensure the quality of mass spectra. In most
HS oligosaccharides, obtained by enzymatic or chemical of these studies, oligosaccharides were rst puried and
depolymerization, were resolved to homogeneity by liquid collected after LC separation and then characterized.
chromatography to identify specic sequence/functional These o-line approaches suer from time-consuming
groups at the origin of its biological activity and then fraction isolation, collection, and purication. Despite
subsequently were characterized by nuclear magnetic res- the fact that on-line approaches hold several advantages,
onance (NMR) or mass spectrometry (MS). The current as of today, only a few reports are available and HS/CS/
existing high-eld NMR instruments require that oligosac- DS disaccharides/hexasaccharides and CS tetradecasac-
charides be available in large quantities, from 100 nmol to charide have been reported [1012].
micromoles, whereas MS analysis requires as little as a few Liquid-phase separation techniques, such as liquid
femtomoles, a billionth of the sample required for NMR. chromatography and electrophoresis, have always played
Hence, mass spectrometry is the method of choice when it a key role in the separation and detection of oligosaccha-
comes to analyzing samples of biological origin, which are rides. Moreover, miniaturized separation techniques, espe-
often procurable only in minute quantities. cially capillary high-performance liquid chromatography
Puried HS or HS-like oligosaccharides have been (cHPLC) and capillary electrophoresis, have had a pro-
analyzed using a number of mass spectrometric techniques. found impact on the modern practice of analyzing minute
HS-like oligosaccharides have been analyzed using fast amounts of biopolymers contained in highly heteroge-
atom bombardment mass spectrometry (FAB-MS), and neous mixtures of biological origin. The characteristic ow
octasaccharides have been detected at the 10-nmol levels. rates are in the range of microliters and nanoliters per
Derivatization of sulfated GAG samples has also been minute for cHPLC, making this separation technique
investigated for FAB-MS. Disaccharides and tetrasaccha- ideally suited for direct conjugation to ESI-MS. Although
rides derived from HS can be analyzed in the amount of anion exchange HPLC has been the most frequently used
<10 Ag by FAB-MS, which can provide information not chromatographic mode for the separation of oligosaccha-
only on molecular ions but also on certain sulfate and rides [13,14], the salt gradients applied to elute the oligo-
linkage positions [4,5]. saccharide from anion exchange columns render them less
Heparan Sulfate Polysaccharides 797
amenable for direct coupling to ESI-MS and mandate on- ESI-MS analysis of HS disaccharides. The volatility of
line microdialysis or a similar ion-removing interface. these ion-pairing agents prevented many of the obstacles
Separation by ion- pair reversed-phase high-performance usually experienced with ion-pairing agents in HPLC-MS.
liquid chromatography (IP-RP-HPLC) is very useful for The retention behavior as well as the inuence of the
sample preparation of oligosaccharides as well because it concentration of the ion pairing on the mass spectrometric
not only enables removal of alkali or alkaline earth metal sensitivity of disaccharide compounds were studied. Final-
cations from oligosaccharide samples but also fractionates ly, the optimized IP-RP-cHPLC-ESI-MS system is used to
oligosaccharides in more complex samples that can other- analyze HS-like oligosaccharides liberated from K5 he-
wise be directly analyzed by ESI-MS. parosan chains. The C18 ion-pair reversed-phase chroma-
Common ion-pairing agents used for IP-RP-HPLC tography (IP-RP-HPLC) LC/MS is shown to be eective
are tetraalkylammonium salts (e.g., tetrabutylammonium for simplifying mass spectra produced from mixtures of
or tetrapropyl ammonium or triethyl ammonium) or tri- heparosan oligosaccharides, thus facilitating extraction of
methyloctadecylammonium salts, which are used mainly neutral masses and size proles of oligosaccharides up to
with ultraviolet (UV) or uorescence detection. Direct tetracontasaccharides. To date, these are the largest oligo-
coupling of ion-pair chromatography to mass spectrome- saccharides of any kind to be chromatographically re-
try is made dicult by the contamination of the interface solved and characterized by mass spectrometry. Next, we
by the unvolatile tetraalkylammonium salts used as mobile demonstrated that the current built in LC/MS system is
phase modiers. Additionally, sensitivity in HPLC electro- also useful in resolving oligosaccharides based on the
spray ionization MS decreases rapidly with rising concen- degree of sulfation and sulfation pattern using antithrom-
trations of ion-pairing agents in the HPLC eluent. The use bin III (ATIII)-binding pentasaccharide.
of low concentrations of ion-pairing agent did result in
insucient retention of the more polar analytes. Another
disadvantage of tetraalkylammonium salts as mobile phase II. INSTRUMENTAL PARAMETERS
modiers in HPLC-ESI-MS is their tendency to form A. Flow Injection Capillary Liquid
cluster ions with anionic analytes. Chromatography
For the coupling to mass spectrometry, volatile buers
are preferred to enhance retention and peak shape. Am- An Ultimatek capillary HPLC workstation (Dionex,
monium acetate is well suited for this purpose because it Sunnyvale, CA) was used for microseparation. Ultimate
does not interfere with mass spectrometric detection in consists of a high-precision reciprocating pump with
concentrations normally applied for HPLC. However, microow processing to achieve precise delivery of micro-
acidic polysaccharides generally require more ecient liter and nanoliter ow rates, a helium sparging device with
ion-pairing agents than ammonium acetate to obtain rea- four separately controlled lines for solvent degassing, a
sonable retention. Nevertheless, it is important to consider quaternary pressure gradient pump that allows to choose
solution chemistry to achieve the best separation together up to four dierent solvent systems for mobile phase
with maximum analyte detectability, which usually implies separation, a column thermostat for controlling column
that HPLC separation conditions need to be adjusted to be temperature (from ambient to 80jC), and a scanning UV
amenable for ESI-MS analysis. Usually, a compromise has VIS absorb ance capable of concurrent monitoring of up to
to be found between optimum chromatographic and mass four selectable wavelengths and requiring as little as 3 nL of
spectrometric conditions. ow cell volume. UltiChrom software was used in data
In this paper, we report on the feasibility of coupling acquisition, analysis, and management. Flow injections
this highly ecient IP-RP capillary high-performance liq- were performed using the above HPLC system. To examine
uid chromatographic separation system to electrospray the eect of ion-pair concentration on ionization eciency,
ionization time-of-ight mass spectrometry. The negative the amines (triethylamine, dibutylamine, tributylamine,
ion detection is the obvious mode of choice for mass tripentylamine, tetrapropylamine, and tetrabutylamine)
spectrometric analysis of HS oligosaccharides. We have were dissolved in watermethanol (1:1) in the desired
applied octadecylated silica stationary phase capillary concentration containing 8 mM acetic acid. A gradient
columns for high-resolution separation of HS disaccha- elution was performed, using a binary solvent system
rides and oligosaccharides. The inuence of solution chem- composed of water (eluent A) and 70% aqueous methanol
istry on chromatographic and mass spectrometric (eluent B), both containing 8 mM acetic acid and 5 mM
performance of disaccharides is studied using dierent ion-pairing agent.
volatile ion-pair reagents. Addition of the volatile amine HPLC separations were performed on a 300 Am 250
suppresses multiply charged analyte ions in favor of less mm C18 polymeric silica column (Vydac, Hesperia, CA).
charged ones, thus resulting in the shift of peak centroids to The column temperature was maintained at 25jC and the
higher masses providing molecular mass information. ow rate was set at 5 AL/min. Sample volumes of 5100 AL
Thus, volatile aliphatic amines seemed to be suitable to were injected. For disaccharides and ATIII pentasacchar-
provide the necessary retention for HPLC and to reduce ide analysis, a 5-AL sample injection loop was used. For
ionic charge, thereby minimizing the occurrence of over- heparosan oligosaccharide analysis, a 100-AL sample in-
lapping charge states in cHPLC-ESI-MS. jection loop was used. The chromatographic conditions
We therefore investigated the applicability of several were optimized for disaccharides, ATIII pentasaccharides,
aliphatic amines as mobile phase modiers for the HPLC- and heparosan oligosaccharides. In brief, nonsulfated di-
798 Kuberan et al.
saccharide was eluted with 100% A, single sulfated di- III. RESULTS AND DISCUSSION
saccharides were eluted with 10% B, isocratic elution were
eluted with 20% B for double sulfated disaccharides, A. Miniaturization of LC/MS
followed by isocratic elution with 35% B for triple sulfated Conventional LC can be coupled with ESI interface by
disaccharide. The pentasaccharides were eluted with 60% using either postcolumn splitting or optimized ESI inter-
B. When heparosan oligosaccharide mix was loaded onto a faces to handle high ow rates. However, the former choice
column using 100 AL of loop, 100% A was used for 20 min results in signicant sample losses and the latter has not
to insure that oligosaccharides were concentrated before entailed noticeable sensitivity gains. Miniaturization of
their elution. The column was washed and reequilibrated chromatographic column parameters brings a key advan-
by further elution with 100% B for 10 min, returning to tage of improved detection sensitivities. A reduction in
100% A for 10 min at the end of the run. The absorbance of column diameter produces a higher sample peak concen-
the column eluate was monitored at 232 nm. The commer- tration in the detector. The maximum peak concentration
cially available standard disaccharides and heparosan- of the sample in the column eluate Cmax is given by:
derived oligosaccharides, obtained by heparitinase diges-
r!
tion, were determined on the basis of their absorbance at N 1
232 nm (e232 = 5500 M1 cm1) due to unsaturated double Cmax m
2p V0 1 k
bond of the uronic acid at the nonreducing end, in con-
junction with the carboxyl group at C5. Cmax depends on the absolute amount of sample loaded on
the column (m) and the column eciency (N) and is
inversely proportional to dead volume (V0) of the column
B. Mass Spectrometry
and the retention factor (k) [20]. The spray performance
Mass spectra were acquired on a Mariner BioSpectrometry can be improved by adjusting the ow rate and tip diam-
Workstation ESI time-of-ight mass spectrometer (Per- eters, which determine the formation and size of the
Septive Biosystems, Framingham, MA). The ion path is 1.3 droplets from which ions will be desorbed. An equation
m in the reectron mode. In the negative ion mode, the that describes droplet formation in ESI is given below:
instrument was calibrated with bis-triuoromethyl benzoic 8 91=3
>
> >
>
acid, heptadecauorononanoic acid, peruorotetradeca- < q = dV 2=3
noic acid, and oligothymidylic acids sodium salt [d(pT)6 re

>
> UT 2 > dt
and d(pT)10]. Nitrogen was used as a desolvation gas as : 4p2 ctan p2 h UA 1 >;
well as a nebulizer. Resolution was measured as full width
at half maximum (FWHM), which was at least 5000 for all
spectra, allowing isotopically resolved signals. The mass where re is the radius of the emission region for droplets at
accuracy in the range from m/z 100 to 2000 Am was better the tip of the Taylor cone, c is the surface tension of the
than 200 ppm. Conditions for ESI-MS were as follows: liquid, h is the liquid cone angle (for the classical Taylor
nebulizer gas ow 0.8 L/min, nozzle temperature 140jC, cone model h = 49.3j), q is the density of the liquid, UT is
drying gas (N2) ow 1.2 L/min, spray tip potential 2.8 kV, the threshold voltage, UA is the applied voltage, and dV/dt
nozzle potential 70 V, and skimmer potential 9 V. Negative is the ow rate [21]. The combination of improved ion
ion spectra were generated by scanning the range of m/z sampling eciency and spray performance will result in a
404000. During analyses, the indicated vacuum was 1.4 dramatic eect on the performance of the ESI source.
105 Torr. Total ion chromatograms (TICs) and mass
spectra were recorded on a personal computer with the B. Influence of (Ion-Pair Agent) on
Data Explorer software version 3.0 (Mariner). The param- Chromatographic Performance
eters studied included the eect of concentration and
In IP-RP-HPLC, retention of analytes is determined by
structures of ion-pairing agents.
several factors such as hydrophobicity of the stationary
phase, charge, hydrophobicity and concentration of the
C. Materials amphiphile, ionic strength and dielectric constant of the
mobile phase, and concentration of organic modier [22].
Standard mix containing six dierent disaccharides and According to the electrostatic model, retention is eected
Heparitinase I derived from Flavobacterium heparinium by the electrostatic interactions between the positive sur-
were purchased from Seikagaku USA (Cape Cod, MA). face potential generated by the amphiphilic ions adsorbed
All ion-pairing agents, chemicals, and PAPS were from at the stationary phase (alkylammonium ions, for instance)
either Sigma (St. Louis, MO) or Aldrich (Milwaukee, WI). and the negative surface potential generated by the caroxy-
K5 Heparosan polysaccharide was prepared from E. coli late/sulfate groups of the HS disaccharides and oligosac-
K5 [15]. 3-OST1 and 6-OST1 sulfotransferases were cloned charides. As a consequence of increasing the concentration
and expressed in baculovirus system [1618]. Glucosamine of an organic modier such as methanol, amphiphilic ions
6-sulfatase was purchased from Glyko (Novato, CA). are desorbed from the stationary phase, resulting in elution
PAP34S was prepared by using a slightly modied proce- of the retained analytes.
dure [19]. DEAE-Sephacel material was purchased from Tetrabutyl ammonium compounds are the most com-
Amersham Pharmacia (Piscataway, NJ). monly used ion-pairing agents in IP-RP-HPLC [23,24]
Figure 1 Ion-pairing agents studied in LC/MS analysis of HS disaccharides.
and tetrabutyl ammonium hydroxide reagent was used in

LC/MS analysis of HS disaccharides [11]. At the outset, it
was decided to study a panel of alkyl ammonium com-
pounds (Fig. 1) along with tetrabutyl ammonium hydrox-
ide as ion-pairing agents to determine how their
concentration and structure would aect LC/MS perfor-
mance of HS fragments.
The inuence of tetrabutylammonium concentration
in the eluent on retention and resolution of disaccharides is
undertaken. Heparitinase acts by eliminative cleavage to
produce these disaccharides with an unsaturated C4C5
bond on the uronic acid residue (Fig. 2). To measure the
retention times and resolution of disaccharides, a mixture
containing six dierent HS disaccharide standards was
eluted with a stepwise gradient of 0100% solvent B (see
Part II. Section A for gradient details) containing 1, 3,
and 5 mM tetraalkylammonium agent (Fig. 3). Retention
time of some disaccharides increased with increasing con-
centration of the ion-pair reagent because of the higher
surface potential at the stationary phase (Fig. 3). Resolu-
tion of disaccharides gradually improved with increasing
ion-pairing reagent concentration as depicted in Fig. 3.
Disaccharides 4 and 5 were poorly resolved at 1 mM
concentration of ion-pairing agents, were partially resolved
at 3 mM concentration, and were resolved fully at 5 mM
concentration. These disaccharides, 4 and 5, contain two
sulfate groups and dier from each other with regard to the
regiospecic positioning of sulfate groups. The peaks
corresponding to disaccharides 2 and 3 have better baseline
separation at higher concentrations of ion-pairing agents.
Disaccharides 2 and 3 have a single sulfate group and
occupy dierent positions in the glucosamine residue.
Although the retention time for disaccharides 13 in-
creased to a small degree at three dierent concentrations
of the tetrabutylammonium agent under the same eluent/
solvent composition, disaccharides 46 were retained more Figure 2 Structures of unsaturated HS disaccharides stud-
tightly at higher concentrations, reecting their stronger ied in capillary reversed-phase high-performance liquid
interaction with the stationary phase. chromatography using various ion-pairing agents.
800 Kuberan et al.
ESI generates a roughly constant ion current, an increase

in the intensity from added acids will reduce the intensity of
the analyte ions. Because ions of higher conductivity (such
as chloride) will suppress the signal signicantly, acetate
ions are preferred in the buer composition used for LC/
MS. Therefore, acetic acid was used to protonate alkyl-
amines, resulting in acetate as counterion.
C. Volatile Ion-Pairing Agents

The eect of a panel of volatile ion-pairing agents on reten-
tion and resolution of six disaccharides was investigated at
the outset. These ion-pairing agents were listed in Fig. 1. Of
all the ion-pairing agents tested, volatile triethylamine, no-
tably, was most widely used in several HPLC-MS analyses
of oligonucleotides, but its suitability as ion-pairing agent
for HS analysis was never investigated [25]. Because 5 mM
tetrabutylammonium ions provided the best chromato-
Figure 3 Capillary HPLC separation of unsaturated HS

disaccharides using dierent concentrations of tetrabutyl
ammonium ion as ion-pairing agent:. (A) 1 mM TBA; (B) 3
mM TBA; and (C) 5 mM TBA. Peak labels correspond to
disaccharides listed in Fig. 2.
A solution of neat dialkylamine or trialkylamine (Fig.

1) does not function as an ion-pair reagent for IP-RP-
HPLC due to the lack of a permanent positive charge on
the amphiphile. Hence, an acidic compound such as acetic
acid is usually added to the mobile phase to protonate the
dialkylamine and trialkylamine. In general, acids of higher
volatility should oer better detectability for ESI-MS. The
dierent counterions in the ion-pair reagent had only a
moderate eect on retention times and chromatographic
separation eciency [25]. However, protonation of alkyl-
amine with an acid has two major eects on solution
properties and hence mass spectra quality. First, the pH
of the solution decreases, and, second, the conductivity of
the solution increases. The addition of acids inuences the
charge state distribution of analytes, which can be
explained on the basis of solution and gas-phase acidbase
equilibria. The more acidic is the solution, the more likely
that the acids will donate protons to analyte anions and
reduce the charge states of analyte species. An increase in Figure 4 Chromatogram of HS disaccharides under dier-
the alkylammonium acetate concentration, which is desir- ent ion-pairing agents: dibutyl ammonium acetate (DBAA),
able to obtain better chromatographic performance, tributyl ammonium acetate (TBAA), and tripentyl ammo-
entailed a further decrease in signal intensity. Because nium acetate (TPAA).
graphic resolution, it was decided to keep all the other ion- oligosaccharide by IP-RP-HPLC-ESI-MS, the inuence of
pairing agents at the same concentration (5 mM) for the the following solution parameters on chromatographic and
current study and their eects on the resolution of disa- mass spectrometric performance was investigated indepen-
ccharides was investigated. Although dibutylammonium dently using IP-RP-HPLC and ESI-MS before their direct
acetate (DBA) resolved less satisfactorily the critical di- coupling for LC/MS analysis: concentration of ion-pair
saccharides 4 and 5, it is important to note that all six disa- reagent and structural features of ion-pair reagent.
ccharides were eluted in less than 20 min while holding good The disaccharides were analyzed using negative ESI-
overall resolution (Fig. 4). The resolution ability of tribu- MS under several dierent ion-pairing reagents and con-
tylammonium acetate was nearly identical to that of dibutyl centrations. Tetrabutylammonium ion suppressed the sig-
ammonium acetate, but it required greater than 60 min to nal of analytes at all concentrations, 1, 3, and 5 mM. Our
elute all six disaccharides under the given set of conditions. eorts to explore the tetrapropylammonium reagent also
Tripentylammonium acetate lengthens even further the were futile. Because 5 mM tetraalkylammonium ion pro-
HPLC run time and, in addition, disaccharides 4 and 5 vided the best chromatographic performance, it was
containing two sulfate groups were hardly resolved (Fig. 4). decided to keep the same concentration (5 mM) for the
subsequent experiments with dierent volatile ion-pairing
D. Coupling of Capillary IP-RP-HPLC agents: tributylammonium, tripentylammonium, triethyl-
with ESI-MS ammonium, and dibutylammonium acetates. Dibutyl-
ammonium acetate, among many ion-pairing agents
To render this chromatographic separation system amena- mentioned above, provided the better chromatographic
ble to ESI-MS, several factors that are related to the resolution and also required a shorter time. Hence, dibu-
solution chemistry of the column euent to be electro- tylammonium acetate was chosen for further LC/MS
sprayed have to be considered. A higher concentration of analysis of complex HS-like oligosaccharides.
an ion-pairing reagent is not suitable for HPLC-ESI-MS
because of the poor detectability of the eluted analytes by E. Partial Digestion of Heparosan
ESI-MS. Tetrabutyl ammonium ion and tetrapropyl am- Polysaccharide
monium ion, even at 1 mM concentration and at the
expense of poor chromatographic separation eciency, Heparan sulfate can be broken down enzymatically into
have suppressed the signal intensity in ESI-MS. The strong oligosaccharides. Three distinct types of lyases are known,
signal-suppressing eect of tetra-alkylammonium ion was with their unique substrate specicity [26,27]. These
attributed to its nonvolatile nature (hence accumulation of enzymes have been used to study the structure of heparan
counterions in the microdroplets) and thus results in sulfate and to prepare heparan sulfate-derived/heparan
lowering of the ionization eciency of the dissolved HS sulfate-like oligosaccharides for the evaluation of biologi-
saccharides. To nd the best compromise for the analysis of cal activity. Flavobacterium heparinum heparitinase cleaves
Scheme 1 Preparation of HS-like oligosaccharides. Partial digestion of Heparosan polysaccharide using heparitinase I to
prepare oligosaccharides for LC/MS analysis. HS-like oligosaccharides varied in size ranging from tetrasaccharide (n = 1) to
tetracontasaccharide (n = 19).
802 Kuberan et al.
-4)-GlcNpAca(14)-GlcAph(1- linkages through an elim- [M-nH]n and [(M-nH)+(1/n)]n. Because monoisotopic

ination reaction, yielding 4,5-unsaturated tetrasaccharides masses are not resolved very well for oligosaccharides from
and higher oligosaccharides as nal products (Scheme 1). triacontasaccharides (DP = 30) to tetracontasaccharides
Heparosan, a high-molecular-weight linear polysaccha- (DP = 40), only average masses are obtained, as shown in
ride, is a glycosaminoglycan found in the Escherichia coli Fig. 7. However, the average mass was sucient for
K5 bacteria [28]. Heparosan was subjected to partial diges- assignment of HS-like oligosaccharide compositions of
tion with heparitinase, and analyzed by a newly developed 30-mers to 40-mers eluting from a C18 column. Table 1
method of high-resolution cHPLC coupled to ESI-MS. summarizes the mass spectral results obtained for all the
HS-like oligosaccharides. Oligosaccharide ions are detected
F. LC/MS Analysis of HS-Like Oligosaccharides as [M-nH]n ions with no adduction by the DBA cation. It
is evident that adduction of oligosaccharides with calcium
The analysis of HS precursor oligosaccharides, obtained ions or other alkaline earth or alkali metal ions was
from the partial digestion of heparosan polymer, by IP-RP- eciently reduced during IP-RP-HPLC with 5 mM dibu-
HPLC coupled to ESI-MS was undertaken to determine the tylammonium acetate as eluent. The intensities of the
greater applicability of the developed methodology. With monosodium adducts were found to be negligible with
an eluent containing 5 mM dibutylammonium acetate and respect to the total ion intensity of all charge states. The
a stepwise gradient of solvent B, all oligosaccharides were presence of small amounts of sodium and potassium
separated. Adduction with alkali and alkaline earth metal adducts was highly advantageous at times for the determi-
ions has always been a major problem in negative ion ESI- nation of the charge state of a signal, especially when only
MS of oligosaccharides. Whereas on-line cation exchange one charge state could be observed due to charge state
enables the trapping of cations on a cation exchanger of reduction with dibutylammonium acetate. Although exces-
relatively high anity for the cations, removal of cations sive adduction is undesirable because the signal intensity is
during IP-RP-HPLC has to take place in the mobile phase distributed among many species, resulting in a low signal-
through competition of an excess of dibutylammonium to-noise ratio for the detected species, and particularly
ions with alkali or alkaline earth metal cations for the accurate mass determinations using the signals of higher
negative charges at the sugarcarboxylate backbone. Total charge states would be hampered because of the overlap-
ion chromatogram of oligosaccharides, eluting from capil- ping of adduct signals, cation adduction would be advan-
lary HPLC, is shown in Fig. 5. The eluent was diverted from tageous when it occurs in a controlled or limited manner.
the mass spectrometer for 20 min at the ow rate of 5 AL/ The doubly charged state was the most abundant for
min because of the presence of buer salts in the digestion oligosaccharides up to decasaccharides, except tetrasac-
buer of 100 AL volume, and the mass spectra of resolved charide, whereas triply charged molecular ion was observed
oligosaccharides are shown in Fig. 6. The bottom panel in as the most abundant charge state for each oligosaccharide
Fig. 6 of a given pair of mass spectra of each oligosaccha- ranging from DP = 12 to DP = 20. Quadruple charge state
ride shows the expanded views of the signals for the most as the abundant charge state for oligosaccharides ranging
abundant charge state of each oligosaccharide ranging in from DP = 22 to DP = 34 and the abundant quintuplet
size from DP 4 to DP 28. The charge state was calculated charge state for oligosaccharides with DP =36, 38, and 40
from the mass dierence between two monoisotopic signals were observed. The result obtained is signicant in that the
Figure 5 TIC from a capillary HPLC-ESI-TOF-MS analysis of a partially digested heparosan. The inset shows the expanded
view of TIC representing oligosaccharides higher than triacontasaccharide.
Figure 6 Electrospray spectra of the heparosan oligosaccharides ranging in size from tetrasaccharide (DP = 4) to
octaicosaccharide (DP = 28). Top panel shows mass spectrum of each oligosaccharide; bottom panel shows the isotope cluster
of parent oligosaccharide ion illustrating its resolution.
804 Kuberan et al.
Figure 6 Continued.
samples used were generated by HS lyases with no puri- mer (Scheme 2) [29,30]. The detailed mass spectrometric
cation step entering the LC/MS system. studies were earlier carried out on pentasaccharide and its
sequence [31,32]. Intensive biochemical and biophysical
G. Enzymatic Modification of Antithrombin studies suggest that the 3-O-sulfate group of N-sulfoglu-
III-Binding Pentasaccharide cosamine residue C and the 6-O-sulfate group of nonre-
ducing terminal glucosamine residue A are critical for
Heparin, more sulfated than HS glycosaminoglycan, is a binding to ATIII [3336]. Indeed, we have recently dem-
widely used anticoagulant drug and elicits its eect through onstrated that heparan sulfate 3-O-sulfotransferase 1 (3-
specic binding with ATIII, which specically recognizes OST-1) isoform catalyzes the rate-limiting biosynthetic
the following structure: GlcNS/Ac(6S)-GlcA-GlcNS(3S reaction, leading to cellular production or cell-free gener-
F 6S)-IdoA(2S)-GlcNS(6S) contained within the poly- ation of anticoagulant heparan sulfate besides other 6-OST
mer. ATIII binding to the above structure triggers a sulfotransferases [17]. The 3-OST1 enzyme recognizes a
conformational change that results in the acceleration of specic precursor structure, corresponding to the anti-
biochemical cascade. A synthetic ATIII-binding pentasac- thrombin-binding site devoid of just the 3-O-sulfate (pen-
charide (pentasaccharide 2) is under clinical trials as an tasaccharide III), and adds this rare substituent to
alternative anticoagulant to animal-derived heparin poly- complete the formation of anticoagulant heparan sulfate
Figure 7 Electrospray mass spectra of oligosaccharides ranging in size from triacontasaccharide (DP = 30) to tetraconta-
saccharide (DP = 40).
806 Kuberan et al.
Table 1 Molecular Masses of HS-Like Oligosaccharides saccharide III by selective removal of the 6-O-sulfate group
Produced by the Partial Digestion of Heparosan from the terminal glucosamine residue and then was treat-
ed with PAP34S, prepared by a slightly modied proce-
Mass (Da)a
Observed dure, to regioselectively resulfate at the same 6-O-position
dp m/z charge state Calculated Theoretical of the terminal glucosamine residue at the nonreducing
end to obtain pentasaccharide V (Scheme 2). We explored
4 0757.28 1 then the newly developed LC/MS system toward resolving
0378.14 2 0758.28 0758.22 these pentasaccharides.
6 0567.71 2 1137.42 1137.33
8 0757.28 2 1516.56 1516.45
10 0946.85 2
0630.90 3 1895.70 1895.56 H. LC/MS of Pentasaccharides
1136.42 2
Pentasaccharides II and III dier from one another only
12 0757.29 3 2274.87 2274.67
by the presence (II) or absence (III) of a 3-O sulfate on
1515.59 3 (dimer)
the internal glucosamine residue C (Scheme 2). Penta-
0883.67 3
14 1326.00 4 (dimer) 2654.01 2653.78 saccharide III diers from pentasaccharide IV by the
1010.05 3 presence of the critical 6-O-sulfate on the nonreducing
16 0757.28 4 3033.15 3032.89 terminal glucosamine residue A. Thus, pentasaccharides
1136.44 3 II, III, and IV have eight, seven, and six sulfate groups,
18 0852.08 4 3412.32 3412.00 respectively. A mixture containing an equimolar amount
1262.82 3 of pentasaccharides II, III, and IV was prepared and
20 0946.87 4 3791.46 3791.11 injected into the cHPLC system. Figure 8 shows the TIC
1389.19 3 of pentasaccharide mix in which three peaks, A, B, and
22 1041.64 4 4170.56 4170.23 C, corresponding to three pentasaccharides, were re-
1515.23 3 solved very well within a short time period of 10 min.
24 1136.17 4 4548.68 4549.34 Peaks A, B, and C were assigned to pentasaccharides IV,
26 1231.20 4 4928.80 4928.45 III, and II respectively, deduced from mass spectral
28 1325.99 4 5307.96 5307.56 values of each peak (Fig. 9). The doubly charged ions
30 1421.54 4 5690.16 5689.82 were observed as the most abundant ions along with
32 1516.53 4 6070.12 6069.14 triple and quadruple charge states as minor ions. Results
1611.41 4 6449.64 are summarized in Table 2. This indicates that the least
34 1288.69 5 6448.45 6448.46 sulfated pentasaccharide IV elutes from the column
1706.14 4 6828.56 ahead, whereas the most sulfated pentasaccharide II,
36 1365.09 5 6830.40 6827.78
containing intact ATIII-binding structure, elutes the last,
38 1440.34 5 7206.70 7207.10
and pentasaccharide III containing seven sulfate groups
40 1516.27 5 7586.35 7586.42
elutes between the least and the most sulfated pentasac-
a
Monoisotopic mass was calculated for oligosaccharides up to charide. Thus, these pentasaccharides can be resolvable
octaicosaccharide (DP = 28) and for triacontasaccharide and higher by capillary reversed-phase columns based on degree of
oligosaccharides up to tetracontasaccharide; only average mass was sulfation. Next, the ability of the LC/MS system to
calculated due to the poor resolution of isotopic clusters of the resolve pentasaccharides I and V was undertaken. Penta-
parent ion. saccharides I and V both contain the same number of
sulfate groupsseven. However, they dier from each
other by sulfation pattern. Resolving such oligosaccha-
[37]. In order to evaluate the biosynthetic pathway of ride mixtures would undoubtedly lead to greater con-
anticoagulant HS, it is necessary to prepare and resolve dence about the homogeneity of any prepared sample for
anticoagulant pentasaccharide and its precursor structures mass spectral analysis. Figure 10 shows the resolution of
to homogeneity. These pentasaccharides closely resemble these pentasaccharides V and I, separated from each
each other and are highly charged, making them dicult to other by 0.6 min, corresponding to peaks A and B,
be characterized. Pentasaccharide II was prepared by respectively. Mass spectra of these pentasaccharides
regioselective sulfation of pentasaccharide III using 3- whose molecular weights dier by 2 Da are shown in
phosphoadenosine-5-phosphosulfate (PAPS) as a sulfate Fig. 11. The observed abundant molecular ion for pen-
donor, catalyzed by baculovirus-cloned and expressed 3- tasaccharide V was 842.71, corresponding to
OST1 sulfotransferase. Pentasaccharide II was then treated [M+2(DBA)-4H]2, and that for pentasaccharide I was
with commercially available recombinant glucosamine-6- 841.71, corresponding to [M+2(DBA)-4H]2. Although
sulfatase (EC 3.1.6.14), a lysosomal hydrolase enzyme DBA adductions were not observed in triple and qua-
whose deciency was implicated in Sanlippo D syndrome druple charge states except for pentasaccharide II, adduc-
in humans, to selectively remove the 6-O-sulfate group tions with DBA were always observed for these
from the terminal N-sulfoglucosamine residue at the non- pentasaccharides in the abundant double charge state.
reducing end (residue A), yielding pentasaccharide I [38]. The results are summarized in Table 2. Note that these
Similarly, pentasaccharide IV was prepared from penta- pentasaccharides, I and V, would have the same molec-
Scheme 2 Enzymatic synthesis of pentasaccharides. Enzymatic modication of antithrombin-binding pentasaccharide variants.
6-OST1 selectively sulfates the 6-OH group at the nonreducing end (residue A), whereas 3-OST1 regioselectively sulfates the 3-
OH group of the middle sugar residue C. PAP34S was used in the preparation of pentasaccharide V for LC/MS analysis.
808 Kuberan et al.
Table 2 Molecular Masses of ATIII-Binding

Pentasaccharide Variants
Observed Charge Quasi-molecular
Pentasaccharide m/z state iona
I 474.70 3 [M 3H]3
777.13 2 [M+1(DBA)3H]2
788.13 2 [M+1(DBA)
+Na4H]2
841.71 2 [M+2(DBA)4H]2
II 375.81 4 [M 4H]4
544.47 3 [M+1(DBA)4H]3
Figure 8 Total ion chromatogram from LC/ESI-MS anal- 544.47 3 [M+1(DBA)4H]-3
ysis of pentasaccharides II, III, and IV having dierent 881.80 2 [M+2(DBA)4H]2
degrees of sulfation. Peaks A, B, and C correspond to III 355.81 4 [M 4H]4
pentasaccharides IV, III, and II, respectively. 474.75 3 [M 3H]3
777.22 2 [M+1(DBA)3H]2
841.81 2 [M+2(DBA)4H]2
IV 335.80 4 [M 4H]4
448.09 3 [M 3H]3
672.64 2 [M 2H]2
737.24 2 [M+1(DBA)3H]2
Vb 356.27 4 [M 4H]4
475.37 3 [M 3H]3
778.12 2 [M+1(DBA)3H]2
842.70 2 [M+2(DBA)4H]2
a
All parent ions were observed in adduction with the ion-pairing
agent, DBA.
b
Pentasaccharide V labeled with 34S containing sulfate as a mass
spectral probe to distinguish it from pentasaccharide I in LC/MS
characterization (see Figs. 10 and 11 for details).
ular weight if pentasaccharide V were otherwise sulfated

using regular PAPS instead of PAP34S as a sulfate donor,
catalyzed by heparan sulfate 6-O-sulfotransferase 1 (6-
OST1). Thus, our current studies demonstrated not only
the ability of LC/MS to resolve HS oligosaccharides
based on sulfation pattern but also the advantage of
introducing 6-O 34SO4 group as a mass spectral probe to
identify each peak corresponding to its respective penta-
saccharide. A pair of HS-like oligosaccharides of com-
position (HexA-GlcN)m and (HexA-GlcN)2m containing
unsaturated uronic acid at the nonreducing end has no
Figure 9 ESI mass spectrum of pentasaccharide IV (A),

pentasaccharide III (B), and pentasaccharide II (C) having
dierent degrees of sulfation. Parent ion of pentasaccharide Figure 10 Total ion chromatograms of pentasaccharide V
IV does not have adduction with ion-pairing agent (DBA). (peak A) and pentasaccharide I (peak B), having the same
Both pentasaccharides III and II form adduction with DBA number of sulfate groups, were resolved based on sulfation
in the parent ion. Results are summarized in Table 2. pattern.
found to enable the ecient on-line coupling of IP-HPLC

to ESI-MS in the analysis of HS. Using capillary columns
packed with C18 particles and gradients of methanol in 5
mM dibutylammonium acetate, HS precursor oligosac-
charides up to at least tetracontasaccharide (40-mer) were
separated. Alkali cation adduction in the separated oligo-
saccharides is eciently reduced, allowing highly accurate
mass determination. To date, these are the largest non-
sulfated HS precursor oligosaccharides to be characterized
by LC/MS. Finally, the current methodology was applied
in characterizing biologically important ATIII-binding
pentasaccharides and its precursors, which dier from each
other by sulfation pattern and/or degree of sulfation. All of
these pentasaccharides were well resolved and character-
ized by LC/MS system with the use of 34SO4 as a mass
spectral probe. Although the biological functions of he-
paran sulfate are well known over several decades, only a
few heparan sulfate structures attributable to their func-
tions are solved thus far. It is primarily due to the dicul-
ties involved in isolating pure HS active oligosaccharides
for characterization. Besides, several HS sequences may be
well capable of binding to a given protein to exert a specic
biological function or event [1,2]. In vitro modication of
Figure 11 Electrospray mass spectrum of pentasaccharide V HS inactive precursors lacking critical functional groups
(panel A) and pentasaccharide I (panel B). Pentasaccharide V and converting them into HS active structures using 34SO4
contains the 6-O-34SO4 group as a mass spectral probe to be or 33SO4 as stable isotopes, catalyzed by a specic cloned
distinguished from its isomer pentasaccharide I and hence its sulfotransferase, would allow one to position or align the
molecular mass is 2 Da more. Isotope clusters of the parent critical functional groups necessary for biological func-
ion of each compound are illustrated as insets in each panel. tions. This newly developed methodology should assist the
Results are summarized in Table 2. chemical synthesis of HS fragments of biological signi-
cance in terms of purication and rapid characterization,
and thus expedite the synthesis of biologically signicant
HS oligosaccharides. Our ndings should undoubtedly
mass dierence between two oligosaccharides of single
pave the way in deciphering multiple functional arrange-
and double charge states. As m (the number of disaccha-
ments, ascribed to many biological activities, which are
ride units) increases, the complexity of the isotopic
predictably embedded in a single, large, chaotic, yet well-
clusters also increases. So it is very important to resolve
organized, HS polysaccharide chain.
the oligosaccharides based on charge, size, and hydro-
phobic or sulfation pattern into as much homogeneity as
possible before entering the mass spectrometry. It is also
important to note that no fragmentation is observed ACKNOWLEDGMENTS
under current LC/MS experimental conditions. This
result has implications for the analysis of HS oligosac- This study was supported by grants from the National
charides of larger sizes by electrospray MS that would Institutes of Health (P01 HL66105 and P01 HL41484).
denitely lead to determine the alignment of critical We thank the Rosenberg group and Dr. Zaia for their
functional groups necessary for elucidating structure comments on the manuscript, and also Gail Monahan
function relationships. and Bill Lombardi for providing various assistance.
IV. CONCLUSIONS REFERENCES

In this work, we have demonstrated the utility of IP-RP- 1. Berneld, M.; Gotte, M.; Park, P.W.; Reizes, O.; Fitzger-
capillary HPLC-ESI-TOF-MS for HS oligosaccharide ald, M.L.; Lincecum, J.; Zako, M. Functions of cell surface
analysis. Microcapillary HPLC columns with integrated heparan sulfate proteoglycans. Annu. Rev. Biochem. 1999,
electrospray emitters provide ecient temporal separation 68, 729.
and ionization of oligosaccharide species, whereas MS 2. Rosenberg, R.D.; Shworak, N.W.; Liu, J.; Schwartz, J.J.;
operation in external ion accumulation mode maintains Zhang, L.J. Heparan sulfate proteoglycans of the cardio-
vascular system. Specic structures emerge but how is
chromatographic resolution during mass spectral acqui- synthesis regulated? J. Clin. Invest. 1997, 99, 2062.
sitions. It is concluded that solution parameters have 3. Conrad, H.E. Heparan sulfate proteoglycanspresent and
opposite eects on the performance of IP-RP-HPLC and future. Trends Glycosci. Glycotechnol. 1998, 10, 51.
negative ion ESI-MS. Therefore, a compromise has to be 4. Lamb, D.J.; Wang, H.M.; Mallis, L.M.; Linhardt, R.J.
810 Kuberan et al.
Negative-ion fast-atom-bombardment tandem mass-spec- gas-phasethe mechanism of electrospray mass-spectro-

trometry to determine sulfate and linkage position in metry. Anal. Chem. 1993, 65, A972.
glycosaminoglycan-derived disaccharides. J. Am. Soc. 22. El Rassi, Z. Recent progress in reversed-phase and hydro-
Mass Spectrom. 1992, 3, 797. phobic interaction chromatography of carbohydrate spe-
5. Khoo, K.H.; Morris, H.R.; McDowell, R.A.; Dell, A.; cies. J. Chromatogr., A 1996, 720, 93.
Maccarana, M.; Lindahl, U. FABMS derivatization 23. Zhu, Y.; Wong, P.S.; Cregor, M.; Gitzen, J.F.; Coury,
strategies for the analysis of heparin-derived oligosaccha- L.A.; Kissinger, P.T. In vivo microdialysis and reverse
rides. Carbohydr. Res. 1993, 244, 205. phase ion pair liquid chromatography/tandem mass spec-
6. Juhasz, P.; Biemann, K. Mass-spectrometric molecular- trometry for the determination and identication of
weight determination of highly acidic compounds of acetylcholine and related compounds in rat brain. Rapid
biological signicance via their complexes with basic Commun. Mass Spectrom. 2000, 14, 1695.
polypeptides. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4333. 24. Legrand-Defretin, V.; Juste, C.; Henry, R.; Corring, T. Ion-
7. Rhomberg, A.J.; Ernst, S.; Sasisekharan, R.; Biemann, K. pair high-performance liquid chromatography of bile salt
Mass spectrometric and capillary electrophoretic investiga- conjugates: application to pig bile. Lipids 1991, 26, 578.
tion of the enzymatic degradation of heparin-like glycos- 25. Huber, C.G.; Krajete, A. Comparison of direct infusion
aminoglycans. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4176. and on-line liquid chromatography/electrospray ionization
8. Chai, W.G.; Luo, J.L.; Lim, C.K.; Lawson, A.M. mass spectrometry for the analysis of nucleic acids. J. Mass
Characterization of heparin oligosaccharide mixtures as Spectrom. 2000, 35, 870.
ammonium salts using electrospray mass spectrometry. 26. Linhardt, R.J.; Turnbull, J.E.; Wang, H.M.; Loganathan,
Anal. Chem. 1998, 70, 2060. D.; Gallagher, J.T. Examination of the substrate-specicity
9. Yang, H.O.; Gunay, N.S.; Toida, T.; Kuberan, B.; Yu, of heparin and heparan-sulfate lyases. Biochemistry (US)
G.L.; Kim, Y.S.; Linhardt, R.J. Preparation and structural 1990, 29, 2611.
determination of dermatan sulfate-derived oligosaccha- 27. Desai, U.R.; Wang, H.M.; Linhardt, R.J. Substrate
rides. Glycobiology 2000, 10, 1033. specicity of the heparin lyases from Flavobacterium
10. Zaia, J.; Costello, C.E. Compositional analysis of glycos- heparinum. Arch. Biochem. Biophys. 1993, 306, 461.
aminoglycans by electrospray mass spectrometry. Anal. 28. Vann, W.F.; Schmidt, M.A.; Jann, B.; Jann, K. The
Chem. 2001, 73, 233. structure of the capsular polysaccharide (K5 antigen) of
11. Oguma, T.; Toyoda, H.; Toida, T.; Imanari, T. Analytical urinary-tract-infective Escherichia coli 010:K5:H4. A poly-
method of chondroitin/dermatan sulfates using high- mer similar to desulfo-heparin. Eur. J. Biochem. 1981, 116,
performance liquid chromatography/turbo ion spray ion- 359.
ization mass spectrometry: application to analyses of the 29. Duchaussoy, P.; Lei, P.S.; Petitou, M.; Sinay, P.; Lormeau,
tumor tissue sections on glass slides. Biomed. Chromatogr. J.C.; Choay, J. The 1st total synthesis of the antithrombin-
2001, 15, 356. III binding-site of porcine mucosa heparin. Bioorg. Med.
12. Dacol, R.; Silvestro, L.; Naggi, A.; Torri, G.; Baiocchi, C.; Chem. Lett. 1991, 1, 99.
Moltrasio, D.; Cedro, A.; Viano, I. Characterization of the 30. Sinay, P.; Jacquinet, J.C.; Petitou, M.; Duchaussoy, P.;
chemical-structure of sulfated glycosaminoglycans after Lederman, I.; Choay, J.; Torri, G. Total synthesis of a
enzymatic digestionapplication of liquid-chromatogra- heparin pentasaccharide fragment having high-anity for
phy mass-spectrometry with an atmospheric-pressure inter- antithrombin-III. Carbohydr. Res. 1984, 132, C5.
face. J. Chromatogr. 1993, 647, 289. 31. Pope, R.M.; Raska, C.S.; Thorp, S.C.; Liu, J. Analysis of
13. Loganathan, D.; Wang, H.M.; Mallis, L.M.; Linhardt, R.J. heparan sulfate oligosaccharides by nano-electrospray
Structural variation in the antithrombin-III binding-site ionization mass spectrometry. Glycobiology 2001, 11, 505.
region and its occurrence in heparin from dierent sources. 32. Shriver, Z.; Sundaram, M.; Venkataraman, G.; Fareed, J.;
Biochemistry (US) 1990, 29, 4362. Linhardt, R.J. Cleavage of the antithrombin III binding
14. Pervin, A.; Gallo, C.; Jandik, K.A.; Han, X.J.; Linhardt, site in heparin by heparinases and its implication in the
R.J. Preparation and structural characterization of large generation of low molecular weight heparin. Proc. Natl.
heparin-derived oligosaccharides. Glycobiology 1995, 5, 83. Acad. Sci. U.S.A. 2000, 97, 10365.
15. Fritz, T.A.; Gabb, M.M.; Wei, G.; Esko, J.D. Two N- 33. Atha, D.H.; Lormeau, J.C.; Petitou, M.; Rosenberg, R.D.;
acetylglucosaminyltransferases catalyze the biosynthesis of Choay, J. Contribution of monosaccharide residues in
heparan sulfate. J. Biol. Chem. 1994, 269, 28809. heparin binding to antithrombin-III. Biochemistry 1985,
16. Liu, J.; Shworak, N.W.; Fritze, L.M.S.; Edelberg, J.M.; 24, 6723.
Rosenberg, R.D. Purication of heparan sulfate D-glucosa- 34. Atha, D.H.; Stephens, A.W.; Rosenberg, R.D. Evaluation
minyl 3-O-sulfotransferase. J. Biol. Chem. 1996, 271, 27072. of critical groups required for the binding of heparin to
17. Zhang, L.; Beeler, D.L.; Lawrence, R.; Lech, M.; Liu, J.; antithrombin. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 1030.
Davis, J.C.; Shriver, Z.; Sasisekharan, R.; Rosenberg, R.D. 35. Desai, U.R.; Petitou, M.; Bjork, I.; Olson, S.T. Mechanism
6-o-Sulfotransferase-1 represents a critical enzyme in the of heparin activation of antithrombinrole of individual
anticoagulant heparan sulfate biosynthetic pathway. J. residues of the pentasaccharide activating sequence in the
Biol. Chem. 2001, 276, 42311. recognition of native and activated states of antithrombin.
18. Shworak, N.W.; Liu, J.; Fritze, L.M.; Schwartz, J.J.; J. Biol. Chem. 1998, 273, 7478.
Zhang, L.; Logeart, D.; Rosenberg, R.D. Molecular 36. Wu, Z.L.; Zhang, L.; Beeler, D.L.; Kuberan, B.; Rosen-
cloning and expression of mouse and human cDNAs berg, R.D. A new strategy for dening critical functional
encoding heparan sulfate D-glucosaminyl 3-O-sulfotrans- groups on heparan sulfate. FASEB J. 2002, 16, 539.
ferase. J. Biol. Chem. 1997, 272, 28008. (272). 37. Zhang, L.; Lawrence, R.; Schwartz, J.J.; Bai, X.; Wei, G.;
19. Renosto, F.; Segel, I.H. Choline sulfokinase of penicillium Esko, J.D.; Rosenberg, R.D. The eect of precursor
chrysogenumpartial-purication and kinetic mechanism. structures on the action of glucosaminyl 3-O-sulfotransfer-
Arch. Biochem. Biophys. 1977, 180, 416. ase-1 and the biosynthesis of anticoagulant heparan sulfate.
20. Chervet, J.P.; Ursem, M.; Salzmann, J.B. Instrumental J. Biol. Chem. 2001, 276, 28806.
requirements for nanoscale liquid chromatography. Anal. 38. Freeman, C.; Clements, P.R.; Hopwood, J.J. Human liver
Chem. 1996, 68, 1507. N-acetylglucosamine-6-sulphate sulphatase. Purication
21. Kebarle, P.; Tang, L. From ions in solution to ions in the and characterization. Biochem. J. 1987, 246, 347.
35
*
Enzymatic Synthesis of Heparan Sulfate*
Balagurunathan Kuberan, David L. Beeler, and Robert D. Rosenberg
Massachusetts Institute of Technology, Cambridge, Massachusetts, U.S.A. and
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, U.S.A.
I. INTRODUCTION potential of cell-free, in vitro chemoenzymatic synthesis of

biologically active HS polysaccharide. Because anticoagu-
In the case of proteins and DNA, the ability to rapidly and lant HS biosynthesis and their biological functions are well
easily synthesize the fragments of these components facil- characterized, we initiated our explorations of this new
itated the establishment of structurefunction relationship approach by carrying out the synthesis of HS like antico-
of these molecules and generated necessary reagents that agulant polysaccharide at the outset.
permitted the delineation of the biologic roles of these Heparin, a strongly acidic, linear sulfated polysaccha-
macromolecules. In the case of Heparan Sulfate (HS) and ride, is used in the prevention and treatment of thrombosis.
other carbohydrates, the total chemical synthesis of these Heparin was rst isolated from the liver from which it
components is challenging because of the following reas- derives its name [1]. Heparin-like polysaccharides are
ons: (1) orthogonal glycosylation conditions required for shown to interact with numerous proteins and orchestrate
alpha- and beta-selective glycosidic linkage formation; (2) many dierent biologic functions [2]. A unique pentasac-
successful stereo inversion at C-5 of uronic acids; (3) charide domain present within heparin was found to bind
diligent stepwise deprotection and regioselective sulfation; to Antithrombin III (ATIII) in a highly specic manner to
achieving selective discrimination between the 2- and 3-OH induce a conformational change that is sucient to pro-
groups of uronic acid residues. mote rapid inhibition of blood coagulation [3,4]. Sinay and
On the other hand, a general enzymatic method for HS coworkers pioneered the original chemical synthesis of the
assembly would allow for the rapid synthesis of structures ATIII binding pentasaccharide and analogs [5,6].
of interest to delineate structurefunction relationships of Heparin-induced thrombocytopenia (HIT) is an im-
this class of molecules. In addition, it would allow us to munologic disorder associated with heparin treatment [7].
understand HS biosynthesis and determine how specic HIT paradoxically increases thrombosis, which occurs in
structures are assembled, and thus it might establish a rm about 30% of the recognized HIT cases, and is a major
foundation for the glycobiology of proteoglycans. Even cause of morbidity and mortality in patients undergoing
enzyme-assisted synthesis is challenging because there are treatment with heparin. It has been shown that HIT is
at least a dozen enzymes/isoforms involved in HS biosyn- induced by antibodies against PF4heparin complex. The
thesis and this approach requires most of them to complex formation requires a 2-O sulfated iduronic acid
be available. In particular, the combination of enzymes residue [8]. Engineering new heparin that is unable to form
needed to generate a particular bioactive HS structure is heparinPF4 complexes would be a major advance in
unknown. Ultimately, a synthetic strategy comprising of anticoagulation therapy. There is also an increased concern
chemical and enzymatic methods based on the merits of for the potential spread of diseases of animal origin to
each step would optimally expedite the synthesis of bioac- humans, such as bovine encephalopathy, due to the use of
tive HS structures. To achieve this end, we examined the animal-derived heparin. The abovementioned potential
side eects of animal-derived heparin prompted the chem-
ical synthesis of heparin-based anticoagulants. Despite
* Reproduced with permission from American Chemical Society; J. many advances made in chemical synthesis, this approach
Am. Chem. Soc., 125, 1242412425, 2003. is cumbersome and time-consuming. The limitations of
811
812 Kuberan et al.
chemical approaches prompted us to undertake a rapid cosamine units, which clearly suggests that epimerization
approach to synthesize a heparin-like anticoagulant with occurs immediately after N-deacetylation and N-sulfation,
improved therapeutic characteristics using cloned enzymes but before O-sulfation [10,14]. Therefore, our synthetic
in a relatively simple manner. strategy was devised in such a manner that NDST2 and
We termed this engineered anticoagulant polysaccha- C5 epimerase are coupled to prepare in a single step
ride as Mitrin because of its origin at Massachusetts predominately N-sulfated polysaccharide 2 containing a
Institute of Technology (MIT) in analogy to Heparin small percentage of unmodied GlcNAc residues in addi-
whose origin is hepatic [1]. A nonsulfated N-acetyl hepar- tion to both unmodied glucuronic acid residues and newly
osan polysaccharide 1 was isolated with an average molec- generated iduronic acid residues without 2-O sulfation.
ular weight of 7000 Da from K5 Escherichia coli [9]. The second and nal step in the synthesis of anticoag-
Polysaccharide 1 resembles the unmodied nascent HS ulant Mitrin 3 was catalyzed by 6-O sulfotransferase
chain. It is used as a starting material in the enzymatic (6-OST) and 3-O sulfotransferase (3-OST) (Scheme 1).
synthesis of Mitrin. The rst step is the synthesis of There are three heparan sulfate 6-O sulfotransferase iso-
N-sulfated polysaccharide 2 enriched with iduronic acid forms: 6-OST1, 6-OST2 (6-OST2a and 6-OST2b are two
catalyzed by N-deacetylase-N-sulfotransferase (NDST) splice variants), and 6-OST3 [15]. It is known that all three
and C-5 epimerase (Scheme 1). These two initial modica- isoforms sulfate completely desulfated N-sulfated
tions are the essential gateway for subsequent enzymatic (CDSNS)heparin equally well [15]. However, N-sulfo-
modications [10]. A single protein catalyzes both N-deace- heparosan was shown to be preferentially sulfated by these
tylation and N-sulfation. These two reactions are tightly isoforms in the following order: 6-OST2 > 6-OST3 > 6-
coupled in vivo, because free glucosamine residues are OST1. Thus, in essence, we have utilized the 6-OST2a
rarely found in HS and Heparin, although each activity isoform to catalyze the 6-O sulfation of glucosamine units.
can be separately studied in vitro. This enzyme exists as four The 6-O sulfation was coupled with 3-O sulfation, which is
isoforms in humans: NDST1, NDST2, NDST3, and catalyzed by 3-OST1 sulfotransferase to generate Mitrin
NDST4 [11]. We utilized the NDST2 isoform to selectively anticoagulant [16]. There are as many as six isoforms of
N-deacetylate and N-sulfate glucosamine units [12]. This heparan sulfate 3-O sulfotransferases: 3-OST1, 3-OST2, 3-
step was carried out in conjunction with HS C-5 epimerase OST3 (3-OST3a and 3-OST3b are two variants), 3-OST4,
to generate the iduronic acid-enriched polysaccharide 2 3-OST5, and 3-OST6 [17,18]. It was demonstrated earlier
[13,14]. The stereochemical nature at C-5 carbon of uronic that 3-OST1 is primarily involved in generating anticoag-
acid is reversed in this transformation (Scheme 1). Epimer- ulant heparan sulfate [19]. It was also shown that 3-OST1
ization proceeds only when these residues are placed im- generally acts on glucosamine units anked by the reducing
mediately adjacent to the reducing side of the glycosidic side of GlcA and the nonreducing side of IdoA2S to
bond of the N-sulfated glucosamine residues, but it will not generate ATIII binding structures containing GlcA
react with glucuronic acids that are O-sulfated, or that are GlcNS3S and GlcAGlcNS3S6S [1921]. Because 6-O
adjacent to O-sulfated glucosamine residues or adjacent to sulfation and 3-O sulfation are not coupled to generate
the reducing side of the glycosidic bond of the N-acetylglu- anticoagulant structures in vivo, it was anticipated that
Scheme 1 Enzymatic synthesis of Mitrin anticoagulants.

Enzymatic Synthesis of Heparan Sulfate 813
coupling both modications in vitro would dramatically

shorten the time required for Mitrin synthesis. Mitrin was
successfully prepared from polysaccharide 2 by using
3-OST1 and 6-OST2a sulfotransferases. The nal step
was also carried out in the presence of radioactive PAP35S
to prepare the radiolabeled Mitrin in order to test its ability
to bind to ATIII by gel mobility shift assay [22]. The
synthesized Mitrin polysaccharide 3 was found to bind to
ATIII. In the presence of ATIII, Mitrin binds specically to
ATIII and hence its mobility is retarded, whereas in the
absence of ATIII, Mitrin migrates more rapidly (Fig. 1). A
greater percentage of Mitrin binds to ATIII as compared to
in vitro modied commercial heparin. This result is con- Figure 2 Biological activity of Mitrin. Human factor Xa
rmed by a heparin-dependent factor Xa inhibition assay was incubated with antithrombin III in the presence of
(Fig. 2). The specic activity of Mitrin is approximately 45 Mitrin (polysaccharide 3) or commercial heparin or poly-
times that of commercial heparin. Finally, Mitrin was saccharide 2 as a negative control. The percentage of
cleaved by heparitinases I, II, and III for structural analysis inhibition of thrombin activity was calculated from three
by capillary liquid chromatography coupled to electro- experiments performed in triplicate.
spray ionization mass spectrometry (LC/MS) [23]. The
LC/MS analysis showed one major trisulfated disaccharide
containing 3-O sulfated glucosamine unit, DU-GlcNS3S6S, because of the presence of 3-O sulfate groups. These 3-O
corresponding to molecular ion 576.0 [M-1H] 1 and two sulfate containing tetrasaccharides are: DU-GlcNAc6S
other minor disaccharides, DU-GlcNS and DU-GlcNS6S, GlcAGlcNS3S6S with molecular ion 517.0 [M-2H] 2,
corresponding to molecular ion 416.1 and 496.1 [M-1H] 1, DU-GlcNAc6SGlcAGlcNS3S with molecular ion 477.1
respectively. It was demonstrated earlier by us that absence [M-2H] 2, and DU-GlcNS6SGlcAGlcNS3S6S with mo-
of 2-O sulfation could signicantly increase the number of lecular ion 536.0 [M-2H] 2. This result demonstrates that
3-O sulfation sites within the polymer which may account Mitrin consists of multiple ATIII-binding sites within the
for the presence of 3-O sulfate containing trisulfated disac- polymer and explains why Mitrin has greater ability to
charide as a major component [21]. The LC/MS analysis inhibit factor Xa. Because Mitrin is free of 2-O sulfated
also conrmed the presence of many tetrasaccharides, iduronic acid residues, we expect that it will have a reduced
which are resistant to any further cleavage by heparitinases, ability to bind to PF4 which should decrease its ability to
cause HIT, and at the same time, increase its anticoagulant
activity against the platelet-rich thrombi present on the
arterial side of the circulation. It seems likely that Mitrin
will be relatively more resistant to be cleaved by endoge-
nous heparanases because it lacks 2-O sulfated iduronic
acid, and hence it could exhibit longer in vivo half-life [24].
Furthermore, this engineered polysaccharide would be free
of animal-derived pathogens. Molecular weight of Mitrin
can be tailored at any stage during this two-step synthesis
by standard chemical or enzymatic cleavage techniques that
have been utilized in similar fashion to produce low molec-
ular weight heparin. Engineered Mitrin with the desired
pharmacological properties is predicted to emerge as a
replacement drug for the animal-derived low molecular
weight heparins that are currently in use. We acknowledge
that the use of regenerating PAPS system would substan-
tially reduce the cost of producing Mitrin in large scales.
This approach permits us to develop the model synthetic
schemes for designing the polysaccharides with specic
biologic functions and altering their structures to avoid
detrimental side eects. In addition, we have been able to
synthesize multiple biologic sites within the polymer, which
increases the potency of Mitrin and may permit the poly-
Figure 1 Gel shift analysis of polysaccharide 3. PAP35S- saccharides to act on in discreet regions of the body.
radiolabeled Mitrin (10,000 counts) was reacted with 5 Ag Next, we chose to synthesize, evaluate, and character-
ATIII. Complex formation was analyzed by nondenaturing ize the ATIII-binding pentasaccharide, because we recog-
gel electrophoresis (4% polyacrylamide). The mobility of nize that the synthesis of the rst bioactive oligosaccharide
radiolabeled Mitrin was compared with and without ATIII. would prove the feasibility of this approach to synthesize
814 Kuberan et al.
any oligosaccharide [25]. The ATIII-binding pentasaccha- data acquisition and analysis. A gradient elution was
ride was chosen because more is known about this product performed, using a binary solvent system composed of
than any other oligosaccharide. This includes not only the water (eluent A) and 70% aqueous methanol (eluent B),
structure of the oligosaccharide but also functionally im- both containing 8 mM acetic acid and 5 mM dibutylamine
portant residues within the oligosaccharide. For example, as an ion-pairing agent. Alternatively isocratic elution with
binding of synthesized pentasaccharide to ATIII, as visu- 0% B and followed by 40% B was carried out to determine
alized by gel shift assay, demonstrated that 3-O and 6-O the total disaccharide composition. HPLC separations
sulfate groups were correctly placed within the synthesized were performed on a 0.3250 mm C18 silica column. The
product [22]. Furthermore, the use of stable isotopes and ow rate was set at 5 Al min 1. Mass spectra were acquired
LC/MS permitted us to monitor the generation of the nal on a Mariner BioSpectrometry Workstation ESI time-of-
product and the conrmation of its structure and purity ight mass spectrometer (PerSeptive Biosystems). In the
[23]. None of this type of structural and molecular data is negative-ion mode, nitrogen was used as a desolvation gas
available for any other bioactive oligosaccharides. We as well as a nebulizer. Conditions for ESI-MS were as
envision that the next step in this eld will require the follows: nebulizer ow 0.75 l/min, nozzle temperature
production of an oligosaccharide library (by using this 140jC, drying gas (N2) ow 1.2 l/min, spray tip potential
enzymatic assembly approach). Generalization of our ap- 2.8 kV, nozzle potential 70 V, and skimmer potential 12 V.
proach to oligosaccharide of structures dierent from that Negative ion spectra were generated by scanning the range
of ATIII-binding pentasaccharide can be carried out by of m/z 402000.
minor alterations in our enzymatic synthetic method,
which include changes in the order of the addition of
enzymes or the use of dierent isoforms or the use of B. Expression of Heparan Sulfate
exo-catabolic enzymes along with glycosyl transferases to Sulfotransferases and Epimerase
remodel oligosaccharides from the nonreducing end. These
All of the HS biosynthetic enzymes were expressed and
latter enzymes are all cloned, expressed, and characterized.
puried in baculovirus system as described in our earlier
In addition, EXT polymerase could be employed to syn-
work [19,21]. In brief, a donor plasmid for the preparation
thesize the starting E. coli K5 polysaccharide or oligosac-
of recombinant baculovirus expressing a soluble form of
charide. Thus our approach can be generalized to allow
the epimerase was constructed in pFastBac HT plasmid
oligosaccharides of virtually any size or structure to be
modied by the insertion of honeybee mellitin signal
synthesized. The ready availability of these structures
peptide ahead of the histidine tag. HS biosynthetic enzyme
should permit us to identify ligand proteins, which recog-
recombinant baculovirus was prepared by using the donor
nize specic HS structures, and to use these ligand proteins
and the Bac-to-Bac baculovirus expression system (Life
to further our understanding of various biological systems.
Technologies) according to the manufacturers protocol,
This library can then be used to dene critical biological
except that recombinant bacmid DNA was puried using
groups on the oligosaccharides, which interact with their
an endotoxin-free plasmid purication kit (Qiagen) and
target ligands other than ATIII. In this fashion, structural
transfection of Sf9 cells was scaled up to employ f15 Ag of
knowledge can be developed which will eventually rival the
bacmid DNA and f2.5107 exponentially growing cells in
ATIII pentasaccharide system. This foundation of knowl-
four 100-mm dishes and amplied twice. The resulting
edge is required before other oligosaccharides can be
high-titer viral stock was stored in aliquots (0.75 mL)
evaluated with regard to their structures and functions.
sucient to infect f3108 cells, as determined by Western
blotting of medium from infected cells using (his)4 antibody
(Qiagen). Infected cells were plated and incubated at 26jC
II. MATERIALS AND METHODS for 9096 h. The pooled medium was subjected to further
HS precursor polysaccharide was prepared from E. coli purication to obtain pure enzymes.
K5 strain [9]. Heparan Sulfate C-5 epimerase, 3-OST1,
6-OST2a, and NDST2 sulfotransferases were all cloned
C. Preparation of [35S]PAPS
and expressed in baculovirus system [12,13,1517]. The
radioactive PAP35S was prepared as reported earlier, PAPS was prepared following the modied procedure of
whereas PAPS was purchased from Calbiochem. All chem- Renosto and Segel using a yeast extract [22]. Briey, the
icals were purchased from Sigma. ATIII and Factor Xa yeast extract (15 mg) was dialyzed against 10 mM Tris
were from Haematologic Technologies Inc. Chromogenic HCl, pH 7.4, with 1 mM EDTA and then mixed with 5 mL
substrate S-2765 was from Chromogenix. Heparitinase I, of 120 mM TrisHCl, pH 8.5, 36 mM MgCl2, 16 mM ATP,
II, and III were obtained from Seikagagu. APS kinase was and 2 mCi Na235SO4. The reaction mixture was incubated
a generous gift from Professor I. H. Segel (Univ. of at 37jC for 1 h, heated at 100jC for 2 min, and spun at
California, Davis). 10,000g for 10 min. The supernatant was charged to a 1
mL-bed DEAESephacel column equilibrated with 10 mM
A. LC-MS Analysis triethylammonium carbonate, pH 8.15. The column was
washed with 3 mL of 250 mM triethylammonium carbon-
An ultimate capillary HPLC workstation (Dionex) was ate, pH 8.15, and eluted with 400 mM triethylammonium
used for microseparation. UltiChrom software was used in carbonate, pH 8.15. The eluted product was dried, recon-
Enzymatic Synthesis of Heparan Sulfate 815
stituted in 500 Al of 30 mM KH2PO4, and ltered through a F. Purification of Polysaccharides

Whatman Partisil 5 SAX anion exchange HPLC column
(0.4625 cm). The matrix was washed for 30 min at a ow The reaction mixture, after termination of the reaction, was
rate of 0.8 mL/min with a linear gradient of 30400 mM diluted to 5 mL with 0.25 M NaCl, 20 mM NaAc, 0.01%
KH2PO4 at 24jC, and [35S]PAPS was eluted over 30 min TX-100, pH 6.0, and then 1 mg of glycogen was added to
at a ow rate of 0.8 mL/min with 400 mM KH2PO4 at minimize nonspecic interaction of polysaccharides with
24jC. The radiolabeled product (61 Ci/mmol) was neutral- the column matrix. The diluted reaction mixture was
ized with 10 N NaOH and diluted 100-fold with H2O and loaded on 0.1 mL of DEAESephacel column, pre-equil-
then charged to a 1-mL bed DEAESephacel column ibrated with 2 mL of washing buer containing 0.25 M
equilibrated with 10 mM triethylammonium carbonate, NaCl, 20 mM NaAc, 0.01% TX-100, pH 6.0. The column
pH 8.15, and the column was washed and eluted as was washed with 20 column volumes of washing buer and
described above. The radiolabeled puried product was the polysaccharide was eluted from the column with 2 mL
dried, dissolved in H2O, and stored at 80jC. In a similar of 1 M NaCl in 20 mM NaAc, pH 6.0. Absolute ethanol (8
manner, as mentioned above, PAPS with other stable mL) and 1 mg of glycogen were added to 2 mL of eluent in a
isotopes were prepared. 50-mL disposable polystyrene tube and incubated at 4jC
overnight to facilitate the precipitation of polysaccharides
(Scheme 1). The precipitate was obtained by centrifuging in
D. Production and Purification of K5
a RC3B centrifuge for 15 min at 3000 rpm. The obtained
Polysaccharide 1 pellet was washed with 1 mL of 70% ethanol twice, and
E. coli K5 bacterial cells were grown, as reported earlier nally dissolved in 200 Al of double-distilled water for
with slight modication, overnight in 1 L of growth media subsequent characterization.
containing following ingredients: casaminoacids (20 g),
yeast extract (10 g), NaH2PO4 (4.8 g), KH2PO4 (4.2 g), G. Gel Mobility Shift Assay
K2HPO4 (5.3 g), MgCl2 (0.5 g), glucose (2 g), FeSO4 (20
mg). The bacterial culture was adjusted to pH 6 with acetic The heparinATIII binding buer contained 12% glyc-
acid, solid protease (200 mg/l) was added and maintained erol, 20 mM TrisHCl (pH 7.9), 100 mM KCl, 1 mM
at 37jC for 24 h. Insoluble material was removed by EDTA, and 1 mM DTT. For a typical 20-Al binding
centrifugation at 3000 rpm. The supernatant was diluted reaction, radiolabeled polysaccharide (f10,000 cpm) was
with an equal volume of double distilled water and applied mixed with AT-III (5 Ag) in the binding buer. The
to a DEAESephacel column (50 mL) that was previously reaction mixture was incubated at room temperature
equilibrated with washing buer, 0.2 M NaCl in 20 mM (23jC) for 20 min and then subsequently applied to a
sodium acetate (pH 6). The column was washed with 20 4.5% native polyacrylamide gel (with 0.1% of bis-acryl-
bed volumes of washing buer and K5 polysaccharide was amide). The gel buer was 10 mM Tris (pH 7.4) and 1 mM
eluted then with 0.5 M NaCl in 20 mM sodium acetate EDTA, and the electrophoresis buer was 40 mM Tris
containing 0.01% TRX-100 (pH 6). The eluate was (pH 8.0), 40 mM acetic acid, 1 mM EDTA. The gel was
adjusted to 1 M NaCl with solid sodium chloride and then run at 6 V/cm for 12 h with an SE 250 Mighty Small II
4 volumes of cold ethanol was added and left overnight at gel apparatus (Hoefer Scientic Instruments). After elec-
4jC to precipitate N-acetyl heparosan, which was trophoresis, the gel was transferred to 3 MM paper and
obtained by centrifugation at 3000 rpm for 30 min, dried under vacuum. The dried gel was autoradiographed
subsequently vacuum-dried. The isolated polysaccharide by a PhosphorImager 445SI (Molecular Dynamics). The
1 (Scheme 1) was digested with heparitinases and analyzed image was analyzed with NIH Image 1.60 and the band
by LC/MS. The m/z of 378 corresponding to [M-1H]1 intensities were evaluated.
and m/z of 757 [M-1H]1 were observed for disaccharide
and tetrasaccharide molecular ions, respectively. H. Factor Xa Assay
E. Enzymatic Modification with Recombinant Human factor Xa (10.4 mg/mL 50% glycerol, 820 units/
Enzymes: NDST2, C5 Epimerase, 6-OST2a, mg) was used for assay. Factor Xa was diluted 1:200 with
and 3-OST1 PBS containing 1 mg of bovine serum albumin (4 and 15
units/mL, respectively). ATIII (2.5 mg/mL) was diluted
The labeling 2 buer contains 50 mM MES (pH 7.0), 1% 1:200 to give a 2107 M stock solution. The chromogenic
(W/V) Triton X-100, 5 mM MgCl2, 5 mM MnCl2, 2.5 mM substrate S-2765 was from Chromogenix and the stock
CaCl2, 0.075 mg/mL protamine chloride, 1.5 mg/mL BSA solution of 1 mM with 1 mg/mL Polybrene in water was
or 25 mM HEPES, 40 mM CaCl2, pH 6.5. For a 2500- prepared. Heparin (174 international units/mg; Sigma) was
Al reaction, the following were assembled: polysaccharide used as a standard. Mitrin was used for factor Xa (10 ng).
(nal concentration was 0.1 mM equivalent of unmodied The protocol involved adding 25 Al of ATIII (2107 M) to
disaccharide), 1250 Al of 2 buer, sulfotransferase or 25 Al of a serial dilution of heparin standards or Mitrin in
epimerase, [35S]PAPS (1.0107 cpm), or [32S]PAPS (nal TrisEDTA (50 mM Tris, 7.5 mM EDTA, and 175 mM
concentration of 20 AM), and water was added to make NaCl (pH 8.4)) buer. The reaction was incubated at 37jC
volume 2.5 mL. The reaction was incubated at 37jC for 12 for 75 sec. Factor Xa (25 Al, 4 units/mL) was added. After
h and then subjected to further purication. incubating at 37jC for 195 sec, 25 Al of S-2765 was added.
816 Kuberan et al.
The absorbance at 405 nm was read every minute for 10 sulfate. cDNA cloning and expression of D-glucuronyl C5-
min using a Beckman UV spectrometer. epimerase from bovine lung. J. Biol. Chem. 1997, 272,
28158.
14. Campbell, P.; Hannesson, H.H.; Sandback, D.; Roden, L.;
Lindahl, U.; Li, J.P. Biosynthesis of heparin/heparan
ACKNOWLEDGMENT sulfate. Purication of the D-glucuronyl C-5 epimerase
from bovine liver. J. Biol. Chem. 1994, 269, 26953.
This study was supported by grants from the National 15. Habuchi, H.; Tanaka, M.; Habuchi, O.; Yoshida, K.;
Institutes of Health (P01HL66105 and P01 HL41484). Suzuki, H.; Ban, K.; Kimata, K. The occurrence of three
isoforms of heparan sulfate 6-O-sulfotransferase having
dierent specicities for hexuronic acid adjacent to the
targeted N-sulfoglucosamine. J. Biol. Chem. 2000, 275,
REFERENCES 2859.
16. Liu, J.; Shworak, N.W.; Fritze, L.M.S.; Edelberg, J.M.;
1. McLean, J. The thromboplastic action of cephalin. Am. J. Rosenberg, R.D. Purication of heparan sulfate D-gluco-
Physiol. 1916, 41, 250. saminyl 3-O-sulfotransferase. J. Biol. Chem. 1996, 271,
2. Capila, I.; Linhardt, R.J. Heparinprotein interactions. 27072.
Angew. Chem. Inter. Ed. 2002, 41, 391. 17. Shworak, N.W.; Liu, J.A.; Petros, L.M.; Zhang, L.;
3. Damus, P.S.; Hicks, M.; Rosenberg, R.D. Anticoagulant Kobayashi, M.; Copeland, N.G.; Jenkins, N.A.; Rosen-
action of heparin. Nature 1973, 246, 355. berg, R.D. Multiple isoforms of heparan sulfate D-
4. Rosenberg, R.D.; Damus, P.S. The purication and glucosaminyl 3-O-sulfotransferaseIsolation, character-
mechanism of action of human antithrombinheparin ization, and expression of human cDNAs and identication
cofactor. J. Biol. Chem. 1973, 248, 6490. of distinct genomic loci. J. Biol. Chem. 1999, 274, 5170.
5. Sinay, P.; Jacquinet, J.C.; Petitou, M.; Duchaussoy, P.; 18. Xia, G.; Chen, J.; Tiwari, V.; Ju, W.; Li, J.P.; Malmstrom,
Lederman, I.; Choay, J.; Torri, G. Total synthesis of a A.; Shukla, D.; Liu, J. Heparan sulfate 3-O-sulfotransfer-
heparin pentasaccharide fragment having high-anity for ase isoform 5 generates both an antithrombin-binding site
antithrombin-III. Carbohydr. Res. 1984, 132, C5. and an entry receptor for herpes simplex virus, type 1. J.
6. Petitou, M.; Herault, L.P.; Bernat, A.; Driguez, P.A.; Biol. Chem. 2002, 277, 37912.
Duchaussoy, P.; Lormeau, J.C.; Herbert, J.M. Synthesis of 19. Liu, J.A.; Shworak, N.W.; Sinay, P.; Schwartz, J.J.; Zhang,
thrombin-inhibiting heparin mimetics without side eects. L.; Fritze, L.M.; Rosenberg, R.D. Expression of heparan
Nature 1999, 398, 417. sulfate D-glucosaminyl 3-O-sulfotransferase isoforms re-
7. Chong, B.H.; Fawaz, I.; Chesterman, C.N.; Berndt, M.C. veals novel substrate specicities. J. Biol. Chem. 1999, 274,
Heparin-induced thrombocytopenia: mechanism of inter- 5185.
action of the heparin-dependent antibody with platelets. 20. Razi, N.; Lindahl, U. Biosynthesis of heparin/heparan
Br. J. Haematol. 1989, 73, 235. sulfate. The D-glucosaminyl 3-O-sulfotransferase reaction:
8. Stringer, S.E.; Gallagher, J.T. Specic binding of the target and inhibitor saccharides. J. Biol. Chem. 1995, 270,
chemokine platelet factor 4 to heparan sulfate. J. Biol. 11267.
Chem. 1997, 272, 20508. 21. Zhang, L.; Lawrence, R.; Schwartz, J.J.; Bai, X.; Wei, G.;
9. Vann, W.F.; Schmidt, M.A.; Jann, B.; Jann, K. The struc- Esko, J.D.; Rosenberg, R.D. The eect of precursor
ture of the capsular polysaccharide (K5 antigen) of urinary- structures on the action of glucosaminyl 3-O-sulfotransfer-
tract-infective Escherichia coli 010:K5:H4. A polymer ase-1 and the biosynthesis of anticoagulant heparan sulfate.
similar to desulfo-heparin. Eur. J. Biochem. 1981, 116, 359. J. Biol. Chem. 2001, 276, 28806.
10. Rosenberg, R.D.; Shworak, N.W.; Liu, J.; Schwartz, J.J.; 22. Wu, Z.L.; Zhang, L.; Beeler, D.L.; Kuberan, B.; Rosen-
Zhang, L.J. Heparan sulfate proteoglycans of the cardio- berg, R.D. A new strategy for dening critical functional
vascular system. Specic structures emerge but how is groups on heparan sulfate. FASEB J. 2002, 16, 539.
synthesis regulated? J. Clin. Invest. 1997, 99, 2062. 23. Kuberan, B.; Lech, M.; Zhang, L.J.; Wu, Z.L.; Beeler,
11. Aikawa, J.; Grobe, K.; Tsujimoto, M.; Esko, J.D. Multiple D.L.; Rosenberg, R.D. Analysis of heparan sulfate
isozymes of heparan sulfate/heparin GlcNAc N-deacety- oligosaccharides with ion pair-reverse phase capillary high
lase/GlcN N-sulfotransferase. Structure and activity of the performance liquid chromatographymicroelectrospray
fourth member, NDST4. J. Biol. Chem. 2001, 276, 5876. ionization time-of-ight mass spectrometry. J. Am. Chem.
12. Orellana, A.; Hirschberg, C.B.; Wei, Z.; Swiedler, S.J.; Soc. 2002, 124, 8707.
Ishihara, M. Molecular cloning and expression of a 24. Bame, K.J. Heparanases: endoglycosidases that degrade
glycosaminoglycan N-acetylglucosaminyl N-deacetylase/ heparan sulfate proteoglycans. Glycobiology 2001, 11,
N-sulfotransferase from a heparin-producing cell line. J. 91R.
Biol. Chem. 1994, 269, 2270. 25. Kuberan, B.; Lech, M.; Beeler, D.L.; Wu, Z.; Rosenderg,
13. Li, J.; Hagner-McWhirter, A.; Kjellen, L.; Palgi, J.; R.D. First enzymatic synthesis of ATIII-binding heparan
Jalkanen, M.; Lindahl, U. Biosynthesis of heparin/heparan sulfate pentasaccharide. Nature Biotechnology 2003.
36
Polysaccharide-Based Hydrogels in Tissue Engineering
Hyunjoon Kong and David J. Mooney
University of Michigan, Ann Arbor, Michigan, U.S.A.
I. INTRODUCTION create a space into which cells in the neighboring tissue can
migrate. These cells can then regenerate the tissue, while the
Every day, thousands of people unexpectedly suer the loss material degrades. This approach is used clinically in
or malfunction of organs or tissues due to accidents or dentistry (guided tissue regeneration) and orthopedics
various diseases. Together, the medical costs related to (guided bone regeneration), but success in this approach
these problems are estimated to exceed $40 billion per year is not always consistent, nor predictable [7,8]. To more
in the United States. To treat these people, a variety of specically guide the regeneration of the desired tissue via
surgical procedures combined with medical treatments stimulating specic cell populations, various bioactive
have been developed to date. These include transplantation molecules have been delivered to the tissue defect site to
of patients or other donors tissues or organs (e.g., liver), stimulate the target cells and induce regeneration by these
implantation of articial prosthesis (e.g., hip joints), and cells (induction) [9]. The molecules may be released out of a
utilization of extracorporeal support devices (e.g., kidney material carrier in a sustained manner to provide long-term
dialysis). While saving countless lives and improving the stimulation of the cells in the tissues neighboring the
lives of millions, these treatments are still associated with materials. Finally, in place of bioactive molecules, tissue-
several problems. For example, transplanting a patients specic cells or stem cells may be transplanted to the
healthy tissue to another part of the body (e.g., from rib to desired site, using an appropriate vehicle to grow the
reconstruct the face) to treat a defect may cause various desired tissue (cell transplantation). The transplanted cells
complications, including morbidity of the donor site [1]. are typically rst harvested from a small biopsy of tissue
Transplantation of tissues or organs from one individual to from the patient or a donor, and expanded to a desired
another (allotransplantation) is limited by the extreme number in vitro. The desired tissue or organ structure can
shortage of donated tissues [2], the immunological re- develop via the proliferation and dierentiation of the
sponse to the transplants, and the possibility of transmis- transplanted cells and interacting host cells. Induction
sion of infectious diseases [3]. Utilizing xenografts from and cell transplantation approaches have been widely
animals is extremely limited due to hyperacute rejection of utilized in recreating various tissues, including the artery
the animal tissues [4], infection, and the possible introduc- [10], capillary blood vessels [11], skin, cartilage [12], bone
tion of new diseases into humans. The long-term implan- [13], bladder [14], liver [15], ligament [16], nerve [17],
tation of nondegradable synthetic materials into the body intestine [18], heart valve [19], and tendon [20]. Some tissue
can lead to inammation around the implants and eventual engineering products have already been commercialized
mechanical failure of the materials, requiring resurgery. under the approval of the Food and Drug Administration
A recently emerged approach to replace the structure (FDA). Carticel R, autologous chondrocyte transplanta-
and function of lost tissues, while circumventing the com- tion, and ApligrafR, living skin equivalents, are examples
plications of current therapies, is to regrow or engineer of these new therapies [21].
these structures using combination of materials, bioactive In virtually all tissue engineering approaches, an exog-
molecules, and cells [2,5]. This tissue engineering concept is enous three-dimensional polymer matrix is critical to the
classied into three approaches: conduction (Fig. 1a), success of the approach. The material should perform as a
induction (Fig. 1b), and cell transplantation (Fig. 1c) [6]. vehicle to transport the cells and bioactive molecules to the
In conduction approaches, biodegradable materials are desired sites in the body, a synthetic extracellular matrix
surgically implanted at the site of the damaged tissues to (ECM) to regulate the function of cells, and a template to
817
818 Kong and Mooney
Figure 1 Approaches in tissue engineering, (a) conduction to promote the migration of host cells by providing a space with
biodegradable materials, (b) induction to stimulate target cells by releasing bioactive molecules from the biodegradable materials,
(c) cell transplantation of desired cell populations in biodegradable materials to drive growth of new tissue. (From Ref. 6.)
guide the growth of new tissues. For this purpose, a variety biocompatibility [26]. The chemical structures of polysac-
of biodegradable synthetic or naturally derived polymers charides are similar to the bioactive glycosaminoglycan
have been employed to date [22]. For example, synthetic (GAG) molecules (e.g., heparin sulfate, chondroitin sul-
polyesters [e.g., poly(glycolic acid)] have found utility due fate, hyaluronan) present in the ECM of mammalian
to their FDA approval in a number of medical applications tissues [27]. Furthermore, polysaccharides are readily ac-
[23]. The materials utilized in tissue engineering can be cessible at low cost. This chapter will focus on polysaccha-
processed into various physical forms, including a nonwo- ride-based hydrogels as tissue engineering matrices, and
ven mesh or brils, porous scaolds, and hydrogels. Cells alginate, chitosan, hyaluronic acid, and agarose hydrogels,
are seeded onto the nonwoven or porous scaold followed in particular, will be reviewed.
by implantation (Fig. 2). Alternatively, if a hydrogel is the
desired form, the cells are often mixed with polymer so-
lutions prior to gelling, followed by injection into the body. II. DESIGN OF HYDROGELS FOR TISSUE
Hydrogels are an appealing form for biomaterials used ENGINEERING
in tissue engineering because they completely surround
To achieve the desired performance from hydrogels in
cells and may be injected into a patient using minimally
tissue engineering applications, the chemical and physical
invasive surgical procedures. In addition, as hydrogels are
properties of hydrogels should meet the following de-
mainly composed of water, they have a structural similarity
sign criteria:
to the normal ECMs of tissues and often exhibit good
biocompatibility. These advantageous features have made 1. Good biocompatibility is essential, since it deter-
hydrogels attractive as biomaterials to carry cells and mines the degree of foreign body response by host
bioactive molecules [24]. Various types of hydrogel-form- cells (e.g., lymphocytes) and will aect the viability
ing polymers, including polysaccharides (i.e., alginate, of cells immobilized in the material.
chitosan, hyaluronic acid, agarose), poly(acrylic acid), 2. The rheological properties of pregelled solutions
poly(vinyl alcohol), poly(ethylene glycol) and its copoly- and gelation kinetics should be readily controlled
mers, and polypeptides (e.g., collagen and gelatin) to allow exibility in their use. This is especially
have been investigated [25]. Hydrogels based on naturally critical when one desires an injectable gel.
occurring polymers (e.g., polysaccharides) have many ad- 3. Hydrogels should be readily fabricated into
vantageous features, including low toxicity and good various structures (e.g., nanoporous or micro-
Polysaccharide-Based Hydrogels in Tissue Engineering 819
Figure 2 Schematic illustration of methods to transplant cells via material carriers; Cells are mixed with a polymer solution
which has a capacity to form a hydrogel, followed by the delivery of mixture via injection. Alternatively, cells are seeded onto
macroporous solid materials, followed by delivery via implantation. (From Ref. 25.)
porous scaolds), depending on the specic material or release of cell-interacting molecules

application. For example, micron-sized gel par- from the gel.
ticles are favorable for delivering pregelled 6. Hydrogels should undergo degradation in a
matrices via injection. The pore size will regulate controllable and predictable manner. As newly
diusion through the gels and migration of cells developed tissues propagate through the gels, the
into the gels. gel should degrade to provide a space for new
4. A wide range of mechanical properties (i.e., elastic tissue formation. It is important to match the
modulus, ultimate strength, ultimate strain at degradation rate of the gel to the growth rate of
break under compressive, shear, and tensile new tissues, which may vary with the specic tissue
deformation) should be available. Once hydrogels being engineered. Degradation of gels also pre-
are placed at a desired site in the body, they must vents chronic foreign body responses.
maintain their structural integrity in the face of
mechanical deformation exerted by neighboring
tissues and the contractional forces of cells within III. POLYSACCHARIDE-BASED HYDROGELS
the gels [28]. In addition, they must act as a
template to direct new tissue growth. Therefore, In general, polysaccharides are composed of saccharide
broad ranges of mechanical properties are consid- rings having dierent functional groups and conforma-
ered benecial, as dierent sites in the body may tions. These monomeric residues are connected to form
have dierent loading conditions. In addition, the alternating or block copolymers. The dierences in chem-
stiness of the gel may directly aect the adhesion ical structure and conformation of the residues result in
and gene expression of interacting cells [29]. dierent gelation mechanisms, degradation behaviors, and
5. Hydrogels used as synthetic ECMs should have interactions with cells. The cell interaction ability of poly-
specic interactions with cells to elicit an appro- saccharide gels is often manipulated by the coupling of
priate function from the cells [30]. These inter- small synthetic cell adhesion ligands to the polymers or
actions may involve adhesion of cells to the immobilization of growth factors within the gel. This
820 Kong and Mooney
section reviews various polysaccharide-based hydrogels

utilized, to date, in tissue engineering, and chemical and
physical modications developed to enhance their roles in
tissue regeneration.
A. Alginate Hydrogels
Alginate and its derivatives have been widely utilized in
humans (e.g., food additives, dental impression materials,
drug delivery vehicles [31]) in the past due to their biocom- Figure 4 Selective ionic cross-linking (e.g., Ca2+) between
patibility and low toxicity. Hydrogels are formed from guluronic (G) acid residues on adjacent polymer chains of
alginate via cross-linking for use as cell immobilization alginate is responsible for the formation of ionically cross-
matrices [32] and wound dressings [33]. PolyMem R wound linked hydrogels.
dressing is one alginate-based material approved as a safe
material by the FDA. The physical properties of alginate
hydrogels, including mechanical properties, are readily
model (Fig. 4). The mechanical properties and porosities
controlled with modication of the chemical structure of
of the gels are generally determined from the types of
the sugar residues and use of various cross-linking mole-
binding cations and their concentrations, proportions of
cules. In addition, interactions of cells with this material are
G acid residues, and their block length [37,38]. However,
often modied via conjugation of small bioactive mole-
only a limited range of mechanical properties is available
cules. These materials have recently been utilized to engi-
from ionically cross-linked gels.
neer cartilage, bone, liver, muscle, blood vessels, and nerve
The range of mechanical properties available from
tissue. For these purposes, tissue-specic cells and specic
alginate hydrogels can be broadened with several
growth factors were frequently immobilized in the gels
approaches. First, utilization of other gelation modes,
either separately or together.
which result from covalently modifying alginate molecules,
or reinforcing the gels with other molecules to form poly-
1. Alginate
electrolyte complexes can lead to a wide range of available
Alginates harvested from sea brown algae are anionic properties [37,39]. Alginate molecules can be covalently
block copolymers consisting of 1,4-linked h-D-mannuronic cross-linked using various di- or multiamines or hydrazide-
(M) acid residues and 1,4-linked a-L-guluronic (G) acid containing cross-linking molecules. Typically, carboxylic
residues, which have dierent conformations (Fig. 3). The groups on alginate molecules are activated by N-hydroxy-
sugar residues are organized into blocks of GGG, sulfosuccinimide (sulfo-NHS) and 1-ethyl-3-[3-(dimethyl-
MMM, or alternating MGMG copolymers, which amino)propyl] carbodiimide (EDC) in this approach (Fig.
have dierent magnitudes of chain exibility [34]. The 5). Ethylenediamine [40], L-lysine, poly(ethylene glycol)
proportion of these three dierent blocks, length of each diamine, and adipic acid dihydrazides have been used as
block, and overall molecular weights of the polymer chains cross-linkers (Fig. 6) [41]. As the length of G acid units and
are dependent on their natural sources. However, they also their proportions in alginate are not critical in the forma-
can be further modied via chemical or radiation treat- tion of covalently cross-linked hydrogels, formation of
ments [35]. hydrogels is possible at much lower concentrations of
alginate and cross-linking components. Alginate can also
2. Preparation of Hydrogels and Control be cross-linked by gamma cross-linking reactions between
of Mechanical Properties alginate chains having grafted acrylate or allyl groups.
Alginates readily form hydrogels via a preferential binding Another approach to modify the mechanical proper-
of certain divalent cations (i.e., Ca2+, Sr2+, Ba2+) to G ties of hydrogels is to use a combination of high and low
blocks containing more than 20 monomers [36]. This cross- molecular weight (MW) alginates, as this allows a higher
linking structure has been described using an egg-box loading of alginate in the gel (Fig. 7) [42]. For example,
Figure 3 Molecular structure of alginate molecules, G: a-L-guluronic acid, M: h-D-mannuronic acid.

Figure 5 Formation of covalently cross-linked alginate hydrogels with carbodiimide reagents [EDC:1-(3-ethyl)-3-[3-
(dimethylamino)prophyl] carbodiimide, Sulfo-NHS: N-hydroxysulfosuccinimide]. Sulfo-NHS and EDC activate carboxylic
groups in alginate molecules, leading to a reaction with amine groups in cross-linking molecules (H2N-R-NH2). R can be a
variety of structures, as shown in Figure 6.
hydrogels prepared from a mixture of high-MW (f3 105 Variations in the gel cross-linking mechanism or
g/mol) and low-MW (f6 104 g/mol) alginates demon- structure of alginate itself have been pursued to regulate
strates a continuous increase in the elastic moduli with total gel degradation. Hydrogels prepared by covalent cross-
solids concentration. This is accomplished with minimal linking or photo cross-linking are typically more resistant
increases in the viscosity of the pregelled solution. In to structural disintegration than ionically cross-linked gels.
contrast, increasing the solids concentration of gels formed Alternatively, partial oxidation of alginate molecules can
solely with high-MW alginate leads to a reduction in the introduce acetal groups labile to hydrolysis, which leads to
elastic moduli at intermediate solids concentration, likely chain scission of alginate molecules over time (Fig. 8).
due to poor mixing of the high-viscosity pregelled solution Alginates with chains smaller than the renal clearance of
with the gelling cation. the kidney [48] can also be covalently cross-linked with
Alginate gels may also be reinforced by the formation molecules that form or contain degradable linkages. For
of complexes with polycations such as poly-L-lysine, poly(- example, hydrogels prepared from fully oxidized poly(al-
ethyleneimine), and chitosan [37,4345]. If these polymers dehyde guluronates), via cross-linking with adipic acid
coat the outside of the alginate gel, the porosity and dihydrazide (AAD), demonstrated a degradation prole
structural stability of gels are also altered. This approach regulated by the extent of cross-linking. Interestingly, the
has been used to modulate the release of drug molecules mechanical properties and degradation rates of these gels
encapsulated in alginate beads [46]. In addition, the for- can be decoupled once the cross-linking density exceeds a
mation of complex layers enhances the resistance of gels to critical point where dangling cross-linking molecules are
ion-exchange, thus increasing the lifetime of the gels [37]. introduced into the system. These molecules retard the
degradation of hydrogels due to re-cross-linking (Fig. 9)
3. Controlling Degradation of Alginate Hydrogels [49,50].
Unmodied alginate is not believed to undergo enzymatic
or hydrolytic degradation in the body. Rather, ionically 4. Controlling the Cell and Tissue Interactions
cross-linked hydrogels undergo an ion exchange process, of Alginate Hydrogels
which results in, rst, the release of cross-linking cations, Adhesion of cells to a material can be regulated to induce a
and second, the uncross-linked alginate chains. This process change in cell function, such as proliferation, migration,
occurs in an unpredictable and uncontrollable manner [47]. dierentiation, and apoptosis [51]. However, cells do not
Figure 6 Cross-linking molecules utilized to form covalently cross-linked hydrogels, (a) L-lysine, (b) adipic acid dihydrazide,
and (c) poly(ethylene glycol)diamine.
822 Kong and Mooney
Figure 7 Decoupling the eects of solid concentration on pre-and postgel properties of alginate, (a) the viscosity (D) of pre-gelled
solutions and (b) shear modulus (G) of post-hydrogels was varied by the concentration of alginates in the solution used to form
the gel; decreasing the weight fraction of high-molecular weight alginate as a function of the total solids [W(H)] limits the increase
in viscosity of pregelled solutions upon increasing the total alginate concentration, while it does not interfere with the increase in
the shear modulus.
directly bind to alginates, and the negatively charged 5. Processing of Alginate Hydrogels
alginate hydrogels discourage the adsorption of proteins Alginate hydrogels can be fabricated into in situ forming
mediating cell adhesion. To incorporate specic cell inter- injectable gels, nanoporous microbeads, and microporous
action ability into alginate hydrogels and regulate cell constructs, depending on the specic application. Micro-
function and tissue development, numerous sugar- or porous hydrogels [63] can be formed via several processes
amino acid-based small molecules have been covalently to provide signicant control of the total porosity and pore
coupled to alginate (Fig. 10a) [52,53]. Specically, alginate size distribution [64]. Gel beads have been the most widely
hydrogel surfaces modied with small synthetic peptides
lead to enhanced adhesion, proliferation, and dierentia-
tion of several cell types, including C2C12 skeletal myo-
blasts, chondrocytes, osteoblasts, and preadipocytes (Fig.
11) [5458]. The biological response of cells to the material
can be controlled with the density of peptides in the gel,
length of spacers connecting the oligopeptides to the gel,
and the physical properties of the gels [54]. Bioactive
carbohydrates (e.g., galactose derivatives) containing eth-
ylenediamine as a spacer were also covalently grafted to
alginate molecules utilizing carbodiimide chemistry (Fig.
10b). Galactose derivatives are recognized by receptors on
liver cells [59], and galactosylated alginate gels improved
the viability, adhesion, and spheroid formation by liver
cells encapsulated in the gels, depending on the degree of
substitution by the bioactive carbohydrates [60]. Bioactive
hyaluronates can also be utilized to infer bioactivity into
alginate gels [61,62].
Figure 9 Decoupling the mechanical properties and degra-

dation properties of oxidized poly(aldehyde guluronate)
hydrogels cross-linked with AAD. The t1/2 indicates the time
when the elastic modulus becomes one-half of its initial
Figure 8 Alginates can be oxidized with sodium periodate value. Gels were incubated in Dulbeccos modied Eagles
to induce acetal groups in the main chain that are labile to medium (DMEM) at 37jC and monitored over time (from
hydrolysis. Ref. 50. Copyright 2000 American Chemical Society).
Figure 10 Modications of alginate to induce cell interaction ability. (a) Conjugation of Arg-Gly-Asp (RGD)-containing
oligopeptides, and (b) conjugation of galactose derivatives having ethylenediamine as chain extender. In both cases, conjugation
is achieved following the activation of carboxylic groups in alginate sugar residues with sulfo-NHS and EDC.
used alginate gel form due to their ease of formation. They combined to form gels with a variety of shapes, sizes, and
can be prepared by extruding droplets of sodium alginate pore sizes and structures.
solution particles into a CaCl2 solution [35]. Smaller and
more homogeneous alginate hydrogel particles can be 6. Alginate Hydrogels as Tissue Engineering Matrices
prepared by using a water/oil emulsion, followed by an For a successful use of alginate hydrogels, which do not
internal gelation [65]. These various techniques can be have cell interaction ability and biodegradability, it may be
Figure 11 Cell adhesion to alginate gels can be controlled by coupling cell adhesion peptides, (a) no adhesion of myoblasts is
observed to unmodied alginate hydrogels, (b) while signicant adhesion and spreading of cells is observed on RGD-modied
alginate hydrogels (from Ref 53 Copyright 1999 Elsevier Sciences).
824 Kong and Mooney
critical to provide hydrogels with both the ability to to their function in this application. Grafting RGD-con-
interact with specic cells and the capacity to degrade. taining oligopeptides to alginate molecules increased the
Alginate hydrogels have been utilized to regenerate carti- dierentiation of bone-forming cells in alginate gels, and
lage, bone, nerve, and liver tissues via conjugation of resulted in an enhanced formation of bone tissue following
oligopeptides to mimic adhesion ligands, incorporation gel/bone precursor cell implantation [57]. Strikingly,
of bioactive molecules for subsequent release, and modi- cotransplantation of bone and cartilage-forming cells in
cation to control their degradation. The gels also have been RGD-modied alginate hydrogels led to the development
utilized as stimulatoryresponsive carriers of growth fac- of structures that structurally and functionally resembled
tors to promote growth of blood vessels. It has been normal growth plates (Fig. 12) [77]. Accelerating the
thought that alginate may invoke a signicant inamma- degradation of RGD-modied alginate-derived gels has
tory response, depending on its composition [66]. Recently, also been reported to contribute to the formation of
newly developed purication techniques suggest these con- mineralized bone tissue [78]. To stimulate the growth of
cerns may have arisen due to contaminants, not the algi- bone, naturally derived or synthetic peptides mimicking the
nate itself. Reduction of endotoxin levels via treatment function of bone morphogenic proteins (BMPs) have also
with weak acids, citrate, and ethanol led to no demonstra- been incorporated into alginate gels. BMP-2-derived oli-
ble mitogen-induced foreign body reaction, even upon the gopeptides have been covalently coupled to alginate to
implantation of M acid residue-rich alginates, which have control their availability, as simply dispersing the oligo-
been hypothesized to stimulate more foreign body response peptides would likely result in a rapid diusion out of the
than high G acid-content alginates [67]. gels [79].
Regeneration of Cartilage Development of Capillary Blood Vessels
One of the rst applications of alginate hydrogels in Development of an appropriate network of blood
tissue engineering was to engineer cartilage tissue in vivo to vessels is critical to the creation of large tissues, as they
serve as a bulking tissue in the treatment of vesicoureteral require large amounts of oxygen and other nutrients. New
reux (reux of uid from bladder to kidney) via a mini- blood vessel formation may also be important in the repair
mally invasive surgical procedure [68]. Autologous chon- of damaged blood vessels. In general, the formation of new
drocytes harvested from auricular (ear) cartilage were blood vessels is driven by the action of various growth
loaded into alginate hydrogels, and the mixture subse- factors, including vascular endothelial growth factor
quently injected. Cartilaginous tissues formed at the injec- (VEGF) [80]. One approach to control the process of new
tion site [69]. Recently, chondrocytehydrogel mixtures blood vessel formation is to immobilize these factors in
injected into patients with vesicoureteral reux led to a alginate gels. Following injection at the desired site, the
successful clinical outcome in the majority of patients alginate allows for a sustained release of the factor at that
[70,71]. Chondrocyte-including alginate hydrogels have site. Encapsulation of bFGF and VEGF in alginate beads
also been used in animal studies to prepare facial implants has led to its sustained release, while maintaining its
with complex shapes [72]. An injection molding process biological function [8183]. The release rate of the factors
was developed to allow complex structures to be readily from the gels may also be amplied by externally applied
prepared in this latter study. Alginate hydrogels have also mechanical stimulation, such as compression to the gel
been used to transplant chondrocytes and regenerate new matrices [84]. This approach has been used to enhance
tissues resembling the human ear and to repair defects in
knee joints [73]. A major issue with the use of alginate in
cartilage engineering is the resultant phenotype (function)
of the chondrocytes in the gel. Some studies have reported
the expression of type I collagen by the cells and formation
of brous tissue. These undesirable features have raised
concern on the long-term stability of engineered cartilage
[74]. However, incorporation of appropriate adhesion
factors, growth factors [e.g., transforming growth factors
(TGF)-h-2 or insulin-like GF], or components of the
natural extracellular matrix (e.g., elastin, brin, hyaluronic
acid) into the gels may allow one to manipulate the cell
function and tissue formation [75,76]. For example, inclu-
sion of Arginine-Gelysin Aspartic Acid (RGD) peptides
enhances chondrocyte proliferation and accelerates carti-
lage formation [58].
Figure 12 Cotransplanting osteoblasts and chondrocytes in
Regeneration of Bone RGD-modied alginate hydrogels leads to formation of
Alginate has been used to regenerate bone tissue, and growthlike plate structures at the interface between the
optimization of the cellular interaction ability and degrada- cartilagenous (A) and bony regions (B), which is similar to
tion behavior of alginate hydrogels has greatly contributed that seen in the developing long bones.
blood vessel formation in ischemic tissues, and has been

found to be critically dependent on the interaction of the
factor with the alginate. Thus, activated release of VEGF
by repetitive compression conducted on a regular basis has
been found to accelerate the formation of blood vessels,
compared with gels which did not experience the stimula-
tion (Fig. 13) [85].
Regeneration of Nerve and Liver

Alginate gels with microporous structures have also
been used to regenerate nerves and liver. A macroporous Figure 14 Structure of chitosan; D: D-glucosamine, A: N-
gel structure has been reported to guide regeneration of acetyl-D-glucosamine.
nerves in the spine of various animal models [86,87]. The
growth of neurites could further be enhanced when the gels
were supplemented with Schwann cells [88]. Covalently cules. Hydrogels processed into microspheres and porous
connecting galactose moieties to alginate gels could en- scaolds have been investigated for use in the regeneration
hance the adhesion of hepatocytes, leading to improve- of cartilage, bone, liver, and nerve tissues. In addition,
ments in the viability of the cells and their clustering to chitosan gels have been used as a coating material for
form spheroids [62]. tracheal prosthesis, since they support the growth of respi-
ratory epithelial cells [94].
B. Chitosan Hydrogels 1. Chitosan
Chitosan and its derivatives have been used in various Chitosan is attained by deacetylating chitin, which is a
biomedical applications, including drug delivery and cell naturally occurring polysaccharide typically isolated
transplantation. It has also been used as a diet food to from arthropod exoskeletons. It is generally composed of
prevent the absorbance of fat into the body, wound dressing, D-glucosamine residues and N-acetyl-D-glucosamine resi-
and contact lens material. It is attractive for these applica- dues having varying degrees of deacetylation (Fig. 14). It is
tions due to its good biocompatibility, low toxicity, and generally soluble in acidic pH but insoluble in physio-
structural similarity to GAG molecules naturally existing in logical pH. Varying the degree of deacetylation aects
the human body [8993]. Chitosan solutions readily form the crystallinity and crystal structure of the chains and
hydrogels by adjustments of the pH or cross-linking with the solubility of the polymer chains in water [95]. The solu-
various functional molecules. The material properties of bility in water can also be improved by attaching hydro-
these gels are controlled with modication of the molecular philic groups to chitosan molecules (e.g., glycol chitosan,
structure of chitosan, use of a variety of cross-linking N-carboxymethyl chitosan, Fig. 15). In the same context,
molecules, and formation of an interpenetrating network the solubility of chitosan in organic solvents is enhanced
with other polymers. Nonspecic interactions with cells, by alkylation, carboxyalkylation, and acylation [9698].
which may induce a severe foreign body response, may be Chitosan undergoes degradation in physiological environ-
modulated with incorporation of specic bioactive mole- ments by a variety of enzymes, including chitosanase and
Figure 13 Blood vessel growth is enhanced by compressive stimulation-controlled release of VEGF from alginate hydrogels
implanted into the muscle tissue of mice at the site of femoral artery ligation. (a) A low density of vessels was observed (arrows
point to blood vessels) when mechanical stimulation was not applied, and (b) a much higher density was observed when
mechanical stimulation was applied to the VEGF-containing gel (from Ref. 85 Copyright 2000 Macmillan Magazine Ltd).
826 Kong and Mooney
Figure 15 Structures of chitosans modifed to enhance water solubility. (a) Glycol chitosan and (b) a-carboxymethyl chitosan.
lysozyme, which selectively attack the acetylated residues. lybdate, or phosphate) or polyanions (e.g., dextran sulfate,
Increasing the degree of deacetylation has been reported to carboxymethylcellulose, or polyphosphoric acid) to form
enhance the biocompatibility of this polymer [99]. gels [102104]. Alternatively, chitosan can form gels via
chemical cross-linking with various functional molecules
including glutaraldehyde, carbohydrate scleroglucandial-
2. Preparation of Hydrogels and Control dehyde, ethylenediaminetetraacetic acid (Fig 16b), and
of Mechanical Properties genipin [105,106]. However, the use of cross-linking mol-
Chitosan hydrogels have been prepared by utilizing phys- ecules having aldehyde groups may deteriorate the bio-
ical interactions between the polymer chains, ionic or compatibility of materials. Grafting azides to chitosan
chemical cross-linking with reactive cross-linking mole- molecules allows in situ gel formation via UV irradiation
cules, and photo cross-linking. Strong physical interac- (Fig. 16c) [107].
tions between chains can be induced by changes in pH, The mechanical properties of chitosan hydrogels may
temperature, or grafting of hydrophobic molecules. be dependent on the degree of deacetylation or other
Increasing the pH reduces the solubility of partially deacet- covalent modication, types of cross-linking molecules,
ylated chitosan in water, leading to the formation of pH- and concentration of polymers, although there has not been
dependent hydrogels. In addition, this ready control over a systematic study on the mechanical properties of chitosan
the charge density of chitosan molecules by varying pH hydrogels. Varying the substitution degree by deacetyla-
can lead to gels that release negatively charged macro- tion may aect the crystallinity, which is an important
molecules, like DNA, in a controlled manner [100,101]. factor to attain high stiness from a material [108].
Attaching hydrophobic molecules (e.g., palmitic acid-con- The properties of chitosan hydrogels can also be
taining moieties) to water-soluble glycol chitosan also modied by forming semi-interpenetrating networks
enhances hydrophobic interactions between polymer mol- (IPNs) of chitosan with specic polymers, or grafting these
ecules, leading to gel formation (Fig. 16a). polymers to chitosan. Variables that have been manipu-
Positively charged amine groups in chitosan electro- lated with these approaches include the crystallinity, re-
statically interact with anions (e.g., sulfate, oxalate, mo- sponse to temperature, swelling ratio, and mechanical
Figure 16 Molecules used to form cross-linked chitosan hydrogels. (a) Palmitic acid N-hydroxysuccimide ester for physical
cross-linking. (b) Ethylenediamine tetraacetic acid for chemical cross-linking. (c) P-azidebenzoic acid for photo cross-linking.
properties. IPNs are prepared by blending specic poly- san similar to the bioactive polysaccharide molecules pres-
mers [e.g., poly(acrylic acid), poly(ethylene oxide)] with ent in the normal extracellular matrix of mammalian
chitosan solutions or suspensions, followed by gelation. tissues. However, the interactions between chitosan mole-
Alternatively, monomers or macromers can be used in the cules and cells tend to be nonspecic [120]. Formation of
formation of IPN, via simultaneous or stepwise polymer- brous tissues after an inammatory response would likely
ization of these molecules and gelation of chitosan [109]. inhibit tissue formation, and plain chitosan-based gels may
Strong electrostatic attractions or hydrogen bonding pres- not be desirable for tissue engineering.
ent in IPN structures tend to reduce crystallinity while Various modications of chitosan molecules or incor-
increasing the swelling ratio of the gels and their mechan- poration of specic bioactive molecules into the gels have
ical properties [110]. Grafting of reactive monomers to been performed to mediate the specic interactions of cells
chitosan followed by polymerization also can be exploited with these materials. Since the inammatory responses to
to alter the properties of hydrogels [110,111]. For example, these materials may be related to the N-acetylglucosamine
grafting poly(acrylic acid) or poly(lactic acid-co-glycolic residues in chitosan molecules, chitosan has been deacety-
acid) (PLGA) to chitosan has been demonstrated to con- lated to reduce the degree of biological response [121].
trol the swelling ratio of chitosan hydrogels in a rened Alternatively, chitosan has been modied with GAG mol-
manner, depending on the degree of grafting. When PLGA ecules present in extracellular matrices via an electrostatic
is grafted to chitosan molecules, the proportion of lactic immobilization process to accomplish the same goal [122].
acid/glycolic acid in the copolymer also inuences the In addition, various proteins including collagen, gelatin,
properties of the hydrogels. and albumin [123], RGD-containing oligopeptides, and
sugar residues such as galactose and fructose have been
3. Control of Degradation Properties conjugated to chitosan molecules [124126]. Hydrogels
prepared from mixtures of chitosan and polypyrrolidone
A variety of approaches have been developed to control the have been reported to support the growth of endothelial
degradation of chitosan hydrogels. Chitosan molecules cells while restricting the growth of broblasts [127].
undergo an enzymatic degradation, and this enzymatic
digestion tends to slow with increases in the crystallinity 5. Processing of Chitosan Gels
of the polymers. Therefore, increasing the degree of deacet-
ylation to 50% accelerates the degradation rate [112115]. Chitosan hydrogels can be processed into beads and po-
However, fully deacetylating (up to 97%) induces the rous scaolds. Gel beads are prepared by dropping so-
formation of a new crystalline structure, which does not lutions of chitosan and cross-linking molecules into NaOH
degrade. In addition, modifying chitosan molecules via solutions, followed by cross-linking [128]. Smaller hydrogel
acylation makes them more labile to enzymatic degrada- particles can be prepared by forming a water/oil emulsion,
tion [116]. Formation of hydrogels via chemical cross- followed by an ionotropic gelation process [129]. Porous
linking, in contrast, increases the resistance to degradation hydrogels are prepared via freeze-drying of previously
[117]. Forming IPN structures with xanthan or chondroitin formed gels [130]. The porosity can be adjusted in this
sulfate also decreases the degradation rate [118,119]. latter form by varying the solids concentration and the
freezing conditions to alter the size of ice crystals formed
during the freezing process. The resultant structure is less
4. Modication of Biological Activity sti than nonporous gels but more extendible [130]. To
Mammalian cells strongly interact with chitosan, perhaps produce three-dimensional porous scaolds in a rened
due to the N-acetylglucosamine groups in the chitosan manner, a microfabrication technique utilizing a robotic
molecules. This may make the chemical structure of chito- dispensing system has been developed (Fig. 17) [131].
Figure 17 Chitosan gel prepared by a robotic dispensing system. (a) Overview of scaold just after dispensing pregelled
solutions, and (b) top view (Scanning Electron Microscopy) of the chitosan scaold after freeze-dying (from Ref 131, Copyright
2002 Elsevier Science).
828 Kong and Mooney
6. Applications of Chitosan Hydrogels in Tissue have utility in regenerating tissues containing these cell
Engineering types [143,144]. Further enhancements have been achieved
To utilize chitosan hydrogels (which interact with cells in a by the conjugation of bioactive molecules (e.g., carbohy-
nonselective manner and degrade in a physiological envi- drates, proteins) to chitosan molecules, or by immobiliza-
ronment) in tissue engineering it may be essential to induce tion of growth factors with the gels. Fructose and
specic interactions with target cells and optimize degra- galactose, for example, which are known to mediate the
dation rates of hydrogels. Chitosan hydrogels have been adhesion and proliferation of hepatocytes, have been con-
utilized to engineer cartilage, bone, and nerve tissues via jugated to chitosan molecules. The liver-specic function of
supplementation with growth factors or glycosaminogly- the cells was maintained in these matrices. In addition,
can molecules. In particular, the combined use of chitosan galactosylated chitosangraftdextran complexes with
hydrogels with bioactive inorganic particles or synthetic plasmid DNA, and these complexes may be used to genet-
polymers has been widely attempted to optimize the prop- ically modify hepatocytes [145,146]. The liver is highly
erties of these hydrogels for tissue engineering. vascularized, and incorporation of endothelial cell growth
factor to stimulate neovascularization has been shown to
Regeneration of Cartilage improve the viability of hepatocytes within the gels [147].
Chitosan solutions containing chondrocytes have been Blending chitosan gels with collagen similarly enhanced the
injected into the articular cartilage of rats to regenerate attachments of chroman cells to the gels and the subse-
cartilage [132], and chitosan coupled with BMP-7 has been quent viability of the cells [123]. Chitosan gels coated with
shown to enhance the repair of lesions in particular carti- poly(lysine) or chitosanpoly(lysine) mixture have also
lage [133]. In this latter treatment, BMP-7 was proposed to been reported as promising materials for the repair of
contribute to the proliferation of the chondrocytes, while damaged neural tissues [145,148].
the chitosan was proposed to partially heal the surface of
the cartilage. However, chitosan can induce the growth of
brous tissues, leading to concerns regarding the structure C. Hyaluronic Acid Hydrogels
and function of new tissues [132]. To enhance cartilage
formation, chondroitin sulfate, a GAG molecule found in Hyaluronic acid (also named hyaluronate and hyaluronan)
cartilage, has been immobilized in the chitosan gel matrix present in the normal ECM has found applications in
in an eort to more closely mimic normal cartilage extra- biomedical engineering due to its intrinsic cell interaction
cellular matrix [122,133,134]. These GAGchitosan matri- ability. Hyaluronic acid has been used in a number of tissue
ces have been reported to enhance the proliferation of cells. engineering applications, including the treatment of oste-
Regeneration of Bone oarthritis [149151]. Hyaluronic acid hydrogels formed via
cross-linking with a variety of functional molecules have
Chitosan has been recently reported to direct the
been utilized to recreate cartilage, bone, and skin.
dierentiation of osteoprogenitor cells and support the
adhesion of human osteoblasts and expression of type I
1. Hyaluronic Acid
collagen by the cells [135]. These ndings suggest that
chitosan may be a desirable material for bone regeneration Hyaluronic acid is one of the proteoglycans normally
[136]. To enhance the osteoconductivity of chitosan gels present in synovial joint uids and the ECM of mammalian
and mechanically reinforce the gels, chitosan has been tissues [152]. It is composed of N-acetyl-D-glucosamine
blended with bioactive inorganic particles, such as hy- units and D-glucuronic acid units connected in an alter-
droxyapatite [137] or calcium phosphates, which induce nating manner (Fig. 18). This molecule is typically isolated
the formation of apatites [138140]. These composites were from rooster comb. It undergoes degradation in the pres-
usually processed to provide an in situ forming injectable ence of hyaluronidase, which is found in cells and serum
gel or porous scaold. Porous chitosanceramic compos- [153]. However, hyaluronic acid obtained from animals
ites exhibited an enhanced compressive modulus and yield
strength, potentially allowing the use of these composites
under load-bearing conditions. In addition, cells within
composites demonstrated high expression of bone-specic
genes and deposition of mineralized phases, in vitro.
Alternatively, specic growth factors [e.g., BMP-7, plate-
let-derived growth factor (PDGF)] have been immobilized
in chitosan gels to enhance the osteoinductivity of the
chitosan. Release of the growth factors was mainly con-
trolled by the degradation rate of the gels, and regeneration
of bone tissues in defect sites was demonstrated with these
materials [141,142].
Regeneration of Nerve and Liver
Unmodied chitosan molecules readily promote ad- Figure 18 Chemical structure of hyaluronic acid; G: D-
hesion of hepatocytes and neural cells, suggesting they may glucuronic acid, A: N-acetyl-D-gucosamine.
may induce an inammatory response in humans [154,155]. to glucuronic residues in hyaluronate molecules as a pen-
Therefore, a thorough purication process may be re- dant group, allowing cross-linking with sulfosuccinimide-
quired. Nonanimal-derived hyaluronic acid (Restlanek), containing molecules of varying spacer length and exibility
produced by isolating this molecule from cultural strepto- (Fig. 19) [166,167]. Attaching photosensitive molecules, in-
cocci bacteria, has been investigated to bypass this issue cluding cinnamate or thymine groups, to hyaluronic acid al-
[156,157]. lows for the formation of hydrogels via photo cross-linking.
Hyaluronic acid can also be processed to create in situ
2. Preparation and Control of Physical Properties forming gels and porous scaolds. By utilizing various
of Hyaluronic Acid Hydrogels cross-linkers as a spacer, hydrogels can be used as carriers
Hyaluronic acid solutions form hydrogels due to inter- and of drugs, proteins, and peptides [168]. Porous structures
intramolecular interactions, chemical cross-linking, and can also be formed by a freeze-drying process [166]. In
photo cross-linking. In solution, hyaluronic acid and its general, the mechanical properties and water uptake of
oligosaccharides show extensive intramolecular hydrogen hyaluronic acid gels are regulated by the cross-linking
bonding as well as secondary (helical) and tertiary inter- mechanism and properties of the cross-linking molecules.
actions [158]. Therefore, annealing hyaluronic solutions at Degradation of hyaluronic hydrogels is typically reg-
appropriate temperatures can lead to gel formation [159]. ulated by enzymatic degradation of the hyaluronic acid
The temperatures required to induce the solgel transition backbone. Chemical cross-linking has been reported to be
can be reduced by grafting poly(N-isopropylacrylamide) to eective in decreasing the degradation rate [163]. The
hyaluronic acid molecules [160]. Various hydrazide-con- degradation rate of chemically cross-linked hydrogels does
taining molecules having multiarms [161], glycididylether- not appear to be aected by the cross-linking density [165],
containing molecules [162], carbodiimides [163], and but is aected by the type of cross-linkers [161].
divinylsulfone [164], have also been utilized to prepare
covalently cross-linked hydrogels (Fig. 19). Novel poly- 3. Applications of Hyaluronic Acid Hydrogels
merization techniques allow one to readily alter the number in Tissue Engineering
of reactive hydrazides in the hydrazide-type cross-linking The main applications of hyaluronic acid hydrogels in
molecules [165]. Alternatively, dihydrazides can be attached tissue engineering have been in the regeneration of cartilage
Figure 19 Cross-linking molecules used to form hyaluronate hydrogels. (a) 3,3V-Dithiobis(propanic dihydrazide), (b)
polyethylene glycol bis(succinimidyl propionate), (c) polyhydrazides, (d) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and
(e) 3,3V-dithiobis(sulfosuccinimidylpropionate).
830 Kong and Mooney
and bone. Porous hyaluronic acid scaolds transplanted porous scaolds have been utilized in attempts to regener-
into osteochondral defects led to bony lling of the defects ate cartilage and nerve.
and growth of hyaline cartilage on the surface [169,170].
Supplementing hydrogels with insulin-like growth factor-1 1. Formation of Agarose Hydrogels
and BMP-2 have been reported to promote cartilage Although agarose molecules behave as sti coils in solu-
formation, while supplementing the gels with transforming tion, decreasing the temperature results in aggregation of
growth factor-h and BMP-2 may enhance bone formation molecules having a double helix conformation, leading to
[167]. Encapsulating chondrocytes in hyaluronic acid gels formation of thermoreversible hydrogels [183]. Puried
leads to the formation of a structure containing aggrecan agarose has a lower solgel transition temperature
and large proteoglycans, having similar viscoelastic prop- (f30jC) than agar (f40jC). In addition, the gels have a
erties as native cartilage [171]. In addition, hyaluronic acid much higher melting temperature (f90jC) than the criti-
may prevent dedierentiation of chondrocytes to bro- cal temperature to form gels upon cooling. The mechanical
blast-like cells and thus be specically useful in these properties and pore structure of hydrogels are determined
applications [171,172]. Hyaluronic acid hydrogels have by the solids concentration, molecular weight, and thermal
also been used as facial intradermal implants to augment history of the materials [179,184]. In particular, a low solids
wrinkles and bulk lips [173], and as an articial skin [174]. concentration results in soft hydrogels having large pores,
Typical cross-linked hydrogels have limited mechani- facilitating the inltration of cells [185]. Upon mixing with
cal properties and fast degradation rates, invoking con- cells, agarose solutions can be processed into spherical
cerns as to the maintenance of the structural integrity of the beads by pumping the mixture of agarose and cells drop-
gels for a desired time [169]. Modied hyaluronan benzyl wise into cold hydrophobic solutions. To prevent the
ester processed into micro perforated lms (HYAFFR 11) protrusion of cells out of gel beads, gels have been rein-
has been reported to extend the time to undergo structural forced by poly(styrene sulfonic acid) or carboxymethyl
disintegration [175178]. This material appears to have an cellulose [186]. To improve the adhesion of cells to these
enhanced eectiveness in the treatment of burns and gels, synthetic oligopeptides (e.g., Tyr-Ile-Gly-Ser-Arg,
chronic ulcers [175177], and also maintains the capacity YIGSR) have been covalently attached to agarose mole-
to support the regeneration of cartilage [178]. cules. This conjugation can be conducted with a photo-
coupling agent (e.g., 1,1-carbonyldiimidazole) or
D. Agarose Hydrogels thermocoupling agents (e.g., benzophenone) [187].
Agarose is a fraction of agar harvested from marine red
macroalgae that can be gelled. It is an alternating copoly- 2. Applications of Agarose Hydrogels in Tissue
mer of h-D-galactopyranosyl units and 3,6 anhydro a-L- Engineering
galactopyranosyl units (Fig. 20). Agarose forms hydrogels Agarose hydrogels have been utilized to encapsulate islets
via intermolecular aggregations prompted by thermal of Langerhans, which are used to form bioarticial pan-
changes [179]. The properties of agar hydrogels, including creas [186,188]. Encapsulating the cells in agarose gels may
mechanical properties, degradation rates, and cell interac- prolong the function of the cells following transplantation,
tion ability, can be controlled with modication of the even without the use of immunosuppressing drugs [188].
molecular structure, gelation conditions, and mixing with Agarose hydrogels have also been utilized as a culture
various synthetic polymers. Due to its biocompatibility, it system for chondrocytes to induce a redierentiation of
has generally been used as a cell immobilization matrix, the cells to a chondrocytic phenotype [189,190]. However,
drug delivery vehicle, or dental impression material agarose gels may not be ideal materials to use under load-
[180,181]. In addition, agarose gels have been used in bearing conditions due to their limited mechanical proper-
analyzing DNA molecules and proteins via electrophoresis ties [191]. Therefore, encapsulating a mixture of agarose
[182]. Agarose hydrogels processed to form beads and gels and chondrocytes within biodegradable poly(glycolic
acid-co-lactic acid) scaolds has been performed to en-
hance the structural stability and degradation properties of
the composite structure [192].
Agarose gels have also been used by several groups to
promote nerve regeneration. The absence of cell adhesion
ligands in pure agarose gels may lead to a low viability of
encapsulated cells [193], and laminin-derived oligopeptides
(e.g., CDPYISGSR) have been incorporated into agarose
gels to promote cell adhesion. The presence of these
oligopeptides has been shown to support outgrowth of
neurites from dorsal root ganglia into these gels (Fig. 21)
[194]. Alternatively, immobilizing chitosan molecules in
the agarose gel matrices can also enhance the outgrowth of
neurites, likely due to the positive charge of the chitosan
Figure 20 Chemical structure of agarose. molecules [185]. The outgrowth rate of neurites in this
Figure 21 Outgrowth of neurites from dorsal root ganglia (DRG) cultured for 6 days on (a) plain agarose gels, or (b)
CDPGYIGSR-containing agarose gels. A signicant increase in neurite outgrowth was achieved with peptide coupling (from Ref
194, Copyright 1997 John Wiley & Sons Inc.).
latter system was inversely related to the stiness of gels, has dierent gelation mechanisms, mechanical properties,
which can be altered by the solids concentration [195]. degradation behavior, and biological activity, depending
on the chemical structures of the polysaccharide chains,
how they are induced to form a gel, and their physical form.
IV. FUTURE PERSPECTIVES To satisfy the varied design criteria for the large number of
dierent applications of these materials, physical and
In this chapter, we described the use of polysaccharide- biological properties of these materials are often altered
based hydrogels as tissue engineering matrices. In general, via chemical modication of their molecular structure, use
hydrogels exhibit good biocompatibility, and these gels can of biocompatible cross-linking molecules, immobilization
be delivered into patients using injection processes, which of adhesion or growth factors in the gels, and control of the
avoid open surgical procedures. Each hydrogel discussed pore structure.
Figure 22 Enzymatic polymerization of (a) articial chitin; chitinase promotes a ring-opening polyaddition of a chitobiose
oxazoline derivative, and (b) articial hyaluronic acid; hyaluronidase activates the oxazoline derivatives in gulucuronic acid
h(1!3) 2-acetoamido-2-deoxy-D-glucose dissacharide via protonation, followed by polymerization with 4-hydroxyl groups in
gulucuronic acid of another disacharide.
832 Kong and Mooney
Hydrogels formed from naturally derived polysac-

charides have many advantageous features for use in tissue
engineering, but any given hydrogel also has some disad-
vantageous features, including batch-to-batch variations
and limited mechanical properties. The molecular weights
and proportion of various residues in the polymers may
vary with the source of the polymers, resulting in variations
in the properties of hydrogels. The limited modication of
molecular structure achieved to date may not achieve a
sucient range of mechanical properties for all desired
applications. In particular, polysaccharide-based hydro-
gels are regarded as brittle materials, and may fail under
dynamic loading environments that require high toughness
and resistance to fatigue [196,197]. Clearly, one cannot
satisfy the design criteria for all of the varied tissue
engineering applications with a single type of materials.
Rather, we propose that a variety of materials will nd
utility in this eld. Recreation of complex tissues composed
of dierent cell types adds further complexity. Hybridiza- Figure 23 Unnatural polysaccharides synthesized by enzy-
tion and complex formation between dierent polysac- matic polymerization. (a) Alternatively methylated cellulose;
charides, or polysaccharides and synthetic polymers, may cellulase catalyzes the polymerization of 6-O-methyl-h-
allow one to optimize the properties of materials for cellobiosyl uoride. (b) Cellulosexylan hybrid polysacchar-
diering uses. ides; xylase catalyzes the polymerization of h-xylopyranosyl-
To enhance the reproducibility of polysaccharide- glucopyranosyl uoride.
based hydrogel properties, polysaccharides have been har-
vested from a biosynthesis of bacteria, instead of obtaining
them from the natural source. In the case of alginate,
cooperation and communication between people working
mannuronic acid residues can be produced from the bac-
in diverse research elds (e.g., chemists, material scientists,
teria Pseudomonas. Dierent proportions of guluronic acid
biologists, and clinicians) will be critical to realize the full
residues can then be introduced by genes encoding for
potential of these materials.
dierent epimerases [198,199]. Utilizing this approach,
alginates having a higher fraction of guluronic acid resi-
dues than the naturally derived alginates could be achieved REFERENCES
[38]. Hyaluronic acid could also be cultivated from the
bacteria Streptococcus zooepidemicus [200]. Coupled with 1. Lovice, D.B.; Mingrone, M.D.; Toriumi, D.M. Grafts and
enhanced reproducibility, this approach may allow mass implants in rhinoplasty and nasal reconstruction. Otola-
ryngol. Clin. North Am. 1999, 32, 113.
production at a low cost while avoiding the ultrapurica- 2. Langer, R.; Vacanti, J.P. Tissue engineering. Science 1993,
tion required when this material is isolated from animal 160, 920.
tissues. Another exciting approach to enhancing the mate- 3. Mellonig, J.T.; Prewett, A.B.; Moyer, M.P. HIV inactiva-
rial properties of hydrogels is the synthesis of polysaccha- tion in a bone allograft. J. Periodontol. 1992, 63, 979.
rides via an enzymatic polymerization [201]. This method 4. Bach, F.H.; Turman, M.A.; Vercellotti, G.M.; Platt, J.L.;
utilizes enzymatic catalysis to synthesize the novel polymers, Dalmasso, A.P. Accommodation: A working paradigm for
progressing toward clinical discordant xenografting.
which cannot be prepared with a conventional chemical
Transplant. Proc. 1991, 23, 205.
catalysis. Adopting appropriate donor molecules, acceptor 5. Grith, L.G.; Naughton, G. Tissue engineeringCurrent
molecules, and enzymes (e.g., hyaluronidase, chitinase) challenges and expanding opportunities. Science 2002, 295,
leads to the desired polymeric structure, including hyalur- 1009.
onan and chitin (Fig. 22) [202,203]. This technique thus 6. Alsberg, E.; Hill, E.E.; Mooney, D.J. Cranofacial tissue
may be able to synthesize naturally occurring polysacchar- engineering. Crit. Rev. Oral Biol. Med. 2001, 12, 64.
ides, including those present in mammalian ECMs. Fur- 7. Jackson, D.W.; Simon, T.M. Tissue engineering principles
in orthopaedic surgery. Clin. Orthop. Relat. Res. 1999,
thermore, this synthetic technique allows the synthesis of 367, S31.
unnatural polysaccharides, such as alternating methylated 8. Hunziker, E.B.; Rosenberg, L.C. Repair of partial-thick-
cellulose or cellulosexylan hybrid polysaccharides (Fig. ness defects in articular cartilage: Cell recruitment from the
23). The recent development of various articial enzymes synocial membrane. J. Bone Joint Surg. Am. 1996, 78A,
may provide further exibility to alter the molecular struc- 721.
ture of hybrid polysaccharides. 9. Shea, L.D.; Smiley, E.; Bonadio, J.; Mooney, D.J. DNA
delivery from polymer matrices for tissue engineering. Nat.
In summary, a large number of studies to date clearly Biotechnol. 1999, 17, 551.
indicate naturally derived polysaccharide gels have great 10. Niklason, L.E.; Gao, J.; Abbott, W.M.; Hirschi, K.K.;
utility in tissue engineering. These studies broaden the Houser, S.; Marini, R.; Langer, R. Functional arteries
range of applications for these materials, and an intensive grown in vitro. Science 1999, 284, 489.
11. Richardson, T.; Peters, M.C.; Ennett, A.B.; Mooney, D.J. 33. Suzuki, Y.; Tanihara, M.; Nishimura, Y.; Suzuki, K.;
Polymeric system for dual growth factor delivery. Nat. Yamawaki, Y.; Kudo, H.; Kakimaru, Y.; Shimizu, Y. In
Biotechnol. 2001, 19, 1029. vivo evaluation of a novel alginate dressing. J. Biomed.
12. Ma, P.X.; Langer, R. Morphology and mechanical Mater. Res. 1999, 48, 522.
function of long-term in vitro engineered cartilage. J. 34. Whittington, S.G. Conformational energy calculations on
Biomed. Mater. Res. 1999, 44, 217. alginic acid: 2. Conformational statistics of copolymers.
13. Service, R.F. Tissue engineers build new bone. Science Biopolymers 1971, 10, 1617.
2000, 289, 1498. 35. Leo, W.J.; McLoughlin, A.J.; Malone, D.M. Eects of
14. Oberpenning, F.; Meng, J.; Yoo, J.J.; Atala, A. De novo sterilization treatments on some properties of alginate
reconstitution of a functional mammalian urinary bladder solutions and gels. Biotechnol. Prog. 1990, 6, 51.
by tissue engineering. Nat. Biotechnol. 1999, 17, 149. 36. Haug, A.; Larsem, B.; Smidsrd, O. A study of constitution
15. Davis, M.W.; Vacanti, J.P. Toward development of an im- of alginic acid by partial acid hydrolysis. Acta Chem.
plantable tissue engineered liver. Biomaterials 1996, 17, 365. Scand. 1966, 20, 183.
16. Lin, V.S.; Lee, M.C.; ONeal, S.; McKean, J.; Sung, K.L.P. 37. Smidsrd, O.; Skjak-Brk, S. Alginate as immobilization
Ligament tissue engineering using synthetic biodegradable matrix for cells. Trends Biotechnol. 1990, 8, 71.
ber scaolds. Tissue Eng. 1999, 5, 443. 38. Draget, K.I.; Skjak-Brk, G.G.; Smidsrd, O. Alginate
17. Schense, J.C.; Bloch, J.; Aebischer, P.; Hubbell, J.A. based new materials. Int. J. Biol. Macromol. 1997, 21, 47.
Enzymatic incorporation of bioactive peptides into brin 39. Eiselt, P.; Lee, K.Y.; Mooney, D.J. Rigidity of two-com-
matrices enhances neurite extension. Nat. Biotechnol. ponent hydrogels prepared from alginate and poly (ethyl-
2000, 18, 415. ene glycol)-diamines. Macromolecules 1999, 32, 5561.
18. Organ, G.M.; Mooney, D.J.; Hansen, L.K.; Schloo, B.; 40. Tanihara, M.; Suzuki, Y.; Yamamoto, E.; Noguchi, A.;
Vacanti, J.P. Enterocyte transplantation using cell-poly- Mizushima, Y. Sustained release of basic broblast growth
mer devices to create intestinal epithelial-lined tubes. factor and angiogenesis in a novel covalently crosslinked
Transplant. Proc. 1993, 25, 998. gel of heparin and alginate. J. Biomed. Mater. Res. 2001,
19. Shinoka, T.; Breuer, C.K.; Tanel, R.E.; Zund, G.; Miura, 56, 216.
T.; Ma, P.X.; Langer, R.; Vacanti, J.P.; Mayer, J.E., Jr. 41. Lee, K.Y.; Rowley, J.A.; Eiselt, P.; Moy, E.M.; Bouhadir,
Tissue engineering heart valves: Valve leaet replacement K.H.; Mooney, D.J. Controlling mechanical and swelling
study in a lam model. Ann. Thorac. Surg. 1995, 60, S513. properties of alginate hydrogels independently by cross-
20. Cao, Y.; Vacanti, J.P.; Ma, P.X.; Ibarra, C.; Paige, K.T.; linker type and cross-linking density. Macromolecules
Upton, J.; Langer, R.; Vacanti, C.A. Tissue engineering of 2000, 33, 4291.
tendon. Mater. Res. Soc. Symp. Proc. 1995, 394, 83. 42. Kong, H.J.; Lee, K.Y.; Mooney, D.J. Decoupling the
21. Lysaght, M.J.; Reyes, J. The growth of tissue engineering. dependence of rheological/mechanical properties of hydro-
Tissue Eng. 2001, 7, 485. gels from solids concentration. Polymer. 2002, 43, 6239.
22. Wong, W.H.; Mooney, D.J. Synthesis and properties of 43. Thu, B.; Bruheim, P.; Espevik, T.; Smidsrd, O.; Soon-
biodegradable polymers used as synthetic matrices for tis- Shiong, P.; Skjak-Brk, G. Alginate polycation micro-
sue engineering. In Synthetic Biodegradable Polymer Scaf- capsules: 1. Interaction between alginate and polycation.
folds; Atala, A., Mooney, D.J., Eds.; Birkauser: Boston, Biomaterials 1996, 17, 1031.
1997; 51 pp. 44. Joung, J.J.; Akin, C.; Royer, G.P. Immobilization of
23. Huang, S.J. Biodegradable polymers. In Polymers growing-cells by polyethyleneimine-modied alginate.
Biomaterials and Medical Application; Kroshwitz, I.J., Appl. Biochem. Biotechnol. 1987, 14, 259.
Ed.; Wiley & Sons: New York, 1989. 45. Murata, Y.; Maeda, T.; Miyamoto, E.; Kawashima, S.
24. Ratner, B.D.; Homan, A.S. In Hydrogels for Medical and Preparation of chitosan-reinforced alginate gel beads
Related application; Andrade, J.D., Ed.; American Chem- Eects of chitosan on gel matrix erosion. Int. J. Pharm.
ical Society: Washington, DC, 1976; Vol. 31, 1. 1991, 96, 139.
25. Lee, K.Y.; Mooney, D.J. Hydrogels for tissue engineering. 46. Gaserd, O.; Jollie, I.G.; Hampson, F.C.; Dettmar, P.W.
Chem. Rev. 2001, 101, 1869. The enhancement of the bioadhesive properties of calcium
26. Clark, A.H.; Ross-Murphy, S.B. Structural and mechan- alginate gel beads by coating with chitosan. Int. J. Pharm.
ical properties of biopolymer gels. Adv. Polym. Sci. 1987, 1998, 175, 237.
83, 57. 47. Kikuchi, A.; Kawabuchi, M.; Sugihara, M.; Sakurai, Y.;
27. Yannas, V. Biologically active analogues of the extra- Okano, T. Pulsed dextran elease from calcium-alginate gel
cellular matrix: Articial skin and nerves. Angew. Chem. beads. J. Control. Release 1997, 47, 21.
Int. Ed. Engl. 1990, 29, 20. 48. Al-Shamkhani, A.; Duncan, R. Radioiodination of algi-
28. Menard, C.; Mitchell, S.; Spector, M. Contractile behavior nate via covalently-bound tyrosinamide allows monitoring
of smooth muscle actin-containing osteoblasts in collagen of its fate in vivo. J. Bioact. Compat. Polym. 1995, 10, 4.
GAG matrices in vitro: Implant-related cell contraction. 49. Bouhadir, K.H.; Lee, K.Y.; Alsberg, E.; Damm, K.L.;
Biomaterials 2000, 21, 1867. Anderson, K.W.; Mooney, D.J. Degradation of partially
29. Ingber, D.; Karp, S.; Plopper, G.; Hansen, L.; Mooney, D.J. oxidized alginate and its potential application for tissue
In Physical Forces and the Mammalian Cell; Frangos, J.A., engineering. Biotechnol. Prog. 2001, 17, 945.
Ed.; Academic Press: New York, 1993; 6179. Chap. 2. 50. Lee, K.Y.; Bouhadir, K.H.; Mooney, D.J. Degradation
30. Giancotti, F.G.; Ruoslahti, E. TransductionIntegrin behavior of covalently cross-linked poly(aldehyde guluro-
signaling. Science 1999, 285, 1028. nate) hydrogels. Macromolecules 2000, 33, 97.
31. Gombotz, W.R.; Wee, S.F. Protein release from alginate 51. Ingber, D.E. Mechanochemical switching between growth
matrices. Adv. Drug. Deliv. Rev. 1998, 31, 267. and dierentiation by extracellular matrix. In Principles of
32. Goosen, M.F.A.; Oshea, G.M.; Gharapetian, H.M.; Chou, Tissue Engineering; Lanza, R.P. Langer, R., Chick, W.L.
S.; Sun, A.M. Optimization of microencapsulation param- Eds.; R.G. Landes Co.: Austin, 1997.
eters: Semipermeable microcapsules as a bioarticial 52. Sultzbaugh, K.J.; Speaker, T.J. A method to attach lectins
pancreas. Biotechnol. Bioeng. 1985, 27, 146. to the surface of spermine alginate microcapsules based on
834 Kong and Mooney
the avidin biotin interaction. J. Microencapsul. 1996, 13, sphincter deciency using autologous ear chondrocytes as
363. a bulking agent. Neurourol. Urodyn. 2001, 20, 157.
53. Rowley, J.A.; Madlambayan, G.; Mooney, D.J. Alginate 72. Chang, S.C.N.; Rowley, J.A.; Tobias, G.; Genes, N.G.;
hydrogels as synthetic extracellular materials. Biomaterials Roy, A.K.; Mooney, D.J.; Vacanti, C.A.; Bonassar, L.J.
1999, 20, 45. Injection molding of chondrocyte/alginate constructs in the
54. Rowley, J.A.; Mooney, D.J. Alginate type and RGD shape of facial implants. J. Biomed. Mater. Res. 2001, 55,
density control myoblast phenotype. J. Biomed. Mater. 503.
Res. 2002, 60, 217. 73. Paige, K.T.; Cima, L.G.; Yaremchuk, M.J.; Vacanti, J.P.;
55. Alsberg, E.; Anderson, K.W.; Albeiruti, A.; Franceschi, Vacanti, C.A. Injectable cartilage. Plast. Reconstr. Surg.
R.T.; Mooney, D.J. Cell-interactive alginate hydrogels for 1995, 96, 1390.
bone tissue engineering. J. Dent. Res. 2001, 80, 2025. 74. Gregory, K.E.; Marsden, M.E.; Anderson-MacKenzie, J.;
56. Prieto, A.L.; Edelman, G.M.; Crossin, K.L. Multiple Bard, J.B.; Bruckner, P.; Farjanel, J.; Robins, S.P.;
integrins mediate cell attachment to cytoactin tenascin. Hulmes, D.J. Abnormal collagen assembly, though normal
Proc. Natl. Acad. Sci. USA 1993, 90, 10154. phenotype, in alginate bead cultures of chick embryo
57. Wong, W.H.; Mooney, D.J. Synthesis and properties of chondrocytes. Exp. Cell Res. 1999, 246, 98.
biodegradable polymers used as synthetic matrices for 75. De Chalain, T.; Phillips, J.H.; Hinek, A. Bioengineering
tissue engineering. In Bioactive Polymers in Synthetic of elastic cartilage with aggregated porcine and human
Biodegradable Polymer Scaolds; Atala, A., Mooney, auricular chondrocytes and hydrogels containing alginate,
D.J., Eds.; Birkauser: Boston, 1997; 83. collagen, and n-elastin. J. Biomed. Mater. Res. 1999, 44,
58. Halberstadt, C.; Austin, C.; Rowley, J.; Culberson, C.; 280.
Loebsack, A.; Wyatt, S.; Coleman, S.; Blacksten, L.; Burg, 76. van Susante, J.L.C.; Pieper, J.; Buma, P.; van Kuppervelt,
K.; Mooney, D.J.; Holder, W. A hydrogel material for H.; van Beuningen, H.; van der Kraan, P.M.; Veerkamp,
plastic and reconstructive applications injected into the J.H.; van den Berg, W.B.; Veth, R.P.H. Linkage of
subcutaneous space of a sheep. Tissue Eng. 2002, 8, 309. chondroitin-sulfate to type I collagen scaolds stimulates
59. Weigel, P.H. Rat hepatocytes bind to synthetic galactoside the bioactivity of seeded chondrocytes in vitro. Biomate-
surfaces via a patch of asialoglycoprotein receptors. J. Cell rials 2001, 22, 2359.
Biol. 1980, 87, 855. 77. Alsberg, E.; Anderson, K.W.; Albeiru, A.; Rowley, J.A.;
60. Yang, J.; Goto, M.; Ise, H.; Cho, C.S.; Akaike, T. Mooney, D.J. Engineering growing tissues. Proc. Natl.
Galactosylated alginate as a scaold for hepatocytes Acad. Sci. USA 2002, 99, 12025.
entrapment. Biomaterials 2002, 23, 471. 78. Lee, K.Y.; Alsberg, E.; Mooney, D.J. Degradable and
61. Oerther, S.; Le Gall, H.; Payan, E.; Lapicque, F.; Presle, N.; injectable poly(aldehyde guluronate) hydrogels for bone
Hubert, P.; Dexheimer, J.; Netter, P.; Lapicque, F. tissue engineering. J. Biomed. Mater. Res 2001, 56, 228.
Hyaluronatealginate gel as a novel biomaterial: Mechan- 79. Suzuki, Y.; Tanihara, M.; Suzuki, K.; Saitou, A.; Sufan,
ical properties and formation mechanism. Biotechnol. W.; Nishimura, Y. Alginate hydrogel linked with
Bioeng. 1999, 63, 206. synthetic oligopeptide derived from BMP-2 allows ectopic
62. Lindenhayn, K.; Perka, C.; Spitzer, R.S.; Heilmann, H.H.; osteoinduction in vivo. J. Biomed. Mater. Res. 2000, 50,
Pommerening, K.; Mennicke, J.; Sittinger, M. Retention of 405.
hyaluronic acid in alginate beads: Aspects for in vitro 80. Elcin, Y.M.; Dixit, V.; Gitnick, T. Extensive in vivo angio-
cartilage engineering. J. Biomed. Mater. Res. 1999, 44, 149. genesis following controlled release of human vascular
63. Shapiro, L.; Cohen, S. Novel alginate sponges for cell endothelial cell growth factor: Implications for tissue
culture and transplantation. Biomaterials 1997, 18, 583. engineering and wound healing. Artif. Organs 2001, 25,
64. Eiselt, P.; Yeh, J.; Latvala, R.K.; Shea, L.D.; Mooney, D.J. 558.
Porous carriers for biomedical applications based on 81. Peters, M.C.; Isenberg, B.C.; Rowley, J.A.; Mooney, D.J.
alginate hydrogels. Biomaterials 2000, 21, 1921. Release from alginate enhances the biological activity of
65. Poncelet, D.; Babak, V.; Dulieu, C.; Picot, A. A physico- vascular endothelial growth factor. J. Biomater. Sci.
chemical approach to production of alginate beads by Polym. Eng. 1998, 9, 1267.
emulsicationinternal ionotropic gelation. Colloids Surf. 82. Downs, M.L.; Robertson, N.E.; Riss, T.L.; Plunkett, M.L.
A 1999, 155, 171. Calcium alginate beads as a slow-release system for
66. Soon-Shiong, P.; Otterlie, M.; Skjak-Brk, S.; Smidsrd, delivering angiogenic molecules in vivo and in vitro. J.
O.; Heintz, R.; Lanza, R.P.; Espevik, T. An immunologic Cell. Physiol. 1992, 152, 422.
basis for the brotic reaction to implanted micro-capsules. 83. Nugent, M.A.; Chen, O.S.; Edelman, E.R. Controlled
Transplant. Proc. 1991, 23, 758. release of broblast growth factor: Activity in cell culture.
67. Klock, G.; Pfeermann, A.; Ryser, C.; Grohn, P.; Kuttler, Mater. Res. Soc. Symp. Proc. 1992, 252, 273.
B.; Hahn, H.J.; Zimmermann, U. Biocompatibility of 84. Lee, K.Y.; Peters, M.C.; Mooney, D.J. Controlled drug
mannuronic acid-rich alginates. Biomaterials 1997, 18, 707. delivery from polymers by mechanical signals. Adv. Mater.
68. Atala, A.; Kim, W.; Paige, K.T.; Vacanti, C.A.; Retik, A.B. 2001, 13, 837.
Endoscopic treatment of vesicoureteral reux with a 85. Lee, K.Y.; Peters, M.C.; Anderson, K.W.; Mooeny, D.J.
chondrocytealginate suspension. J. Urol. 1994, 152, 642. Controlled growth factor release from synthetic extracel-
69. Paige, K.T.; Cima, L.G.; Yaremchuk, M.J.; Schloo, B.L.; lular matrices. Nature 2000, 408, 998.
Vacanti, J.P.; Vacanti, C.A. De novo cartilage generation 86. Suzuki, Y.; Tanihara, M.; Ohnishi, K.; Suzuki, K.; Endo,
using calcium alginatechondrocyte constructs. Plast. K.; Nishimura, Y. Cat peripheral nerve regeneration across
Reconstr. Surg. 1996, 97, 168. 50 mm gap repaired with a novel nerve guide composed of
70. Diamond, D.A.; Caldamone, A.A. Endoscopic treatment freeze-dried alginate gel. Neurosci. Lett. 1999, 259, 75.
of vesicoureteral reux in children using autologous 87. Suzuki, K.; Suzuki, Y.; Tanihara, M.; Ohnishi, K.;
chondrocytes: Preliminary results. Pediatrics 1998, 102 Hashimoto, T.; Endo, K.; Nishimura, Y. Reconstruction
(suppl.), 868. of rat peripheral nerve gap without sutures using freeze-
71. Bent, A.E.; Tutrone, R.T.; McLennan, M.T.; Lloyd, K.K.; dried alginate gel. J. Biomed. Mater. Res. 1999, 49, 528.
Kennelly, M.J.; Badlani, G. Treatment of intrinsic 88. Mosahebi, A.; Simon, M.; Wiberg, M.; Terenghi, G. A
novel use of alginate hydrogels as Schwann cell matrix. 109. Lee, J.W.; Kim, S.Y.; Kim, S.S.; Lee, Y.M.; Lee, K.H.;
Tissue Eng. 2001, 7, 525. Kim, S.J. Synthesis and characteristics of interpenetrating
89. Skjak-Brk, S.; Anthonsen, T.; Sandford, P. Chitin and polymer network hydrogel composed of chitosan and
Chitosan Sources, Chemistry, Biochemistry, Physical Prop- poly(acrylic acid). J. Appl. Polym. Sci. 1999, 73, 113.
erties, and Applications; Elsevier: London, 1992. 110. Borzacchiello, A.; Ambrosio, L.; Netti, P.A.; Nicolasis, L.;
90. Muzzarelli, R.A.A., Jeuniaux, C., Gooday, W., eds.; Chitin Peniche, C.; Gallardo, A.; San Roman, J. Chitosan-based
in Nature and Technology; Plenum Press: New York, 1986. hydrogels: Synthesis and characterization. J. Mater. Sci.
91. Chandy, T.; Sharma, C. ChitosanAs a biomaterial. Mater. Med. 2001, 12, 861.
Biomater. Artif. Cells Artif. Organs 1990, 18, 1. 111. Yazdani-Pedram, M.; Retuert, J.; Quijada, R. Hydrogels
92. Drohan, W.N.; MacPhee, M.J.; Miekka, S.I.; Singh, M.S.; based on modied chitosan: 1. Synthesis and swelling
Elson, C.; Taylor, J.R., Jr. Chitin Hydrogels, Methods of behavior of poly(acrylic acid) grafted chitosan. Macromol.
Their Production and Use. US Patent 6,124,273, 1997. Chem. Phys. 2000, 201, 923.
93. Koite, S.S.; Koide, S.S. Chitinchitosan: Properties, 112. Qu, X.; Wirsen, A.; Albertsson, A.C. Synthesis and
benets and risks. Nutr. Res. 1998, 18, 1091. characterization of pH-sensitive hydrogels based on
94. Risbud, M.; Endres, M.; Ringe, J.; Bhonde, R.; Sittinger, chitosan and D,L-lactic acid. J. Appl. Polym. Sci. 1999,
M. Biocompatible hydrogel supports the growth of 74, 3193.
respiratory epithelial cells: Possibilities in tracheal tissue 113. Singh, D.K.; Ray, A.R. Biomedical applications of chitin,
engineering. J. Biomed. Mater. Res. 2001, 56, 120. chitosan, and their derivatives. J. Macromol. Sci. Rev.
95. Cho, Y.W.; Jang, J.; Park, C.R.; Ko, S.W. Preparation and Macromol. Chem. Phys 2000, C40, 69.
solubility in acid and water of partially deacetylated chitins. 114. Onishi, H.; Machida, Y. Biodegradation and distribution
Biomacromolecules 2000, 1, 609. of water-soluble chitosan in mice. Biomaterials 1999, 20,
96. Yalpani, M.; Hall, L.D. Some chemical and analytical 175.
aspects of polysaccharide modications: 3. Formation of 115. Kurita, K.; Kaji, Y.; Mori, T.; Nishiyama, Y. Enzymatic
branched-chain, soluble chitosan derivatives. Macromole- degradation of beta-chitin: Susceptibility and the inuence
cules 1984, 17, 272. of deacetylation. Carbohydr. Polym. 2000, 42, 19.
97. Moore, G.K.; Roberts, G.A.F. Reactions of chitosan: 2. 116. Lee, K.Y.; Ha, W.S.; Park, W.H. Blood compatibility and
Preparation and reactivity of N-acyl derivatives of chito- biodegradability of partially N-acetylated chitosan deriv-
san. Int. J. Biol. Macromol. 1981, 3, 292. atives. Biomaterials 1995, 15, 1211.
98. Muzzarelli, R.A.A.; Tanfani, F. N-(Ortho-carboxybenzyl) 117. Jameela, S.R.; Jayakrishnan, A. Glutaraldehyde cross-
chitosan, N-carboxymethyl chitosan and dithiocarbamate linked chitosan microspheres as a long-acting biodegrad-
chitosanNew chelating derivatives of chitosan. Pure able drug-delivery vehicleStudies on the in-vitro release
Appl. Chem. 1982, 54, 2141. of mitoxantrone and in-vivo degradation of microspheres
99. Molinaro, G.; Leroux, J.C.; Damas, J.; Adam, A. in rat muscle. Biomaterials 1995, 16, 769.
Biocompatibility of thermosensitive chitosan-based hydro- 118. Chellat, F.; Tabrizian, M.; Dumitriu, S.; Chornet, E.;
gels: An in vivo experimental approach to injectable Rivard, C.H.; Yahia, L. Study of biodegradation behavior
biomaterials. Biomaterials 2002, 23, 2717. of chitosanxanthan microspheres in simulated physiolog-
100. Mao, H.Q.; Roy, K.; Troung-Le, V.L.; Janes, K.A.; Lin, ical media. J. Biomed. Mater. Res. 2000, 53, 592.
K.Y.; Wang, Y.; August, J.T.; Leong, K.W. Chitosan 119. Kofuji, K.; Ito, T.; Murata, Y.; Kawashima, S. The
DNA nanoparticles as gene carriers: Synthesis, character- controlled release of a drug from biodegradable chitosan
ization and transfection eciency. J. Control. Release gel beads. Chem. Pharm. Bull. 2000, 48, 579.
2001, 70, 399. 120. Ueno, H.; Mori, T.; Fujinaga, T. Topical formulations and
101. MacLaughlin, F.C.; Mumper, R.J.; Wang, J.J.; Tagliaferri, wound healing applications of chitosan. Adv. Drug Del.
J.M.; Gill, I.; Hinchclie, M.; Rolland, A.P. Chitosan and Rev. 2001, 52, 105.
depolymerized chitosan oligomers as condensing carriers for 121. Peluso, G.; Petillo, O.; Ranieri, M.; Santin, M.; Ambrosio,
in vivo plasmid delivery. J. Control. Release 1998, 56, 259. L.; Calabro, D. Chitosan-mediated stimulation of macro-
102. Janes, K.A.; Fresneau, M.P.; Marazuela, A.; Fabra, A.; phage function. Biomaterials 1994, 15, 1215.
Alonso, M.J. Chitosan nanoparticles as delivery systems 122. Sechriest, V.; Miao, Y.J.; Niyibizi, C.; Westerhausen-
for doxorubicin. J. Control. Release 2001, 73, 255. Larson, A.; Matthew, H.W.; Evans, C.H.; Fu, F.H.; Suh,
103. Back, J.F.; Oakenfull, D.; Smith, M.B. Increased thermal J.K. GAG-augmented polysaccharide hydrogel: A novel
stability of proteins in the presence of sugars and polyols. biocompatible and biodegradable material to support
Biochemistry 1979, 18, 5191. chondrogenesis. J. Biomed. Mater. Res. 2000, 49, 534.
104. Long, D.D.; VanLuyen, D. Chitosancarboxymethylecel- 123. Elcin, A.E.; Elcin, Y.M.; Pappas, G.D. Neural tissue
lulose hydrogels as supports for cell immobilization. J. engineering: Adrenal chroman cell attachment and
Macromol. Sci. A Pure Appl. Chem. 1996, A33, 1875. viability on chitosan scaolds. Neurol. Res. 1998, 20, 648.
105. Mi, F.L.; Sung, H.W.; Shyu, S.S. The study of gelation 124. Yang, J.; Chung, T.W.; Nagaoka, M.; Goto, M.; Cho, C.S.;
kinetics and chain-relaxation properties of glutaraldehyde Akaike, T. Hepatocyte-specic porous polymer-scaolds
cross-linked chitosan gel and their eects on microspheres of alginate/galactosylated chitosan sponge for liver-tissue
preparation and drug release. Carbohydr. Polym. 2000, 41, engineering. Biotechnol. Lett. 2001, 23, 1385.
389. 125. Yagi, K.; Michibayashi, N.; Kurikawa, N.; Nakashima, Y.;
106. Valenta, C.; Christen, B.; Nernkop-Schnurch, A. Chito- Mizoguchi, T.; Harada, A. Eectiveness of fructose-
sanEDTA conjugate: A novel polymer for topical gels. J. modied chitosan as a scaold for hepatocyte attachment.
Pharm. Pharmacol. 1998, 50, 45. Biol. Pharm. Bull. 1997, 20, 1290.
107. Ono, K.; Saito, Y.; Yura, H.; Ishikawa, K.; Kurita, A.; 126. Komazawa, H.; Saiki, I.; Nishikawa, N.; Yoneda, J.; Yoo,
Akaike, T.; Ishihara, M. Photocrosslinkable chitosan as a Y.C.; Kojima, M.; Ono, M.; Itoh, I.; Nishi, N.; Tokura, S.;
biological adhesive. J. Biomed. Mater. Res. 2000, 49, 289. Azuma, I. Inhibition of tumor metastasis by RGDs peptide
108. Nishino, T.; Matsui, R.; Nakamae, K. Elastic modulus of conjugated with sulfated chitin derivatives. Clin. Exp.
the crystalline regions of chitin and chitosan. J. Polym. Sci. Metastasis 1993, 11, 482.
B Polym. Phys. 1999, 27, 1191. 127. Risbud, M.; Hardikar, A.; Bhonde, R. Growth modulation
836 Kong and Mooney
of broblasts by chitosanpolyvinyl pyrrolidone hydrogel: chitosan conjugate having antennary galactose residues as
Implications for wound management? J. Biosci. 2000, 25, a gene delivery tool. Carbohydr. Polym. 1997, 32, 105.
25. 146. Park, Y.K.; Park, Y.H.; Shin, B.A.; Choi, E.S.; Park, Y.R.;
128. Guibal, E.; Milot, C.; Tobin, J.M. Metal-anion sorption by Akaike, T.; Cho, C.S. Galactosylated chitosangraft
chitosan beads: Equilibrium and kinetic studies. Ind. Eng. dextran as hepatocyte-targeting DNA carrier. J. Control.
Chem. Res. 1998, 37, 1454. Release 2000, 69, 97.
129. Lim, L.Y.; Wan, L.S.C.; Thai, P.Y. Chitosan microspheres 147. Elcin, Y.M.; Dixit, V.; Lewin, K.; Gitnick, G. Xenotrans-
prepared by emulsication and ionotropic gelation. Drug plantation of fetal porcine hepatocytes in rats using a tissue
Dev. Ind. Pharm. 1997, 23, 981. engineering approach. Artif. Organs 1999, 23, 146.
130. Madihally, S.V.; Matthew, H.W. Porous chitosan scaolds 148. Gong, H.P.; Zhong, Y.H.; Li, J.C.; Gong, Y.D.; Zhao,
for tissue engineering. Biomaterials 1999, 20, 1133. N.M.; Zhang, X.F. Studies on nerve cell anity of
131. Ang, T.H.; Sultana, F.S.A.; Hutmacher, D.W.; Wong, chitosan-derived materials. J. Biomed. Mater. Res. 2000,
Y.S.; Fuh, J.Y.H.; Mo, X.M.; Loh, H.T.; Burdet, E.; Teoh, 52, 285.
S.H. Fabrication of 3D chitosanhydroxyapatite scaolds 149. Entwistle, J.; Hall, C.L.; Turley, E.A. Hyaluronan recep-
using a robotic dispensing system. Mater. Sci. Eng. C 2002, tors: Regulators of signaling to the cytoskeleton. J. Cell
20, 35. Biochem. 1996, 61, 569.
132. Lu, J.X.; Prudhommeaux, F.; Meunier, A.; Sedel, L.; 150. Rudert, M.; Wirth, C.J. Cartilage cell transplantation.
Guillemin, G. Eects of chitosan on rat knee cartilages. Experimental principles and clinical applications. Ortho-
Biomaterials 1999, 20, 1937. pade 1997, 26, 741.
133. Mattioli-Belmonte, M.; Gigante, A.; Muzzarelli, R.A.A.; 151. Goa, K.L.; Beneld, P. Hyaluronic acid. A review of its
Politano, R.; De Benedittis, A. N,N-Dicarboxymethyl pharmacology and use as a surgical aid in ophthalmology,
chitosan as delivery agent for bone morphogenetic protein an its therapeutic potential in joint disease and wound
in the repair of articular cartilage. Med. Biol. Eng. Comput. healing. Drugs 1994, 47, 536.
1999, 37, 130. 152. Brekke, H.; Toth, J.M. Principles of tissue engineering
134. Suh, J.K.; Matthew, H.W.T. Application of chitosan-based applied to programmable osteogenesis. J. Biomed. Mater.
polysaccharide biomaterials in cartilage tissue engineering: Res. 1998, 32, 380.
A review. Biomaterials 2000, 21, 2589. 153. Orlidge, A.; Damore, P.A. Cell specic eects of glyco-
135. Lahiji, A.; Sohrabi, A.; Hungerford, D.S.; Frondoza, C.G. saminoglycans on the attachment and proliferation of
Chitosan supports the expression of extracellular matrix vascular wall components. Microvasc. Res. 1986, 31, 41.
proteins in human osteoblasts and chondrocytes. J. 154. Iwata, S. Pharmacological and clinical aspects of intra-
Biomed. Mater. Res. 2000, 51, 586. articular injection of hyaluronate. Clin. Orthop. Relat. Res.
136. Klokkevoid, P.R.; Vandemark, L.; Kenney, E.B. Bernard, 1993, 289, 285.
G.W. Osteogenesis enhanced by chitosan (poly-N-acetyl- 155. Laurent, T.C. Biochemistry of hyaluronan. Acta Otola-
glucosaminoglycan) in vitro. J. Periodontol. 1996, 67, 1170. ryngol. 1987, 442S, 7.
137. Zhao, F.; Yin, Y.J.; Lu, W.W.; Leong, J.C.; Zhang, W.J.; 156. Afy, A.M.; Stern, M.; Guntenhoner, M.; Stern, R.
Zhang, J.Y.; Zhang, M.F.; Yao, K.D. Preparation and Purication and characterization of human serum hyalu-
histological evaluation of biomimetic 3-dimensional hy- ronidase. Arch. Biochem. Biophys. 1993, 305, 434.
droxyapatite/chitosangelatin network composite scaf- 157. Filion, M.C.; Phillips, N.C. Pro-inammatory contami-
folds. Biomaterials 2002, 23, 3227. nating DNA in hyaluronic acid preparations. J. Pharm.
138. Gutosawa, A.; Song, L.; Armstrong, B.L.; Campbell, A.A. Pharmacol. 2001, 53, 555.
Injectable stimuli-sensitive polymer ceramic composites for 158. Scott, J.E. Secondary structures in hyaluronan solutions
bone regeneration. Trans. Soc. Biomater. 1998, 21, 450. Chemical and biological implications. CIBA Found. Symp.
139. Zhang, Y.; Zhang, M.Q. Synthesis and characterization of 1989, 143, 6.
macroporous chitosan/calcium phosphate composite scaf- 159. Fujiwara, J.; Takahashi, M.; Hatakeyama, T.; Hatakeya-
folds for tissue engineering. J. Biomed. Mater. Res. 2001, ma, H. Gelation of hyaluronic acid through annealing.
55, 304. Polym. Int. 2000, 49, 1604.
140. Lee, Y.M.; Park, Y.J.; Lee, S.J.; Ku, Y.; Han, S.B.; Choi, 160. Ohya, S.; Nakayam, Y.; Matsuda, T. Thermoresponsive
S.M.; Klokkevold, P.R.; Chung, C.P. Tissue engineered articial extracellular matrix for tissue engineering: Hyal-
bone formation using chitosan/tricalcium phosphate uronic acid bioconjugated with poly(N-isopropylacryla-
sponges. J. Periodontol. 2000, 71, 410. mide) grafts. Biomacromolecules 2001, 2, 856.
141. Muzzarelli, R.A.A.; Biagini, G.; Belmonte, M.A.; Talassi, 161. Prestwich, G.D.; Marecak, D.M.; Marecek, J.F.; Ver-
O.; Gandol, M.G.; Solmi, R.; Carraro, S.; Giardino, R.; cruysse, K.P.; Ziebell, M.R. Controlled chemical modi-
Fini, M.; Nicolialdini, N. Osteoinduction by chitosan- cation of hyaluronic acid: Synthesis, applications and
complexed BMP: Morpho-structural responses in an osteo- biodegradation of hydrazide derivatives. J. Control.
porotic model. J. Bioact. Compat. Polym. 1997, 12, 321. Release 1998, 53, 93103.
142. Lee, Y.M.; Park, Y.J.; Lee, S.J.; Ku, Y.; Han, S.B.; 162. Yui, N.; Nishira, J.; Okano, T.; Sakurai, Y. Regulated
Klokkevold, P.R.; Chung, C.P. The bone regenerative release of drug microspheres from inammation responsive
eect of platelet-derived growth factor-BB delivered with a degradable matrices of cross-linked hyaluronic acid. J.
chitosan/tricalcium phosphate sponge carrier. J. Periodon- Control. Release 1993, 25, 133.
tol. 2000, 71, 418. 163. Tomihiara, K.; Ikada, Y. Crosslinking of hyaluronic acid
143. Yura, H.; Goto, M.; Okazaki, H.; Kobayashi, K.; Akaike, with water-soluble carbodiimide. J. Biomed. Mater. Res.
T. Structural eect of galactose residue in synthetic 1997, 37, 243.
glycoconjugates on interaction with rat hepatocytes. J. 164. Balazs, E.A.; Leshchiner, A. Cross-linked Gels of Hyaluro-
Biomed. Mater. Res. 1995, 29, 1557. nic Acid and Products Containing Such Gels. US Patent
144. Gong, H.P.; Zhong, Y.H.; Li, J.C.; Gong, Y.D.; Zhao, 4,605,691, 1986.
N.M.; Zhang, X.F. Studies on nerve cell anity of 165. Vercruysse, K.P.; Marecak, D.M.; Marecek, J.F.; Pre-
chitosan-derived materials. J. Biomed. Mater. Res. 2000, stwich, G.D. Synthesis and in vitro degradation of new
52, 282. polyvalent hydrazide cross-linked hydrogels of hyaluronic
145. Murata, J.; Ohya, Y.; Ouchi, T. Design of quaternary acid. Bioconjug. Chem. 1997, 8, 686.
166. Pouyani, T.; Harbison, G.S.; Prestwich, G.D. Novel Aymard, P. New insight into agarose gel mechanical
hydrogels of hyaluronic acid: Synthesis, surface morphol- properties. Biomacromolecules 2001, 1, 730.
ogy, and solid-state NMR. J. Am. Chem. Soc. 1994, 116, 185. Dillon, G.P.; Yu, X.J.; Sridharan, A.; Ranieri, J.P.;
7515. Bellamkonda, R.V. The inuence of physical structure
167. Bulpitt, P.; Aeschlimann, D. New strategy for chemical and charge on neurite extension in a 3D hydrogel scaold.
modication of hyaluronic acid: Preparation of function- J. Biomater. Sci. Polym. Ed. 1998, 9, 1049.
alized derivatives and their use in the formation of novel 186. Tun, T.; Inoue, K.; Hayashi, H.; Aung, T.; Gu, Y.J.; Doi,
biocompatible hydrogels. J. Biomed. Mater. Res. 1999, 47, R.; Kaji, H.; Echigo, Y.; Wang, W.J.; Setoyama, H.; Ima-
152. mura, M.; Maetani, S.; Morikawa, N.; Iwata, H.; Ikada, Y.
168. Drobnik, J. Hyaluronan in drug delivery. Adv. Drug. A newly developed three-layer agarose microcapsule for a
Deliv. Rev. 1991, 7, 295. promising biohybrid articial pancreas: Rat to mouse
169. Solchaga, L.A.; Dennis, J.E.; Goldberg, V.M.; Caplan, A.I. xenotransplantation. Cell Transplant. 1996, 5, S59.
Hyaluronic acid-based polymers as cell carriers for tissue- 187. Bellamkonda, R.; Ranieri, J.P.; Bouche, N.; Aebischer, P.
engineered repair of bone and cartilage. J. Orthop. Res. Hydrogel-based-3-dimensional matrix for neural cells. J.
1999, 17, 205. Biomed. Mater. Res. 1995, 29, 663.
170. Liu, L.S.; Thomson, A.Y.; Heidaran, M.A.; Poster, J.W.; 188. Iwata, H.; Takagi, T.; Amemiya, H.; Shimizu, H.;
Spiron, R.C. An osteoconductive collagen hyaluronate Yamashita, K.; Kobayashi, K.; Akutsu, T. Agarose for a
matrix for bone regeneration. Biomaterials 1999, 20, 1097. bioarticial pancreas. J. Biomed. Mater. Res. 1992, 26, 967.
171. Brun, J.P.; Abatangelo, G.; Radice, M.; Zacchi, V.; 189. An, Y.H.; Webb, D.; Gutoswska, A.; Mironov, V.A.;
Guidolin, D.; Gordini, D.D.; Cortivo, R. Chondrocyte Friedman, R.J. Regaining chondrocyte phenotype in
aggregation and reorganization into 3-dimensional scaf- thermosensitive gel culture. Anat. Rec. 2001, 263, 336.
folds. J. Biomed. Mater. Res. 1999, 46, 337. 190. Benya, P.D.; Shaer, J.D. Dedierentiated chondrocytes
172. Homandberg, G.A.; Hui, F.; Wen, C.; Kuettner, K.E.; reexpress the dierentiated collagen phenotypes. Cell 1982,
Williams, J.M. Hyaluronic acid suppresses bronectin 20, 215.
fragment mediated cartilage chondrolysis: I. In vitro. 191. Sittinger, M.; Bujia, J.; Minuth, W.W.; Hammer, C.;
Osteoarthr. Cartil. 1997, 5, 309. Burmester, G.R. Engineering of cartilage tissue using
173. Duranti, F.; Salti, G.; Bovani, B.; Calandra, M.; Rosati, bioresorbable polymer carriers in perfusion culture. Bio-
M.L. Injectable hyaluronic acid gel for soft tissue materials 1994, 15, 451.
augmentationA clinical and histological study. Derma- 192. Bujia, J.; Sittinger, M.; Minuth, W.W.; Hammer, C.;
tol. Surg. 1998, 24, 1317. Burmester, G.R. Engineering of cartilage tissue using
174. Choi, Y.S.; Hong, S.R.; Lee, Y.M.; Song, K.W.; Park, bioresorbable polymer eeces and perfusion culture. Acta
M.H.; Nam, Y.S. Study on gelatin-containing articial Otolaryngol. 1995, 115, 307.
skin: I. Preparation and characteristics of novel gelatin- 193. OConnor, S.M.; Stenger, D.A.; Shaer, K.M.; Ma, W.
alginate sponge. Biomaterials 1999, 20, 409. Survival and neurite outgrowth of rat cortical neurons in
175. Cortivo, R.; Brun, P.; Rastrelli, A.; Abatangelo, G. In vitro three-dimensional agarose and collagen gel matrices.
studies on biocompatibility of hyaluronic-acid esters. Neurosci. Lett. 2001, 304, 189.
Biomaterials 1991, 12, 727. 194. Borkenhagen, M.; Clemence, J.F.; Sigrist, H.; Aebischer, P.
176. Della, V.F.; Romeo, A. New Polysaccharide Esters and Three-dimensional extracellular matrix engineering in the
Their Salts. European Patent 216,453, 1987. nervous system. J. Biomed. Mater. Res. 1998, 40, 392.
177. Milella, E.; Brescia, E.; Massaro, C.; Ramires, P.A.; 195. Balgude, A.P.; Yu, X.; Szymanski, A.; Bellamkonda, R.V.
Miglietta, M.R.; Fiori, V.; Aversa, P. Physico-chemical Agarose gel stiness determines rate of DRG neurite
properties and degradability of non-woven hyaluronan extension in 3D cultures. Biomaterials 2001, 22, 1077.
benzylic esters as tissue engineering scaolds. Biomaterials 196. Oates, C.G.; Lucas, P.W.; Lee, J.P. How brittle are gels.
2002, 23, 1053. Carbohydr. Polym. 1993, 20, 189.
178. Grigolo, B.; Lisignoli, G.; Piacentini, A.; Fiorini, M.; 197. dosSantos, V.A.P.M.; Leenen, E.J.T.M.; Rippoll, M.M.;
Bobbi, P.; Mazzotti, G.; Ducan, M.; Pavesio, A.; Facchini, vanderSluis, C.; vanVliet, T.; Tramper, J.; Wijels, R.H.
A. Evidence for redierentiation of human chondrocytes Relevance of rheological properties of gel beads for their
grown on a hyaluronan-based biomaterial (HYAFFR 11): mechanical stability in bioreactors. Biotechnol. Bioeng.
Immunohistochemical and ultrastructural analysis. Bio- 1997, 56, 517.
materials 2002, 23, 1187. 198. Ertesvag, H.; Valla, S. Biosynthesis and applications of
179. Rees, D.A. Secondary and tertiary structure of polysac- alginates. Polym. Degrad. Stab. 1998, 59, 85.
charides in solutions and gels. Angew Chem. Int. Ed. Engl. 199. Ott, C.M.; Day, D.F. Bacterial alginateAn alternative
1977, 16, 214. industrial polymer. Trends Polym. Sci. 1995, 3, 402.
180. Uludag, H.; De Vos, P.; Tresco, P.A. Technology of 200. Cooney, M.J.; Goh, L.T.; Lee, P.L.; Johns, M.R.
mammalian cell encapsulation. Adv. Drug Deliv. Rev. Structured model-based analysis and control of the
2000, 42, 29. hyaluronic acid fermentation by Streptococcus zooepide-
181. Wang, N.; Wu, X.S. A novel approach to stabilization of micus: Physiological implications of glucose and complex
protein drugs in poly(lactic-co-glycolic acid) microspheres nitrogen-limited growth. Biotechnol. Prog. 1999, 15, 898.
using agarose hydrogel. Int. J. Pharm. 1998, 166, 1. 201. Kobayashi, S.; Uyama, H.; Kimura, S. Enzymatic poly-
182. Liu, M.S.; Zang, J.; Evangelista, R.A.; Rampal, S.; Chen, merization. Chem. Rev. 2001, 101, 3793.
F.T.A. Double-stranded DNA analysis by capillary 202. Kobayashi, S.; Morii, H.; Itoh, R.; Kimura, S.; Ohmae, M.
electrophoresis with laser-induced uorescence using ethid- Enzymatic polymerization to articial hyaluronan: A novel
ium-bromide as an intercalator. Biotechniques 1995, 18, method to synthesize a glycosaminoglycan using a tran-
316. sition state analogue monomer. J. Am. Chem. Soc. 2001,
183. Aymard, P.; Martin, D.R.; Plucknett, K.; Foster, T.J.; 123, 11825.
Clark, A.H.; Norton, I.T. Inuence of thermal history on 203. Kobayashi, S.; Kiyosada, T.; Shoda, S. Synthesis of
the structural and mechanical properties of agarose gels. articial chitin: Irreversible catalytic behavior of a glycosyl
Biopolymers 2001, 59, 131. hydrolase through a transition state analogue substrate. J.
184. Normand, V.; Lootens, D.L.; Amici, E.; Plucknett, K.P.; Am. Chem. Soc. 1996, 118, 13113.
37
Synthetic and Natural Polysaccharides Having Specic
Biological Activities
Takashi Yoshida
Kitami Institute of Technology, Kitami, Japan
I. INTRODUCTION In this review, recent developments in the study of

biological polysaccharides and oligosaccharides are pre-
Naturally occurring polysaccharides and oligosaccharides sented. Synthetic polysaccharides with biological activities
are recently getting an increase of attention because of their will be not presented in detail here; details may be found
important roles in specic biological recognitions. How- in the authors recent report [1] and many other exact
ever, because polysaccharides in nature have complex reviews [913].
structure, it is dicult to elucidate the relationship between
their structures and biological activities. Therefore we have
presented many papers on the synthesis of polysaccharides
having dened structures by the ring-opening polymeriza- II. ANTITUMOR ACTIVITY OF 1,3-h
h -GLUCAN
tion of anhydrosugar derivatives and examined the struc- A. Lentinan as a Potent Antitumor Polysaccharide
ture and biological activity relationship [1,2]. Especially,
we found that curdlan sulfate had potent anti-HIV and low Lentinan is a naturally occurring polysaccharide in mush-
blood anticoagulant activities [3,4]. Curdlan is a naturally room, from L. edodes Sing., and has 1,3-h-glucopyranosidic
occurring polysaccharide having linear 1,3-h-linked gluco- structure with two 1,6-h-D-glucopyranosidic branches ev-
pyranose structure and is produced by a bacterial strain, ery ve glucose units in the main chain. In 1969, Chihara
Alcaligenes faecalis var. myxogenes 10C3. Curdlan was found for the rst time the strong antitumor activity of
sulfated with piperidine-N-sulfonic acid in dimethyl sulf- lentinan [14,15], and in 1985 lentinan was approved as an
oxide (DMSO) to give curdlan sulfates having several mo- antitumor drug against stomach cancer in Japan [8]. In the
lecular weights and dierent sulfur contents. We found that structure and biological activity relationship, the high-
curdlan sulfate with a sulfur content of 14.4% completely order structure with a right-handed triple helix and micelle
inhibited the infection of HIV in the concentration of as formation as well as 1,3-h-D-glucopyranosidic structure
low as 3.3 Ag/mL and low cytotoxicity. This was one of the are considered to play important roles in the strong anti-
most potent anti-HIV polysaccharides. The Phase I/II tumor activity (Fig. 1).
clinical trial was carried out in the United States [57]. Schizophyllan isolated from a mushroom S. commune
In general, 1,3-h-glucan has strong antitumor activity; has similar structure to lentinan, 1,3-h-D-glucan having a
lentinan, produced by the fungus Lentinus edodes, and 1,6-h-D-glucopyranosidic branch in every three glucose
schizophyllan, isolated from Schizophyllan commune, were units in the main chain and also has strong antitumor
used clinically against cancer in Japan [8]. Interestingly, activity [16].
1,3-a-glucans were recently reported to induce IL-12 in In general, the polysaccharides in the mushroom have a
vivo by the interaction of immune cells such as NK and 1,3-h-D-glucopyranosidic structure, which is highly ordered
NKT cells to decrease the size of cancer. The anticancer and gave strong antitumor activity. However, in 1998, Nor-
mechanism was reported. A new biological activity of isue reported a dierent structure of polysaccharide from
chitin and chitosan has been reported, and the clinical 1,3-h-D-glucanthe water-insoluble polysaccharide as a
application of chitin was also presented. major extract in the fruiting body of Ganoderma lucidum,
839
840 Yoshida
Figure 1 Structure of lentinan.
called lingzhi in China, had 1,3-a-D-glucopyranosidic III. INTERLEUKIN-12 PRODUCTION BY 1,3-a-

structure [17]. The extract has been used in China and D-GLUCOSIDIC OLIGOSACCHARIDE
Japan as a Chinese medicine for cancer. Mizuno found
water-soluble and -insoluble polysaccharides in the same The results of many immunological studies so far revealed
mushroom. The structure of water-soluble polysaccharide the two eective cells against cancer: The rst is cytotoxic
was 1,3-h- D -glucan with 1,6-h- D -linked glucose side T-lymphocyte (CTL) activated by 1,3-h-glucan immuno-
chains; this was concluded to be responsible for the anti- modulators, such as lentinan, schizophyllan, PSK (Corio-
tumor activity of G. lucidum [18,19]. olus), and OK-432 (Streptococcal). By the activation of
The strong antitumor activity of a pharmaceutical immunological system, the cytokines and interleukins of
composition comprising activated hemicellulose was TNF-a, interferon-g (IFN-g), and IL-12 are released [22]
reported [20]. Activated hemicellulose compound is a and then the CTL is activated to attack cancer cells. The
known biologically active substance obtained by enzyme second is the natural killer T-cell (NKT cell), which is called
treatments of plant ber existing in the cell wall of fungal the 4th lymphocyte in addition to T, B, and NK cells. The
mycelia and a mixture of some polysaccharides and pepti- NKT cell is activated by 1,3-a-glucans such as nigerooli-
doglycan, proteoglucan, lectin, and nucleic acid. The acti- gosaccharides, a mixture of nigerose, nigerosyl glucose,
vated hemicellulose compound induced interleukin-12 (IL- and nigerosyl maltose. The NKT cell has two glycoproteins
12) in vivo and is used clinically as a strong antitumor drug as receptors on the surface of cells: these are the T cell
without dangerous side eects for the treatment of cancer. receptor (TCR, Va24Vb11), which binds to a a-galacto-
We reported that a new polysaccharide was found in sylceramide, and the NK cell receptor (NKR-P1,
the extracts of the activated hemicellulose and the structure CD3CD161), which recognized 1,3-a oligosaccharides to
was determined by chemical and spectroscopic methods, activate the NKT cell. Yagita (Kinki University Hospital
indicating that the polysaccharide had 1,3-h-D-glucan hav- Institute of Immunotherapy for Cancer, Japan) found for
ing a single 1,6-h-D-glucopyranosidic branch in every 10 the rst time that the NKR-P1 was stimulated by 1,3-a
12 units. Another polysaccharide having 1,4-a-D-gluco- oligosaccharides to produce IL-12 [23]. Nigerooligosac-
pyranosidic structure such as amylopectin was also found. charide having 1,3-a-D-glucopyranosidic structure was
In addition, it was revealed that 1,3-h-D-glucan inhibited produced by Acremonium sp. S4G13 [24] and has been
the multiplication of Sarcoma 180 and Lewis lung cancer in manufactured industrially as a sweetener (Fig. 3). In 1994,
rat and had the ability to produce interleukin-12 (IL-12) in interactions of the oligosaccharides on the target cell
vivo [21] (Fig. 2). surface with NKR-P1 were crucial both for target cell
recognition and for delivery of stimulatory of inhibitory
signals linked to the NK cytolytic machinery, suggesting
that purging of tumor cells in vivo may be a therapeutic
possibility [25]. Several specic biological activities of
nigerooligosaccharides were reported. Mitogen-induced
Figure 2 Structure of a new 1,3-h-D-glucan. Figure 3 Structure of nigerose.

Synthetic and Natural Polysaccharides 841
proliferation of splenocytes from normal mice was aug- migration not only delay wound healing but also aggravate
mented in a dose-dependent manner by nigerose. Nigero- infection. However, it has not been clear whether chitin and
oligosaccharides enhanced IL-12 and IFN-g production by chitosan induce arachidonic acid products (AAPs) such as
normal splenocytes in the presence of the potent IL-12 leukotriene B4 (LTB4), which promote PMN migration and
inducer, heat-killed Lactobacillus plantarum L-137, in upregulation of endothelial leukocyte adhesion molecule
vitro. These results suggest that nigerooligosaccharides expression. The eects of chitin and chitosan on the release
may exert immunopotentiating activity through the acti- of AAPs were investigated by Minami (Tottori University,
vation of IL-12-dependent T helper 1 (Th1)-like immune Japan) [29]. Supernatants of canine PMN cell suspensions
response [26]. Recently, the results of augmentation of incubated with chitin and chitosan contained a LTB4
natural killer activity by nigerooligosaccharides appeared concentration high enough to induce canine PMN migra-
in in vitro and in vivo studies. In vitro treatment of hepatic tion in vitro. The supernatants also contained the same
mononuclear cells (MNC) from normal mice with 1 mg/mL concentration of prostaglandin E2 (PGE2) as that normally
nigerooligosaccharides for 17 hr enhanced their cytotox- found in the peripheral blood of dogs. Intraperitoneal
icity against YAC-1 cells. NK activity of hepatic MNC was administration of chitosan to dogs induced peritoneal
also enhanced in mice injected intraperitoneally with 0.4 exudative uid (PEF); however, this is not true for chitin.
mg of nigerooligosaccharides. Drinking of 1% nigerooli- These results suggested that chitin and chitosan stimulated
gosaccharides signicantly improved the survival curve of canine PMNs to release LTB4 in vitro and that chitosan
mice intravenously inoculated with EL-4 cells [27]. In 2000, induced PEF that contained enough LTB4 to enhance
Yagita showed for the rst time the high clinical eects of canine PMN migration in vitro.
nigerooligosaccharides on several human cancers [22]. Many specic biological activities of chitin and chito-
In addition, liver injury because of sequential activa- san were reported by Minami: eects on granulation tissue
tion of NK and NKT cells by carrageenan was reported in cats [30], on canine PMN cells [31], on canine gastroin-
[28]. Carrageenan is a high molecular weight polysaccha- testinal tract [32], on migrations of broblasts [33], on
ride and is widely used as a food additive for the solidi- vascular endothelium [33], on complement [34], on analge-
cation of plant oils. A time-kinetic study showed sequential sic [35], on enhanced healing of cartilaginous injuries [36],
activation of NK and NKT cells in the liver. NK and NKT and on a mice model of acute respiratory distress syndrome
cytotoxicities were augmented. The in vivo elimination of (ARDS) [37]. The chemical modication of chitin at the N-
NK cells reduced the liver injury induced by carrageenan. position for avoiding intermolecular and intramolecular
Direct binding of carrageenan onto NK cells was also hydrogen linkages was carried out to become water-solu-
demonstrated, and the binding induced a subsequent pro- ble, lm-forming materials. Dung (NCST of Vietnam) [38]
duction of IFN-g. Not only phagocytic cells but also reported that some new derivatives including N-carboxy-
primitive lymphocytes subsets might be important targets methyl, N-carboxylbutyl, and 5-methyl pyrolidinone chi-
for the acute toxicity of carrageenan. tosans have been investigated to form membrane having
wound-healing activity in vivo. More than 200 clinical
trials in Vietnam have been treated positively for deep
IV. CHITIN AND CHITOSAN, NEW burn, trauma, ulcer, and orthopedic patients, including
BIOLOGICAL ACTIVITIES 14 serious cases that have been saved.
Chitin and chitosan are the second most plentiful natural
polymers next to cellulose and draw much attention be-
cause of their high biocompatibility. The structure consist V. STRUCTUREANTI-HIV ACTIVITY
of 2-acetoamide-2-deoxy-h-D-glucose for chitin and 2-ami- RELATIONSHIP OF SULFATED
no-2-deoxy-h-D-glucose for chitosan through 1,4-h glyco- POLYSACCHARIDES
side linkage (Fig. 4). Many reports have been published on
the acceleration of wound healing by chitin and chitosan. A. Anti-HIV Activity of Sulfated Polysaccharides
Polymorphonuclear (PMN) migration is one of the
The year 1987 was a commemorative year for the discovery
important steps in wound healing, and defects in PMN
of polysaccharides having anti-HIV activity and several
papers appeared. The rst report on anti-HIV activity of
natural polysaccharides was reported by Nakashima and
Yamamoto in 1987 [39,40]. An aqueous extract from the
marine red alga Schizymenia pacica has been tested in a
cell-free system for its eect on reverse transcriptase (RT)
from avian retrovirus and mammalian retrovirus. The
extract inhibited the RT from both retroviruses. This paper
described no information on the structure of the new
inhibitor against RT. However, the authors presumed that
this inhibitor is a polysaccharide. In the next paper in the
Figure 4 Structure of chitin and chitosan. same year, this inhibitor was revealed to be a sulfated
842 Yoshida
polysaccharide, E-carrageenan, having a molecular weight synthetic polysaccharides having potent anti-HIV activity.
of around 2 million and composed of galactose and 3,6- The nonsulfated polysaccharides did not prevent them at
anhydrogalactose having degree of sulfation of 20%. It was any concentration tested (Fig. 5). The anti-HIV eect of
found that the sulfated polysaccharide inhibited selectively these polysaccharides was conrmed by measuring HIV-
HIV RT and replication in vitro. Chondroitin, dermatan, specic antigen expression in infected MT-4 cell, which is a
and keratan sulfates and heparin also inhibited the RT of human T4-positive cell line carrying human T-cell lympho-
avian mycloblastosis virus [41]. In addition, we found in tropic virus type-I, HTLV-1, and sensitive to HIV. Dextran
1987 that the synthetic polysaccharides dextran, xylofuran, sulfate was also found to have inhibitory eects on RT
and ribofuranan sulfates completely prevented HIV-in- activity and to possibly possess anti-HIV activity (Fig. 6).
duced cytophathic eects (CPE) at concentrations of less The inhibitory eects in vitro of the virus infection to T
than 10 and 100 Ag/mL [42]. This is the rst report on the cells were demonstrated.
Figure 5 Anti-HIV activity of sulfated polysaccharides.

Figure 6 Photograph of anti-HIV activity of sulfated polysaccharides.
Drugs are classied into four groups: (1) vaccines, (2) dextran sulfate (20.6 unit/mg) were used as standards for
inhibitors of reverse transcriptase, (3) fusion inhibitors of anti-HIV and blood anticoagulant activities, respectively.
the virus to T cells, and (4) protease inhibitors. Azidothy- Both sulfated ribofuranan having M n = 17 103 and
midine (AZT), dideoxyinosine (DDI), and dideoxycytidine ribopyranan having M n = 14 103 had high anti-HIV
(DDC) belonging to the nucleoside drugs classied under activity of 3.3 Ag/mL as a complete inhibitory eect on the
(2) and some protease inhibitors (4) have been used clini- replication of HIV on MT-4 cell in vitro and 0.3 Ag/mL as
cally. During the search for AIDS drugs among existing EC50. Furthermore, sulfated branched ribofuranan and
drugs and chemicals, we attracted attention to ribopoly- ribopyranan having 23 mol% of glucose branch also
saccharides as potent anti-HIV polysaccharides. Because showed potent anti-HIV activity. The activity increased
D-ribose is a component of the gene and is a constituent of with an increase in the proportion of branches, suggesting
vitamin B2 and some antibiotics. Benzylated 1,4-anhydro- that the introduction of branches lead to an increase in the
ribose, 1,4-anhydro-2,3-di-O-benzyl-a-D -ribopyranose proportion of sulfate groups in the polysaccharides. The
(ADBR), was synthesized and underwent ring-opening blood anticoagulant activity is an important biological
polymerization with Lewis acid catalysts such as boron activity of sulfated polysaccharides, and heparin and dex-
triuoride etherate and phosphorus pentauoride to give tran sulfate were used clinically during operation. The
the corresponding 1,5-a furanosidic polysaccharide, blood anticoagulant activity in vitro was determined by
(1!5)-a-D-ribofuranan, after removal of the protective using bovine plasma according to the United States Phar-
benzyl groups into hydroxyl groups [43]. The ring-opening macopoeia [47]. It was found that sulfated ribofuranan had
polymerization of 1,4-anhydro-2,3-O-benzylidene-a-D- higher anticoagulant activity, 56 unit/mg, than that of
ribopyranose (ABRP) with antimony pentachloride as a sulfated ribofuranan (29 unit/mg), probably because of
catalyst gave a stereoregular 1,4-h pyranosidic polymer, the high activity of ribofuranan, which may be due to the
2,3-di-O-benzylidene-(1!4)-h-D-ribopyranan having a exibility of the main chain [48]. These results suggest that
cellulose-type polymer backbone. After debenzylidena- (1) both furanan- and pyranan-type polysaccharides had
tion, (1!4)-h-D-ribopyranan was obtained [44]. After potent anti-HIV activity, and especially, (2) the furanan-
sulfation, we examined the relationship between structure type polysaccharides and branches in polysaccharides were
of polysaccharides and biological activities such as anti- contributed to high blood anticoagulant activity. However,
HIV and blood anticoagulant activities [45,46]. the high blood anticoagulant activity is a side eect for the
anti-HIV activity. Therefore we investigated sulfated poly-
B. Relationship Between Structure and Biological saccharides having high anti-HIV activity and low blood
Activities of Ribopolysaccharides anticoagulant activity.
First of all, the structurebiological activity relationship of C. Relationship Between Molecular Weights and
sulfated ribofuranan and ribopyranan was examined and Biological Activities of Ribopolysaccharides
the result is shown in Table 1.
Curdlan sulfate (3.3 Ag/mL for complete inhibitory The relationship between molecular weights and anti-HIV
concentration of HIV or 0.1 Ag/mL for 50% protective and blood anticoagulant activities were examined by sul-
concentration of HIV infection to MT-4 cell, EC50) and fated ribofuranans and alkyl ribofuranans having molecu-
844 Yoshida
Table 1 Structure and Biological Activity Relationship of Sulfated

Ribopolysaccharide
Mn EC50 CC50 AA
(103) (Ag/ml) (Ag/ml) (unit/mg)
1 17 3.3a >1000 56
2 12 0.3 783 55
3 17 0.5 >1000 29
4 12 0.9 >1000 47
a
EC100 = minimum eective concentration of curdlan sulfate (CS) for complete
inhibition of HIV infection.
Std EC50 = 0.43 mg/ml (cs); AA = 22.7 unit/mg (DS); CC50 = 50% cytotoxic
concentration.
lar weights of Mn = 3 10317 103. As shown in Table 2, hydrophobic structure by which the sulfated alkyl oligo-
sulfated ribofuranan having a high molecular weight of saccharide molecules are easily oriented and aggregated.
Mn = 23 103 had both high anti-HIV of EC100 = 3.3 Ag/ The atmosphere near HIV might be changed to hydrophilic
mL (EC50 = 0.1 Ag/mL) and blood anticoagulant activities and then HIV reduced the infectivity to MT-4 cell [49].
of 56 unit/mg, respectively. The activities decreased with
decrease in molecular weights. Sulfated ribofuranan having
Mn = 6 103exhibited low anti-HIV (EC50 = 68.6 Ag/mL) D. Relationship Between Degree of Sulfation and
and blood anticoagulant (14 unit/mg) activities. However, Biological Activities of Ribopolysaccharides
sulfated octadecyl ribofuranans having M n = 6 103 and
M n = 3 103 had a high anti-HIV activity of EC50 = 0.6 To examine the relationship between degree of sulfation
and 2.5 Ag/mL, respectively. The blood anticoagulant and anti-HIV activity, sulfated polydeoxyriboses were
activity decreased with decrease in molecular weights. The synthesized by the ring-opening polymerization of 1,4-
enhancement of anti-HIV activity by binding of the alkyl anhydro-deoxyribose derivatives [50]. The copolymeriza-
groups might be ascribed to the formation of a hydrophilic tion of the deoxyribose monomers and 1,4-anhydro-2,3-di-
Table 2 Molecular Weight and Biological Activity

Relationship of Sulfated Ribopolysaccharide
Mn EC50 CC50 AA
( 103) (Ag/ml) (Ag/ml) (unit/mg)
1 17 3.3a >1000 56
2 9 0.6 >1000 17
3 6 68.6 >1000 14
4 6 0.6 >1000 11
5 3 2.5 >1000 7
a
EC100 = minimum eective concentration of curdlan sulfate
(CS) for complete inhibition of HIV infection.
Std EC50 = 0.43 mg/ml (CS); AA = 22.7 unit/mg (DS);
CC50 = 50% cytotoxic concentration.
Table 3 Degree of Sulfation and Biological Activity we carried out the synthesis of sulfated polysaccharides
Relationship of Sulfated Ribopolysaccharide having both potent anti-HIV activity and low blood anti-
coagulant activity.
Mn EC50 CC50 AA
Curdlan is a naturally occurring linear polysaccharide
( 103) (Ag/ml) (Ag/ml) (unit/mg)
from A. faecalis var. myxogenes 10C3 strain and is made up
1 5.8 >1000 561 2 of chains of 1,3-h linked D-glucose units [51]. Curdlan was
2 6.4 16.6 715 17 sulfated by piperidine-N-sulfonic acid in dimethyl sulfox-
3 20 0.6 >1000 18 ide to give curdlan sulfates with several molecular sizes and
dierent sulfur contents [3] (Fig. 7). We found that curdlan
Std EC50 = 0.43 mg/ml (CS); AA = 22.7 unit/mg (DS); CC50 = sulfate with a sulfur content of 14.4% completely inhibited
50% cytotoxic concentration.
the infection of HIV in the concentration as low as 3.3 Ag/
mL. Curdlan sulfate is one of the most potent anti-HIV
polysaccharides and is used as a standard for the anti-HIV
activity (Table 4). The blood anticoagulant activity was less
than 10 unit/mg compared to the standard dextran sulfate
(Meito Sangyo, NC-1032, 20.6 unit/mg). The high resolu-
tion nuclear magnetic resonance (NMR) analysis including
COSY and RELAYED-COSY experiments revealed that
for curdlan sulfate with 1.6 sulfate groups per glucose unit
(DS), the sulfate group was introduced to the C6, C4, and
C2 positions in the glucose unit in the proportion of
O-tert-butyldimethylsilyl-a-D-ribopyranose (ADSR) were f100%, f5%, and f40%, respectively.
carried out to give copolysaccharides having various ratio Furthermore, the therapeutical availability of curdlan
of the deoxymonomeric unit after deprotection. After sulfates was examined [4]. Cytotoxic activity of curdlan
sulfation, we examined the relationship between degree of sulfate on uninfected MT-4 cells has not been observed at a
sulfation and anti-HIV and blood anticoagulant activities concentration of up to 5000 Ag/mL, suggesting that curdlan
as shown in Table 3. It was found that both sulfated 2- and sulfates had low cytotoxicity. In the animal model in vivo,
3-deoxy ribofuranans having degree of sulfation of 1 (S the LD50 of curdlan sulfate in intravenous injection was
content, 10.64%) had no anti-HIV activity. The anti-HIV found to be around 2000 mg/kg, employing mice and rats,
activity increased with a decrease of the proportion of the and neither death nor hemorrhage was observed in con-
deoxy unit (= an increase of degree of sulfation) to give an secutive administration of curdlan sulfate for 2 weeks at
EC50 as high as 0.6 Ag/mL, suggesting that the number of doses of 50 mg/kg/day in SpragueDawley rats. The total
sulfate groups in the polysaccharides is important for the dosage required per day is calculated to be approximately
high anti-HIV activity. The sulfated deoxyribofuranan 160470 mg/day/man, i.e., 3.29.4 mg/kg/day. On the
having high anti-HIV activity, with EC50 = 0.6 Ag/mL, other hand, we assayed HIV infectivity in the curdlan-
had relative low blood anticoagulant activity, 17 unit/mg, sulfate-depleted cell culture following cocultivation of
compared to that of sulfated ribofuranan (56 unit/mg) as HIV-infected MT-4 cell, MT-4 cell, and curdlan sulfate at
shown in Table 1. cultivation times of 24, 48, 72, or 168 hr under the cell
number ratio of (MT-4/HIV)/(MT-4) of 0.002 or 0.5. The
E. Curdlan Sulfate Having High Anti-HIV antigen expression of HIV on the cell surface in curdlan-
and Low Blood Anticoagulant Activities sulfate-depleted cell culture completely disappeared 12
days after cocultivation of MT-4/HIV, MT-4, and curdlan
We found that sulfated ribofuranan and ribopyranan had sulfate for 168 hr, at 5 Ag/mL concentration of curdlan
both potent anti-HIV and high blood anticoagulant activ- sulfate in (MT-4/HIV)/(MT-4) of 0.5. The half-life of
ities. The anticoagulant activity is an important biological curdlan sulfate in plasma was found to vary depending
activity for sulfated polysaccharides; for example, heparin on its molecular weights, i.e., 60 min for Mw of 7 104 and
is used to avoid blood coagulation during surgery. How- 180 min for 17 104, by employing a rat model. The
ever, in using AIDS drug, the anticoagulant activity of experimental results on the LD50 by intravenous injection
sulfated polysaccharides is a serious side eect. Therefore administration, the anticoagulant activity, the antigenicity,
Figure 7 Structure of curdlan sulfate.

846 Yoshida
Table 4 Anti-HIV Activity of Curdlan Sulfate After mixing of the two compounds in aqueous solutions, a
gel formed in the sample tube. The gel isolated was found to
No. S content DS Mn [a]D25 EC100a be primarily composed of curdlan sulfate and poly-L-
1 5.6 0.35 Not eective lysine, having excluded most of the sodium and bromide
2 8.9 6.8 1000 ions. Nuclear magnetic resonance analysis revealed a single
3 12.1 1.1 8.1 1.7 10 species that is quite dierent from either of the two original
4 12.1 1.1 11.8 3.8 3.3 species. The extent of gelling was aected by pH, molecular
5 12.5 1.3 15.7 2.3 3.3 weight of poly-L-lysine, temperature, and absolute concen-
6 13.6 1.4 3.4 0.8 3.3 tration. Maximal gelling was attained at pH 4.08.4,
7 14.1 1.6 2.1 1.9 3.3 molecular weight of 10 103, and temperature of 37jC.
8 14.4 1.6 4.6 0.1 3.3 These results suggest that curdlan sulfate can bind strongly
9 14.7 1.6 2.0 1.5 3.3 with poly-L-lysine by the ionic interactions in such way as
a to produce a polyion complex with a conformation distinct
Minimum eective concentration of curdlan sulfate for complete
inhibition of HIV infection.
from either of the two original compounds. This interac-
tion may help explain the observed inhibition of HIV and
other viruses by curdlan sulfate.
Furthermore, to elucidate the inhibitory mechanism of
and the half-life suggested that it would be worthwhile to curdlan sulfate on HIV, Uryu also reported that a partial
investigate curdlan sulfate in AIDS clinical research with sequence in gp120 was synthesized and measured the
further toxicological examinations. interaction with curdlan sulfate by NMR [56]. The se-
Kozbor et al. (Allegheny University of the Health quence consisted of a dimer of the sequence from 506 to
Sciences, U.S.A.) showed that T cell tropic HIV is over 518, which is one of putative reaction sites for sulfated
10-fold more sensitive to neutralization by curdlan sulfate polysaccharides. When curdlan sulfate and the dimeric
than macrophage tropic HIV, which possessed a relatively sequence were mixed in appropriate molar ratios, gel-like
less-charged amino acid composition in the V3 sequence materials were formed. The 1H NMR spectra revealed the
[52]. In addition, they synthesized the monomeric and formation of interpolymeric ionic interactions between
oligomeric gp120 mutants and examined the interaction negatively charged curdlan sulfate and the positively
of curdlan sulfate with the V2-, V3-, and CD4-binding charged dimeric sequence. For a V3 region peptide se-
domains on the gp120. The presence and the amino acid quence, amino acid residue 309331, the mixing of curdlan
composition of the V3 loop appeared to determine the sulfate with the V3 peptide did not provide gels, but yielded
extent of interaction of curdlan sulfate with the V2- and precipitates that aorded no structural information by the
CD4-binding regions on native gp120 monomers. The solution NMR.
positive charge of V3 in the oligomeric gp120 had higher
eective interaction with curdlan sulfate than that in the
monomeric gp120, suggesting that the curdlan-sulfate- VI. SPECIFIC BIOLOGICAL ACTIVITY OF
induced masking of V3 on oligomeric gp120 appears to LACQUER (URUSHI) POLYSACCHARIDES
be associated with the anti-HIV activity of curdlan sulfate
in vitro [53]. Lacquer, which originated in Asia, is the only natural
Taking into account the interaction mechanism of product that is polymerized by an enzyme, laccase, to give
heparin with antithronbin III, a coagulant protein, ionic a beautiful coating surface [57]. In the sap of lacquer tree,
interactions play an important role in the interaction there are three major components: urushiols (3- or 4-
between negatively charged polysaccharide heparin and alkenyl catechol derivatives), laccase, and polysaccharides.
positively charged lysine residues in antithronbin III. HIV Oshima and Kumanotani revealed the partial structure of a
infects T cell by binding of the envelope glycoproteins Chinese lacquer polysaccharide by the chemical methods
gp120 on HIV and CD4 on T cell. According to the such as sugar analysis, methylation analysis, and Smith
secondary structure of HIV envelope glycoprotein gp120, degradation to aord a branched 1,3-h-galactopyranan
a helical portion containing several basic amino acids such having 4-O-methyl glucuronic acid in the terminal of
as lysine and arginine were found in the amino acid residues complex branches [58]. We reported the structural charac-
506 through 518 in the a6 helical region, Thr-Lys+-Ala- terization of a Chinese lacquer polysaccharide in the sap of
Lys+-Arg+-Arg+-Val-Val-Gln-Arg+-Glu-Arg+-Lys+. Chinese lacquer tree by high-resolution NMR spectrosco-
Negatively charged sulfate groups of sulfated polysaccha- py [59]. Lacquer is an interesting natural product not only
rides might interact with the positively charged regions. By as a coating material but also as a biologically active
these interactions probably causing conformational material. The specic biological activities, e.g., blood co-
change in the gp120, sulfated polysaccharides seems to agulation, and antitumor, anti-HIV, and anticoagulant
inhibit binding of HIV gp120 to CD4 receptor of T cells activities, of a Chinese lacquer polysaccharide, a branched
[54]. A model system for the interaction was constructed acidic polysaccharide, before and after sulfation was in-
using curdlan sulfate and poly-L-lysine.HBr to assimilate vestigated [60]. The lacquer polysaccharide at the concen-
the a6 helix of gp120. Uryu reported the oligomeric tration of 0.016 mg/mL was found to shorten the
nature of both species by using 1H and 13C NMR [55]. coagulation time of bovine plasma more than 1 min by
Figure 8 Coagulant activity of urushi polysaccharide.
comparison with that of a blank, 5 min and 25 sec, saccharides were assayed for antitumor activity by using
suggesting that the lacquer polysaccharide had a blood- Sarcoma 180 tumor in rat. Results are shown in Fig. 9. In
coagulating activity. The linear acidic polysaccharides, this assay system, after 35 days of transplantation, the
sodium hyarulonate and alginate, delayed the coagulation weights of tumor in rat without any polysaccharides in-
time (Fig. 8). Because branched 1,3-h-glucans have potent creased to 20 g. When lentinan (1 mg/kg) was provided to
antitumor activity as mentioned above, the lacquer poly- the rats by an intraperitoneal injection (i.p.), the tumor
Figure 9 Antitumor activity of urushi polysaccharide.

848 Yoshida
disappeared after 35 days of the transplantation. However,

lentinan had no antitumor eects when orally administered
(p.o.). For the natural lacquer polysaccharide, we found
that the weights of tumor decreased to 10, 11, and 13 g by 50
and 5 mg/kg oral administration (p.o.), and 1 mg/kg intra-
peritoneal injection (i.p.), respectively. In particular, it was
interesting that the oral administration of the lacquer poly-
saccharide eectively decreased the weights of tumor,
although lentinan did not inhibit the growth of tumor. Af-
ter sulfation, sulfated lacquer polysaccharides had potent
anti-HIV activity represented by the 50% protecting con-
centration (EC50) around 0.5 Ag/mL and lower blood anti-
coagulant activity than that of standard dextran sulfate.
These results suggested that the lacquer polysaccharides
have specic biological activities by their acidic branched
structure. The lacquer polysaccharides are expected to be
candidate anti-HIV drugs from naturally occurring and
reproductive sources.
Recently, we investigated the structure of Asian urushi
polysaccharides by NMR and GPC measurements [61]. We
revealed that the structures of polysaccharides in China
and Japan, Taiwan and Vietnam, Myanmar and Cambo-
dia were similar, and that the polysaccharides in Myanmar
and Cambodia had larger proportions of L-arabinose and
L-rhamnose than those in other Asian lacquer polysaccha-
rides (Fig. 10). In general, it was previously reported that
the Chinese lacquer polysaccharide had two fractions in the
Figure 11 The GPC proles of the polysaccharide.
GPC prole having molecular weights of M n = 93 103

and 29 103 in the proportion of 25:75 mol%, respectively
[58,59]. The Asian lacquer polysaccharides also had two
molecular weight fractions; however, the shape and pro-
portion in the GPC proles were dierent from each other.
We estimated that the polysaccharides in lacquer tree had
originally one fraction having high molecular weights.
After collection, the polysaccharides were gradually de-
graded to lower molecular weights during the storage of the
sap with contacting air. Therefore, we directly collected the
sap of lacquer tree in the Aizu lacquer eld of Fukushima
prefecture, Japan, and isolated the polysaccharide from the
sap immediately, after 3 and 21 days, and after 7 years. Fig.
11 shows the GPC proles of the polysaccharide isolated
(A) immediately (within 1 min), (B) after 3 days, (C) after
21 days, and (D) after 7 years. In Fig. 11A, it was shown
that the polysaccharide isolated immediately after collec-
tion was revealed to have one fraction having a molecular
weight of Mn = 67 103. The polysaccharide isolated after
3 days was separated into two fractions with Mn = 67 103
and Mn = 23 103 in the proportion of 75:25, respectively
(Fig. 11B). The proportion of the fractions was further
changed into 25:75 in the polysaccharide isolated after 21
Figure 10 NMR spectra of Asian urushi polysaccharides. days of collection (Fig. 11C). After 7 years (Fig. 11D), it
was found that the proportion was not changed anymore. Synthesis and structural analysis of curdlan sulfonate with
Therefore it was found that the lacquer polysaccharide a potent inhibitory eect in vitro of AIDS virus infection.
Macromolecules 1990, 23, 37173721.
originally had one molecular weight fraction having the
4. Kaneko, Y.; Yoshida, O.; Nakagawa, R.; Yoshida, T.;
high molecular weight in the lacquer tree. The molecular Date, M.; Ogihara, S.; Shioya, S.; Matsuzawa, Y.;
weight gradually decreased with an elapse of keeping time Nagashima, N.; Irie, Y.; Mimura, T.; Shinkai, H.; Yasuda,
after collection. After more than 21 days, the proportion of N.; Matsuzaki, K.; Uryu, T.; Yamamoto, N. Inhibitory of
the molecular weights did not change and was kept for at HIV-1 infectivity with curdlan sulfate in vitro. Biochem.
least 7 years without changing. After collection, the sap was Pharmacol. 1990, 39, 797799.
fermented and the bubbles of carbon dioxide was gushed 5. Gordon, M.; Guralnik, M.; Kaneko, Y.; Mimura, T.;
Baker, M.; Lang, T. A phase I study of curdlan sulfonate
out to change the pH. Degradation might proceed by the an HIV inhibitor. Tolerance, pharmacokinetics and eects
dierence of the keeping conditions of the sap. In addition, on coagulation and on CD4 lymphocytes. J. Med. 1994, 25,
the polysaccharides in the two molecular weight fractions 163179.
had the same structure, because the NMR spectra, which 6. Gordon, M.; Guralnik, M.; Kaneko, Y.; Mimura, T.;
corresponded to those in Fig. 11, were not changed, al- Goodgame, J.; Lang, W. Further clinical studies of curdlan
though the degree of degradation was dierent. sulfatean anti-HIV agent. J. Med. 1995, 26, 97131.
7. Gordon, M.; Deeks, S.; Marzo, C.D.; Goodgame, J.;
Guralnik, M.; Lang, W.; Mimura, T.; Pearce, D.; Kaneko,
Y. Curdlan sulfate in a 21-day intravenous tolerance study
VII. CONCLUSIONS in human immunodeciency virus (HIV. and cytomegalo-
virus (CMV. infected patients: indication of anti-CMV
Naturally occurring polysaccharides are interesting not
activity with low toxicity. J. Med. 1997, 28, 108128.
only in their structures but also in their specic biologi- 8. Kaneko, Y.; Yamamoto, N.; Uryu, T. Biological action of
cal activities. However, because natural polysaccharides polysaccharides and those derivatives against cancer and
have complex structures, it is dicult to elucidate the AIDS. Immunother. Prosp. Infect. Dis. 1990, 109119.
relationship between structures and biological activities. 9. Uryu, T. Polysaccharides. In Models of Biopolymers by
The ring-opening polymerization of anhydrosugar Ring-Opening Polymerization; Penczek, S., Ed.; CRC Press:
derivatives is the best method to prepare stereoregular Boca Raton, FL, 1990; 133233.
10. Schuerch, C. The chemical synthesis and properties of
polysaccharides having dened structures and having polysaccharides of biomediated interest. Adv. Polym. Sci.
controlled molecular weights. The biological activities of 1972, 10, 173194.
synthetic polysaccharides with dened structures might 11. Schuerch, C. Synthesis and polymerization of anhydro
describe the relationship between structures and biologi- sugars. Adv. Polym. Sci. 1981, 39, 157212.
cal activities. To solve the dicult problems, we have 12. Okada, M. Ring-opening polymerization of bicyclic and
continued the investigation of both natural and synthetic spiro compounds. Reactivities and polymerization mecha-
nisms. Adv. Polym. Sci. 1992, 102, 146.
polysaccharides. 13. Hatanaka, K. Chemical synthesis of polysaccharides. In
Polysaccharides in Medical Applications; Dumitriu, S., Ed.;
Marcel Dekker, Inc.: New York, 1996; 320.
ACKNOWLEDGMENTS 14. Chihara, G.; Maeda, Y.; Hamuro, J.; Sasaki, T.; Fukuoka,
F. Inhibition of mouse sarcoma 180 by the polysaccharides
The author is particularly indebted to Professor Toshiyu- from Lentinus edodes (Berk) Sing. Nature 1969, 222, 687
ki Uryu of Teikyo University of Science for his many 689.
useful discussions and suggestions on the works described 15. Chihara, G.; Hamuro, J.; Maeda, Y.; Arai, Y.; Fukuoka,
here. The author would like to thank Professor Tetsuo F. Fractionation and purication of the polysaccharides
Miyakoshi of Meiji University for his valuable discus- with marked antitumor activity, especially lentinan, from
Lentinus edodes (Berk) Sing. an edible mushroom. Cancer
sions; and Professor Naoki Yamamoto of Tokyo Medical Res. 1970, 30, 27762781.
and Dental University, and Professor Hideki Nakashima 16. Komatsu, N.; Okubo, S. Host-mediated antitumor action
of St. Marianna University, Mr. Yutaro Kaneko and of Schizophyllan, a glucan produced by Schizophyllum
Toru Mimura of Ajinomoto Co. Ltd., for their valuable commun. Gann 1969, 60, 137144.
discussions and for their help with biological activities. 17. Chen, J.; Zhou, J.; Zhang, L.; Nakamura, Y.; Norisue, T.
The results presented in our papers have been acquired Chemical structure of the water-insoluble polysaccharide
isolated from the fruiting body of Ganoderma iucidum.
through collaboration with a number of students and Polym. J. 1998, 30, 838842.
postdoctoral researchers. The author would also like to 18. Mizuno, T.; Suzuki, E.; Maki, K.; Tamaki, H. Fractiona-
thank the people whose names are cited in the references. tion, chemical modication, and antitumor activity of
water insoluble polysaccharides of the fruiting body of
Ganoderma iucidum. Nippon Nogei Kagaku Kaishi 1985,
REFERENCES 59, 11431151.
19. Mizuno, T.; Osawa, K.; Hagiwara, N.; Kuboyama, R.
1. Yoshida, T. Synthesis of polysaccharides having specic Fractionation and characterization of antitumor polysac-
biological activities. Prog. Polym. Sci. 2001, 26, 379441. charides from Maitake, Grifola frondosa. Agric. Biol.
2. Uryu, T. Articial polysaccharides and their biological Chem. 1986, 50, 16791688.
activities. Prog. Polym. Sci. 1993, 18, 717761. 20. Yagita, A. Interleukin 12 inducer and medical composition,
3. Yoshida, T.; Hatanaka, K.; Uryu, T.; Kaneko, Y.; Suzuki, U.S. Patent: 6238660, 1997.
E.; Miyano, H.; Mimura, T.; Yoshida, O.; Yamamoto, N. 21. Yoshida, T.; Saeki, W. Structure and antitumor activity of
850 Yoshida
polysaccharides from Basidiomycetes. Proceedings of Inter- marine red alga: reverse transcriptase inhibition by an
national Symposium on Polymer Modication and Com- aqueous extract of Schizymenia pacica. J. Cancer Res.
posites (ISPMC2000), Beijing, China, 2000, September . Clin. Oncol. 1987, 113, 413416.
22. Arase, H.; Arase, N.; Saito, T. Interferon g production by 40. Nakashima, H.; Kido, Y.; Kobayashi, N.; Motoki, Y.;
natural killer (NK. cells and NK1.1+ T cells upon NKR Neushul, M.; Yamamoto, N. Purication and character-
p1 cross linking. J. Exp. Med. 1996, 183, 23912396. ization of an avian myeloblastosis and human immunode-
23. Yagita, A. The dierences role of T cell and NK cell receptor ciency virus reverse transcriptase inhibitor, sulfated
in activities of NKT cell. Rinsho Meneki 2002, 37, 1019. polysaccharides extracted from sea algae. Antimicrob.
24. Konishi, Y.; Shindo, K. Production of Nigerose, Nigerosyl Agents Chemother. 1987, 31, 15241528.
Glucose, Nigerosyl Maltose by Acremonium sp. S4G13. 41. Diringer, H. Japan Kokai Tokkyo Koho, 1987-215529.
Biosci. Biotechnol. Biochem. 1997, 61, 439442. 42. Nakashima, H.; Yoshida, O.; Tochikura, T.; Yoshida, T.;
25. Bezouska, K.; Yuen, C.; OBrien, J.; Childs, R.A.; Chai, Mimura, T.; Kido, Y.; Motoki, Y.; Kaneko, Y.; Uryu, T.;
W.; Lawson, A.M.; Drbal, K.; Fiserova, A.; Pospisil, M.; Yamamoto, N. Sulfation of polysaccharides generates
Feizi, T. Oligosaccharide ligands for NKR P1 protein ac- potent and selective inhibitors of human immunodeciency
tivate NK cells and cytotoxicity. Nature 1994, 372, 150157. virus infection and replication in vitro. Jpn. J. Cancer Res.
26. Murosaki, S.; Muroyama, K.; Yamamoto, Y.; Kusaka, H.; (Gann) 1987, 78, 11641168.
Liu, T.; Yoshikai, Y. Immunopotentiating activity of 43. Uryu, T.; Kitano, K.; Ito, K.; Yamanouchi, J.; Matsuzaki,
nigerooligosaccharides for the T helper 1 like immune K. Selective ring-opening polymerization of 1,4-anhydro-a-
response in mice. Biosci. Biotechnol. Biochem. 1999, 63, D-ribopyranose derivative and synthesis of stereoregular
373378. (1!4)-h-D-ribopyranan. Macromolecules 1981, 14, 19.
27. Murosaki, S.; Muroyama, K.; Yamamoto, Y.; Liu, T.; 44. Uryu, T.; Yamanouchi, J.; Kato, T.; Higichi, S.; Matsuza-
Yoshikai, Y. Nigerooligosaccharides augments natural ki, K. Selective ring-opening polymerization of di-O-
killer activity of hepatic mononuclear cells in mice. Int. methylated and di-O-benzylated 1,4-anhydro-h-D-ribo-
Immunopharmacol. 2002, 2, 151159. pyranoses and structure proof of synthetic cellulose-type
28. Abe, T.; Kawamura, H.; Kawabe, S.; Watanabe, H.; Geiyo, polysaccharide (1!4)-h-D-ribopyranan and (1!5)-a-D-
F.; Abo, T. Liver injury due to sequential activation of ribofuranan. J. Am. Chem. Soc. 1983, 105, 68656871.
natural killer cells and natural killer T cells by carrageenan. 45. Yoshida, T.; Wu, C.; Song, L.; Uryu, T.; Kaneko, Y.;
J. Hepatol. 2002, 36, 614623. Mimura, T.; Nakashima, H.; Yamamoto, N. Synthesis of
29. Usami, Y.; Okamoto, Y.; Takayama, T.; Shigemasa, Y.; cellulose-type polyriboses and their branched sulfates with
Minami, S. Chitin and chitosan stimulate canine poly- anti-AIDS virus activity by selective ring-opening copoly-
morphonuclear cells to release leukotriene B4 and prosta- merization of 1,4-anhydro-a-D-ribopyranose derivatives.
glandin E2. Polym. Adv. Technol. 1998, 9, 517522. Macromolecules 1994, 27, 44224428.
30. Kojima, K.; Okamoto, Y.; Miyatake, K.; Kitamura, Y.; 46. Yoshida, T.; Katayama, Y.; Inoue, S.; Uryu, T. Synthesis
Minami, S. Collagen typing of granulation tissue induced by of branched ribofuranans and their sulfates with strong
chitin and chitosan. Carbohydr. Polym. 1998, 37, 109113. anti-AIDS virus activity by selective ring-opening copoly-
31. Usami, Y.; Okamoto, Y.; Takayama, T.; Shigemasa, Y.; merization of 1,4-anhydro-a-D-ribopyranose derivatives.
Minami, S. Eect of N acetyl D glucosamine and D Macromolecules 1992, 25, 40514057.
glucosamine oligomers on canine polymorphonuclear cells 47. U. S. Pharmacopoeia National Formulary, USP XXI,
in vitro. Carbohydr. Polym. 1998, 37, 137141. 1985; 480483.
32. Okamoto, Y.; Nose, M.; Miyatake, K.; Sekine, J.; Oura, 48. Hatanaka, K.; Yoshida, T.; Miyahara, S.; Sato, T.; Ono,
R.; Shigemasa, Y.; Minami, S. Physical changes of chitin F.; Uryu, T.; Kuzuhara, H. Synthesis of new heparinoids
and chitosan in canine gastrointestinal tract. Carbohydr. with high anticoagulant activity. J. Med. Chem. 1987, 30,
Polym. 2001, 44, 211215. 810814.
33. Okamoto, Y.; Watanabe, M.; Miyatake, K.; Morimoto, 49. Choi, Y.; Yoshida, T.; Mimura, T.; Kaneko, Y.; Naka-
M.; Shigemasa, Y.; Minami, S. Eects of chitin/chitosan shima, H.; Yamamoto, N.; Uryu, T. Synthesis of sulfated
and their oligomers/monomers on migrations of broblasts octadecyl ribo-oligosaccharides with potent anti-AIDS
and vascular endothelium. Biomaterials 2002, 23, 1975 virus activity by ring-opening polymerization of a 1,4-
1979. anhydroribose derivative. Carbohydr. Res. 1996, 282, 113
34. Minami, S.; Suzuki, H.; Okamoto, Y.; Fujinaga, T.; Shige- 123.
masa, Y. Chitin and chitosan activate complement via the 50. Choi, Y.; Kang, B.; Lu, R.; Osawa, M.; Hattori, K.;
alternative pathway. Carbohydr. Polym. 1998, 36, 151155. Yoshida, T.; Mimura, T.; Kaneko, Y.; Nakashima, H.;
35. Okamoto, Y.; Kawakami, K.; Miyatake, K.; Morimoto, Yamamoto, N.; Uryu, T. Synthesis of sulfated deoxy-
M.; Shigemasa, Y.; Minami, S. Analgesic eects of chitin ribofuranans having selective anti-AIDS virus activity by
and chitosan. Carbohydr. Polym. 2002, 49, 249252. ring-opening copolymerization of 1,4-anhydro ribose de-
36. Tamaki, Y.; Miyatake, K.; Okamoto, Y.; Takamori, Y.; rivatives. Polym. J. 1997, 29, 374379.
Sakamoto, H.; Minami, S. Enhanced healing of cartilagi- 51. Harada, T.; Harada, A. Curdlan and Succinoglycan. In
nous injuries by glucosamine hydrochloride. Carbohydr. Polysaccharides in Medical Application; Dumitriu, S., Ed.;
Polym. 2002, 48, 369378. Marcel Dekker: New York, 1996, 2158.
37. Khanal, D.R.; Okamoto, Y.; Miyatake, K.; Shinobu, T.; 52. Jagodzinski, P.; Wiaderkiewicz, R.; Kurzawski, G.; Kloc-
Shigemasa, Y.; Tokura, S.; Minami, S. Protective eects of zewiak, M.; Nakashima, H.; Hyjek, E.; Yamamoto, N.;
phosphated chitin (p chitin) in a mice model of acute Uryu, T.; Kaneko, Y.; Posner, M.R.; Kozbor, D.
respiratory distress syndrome (ARDS). Carbohydr. Polym. Mechanism of the inhibitory eect of curdlan sulfate on
2001, 44, 99106. HIV-1 infection in vitro. Virology 1994, 202, 735745.
38. Dumg, P.L.; Dong, N.T.; Mai, P.T.; Son, L.T.; Thanh, 53. Jagodzinski, P.; Wustner, J.; Kmieciak, D.; Wasik, T.J.;
N.K.; Kinh, C.D. Biomaterials based on chitin and its Fertala, A.; Sieron, A.L.; Takahashi, M.; Tsiji, T.; Mimura, T.;
homologs. Adv. Nat. Sci. 2000, 1, 95106. Fung, M.S.; Gorny, M.K.; Kloczewiak, M.; Kaneko,
39. Nakashima, H.; Kido, Y.; Kobayashi, N.; Motoki, Y.; Y.; Koznor, D. Role of the V2, V3, and CD4-binding do-
Neushul, M.; Yamamoto, N. Antiretroviral activity in a mains of gp120 in curdlan sulfonate neutralization sensi-
tivity of HIV-1 during infection of T lymphocytes. Virology 57. Kumanotani, J. Urushi (oriental lacquer) a natural
1996, 226, 217227. aesthetic durable and future promising coating. Prog.
54. Uryu, T.; Ikushima, N.; Katsuraya, K.; Shoji, T.; Org. Coat. 1995, 26, 163195.
Takahashi, N.; Yoshida, T.; Kanno, K.; Murakami, T.; 58. Oshima, R.; Kumanotani, J. Structural studies of plant
Nakashima, H.; Yamamoto, N. Sulfated alkyl oligosac- gum from sap of the lac tree, Rhus vermicifera. Carbohydr.
charides with potent inhibitory eects on human immuno- Res. 1984, 127, 4357.
deciency virus infection. Biochem. Pharmacol. 1992, 43, 59. Lu, R.; Yoshida, T.; Uryu, T. Structural analysis of
23852392. polysaccharides in Chinese lacquer by NMR spectroscopy.
55. Jeon, K.; Katsuraya, K.; Kaneko, Y.; Mimura, T.; Uryu, Seni Gakkaishi 1999, 55, 4756.
T. Studies on interaction mechanism of sulfonated poly- 60. Lu, R.; Yoshida, T.; Nakashima, H.; Premanathan, M.;
saccharides as an AIDS drug by NMR. Macromolecules Aragaki, R.; Mimura, T.; Kaneko, Y.; Yamamoto, N.;
1997, 30, 19972001. Miyakoshi, T.; Uryu, T. Specic biological activities of
56. Jeon, K.; Katsuraya, K.; Inazu, T.; Kaneko, Y.; Mimura, Chinese lacquer polysaccharides. Carbohydr. Polym. 2000,
T.; Uryu, T. NMR spectroscopic detection of interactions 43, 4754.
between a HIV protein sequence and a highly anti-HIV 61. Lu, R.; Yoshida, T. Specic structure and molecular weight
active curdlan sulfate. J. Am. Chem. Soc. 2000, 122, 12536 changing of Asian lacquer polysaccharides. Carbohydr.
12541. Polym. 2003, 54, 419424.
38
Medical Foods and Fructooligosaccharides
Bryan W. Wolf, JoMay Chow, and Keith A. Garleb
Abbott Laboratories, Columbus, Ohio, U.S.A.
Short-chain fructooligosaccharides (scFOS) consist of two medical food is dened by the Food and Drug Adminis-
to four fructose molecules linked by (2!1)-h glycosidic tration [2] as a food that is formulated to be consumed or
bonds and carry a single D-glucosyl molecule at the non- administered enterally under the supervision of a physician
reducing end of the chain linked (1!2)-a as in sucrose. and is intended for the specic dietary management of a
Because of their low molecular weight, scFOS are not disease or condition for which distinctive nutritional
quantied as total dietary ber. However, this attribute requirements, based on recognized scientic principles,
makes them compatible with liquid medical foods, many of are established by medical evaluation. Medical foods
which are fed to patients through a tube. Most sources of are typically liquid; patients may require that the product
dietary ber are not compatible with liquid medical foods: be administered via an enteral feeding tube. The internal
insoluble bers tend to settle and can block the feeding diameter of an enteral feeding tube, for obvious reasons, is
tube, whereas soluble bers increase product viscosity small. If a medical food is fed without the aid of a pump, its
making it dicult to administer through an enteral tube. viscosity must be kept below 100 mPa sec (i.e., 100 cP).
Short-chain fructooligosaccharides have many dietary-- Unfortunately, dietary ber, particularly soluble dietary
ber-like physiological eects. Medical rationales for their ber, has a tendency to increase the viscosity of liquid
use include normalizing bowel function, maintaining large medical foods. Also, insoluble bers settle to the bottom of
bowel integrity, restoring colonization resistance, altering the container, increasing the risk of tube clogging. Hence it
the route of nitrogen excretion, and improving mineral was imperative that sources of ber or ber-like material be
absorption. Overall, compatibility with liquid products identied that provided physiological benets but did not
and numerous physiological benets to the patient justify compromise the physical stability and feeding character-
the use of scFOS in medical foods. istics of the medical food. The fact that these people are
tube-fed should not work to their nutritional detriment.
Nondigestible oligosaccharides are soluble and ferment-
I. INTRODUCTION able and represent an ideal source of dietary ber for use in
medical foods.
Numerous health benets are associated with the con- An oligosaccharide is a carbohydrate consisting of a
sumption of dietary ber. Dietary ber may provide bulk small number, from 2 to 10, of monosaccharides [3].
to the stool, decrease intestinal transit time (e.g., relieve Oligosaccharides can be divided into two broad categories:
constipation), attenuate glycemic response, improve cho- digestible and nondigestible. Many types of nondigestible
lesterol and lipid metabolism, and reduce the risk of colon oligosaccharides are produced commercially from various
cancer. The Life Sciences Research Oce, Federation of sources of food materials (Table 1). Most of these oligo-
American Societies for Experimental Biology [1] recom- saccharides are reducing sugars, thus making them sus-
mends that we consume between 20 and 35 g of total ceptible to the formation of Maillard products during
dietary ber daily of which 7075% should be insoluble liquid product manufacturing. The Maillard reaction
and 2530% soluble. Most individuals can meet this includes all reactions involved when an aldehyde (or
recommendation by incorporating whole grains, fruits, ketone) and an amino group are heated together [7]. This
and vegetables into their diet. However, certain individuals nonenzymatic browning reaction forms linkages that are
must use a medical food to meet their dietary needs. A not hydrolyzed during digestion, resulting in the loss of
853
854 Wolf et al.
Table 1 Nondigestible Oligosaccharides onions, wheat, barley, bananas, tomatoes, garlic, and
artichokes [811]; (see Tables 2 and 3). The isolation and
Oligosaccharide Source/origin Reducing sugar development of scFOS was rst reported in the Japanese
Short-chain Sucrose No literature in 1983 [12 cited from 13]. Although scFOS can
fructooligosaccharides be extracted from a variety of plants, they can also be
Hydrolyzed inulina Inulin Yes produced by adding Aspergillus niger fructosyltransferase
Xylooligosaccharides Xylan Yes (h-fructosyltransferase) to sucrose [14]. Short-chain fruc-
Soybean oligosaccharides Soybeans No tooligosaccharides (e.g., neosugar, Nutraorak,
Galactooligosaccharidesb Lactose Yes MeioligoR, ActilightR) consists of the following oligosac-
Lactulose Lactose Yes charides: 1-kestose, nystose, and 1F-h-fructofuranosyl nys-
a
tose (1-kestotriose, 1,1-kestotetraose, and 1,1,1-
Also referred to as oligofructose [4,5].
b kestopentaose, respectively). These oligosaccharides con-
Also referred to as transgalactosylated oligosaccharides [6].
sist of two to four fructose molecules linked by (2!1)-h
glycosidic bonds and carry a single D-glucosyl molecule at
the nonreducing end of the chain linked (1!2)-a as in
amino acid availability. Any oligosaccharide that contains sucrose (Fig. 1). Short-chain fructooligosaccharides are
reducing sugars would be susceptible to the formation of nonreducing sugars and will not undergo the Maillard
Maillard products with free a-NH2 groups and especially reaction. The Ross Products Division of Abbott Labora-
the q-NH2 group of lysine. tories incorporates scFOS into several medical foods. The
Short-chain fructooligosaccharides (scFOS) occur application of scFOS to medical foods as a fermentable
naturally and have been isolated from foodstus such as dietary ber is discussed below.
Table 2 Fructooligosaccharide Composition of Fruits

mg/g of DM mg/g as is
Ingredient GFa2 GFb3 GFc4 Totald GF2 GF3 GF4 Total

Apple, Red Delicious 0.6 0.0 0.0 0.6 0.1 0.0 0.0 1.1
Apple, Golden Delicious 0.2 0.0 0.0 0.2 0.0 0.0 0.0 0.0
Apple, Granny Smith 0.5 0.0 0.0 0.5 0.1 0.0 0.0 0.0
Apple, Jonagold 0.4 0.0 0.0 0.4 0.1 0.0 0.0 0.1
Apple, Rome 0.3 0.0 0.0 0.3 0.0 0.0 0.0 0.0
Banana 5.9 0.1 0.0 6.0 1.4 0.0 0.0 1.4
Banana, green 3.1 0.0 0.0 3.1 0.7 0.0 0.0 0.7
Banana, red 1.8 0.0 0.0 1.8 0.5 0.0 0.0 0.5
Banana, ripe 8.6 0.0 2.3 10.9 1.6 0.0 0.4 2.0
Blackberry 0.0 0.0 1.2 1.2 0.0 0.0 0.2 0.2
Blueberry 0.2 0.1 0.0 0.3 0.0 0.0 0.0 0.0
Cantaloupe 0.3 0.0 0.4 0.7 0.0 0.0 0.0 0.0
Gooseberry 0.6 tre 0.2 0.8 0.1 tr 0.0 0.1
Grapes, black 0.5 0.0 0.6 1.1 0.1 0.0 0.1 0.2
Grapes, Thompson 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Muskmelon 0.3 0.0 0.9 1.2 0.0 0.0 0.1 0.1
Orange, navel 1.7 0.0 1.1 2.8 0.2 0.0 0.1 0.3
Peach 3.5 0.0 0.0 3.5 0.4 0.0 0.0 0.4
Pear, Bosc 0.8 0.0 0.0 0.8 0.1 0.0 0.0 0.1
Pear, dAnjou 0.3 0.0 1.1 1.4 0.0 0.0 0.2 0.2
Plantain 1.1 0.0 0.0 1.1 0.4 0.0 0.0 0.4
Plum, red 1.8 0.2 0.0 2.0 0.2 0.0 0.0 0.2
Raspberry, red 1.4 0.1 0.0 1.5 0.2 0.0 0.0 0.2
Rhubarb 0.3 0.0 0.0 0.3 0.0 0.0 0.0 0.0
Strawberry tr 0.0 0.0 tr tr 0.0 0.0 tr
Watermelon 2.8 0.0 0.1 3.0 0.2 0.0 0.0 0.2
a
1-Kestose.
b
Nystose.
c F
1 -h-Fructofuranosylnystose.
d
Total fructoooligosaccharide.
e
tr, <12.5 ppm practical detection limit.
Source: Ref. 8.
Medical Foods and Fructooligosaccharides 855
Table 3 Fructooligosaccharide Composition of Vegetables

mg/g of DM mg/g of as is
Ingredient GFa2 GFb3 GFc4 Totald

GF2 GF3 GF4 Total
Acorn squash 1.4 0.0 1.9 3.3 0.2 0.0 0.2 0.4
Artichoke, globe 13.4 5.5 2.8 21.8 1.5 0.6 0.3 2.4
Asparagus 0.3 0.0 0.0 0.3 0.0 0.0 0.0 0.0
Bean, green 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Bean, kidney 0.0 0.1 tre 0.1 0.0 0.1 tr 0.1
Beet, red 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0
Carrot, Bunny Luv 2.2 0.0 0.0 2.2 0.3 0.0 0.0 0.3
Carrot, Dole 1.4 0.0 0.0 1.4 0.2 0.0 0.0 0.2
Celery 0.2 0.0 0.4 0.6 0.0 0.0 0.0 0.0
Chicory root, raw 9.1 6.1 5.9 21.0 1.7 1.1 1.1 3.9
Chicory root, roasted 1.2 2.4 0.8 4.4 1.1 2.2 0.8 4.2
Chinese chive 0.4 0.3 0.4 1.1 0.0 0.0 0.0 0.0
Daikon 0.5 0.0 0.0 0.5 0.0 0.0 0.0 0.0
Eggplant 0.2 0.0 0.4 0.6 0.0 0.0 0.0 0.0
Endive 0.1 0.0 0.2 0.3 0.0 0.0 0.0 0.0
Garlic 8.7 1.2 0.4 10.3 3.3 0.4 0.2 3.9
Garlic powder 0.8 0.6 0.3 1.7 0.7 0.6 0.3 1.6
Ginger root 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Jerusalem artichoke 93.9 94.3 98.1 286.2 19.2 19.2 20.0 58.4
Kiwi 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0
Leek 3.4 0.6 0.7 4.8 0.7 0.1 0.1 0.9
Lettuce 4.9 1.9 1.1 7.9 0.3 0.1 0.1 0.5
Onion, red 11.7 2.1 0.9 14.7 1.1 0.2 0.1 1.4
Onion, Welch 5.8 3.9 3.6 13.4 0.5 0.3 0.3 1.1
Onion, white 17.1 8.8 6.1 32.0 1.7 0.9 0.6 3.1
Onion, yellow 15.5 6.7 4.2 26.4 1.5 0.6 0.4 2.6
Onion, powder 18.5 16.5 12.7 47.7 17.5 15.5 12.0 45.0
Peas 0.2 0.2 8.0 8.4 0.1 0.0 1.0 1.1
Peas, snow 1.0 0.0 5.4 6.5 0.1 0.0 0.5 0.6
Potato, Idaho 0.2 0.0 0.0 0.2 0.0 0.0 0.0 0.0
Potato, sweet 0.8 0.0 0.0 0.8 0.2 0.0 0.0 0.2
Radish, red 0.0 0.0 3.0 3.0 0.0 0.0 0.1 0.1
Shallot 28.2 14.2 10.6 52.9 4.5 2.3 1.7 8.5
Taro root 0.1 0.0 0.1 0.1 0.0 0.0 0.0 0.0
Tomato 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tomato, cherry 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Tomato, Roma 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
Yam 0.9 0.0 0.0 0.9 0.2 0.0 0.0 0.2
Zucchini 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
a
1-Kestose.
b
Nystose.
c F
1 -h-Fructofuranosylnystose.
d
Total fructoooligosaccharide.
e
tr, <12.5 ppm practical detection limit.
Source: Ref. 8.
II. MICROFLORA DISTRIBUTION AND ora. A number of studies have shown that the digestive
FERMENTATION OF SHORT-CHAIN enzymes of vertebrates do not hydrolyze scFOS. Hydro-
FRUCTOOLIGOSACCHARIDES lysis of scFOS could not be demonstrated during in vitro
incubations with either human jejunal homogenates [15]
The physiological eects of scFOS are directly related to or human salivary enzymes [12]. Furthermore, scFOS
the fact that they are not digested in the upper gastroin- were shown to be indigestible in rats [16] and humans
testinal tract but remain intact as they enter the large [12,17]. While mammalian enzymes are unable to degrade
bowel where they are fermented by the indigenous micro- scFOS, there exists within the gastrointestinal tract a large
856 Wolf et al.
Figure 1 Molecular structures of scFOS: (a) 1-kestose, (b) nystose, and (c) 1F-h-fructofuranosylnystose.
and diverse microora population capable of utilizing during fermentation [21], and they are produced in an
scFOS as an energy source. approximate ratio of 60:20:20 [22]. Theoretically, the com-
The population distribution of bacteria diers in the plete fermentation of 1 g of carbohydrate should lead to the
various parts of the gastrointestinal tract. In general, the formation of 10 mmol of SCFA [23]. Daily production of
mouth, stomach, and most of the small intestine are SCFA in the human colon has been estimated to be greater
dominated by aerobes or facultative anaerobes because than 300 mmol/day, yet fecal excretion is only about 10
these locations are not anaerobic, and population densities mmol/day [24]. Most of the SCFA produced during fer-
are relatively low (103108 bacteria/g). Population density mentation are absorbed from the large bowel. Using a
markedly rises at the ileum reaching close to 1011 bacteria/g dialysis bag technique, McNeil et al. [25] found that SCFA
from the cecum to the rectum. In the colon, strict anaerobes are rapidly absorbed from the human rectum.
outnumber aerobic organisms by a factor of 1000:1, and Short-chain fatty acids serve as a source of energy for
the predominant genera include bacteroides, eubacteria, the host. Acetate, the most abundant SCFA, is primarily
peptococci, and bidobacteria [18]. used as a fuel for host tissues. It is the only SCFA found at
Fermentation, the process by which anaerobic orga- appreciable levels in the peripheral blood where it may be
nisms break down dietary and other substrates to obtain oxidized in muscle and adipose tissue. Propionate may be
energy for growth and the maintenance of cellular func- oxidized by the colonocytes as a source of energy but is
tion, is an important component of large bowel activity believed to be primarily used by the liver as a substrate for
[19]. More than 70% of the energy from carbohydrate gluconeogenesis [26]. Butyrate is preferentially oxidized by
fermentation is conserved as short-chain fatty acids colonocytes as a source of energy. Roediger [27] found that
(SCFA) and other fermentation products (lactate, meth- more than 70% of the oxygen consumed by colonocytes
ane, carbon dioxide, and hydrogen); some of these fermen- from the ascending and descending colon could be attri-
tation end-products can be absorbed and utilized by the buted to butyrate oxidation in isolated human colonocytes.
host [20]. The remaining 30% of the energy is used by
bacteria to support growth (synthesis of monomers and
polymerization) and non-growth-related (maintenance of III. PHYSIOLOGICAL EFFECTS
ion gradients, nutrient transport, motility) functions.
The three predominant SCFA, acetate, propionate, As mentioned previously, the structural characteristics
and butyrate, account for 83% of the SCFA produced (i.e., nondigestible in the upper gastrointestinal tract,
nonreducing sugar, and highly soluble) of scFOS make for duction, and (3) increased intraluminal osmolarity and/or
an ideal fermentable ber source in medical foods. Poten- decreased stool pH.
tial physiological benets of scFOS for patients include Short-chain FOS, through fermentation and the pro-
positive eects on bowel function, large bowel integrity, duction of SCFA, may play a positive role in the alleviation
colonization resistance, colon cancer prevention, nitrogen of diarrhea. Wolf et al. [42] compared the fermentability of
excretion, and mineral absorption. several nondigestible oligosaccharides. Short-chain FOS,
xylooligosaccharides (XOS), and hydrolyzed inulin were
A. Bowel Function fermented with human fecal inoculum in vitro. Fermenta-
tion rates of all oligosaccharides were rapid, being essen-
Of all the physiological benets of dietary ber, its eect on tially complete by 6 hr for the scFOS and hydrolyzed
bowel habits is the most noted. The eect of dietary ber inulin, and by 12 hr for the XOS (Fig. 2). A similar study
can best be described as a normalization of bowel evaluating the fermentability of soy ber, gum arabic,
function and has been used for the treatment and preven- scFOS, and lactulose was conducted by Garleb et al. [43].
tion of both constipation [28] and diarrhea [29]. Human Lactulose and scFOS were fermented much more rapidly
studies conrm that the consumption of scFOS can aect (P<0.01) than either gum arabic or soy ber. Furthermore,
stool consistency and subsequently constipation. Hata and the fermentation of scFOS was essentially complete by 6
Nakajima [30] administered varying single doses of scFOS and 12 hr for lactulose.
to 80 healthy adults and determined that the dosage Short-chain fatty acids can improve bowel function by
resulting in 50% of the subjects experiencing diarrhea (a facilitating water absorption. The absorption of 100 mmol
watery stool during the rst defecation following ingestion SCFA is associated with the absorption of 360 mL water
of scFOS) was approximately 0.8 g of scFOS/kg body [44]. Ramakrishna and Mathan [45] found that fecal
weight. Subsequent studies conducted by Briet et al. [31] output of SCFA in patients with acute diarrhea was low
in which healthy human adults received acute or chronic on the rst day of illness, but increased over the next 5 days
dosing with scFOS generated similar results. Tokunaga et as the patients condition improved. Furthermore, they
al. [32] showed that healthy volunteers had softer stools demonstrated that luminal SCFA could restore net water
and increased frequency of bowel movements when they and sodium reabsorption in the rectum of patients with
consumed scFOS for 2 weeks (up to 5 g/day) than during acute diarrhea. In vivo perfusion studies in healthy subjects
baseline or after a 1-week washout period. The adminis- have shown secretion of salt and water in the ascending
tration of scFOS has been reported to relieve moderate colon in response to enteral feeding [46,47]. Bowling et al.
cases of constipation [12,33]; however, severe constipation [48] investigated the eect of SCFA on colonic uid
was not alleviated [33]. We (unpublished observations) secretion induced by enteral feeding. These researchers
designed a study to determine the dose of scFOS at which found that SCFA infusion into the cecum of healthy
50% of children with a history of simple constipation subjects reversed the uid secretion seen in the ascending
produced a stool with pudding-like or watery consistency. colon during enteral feeding and theorized that these
Fifty-ve children (ages 25 years) were enrolled into a ndings could have implications for the management of
double-blind, two group, placebo-controlled, parallel, diarrhea related to enteral feedings.
multicenter, acute-dosage titration study. They were ran-
domly assigned in a 3:2 fashion to receive either scFOS or
sucrose after blocking on sex. Following a 3-day baseline
period, each child received 0.2 g/kg body weight of scFOS
or sucrose once daily at breakfast. The dose was increased
in 0.2 g/kg/day increments every other day just until each
child produced a pudding-like or watery stool. The maxi-
mum possible dosage was 0.8 g/kg/day. Similar to data
reported in adults, the eective dose of scFOS was 0.79 g/
kg/day (95% CI of 0.581.12 g/kg/day). The minimum
dose at which stool consistency of the scFOS group soft-
ened to a greater extent ( P = 0.004) from baseline com-
pared with the sucrose group occurred at the 0.6 g/kg/day
dose. However, administration of scFOS did not change
(P>0.05) the frequency of bowel movements from baseline
compared with sucrose regardless of dose.
Short-chain FOS may promote laxation via a mecha-
nism similar to that of other nondigestible carbohydrates
[34,35] such as lactulose. Based on available evidence from
rats [12,36,37] and from humans [31,35,3840], the laxation Figure 2 Total organic acid (acetate, propionate, butyrate,
eect of scFOS is dose-dependent and is the result of a lactate) production from in vitro fermentation of non-
combination of factors similar to those attributed to digestible oligosaccharides with human feces (n = 3). (From
lactulose [41]: (1) increased bacterial growth, (2) gas pro- Ref. 42.)
858 Wolf et al.
B. Large Bowel Integrity nursing the sow, and intermediate for pigs consuming milk
replacer alone. From these animal experiments, it can be
Short-chain FOS, through the production of SCFA, may concluded that fermentable substrates may play a key role
be useful in maintaining large bowel integrity or serving as in the maintenance of large bowel integrity and function.
adjunctive therapy for the treatment of inammatory Increasing interest has been generated in the use of
bowel disease. The feeding of dietary ber has been asso- enemas/irrigation solutions containing buered, physio-
ciated with the stimulation of colonic cell proliferation. logic levels of SCFA for the treatment of diversion colitis
This stimulatory eect has been attributed to the SCFA and ulcerative colitis. Diversion colitis is an inammatory
produced during ber fermentation. Short-chain fatty process arising in segments of the colorectum at various
acids may stimulate proliferation directly or indirectly by intervals after surgical diversion of the fecal stream. The
mediating blood ow or pH. This phenomenon has been endoscopic appearance is similar to those of active Crohns
observed in both the large and small bowel. For example, disease and ulcerative colitis [55]. The cause of this condi-
Aghdassi et al. [49] found that reducing colonic fermentation is not known, but one mechanism has been postulated;
tion resulted in reduced intestinal adaptation and nutri- a nutritional deciency of the colonic epithelium, speci-
tional recovery in rats with massive small bowel resection. cally because of the absence of SCFA normally present in
Moreover, others have observed stimulation of small bowel colonic contents [56,57]. Harig et al. [58] tested this hy-
mucosal proliferation with SCFA supplementation [50,51]. pothesis by assessing whether SCFA irrigation could ame-
Younes et al. [52] fed rats a ber-free diet or diets contain- liorate inammation in four patients with diversion colitis.
ing 7.5% ber as oat ber, gum arabic, scFOS, or XOS. After 23 weeks of therapy, macroscopic and histological
Cecal wall weight was greater ( P < 0.05) for the ferment- resolution of inammation was evident.
able substrates (i.e., gum arabic, scFOS, and XOS) com- An impaired utilization of SCFA also has been impli-
pared with the poorly fermented oat ber and the ber-free cated in ulcerative colitis, which suggests that diminished
control. Similarly, Campbell et al. [36] fed rats a ber-free intracellular energy production may be important in the
diet or 5% cellulose diets with or without 6% scFOS, inammatory process [59]. It has been demonstrated that
hydrolyzed inulin, or XOS. Nondigestible oligosaccharide fecal water from patients with ulcerative colitis contains
supplementation increased ( P < 0.05) cecum wall weight reduced concentrations of SCFA as well as markedly
compared with the ber-free and cellulose-containing con- increased lactate concentrations and decreased pH
trol diets. Howard et al. [53] fed supplemental (3 g/L) [60,61]. In a study by Breuer et al. [62], the eect of large
scFOS in a liquid diet to neonatal pigs and observed bowel irrigation with SCFA in patients with ulcerative
increased ( P < 0.01) cell proliferation in the proximal colitis was studied. It was found that 9 out of 10 patients
and distal colonic epithelial mucosa. Bourquin et al. [54] completing the study were judged to be at least much
evaluated cecal and colonic development of neonatal pigs improved and showed a signicant change in mean disease
(n = 10) fed sows milk, milk replacer, or milk replacer + 3 activity index score and mucosal histology score. Senagore
g of scFOS/L for 14 days. Cecal tissue weight was greatest et al. [63] conrmed the results of Breuer et al. [62],
( P < 0.05) for pigs consuming milk replacer + scFOS demonstrating an 80% response rate in patients with
compared with pigs consuming either milk replacer alone idiopathic proctosigmoiditis. This study indicates that
or sows milk (Fig. 3). Colonic tissue weight was greatest for administering a solution of SCFA similar to Harig et al.
pigs consuming milk replacer + scFOS, least for pigs [58] for 6 weeks was equally ecacious to corticosteroid or
5-aminosalicylate for the treatment of proctosigmoiditis at
a signicant cost savings. Scheppach et al. [64] investigated
the use of butyrate enemas alone rather than the SCFA
mixture to treat 10 patients with distal ulcerative colitis in a
placebo-controlled, single-blind, randomized trial. The
authors concluded that butyrate, as an end-product of
bacterial fermentation in the large bowel, markedly im-
proved disease activity index and histological parameters.
These data support the hypothesis that the eect of a SCFA
mixture on the inamed mucosa in ulcerative colitis is
largely attributable to butyrate.
It is unlikely that SCFA added directly to an enteral
product would reach the large bowel. Medical foods can
take advantage of the positive eect of SCFA by providing
fermentable ber. For example, Rolandelli et al. [65]
demonstrated a benet of a fermentable ber (pectin) in
the treatment of experimental colitis. Also, Grisham et al.
Figure 3 Cecum weight of neonatal pigs fed sows milk, milk [66] evaluated the eect of enteral diets containing sh oil
replacer, or milk replacer + 3 g of scFOS/L. All values are or the fermentable substrates, scFOS or XOS, on chronic
mean F pooled SEM, n = 10. Bars with unlike superscript colitis induced with peptidoglycan polysaccharide in rats.
letters dier ( P < 0.05). (From Ref. 54.) In this study, rats fed scFOS or XOS had improved
histological and biochemical markers of inammation. and Gibson [69] who showed that inulin or hydrolyzed
Seidner et al. [67] assessed the ecacy of a novel medical inulin selectively enhanced the growth of bidobacteria in
food supplemented with sh oil, scFOS, gum arabic, and fecal slurries with little change in the numbers of either
antioxidants on reducing corticosteroid use in adults with clostridia or coliforms.
mild to moderate ulcerative colitis. Patients given this Enrichment for bidobacteria has also been demon-
formula had a signicantly greater rate of reduction in strated in vivo in both laboratory animals and in humans.
the daily dose of prednisone over 6 mo as compared with For example, Campbell et al. [36] fed rats a control diet or
controls receiving a sucrose-based placebo. control diets that were supplemented with (1) 0% micro-
crystalline cellulose (MCC), (2) 5% MCC, (3) 5% MCC +
C. Prebiotics and Colonization Resistance 6% scFOS, (4) 5% MCC + 6% hydrolyzed inulin, or (5)
5% MCC + 6% XOS for 14 days. They found that those
Short-chain fructooligosaccharides are a prebiotic: A rats that were fed diets containing oligosaccharides
nondigestible food ingredient that improves host health (scFOS, hydrolyzed inulin, or XOS) had greater concen-
by selectively stimulating the growth and/or activity of trations of total anaerobes and bidobacteria and lower
one or a limited number of bacteria in the colon [68]. In concentrations of total aerobes in their ceca compared with
vitro experiments indicate that scFOS are utilized by those fed the control diet. When scFOS were fed to elderly
several benecial species of gastrointestinal bacteria (un- Japanese patients, bidobacteria numbers in feces in-
published data) (Table 4). creased approximately 10-fold [70]. The magnitude of the
Using pure cultures, Hidaka et al. [12] showed that increase depended on the number of bidobacteria that
bacteroides and bidobacteria grew to high optical densi- were initially present; individuals with high initial counts of
ties and produced large amounts of organic acids when bidobacteria showed little, if any, increase in bidobac-
scFOS were provided as an energy source. However, teria in response to treatment with scFOS. Individuals who
potential pathogens such as Escherichia coli and Clostrid- initially had low numbers (less than 108 cfu/g stool) showed
ium perfringens did not. Similar results were noted by Wang two to four log increases in counts [14]. In healthy adult
volunteers, Williams et al. [71] reported an increase in fecal
bidobacteria numbers with the consumption of 4 g
scFOS/day. A study conducted by Garleb et al. [72] further
Table 4 Growth of Common Intestinal Anaerobes Using documented the bidogenic capacity of scFOS. In this
Various Carbohydrates study, healthy subjects were fed a low residue polymeric
DOptical density (600 nm) formula (LRPF), LRPF + 5 g scFOS/L (f15 g scFOS/
day), or LRPF + 10 g scFOS/L (f31 g scFOS/day). The
Organism Glucose Lactose scFOS scFOS had a signicant impact on fecal bidobacteria
levels. On day 14, a greater number ( P < 0.001) of
Bidobacterium
bidobacteria were detected in the feces of subjects con-
B. adolescentis 15703 1.055 0.990 0.839
suming formulas containing scFOS compared with those
B. angulatum 27535 0.186 0.938 0.749
subjects consuming the LRPF without scFOS.
B. bidum 29521 0.834 0.807 0.065
B. breve 15700 0.890 1.035 1.017
B. catenulatum 27539 0.529 0.913 0.935 D. Colonization Resistance
B. infantis 15697 1.022 1.007 0.922
B. longum 15707 0.910 0.945 1.015 Indigenous bacteria confer colonization resistance to the
B pseudocatenulatum 0.093 0.943 0.883 host. In the gastrointestinal tract, this phenomenon refers
27919 to the ability of the indigenous microora to prevent the
colonization, overgrowth, and/or translocation of poten-
Bacteroides tially pathogenic microorganisms. This concept was rst
B. distasonis 8503 1.049 1.024 0.979 described by Van der Waaij et al. [73], who showed that
B. ovatus 8483 0.864 0.956 0.849 indigenous anaerobic organisms prevented colonization
B. thetaiotaomicron 29148 0.825 0.795 0.833 by opportunistic pathogens. Colonization resistance has
B. vulgatus 8482 0.872 0.877 0.870 been attributed to several mechanisms: production of
inhibitory compounds such as SCFA, hydrogen sulde,
Eubacterium and bacteriocins [7476]; reduction of pH and low oxida-
E. aerofaciens 25986 1.020 1.040 0.676 tionreduction potential [77]; and competition for sub-
E. biforme 27806 1.056 1.133 0.127 strates [75,78]. Many researchers [73,79,80] believe that the
E. eligens 27750 1.035 0.012 0.052 anaerobic ora play a primary role in colonization resis-
E. rectale 33656 0.894 0.957 0.089 tance and that the resistance involves the mucosal immune
Ruminococcus
system [81].
R. bromii 27255a 0.006 0.004 0.157
An excellent example of colonization resistance is the
R. productus 27340 0.861 0.904 0.630
relationship between indigenous bacterial microora and
a
A R. bromii control culture that was provided with maltose in- Clostridium dicile. C. dicile, a spore-forming obligate
creased in optical density by 0.641 units. anaerobe, is the leading known cause of nosocomial diar-
860 Wolf et al.
rheal infections [82,83]. Although this organism is a com- setting is questionable. Perhaps, a more acceptable ap-
ponent of the normal intestinal ora of about 35% of proach may be to use fermentable bers such as scFOS to
healthy adults, it can be detected in the stools of up to 15 restore the gastrointestinal microora and environment.
20% of hospitalized adults [84]. Infections due to C. dicile In an in vitro model, May et al. [103] also showed that
are responsible for all cases of pseudomembranous colitis fermentable ber (resulting in increased SCFA concentra-
(PMC) and for up to 20% of cases of antibiotic-associated tions and decreased pH) eectively inhibited the growth of
diarrhea without colitis [85,86]. C. dicile and toxin A production. The in vitro system
When established in the colon, pathogenic strains of C. further demonstrated that pH levels less than 6.0 and/or a
dicile produce exotoxins (toxin A and toxin B) that are SCFA concentration greater than 100 mM could markedly
the cause of diarrhea and colitis [87,88]. Toxin A causes suppress the growth of C. dicile and the production of
uid secretion, mucosal damage, and intestinal inamma- toxin A. In addition, May et al. [104] found that dietary
tion when injected into rodent intestine [89]. Toxin B is a supplementation with oligosaccharides (scFOS or XOS)
more potent cytotoxin in tissue culture than toxin A, but it suppressed the growth of C. dicile, protected cecal epi-
is not enterotoxic in animals [85]. thelial tissue in mice, and reduced the incidence of diarrhea
Use of almost any antibiotic can cause C. dicile in C. dicile-challenged mice. Gaskins et al. [105] demon-
infection, but broad-spectrum antibiotics with activity strated that the administration of scFOS to cefoxitin-
against enteric bacteria are the most frequent agents. treated mice inoculated with C. dicile produced toxin A
Clindamycin is notorious for its propensity to induce the titer in feces signicantly lower ( P < 0.05) than animals
disease [90]. However, in current practice, broad-spectrum not receiving scFOS and similar to mice not compromised
penicillins and cephalosporins are the most common cul- with antibiotics. Wolf et al. [106] demonstrated that dietary
prits, reecting their widespread use [91]. Other factors supplementation with scFOS increased survival time in a
include chemotherapy [92], dietary changes, anesthesia, hamster model of C. dicile colitis (Fig. 4). It is interesting
surgery of the intestinal tract, uremia, and various nonan- to note that the benet of the supplementation of scFOS in
tibiotic medications. Pseudomembranous colitis may occur this study was above any eects of ber contained in the
during the period immediately and almost always within 6 basal diet. In consideration of the research conducted in
weeks after the use of antibiotics has been discontinued. this area, the addition of scFOS to a medical food may be
All of these causative factors suggest that C. dicile benecial for patients at risk of C. dicile infection in long-
infections are associated with the disruption of the normal term care institutions and hospital wards.
bowel microbiota. Wilson et al. [93] noted that C. dicile
could not colonize hamsters in the presence of an undis- E. Colon Cancer
turbed colonic microora, yet C. dicile rapidly attained a
large population size when introduced into antibiotic- Numerous studies using laboratory animals have consist-
treated animals. Likewise, C. dicile was able to achieve ently demonstrated that the feeding of fructans or similar
a population of over 108 cfu per cecum when inoculated carbohydrates inhibits the development of aberrant crypt
into gnotobiotic mice, but colonization was suppressed to foci (ACF) in carcinogen-treated rodents [107]. During
undetectable levels by intestinal ora of conventionally these studies, rats or mice were fed 520% of their diet as
colonized hamsters and mice [94]. hydrolyzed inulin [108,109], inulin [110113], both hydro-
The standard treatment for C. dicile-associated dis- lyzed inulin and inulin [114], or galactooligosaccharides
ease involves administration of the antibiotics vancomycin
or metronidazole. Standard antibiotic therapy is eective
in 80% of patients with C. dicile-associated disease, but
the remaining 20% experience further episodes of diarrhea
or colitis during the permissive period after the antibiotic
has been discontinued [9597]. Once patients have had one
recurrence, they may experience repeated episodes of the
disease over several years [98,99].
Given the causative role of antibiotics in the onset of
C. dicile infection, alternative therapies might oer more
eective strategies for the prevention or treatment of C.
dicile infection. Promising results have been obtained by
restoring normal gut ora. Using a hamster model, Wilson
et al. [100] found that administration (oral plus rectal) of
normal cecal homogenates decreased the numbers of viable
C. dicile and prevented cecitis in antibiotic-challenged
animals. Schwan et al. [101,102] eectively treated recur-
rent C. dicile infection in patients by giving enemas with
fecal contents from healthy adult humans. While direct Figure 4 Survival curves for ciprooxacin treated hamsters
inoculation with gastrointestinal microora appears eec- (n = 8) receiving either 0 or 30 g of scFOS/L of drinking
tive, its aesthetic and hygienic acceptability in a clinical water. (From Ref. 106.)
[115]. Following a brief acclimatization period, they were such as scFOS would reduce the risk of colon cancer in
then treated subcutaneously with azoxymethane or orally humans. First, the animal studies that demonstrated a
with 1,2-dimethylhydrazine, fed for up to 31 additional reduction in ACF were exclusively conducted in one of
weeks, sacriced, and examined for the presence of ACF. two rodent models whereby carcinogenesis was induced via
The mechanism by which indigestible oligosacchar- the subcutaneous administration of either AOM or oral
ides such as scFOS inhibits formation of ACF is not gavage with 1,2-dimethylhydrazine. Furthermore, the
entirely clear, but a variety of explanations have been resulting ACF are only considered to be putative preneo-
proposed (reviewed in Refs. [107,109,116119]). Butyrate, plastic lesions that may or may not develop into colorectal
which is produced as a result of carbohydrate fermentation tumors. Finally, the only feeding study in humans to study
in the colon, serves as a preferred fuel for colonic epithelial the eect of scFOS on selected indices of colon carcino-
cells. According to one theory, this SCFA might also genesis [130] failed to show an eect on fecal pH, enzyme
protect against colon cancer by inhibiting colon cell (nitroreductase, azoreductase, and h-glucuronidase) activ-
proliferation and inducing dierentiation [116] and/or by ity, bile acid, or neutral sterol concentrations in healthy
promoting apoptosis of colon tumor cells [120,121]. Ad- adults despite a signicant increase in fecal bidobacteria
ditionally, butyrate may promote cell proliferation in counts. Admittedly, the dose of scFOS fed was only 12.5 g/
nontransformed colon cells [122] and alter the metabolism day, an amount that was far below those used in laboratory
of human colon tumor cell lines by inducing glutathione S- animal studies.
transferase, an enzyme that helps conjugate reactive com-
pounds and helps to protect cells from these chemicals F. Other Cancers
[123]. Secondly, the fermentation of carbohydrates in the
colon to SCFA causes a reduction in pH, and it has been Dietary fructans might also prove useful for the prevention
proposed that pH could play a signicant role in colonic and treatment of noncolonic cancers (reviewed in Ref.
cell growth, absorption, and secretion. Moreover, pH is [131]). In particular, some data suggest that fructans could
thought to aect the rate of bile degradation and the potentially be used to reduce the incidence and growth of
absorption of ammonia by epithelial cells. Thirdly, the cancer in other locations in the body. For example, Taper
fermentation of carbohydrates such as scFOS in the colon and Roberfroid [109] conducted a study in which they fed
likely aects the quantity and quality of nitrogenous female SpragueDawley rats a basal (control) diet or a
compounds in the colonic lumen. When carbohydrates basal diet that was supplemented with 15% hydrolyzed
are plentiful in the colon, nitrogenous compounds pro- inulin. They subsequently induced mammary tumors by
duced as a result of protein and amino acid fermentation injecting with methylnitrosourea and discovered that fewer
(e.g., ammonia) are used for microbial growth, and less are of the hydrolyzed inulin-supplemented rats developed
absorbed. Because ammonia is more toxic to healthy cells mammary tumors, and those rats who developed mamma-
than to transformed cells and promotes faster cell turn- ry tumors had fewer tumors than rats who had been given
over, higher concentrations of ammonia in the colonic the basal diet alone (12 vs. 19, respectively). In the same
lumen may select for neoplastic growth. Finally, fructans paper, Taper and Roberfroid [109] described another
such as scFOS may reduce the formation of ACF by an experiment in which they provided rats with 15% hydro-
indirect mechanism such as promoting the growth of lyzed inulin, inulin, or pectin, and measured the growth of
Bidobacterium species in the colon as past studies indicate two types of intramuscularly transplanted mouse tumors
that certain species protect against colon tumorigenesis (TLT and EMT6). Results from that experiment showed
[124126] and that a symbiotic combination of pro- that hydrolyzed inulin, inulin, and pectin signicantly
biotic and prebiotic had a synergistic inhibitory eect on inhibited the growth of both tumor cell lines compared
the development of ACF [114,126]. with a control diet. Soon afterward, Taper and Roberfroid
Indigestible oligosaccharides such as scFOS can re- [132] published yet another paper that examined the inhib-
duce fecal concentrations of nitrogenous compounds that itory eect of dietary inulin or hydrolyzed inulin on the
have been linked to cancer. Swanson et al. evaluated the development of cancer metastases. However, in this study,
eects of scFOS and (or) Lactobacillus acidophilus on young male C3H mice were fed one of three diets: a control
fermentation end-products in healthy dogs [127] and diet, a diet containing 15% hydrolyzed inulin, or a diet
healthy humans [128]. Short-chain FOS supplementation containing 15% inulin. Viable neoplastic cells of a trans-
appeared to enhance indices of gut health by positively plantable mouse liver tumor were injected into the right
altering fecal protein catabolite in healthy dogs and healthy thighs of the animals, and the development of lung metas-
humans. By decreasing fecal protein metabolites, scFOS tases was monitored 47 days after transplantation. Results
may improve fecal odor and prevent gut exposure to from this study indicated that supplementation with 15%
potentially carcinogenic compounds. hydrolyzed inulin or inulin reduced the percentage of
Despite the abundance of animal feeding studies animals bearing lung metastases from 59% in the control
showing that prebiotics and probiotics inhibit the develop- group to 36% or 35% in the inulin and hydrolyzed inulin
ment of ACF (reviewed in Refs. [117119,129]) and many groups, respectively.
human clinical studies showing that the feeding of pre- The exact means by which dietary fructans or other
biotics increases fecal bidobacteria concentrations, it may bers inhibit development of noncolonic tumors is also
still be premature to conclude that the feeding of prebiotics unclear. However, some of the same hypothetical mecha-
862 Wolf et al.
nisms that have been used to explain their inhibitory eect mentable ber intake can cause gastrointestinal intoler-
on ACF may apply. Alterations in the colonic microora ance. For this reason, the study by Grin et al. [150] is of
(i.e., an increase in bidobacteria numbers) might act to great interest to the scientic community. They evaluated
suppress tumors by producing inhibitory substances dur- the eects of a mixture of inulin and hydrolyzed inulin on
ing fermentation other than SCFA or through the action of calcium absorption in girls at or near menarche. Dietary
bidobacterial cell walls [133,134]. Secondly, because tu- supplementation with 8 g of inulin + hydrolyzed inulin/
mor cells rely heavily on glucose for energy, alterations in day increased calcium absorption by 18% compared with
glucose availability may also lead to changes in insulin and/ placebo (sucrose).
or insulin-like growth factor concentrations, which could Likewise, dietary supplementation with scFOS has
aect tumor cell growth [131]. Lastly, reduced de novo been shown to enhance apparent magnesium absorption
hepatic synthesis of triglycerides, phospholipids, and very in rats [143145] and true magnesium absorption and
low density lipoproteins might limit cancer cell growth by balance in humans [153]. Tahiri et al. [153] fed moderate
limiting the availability of fatty acids [132]. daily doses of scFOS (10 g/day) to healthy postmenopausal
women. They found that scFOS supplementation in-
creased ( P < 0.05) magnesium absorption by 12.3%,
G. Nitrogen Excretion and Calcium Absorption which was accompanied by an increase in the plasma
magnesium concentration. Recently, Coudray et al. [154]
The addition of ber to a diet can potentially alter total reviewed the eects of dietary bers on magnesium ab-
body nitrogen metabolism by enhancing bacterial metab- sorption in animals and humans. Several mechanisms by
olism and thereby increasing the incorporation of nitrogen which fermentable carbohydrates enhance magnesium ab-
into fecal bacteria. This in turn increases fecal nitrogen sorption were discussed and, similar to some proposed
excretion and subsequently reduces urinary nitrogen ex- mechanisms for the improvement in calcium absorption,
cretion [135,136]. For example, lactulose has been reported were related to SCFA production.
to depress ammonia absorption from the large bowel [137]
and to enhance fecal nitrogen excretion [138]. In addition,
inulin has been shown to enhance urea capture by the rat IV. CONCLUSIONS
cecum [139] and promote fecal nitrogen excretion, partic-
ularly when the level of protein in the diet is moderate [140]. Short-chain fructooligosaccharides represent an ideal
Younes et al. [52] demonstrated in rats that fermentable source of fermentable ber for use in medical foods.
bers such as gum arabic, XOS, and scFOS can increase Short-chain fructooligosaccharides are highly soluble but
fecal nitrogen excretion at the expense of urinary nitrogen are low viscosity bers that will not compromise the tube-
excretion. Such data indicate a potential benet for non- feeding characteristics of a medical food. In vitro, animal,
digestible oligosaccharide therapy in patients with renal and in several instances, human research has been con-
insuciency or chronic renal disease. ducted to identify and support the potential physiological
Considerable eorts are underway to encourage all benets of short-chain fructooligosaccharides.
people, women in particular, to increase their calcium
consumption for improved bone health. Ingredients that
REFERENCES
improve mineral absorption also could be benecial. In
rats, fermentable bers such as inulin [141], hydrolyzed 1. Physiological Eects and Health Consequences of Dietary
guar gum [142], and scFOS [143145] have been shown to Fiber; Bethesda, MD: Federation of American Societies for
enhance apparent calcium absorption. This enhanced Experimental Biology: Life Sciences Research Oce, 1987.
absorption occurs in the large bowel [143145] through a 2. Code of Federal Regulations, 21 CFR 101.9; Food and
Drug Administration: Washington, DC 1993.
variety of mechanisms involving SCFA [147], pH, calbin-
3. Dorlands Illustrated Medical Dictionary. 26th ed.; WB
din-D9K [146], and direct eects of the indigestible car- Saunders Company: Philadelphia, PA, 1981.
bohydrate [148]. Ohta et al. [149] demonstrated in 4. Roberfroid, M. Dietary ber, inulin, and oligofructose: a
ovariectomized mice, a model of osteopenia, that scFOS review comparing their physiological eects. Crit. Rev.
fed with isoavone conjugates increase femoral bone Food Sci. Nutr. 1993, 33, 103.
mineral density. The stimulatory eect of fructans on 5. Roberfroid, M.; Gibson, G.R.; Delzenne, N. The bio-
calcium absorption also has been conrmed in human chemistry of oligofructose, a nondigestible ber: an
approach to calculate its caloric value. Nutr. Rev. 1991,
clinical studies [150152]. Coudray et al. [151] found an 51, 137.
increase ( P < 0.05) in apparent absorption and balance of 6. Tanaka, R.; Takayama, H.; Morotomi, M.; Kuroshima,
calcium in healthy young men fed diets supplemented with T.; Ueyama, S.; Matsumoto, K.; Kuroda, A.; Matai, M.
40 g of inulin/day compared with the control diet. In Eects of administration of TOS and Bidobacterium breve
healthy, male adolescents, Van den Heuvel et al. [152] 4006 on the human fecal ora. Bidobact. Microora 1991,
found that dietary supplementation with 15 g of hydro- 2, 17.
7. Adrian, J. Nutritional and physiological consequences of
lyzed inulin/day increased ( P < 0.05) true fractional the Maillard reaction. World Rev. Nutr. Diet. 1974, 19, 71.
calcium absorption by 10.8 percentage units compared 8. Campbell, J.M.; Bauer, L.L.; Fahey, G.C. Jr; Hogarth,
with placebo (sucrose). Although both of these studies A.J.C.L.; Wolf, B.W.; Hunter, D.E. Selected fructooligo-
found an improvement in calcium absorption, high fer- saccharide (1-kestose, nystose, and 1F-h-fructofuranosyl-
nystose) composition of foods and feeds. J. Agric. Food 30. Hata, Y.; Kakajima, K. Studies of relationship between
Chem. 1997, 45, 3076. intake of fructooligosaccharides and abdominal symp-
9. Spiegel, J.E.; Rose, R.; Karabell, P.; Frankos, V.H.; toms: estimation of the maximum non-eective dose and
Schmitt, D.F. Safety and benets of fructooligosaccharides 50% laxative eect dose. Geriatr. Med. 1985, 23, 817.
as food ingredients. Food Technol. 1994, 48, 85. 31. Briet, F.; Achour, L.; Flourie, B.; Beaugerie, L.; Pellier, P.;
10. Tashiro, Y.; Eida, T.; Hidaka, H. Distribution and Franchisseur, C.; Bornet, F.; Rambaud, J. Symptomatic
quantication of fructooligosaccharides in food materials. response to varying levels of fructooligosaccharides con-
Sci. Rep. Meiji Seiki Kaisha 1992, 31, 35. sumed occasionally or regularly. Eur. J. Clin. Nutr. 1995,
11. Hogarth, A.J.C.L.; Hunter, D.E.; Jacobs, W.A.; Garleb, 49, 501.
K.A.; Wolf, B.W. Ion chromatographic determination of 32. Tokunaga, T.; Nakada, Y.; Tashiro, Y.; Hirayama, M.;
three fructooligosaccharide oligomers in prepared and Hidaka, H. Eects of fructooligosaccharides (FOS) intake
preserved foods. J. Agric. Food Chem. 2000, 48, 5326. on the intestinal microora and defecation in healthy
12. Hidaka, H.; Eida, T.; Takizawa, T.; Tokunaga, T.; Tashiro, volunteers. Bidus (in Japanese) 1993, 6, 143.
Y. Eects of fructooligosaccharides on intestinal ora and 33. Takahashi, Y. Eects of Neosugar in the chronic renal-
human health. Bidobact. Microora 1986, 5, 37. failure patient. Abstract from the report of the 3rd
13. Hidaka, H. Fructooligosaccharides, a newly developed Neosugar Conference, Tokyo, Japan, 1986.
food material for health. Kagaku To Seibutsu (in Japanese) 34. Hidaka, H.; Tashiro, Y.; Eida, T. Proliferation of
1983, 21, 291. bidobacteria by oligosaccharides and their useful eects
14. Hidaka, H.; Hirayama, M.; Sumi, N. A fructooligosac- on human health. Bidobact. Microora 1991, 10, 65.
charide-producing enzyme from Aspergillus niger ATCC 35. Clausen, M.R.; Jorgensen, J.; Mortensen, P.B. Compar-
20611. Agric. Biol. Chem. 1988, 52, 1181. ison of diarrhea induced by ingestion of fructooligosac-
15. Ziesenitz, S.C.; Siebert, G. In vitro assessment of nystose as charide Idolax and disaccharide lactulose: Role of
a sugar substitute. J. Nutr. 1987, 117, 846. osmolarity versus fermentation of malabsorbed carbohy-
16. Oku, T.; Tokunaga, T.; Hosoya, N. Nondigestibility of new drate. Dig. Dis. Sci. 1998, 43, 2696.
sweetener, Neosugar, in the rat. J. Nutr. 1984, 114, 1574. 36. Campbell, J.M.; Fahey, G.C. Jr; Wolf, B.W. Selected
17. Molis, C.; Flourie, B.; Ouarne, F.; Gailing, M.-F.; Lartigue, indigestible oligosaccharides aect large bowel mass, cecal
S.; Guibert, A.; Bornet, F.; Galmiche, J.P. Digestion, and fecal short-chain fatty acids, pH and microora in rats.
excretion, and energy value of fructooligosaccharides in J. Nutr. 1997, 127, 130.
healthy humans. Am. J. Clin. Nutr. 1996, 64, 324. 37. Tashiro, Y.; Satchithanadam, S.; Calvert, R.J.; In Dietary
18. Finegold, S.M.; Sutter, V.L.; Mathisen, G.E. Normal Fiber in Health and Disease. Kritchevsky, D., Boneld, C.,
indigenous intestinal ora. In Human Intestinal Flora in Eds.; Plenum Press: New York 1997; 221233.
Health and Disease; Hentges, D., Ed.; Academic Press: New 38. Hidaka, H.; Hirayama, M.; Tokunaga, T.; Eida, T. In New
York, 1983; 331. Developments in Dietary Fiber; Furda, I., Brine, C.I., Eds.;
19. Cummings, J.H.; Englyst, H.N. Fermentation in the large Plenum Press: New York, 1990; 105117.
intestine and the availability of substrates. Am. J. Clin. 39. Stone-Dorshow, T.; Levitt, M.D. Gaseous response to
Nutr. 1987, 45, 1243. ingestion of a poorly absorbed fructooligosaccharide
20. Smith, C.J.; Bryant, M.P. Introduction to the metabolic sweetener. Am. J. Clin. Nutr. 1987, 46, 61.
activities of intestinalbacteria.Am.J.Clin.Nutr.1979,32,149. 40. Alles, M.S.; Hautvast, J.G.; Nagengast, F.M.; Hartemink,
21. Rombeau, J.L. Short-chain fatty acidsproduction, ab- R.; Van Laere, K.M.; Jansen, J.B. Fate of fructooligosac-
sorption, metabolism, and intestinal eect. In Dietary Fiber, charides in the human intestine. Br. J. Nutr. 1996, 76, 211.
Chemistry, Physiology, and Health Eects; Kritchevsky, D., 41. Bass, P.; Singaram, C. Conn, H.O., Bircher, J., Eds.;
Boneld, C., Anderson, J.; Eds.; Plenum Press: New York, Hepatic Encephalopathy: Syndromes and Therapies; ILMe-
1988; 317337. di-Ed Press: Bloomington, 1994; 387398.
22. Cummings, J.H. Metabolism of dietary ber in the large 42. Wolf, B.W.; Titgemeyer, E.C.; Fahey, G.C. Jr; Garleb,
intestine. The Role of Dietary Fiber in Enteral Nutrition. K.A. Fermentability of selected oligosaccharides. FASEB
Report of the First Abbott Nutritional Research Confer- J. 1994, 8, A186.
ence, Monte Carlo, Monaco, Nov 30Dec 1, 1987. Abbott 43. Garleb, K.A.; Chow, J.; Leyer, G.J.; Johns, P.W.; Wolf,
International, LTD.: Abbott Park, IL, 1989; 113. B.W. In vitro fermentation of soy polysaccharide (soy),
23. Cummings, J.H. Short chain fatty acids in the human gum arabic (ga), fructooligosaccharides (FOS), and lactu-
colon. Gut 1981, 22, 779. lose. FASEB J. 1997, 11, A611.
24. Hoverstad, T. Studies of short-chain fatty acid absorption 44. Caspary, W.F.; Lembcke, B.; Elsenhans, B. Bacterial
in man. Scand. J. Gastroenterol. 1996, 21, 257. fermentation of carbohydrates within the gastrointestinal
25. McNeil, N.I.; Cummings, J.H.; James, W.P.T. Short chain tract. Clin. Res. Rev. 1981, 1, 107.
fatty acid absorption by the human large intestine. Gut 45. Ramakrishna, B.S.; Mathan, V.I. Colonic dysfunction in
1978, 19, 819. acute diarrhoea: The role of luminal short chain fatty acids.
26. Grohn, Y.; Bruss, M.L.; Lindberg, L.A. Propionate Gut 1993, 34, 1215.
loading test for liver function during experimental liver 46. Bowling, T.E.; Raimundo, A.H.; Silk, D.B. Colonic
necrosis in sheep. Am. J. Vet. Res. 1985, 46, 952. secretory eect in response to enteral feeding in man. Gut
27. Roediger, W.E.W. Role of anaerobic bacteria in the 1993, 34 (Suppl. 1), S33.
metabolic welfare of the colonic mucosa in man. Gut 47. Bowling, T.E.; Raimundo, A.H.; Silk, D.B. The colonic
1980, 21, 793. secretory response to enteral feeding: Inuence of high
28. Hull, C.; Greco, R.S.; Brooks, D.L. Alleviation of strength diet. Clin. Nutr. 1993, 12 (Suppl. 2), 23.
constipation in the elderly by dietary ber supplementa- 48. Bowling, T.E.; Raimundo, A.H.; Grimble, G.K.; Silk,
tion. J. Am. Geriat. Soc. 1980, 28, 410. D.B. Reversal by short-chain fatty acids of colonic uid
29. Zimmaro, D.M.; Rolandelli, R.H.; Koruda, M.J.; Settle, secretion induced by enteral feeding. Lancet 1993, 342,
R.G.; Stein, T.P.; Rombeau, J.L. Isotonic tube feeding 1266.
formula induces liquid stool in normal subjects. J. Parenter. 49. Aghdassi, E.; Plapler, H.; Kurian, R.; Raina, N.; Royall,
Enter. Nutr. 1989, 13, 117. D.; Jeejeebhoy, K.N.; Cohen, Z.; Allard, J.P. Colonic
864 Wolf et al.
fermentation and nutritional recovery in rats with massive patients with ulcerative colitis: a prospective, double-
small bowel resection. Gastroenterology 1994, 107, 637. blinded, randomized, placebo controlled multicenter trial.
50. Koruda, M.J.; Rolandelli, R.H.; Settle, R.G.; Zimmaro, Gastroenterology 2000, 118, A782.
D.M.; Rombeau, J.L. Eect of parenteral nutrition 68. Gibson, G.R.; Roberfroid, M.B. Dietary modulation of the
supplemented with short-chain fatty acids on adaptation human colonic microbiota-introducing the concept of
to massive small bowel resection. Gastroenterology 1988, prebiotics. J. Nutr. 1995, 125, 1401.
95, 715. 69. Wang, X.; Gibson, G.R. Eects of the in vitro fermentation
51. Sakata, T. Stimulatory eect of short-chain fatty acids on of oligofructose and inulin by bacteria growing in the
epithelial cell proliferation in the rat intestine: A possible human intestine. J. Appl. Bacteriol. 1993, 75, 373.
explanation for trophic eects of fermentable bre, gut 70. Mitsuoka, T.; Hidaka, H.; Eida, T. Eect of fructo-
microbes and luminal trophic factors. Br. J. Nutr. 1987, 58, oligosaccharides on intestinal microora. Nahrung 1987,
95. 31, 427.
52. Younes, H.; Garleb, K.A.; Behr, S.; Remesy, C.; Demigne, 71. Williams, C.H.; Witherly, S.A.; Buddington, R.K. Inu-
C. Fermentable bers or oligosaccharides reduced urinary ence of dietary neosugar on selected bacterial groups of the
nitrogen excretion by increasing urea disposal in the rat human faecal microbiota. Microb. Ecol. Health Dis. 1994,
cecum. J. Nutr. 1995, 125, 1010. 7, 91.
53. Howard, M.D.; Gordon, D.T.; Pace, L.W.; Garleb, K.A.; 72. Garleb, K.A.; Snook, J.T.; Marcon, M.J.; Wolf, B.W.;
Kerley, M.S. Eect of dietary supplementation with Johnson, W.A. Eect of fructooligosaccharide containing
fructooligosaccharides on colonic microbiota populations enteral formulas on subjective tolerance factors, serum
and epithelial cell proliferation in neonatal pigs. J. Pediatr. chemistry proles, and faecal bidobacteria in healthy
Gastroenterol. Nutr. 1995, 21, 297. adult male subjects. Microb. Ecol. Health Dis. 1996, 9, 279.
54. Bourquin, L.D.; Bennink, M.R.; Rozeboom, K.A.; Wolf, 73. Van der Waaij, D.; Berghuis-de Vries, J.M.; Lekkerkerk-
B.W. Fructooligosaccharides (FOS) enhance cecal and van der Wees, J.E.C. Colonization resistance of the
colonic development of neonatal pigs. FASEB J. 1997, 11, digestive tract in conventional and antibiotic-treated mice.
A601. J. Hyg. 1971, 69, 405.
55. Glotzer, D.J.; Glick, M.E.; Goldman, H. Proctitis and 74. Freter, R.; Brickner, H.; Botney, M.; Cleven, D.; Aranki,
colitis following diversion of the fecal stream. Gastro- A. Mechanisms that control bacterial populations in
enterology 1981, 80, 438. continuous-ow culture models of mouse large intestinal
56. Komorowski, R.A. Histologic spectrum of diversion ora. Infect. Immun. 1983, 39, 676.
colitis. Am. J. Surg. Pathol. 1990, 14, 548. 75. Guiot, H.F.L. Role of competition for substrate in
57. Roediger, W.E.W. The starved colon-diminished mucosal bacterial antagonism in the gut. Infect. Immun. 1982, 38,
nutrition, diminished absorption, and colitis. Dis. Colon 887.
Rectum 1990, 33, 858. 76. Rolfe, R.D. Role of volatile fatty acids in colonization
58. Harig, J.M.; Soergal, K.H.; Komorowski, R.A.; Wood, resistance to Clostridium dicile. Infect. Immun. 1984, 45,
C.M. Treatment of diversion colitis with short-chain-fatty 185.
acid irrigation. NEJM 1989, 310, 23. 77. Hentges, D.J. Enteric pathogennormal ora interactions.
59. Roediger, W.E.W. The colonic epithelium in ulcerative Am. J. Clin. Nutr. 1970, 23, 1451.
colitis: an energy deciency disease? Lancet 1980, 2, 712. 78. Wilson, K.H.; Perini, F. Role of competition for nutrients
60. Vernia, P.; Caprilli, R.; Latella, G.; Barbetti, F.; Maglioc- in suppression of Clostridium dicile by the colonic
ca, F.M.; Cittadini, M. Fecal lactate and ulcerative colitis. microora. Infect. Immun. 1988, 56, 2610.
Gastroenterology 1988, 95, 1564. 79. Brook, I.; Walker, R.I.; Macvittie, T.J. Eect of anti-
61. Vernia, P.; Gnaedinger, A.; Hauck, W.; Breuer, R.I. microbial therapy on bowel ora and bacterial infection in
Organic anions and the diarrhea of inammatory bowel irradiated mice. Int. J. Radiat. Biol. 1988, 53, 709.
disease. Dig. Dis. Sci. 1988, 33, 1353. 80. Wells, C.L.; Maddaus, M.A.; Reynolds, C.M.; Jechorek,
62. Breuer, R.I.; Bjio, S.K.; Christ, M.L.; Bean, J.; Vernia, R.P.; Simmons, R.L. Role of anaerobic ora in the
A.P.; Paoluzi, P.; DiPaolo, M.C.; Caprilli, R. Rectal translocation of aerobic and facultatively anaerobic
irrigation with short-chain fatty acids for distal ulcerative intestinal bacteria. Infect. Immun. 1987, 55, 2689.
colitis (preliminary report). Dig. Dis. Sci. 1991, 36, 185. 81. Van der Waaij, D. The ecology of the human intestine and
63. Senagore, A.J.; MacKeigan, J.M.; Scheider, M.; Ebrom, its consequences for overgrowth by pathogens such as
J.S. Short-chain fatty acid enemas: a cost eective Clostridium dicile. Annu. Rev. Microbiol. 1989, 43, 69.
alternative in the treatment of nonspecic proctosigmoidi- 82. McFarland, L.V.; Surawicz, C.M.; Greenberg, R.N.;
tis. Dis. Colon Rectum 1992, 35, 923. Fekety, R.; Elmer, G.W.; Moyer, K.A.; Mellher, S.A. A
64. Scheppach, W.; Sommer, H.; Kirchner, T.; Paganelli, randomized placebo-controlled trial of Saccharomyces
G.M.; Bartram, P.; Christ, S.; Richter, F.; Dugel, G. Eect boulardii in combination with standard antibiotics for
of butyrate enemas on the colonic mucosa in distal Clostridium dicile disease. JAMA 1994, 271, 1913.
ulcerative colitis. Gastroenterology 1992, 103, 51. 83. Greenough, W.B.; Bennett, R.G. In: Principles of Geriatric
65. Rolandelli, R.H.; Saul, S.H.; Settle, R.G.; Jacobs, D.O.; Medicine and Gerontology; Hazzard, W.R., Andres, R.,
Trerotola, S.O.; Rombeau, J.L. Comparison of parenteral Bierman, E., et al., Eds.; McGraw-Hill: New York, 1990;
nutrition and enteral feeding with pectin in experimental 11681176.
colitis in the rat. Am. J. Clin. Nutr. 1988, 47, 15. 84. Fekety, R. In Diarrheal Diseases; Field, M., Ed.; Elsevier:
66. Grisham, M.B.; DeMichele, S.J.; Garleb, K.A.; Specian, New York, 1991; 293317.
R.D. Sulfasalazine or enteral diets containing sh oil or 85. Kelly, C.P.; Pothoulakis, C.; LaMont, J.T. Clostridium
oligosaccharides attenuate chronic colitis in rats. Inamm. dicile colitis. N. Engl. J. Med. 1994, 330, 257.
Bowel Dis. 1996, 2, 178. 86. McFarland, L.V.; Surawicz, C.M.; Stamm, W.E. Risk
67. Seidner, D.L.; Lashner, B.A.; Brzezinski, A.; DeMichele, factors for Clostridium dicile carriage and C. dicile-
S.J.; Garleb, K.A.; Banks, P.L.; Fiocchi, C.; Katz, J.; associated diarrhea in a cohort of hospitalized patients. J.
Lichtenstein, G.R.; Anton, P.A.; Kam, L.Y. A novel Infect. Dis. 1990, 162, 678.
nutritional formula reduces corticosteroid requirements in 87. Benno, Y.; Kobayashi, T.; Watanabe, K.; Ueno, K.;
Nozawa, Y. Two toxins (D-1 and D-2) of Clostridium 106. Wolf, B.W.; Meulbroek, J.A.; Jarvis, K.P.; Wheeler, K.B.;
dicile causing antibiotic-associated colitis: Purication Garleb, K.A. Dietary supplementation with fructooligo-
and some characterization. Biochem. Int. 1981, 2, 629. saccharides increase survival time in a hamster model of
88. Taylor, N.S.; Thorne, G.M.; Bartlett, J.G. Comparison of Clostridium dicile-colitis. Biosci. Microora 1997, 16, 59.
two toxins produced by Clostridium dicile. Infect. 107. Pool-Zobel, B.; van Loo, J.; Rowland, I.; Roberfroid, M.B.
Immun. 1981, 34, 1036. Experimental evidences on the potential of prebiotic
89. Triadalopoulos, G.; Pothoulakis, C.; OBrien, M.J.; La- fructans to reduce the risk of colon cancer. Br. J. Nutr.
Mont, J.T. Dierential eects of Clostridium dicile toxins 2002, 87 (Suppl 2), S273.
A and B on rabbit ileum. Gastroenterology 1987, 93, 273. 108. Reddy, B.S.; Hamid, R.; Rao, C.V. Eect of dietary
90. Tedesco, F.J.; Barton, R.W.; Alpers, D.H. Clindamycin- oligofructose and inulin on colonic preneoplastic aberrant
associated colitis: a prospective study. Ann. Intern. Med. crypt foci inhibition. Carcinogenesis 1997, 18, 1371.
1974, 81, 429. 109. Taper, H.S.; Roberfroid, M. Inuence of inulin and
91. Nolan, N.P.; Kelly, C.P.; Humphreys, J.F.; Cooney, C.; oligofructose on breast cancer and tumor growth. J. Nutr.
OConnor, R.; Walsh, T.N.; Weir, D.G.; OBrien, D.G. An 1999, 129, 1488S.
epidemic of pseudomembranous colitis: Importance of 110. Verghese, M.; Rao, D.R.; Chawan, C.B.; Williams, L.L.;
person to person spread. Gut 1987, 28, 1467. Shackelford, L. Dietary inulin suppresses azoxymethane-
92. Anand, A.; Glatt, A.E. Clostridium dicile infection induced aberrant crypt foci and colon tumors at the
associated with antineoplastic chemotherapy: A review. promotion stage in young Fisher 344 rats. J. Nutr. 2002,
Clin. Infect. Dis. 1993, 17, 109. 132, 2809.
93. Wilson, K.H.; Sheagren, J.N.; Freter, R.; Weatherbee, L.; 111. Verghese, M.; Rao, D.R.; Chawan, C.B.; Shackelford, L.
Lyerly, D. Gnotobiotic models for study of the microbial Dietary inulin suppresses azoxymethane-induced preneo-
ecology of Clostridium dicile and Escherichia coli. J. plastic aberrant crypt foci in mature Fisher 344 rats. J.
Infect. Dis. 1986, 153, 547. Nutr. 2002, 132, 2804.
94. Wilson, K.H.; Sheagren, N.; Freter, R. Population 112. Rao, C.V.; Chou, D.; Simi, B.; Ku, H.; Reddy, B.S.
dynamics of ingested Clostridium dicile in the gastro- Prevention of colonic aberrant crypt foci and modulation
intestinal tract of the Syrian hamster. J. Infect. Dis. 1985, of large bowel microbial activity by dietary coee, inulin
151, 355. and pectin. Carcinogenesis 1998, 19, 1815.
95. Fekety, R.; Silva, J.; Kauman, C.; Buggy, B.; Deery, H.G. 113. Poulsen, M.; Molck, A.; Jacobsen, B.L. Dierent eects of
Treatment of antibiotic-associated Clostridium dicile short- and long-chained fructans on large intestinal
colitis with oral vancomycin: Comparison of two dosage physiology and carcinogen-induced aberrant crypt foci in
regimens. Am. J. Med. 1989, 86, 15. rats. Nutr. Cancer 2002, 42, 194.
96. Walters, B.A.J.; Roberts, R.; Staord, R.; Seneviratne, E. 114. Femia, A.P.; Luceri, C.; Dolara, P.; Giannini, A.; Biggeri,
Relapse of antibiotic associated colitis: endogenous persis- A.; Salvadori, M.; Clune, Y.; Collins, K.J.; Paglierani, M.;
tence of Clostridium dicile during vancomycin therapy. Caderni, G. Antitumorigenic activity of the prebiotic inulin
Gut 1983, 24, 206. enriched with oligofructose in combination with the
97. Bartlett, J.G.; Tedesco, F.J.; Shull, S.; Lowe, B.; Chang, T. probiotics Lactobacillus rhamnosus and Bidobacterium
Symptomatic relapse after oral vancomycin therapy of lactis on azoxymethane-induced colon carcinogenesis in
antibiotic-associated pseudomembranous colitis. Gastro- rats. Carcinogenesis 2002, 23, 1953.
enterology 1980, 78, 431. 115. Wijnands, M.V.W.; Schoterman, H.C.; Bruijntjes, J.P.;
98. Fekety, R.; Shah, A.B. Diagnosis and treatment of Hollanders, V.M.; Woutersen, R.A. Eect of dietary
Clostridium dicile colitis. JAMA 1993, 269, 71. galacto-oligosaccharides on azoxymethane-induced aber-
99. Kimmey, M.B.; Elmer, G.W.; Surawicz, C.M.; McFarland, rant crypt foci and colorectal cancer in Fischer 344 rats.
L.V. Prevention of further recurrences of Clostridium Carcinogenesis 2001, 22, 127.
dicile colitis with Saccharomyces boulardii. Dig. Dis. 116. Cummings, J.H.; Bingham, S.A. Dietary bre, fermenta-
Sci. 1990, 35, 897. tion and large bowel cancer. Cancer Surv. 1987, 6, 601.
100. Wilson, K.H.; Silva, J.; Fekety, F.R. Suppression of 117. Reddy, B.S. Prevention of colon cancer by pre- and
Clostridium dicile by normal hamster cecal ora and probiotics: evidence from laboratory studies. Br. J. Nutr.
prevention of antibiotic-associated cecitis. Infect. Immun. 1998, 80, S219.
1981, 34, 626. 118. Reddy, B.S. Possible mechanisms by which pro- and
101. Schwan, A.; Sjolin, S.; Trottestam, U.; Aronsson, B. prebiotics inuence colon carcinogenesis and tumor
Relapsing Clostridium dicile enterocolitis cured by rectal growth. J. Nutr. 1999, 129 (Suppl.), 1478S.
infusion of homologous faeces. Lancet 1983, 2, 845. 119. Brady, L.J.; Gallaher, D.D.; Busta, F.F. The role of
102. Schwan, A.; Sjolin, S.; Trottestam, U.; Aronsson, B. probiotic cultures in the prevention of colon cancer. J.
Relapsing Clostridium dicile enterocolitis cured by rectal Nutr. 2000, 130 (Suppl), 410S.
infusion of normal faeces. Scand. J. Infect. Dis. 1984, 16, 120. Hague, A.; Elder, D.J.E.; Hicks, D.J.; Paraskeva, C.
211. Apoptosis in colorectal tumor cells: induction by the short
103. May, T.; Mackie, R.I.; Fahey, G.C.; Cremin, J.C.; Garleb, chain fatty acids butyrate, propionate and acetate and by
K.A. Eect of bre source on short-chain fatty acid the bile salt deoxycholate. Int. J. Cancer 1995, 60, 400.
production and on the growth and toxin production by 121. Hague, A.; Paraskeva, C. The short-chain fatty acid
Clostridium dicile. Scand. J. Gastroenterol. 1994, 19, 916. butyrate induces apoptosis in colorectal tumour cell lines.
104. May, T.; Mackie, R.I.; Garleb, K.A. Eect of dietary Eur. J. Cancer Prev. 1995, 4, 359.
oligosaccharides on intestinal growth of and tissue damage 122. Lupton, J.R. Butyrate and colonic cytokinetics: dierences
by Clostridium dicile. Microecol. Ther. 1995, 23, 158. between in vitro and in vivo studies. Eur. J. Cancer Prev.
105. Gaskins, H.R.; Mackie, R.I.; May, T.; Garleb, K.A. 1995, 4, 373.
Dietary fructooligosaccharide modulates large intestinal 123. Awasthi, Y.C.; Sharma, R.; Singhal, S.S. Human gluta-
inammatory responses to Clostridium dicile in anti- thione S-transferases. Int. J. Biochem. 1994, 26, 295.
biotic-compromised mice. Microb. Ecol. Health Dis. 1996, 124. Kulkarni, N.; Reddy, B.S. Inhibitory eect of Bidobacte-
9, 157. rium longum cultures on azoxymethane-induced aberrant
866 Wolf et al.
crypt foci formation and fecal bacterial h-glucuronidase. carbohydrates on blood urea ux and ammonia absorption
Proc. Soc. Exp. Biol. Med. 1994, 207, 278. in the rat cecum. J. Nutr. 1989, 119, 560.
125. Singh, J.; Rivenson, A.; Tomita, M.; Shimamura, S.; 140. Levrat, M.A.; Remesy, C.; Demigne, C. Inuence of inulin
Ishibashi, N.; Reddy, B.S. Bidobacterium longum, a lactic on urea and ammonia in the rat cecum: consequences on
acid-producing intestinal inhibit colon cancer and modu- nitrogen excretion. J. Nutr. Biochem. 1993, 4, 351.
late the intermediate biomarkers of colon carcinogenesis. 141. Levrat, M.A.; Remesy, C.; Demigne, C. Very acidic
Carcinogenesis 1997, 18, 833. fermentations in the rat cecum during adaptation to a diet
126. Rowland, I.R.; Rumney, C.J.; Coutts, J.T. Eect of rich in amylase-resistant starch (crude potato starch). J.
Bidobacterium longum and inulin on gut bacterial Nutr. Biochem. 1991, 2, 31.
metabolism and carcinogen-induced aberrant crypt foci 142. Hara, H.; Nagata, M.; Ohta, A.; Kasai, T. Increases in
in rats. Carcinogenesis 1998, 19, 281. calcium absorption with ingestion of soluble dietary ber,
127. Swanson, K.S.; Grieshop, C.M.; Flickinger, E.A.; Bauer, guar gum hydrolysate, depend on the caecum in partial
L.L.; Chow, J.; Wolf, B.W.; Garleb, K.A.; Fahey, G.C. Jr nephrectomized and normal rats. Br. J. Nutr. 1996, 76,
Fructooligosaccharides and Lactobacillus acidophilus mod- 773.
ify gut microbial populations, total tract nutrient digesti- 143. Ohta, A.; Osakabe, N.; Yamada, K.; Saito, Y.; Hidaka, H.
bilities and fecal protein catabolite concentrations in Eect of fructooligosaccharides and other saccharides on
healthy adult dogs. J. Nutr. 2002, 132, 3721. Ca, Mg, an P absorption in rats. J. Jpn. Soc. Nutr. Food
128. Swanson, K.S.; Grieshop, C.M.; Flickinger, E.A.; Bauer, Sci. 1993, 46, 123.
L.L.; Wolf, B.W.; Chow, J.; Garleb, K.A.; Williams, J.A.; 144. Ohta, A.; Ohtuki, M.; Takizawa, T.; Inaba, H.; Takashi,
Fahey, G.C. Jr Fructooligosaccharides and Lactobacillus A.; Kimura, S. Eects of frutooligosaccharides on the
acidophilus modify bowel function and protein catabolites absorption of magnesium and calcium by cecectomized
excreted by healthy humans. J. Nutr. 2002, 132, 3042. rats. Int. J. Vitam. Nutr. Res. 1994, 64, 316.
129. Wollowski, I.; Rechkemmer, G.; Pool-Zobel, L. Protective 145. Ohta, A.; Ohtsuki, M.; Baba, S.; Adachi, T.; Sakata, T.;
role of probiotics and prebiotics in colon cancer. Am. J. Sakaguchi, E. Calcium and magnesium absorption from
Clin. Nutr. 2001, 73 (Suppl.), 451S. the colon and rectum are increased in rats fed fructooligo-
130. Bouhnik, Y.; Flourie, B.; Riottot, M.; Bisetti, N.; Gailing, saccharides. J. Nutr. 1995, 125, 2417.
M.F.; Guibert, A.; Bornet, F.; Rambaud, J.C. Eects of 146. Ohta, A.; Motohashi, Y.; Ohtsuki, M.; Hirayama, M.;
fructo-oligosaccharides ingestion on fecal bidobacteria Adachi, T.; Sakuma, K. Dietary fructooligosaccharides
and selected metabolic indexes of colon carcinogenesis in change the concentration of calbindin-D9k dierently in
healthy humans. Nutr. Cancer 1996, 26, 21. the mucosa of the small and large intestine of rats. J. Nutr.
131. Taper, H.S.; Roberfroid, M.B. Non-toxic potentiation of 1998, 128, 934.
cancer radiotherapy by dietary oligofructose or inulin. 147. Trinidad, T.P.; Wolever, T.M.; Thompson, L.U. Eect of
Anticancer Res. 2002, 22, 3319. acetate and propionate on calcium absorption from the
132. Taper, H.S.; Roberfroid, M.B. Inhibitory eect of dietary rectum and distal colon of humans. Am. J. Clin. Nutr.
inulin or oligofructose on the development of cancer 1996, 63, 574.
metastatses. Anticancer Res. 2000, 20, 4291. 148. Mineo, H.; Hara, H.; Kikuchi, H.; Sakurai, H.; Tomita, F.
133. Tsuyuki, S.; Yamazaki, S.; Akashiba, H.; Kamimura, H.; Various indigestible saccharides enhance net calcium
Sekine, K.; Toida, T.; Saito, M.; Kawashima, T.; Ueda, K. transport from the epithelium of the small and large
Tumor-suppressive eect of a cell wall preparation, WPG, intestine of rats in vitro. J. Nutr. 2001, 131, 3243.
from Bidobacterium infantis in germ free and ora bearing 149. Ohta, A.; Uehara, M.; Sakai, K.; Takasaki, M.; Adler-
mice. Bidobact. Microora 1991, 10, 43. creutz, H.; Morohashi, T.; Ishimi, Y. A combination of
134. Sekine, K.; Watanbe-Sekine, E.; Ohta, J.; Toida, T.; dietary fructooligosaccharides and isoavone conjugates
Tatsuki, T.; Kawashimal, T.; Hashimoto, Y. Induction increases femoral bone mineral density and equol produc-
and activation of tumoricidal cells in vitro and in vivo by tion in ovariectomized mice. J. Nutr. 2002, 132, 2048.
the bacterial cell wall of Bidobacterium infantis. Bido- 150. Grin, I.J.; Davila, P.M.; Abrams, S.A. Non-digestible
bact. Microora 1994, 13, 65. oligosaccharides and calcium absorption in girls with
135. Kelsay, J.I.; Behall, K.M.; Prather, E.S. Eect of ber from adequate calcium intakes. Br. J. Nutr. 2002, 87, S187.
fruits and vegetables on metabolic responses of human 151. Coudray, C.; Bellanger, J.; Castiglia-Delavaud, C.; Rem-
subjects. 1. Bowel transit time, numbers of defecations, esy, C.; Vermorel, M.; Rayssignuier, Y. Eect of soluble or
fecal weight, urinary excretions of energy and nitrogen and partly soluble dietary bres supplementation on absorption
apparent digestibilities of energy, nitrogen and fat. Am. J. and balance of calcium, magnesium, iron and zinc in
Clin. Nutr. 1978, 32, 1149. healthy young men. Eur. J. Clin. Nutr. 1997, 51, 375.
136. Calloway, D.H.; Kretsch, M.J. Protein and energy 152. van den Heuvel, E.G.H.M.; Muys, T.; van Dokkum, W.;
utilization in men given a rural Guatemalan diet and egg Schaafsma, G. Oligofructose stimulates calcium absorp-
formulas with and without added oat bran. Am. J. Clin. tion in adolescents. Am. J. Clin. Nutr. 1999, 69, 544.
Nutr. 1978, 31, 1118. 153. Tahiri, M.; Tressol, J.C.; Arnaud, J.; Bornet, F.; Bou-
137. Conn, H.O.; Leevy, C.M.; Vlahcevic, Z.R.; Rodgers, J.B.; teloup-Demange, C.; Feillet-Coudray, C.; Ducros, V.;
Maddrey, W.C.; See, L.; Levy, L.L. Comparison of Pepin, D.; Brouns, F.; Roussel, A.M.; Rayssiguier, Y.;
lactulose and neomycin in the treatment of chronic portal Coudray, C. Five-week intake of short-chain fructo-
systemic encephalopathy. A double blind controlled trial. oligosaccharides increases intestinal absorption and status
Gastroenterology 1977, 4, 573. of magnesium in postmenopausal women. J. Bone Miner.
138. Mortensen, P.B. Eect of oral-administered lactulose on Res. 2001, 16, 2152.
colonic nitrogen metabolism and excretion. Hepatology 154. Coudray, C.; Demigne, C.; Rayssiguier, Y. Eects of
1992, 16, 1350. dietary bers on magnesium absorption in animals and
139. Remesy, C.; Demigne, C. Specic eects of fermentable humans. J. Nutr. 2003, 133, 1.
39
Immobilization of Cells in Polysaccharide Gels
Yunyu Yi and Ronald J. Neufeld
Queens University, Kingston, Ontario, Canada
Denis Poncelet
ENITIAA, Nantes, France
I. INTRODUCTION nontoxic, biocompatible, relatively stable, yet biodegrad-

able. Therefore, natural polysaccharides have gained a
The economic importance of the eld of biotechnology central role as an entrapment matrix for cells and other
worldwide has resulted in considerable research directed biocatalysts.
toward the culture, manipulation, handling, and immobi- Many dierent microorganisms have been encapsu-
lization of living cells. Techniques for the immobilization lated in various matrices for dierent applications, in-
of microbial cells [1], plant cells [2], animal cells [3], and cluding their use in biocontrol [7], bioremediation [8],
embryos [46] have been developed for a variety of scien- biodegradation [912], agriculture [13], metal uptake
tic and industrial applications. [14,15], and wastewater nutrient removal [16]. In gene
A large number of immobilization methods have been therapy delivery systems, alginatepolylysine (PLL)algi-
described for the immobilization of cells, enzymes, and nate capsules are be used to entrap recombinant cells,
other biologically active materials. In general, they can be engineered to secrete a therapeutic agent for the treatment
described as entrapment or microencapsulation methods in of a wide variety of diseases, including brain tumors [17
which the cells or active encapsulant is contained within a 20], liver failure [21], diabetes type I [2224], hemophilia A
polymer matrix or membrane-bound microcapsule, or [25], and Parkinsons disease [26]. Alginate-based micro-
alternatively, as a binding method, where cells or the active capsules have also been applied in tissue engineering [27].
agent is adsorbed or crosslinked to the surface of a support, This review summarizes the techniques of immobili-
or crosslinked cell to cell. These various immobilization zation by entrapment in polysaccharide gels, together with
methodologies are illustrated in Fig. 1. The focus of this directions toward improving polysaccharide gel properties.
chapter will be on entrapment methodologies, whereby the The measurement of the eective diusion coecient of
cells or active biologicals are immobilized by entrapment solutes in polysaccharide gel and the characterization of gel
within a polymer matrix structure consisting of a polysac- mechanical properties will be described. Finally, mathe-
charide gel. This is illustrated by the uppermost image in matical models to simulate the behavior of cells immobi-
Fig. 1. lized in polysaccharide gels will also be outlined.
Cell entrapment in polymeric gels is, by far, the most
widely practiced immobilization method. The procedures
are relatively simple; the materials are biocompatible, II. POLYSACCHARIDES USED FOR CELL
widely available, and acceptable for use in food and IMMOBILIZATION AND ENCAPSULATION
pharmaceutical applications; and the formulation condi- TECHNIQUES
tions are gentle, ensuring the viability and stability of the
encapsulated cultures. Various natural and synthetic poly- The chemical structures of the polysaccharides widely used
mers have been used for this purpose, but because gelation for cell immobilization purposes are shown in Fig. 2. The
is carried out in the presence of cells, natural materials, principle of gelation varies from polymer to polymer, but
primarily polysaccharides, are the materials of choice. can be classied into thermal gelation, ionotropic gelation,
Additionally, polysaccharides have the advantage of being ionotropic/thermal gelation, or precipitation. Examples of
867
868 Yi et al.
diusional access [33]. Agarose gel is not as mechanically

strong as gels made from the more commonly used alginate
or carrageenan [34].
The preparation of agar and agarose gel beads is
carried out by extruding a warm agarcell or agarosecell
suspension dropwise into ice-cold buer [35]. The extruded
droplets gel rapidly upon cooling, immobilizing the cells
within the resulting bead. An alternative technique that is
better suited to large-scale immobilization practice is to
emulsify agar or agarosecell suspension into a warm uid,
which is immiscible with the aqueous polysaccharide
phase. Biocompatible uids that are used to generate
emulsions include vegetable oils such as soybean, corn,
canola, and olive oils. Alternative oils include paran,
mineral, silicone, or tri-n-butylphosphate. Once the emul-
sion is stabilized, it is rapidly cooled. Emulsied polysac-
charide droplets gel, forming small-diameter microspheres
[3640]. The diameter of the microspheres can be readily
controlled by varying the emulsion conditions, but can
range from a few microns to a couple of millimeters in
diameter. It is also possible to form alternate shapes by
forming the gels in molds. Examples of agar or agarose
matrices containing entrapped cells include cubes, disks,
and membranes, which are congured depending on the
particular application [4145].
B. Alginate (Ionotropic Gelation)

Alginate is a naturally occurring polysaccharide commer-
Figure 1 Cell immobilization methods. Cells are entrapped cially extracted from brown algae. A bacterial source of
within a polymer matrix structure or microencapsulation alginate is also available, produced through a fermentation
membrane, or attached to the surface of a particle by process. Alginate is an unbranched heteropolymer consist-
adsorption or covalent attachment, or crosslinked cell to cell. ing of a family of unbranched binary copolymers of 14-
linked h-D-mannuronic (M) and a-L-guluronic acids (G) in
varying compositions and sequences, depending on the
polysaccharides used for each of the categories are shown algal source and part of the plant from which it is extracted
in Table 1. [46]. The proportion as well as the distribution of the two
monomers determine, to a large extent, the physicochem-
A. Agar and Agarose (Thermal Gelation) ical properties of the resulting gel [4648].
Gelation of sodium alginate is achieved through the
Agarose is a puried preparation of agar. Both polysac- addition of polyvalent cations, which displace the sodium
charides have been mainly used as media to culture and and ionically crosslink guluronic acid residues on neigh-
identify microorganisms, but are also used in cell encapsu- boring polymer chains, as illustrated in Fig. 4. Gelation is
lation. Agar is isolated from marine red algae and consists carried out at room temperature under mild conditions
of a complex family of water-soluble polysaccharides with with biocompatible reagents, making this the most widely
a variety of dierent structures [28]. used cell immobilization procedure.
Agarose is the basic gel-forming component of agar. Cations used to trigger the gelation of alginate include
It is a linear polysaccharide in which 1,3-linked h-D- Ca2+, Cu2+, Ba2+, Sr2+, Pb2+, Mn2+, Cd2+, Zn2+,
galactopyranose and 1,4-linked 3,6-anhydro-a-L-galacto- Ni2+, and Al3+. Ca2+ is the most widely used because it
pyranose, (i.e., L-galactose anhydride) are arranged alter- is nontoxic and forms strong gels with alginate. One of the
natively [29]. The solgel transition of agarose upon advantages in the use of alginate to encapsulate cells is that
cooling was studied by optical rotation, dierential scan- the gel is reversible in the presence of calcium chelators,
ning calorimetry, x-ray diraction, and computerized mo- such as ethylenediaminetetraacetic acid (EDTA), phos-
lecular model building. A double-helix model was phate or citrate buer, or antigelling cations (Na+ and
proposed as the conformation of agarose gel [30,31] as Mg2+) [33]. However, this may also be seen as a disadvan-
shown in Fig. 3. The gel is thermally reversible, showing tage, as alginate gels can be unstable if immobilized cells are
marked hysteresis at the melting and gelling points [30,32]. cultured in media containing one or more of these chela-
The gel has pentagonal pores, which are large enough to tors. Alternatively, more stable gels can be obtained with
allow proteins with molecular weights into the millions, Ba2+, Pb2+, Sr2+, and Cu2+, which have a greater anity
Immobilization of Cells in Polysaccharide Gels 869
Figure 2 Chemical structures of the most widely used polysaccharides in cell encapsulations.
870 Yi et al.
Table 1 Gel Formation Mechanisms for Cell Entrapment [36,59,60]. Alternative droplet extrusion techniques have
thus been developed to produce smaller beads on large
Principle of gelation Polysaccharides scale. These techniques include air jet extrusion [61,62],
Thermal gelation Agar, agarose controlled drying [6264], atomization [6567], rotating
Ionotropic gelation Alginate, chitosan, pectate disc atomization [6870], resonance nozzle extrusion [71],
Ionotropic/thermal n-Carrageenan, gellan gum vibrating nozzle extrusion [7276], mechanical cutting of
gelation liquid jets [77,78], and electrostatic droplet generation [79
Precipitation Cellulose, cellulose triacetate 84]. As an example, electrostatic droplet generation diers
from traditional droplet extrusion methods in that the
needle and gelation bath are oppositely charged; therefore,
polymercell solution accumulates charge when it passes
for alginate; however, they are not used as often in cell through the needle. The charge dierential between the
immobilization because of potential toxicity. As an exam- extruded droplet and the gelation bath pulls the droplet
ple, Ba2+ has been used for the immobilization of yeast from the needle earlier than would be the case in the
[49] and pancreatic islet cells [50]. Stabilization of calcium absence of the charge.
alginates can also be achieved by crosslinking with An alternate technique has been developed to produce
reagents that are not inuenced by chelator/exchangers. alginate microspheres on an industrial scale, without the
Polymers, including chitosan, polyacrylamide, polyacry- need to rely on individual droplet extrusion methodologies.
late, polyacrylic acid, polyvinyl alcohol (PVA), protein, The technique, termed emulsication/internal gelation, is
poly(vinylamine), poly(allylamine), and polycations (poly- based on the emulsion dispersion of alginate sol into an
ethylenimine, PLL), have been reported to form a more immiscible uid [85,86]. Calcium is added to the alginate
stable, lower-porosity complex with alginate [5154]. sol as insoluble salt microcrystals (typically carbonate),
Bodalo et al. [55] improved retention of Ca-alginate- and gelation of the dispersed alginate microdroplets is
entrapped Pseudomonas sp. BA2 by treatment of cells with triggered by adjusting the pH of the emulsion (7.56.5),
glutaraldehyde before immobilization, or treatment of rapidly liberating soluble calcium internally from within
cell-loaded alginate beads with glutaraldehyde or a com- the emulsied droplets, as illustrated in Fig. 5. Microsphere
bination of glutaraldehyde and hexamethylenediamine, or diameters can now range down to a few microns, and
activation of alginate pregelation sol with periodate before microspheres can be produced on a large scale, with mean
dropping the cellalginate slurry into a calcium gelation diameters that are readily controlled through a control of
bath. The resulting beads were then incubated in poly- the emulsion conditions. A drawback to this technique is
ethyleneimine solution for a specied time. The alginate- that emulsion dispersions generate size distributions in the
entrapped cells prepared by treating cell-loaded alginate emulsied droplets; thus, the resulting size dispersion of the
beads with glutaraldehyde showed better performance in microsphere preparations is more dicult to control than is
terms of activity and stability than its control. the case with single droplet extrusion methods.
More homogeneous alginates, necessary for applica- Membrane-coated alginate core capsules can be pre-
tion as tissue engineering scaolds, were achieved by using pared by the association of complementary polymers, such
CaCO3GDL (D-glucono-y-lactone) and CaCO3GDL as poly-L-lysine, chitosan, or polyethyleneimine, which
CaSO4 as counterions to control the gelation rate [56]. form polyelectrolyte complex membranes as a result of
The gelation rate is seen to be a function of total calcium electrostatic interactions with polyanionic alginate. Poly-
content, ratio of CaCO3 and CaSO4, gelation temperature, electrolyte complex membranes are formed quickly at
and alginate concentration. A more structurally uniform ambient or lower temperatures under gentle, well-con-
and mechanically stronger alginate gel was obtained at a trolled conditions [87]. In some cases, the alginate core is
slower gelation rate. then liqueed using a calcium chelator, leaving a poly-L-
The most commonly used method to immobilize cells lysine membrane-bound liquid core microcapsule contain-
within alginates is to extrude the cellpolymer suspension ing the immobilized cells. These microcapsules have gained
dropwise into a gently mixed gelation bath, typically importance for medical applications such as articial liver
CaCl2. The procedure is simple and used routinely in the
formation of uniform-diameter alginate beads at the labo-
ratory scale. Depending on alginate concentration and
viscosity, and the manner in which the droplets are formed,
alginate beads can be formed with a controlled diameter
within the range of 0.53.5 mm. Although simple, this
procedure is not well suited for large-scale production
due to the production limitation imposed by the speed of
droplet formation [57], which depends on the needle diam-
eter, viscosity of alginate solution, and pressure applied on
the solution [58]. An additional disadvantage is that
smaller-diameter beads are often desired, with diameters Figure 3 Random coil to helix transition of agarose upon
under 500 Am, to minimize mass transfer limitations cooling.
Figure 4 Alginate solgel transition to form Ca-alginate polyionic gel (egg box model).
support [88], articial pancreas [89], gene therapy [90], and lyte complexes. These complexes are commonly prepared
tissue engineering [56]. by extruding the core polymer sol containing cells into a
Considerable progress has been made in the develop- solution of the counterion. A membrane is rapidly formed
ment of membrane-bound polysaccharide core microcap- around the droplet via electrostatic interaction.
sules and microspheres as can be seen by the numerous There are a number of challenges that must be over-
examples in Table 2. An examination of the examples come before the clinical application of polyelectrolyte
presented would lead to the conclusion that alginate has complex capsules. For example, based on the recommen-
been the main core polymer used, but other anionic poly- dation of the Islet Transplantation Registry, recipients
saccharides have also been employed to form polyelectro- should be implanted with 6000 islets per kilogram body
Figure 5 Schematic illustration of emulsication/internal gelation to obtain alginate beads, accompanying the solgel transition
process of alginate.
872 Yi et al.
Table 2 Membrane-Bound Polysaccharide Core Microcapsules

Gel core/coating material Immobilized cell line References
Agarose/poly(styrenesulfonic acid) (PSSa) Xenogeneic islets of Langerhans 91,

Agarose/poly(styrenesulfonic acid) (PSSa) Dopamine-secreting PC12 cells 92,93
Agarose/poly-L-lysine/alginate 1B5 hybridoma cells 94
Alginate/poly-L-lysine Pancreatic islets 89
Alginate/poly-L-lysine 293 endo cells and JJN3 myeloma cells 95
Alginate/poly-L-lysine Goat hepatocyte 96
Alginate/poly-L-lysine iNOS-expressing cells 18
Alginate/poly-L-lysine Porcine and bovine islets 97
Alginate/poly-L-lysine Mouse insulinoma cell line (MIN6) 98
Alginate/poly-L-lysine Insulin-secreting h TC6-F7 cells 99
Alginate/poly-L-lysine/alginate Recombinant mouse myoblast 17
Alginate/poly-L-lysine/alginate Murine broblast 77
Alginate/poly-L-lysine/alginate Hepatoma cells 100
Alginate/poly-L-lysine/alginate Engineered human growth hormone 101
(hGH)-secreting cells
Alginate/poly-L-lysine/alginate Hepatocytes 102
Alginate/poly-L-lysine/alginate Leydig cells 103
Alginate/poly-L-lysine/alginate Porcine fetal broblast (PFF) cells 104
Alginate/poly-L-lysine/alginate Genetically engineered Escherichia coli 105
DH 5 cells
Alginate/poly-L-lysine/alginate Mouse C2C12 myoblasts 106
Alginate/poly-L-lysine/alginate/PEG amine Islet cells 107
Alginate/poly-L-lysine/polyethylenimine Islets of Langerhans 108
Alginate/poly-L-lysine/polyethylenimine Hybridoma cells 109
Alginate/poly-L-lysine/polyethylenimine Islet cells 110
Alginate/poly-L-lysine/polyvinylamine Escherichia coli 111
Alginate/aminopropyl-silicate/alginate Islets 112
Alginate cellulose sulfate/ Pancreatic islets 24,87,113
poly(methylene coguanidine)
Alginatecellulose sulfate/poly(methylene coguanidine) Murine hepatocyte 114
Alginate/chitosan Lactobacillus casei YIT 9018 115
Alginate/chitosan Islets 116
Alginate/chitosan Hepatocyte 21
Alginate/chitosan Mouse insulinoma cell line (MIN6) 98
Alginate/chitosan/polyethylene glycol Islets of Langerhans 117
Alginate/glycol chitosan/alginate/glyco chitosan Islets 118
Alginate/spermine N/A 119
Alginateprotamine sulphate Sperm 120
Alginate/poly-L-ornithine BDNF-producing broblasts 121
Alginate/poly(vinylamine) Mouse erythroleukemia cell line 122
Alginate/a-phenoxycinnamylidene-acetylated IW32 mouse leukemia cells 123
poly(allylamine)
Alginate/poly(allylamine a-cyanocinnamylideneacetate Mouse leukemia cell 124
Alginate/a-phenylcinnamyldiene acetylatedpoly-L-lysine GH3 (a rat pituitary tumor cell line) cells 53,125
Na-cellulose sulfate/polydiallyldimethyl Hybridoma cells 126
ammonium chloride
CM-cellulose/poly-L-lysine/poly(ethyleneimine) Hybridoma cells 109
Sodium cellulose sulfate/ Hybridoma cells 126
polydiallyldimethylammonium chloride
Sodium cellulose sulfate/ Serratia marcescens B345 127
poly(dimethyldialylammonium chloride)
Chitosan/acrylonitrile-vinylchloride copolymer Phaeochromocytoma-derived cell line 128
and broblast cell lines
Chitosan/CMC Hybridoma cells 129
Starch/alginate Lactic acid bacteria 130
Starch/alginate Lactobacillus casei 131
Starch/alginate Lactobacillus rhamnosus 132
weight to control blood glucose metabolism. Therefore, be soft. Gel-inducing agents, such as K+, Rb+, Cs+,
hundreds of milliliters of microencapsulated islets would Ca2+, NH+ 4, Cu
2+
, Mg2+, Fe3+, Al3+, aliphatic amines,
need to be implanted to a normal adult recipient (above 50 aromatic amines, and water-miscible organic solvents,
kg) if traditional microencapsulation techniques were were employed to improve mechanical stability [135, 139
usedlevels that are unacceptably high. A method was 141]. The properties of the nal gel matrix are strongly
developed by Park et al. [50] with the aim of minimizing the aected by gelation conditions. For example, the mechan-
size of microencapsulated islets and the number of empty ical strength of the gel increases with increasing carrageen-
microcapsules. Empty capsules are encountered with tra- an concentration and stronger gels were obtained with
ditional microencapsulation techniques, which leads to a monovalent cations such as potassium, cesium, and am-
higher volume of implanted microcapsules. A novel cen- monium, compared to the gels formed with monovalent
trifugation-based technique was developed, in which each and bivalent metal ions [139,142]. Carrageenan beads show
islet in an upper solution layer was covered with a thin better strength when incubating in some curing agents,
alginate layer while passing successively through a dextran such as potassium and polyethyleneimines [143]. A study
solution (d = 1.032 g/mL), a BaCl2 and dextran solution by Chao et al. [139] on the eect of curing agent on the
(d = 1.045 g/mL), and a dextran solution (d = 1.058g/mL) resulting carrageenan strength was carried out by using 10
drawn by centrifugal force. Gelation of a thin alginate layer amines to treat cell-containing n-carrageenan beads pre-
around the islets occurred in the BaCl2 and dextran solu- pared by an emulsication method. They found that both
tion layer. linear and branched polyethyleneimines are eective in
One of the inherent diculties in using polysaccharide enhancing heat and abrasion resistance of beads.
gels for cell encapsulation is that the encapsulating poly- Carrageenan beads are prepared in a fashion similar to
mers are natural materials, and thus are often available that of alginate. Encapsulation of cells in carrageenan is
uncharacterized and have chemistries that can vary widely achieved by either extruding carrageenancell suspension
from source to source, and even from batch to batch. This dropwise into KCl solution [144], or by using an emulsion/
creates diculties when attempting to compare data from dispersion process [145148]. Various shapes of gels con-
laboratory to laboratory, and researchers often encounter taining immobilized cells include beads, cubes, blocks, and
dierences in the mechanical and diusional properties of membranes, depending on the specic purpose [135,
the gels when changing the source or batch of polymer. One 149,150].
of the most interesting developments in the eld has been
the work being carried out at the Norwegian Biopolymer
Laboratory (NOBIPOL), involving enzymatic modica- D. Chitin and Chitosan (Ionotropic Gelation)
tions to alginate chemistry. Skjak-Brk et al. [133] modi-
ed alginates with a mannuronanC5 epimerase from Chitosan is a cationic polysaccharide consisting of gluco-
Azotobacter vinelandii, which resulted in alginates with a samine and N-acetylglucosamine residues with a 1,4-h
high guluronic acid content. The enzymatically modied linkage (Fig. 2). Chitosan can be prepared by deacetylation
alginates were better able to form gels with calcium, thus of chitin, obtained from crustacean shell wastes [151,152].
enhancing its mechanical properties. AlgE4, a C5 epimer- The amino groups of chitosan are protonated at pH
ase, converts homopolymeric mannuronate blocks of algi- levels lower than 6, which allows an ionic gel network to
nate into alternating mannuronate/guluronate sequences form via crosslinking with counterions. A number of
[134]. A number of dierences were observed in the prop- dierent multivalent anionic counterions have been used
erties of the epimerized alginates including reduced elastic- to crosslink chitosan as reviewed by Vorlop and Klein
ity, higher degree of syntheresis, and increase in solgel [153]. Cell encapsulation in chitosan microspheres can be
transitional rate. achieved by adding chitosancell suspension (pH<6)
dropwise into a solution containing counterions with low
molecular weight [154]. Chitosan microcapsules can be
C. Carrageenan (Ionotropic/Thermal Gelation) obtained by extruding cellchitosan solution into counter-
ions with high molecular weight, such as alginate [21], or by
Carrageenan consists of alternating 1,3-linked h- D - emulsifying chitosancell slurry into an organic phase,
galactose and 1,4-linked 3,6-anhydro-a-D-galactose. The whereby membrane formation is initiated by addition of
structures of the three main types of natural carrageenan, terephthaloyl chloride, hexamethylene diisocyanate, or
kappa (n), lambda (E), and iota (L), are shown in Fig. 2. glutaraldehyde [155].
Among the three types, n-carrageenan is considered the Although alginate and n-carrageenan gels have been
most suitable polymer for cell encapsulation [135]. Gela- most frequently used for immobilizing viable cells for
tion of carrageenan occurs under a variety of conditions. A fermentation [156], chitosan beads can be a good alterna-
thermally reversible solgel transition is accompanied by a tive when the drawbacks of alginate or carrageenan limit
corresponding coilhelix transition [136,137], with the their application for some specic applications. For exam-
subsequent formation of double helices and association ple, chitosan beads are stable in phosphate-buered so-
of the helices promoted by potassium ions [138]. Although lutions [157] and showed better mechanical property than
cooling the solution to the gelication temperature (10jC) alginate in a comparative study carried out by Ridout and
is the simplest way to obtain gels, the resulting gels tend to Brownsey [158].
874 Yi et al.
E. Gellan Gum (Ionotropic/Thermal Gelation) with polyethyleneimine and glutaraldehyde successively

[172].
Gellan gum is a bacterial gel-forming polysaccharide The ionotropic pectate gel beads can be prepared with
produced by Sphingomonas paucimobilis in aerobic a simple and gentle procedure. Beads are produced by
fermentation [159]. As shown in Fig. 2, gellan gum is a extruding the polymercell suspension into a gelation bath.
high-molecular-mass anionic homopolymer with tetra- The cations that have been used to trigger the gelation of
saccharide repeating units [34] consisting of h-D-glucose, pectin with low esterication, or pectate are Ca2+, Al3+,
h-D-glucose, h-D-glucuronic acid, and a-L-rhamnose. Zn2+, Co2+, and Mg2+ [140,170,173175].
Gelation of deacetylated gellan gum can be initiated
by either adding salts or decreasing temperature [136].
G. Cellulose (Precipitation)
In the absence of gel-promoting cations, brils are pro-
duced by the formation of a double helix between the Cellulose consists of 14-linked h-D-glucopyranosyl units
ends of neighboring molecules upon heating and cooling. with additional interchain interaction through hydrogen
Gel-promoting cations lead to lateral crystallization of bonding. As cellulose is water-insoluble, organic liquids
brils and formation of permanent gel [159,160]. Factors that have been used to dissolve cellulose were reviewed by
that are reported to aect the gel rheological properties are Linko and Linko [176]. Most of those organic solvents are
gum concentration, ionic strength, and type of stabilizing too harsh for entrapment of viable cells. Thus, fully formed
cation [161163]. Deacylated gellan gum showed better cellulose carriers are more frequently used for the adsorp-
thermal stability and resistance to enzymes [161,162], and tion of living cells than as an entrapment matrix.
demonstrated better rheological properties than those of Cells were entrapped in cellulose beads formed by a
agar, n-carrageenan, and alginate at the same concentra- precipitation mechanism, by transferring the polymercell
tion [164]. suspension from the organic solvent to water. Linko and
Gellan beads are produced by droplet extrusion or Linko [176] and Linko et al. [177] entrapped various
emulsion/dispersion of warm gellan gum-cell suspension. bacteria and yeast cells inside cellulose beads by means of
Gelation is initiated upon cooling [162,165167]. Gellan this procedure. Actinoplanes missouriensis (glucose isomer-
gum is a thermoresistant gelling polysaccharide, showing ase activity), Kluyveromyces fragilis (h-galactosidase activ-
rheological stability for 15 days [162]. The gel setting ity), and Saccharomyces cerevisiae (invertase activity) were
temperature of gellan gum is high (over 50jC), but can encapsulated within cellulose and cellulose diacetate and
be reduced with citrate, metaphosphate, EDTA [163] or triacetate beads with the aim of retaining catalytic enzy-
xanthan gum [156], to a temperature range better suited to matic activity. Thus, cell viability was a secondary concern,
cell encapsulation. as long as the enzyme activity was present. Organic solvents
that have been used by Linko and Linko [176] and Linko et
al. [177] to prepare cellulose diacetate and triacetate beads
F. Pectin and Pectate (Ionotropic Gelation)
were dimethylsulfoxide (DMSO), N-ethylpyridinium chlo-
Pectins, as important constituents of the cell wall of higher ride (NEPC), or a mixture of DMSO with acetone, or a
plants, have a linear 1,4-linked a-D-galacturonate back- combination of NEPC and DMSO, and a mixture of
bone, with its galacturonic acid subunits partly methyl NEPC and dimethyformamide. Alternative solvents are
esteried, depending on the source. The degree of esteri- acetone [178180], n-hexane [181], or methylene chloride
cation inuences, to a large extent, the solubility and the [182,183], as reviewed by Willaert and Baron [33].
gelation process of pectin [34]. The gelation of pectin with
high degree of esterication (>50%) requires a low water
activity environment, which is often achieved by adding III. MECHANICAL PROPERTIES OF
sucrose, glycerol, or other dehydrating agents at a pH of POLYSACCHARIDE GELS
around 3. These low pH levels are encountered in fruit
juices and pulps; thus, this polymer nds application in the The mechanical stability of gels used to entrap cells is of
manufacture of jams. Although gelation of pectins with a critical importance when gels are subject to bioreactor
low degree of esterication forms gels in the presence of environments, particularly in scaled-up applications. Ex-
calcium ion, the resulting gel strength increases with in- perimentally, mechanical properties of polysaccharide gel
creasing calcium concentration. It was reported that the beads are most frequently measured by uniaxial compres-
gelation of pectin was governed by a mechanism similar to sion at rates in the range of 0.3200 mm/min, as shown in
that of alginate. An egg box model was proposed for the Fig. 6a. The full stressstrain prole during controlled
gelation process similar to that used to describe the gelation compression is recorded. Youngs modulus can be calcu-
of alginate [168,169]. lated from the stressstrain ratio at a very small deforma-
It is reported that Ca-pectate gel might be a better tion, whereas fracture properties are revealed from the
alternative to Ca-alginate because a comparative study fracture point, and fracture stress and strain, and the
showed that pectate gel beads were less sensitive to ion fracture energy are calculated.
and chemical agents, and showed remarkable mechanical Viscoelastic properties of polysaccharide gels were
and operational properties [170, 171]. Furthermore, stabi- examined by a creep or compression and recovery
lization of pectate beads could be achieved by treatment experiment. Cylindrical gels are subject to a constant load
Figure 6 Approaches commonly used in measuring mechanical properties of polysaccharide gel. (a) Schematic illustration of
uniaxial compression test, a stressstrain curve commonly obtained. (b) A simple test to measure the polysaccharide gel beads
fracture ratio after they were placed in hypotonic solution, or agitated in NaCl solution, or with glass beads for specied time
period. The percentages of intact beads were recorded and compared.
for a number of hours, with the gel height before and after relevance of gel mechanical properties (such as stiness,
compression recorded and compared. An oscillating ex- fracture resistance, and elasticity) to the gels resistance to
periment is also carried out to reveal the fatigue pattern of abrasion. In their experiment, abrasion resistance of agar
the gel. A gel cylinder is subject to cyclic compressions up to and n-carrageenan gel beads was evaluated in a 1-m-high
1000 times, and maximum stress resistance is recorded and bubble column. Abrasion resistance was found to be related
compared with that after the rst compression [10,47, more to gel fatigue properties than to fracture properties.
56,167,184187]. Gel strength is also examined by measur- Youngs modulus for various polysaccharide gels is
ing the force required to compress gel beads to a certain shown in Table 3. In general, the mechanical strength of
degree at a constant rate [46,47,188]. Because polysaccha- polysaccharide gel increases with increasing polymer con-
ride gels are viscoelastic materials, the mechanical proper- centration and/or iontropic counterion. The polymer
ties are thus dependent on the rate of compression and composition and counterion type can also aect the
tension. Additionally, polysaccharide gel, as a hydrogel, mechanical strength of the gel. For example, the highest
might suer water loss during the compression test, which mechanical strength of alginate beads is found when the
tends to change the gel structure and subsequent mechan- guluronic monomer content is more than 70% and the
ical behavior. This eect will be even more signicant at average length of the guluronic monomer blocks is about
higher test temperatures [189]. Therefore, the mechanical 15 [46]. In a certain concentration range, Youngs mod-
strength of the gel is only comparable at the same test speed ulus of alginate and gellan gum was found to be propor-
and temperature. tional to the polymer concentration squared [167,184],
The qualitative strength of an individual bead or whereas Youngs modulus increased almost linearly with
capsule can be obtained by its ability to withstand a simple concentration of agar and carrageenan [195]. There is also
compression test applied with tweezers, micrometers, or a correlation between the mechanical gel strength of
microscope slides [190,191]. Quantitative measurements on alginate beads and the polymer anity for cations. As
individual capsules are conducted using a more advanced alginate forms gels in the presence of various multivalent
compression test with a sensitive device designed to mea- cations, gel strength decreases in the following cation
sure resistance to compressive force [46]. Alternative tech- order [46]:
niques involve determining the percentage of intact
capsules remaining after agitation in NaCl solution [53],
agitation with glass beads [192], shearing in a cone and Pb2 > Cu2 Ba2 > Sr2 > Cd2 > Ca2 > Zn2
plate viscometer to determine resistance to uid shear force > Co2 > Ni2
[193], or via an osmotic pressure test [194], as illustrated in
Fig. 6b. In the osmotic pressure test, alginate capsules were
exposed to a gradation of hypotonic solutions and the More recently, Ouwerx et al. [184] reported the
percentages of broken capsules were quantied. Youngs modulus of alginate gel, which decreased in a
Gel abrasion resistance is an important factor in slightly dierent cation order:
considering its applicability in an agitated bioreactor,
where gel beads experience collisions with other beads, Cd2 > Ba2 > Cu2 > Ca2 > Ni2 > Co2
mixing devices, baes, bubbles, or the vessel wall, or are
exposed to liquid shear. Dos Santo et al. [195] examined the > Mn2
876 Yi et al.
Table 3 Comparison of Youngs Moduli of Various Polysaccharide Gels

Crosshead Youngs
Gel material Gel geometry Gelling solution speed modulus (N/m2) References
2% Ca-alginate Gel cylinder (height 20.0 mm; 0.1 M CaCl2 and 6 mm/sec 108,000 F 2000 187
inner diameter 14.2 mm) 0.2 M NaCl
5% PVA-SbQ/2% Gel cylinder (height 20.0 mm; 0.1 M CaCl2 and 6 mm/sec 188,000 F 7000 187
Ca-alginate inner diameter 14.2 mm) 0.2 M NaCl
2% Agar Gel cylinder with a height and N/A 3 mm/min 110,000 (estimated 195
diameter of 20 F 0.5 mm from gure)
2% Carrageenan Gel cylinder with a height and 0.134 M KCl 3 mm/min 120,000 (estimated 195
diameter of 20 F 0.5 mm from gure)
1.5% Alginate Beads with diameter 0.05 M CaCl2 2 mm/min 48,000 (estimated 184
of 2.43 mm from gure)
1.5% Alginate Beads with diameter 0.05 M CdCl2 2 mm/min 80,500 (estimated 184
of 2.43 mm from gure)
0.5% Agarose Gel cylinder with 17 mm N/A 5 mm/min 10,000 186
diameter and 150 mm height
1.0% Agarose Gel cylinder with 17 mm N/A 5 mm/min 36,000 186
diameter and 150 mm height
0.5% Agarose/0.5% Gel cylinder with 17 mm N/A 5 mm/min 14,000 186
guar gum diameter and 150 mm height
1.5% Gellan gum Gel cylinder with height 0.1 M KCl 2 mm/min 290,000 167
of 15 mm and cross-sectional
area of 155 mm2
1.5% Chitosan Beads with diameter of 3 mm 1.5% (wt/vol) 3 mm/min 27,500 (estimated 157
Na4P2O7 from gure)
The cation anity series for pectin gel was reported as IV. HYBRID GEL/COATING/FILLER
[184]:
There is an increasing tendency to improve polysaccharide
gel properties by incorporating other polysaccharides,
Cu2 > Cd2 > Ba2 > Ni2 > Ca2 > Co2 polymers, or llers into the primary polysaccharide gel,
or by applying a coating layer to the gel to enhance its
> Mn2 mechanical, physical, and chemical properties, or to con-
trol the retention or release of various encapsulants.
For gellan gum gel, when monovalent cations were used to
initiate gelation, the strength of the resulting gel increased
in the order: A. Fillers
The addition of ller to a polymer network, like a gel, can
TMA tetramethylammonium < Li < Na < K alter many of its physical characteristics. The changes that
might occur due to the presence of a ller in a primary
< Cs < H polysaccharide gel are:
Increase in density and mechanical properties in terms
In the case of divalent cations, the order was [167]: of hardness, elasticity, shear resistance, and ten-
sile strength
Mg2 ; Ca2 ; Sr2 ; Ba2 < Zn2 < Cu2 < Pb2 Magnetized matrix
Reinforcement of gel
Nutritional supplement
At a crosshead speed of 3 mm/min, agar and carra-
Increased gas permeability.
geenan had almost the same Youngs modulus at the same
concentration, and their Youngs moduli increased in a The llers that have been added to polysaccharide gels
similar way. However, gel response under larger deforma- can be classied into mineral, metallic, and organic llers.
tion exhibited considerable dierences [195]. It was dem- Mineral llers such as activated carbon, silica, kaolin,
onstrated that the rheological properties of deacetylated calcium carbonate, and celite were applied for the purpose
gellan gels are superior to those of other common polysac- of reinforcing the matrix. Carbony iron powder and Fe2O3
charide gels such as agar, n-carrageenan, and alginate at are used to modify the density, or impart magnetic prop-
similar concentrations [37,164]. erties to the gel matrix. Although organic llers such as
starch, skim milk powder, or collagen are mostly used as crobial populations [208]. Skim milk was added into bead
nutrient sources for entrapped cells, additional advantages formulations, providing a nutrient source that may in-
sometimes occur, such as increased mechanical strength. A crease activity, growth, and/or cell survival [203,206].
summary of llers that have been applied in polysaccharide When Pseudomonas uorescens R2f cells were encapsulated
gels containing cells is shown in Table 4. in alginate beads amended with skim milk, higher survival
When Leuconostoc oenos or Lactobacillus was immo- rates were observed compared to either free cells or cells
bilized in carrageenan, the addition of silica gel to the immobilized in alginate alone. Interestingly, even higher
carrageenan matrix improved operational stability in con- survival rates were observed when both bentonite clay and
tinuous fermentation. It was found that silica gel increased skim milk powder were added to alginate beads [204].
the half-life of immobilized L. oenos and induced a higher
conversion rate of L-malic acid to L-lactic acid. This B. Mixed Gel
increase was explained by the increased gel porosity caused
by the doped silica gel [201]. Tal et al. [200] demonstrated Synergistic eects may be observed when two polymers or
that freeze-dried alginate beads containing high concen- polysaccharides are mixed, showing better performance
trations of starch were found to have better mechanical than that of individual polysaccharide gel [209]. Various
properties than beads containing low concentrations of this combinations of polymer and polysaccharide have been
ller. Addition of mineral elements (silica and kaolin) to reported for dierent applications, as summarized in Table
alginate solutions increased resistance to rupture, thus 5. Some specic examples follow.
preserving the viability of somatic embryos at low relative The partition coecient of hydrophobic substrate to
humidities to a greater extent than other matrices [4,6]. It hydrophilic polysaccharide gel matrix was modied by the
was demonstrated that the stability of alginate gel ber was addition of hydrophobic silicone polymer to an alginate
enhanced by adding celite and pectin [198]. To facilitate the gel matrix [214217]. Alginate matrix doped with colloidal
recovery of chitosanalginate microcapsules containing silica showed higher strength than in its absence. The
DNA, carbony iron powder was coencapsulated [199]. A physical strength of the resulting mixed gel increased with
dense, inert material such as Fe2O3 is sometimes added to the amount of colloidal silica added [212]. The solgel
carrageenan matrix to control the density of the gel beads process is an approach used to synthesize porous silica
[207]. glass at room temperature. This process has been widely
For soil applications, clay and skim milk powder studied in the past decade to entrap a large variety of
provide protection and nutrients in both alginate and n- biological materials, including enzymes, antibodies, anti-
carrageenan gel beads [205,206]. Addition of clay improved gens, DNA, RNA, regulatory proteins, membrane-bound
cell survival and mechanical strength of the gel matrix. proteins, and whole cells [225,226]. Rietti-Shati et al. [223]
These positive eects were thought to be due to protection combined the solgel process with alginate entrapment to
of the cells during drying and rehydration due to the conne Pseudomonas in alginatesilicate mixed gel. In
protection provided by a clay layer around the cell, and their experiment, cell-loaded calcium alginate beads were
resulting modied physicochemical characteristics of mi- rst prepared by droplet extrusion. The resulting beads
Table 4 Fillers Used in Polysaccharide Gels Containing Cells

Filler/gel Cell Purpose References
Alumina/alginate Cephalosporium acremonium Cephalosporin C production 196,
Activated carbon/alginate or Sewage sludge Improve matrix gas permeability, 197
acrylic/silicone/alginate density, and mechanical strength
Celite pectin/alginate Yeast cell Good mechanical strength 198
Kaolin/alginate Carrot somatic embryos Articial seeds 4, 6
Carbony iron powder/alginate DNA Magnetize matrix 199
Starch/alginate Pseudomonas sp. Matrix enhancer and carbon source 200
Silica gel/carrageenan L. oenos or Lactobacillus Malolactic fermentation in wine 201
Skim milk/alginate beads Enterobacter sp. Soil application 202
Skim milk powder/alginate Azospirillum Soil application 203
Skim milk powder/alginate Pseudomonas uorescens Soil application 204
Clay/alginate
Skim milk powder/clay/alginate
Clay/alginate or carrageenan Bacteria Soil application 205, 206
Skim milk/alginate or carrageenan
Clay/skim milk/alginate or
carrageenan
878 Yi et al.
Table 5 Mixed Gel

Material Cell Purpose References
Agarpolyethyleneimine Rhodobacter sphaeroides Hydrogen production 45,

Agarpoly-L-lysine R. sphaeroides Hydrogen production 45
Agarosecollagen Human breast Inhibit renal tumor growth 210
adenocarcinoma cells
Agarosepoly (N-p-vinylbenzyl-D- MIN 6 cells Insulin production 211
maltonamide-co-SU)
Agarosepoly(N-p-vinylbenzyl-D- MIN 6 cells Insulin production 211
lactonamide-co-SU)
Alginate/colloidal silica Saccharomyces cerevisiae Enhance mechanical property 212
Alginate/gelatin Recombinant Sustained release of adenovirus 213
adenoviruses (rAds)
Alginategellan Carrot somatic embryos Articial seeds 46
Alginatepolyurethane Sewage sludge Improve matrix gas permeability, 197
density, and mechanical strength
Alginate/silicone prepolymer Pichia pastoris Enhance partition coecient 214,215
of hydrophobic substrate
Alginate/silicone prepolymer Nocardia corallina Enhance partition coecient 216
Alginate/silicone prepolymer Nocardia corallina Enhance partition coecient 217
Alginate/polyacrylate Mammalian cell Enhance mechanical property 218
Alginate/PEG Hybridoma cell Minimize cell leakage and 74,219
high-density growth
Alginate/PEG acrylate Islets of Langerhans Maintain integrity of gel 220
Alginate/PVA Yeast cells Stronger mechanical strength 221
Alginate/PVA-SbQ Nitrogen-reducing More stable and stronger gel 10,185
microorganism
Alginatepoly(N-vinyl pyrrolidone) Activated sludge Better physical property 9
Chlmydomonas reinhardtii Improve mechanical stability 51
Alginate/PVCL Chlmydomonas reinhardtii Improve mechanical stability 51
(poly-vinylcaprolactam)
Collagenalginate/ GH3 rat pituitary GH3 rat pituitary tumor cell 222
poly-L-lysine/alginate tumor cell
Alginatesilicate Pseudomonas Atrizine degradation 223
n-Carrageenangelatin Aerobic and anaerobic Degrading trichlorophenol 144
communities
n-Carrageenan locust bean gum Propionibacterium Production of propionic acid 224
freudenreicgii and acetic acid
Bidobacterium longum Dairy product 146
Chitosankonjac our Scenedesmus bicellularis Wastewater biotreatment 157
Gellanxanthan Bidobacteria Dairy process 156
were then air-dried on lter paper followed by incubation cells within the bead with access to radial diusion chan-
with a mixture of hexane and silicate monomer nels, minimizing local diusional limitations expected with
tetramethoxysilane for 1 day at room temperature. During dense cell masses. PEGalginate hybrid gels are thought to
the incubation, tetramethoxysilane penetrated into algi- provide a void network for high-density cell growth within
nate matrix, using the gelled water for its hydrolysis. The a gel matrix, while at the same time minimizing cell leakage
resulting polymerization of the hydrolyzed tetramethoxy- [219]. AlginatePEG acrylated hybrid gel encapsulating
silane led to the formation of an alginatesilicate inter- islets of Langerhans showed greater mechanical stability
penetrating gel network. Unfortunately, entrapped cells compared to alginate alone. This hybrid gel was stabilized
were inactive following entrapment. both by calciumalginate ionotropic interactions, and
Limitations of the alginate matrix on the growth of through the crosslinking of PEG by photoactivated free
hybridoma cells were minimized by creating a polyethylene radical polymerization. The major advantage anticipated
glycol (PEG)alginate interpenetrating network with radi- with the double-complexed alginate hybrid gel was
al pores. The radial pores provide a means to distribute enhanced chemical stability due to the presence of covalent
bonds. Encapsulated islets were viable and demonstrated medium additive, was then added to the gel. As the cells
insulin secretory function [220]. consumed the gelatin, the gel became more porous, liber-
Polyvinyl pyrrolidonealginate hybrid gel was pre- ating the biogas and reducing the tendency of the beads to
pared to immobilize Chlamydomonas reinhardtii for the oat [144].
purpose of nitrate consumption and removal from nitrate- The rheological and mechanical properties of a poly-
contaminated water [51]. PVA bearing photosensitive stil- saccharide gel can be enhanced by mixing with another
bazolium groups blended with alginate were applied as polysaccharide, such as galactomannan, which includes
carrier in a denitrication process. The hybrid gel showed locust bean gum, carob bean gum, tara gum, and guar
a longer lifespan compared to alginate alone [10,185]. gum [227]. Locust bean gum, extracted from the plant
Polyacrylate was incorporated into alginate gel matrix to Ceratonia siliqua, has been blended frequently with n-
entrap mousemouse hybridoma cells. The resulting mixed carrageenan [145,146,224]. Carob bean gum has also been
gel showed minimized gel destruction in a uidized bed incorporated into carrageenan beads to improve bead
reactor [218]. strength and stability in a bioreactor [228,229]. Guar
In order to lower the gellan gum setting temperature, gum, from the bean plant Cyamopsis tetragonobus, is a
a small amount of xanthan was added to entrap tem- highly substitute galactomannan. Although no signicant
perature-sensitive Bidobateria to be used in food products enhancement of properties has been observed in mixtures
or health supplements. The encapsulated cells showed with n-carrageenan [230], synergistic aects with n-carra-
high tolerance to an acidic environment [156], simulating geenantara gum [231], xanthanguar gum [232], and
the conditions that the cells would be exposed to in the agaroseguar gum gels [186] have been reported.
stomach.
A mixed microbial population degrading 2,4,6-tri- C. Coating
chlorophenol produced biogas, which, when trapped in
n-carrageenan, caused the beads to be buoyant and thus Xerotransplantation is an application that requires the
oat in a bioreactor. Gelatin, which is a common cell introduction of mammalian cells into a host. To immuno-
Table 6 Coating Materials Used in Cell Immobilization
Core materials/coating materials Cell Purpose References

Agarose/polystyrene sulfonic acid/ Pancreatic B cell line MIN6 Biohybrid articial pancreas 233,
polybrene/carboxymethyl cellulose
Alginate/urethane prepolymer Bakers yeast Increase cell viability in 234
(ENT-2000 or PU-6) organic solvent
Alginate/glycol chitosan Islet Immunoisolation 118
Alginate/collagen Human liver (CCL-13) and Mammalian cell carrier 235
mouse broblast (L929) cell
Alginate/solgel-based siliceous layer HepG2 cells Better mechanical property 236
Alginate/poly-L-ornithine/alginate Coencapsulation of rat islets Immunoisolation 237
and Sertolis cells
Alginate/alginate Yeast cells Prevent cell leakage 238
Alginate/ethylene-vinyl Horseradish hairy roots An articial seed 239
acetateacrylic acid terpolymer
Alginate/paran
Alginate/polyorganosiloxane
Alginate/glycidyl methacrylate Guinea pig red blood cells Immunoisolation 240
N-vinylpyrrolidinone
copolymers-2-hydroxyethyl
methacrylate-methacrylic
acid copolymers
Alginate/Eudragit RL 100 Erythrocytes Immunoisolation 241,242
Alginate/PEI/polyacrylacid Pancreatic islets Immunoisolation 243
acid/CMC/alginate
Ba-alginate/polyacrylic acid Parathyroid tissue of pig Xerotransplantation 244
Agartrimethylammonium glycol Rhodobacter sphaeroides Hydrogen production 45
chitosan iodide (TGCI)
Alginate and urethane polymer Hybridoma cells Monoclonal antibody 245
production
Alginate/polyurea Aspergillus ochraceus Hydroxylation of 246
progesterone
880 Yi et al.
isolate the implanted cells, a selective permeable coating is measurement, a diaphragm cell with two well-mixed com-
commonly applied to the capsule. Coating of gel capsules is partments separated by a gel disc is used, where a pseudo-
also intended to control or reduce cell release, but also to steady-state diusional ow is established (method I),
increase mechanical and chemical properties of the gels yielding the eective diusion coecient De [253]. In
[57]. Various coating materials have been used with this nonsteady-state measurements, the diusion of solutes is
purpose as outlined in Tables 2 and 6. Encapsulation of recorded by concentration changes in the supernatant
tissues or cells within a semipermeable membrane presents uid. The diusion coecient (D) and partition coecient
opportunities for cell implantation or transplantation be- (k) are then obtained. Nonsteady-state measurements can
cause the membrane permits the passage of low-molecular- be carried out in a time lag diaphragm cell [254] (method
weight substrates, such as oxygen, nutrients, metabolites, II), nonsteady-state diusion out of a gel sphere or disk
and cell-generated hormones and other products, but not into an innite solution [256] (method III), nonsteady-state
passage of high-molecular-weight immune response anti- diusion into gel spheres [257,261] (method IV), or gel slab
bodies and complements. from a nite solution [262] (method V).
Polyion complexes are commonly used as coating For the case of diusion in gels containing cells,
material. Alginate can form polyion complex with glyco Westrin and Axelsson [251] described two theoretical
chitosan, which is a positively charged polysaccharide and approaches. The rst approach is to regard the cells as
is water-soluble at pH 7.4. The number of layers of glycol impermeable to the diusing solute, or to assume a very
chitosanalginate polyion complex was optimized to pro- low value of the eective diusion coecient within cells.
tect encapsulated islets from host immune reaction [118]. The second approach considers the possibility of a signif-
Alginate beads containing hybridoma cells were coat- icant diusional ux through the cells, and the eective
ed with urethane polymer, showing enhanced gel strength diusion coecient within cells (Dc) will thus be an addi-
and reduced cell leakage compared to that of uncoated tional parameter. They indicated that the rst approach is
beads [245]. Kanda et al. [234] showed that yeast encapsu- the most common and has the advantage of not requiring
lated in alginate can be eectively protected from the Dc values. However, the second is a more general approach
toxicity of organic solvent by coating with a polyurethane because setting Dc to zero is equivalent to the rst ap-
layer derived from hydrophilic photo-crosslinkable resin proach. There are four models (exclusion models, models
prepolymer (ENT-2000) and hydrophilic urethane pre- of suspended impermeable spheres, capillary models, and
polymer (PU-6). Encapsulated yeast were used for the empirical models) developed based on these two theoretical
stereoselective reduction of ethyl-3-oxobutanoate to approaches with dierent assumptions. Their mathemati-
ethyl-(S)-3-hydroxybutanoate in isooctane. Double en- cal representation is shown in Table 7.
trapment ensured yeast viability in isooctane, whereas Korgel et al. [253] examined the eective diusivity of
single entrapment did not provide the needed protection. galactose in calcium alginate beads containing cells. The
Houng et al. [246] coated alginate beads with polyurea with results were compared with other literature data, and it was
the aim of increasing substrate partition coecient from found that the random pore model originally developed by
biphasic media to alginate matrix. Wakao and Smith [264] was the best model to predict the
Chitosan has been reported to increase the mechanical eective diusivity of the sugar in the cell-loaded gel. More
resistance of alginate gel through ionic interactions recently, Mota et al. [265] proposed a homogeneous porous
[247,248]. Hence, Serp et al. [72] showed that the mechan- media model, in which tortuosity terms (Tc and Tg for cell
ical strength of alginate beads could be doubled by coating and gel) were introduced to take into account the eects of
with 510 kDa chitosan. In addition, coated beads resulted cell and gel matrix on eective diusivity.
in reduced loss of cells. In the case of solute transport in cell-free polymer gel,
Poly(methylene coguanidine) was used as a novel Muhr and Blanshard [266] pointed out that the polymer
material for the coating of alginatecellulose sulfate micro- may inuence solute diusion in a variety of ways:
capsules containing islet cells [87,249]. The longer path length and reduced free volume for
solute diusion due to the presence of impene-
trable and slow-moving polymer chains.
V. MASS TRANSFER Increased hydrodynamic drag of the polymersolvent
interface on the moving solute molecules.
It is often important to know the mass transport properties Altered solvent properties due to the presence of
of substrates and products within gel beads to be able to impenetrable and slow-moving polymer chain.
predict or improve the growth or productivity of immobi- The polymer may be involved in the shearing of
lized cells. There are several excellent reviews on the subject polymersolvent and polymersolute bonds and
[33,250252]. Measurements of eective diusivities for bending of polymer chain during solute diusion.
substrate or metabolic products within polysaccharide gels
in the form of beads or membranes have been widely A better understanding of the parameters governing
reported [207,252263]. Experimental methods were clas- solute diusion within the polysaccharide gel matrix and
sied into two main approaches: steady-state and non- the way by which gel matrix aects diusion has been
steady-state measurements, which can be further approached through the development of a number of
subdivided into ve main methods [251]. In steady-state mathematical expressions to model solute diusion in
Table 7 Models Predicting the Dependence of Eective Diusion Coecient on the Cell
Concentration
Model Equation References
Exclusion model De 251
1 /c
De0
Models of suspended De 1 /c 251
impermeable spheres
De0 1 /c =2
Models of suspended De 2=Dc 1=De0 2/c 1=Dc 1=De0 251
permeable spheres
De0 2=Dc 1=De0 /c 1=Dc 1=De0
Capillary models De 251
1 /c 2
De0
Empirical models Fit experiment data to a 251
second-order polynomial
Random pore model De 1 253

De0 1 2:23/c 1:40/2c
Homogeneous porous De ec ec 265
media model
De0 TR Tc ec Tg ec
De is eective diusion coecient in gel; De0 is eective diusion coecient in the parts of a cell-
containing gel that are not occupied by cells; Dc is eective coecient in cells (m2/sec); /c is the cell
volume fraction of gel; /p is the polymer volume fraction of gel; ec = 1 /p is the gel void fraction
(porosity); Tg is tortuosity of gel matrix (e.g., the molecule path tortuosity in a gel matrix for a
dened structure and a dened diusing molecule); Tc is tortuosity due to the presence of cells.
hydrogels. These models, based on either free volume VI. IMMOBILIZATION EFFECTS ON
theory, hydrodynamic theory, obstruction theory, or com- CELL PHYSIOLOGY
bined obstruction and hydrodynamic eects, were reviewed
and tested against literature data [267]. Amsden separated There are many examples in the literature which demon-
these models into those that are best suited to homogeneous strate that cell physiology and morphology are aected by
hydrogels consisting of exible polymers and those appli- immobilization. The factors that may contribute to these
cable to heterogeneous hydrogels, made up of rigid poly- changes include: dierent microenvironments (such as
mer chains. A scaling hydrodynamic model best described ionic strength, ionic charges, pH, and water activity)
solute diusion data with homogeneous gels and obstruc- created by the gel matrix compared to that which cells
tion models best described data with heterogeneous gels. encounter in suspension culture, physical stress exerted by
Diusion coecient data for specic solutes in poly- closely packed growing cells on one another, and mass
saccharide gels, as reported in the literature, are often transfer limitation (oxygen, substrate, and product) im-
inconsistent and thus dicult to compare from case to posed by the gel matrix [57,206,268]. Because polysaccha-
case. A large part of these dierences in results from ride gels are generally regarded as biocompatible, the
laboratory to laboratory may be explained by the fact that factor that most likely inuences cell behavior would be
the polymers used to form gels and coatings are natural mass transfer limitation, which leads to oxygen, nutrient,
materials, and, as such, vary widely in terms of their and product gradients through the gel matrix. This can be
quality, purity, molecular weight (even from batch to seen by the spatial cell distribution changes through the gel
batch), and chemical composition (guluronic acid content matrix before and after fermentation [269,270]. There is
and guluronic block size in alginate, degree of deacetyla- always controversy over the eect of immobilization on cell
tion for chitosan, etc.). In addition, there are often subtle physiology and performance, and the mechanisms behind
dierences in the methodologies used to form the gels, such cell behavioral alterations upon immobilization are often
as concentrations of polymers, use of counterions and their poorly described. Thus, the eects may be considered on a
concentration, type of buer, pH, temperature, and length case-to-case basis.
of gelation period. All of these factors can play a role in Encapsulation techniques may lead to changes in the
determining the structure of the resulting gel and, as a physicochemical properties of the microenvironment, in-
result, the diusion characteristics. There is denitely a uencing cell metabolism. Gillet et al. [271] observed that
need within the discipline to chemically characterize and immobilization of plant cells could substantially enhance
standardize the polymers being used, and their methodol- the production and removal of scopolin. Scopolin-produc-
ogy of gelation and coating. ing cell suspensions accumulate scopolin within cytoplas-
882 Yi et al.
mic compartments, and cell disruption was necessary to exhibit reduced growth and productivities, growth rates
recover this product. In contrast, cells immobilized in and cell densities that are dependent on time, and their
alginate excreted considerable amounts of scopolin, which position in the gel matrix. The microenvironments created
diused out of the gel matrix into the culture media without by a gel matrix are likely dierent than that experienced by
the need to disrupt the cells. Immobilized cells were mor- free cells, leading to changes in cell physiology, growth, and
phologically and physiologically distinct. For example, a biocatalytic activity. The overall eect of immobilization
spherical cell shape was more frequently encountered with on the cell behavior can be estimated using an eectiveness
immobilized cells in contrast to suspension cultures in factor. This eectiveness factor for immobilized cells is
which the cells were more likely to be elongated. based on steady-state reaction diusion models with the
Results similar to that described above were observed, assumption of a homogeneous distribution of cells over the
with other types of immobilized cell lines, in which immo- carrier [285]. In the case of growing immobilized cells, a
bilization enhanced excretion of metabolites: Streptomyces calculation of the eectiveness factor is coupled to a
aureofaciens in n-carrageenan [272], Gibberella fujikuroi in transient reactiondiusion model, which leads to a time-
alginate [273], and Solanum aviculare in alginate [274]. dependent eectiveness factor [285].
The type and concentration of gel may also aect the Prediction of the behavior of immobilized cells within
growth of the immobilized cells. Walsh et al. [275] observed polysaccharide gel, including its growth, substrate con-
that S. cerevisiae demonstrated elongated and lens-shaped sumption, and product formation, is necessary for the
microcolonies in alginate beads produced by external understanding, design, and optimization of immobilized
gelation, in which the alginate concentration increased cell systems. Therefore, dierent models have been devel-
from the core to the periphery of the gel bead. In contrast, oped and subsequently reviewed by Willaert and Baron
S. cerevisiae demonstrated spherical microcolonies in algi- [33]. In their review, the models used to describe the growth
nate beads formed by internal gelation, in which the of single cells and their production kinetics were classied
alginate concentration was uniform throughout the bead, into the unstructured model, in which no intracellular
whereas S. cerevisiae showed nonspherical and irregular- components are considered, or the structured model, in
shaped microcolonies in carrageenan gel beads. which intracellular components are considered. The un-
hTC3 cells experienced a short hindrance in their structured model is commonly used [286,287]. The struc-
metabolic and secretory activity due to growth inhibition tured model was developed by Monbouquette and Ollis
after entrapment in alginate with a high guluronic acid [288]. In their study, intracellular RNA was used as a
content. In contrast, hTC3 cells encapsulated in alginate marker to reect the physiological state of bacteria, hence
with a high mannuronic acid content showed rapid cell intrinsic biocatalytic activity.
growth [276]. The ciliated protozoan Tetrahymena thermo- Alternatively, various models can be classied into
phila, entrapped in solid alginate beads, survived but was steady-state, dynamic, and pseudo-steady-state models
incapable of growth. However, when encapsulated in [33]. Early models only considered steady-state concentra-
hollow alginate spheres, Tetrahymena grew well, reaching tion proles in a gel matrix when cell mass varied slowly or
0.9 107 cells/mL [277]. was uniform throughout the gel [289,290]. Although some-
Immobilization could have either positive or negative times useful for design, steady-state models cannot de-
eects on product formation [278]. Alginate-encapsulated scribe the transient conditions of startup or be responsive
Azotobacter showed around 60 times higher nitrogenase to changing bioreactor conditions [33].
activity than that of free cells [279]. Dembzynski and Dynamic models then are designed to predict the
Jankowski found that Lactobacillus rhamnosus showed evolution of substrate, product, and cells with time. Fac-
lower cell productivity in alginate/starch core capsules than tors that have been taken into account in the dynamic
in free cell culture. Mass transfer limitation was found to models are cell growth, reaction and mass transfer of
account for this decrease [132]. solutes involved, and biomass leakage. Most of those
Immobilized cells showed better resistance against models were constructed based on the rst one or two
toxic compounds such as alcohol [280], external pH factors [281294]. Only a few models considered biomass
[281], repressor [282], or toxic hydrocarbon substrate leakage [295297], although cell losses due to leakage have
[283]. This could be explained by the mass transfer limi- been found experimentally [298,299]. Dierent models to
tation imposed by the immobilization matrix. Cell po- describe polysaccharide gel-entrapped cell systems were
pulations often exhibit better plasmid stability upon constructed and supported by experimental results. How-
encapsulation than suspended cells [284]. This improve- ever, their capability to extrapolate results are an issue
ment was explained by restricted growth in the gel beads [300]. More recently, Laca et al. [297] developed models by
that prevented plasmid loss. coupling cell growth and substrate consumption that oc-
curred in both the gel matrix and liquid medium. To obtain
a more generally applicable model for cells immobilized via
VII. MODELING OF IMMOBILIZED various approaches, they introduced the pore diusion
CELL SYSTEMS model and the homogeneous model. The former model
takes the cell carrier as a heterogeneous structure with
Immobilization matrices impose mass transfer resistances uniformly distributed pores, and the external transfer
involving substrates and products. Conned cells may thus resistant of substrate is regarded as zero. This model is
more applicable for cell conglomerate or adsorbed cell eectiveness factors. With the aim of understanding the
systems. The later model takes the cell carrier as a contin- design and optimization of immobilized cells, models to
uous medium through which diusion is occurring, which describe the growth of single cells and their production
is more suitable in describing cells conned in microcap- kinetics, including its growth, substrate consumption, and
sules and polysaccharide gels. When applied to cells con- product formation, have also been reviewed in this article.
ned in microcapsules, the external transfer resistance of
substrate is regarded as nonzero, whereas in the case of
polysaccharide gel entrapment, the external transfer resis- REFERENCES
tance of substrate is regarded as nonzero. In all cases,
external transfer resistance of immobilized cells is nonzero. 1. Becerra, M.; Baroli, B.; Fadda, A.M.; Blanco Mendez, J.;
When establishing the mass balance of cell and substrate Gonzalez Siso, M.I. Lactose bioconversion by calcium-
alginate immobilization of Kluyveromyces lactis cells.
for their models, they treated cell mass change in the same Enzyme Microb. Technol. 2001, 29, 506512.
way as that with substrate by introducing the term cell 2. Bodeutsch, T.; James, E.A.; Lee, J.M. The eect of
diusivity, which allows the consideration of position immobilization on recombinant protein production in
change of cells incorporated in the nal model. By inserting plant cell culture. Plant Cell Rep. 2001, 20, 562566.
dierent initial and boundary conditions of mass concen- 3. Joki, T.; Machluf, M.; Atala, A.; Zhu, J.; Seyfried, N.T.;
tration and substrate concentration, and by considering the Dunn, I.F.; Abe, T.; Carroll, R.S.; Black, P.M. Contin-
uous release of endostatin from microencapsulated
dierent external resistances to biomass and substrate in engineered cells for tumor therapy. Nat. Biotechnol.
dierent immobilization techniques, those two general 2001, 19, 3539.
models (pore diusion and homogeneous model) have 4. Timbert, R.; Barbotin, J.N.; Thomas, D. Enhancing
been demonstrated to be suitable in describing the dierent carrot somatic embryos survival during slow dehydration,
types of cell immobilization techniques. by encapsulation and control of dehydration. Plant Sci.
Dynamic modeling to describe transient behavior may 1996a, 120, 215222.
5. Timbert, R.; Barbotin, J.N.; Thomas, D. Eect of sole
be reduced to what is described as pseudo-steady-state
and combined pre-treatments on reverse accumulation,
modeling [295,301], where growth and substrate consump- survival and germination of encapsulated and dehydrated
tion and/or product formation rates are treated separately. carrot somatic embryos. Plant Sci. 1996b, 120, 223231.
This approach is valid when the time scale for growth is 6. Timbert, R.; Barbotin, J.N.; Kersulec, A.; Bazinet, C.;
much larger than the time scale for consumption and Thomas, D. Physico-chemical properties of encapsulation
product formation [33]. Under these conditions, the system matrix and germination of carrot somatic embryos.
of partial dierential equations is reduced to ordinary Biotechnol. Bioeng. 1995, 46, 573578.
7. Axtell, R.C.; Guzman, D.R. Encapsulation of the
dierential equations facilitating numerical solution. mosquito fungal pathogen Lagenidium giganteum in
calcium alginate. J. Am. Mosq. Control Assoc. 1987, 3,
450459.
8. Weir, S.C.; Dupuis, S.P.; Providenti, M.A.; Lee, H.;
VIII. CONCLUSIONS
Trevors, J.T. Nutrient-enhanced survival of and phenan-
As described above, cell encapsulation in polysaccharide threne mineralization by alginate-encapsulated and free
Pseudomonas sp. UG14Lr cells in creosote-contaminated
gel has been extensively used in biocontrol, bioremedia- soil slurries. Appl. Microbiol. Biotechnol. 1995, 43, 946
tion, agriculture, metal uptake, and wastewater nutrient 951.
removal. Most recently, considerable eort has been made 9. Doria-Serrano, M.C.; Riva-Palacio, G.; Ruiz-Trevino,
to develop alginate-based microcapsules as gene therapy F.A.; Hernandez-Esparza, M. Poly(N-vinyl pyrrolidone)
delivery system, in which a cell is engineered to secrete a calcium alginate (PVPCa-alg) composite hydrogels:
therapeutic agent for treatment of various diseases. physical properties and activated sludge immobilization
for wastewater treatment. Ind. Eng. Chem. Res. 2002, 41,
A number of improvements of the physicochemical
31633168.
and mechanical properties of polysaccharide gels have 10. Vogelsang, C.; Husby, A.; Ostgaard, K. Functional
been made, in which polysaccharide gels were doped with stability of temperature-compensated nitrication in
various llers, or copolymerized with other polymers or domestic wastewater treatment obtained with PVA-
polysaccharide gel to form mixed gels, or coated with SBQ/alginate gel entrapment. Water Res. 1997, 31,
other polymers. 16591664.
Properties of polysaccharide gels, with or without cell, 11. Dias, J.C.T.; Rezende, R.P.; Linardi, V.R. Biodegrada-
tion of acetonitrile by cells of Candida guilliermondii
such as mechanical property and mass transport property, UFMG-Y65 immobilized in alginate, n-carrageenan and
which are important for its application, have been widely citric pectin. Braz. J. Microbiol. 2000, 31, 6166.
investigated. Approaches to measure eective diusivities 12. Bandhyopadhyay, K.; Das, D.; Maiti, B.R. Solid matrix
and mechanical property were reviewed in this article. characterization of immobilized Pseudomonas putida
Models that have been developed to describe substrate or MTCC 1194 used for phenol degradation. Appl. Micro-
metabolic products transport in polysaccharides have been biol. Biotechnol. 1999, 51, 891895.
13. Hall, B.M.; McLoughlin, A.J.; Leung, K.T.; Trevors, J.T.;
outlined and compared. Lee, H. Transport and survival of alginate-encapsulated
The eect of immobilization on cell physiology and and free luxlac marked Pseudomonas aeruginosa UG2Lr
morphology has been given, and the overall eect of cells in soil. FEMS Microbiol. Ecol. 1998, 26, 5161.
immobilization on cell behavior has been evaluated by 14. Bang, S.S.; Pazirandeh, M. Physical properties and heavy
884 Yi et al.
metal uptake of encapsulated Escherichia coli expressing a formation of polysaccharide gels and network. In
metal binding gene (NCP). J. Microencapsul. 1999, 16, Advances in Carbohydrate Chemistry and Biochemistry;
489499. Wolrom, M.L., Tipson, R.S., Eds.; Academic Press:
15. Fry, I.V.; Mehhorn, R.J. Polyurethane and alginate- New York, 1969; Vol. 24, 267332.
immobilized algal biomass for the removal of aqueous 33. Willaert, R.G.; Baron, G.V. Gel entrapment and micro-
toxic metals. In Emerging Technology for Bioremediation encapsulation: methods, applications and engineering
for Metals; Means, J.L., Hinchee, R.E., Eds.; CRC Press, principles. Rev. Chem. Eng. 1996, 12, 1205.
Inc.: Boca Raton, FL, 1994; 130134. 34. Murano, E. Use of natural polysaccharides in the micro-
16. Chevalier, P.; Noue de la, J. Wastewater nutrient removal encapsulation techniques. J. Appl. Ichthyol. 1998, 14, 245
with microalgae immobilized in carrageenan. Enzyme 249.
Microb. Technol. 1985, 7, 621624. 35. Brodelius, P.; Nilsson, K. Entrapment of plant cells in
17. Cirone, P.; Bourgeois, J.M.; Austin, R.C.; Chang, P.L. A dierent matrices. A comparative study. FEBS Lett. 1980,
novel approach to tumor suppression with microencapsu- 122, 312316.
lated recombinant cells. Hum. Gene Ther. 2002, 13, 1157 36. Neufeld, R.J.; Peleg, Y.; Rokem, J.S.; Pines, O.; Gold-
1166. berg, I. L-malic acid formation by immobilized Saccha-
18. Xu, W.; Liu, L.; Charles Ian, G. Microencapsulated romyces cerevisiae amplied for fumarase. Enzyme
iNOS-expressing cells cause tumor suppression in mice. Microb. Technol. 1991, 13, 991996.
FASEB J. 2002, 16; 213215. 37. De Smet Poncelet, B.; Poncelet, D.; Neufeld, R.J.
19. Thorsen, F.; Read, T.A.; Lund-Johansen, M.; Tysnes, Emerging techniques, materials, and applications in cell
B.B.; Bjerkvig, R. Alginate-encapsulated producer cells: a immobilization. In Fundamentals of Animal Cell Encap-
potential new approach for the treatment of malignant sulation and Immobilization; Goosen, M.F.A., Ed.; CRC
brain tumors. Cell Transplant. 2000, 9, 773783. Press, Inc.: Boca Raton, FL, 1993; 297314.
20. Read, T.-A.; Farhadi, M.; Bjerkvig, R.; Olsen, B.R.; 38. Nilsson, K.; Brodelius, P.; Mosbach, K. Entrapment of
Rokstad, A.M.; Huszthy, P.C.; Vajkoczy, P. Intravital microbial and plant cells in beaded polymers. Methods
microscopy reveals novel antivascular and antitumor Enzymol. 1987, 135, 222228.
eects of endostatin delivered locally by alginate-encap- 39. Shimada, A.; Koda, T.; Nakamura, I. Concanavalin A
sulated cells. Cancer Res. 2001, 61, 68306837. agarose gel system capable of accumulating extracelluar
21. Yu, S.-H.; Buchholz, R.; Kim, S.-K. Encapsulation of rat glucoamylase produced by immobilized Saccharomycopsis
hepatocyte spheroids for the development of articial buligera. J. Ferment. Bioeng. 1998, 85, 542545.
liver. Biotechnol. Tech. 1999, 13; 609614. 40. Nilsson, K.; Birnbaum, S.; Flygare, S.; Liuse, L.;
22. Papas, K.K.; Long, R.C. Jr.; Sambanis, A.; Constantini- Schroder, U.; Jeppsson, U.; Larsson, P.O.; Mosbach,
dis, I. Development of a bioarticial pancreas: II. Eects K.; Brodelius, P. A general method for the immobilization
of oxygen on long-term entrapped hTC3 cell cultures. of cells with preserved viability. Eur. J. Appl. Microbiol.
Biotechnol. Bioeng. 1999, 66, 231237. Biotechnol. 1990, 17, 319326.
23. Soon-Shiong, P. Treatment of type I diabetes using en- 41. Yokoi, H.; Tokushige, T.; Hirose, J.; Hayashi, S.;
capsulated islets. Adv. Drug Deliv. Rev. 1999, 35, 259270. Takasaki, Y. Hydrogen production by immobilized cells
24. Brissova, M.; Anilkumar, A.V.; Shahrokhi, K.; Wang, T.; of aciduric Enterobacter aerogenes strain HO-39. J.
Powers, A.C. Pancreatic islet transplantation: device Ferment. Bioeng. 1997, 83, 481484.
biocompatibility and cell function. Polym. Prep. 1998, 42. Perrot, F.; Jouenne, T.; Feuilloley, M.; Vaudry, H.;
39, 253254. Junter, G.A. Gel immobilization improves survival of
25. Garcia-Martin, C.; Chuah Marinee, K.L.; Van Damme, Escherichia coli under temperature stress in nutrient-poor
A.; Robinson, K.E.; Vanzieleghem, B.; Saint-Remy, J.- natural water. Water Res. 1998, 32, 35213526.
M.; Gallardo, D.; Ofosu, F.A.; Vandendriessche, T.; 43. Lebeau, T.; Junter, G.A.; Jouenne, T.; Robert, J.M.
Hortelano, G. Therapeutic levels of human Factor VIII in Marennine production by agar-entrapped Haslea ostrea-
mice implanted with encapsulated cells: potential for gene ria Simonsen. Biores. Technol. 1999, 67, 1317.
therapy of haemophilia A. J. Gene Med. 2002, 4, 215223. 44. Lebeau, T.; Gaudin, P.; Junter, G.A.; Mignot, L.; Robert,
26. Tsang, P.W.; Kwan, H.C.; Sun, A.M. Striatal transplants J.M. Continuous marennine production by agar-entrap-
of microencapsulated bovine chroman cells reduces ped Haslea ostrearia using a tubular photobioreactor with
rotational behavior in the rat model of parkinsonism. internal illumination. Appl. Microbiol. Biotechnol. 2000,
23rd Proceedings of the International Symposium on 54, 634640.
Controlled Release of Bioactive Materials; 1996; 67. 45. Zhu, H.; Wakayama, T.; Suzuki, T.; Asada, Y.; Miyake,
27. De Haan, B.J.; van Goor, H.; De Vos, P. Processing of J. Entrapment of Rhodobacter sphaeroides RV in cationic
immunoisolated pancreatic islets: implications for histo- polymer/agar gels for hydrogen production in the
logical analyses of hydrated tissue. Biotechniques 2002, presence of NH4+. J. Biosci. Bioeng. 1999, 88, 507
32, 612614, 616, 618629. 512.
28. Duckworth, M.; Yaphe, W. The structure of agar: Part 1. 46. Smidsrd, O.; Skjak-Brk, G. Alginate as immobilization
Fractionation of a complex mixture of polysaccharides. matrix for cells. Tibtech 1990, 8, 7178.
Carbohydr. Polym. 1971, 29, 209215. 47. Martinsen, A.; Skjak-Brk, G.; Smidsrd, O. Alginate as
29. Clark, A.H.; Ross-Murphy, S.B. Structure and mechan- immobilization material: 1. Correlation between chemical
ical properties of biopolymer gels. Advances in Polymer and physical properties of alginate gel beads. Biotechnol.
Science; Springer-Verlag: Berlin, 1987; Vol. 83, 57192. Bioeng. 1989, 33, 7989.
30. Arnott, S.; Fulmer, A.; Scott, W.E.; Dea, I.C.M.; Moor- 48. Draget, K.I.; Skjak-Brk, G.; Christensen, B.E.; Gaserd,
house, R.; Rees, D.A. The agarose double helix and its O.; Smidsrd, O. Swelling and partial solubilization of
function in agarose gel structure. J. Mol. Biol. 1974, 90, alginic acid gel beads in acidic buer. Carbohydr. Polym.
269284. 1996, 29, 209215.
31. Reid, D.S.; Bryce, T.A.; Clark, A.H.; Rees, D.A. Helix 49. Park, H.-J.; Khang, Y.-H. Production of cephalosporin C
coil transition in gelling polysaccharides. Faraday Dis- by immobilized Cephalosporin acremonium in polyethyle-
cuss. 1974, 57, 230237. neimine-modied barium alginate. Enzyme Microb. Tech-
32. Rees, D.A. Structure, conformation and mechanism in the nol. 1995, 17, 408412.
50. Park, Y.G.; Iwata, H.; Ikada, Y. Microencapsulation of 69. Begin, F.; Castaigne, F.; Goulet, J. Production of alginate
islets and model beads with a thin alginate-Ba++ gel layer beads by a rotative atomizer. Biotechnol. Tech. 1991, 5,
using centrifugation. Polym. Adv. Technol. 1998, 9, 734 459464.
739. 70. Ogbanna, J.C.; Matsumura, M.; Kataoka, H. Eective
51. V lchez, C.; Gatbayo, I.; Markvicheva, E.; Galvan, F.; oxygenation of immobilized cells through the reduction in
Leon, R. Studies on the suitability of alginate-entrapped bead diameter: a review. Process Biochem. 1991, 26, 109
Chlamydomonas reinhardtii cells for sustaining nitrate 121.
consumption processes. Biores. Technol. 2001, 78, 5561. 71. Hulst, A.C.; Tramper, J.; Vant Riet, K.; Westerbeek,
52. Larroche, C.; Gros, J.B. Special transformation processes J.M.M. A new technique for the production of immobi-
using fungal spores and immobilized cells. Adv. Biochem. lized biocatalyst in large quantities. Biotechnol. Bioeng.
Eng. Biotechnol. 1997, 55, 179220. 1985, 27, 870876.
53. Wang, Y.J. Development of new polycations for cell 72. Serp, D.; Cantana, E.; Heinzen, C.; von Stockar, U.;
encapsulation with alginate. Mater. Sci. Eng., 2000, C 13, Marison, I.W. Characterization of an encapsulation de-
5963. vice for the production of monodisperse alginate beads for
54. Park, H.-J.; Khang, Y.-H. Studies of repeated fed-batch cell immobilization. Biotechnol. Bioeng. 2000, 70, 4153.
fermentation of cephalosporin C in an immobilized cell 73. Brandenberger, H.; Nussli, D.; Piech, V.; Widmer, F.
bioreactor. J. Microbiol. Biotechnol. 1995b, 5, 229233. Monodisperse particle production: a new method to
55. Bodalo, A.; Bastida, J.; Gomez, J.L.; Alcaraz, I.; Asanza, prevent drop coalescence using electrostatic forces. J.
M.L. Stabilization studies of L-aminoacylase-producing Electrost. 1997, 45, 227238.
Pseudomonas sp. BA2 immobilized in calcium alginate gel. 74. Seifert, D.; Band Philips, J.A. Production of small,
Enzyme Microb. Technol. 1997, 21, 6469. monodisperse alginate beads for cell immobilization.
56. Kuo, C.K.; Ma, P.X. Ionically crosslinked alginate Biotechnol. Prog. 1997, 13, 562568.
hydrogels as scaolds for tissue engineering: Part 1. 75. Fagerquist, R. Jet, wave, and droplet velocities for
Structure, gelation rate and mechanical properties. Bio- continuous uid jet. J. Imag. Technol. 1996, 40, 405411.
materials 2001, 22, 511521. 76. Gotoh, T.; Honda, H.; Shiragami, N.; Unno, H. Forced
57. Groboillot, A.F.; Boadi, D.K.; Poncelet, D.; Neufeld, R.J. breakup of a power-law uid jet discharged from an
Immobilization of cells for application in the food orice. J. Chem. Eng. Jpn. 1991, 24, 799801.
industry. Crit. Rev. Biotechnol. 1994, 14, 75107. 77. Schwinger, C.; Koch, S.; Jahnz, U.; Wittlich, P.; Rainov,
58. Poncelet, D.; de Smet, B.; Beaulieu, C.; Neufeld, R.J. N.G.; Kressler, J. High throughput encapsulation of
Scale-up of gel bead and microcapsule production in cell murine broblasts in alginate using the JetCutter tech-
immobilization. In Fundamentals of Animal Cell En- nology. J. Microencapsul. 2002, 19, 273328.
capsulation and Immobilization; Goosen, M.F.A., Ed.; 78. Prusse, U.; Fox, B.; Kirchhof, M.; Bruske, F.; Breford, J.;
CRC Press, Inc.: Boca Raton, FL, 1993, 113142. Vorlorp, K.D. New process (jet cutting method) for the
59. Su, H.; Bajpai, R.; Preckshot, G.W. Characterization of production of spherical beads from highly viscous
alginate beads formed by a two uid annular atomizer. polymer solutions. Chem. Eng. Technol. 1998, 21, 2933.
Appl. Biochem. Biotechnol. 1989, 20/21, 561569. 79. Hamad, A.; Al-Hajry, H.A.; Al-Maskry, S.A.; Al-
60. Ryu, D.D.Y.; Kim, H.S.; Taguchi, H. Intrinsic fermenta- Kharousi, L.M.; El-Mardi, O.; Shayya, W.H.; Goosen,
tion kinetic parameters of immobilized yeast cells. J. M.F.A. Electrostatic encapsulation and growth of plant
Ferment. Technol. 1984, 62, 255261. cell cultures in alginate. Biotechnol. Prog. 1999, 15, 768
61. Levee, M.G.; Lee, G.M.; Paek, S.H.; Palsson, B.O. 774.
Microencapsulated human bone-marrow cultures: a po- 80. Goosen, M.F.A.; Al-Ghafri, A.S.; El-Mardi, O.; Al-
tential culture system for the clonal outgrowth of Belushi, M.I.J.; Al-Hajri, H.A.; Mahmoud, E.S.E.; Con-
hematopoietic progenitor cells. Biotechnol. Bioeng. solacion, E.C. Electrostatic droplet generation for encap-
1994, 43, 734739. sulation of somatic tissue: assessment of high voltage
62. Klein, J.; Stock, J.; Vorlop, K.D. Pore size and properties power supply. Biotechnol. Prog. 1997, 13, 497502.
of spherical Ca-alginate biocatalysts. Eur. J. Appl. 81. Goosen, M.F.A.; Mahmoud, E.S.E.; Al-Ghafri, A.S.; Al-
Microbiol. Biotechnol. 1983, 18, 8691. Hajri, H.A.; Al-Sinani, Y.S.; Bugarski, B. Immobilization
63. Klein, J.; Eng, H. Immobilization of microbial cells in of cells using electrostatic droplet generation. In Methods in
epoxy carrier system. Biotechnol. Lett. 1979, 1, 171176. Molecular Biology: Immobilization of Enzymes and Cells;
64. Klein, J.; Hackel, U.; Wagner, F. Phenol degradation by Bickersta, G., Ed.; Humana Press: New Jersey, 1997; 167
Canida tropicalis whole cells entrapped in polymer anionic 174.
network. ASC Symp Ser. 1979, 106, 101118. 82. Poncelet, D.; Bugarski, B.; Amsden, B.G.; Zhu, J.;
65. Champagne, C.P.; Blahuta, N.; Brion, F.; Gagnon, C. A Neufeld, R.J.; Goosen, M.F.A. A parallel-plate electro-
vortex-bowl disk atomizer system for the production of static droplet generator: parameters aecting microbeads
alginate beads in a 1500-liter fermenter. Biotechnol. size. Appl. Microbiol. Biotechnol. 1994, 42, 251255.
Bioeng. 2000, 68, 681688. 83. Bugarski, B.; Li, Q.; Goosen, M.F.A.; Poncelet, D.;
66. Kwok, K.K.; Groves, M.J.; Burgess, D.J. Production of Neufeld, R.J.; Vunjak, G. Electrostatic droplet genera-
515 Am diameter alginate polylysine microcapsules by tion: investigation of mechanism of polymer droplet
air-atomization technique. Pharm. Res. 1991, 8, 341344. formation. AIChE J. 1994, 40, 10261031.
67. Siemann, M.; Muller-Hurting, R.; Wagner, F. Character- 84. Bugarski, B.; Amsden, B.; Neufeld, R.J.; Poncelet, D.;
ization of the rotating nozzle-ring technique for the Goosen, M.F.A. Eect of electrode geometry and charge
production of small spherical biocatalysts. Physiological on the production of polymer microbeads by electro-
of immobilized cells. Proceedings of an International statics. Can. J. Chem. Eng. 1994, 72, 517521.
Symposium, Wageningen, The Netherlands, 1990; 275 85. Poncelet, D.; Lencki, R.; Beaulieu, C.; Halle, J.P.;
282. Neufeld, R.J.; Fournier, A. Production of alginate beads
68. Senuma, Y.; Lowe, C.; Zweifel, Y.; Hilborn, J.G.; by emulsication/internal gelation: I. Methodology. Appl.
Marison, I. Alginate hydrogel microspheres and micro- Microbiol. Biotechnol. 1992, 38, 3945.
capsules prepared by spinning disk atomization. Biotech- 86. Poncelet, D.; De Smet, B. Poncelet; Beaulieu, C.; Huguet,
nol. Bioeng. 2000, 67, 616622. M.L.; Fournier, A.; Neufeld, R.J. Production of alginate
886 Yi et al.
beads by emulsication/internal gelation: Part 2. Physico- cyte viability and CYPIA1 activity during encapsulation.
chemistry. Appl. Microbiol. Biotechnol. 1995, 43 (4), 344 Artif. Cells Blood Substit. Immobil. Biotechnol. 2000, 28,
350. 215227.
87. Lac k, I.; Bris s ova, M.; Anilkumar, A.V.; Powers, A.C.; 103. Machluf, M.; Orsola, A.; Atala, A. Controlled release of
Wang, T. New capsule with tailored properties for the therapeutic agents: slow delivery and cell encapsulation.
encapsulation of living cells. J. Biomed. Mater. Res. 1998, World J. Urol. 2000, 18, 8083.
34, 5260. 104. Cheng, W.T.K.; Chen, B.-C.; Chiou, S.-T.; Chen, C.-M.
88. Stange, J.; Mitzner, S.; Dautzenberg, H.; Ramlow, W.; Use of nonautologous microencapsulated broblasts in
Knippel, M.; Steiner, M.; Ernst, B.; Klinkmann, R.; growth hormone gene therapy to improve growth of
Klinkmann, H. Prolonged biochemical and morphological midget swine. Hum. Gene Ther. 1998, 9, 19952003.
stability of encapsulated liver cellsa new method. Bio- 105. Prakash, S.; Chang, T.M. Growth kinetics of genetically
mater. Artif. Cells Immobil. Biotechnol. 1993, 21, 343352. engineered E. coli DH 5 cells in articial cell APA
89. Lim, F.; Sun, A.M. Microencapsulated islets as bioarti- membrane microcapsules: preliminary report. Artif. Cells
cial endocrine pancreas. Science 1980, 210, 908910. Blood Substit. Immobil. Biotechnol. 1999, 27, 291301.
90. Basic, D.; Vacek, I.; Sun, A.M. Microencapsulation and 106. Ross, C.J.; Ralph, M.; Chang, P.L. Delivery of recombi-
transplantation of genetically engineered cells: a new ap- nant gene products to the central nervous system with
proach to somatic gene therapy. Artif. Cells Blood Substit. nonautologous cells in alginate microcapsules. Hum.
Immobil. Biotechnol. 1996, 24, 219255. Gene Ther. 1999, 10, 4959.
91. Morikawa, N.; Iwata, H.; Fujii, T.; Ikada, Y. An 107. Chen, J.-P.; Chu, I.-M.; Shiao, M.-Y.; Hsu, B.R.-S.; Fu,
immuno-isolative membrane capable of consuming cyto- S.-H. Microencapsulation of islets in PEG-amine modied
lytic complement proteins. J. Biomater. Sci. Polym. Ed. alginate-poly(L-lysine)-alginate microcapsules for con-
1996, 8, 225236. structing bioarticial pancreas. J. Ferment. Bioeng. 1998,
92. Date, I.; Miyoshi, Y.; Ono, T.; Imaoka, T.; Furuta, T.; 86, 185190.
Asari, S.; Ohmoto, T.; Iwata, H. Preliminary report of 108. Lim, F. Substances with encapsulated cells. US Patent
polymer-encapsulated dopamine-secreting cell grafting 4409331, 1983.
into the brain. Cell Transplant. 1996, 5 (5 Suppl 1), S17 109. Hsu, Y.L.; Chu, I.M. Poly(ethylenimine)-reinforced
S19. liquid-core capsules for the cultivation of hybridoma
93. Miyoshi, Y.; Date, I.; Ohmoto, T.; Iwata, H. Histological cells. Biotechnol. Bioeng. 1992, 40, 13001308.
analysis of microencapsulated dopamine-secreting cells in 110. Sun, A.M.; OShea, G.M.; Goosen, M.F.A. Injectable
agarose/poly (styrene sulfonic acid) mixed gel xenotrans- microencapsulated islet cells as a bioarticial pancreas.
planted into the brain. Exp. Neurol. 1996, 138, 169175. Appl. Biochem. Biotechnol. 1984, 10, 8799.
94. Orive, G.; Hernandez, R.M.; Gascon, A.R.; Igartua, M.; 111. Jarvis, A.P., Jr.; Demande, F. Recovery and purication
Rojas, A.; Pedraz, J.L. Microencapsulation of an anti-VE- of a substance developed but not excreted by cells, by
cadherin antibody secreting 1B5 hybridoma cells. Bio- encapsulation and lysis of the cell membrane. US Patent
technol. Bioeng. 2001, 76, 285294. 4582799, 1984.
95. Rokstad, A.M.; Holtan, S.; Strand, B.; Steinkjer, B.; 112. Sakai, S.; Ono, T.; Ijima, H.; Kawakami, K. Synthesis and
Ryan, L.; Kulseng, B.; Skjak-Braek, G.; Espevik, T. transport characterization of alginate/aminopropyl-sili-
Microencapsulation of cells producing therapeutic pro- cate/alginate microcapsule: application to bioartical
teins: optimizing cell growth and secretion. Cell Trans- pancreas. Biomaterials 2001, 22, 28272834.
plant. 2002, 11, 313324. 113. Hunkeler, D.; Prokop, A.; Powers, A.; Haralson, M.;
96. Khan, A.A.; Capoor, A.K.; Parveen, N.; Naseem, S.; DiMari, S.; Wang, T. A screening of polymers as
Venkatesan, V.; Habibullah, C.M. In vitro studies on a biomaterials for cell encapsulation. Polym. News 1997,
bioreactor module containing encapsulated goat hepato- 22, 232240.
cytes for the development of bioarticial liver. Indian J. 114. Canaple, L.; Nurdin, N.; Angelova, N.; Hunkeler, D.;
Gastroenterol. 2002, 21, 5558. Desvergne, B. Development of a coculture model of en-
97. Lanza, R.P.; Jackson, R.; Sullivan, A.; Ringeling, J.; capsulated cells. Ann. N.Y. Acad. Sci. 2001, 944, 350361.
McGrath, C.; Kuhtreiber, W.; Chick, W.L. Xenotrans- 115. Koo, S.M.; Cho, Y.-H.; Huh, C.-S.; Baek, Y.-J.; Park, J.
plantation of cells using biodegradable microcapsules. Improvement of the stability of Lactobacillus casei YIT
Transplant 1999, 67, 11051111. 9018 by microencapsulation using alginate and chitosan.
98. Kompala, D.S.; Elias, C.B.; Perry, W.B.; Plant, J.K. J. Microbiol. Biotechnol. 2001, 11, 376383.
Characterization of insulin secretion from islet cell lines 116. Hardikar, A.A.; Risbud, M.V.; Bhonde, R.R. Improved
encapsulated in P-L-L and chitosan microspheres. Pro- post-cryopreservation recovery following encapsulation of
ceedings of the 216th ACS National Meeting, Washing- islets in chitosanalginate microcapsules. Transplant.
ton, DC (BIOT-41), 1998. Proc. 2000, 32, 824825.
99. Mamujee, S.N.; Zhou, D.; Wheeler, M.B.; Vacek, I.; Sun, 117. Chandy, T.; Mooradian, D.L.; Rao, G.H.R. Evaluation
A.M. Evaluation of immunoisolated insulin-secreting of modied alginatechitosanpolyethylene glycol micro-
hTC6-F7 cells as a bioarticial pancreas. Ann. Trans- capsules for cell encapsulation. Artif. Organs 1999, 23,
plant. 1997, 2, 2732. 894903.
100. Yu, S.-C.; Chen, J.-P.; Liu, H.-S.; Hsu, B.R.-S.; Fu, S.-H. 118. Sakai, S.; Ono, T.; Ijima, H.; Kawakami, K. Control of
Macrophages as an eector mechanism to reject encapsu- molecular weight cut-o for immunoisolation by multi-
lated hepatoma cells. Transplant. Proc. 2000, 32, 958959. layering glycol chitosanalginate polyion complex on
101. Stockley, T.L.; Robinson, K.E.; Delaney, K.; Ofosu, F.A.; alginate-based microcapsules. J. Microencapsul. 2000,
Frederick, A.; Chang, P.L. Delivery of recombinant 17, 691699.
product from subcutaneous implants of encapsulated 119. Speaker, T.J.; Sultzbaugh, S.; Kenneth, J. Microcapsules
recombinant cells in canines. J. Lab. Clin. Med. 2000, of pre-determined peptide(s) specicity(ies), their prepara-
135, 484492. tion and uses. PCT International Application, WO Patent
102. Wang, L.; Sun, J.; Li, L.; Harbour, C.; Mears, D.; 9629059, 1996.
Koutalistras, N.; Sheil, A.G.R. Factors aecting hepato- 120. Munkittrick, T.W.; Nebel, R.L.; Saacke, R.G. Accessory
sperm numbers for cattle inseminated with protamine junction zones in gels of iota and kappa carrageenan. In
sulfate microcapsules. J. Dairy Sci. 1992, 75, 725731. Gums and Stabilizer for the Food Industry 4; Phillips, G.O.,
121. Tobias, C.A.; Dhoot, N.O.; Wheatley, M.A.; Tessler, A.; Williams, P.A., Wedlock, D.J., Eds.; IRL Press: Wash-
Murray, M.; Fischer, I. Grafting of encapsulated BDNF- ington, DC, 1988; 127134.
producing broblasts into the injured spinal cord without 139. Chao, K.C.; Haugen, M.M.; Royer, G.P. Stabilization of
immune suppression in adult rats. J. Neurotrauma 2001, n-carrageenan gel with polymeric amines: use of immobi-
18, 287301. lized cells as biocatalysts at elevated temperatures.
122. Wang, F.F.; Wu, C.R.; Wang, Y.J. Preparation and Biotechnol. Bioeng. 1986, 28, 12891293.
application of poly (vinylamine)/alginate microcapsules to 140. Klein, J.; Vorlop, K.D. Immobilization techniquescells.
culturing of a mouse erythroleukemia cell line. Biotechnol. In Comprehensive Biotechnology; Moo-Young, M., Coo-
Bioeng. 1992, 40, 11151118. ney, C.L. Humphrey, A.E. Eds.; Pergamon Press, Ltd.:
123. Lu, M.Z.; Lan, H.L.; Wang, F.F.; Chang, S.J.; Wang, UK, 1985; Vol. 2, 203224.
Y.J. Cell encapsulation with alginate and a-phenoxycin- 141. Guiseley, K.B. Chemical and physical properties of algal
namylidene-acetylated poly-(allylamine). Biotechnol. Bio- polysaccharides used for cell immobilization. Enzyme
eng. 2000, 70, 479483. Microbiol. Technol. 1989, 11, 706716.
124. Lu, M.Z.; Lan, H.L.; Wang, F.F.; Wang, Y.J. A novel cell 142. Stanley, N. Production, properties and uses of carra-
encapsulation method using photosensitive poly (allyl- geenans. In Production and Utilization of Products from
amine a-cyanocinnamylideneacetate). J. Microencapsul. Commercial Seaweeds; McHugh, D.J., Ed.; FAO Fishing
2000, 17, 245251. Technical Paper; Food and Agriculture Organization of
125. Chang, S.J.; Lee, C.H.; Wang, Y.J. Microcapsules the United Nations: Rome, Italy; 1987:116146.
prepared from alginate and a photosensitive poly(L- 143. Takata, I.; Kayashima, K.; Tosa, T.; Chitaba, I. Improve-
lysine). Biomater. Sci. Polym. Ed. 1999, 10, 531542. ment of stability of fumarase activity of Brevibacterium
126. Dautzenberg, H.; Schuldt, U.; Grasnick, G.; Karle, P.; avum by immobilization with n-carrageenan and poly-
Muller, P.; Lohr, M.; Pelegrin, M.; Piechaczyk, M.; ethyleneimine. J. Ferment. Technol. 1982, 60, 431437.
Rombs, K.V.; Gunzburg, W.H.; Salmons, B.; Saller, 144. Gardin, H.; Pauss, A. n-Carrageenan/gelatin gel beads for
R.M. Development of cellulose sulfate-based polyelectro- the co-immobilization of aerobic and anaerobic microbial
lyte complex microcapsules for medical applications. Ann. communities degrading 2,4,6-trichlorophenol under air-
N.Y. Acad. Sci. 1999, 875, 4663. limited conditions. Appl. Microbiol, Biotechnol. 2001, 56,
127. Foerster, M.; Mansfeld, J.; Dautzenberg, H.; Schellen- 517523.
berger, A. Immobilization in polyelectrolyte complex 145. Lamboley, L.; Lacroix, C.; Artignan, J.M.; Champagne,
capsules: encapsulation of a gluconate-oxidizing Serratia C.P.; Vuillemard, J.C. Long-term mechanical and bio-
marcescens strain. Enzyme Microb. Technol. 1996, 19, logical stability of an immobilized cell reactor for con-
572577. tinuous mixed-strain mesophilic lactic starter production
128. Zielinski, B.A.; Aebischer, P. Chitosan as a matrix for in whey permeate. Biotechnol. Prog. 1999, 15, 646654.
mammalian cell encapsulation. Biomaterials 1994, 15, 146. Maitrot, H.; Paquin, C.; Larcoix, C.; Champagne, C.P.
10491056. Production of concentrated freeze-dried cultures of
129. Yoshioka, T.; Hirano, R.; Shioya, T.; Kako, M. Bidobacterium longum in n-carrageenan-locust bean
Encapsulation of mammalian cell with chitosan-CMC gum gel. Biotechnol. Tech. 1997, 11, 527531.
capsule. Biotechnol. Bioeng. 1990, 35, 6672. 147. Lamboley, L.; Lacroix, C.; Champagne, C.P.; Vuillemard,
130. Jankowski, T.; Zielinska, M.; Wysakowska, A. Encapsu- J.C. Continuous mixed strain mesophilic lactic starter
lation of lactic acid bacteria with alginate/starch capsules. production in supplemented whey permeate medium using
Biotechnol. Tech. 1997, 11, 3134. immobilized cell technology. Biotechnol. Bioeng. 1997, 56,
131. Yoo, I.; Seong, G.H.; Chang, H.N.; Park, J.K. Encapsu- 502516.
lation of Lactobacillus casei cells in liquid-core alginate 148. Lacroix, C.; Paquin, C.; Arnaud, J.P. Batch fermentation
capsules for lactic acid production. Enzyme Microb. with entrapped growing cells of Lactobacillus casei.
Technol. 1996, 19, 428433. Optimization of rheological properties of the entrapped
132. Dembzynski, R.; Jankowski, T. Growth characteristics gel matrix. Appl. Microbiol. Biotechnol. 1990, 32, 403
and acidifying activity of Lactobacillus rhamnosus in 408.
alginate/starch liquid-core capsules. Enzyme Microb. 149. Czaczyk, K.; Olejnik, A.; Trojanowska, K. The inuence
Technol. 2002, 31, 111115. of LBG addition to carrageenan on the mechanical
133. Skjak-Brk, G.; Smidsrod, O.; Larsen, B. Tailoring of stability of the gel and the fermentative activity of
alginates by enzymatic modication in vitro. Int. J. Biol. immobilized propionic acid bacteria. Acta Biotechnol.
Macromol. 1986, 8, 330336. 1999, 19, 147156.
134. Draget, K.I.; Strand, B.; Hartmann, M.; Valla, S.; 150. Moon, S.H.; Parulekar, S.J. Characterization of n-
Smidsrod, O.; Skjak-Braek, G. Ionic and acid gel carrageenan gels used for immobilization of Bacillus
o
formation by epimerised alginates; the eect of AlgE4. rmus. Biotechnol. Prog. 1991, 7, 516525.
Int. J. Biol. Macromol. 2000, 27, 117122. 151. Suzuki, T.; Mizushima, Y. Characteristics of silica
135. Chibata, I.; Tosa, T.; Sato, T.; Takata, I. Immobilization chitosan complex membrane and their relationships to
of cells in carrageenan. Methods Enzymol. 1987, 135, 189 the characteristics of growth and adhesiveness of L-929
198. cells cultured on the biomembrane. J. Ferment. Bioeng.
136. Rinaudo, M. Gelation of ionic polysaccharides. In Gums 1997, 84, 128132.
and Stabilizer for the Food Industry 4; Philips, G.O., 152. Dumitriu, S.; Vidal, P.; Chornet, S. Enzyme immobiliza-
Williams, P.A., Wedlock, D.J., Eds.; IRL Press: Oxford, tion using chitosanxanthan complexes. In Methods in
1988; 119134. Biotechnology: Vol. 1. Immobilization of Enzymes and
137. Borchard, W. Thermoreversible gelation. In Chemistry Cells; Bickersta, G.F., Ed.; Humana Press. Inc.: Totowa,
and Technology of Water-Soluble Polymers; Finch, C.A., NJ, 1997, 229238.
Ed.; Plenum Press: New York, 1983; 113124. 153. Vorlop, K.D.; Klein, J. Entrapment of microbial cells in
138. Oakenfull, D.G.; Scott, A. Size and stability of the chitosan. Methods Enzymol. 1987, 135, 259268.
888 Yi et al.
154. Shinonaga, M.-K.; Kawamura, Y.; Yamane, T. Immobi- and hardened calcium pectate gel beads with and without
lization of yeast cells with cross-linked chitosan beads. J. cells. Biotechnol. Appl. Biochem. 1992, 15, 236251.
Ferment. Bioeng. 1992, 74, 9094. 173. Giordano, R.L.C.; Hirano, P.C.; Goncalves, L.R.B.;
155. Groboillot, A.F.; Champagne, C.P.; Darling, G.D.; Netto, W.S. Study of biocatalyst to produce ethanol
Poncelet, D.; Neufeld, R.J. Membrane formation by from starch. Appl. Biochem. Biotechnol. 2000, 8486,
interfacial cross-linking of chitosan for microencapsula- 643654.
tion of Lactococcus lactis. Biotechnol. Bioeng. 1993, 42, 174. Navarro, A.R.; Marangoni, H.; Plaza, I.M.; Callieri,
11571163. D.A.S. Horizontal reactor for the continuous product of
156. Sun, W.; Griths, M.W. Survival of bidobacteria in ethanol by yeasts immobilized in pectin. Biotechnol. Lett.
yogurt and simulated gastric juice following immobiliza- 1984, 6, 465470.
tion in gellanxanthan beads. Int. J. Food Microbiol. 175. Navarro, A.R.; Rubio, M.C.; Callieri, D.A.S. Production
2000, 61, 1725. of ethanol by yeasts immobilized in pectin. Eur. J. Appl.
157. Kaya, V.M.; Picard, G. Stability of chitosan gel as Microbiol. Biotechnol. 1983, 17, 148151.
entrapment matrix for viable Scenedesmus bicellularis cells 176. Linko, Y.-Y.; Linko, P. Entrapment of microbial cells in
immobilized on screens for tertiary treatment of waste- cellulose gel. Methods Enzymol. 1987, 135, 268282.
water. Biores. Technol. 1996, 56, 147155. 177. Linko, Y.-Y.; Poutanen, K.; Weckstrom, L.; Linko, P.
158. Ridout, M.J.; Brownsey, G.J. Mechanical properties of Preparation and kinetic behavior of immobilized whole
chitosan gels. In Gums and Stabilizers for the Food In- cell biocatalysts. Biochemie 1980, 62, 387394.
dustry; Phillips, G.O., Wedlock, D.J., Williams, P.A., 178. Joshi, S.; Yamazaki, H. Cellulose acetate entrapment of
Eds.; Elsevier Applied Science: London, 1986; 589595. Escherichia coli on cotton cloth for aspartate production.
159. Banik, R.M.; Kanari, B.; Upadhyay, S.N. Exopolysac- Biotechnol. Lett. 1986, 8, 277282.
charide of the gellan family: prospects and potential. 179. Giovenco, S.; Marconi, W.; Pansolli, P. Microbial cells
World J. Microbiol. Biotechnol. 2000, 16, 407414. entrapped in cellulose acetate beads. Methods Enzymol.
160. Gunning, A.P.; Morris, V.J. Light scattering studies of 1987, 135, 282293.
hylammonium gellan. Int. J. Biol. Macromol. 1990, 12, 180. Marconi, W.; Pansolli, P.; Giovenco, S. A new technique
338341. for cell entrapment in cellulose acetate beads. J. Mol.
161. Moorhouse, R.; Colegrove, G.T.; Sanford, P.A.; Barid, Catal. 1987, 40, 261265.
J.K.; Kang, K.S. In Solution Properties of Polysacchar- 181. Sakimae, A.; Onishi, H. Preparation of immobilized en-
ides; Brandt, P.A., Ed.; ACS Symposium Series 150; zymes of microorganisms. US Patent 4276381, 1981.
American Chemical Society: Washington, DC, 1981; 111 182. Dinelli, D. Fiber entrapped enzymes. Process Biochem.
124. 1972, 7, 912.
162. Norton, S.; Lacroix, C. Gellan gum as entrapment matrix 183. Ghose, T.K.; Kannan, V. Studies on ber entrapped
for high temperature fermentation process: a rheological whole microbial cells in urea hydrolysis. Enzyme Microb.
study. Biotechnol. Tech. 1990, 4, 351356. Technol. 1979, 1, 4750.
163. Camelin, L.; Lacroix, C.; Paquin, C.; Prevost, H.; Cachon, 184. Ouwerx, C.; Veling, N.; Mestdagh, M.M.; Axelos, M.A.V.
R.; Divies, C. Eects of chelants on gellan gum rheological Physico-chemical properties and rheology of alginate gel
properties and setting temperature for immobilization of beads formed with various divalent cations. Polym. Gels
living bidobacteria. Biotechnol. Prog. 1993, 9, 291297. Netw. 1998, 6, 393408.
164. Sanderson, G.R.; Bell, V.L.; Ortega, D. A comparison of 185. Hertzberg, S.; Moen, E.; Vogelsang, C.; stgaard, K.
gellan gum, agar, n-carrageenan and algine. Cereal Foods Mixed photo-cross-linked polyvinyl alcohol and calcium-
World 1989, 34, 991998. alginate gels for cell entrapment. Appl. Microbiol.
165. Moslemy, P.; Guiot, S.R.; Neufeld, R.J. Production of Biotechnol. 1995, 43, 1017.
size-controlled gellan gum microbeads encapsulating 186. Garcia, R.B.; De Boinis, M.; Andrade, C.T. Mechanical
gasoline-degrading bacteria. Enzyme Microb. Technol. and morphological features of agaroseguar gum gels.
2002, 30, 1018. Polym. Bull. 1994, 32, 111116.
166. Audet, P.; Lacroix, C. Two phase process for the 187. Vogelsang, C.; Wijels, R.H.; stgaard, K. Rheological
production of biopolymer gel beads. Appl. Microbiol. properties and mechanical stability of new gel-entrapment
Biotechnol. 1989, 24, 217226. systems applied in bioreactor. Biotechnol. Bioeng. 2000,
167. Grasdalen, H.; Smidsrod, O. Gelation of gellan gum. 70, 247253.
Carbohydr. Polym. 1987, 7, 371393. 188. Schoichet, M.S.; Li, R.H.; White, M.L.; Winn, S.R.
168. Rolin, C.; De Vries, J. Pectin. In Food Gel; Harris, P., Ed.; Stability of hydrogels used in cell encapsulation: an in
Elsevier Applied Science: London, 1990; 401434. vitro comparison of alginate and agarose. Biotechnol.
169. Morris, E.R.; Powell, D.A.; Gidley, M.J.; Rees, D.A. Bioeng. 1995, 50, 374381.
Conformations and interactions of pectins: I. Polymor- 189. Anseth, K.S.; Bowman, C.N.; Brannon-Peppas, L.
phism between gel and solid states of calcium polygalact- Mechanical properties of hydrogels and their experimen-
onate. J. Mol. Biol. 1982, 155, 507516. tal determination. Biomaterials 1996, 17, 16471657.
170. Smogrovicova, D.; Domeny, Z.; Gemeiner, P.; Malov - 190. Ma, X.; Vacek, I.; Sun, A. Studies on the parameters of
kova, A.; Sturd k, E. Reactors for continuous primary making alginatepoly-L-lysinealginate biomicrocapsules
beer fermentation using immobilized yeast. Biotech. Tech. for cell transplantation. Artif. Organs 1991, 15, 274.
1997, 11, 261264. 191. Prokop, A.; Hunkeler, D.; DiMari, S.; Haralson, M.A.;
171. Gemeiner, P.; Kurillova, L.; Markovic, O.; Malov kova, Wang, T.G. Water soluble polymers for immunoisolation:
A.; Uhr n, D.; Ilavsky, M.; Stefuca, V.; Polakovic, M.; I. Complex coacervation and cytotoxicity. Adv. Polym.
Bales, V. Calcium pectate gel beads for cell entrapment: 3. Sci. 1998, 136, 149.
Physical properties of calcium pectate and calcium alginate 192. Leblond, F.A.; Tessier, J.; Halle, J.P. Quantitative method
gel beads. Biotechnol. Appl. Biochem. 1991, 13, 335345. for the evaluation of biomicrocapsule resistance to
172. Kurillova, L.; Gemeiner, P.; Ilavsky, M.; Stefuca, V.; mechanical stress. Biomaterials 1996, 17, 20972102.
Polakovic, M.; Welwardova, A.; Toth, D. Calcium pectate 193. Peirone, M.A.; Delaney, K.; Kwiecin, J.; Fletch, A.;
gel beads for cell entrapment: 4. Properties of stabilized Chang, P.L. Delivery of recombinant gene product to
canines with nonautologous microencapsulated cells. carrying polymers and their eects on insulin secretion
Hum. Gene Ther. 1998, 9, 195206. from MIN6 cells in a solution state. J. Biomater. Sci.
194. Van Raamsdonk, J.M.; Chang, P.L. Osmotic pressure Polym. Ed. 2001, 12, 911920.
test: a simple, quantitative method to assess the mechan- 212. Fukushima, Y. A new immobilization technique of whole
ical stability of alginate microcapsules. J. Biomed. Mater. cells and enzymes with colloidal silica and alginate.
Res. 2001, 51, 264271. Biotechnol. Bioeng. 1988, 32, 584594.
195. dos Santo, V.A.P.M.; Leenen, E.J.T.M.; Rippol, M.M.; 213. Kalyanasundaram, S.; Feinstein, S.; Nicholson, J.P.;
van der Sluis, C.; van Viliet, T.; Tramper, J.; Wijels, Leong, K.W.; Garver, R.I.J. Coacervate microspheres as
R.H. Relevance of rheological properties of gel beads for carriers of recombinant adenoviruses. Cancer Gene Ther.
their mechanical stability in bioreactors. Biotechnol. 1999, 6, 107112.
Bioeng. 1997, 56, 517529. 214. Kawakami, K.; Furukawa, S.-Y. Alcohol-oxidation ac-
196. Araujo, M.L.G.C.; Giordano, R.C.; Hokka, C.O. Studies tivity of whole cells of Pichia pastoris extrapped in hybrid
on the respiration rate of free and immobilized cells of gels composed of Ca-alginate and organic solicate. Appl.
Cephalosporium acremonium in cephalosporin C produc- Biochem. Biotechnol. 1997, 67, 2331.
tion. Biotechnol. Bioeng. 1999, 63, 593600. 215. Kawakami, K.; Nakahara, T. Importance of solute
197. Wu, S.-Y.; Lin, C.-N.; Chang, J.-S.; Lee, K.-S.; Lin, P.-J. partitioning in biphasic oxidation of benzyl alcohol by
Microbial hydrogen production with immobilized sewage free and immobilized whole cells of Pichia pastoris. Bio-
sludge. Biotechnol. Prog. 2002, 18, 921926. technol. Bioeng. 1994, 43, 918924.
198. Kim, S.W.; Kim, E.Y. Development of new alginate ber 216. Kawakami, K.; Tsuruda, S.; Miyagi, K. Immobilization
for the immobilization of yeast. Biotechnol. Tech. 1993, of microbial cells in a mixed matrix of silicone polymer
10, 579584. and calcium alginate gel: epoxiation of 1-octene by
199. Alexakis, T.; Baodi, D.K.; Quong, D.; Groboillot, A.; Nocardia corallina B-276 inorganic media. Biotechnol.
ONeill, I.; Poncelet, D.; Neufeld, R.J. Microencapsula- Prog. 1990, 6, 357361.
tion of DNA within alginate microspheres and crosslinked 217. Kawakami, K.; Abe, T.; Yoshida, T. Silicone-immobilized
chitosan membranes for in vivo application. Appl. biocatalysts eective for bioconversions in nonaqueous
Biochem. Biotechnol. 1995, 50, 93106. media. Enzyme Microb. Technol. 1992, 14, 371375.
200. Tal, Y.; van Rijn, J.; Nussinovitch, A. Starch as ller, 218. Mano, T.; Mitsuda, S.; Kumazawa, E.; Takeshita, Y.
matrix enhancer and a carbon source in freeze-dried Immobilization method of mammalian cells using alginate
denitrifying alginate beads. Proc. 4th Int. Conf. Hydro- and polyacrylate. J. Ferment. Bioeng. 1992, 73, 486489.
colloids, Osaka, 2000, 347354. 219. Seifert, D. Bioactive cells immobilized in alginate beads
201. Spettoli, P.; Nuti, M.P.; Crapisi, A.; Zamorani, A. containing voids formed with polyethylene glycol. US Pat-
Technological improvement of malolactic fermentation ent 5175093, 1992.
in wine by immobilized microbial cells in a continuous 220. Desai, N.P.; Sojomihardjo, A.; Yao, Z.; Ron, N.; Soon-
ow reactor. Ann. N.Y. Acad. Sci. 1987, 501, 386389. Shiong, P. Interpenetrating polymer networks of alginate
202. Vassileva, M.; Azcon, R.; Barea, J.-M.; Vassilev, N. Eect and polyethylene glycol for encapsulation of islets of
of encapsulated cells of Enterobacter sp. on plant growth Langerhans. J. Microencapsul. 2000, 17, 677690.
and phosphate uptake. Biores. Technol. 1998, 67, 229232. 221. Li, J.; Zhou, J. A new complex PVAalginate gel for
203. Fages, J. An optimized process for manufacturing an immobilization of yeast cells. Weishengwu Xuebao 1995,
Azospirillum inoculant for crops. Appl. Microbiol. Bio- 35, 232234.
technol. 1990, 32, 473478. 222. Hsu, F.Y.; Tsai, S.W.; Wang, F.F.; Wang, Y.J. The
204. Van Elsas, J.D.; Trevors, J.T.; Jain, D.; Wolter, A.C.; collagen-containing alginate/poly(L-lysine)/alginate mi-
Heijnen, C.E.; Overbeek, V. Survival of, and root crocapsules. Artif. Cells. Blood Substit. Immobil. Bio-
colonization by, alginate encapsulated Pseudomonas technol. 2000, 28, 147154.
uorescens cells following introduction into soil. Biol. 223. Rietti-Shati, M.; Ronen, D.; Mandelbaum, R.T. Atrazine
Fertil. Soils 1992, 14, 1422. degradation by Pseudomonas strain ADP entrapped in
205. Cassidy, M.B.; Mullineers, H.; Lee, H.; Trevors, J.T. solgel glass. J. SolGel Sci. Technol. 1996, 7, 7779.
Mineralization of pentachlorophenol in a contaminated 224. Zazczyk, K.; Olejnik, A.; Trojanowska, K. The inuence
soil by Pseudomonas sp. UG30 cells encapsulated in n- of LBG addition to carrageenan on the mechanical
carrageenan. J. Ind. Microbiol. Biotechnol. 1997, 19, 43 stability of the gel and the fermentative activity of
48. immobilized propionic acid bacteria. Acta Biotechnol.
206. Cassidy, M.B.; Lee, T.; Trevors, J.T. Environmental 1999, 2, 147156.
applications of immobilized microbial cells: a review. J. 225. Gill, I.; Ballestero, A. Bioencapsulation within synthetic
Ind. Microbiol. 1996, 16, 79101. polymers: Part 1. Solgel encapsulated biologicals. Trends
207. Scott, C.D.; Woodward, C.A.; Thompson, J.E. Solute Biotechnol. 2000, 18, 282296.
diusion in biocatalyst gel beads containing biocatalysis 226. Gill, I. Bio-doped nanocomposite polymers: solgel
and other additives. Enzyme Microb. Technol. 1989, 11, bioencapsulates. Chem. Mater. 2001, 13, 34043421.
258263. 227. Dea, I.C.M. Interactions of ordered polysaccharide struc-
208. England, L.S.; Lee, H.; Trevors, J.T. Bacterial survival in turessynergism and freezethaw phenomena. In Poly-
soil: eect of clays and protozoa. Soil Biol. Biochem. saccharides in Food; Blanshard, J.M.V., Mitchell, J.R.,
1993, 25, 525531. Eds.; Butterworth: London, 1979 (Part IV, c15); 229247.
209. Morris, E.R. Mixed polymer gels. In Food Gels; Harris, P., 228. Turquois, T.; Rochas, C.; Taravel, F.R. Rheological
Ed.; Elsevier: London, 1990; 291359. studies of synergistic kappa-carrageenancarob galacto-
210. Asina, S.; Jain, K.; Rubin, A.; Smith, B.; Stenzel, K. Can- mannan gels. Carbohydr. Polym. 1992, 17, 263268.
cer-cell proliferation-suppressing material produced by 229. Casas, L.T.; Dominguez, F.; Brito, E. Characterization
cancer cells restricted by entrapment. US Patent 5888497, and optimization of a new immobilized system of n-
2001. carrageenan through interaction with carob bean gum and
211. Park, K.-H.; Goto, M.; Miyazaki, J.-I.; Cho, C.-S.; polyols. J. Ferment. Bioeng. 1990, 69, 98101.
Akaike, T. Incorporation of sulfonylurea into sugar- 230. Fernandes, P.B.; Goncalves, M.P.; Doublier, J.L. Rheo-
890 Yi et al.
logical behavior and solgel transition of galactomannan/ 246. Houng, J.-Y.; Chiang, W.-P.; Chen, K.-C. 11a-Hydroxy-
kappa-carrageenan blends. In Gums and Stabilizers for the lation of progesterone in biphasic media using alginate
Food Industry 6; Phillips, G.O., Williams, R.A., Wedlock, entrapped Aspergillus ochraceus gel beads coated with
D.J., Eds.; IRL Press: Oxford, 1991; 181190. polyurea. Enzyme Microb. Technol. 1994, 16, 485491.
231. Cairns, P.; Morris, V.J.; Miles, M.J.; Brownsey, G.J. 247. Bartkowiak, A.; Hunkeler, D. Alginate-oligochitosan
Synergistic behavior in kappa-carrageenantara gum microcapsules: a mechanistic study relaying membrane
mixed gels. In Gums and Stabilizers for the Food Industry and capsule properties to reaction conditions. Chem.
3; Phillips, G.O., Wedlock, D.J., Williams, P.A., Eds.; Mater. 1999, 11, 24862492.
Elsevier Applied Science Publisher: London, 1985; 597 248. Gaserd, O.; Smidsrd, O.; Skjak-Brk, G. Microcap-
604. sules of alginate chitosan: I. A quantitative study of the
232. Tako, M.; Nakamura, S. Synergistic interaction between interaction between alginate and chitosan. Biomaterials
xanthan and guar gum. Carbohydr. Res. 1985, 138, 207213. 1998, 19, 18151825.
233. Kawakami, Y.; Inoue, K.; Hayashi, H.; Wang, W.J.; 249. Brissova, M.; Lacik, I.; Power, A.C.; Anilkumar, A.V.;
Setoyama, H.; Gu, Y.J.; Imamura, M.; Iwata, H.; Ikada, Wang, T. Control and measurement of permeability of
Y.; Nozawa, M.; Miyazaki, J. Subcutaneous xenotrans- design of microcapsule cell delivery system. J. Biomed.
plantation of hybrid articial pancreas encapsulating Mater. Res. 1998, 34, 6170.
pancreatic B cell line (MIN6): functional and histological 250. Libicki, S.B.; Salmon, P.M.; Robertson, C.R. The
study. Cell Transplant. 1997b, 6, 541545. eective diusive permeability of a non-reacting solute
234. Kanda, T.; Miyata, N.; Fukui, T.; Kawamoto, T.; in microbial cell aggregate. Biotechnol. Bioeng. 1988, 32,
Tanaka, A. Doubly entrapped bakers yeast survives 6885.
during the long-term stereoselective reduction of ethyl 3- 251. Westrin, B.A.; Axelsson, A. Diusion in gels containing
oxobutanoate in an organic solvent. Appl. Microbiol. immobilized cells: a critical review. Biotechnol. Bioeng.
Biotechnol. 1998, 49, 377381. 1991, 38, 439446.
235. Grohn, P.; Klock, G.; Zimmermann, U. Collagen-coated 252. Zhang, W.; Furusaki, S. On the evaluation of diusivities
Ba2+-alginate microcarriers for the culture of anchorage- in gels using the diusion cell technique. Biochem. Eng.
dependent mammalian cells. BioTechniques 1997, 22, 970 Sci. 2001a, 9, 7382.
972, 974975. 253. Korgel, B.A.; Rotem, A.; Monbouquette, H.G. Eective
236. Boninsegna, S.; Dal Toso, R.; Dal Monte, R. Alginate diusivity of galactose in calcium alginate gels containing
microspheres loaded with animal cells and coated by a immobilized Zymomonas mobilis. Biotechnol. Prog. 1992,
siliceous layer. J. SolGel Sci. Technol. 2003, 26, 11511157. 8, 111117.
237. Luca, G.; Calaore, R.; Basta, G.; Ricci, M.; Calvitti, M.; 254. Hannoun, B.J.M.; Stephanopoulos, G. Diusion coe-
Luca, N.; Nastruzzi, C.; Becchetti, E.; Capitani, S.; cients of glucose and ethanol in cell-free and cell-occupied
Brunetti, P.; Rossi, C. Improved function of rat islets calcium alginate membrane. Biotechnol. Bioeng. 1986, 28,
upon comicroencapsulation with Sertolis cells in alginate/ 829835.
poly-L-ornithine. AAPS PharmSciTech 2001, 2 (3) (on- 255. Tanaka, H.; Matsumura, M.; Veliky, I.A. Diusion
line computer le). characteristics of substrates in Ca-alginate gel beads.
238. Uemura, Y.; Hamakawa, N.; Yoshizawa, H.; Ando, H.; Biotechnol. Bioeng. 1984, 24, 5358.
Ijichi, K.; Hatate, Y. Eect of calcium alginate coating on 256. Amsden, B.; Turner, N. Diusion characteristics of
the performance of immobilized yeast cells in calcium calcium alginate gel. Biotechnol. Bioeng. 1999, 65, 605
alginate beads. Chem. Eng. Commun. 2000, 177, 114. 610.
239. Repunte, V.P.; Taya, M.; Tone, S. Conservation of root 257. Jovetica, S.; Beeftink, H.H.; Tramper, J.; Marinelli, F.
regeneration potential of cell aggregates from horseradish Diusion of (de)acylated antibiotic A40926 in alginate
hairy roots used as articial seeds. J. Chem. Eng. Jpn. and carrageenan beads with or without cells and/or
1996, 29, 874880. soybean meal. Enzyme Microb. Technol. 2001, 28, 510
240. Wen, S.; Stevenson, W.T.K. Microcapsules for cell 514.
entrapment by template polymerization of a synthetic 258. Venancio, A.; Teixeira, J.A. Characterization of sugar
hydrogel coating around calcium alginate gel: preliminary diusion coecients in alginate membranes. Biotechnol.
development. J. Mater. Sci. Mater. Med. 1993, 4, 2331. Tech. 1997, 11, 183185.
241. Lamberti, F.V.; Wheatley, M.A.; Evangelista, R.A.; 259. Converti, A.; Casagrande, M.; Giovanni, M.D.; Rovatti,
Sefton, M.V. The use of polyacrylates in the micro- M.; Borghi, M.D. Evaluation of glucose diusion
encapsulation of viable tissue cells. Polym. Prep. 1983, 24, coecient through cell layers for the kinetic study of an
7576. immobilized cell bioreactor. Chem. Eng. Sci. 1996, 51,
242. Lamberti, F.V.; Sefton, M.V. Microencapsulation of 10231026.
erythrocytes in Eudragit-RL-coated calcium alginate. 260. Beuling, E.E.; van den Heuvel, J.C.; Ottengraf, S.P.P.
Biochim. Biophys. Acta 1983, 759, 8191. Diusion coecients of metabolites in active biolms.
243. Schneider, S.; Feilen, P.; Slotty, V.; Kampfner, D.; Preuss, Biotechnol. Bioeng. 2000, 67, 5360.
S.; Berger, S.; Beyer, J.; Pommersheim, R. Multilayer 261. yaas, J.; Storr, I.; Svendsen, H.; Kevine, D.W. The
capsules: a promising microencapsulation system for eective diusion coecient and the distribution constant
transplantation of pancreatic islets. Biomaterials 2001, for small molecules in calcium-alginate gel beads. Bio-
22, 19611970. technol. Bioeng. 1995, 47, 492500.
244. Gaumann, A.; Laudes, M.; Jacob, B.; Ommersheim, R.; 262. Chresand, T.J.; Dale, B.E.; Hanson, S.I.; Gillies, R.J. A
Laue, C.; Vogt, W.; Schrezenmeir, J. Xenotransplantation stirred bath technique for diusivity measurement in cell
of parathyroids in rats using barium-alginate and poly- matrices. Biotechnol. Bioeng. 1988, 32, 10291036.
acrylic acid multilayer microcapsules. Exp. Toxicol. 263. Axelsson, A.; Persson, B. Determination of eective
Pathol. 2001, 53, 3543. diusion coecients in alginate gel plates with varying
245. Iijima, S.; Mano, T.; Taniguchi, M.; Kobayshi, T. yeast cell content. Appl. Biochem. Biotechnol. 1988, 18,
Immobilization of hybridoma cells with alginate and ure- 231250.
thane polymer and improved monoclonal antibody pro- 264. Wakao, N.; Smith, J. Diusion in catalyst pellets. Chem.
duction. Appl. Microbiol. Biotechnol. 1988, 28, 572576. Eng. Sci. 1962, 17, 825834.
265. Mota, M.; Teixeira, J.A.; Yelshin, A. Immobilized Immobilized Cells; De Bont, J.A.M., Visser, J., Mattias-
particles in gel matrix-type porous media. Homogeneous son, B., Tramper, J., Eds.; Elsevier Science Publishers:
porous media model. Biotechnol. Prog. 2001, 17, 860 Amsterdam, 1990; 415420.
865. 283. Moslemy, P.; Neufeld, R.J.; Guiot, S. Biodegradation of
266. Muhr, A.H.; Blanshard, J.M.V. Diusion in gels. Polymer gasoline by gellan gum encapsulated bacterial cells.
1982, 23, 10121026. Biotechnol. Bioeng. 2002, 80, 175184.
267. Amsden, B. Solute diusion within hydrogels. Mecha- 284. Sayadi, S.; Nasri, N.; Barbotin, J.N.; Thomas, D. Eect of
nisms and models. Macromolecules 1998, 31, 83828395. environmental growth conditions on plasmid stability,
268. Karel, S.F.; Libicki, S.B.; Robertson, C.R. The immobi- plasmid copy number and catechol 2,3-dioxygenase
lization of whole cells: engineering principles. Chem. Eng. activity in free and immobilized E. coli cells. Biotechnol.
Sci. 1985, 40, 13211354. Bioeng. 1989, 33, 801808.
269. Zhang, W.; Berry, A.; Franco, C.M.M. An improved 285. Willaert, R.G.; Baron, G.V. Eectiveness factor calcu-
procedure for characterization of spatial and temporal lation for immobilized growing cell systems. Biotechnol.
evolution of immobilized cells in gel membranes. Appl. Tech. 1994, 8, 695700.
Microbiol. Biotechnol. 2001, 56, 693699. 286. Smogrovicova, D.; Domeny, Z.; Svitel, J. Modeling of
270. Harder, A.; Roels, J.A. Application of simple structured saccharide utilization in primary beer fermentation with
models in bioengineering. Adv. Biochem. Eng. 1982, 21, yeasts immobilized in calcium alginate. Appl. Biochem.
55107. Biotechnol. 2001, 94, 147158.
271. Gillet, F.; Roisin, C.; Fliniaux, M.A.; Jacquin-Dubreuil, 287. Dincbas, V.; Hortacsu, A.; Camurdan, A. Plasmid
A.; Barbotin, J.N.; Nava-Saucedo, J.E. Immobilization stability in immobilized mixed cultures of recombinant
of Nicotiana tobacum plant cell suspensions within cal- Escherichia coli. Biotechnol. Prog. 1993, 9, 218220.
cium alginate gel beads for the production of enhanced 288. Monbouquette, H.G.; Ollis, D.F. A structured model for
amount of acopolin. Enzyme Microb. Technol. 2000, 26, immobilized cell kinetics. Ann. N.Y. Acad. Sci. 1986, 469,
229234. 230244.
272. Asanza Teruel, M.L.; Gontier, E.; Bienaime, C.; Nava 289. Godia, F.; Cacas, C.; Sola, C. Mathematic modelization
Saucedo, J.E.; Barbotin, J.-N. Response surface analysis of a packed-bed reactor performance with immobilized
of chlortetracycline and tetracycline production with n- yeast for ethanol fermentation. Biotechnol. Bioeng. 1987,
carrageenan immobilized Streptomyces aureofaciens. En- 30, 836843.
zyme Microb. Technol. 1997, 21, 314320. 290. Chang, H.N.; Park, T.H. A theoretic model for immobi-
273. Roisin, C.; Nava Saucedo, J.E.; Barbotin, J.-N. Diversi- lized whole cell enzyme. J. Theor. Biol. 1985, 116, 920.
ed shunt to the production of dierent proportions of 291. Nakasaki, K.; Murai, T.; Akiyama, T. Dynamic modeling
secondary metabolites (polyketides and terpenes) induced of immobilized cell reactor: application to ethanol
by varying immobilization constraints on Gibberella fermentation. Biotechnol. Bioeng. 1989, 33, 13171323.
fujikuroi. Ann. N.Y. Acad. Sci. 1996, 782, 6169. 292. Sayles, G.D.; Ollis, D.F. Periodic operation of immobi-
274. Roisin, C.; Gillet-Manceau, F.; Nava Saucedo, J.E.; lized cell system: analysis. Biotechnol. Bioeng. 1989, 34,
Fliniaux, M.; Jacquin-Dubreuil, A.; Barbotin, J.-N. 160170.
Enhanced production of scopolin by Solanum aviculare 293. de Gooijer, C.D.; Wijels, R.H.; Tramper, J. Growth and
cells immobilized within Ca-alginate gel beads. Plant Cell substrate consumption of Nitrobacter agilis cells immobi-
Rep. 1997, 16, 349353. lized in carrageenan: Part 1. Dynamic modeling. Bio-
275. Walsh, P.K.; Isdell, F.V.; Noone, S.M.; Odonovan, M.G.; technol. Bioeng. 1990, 38, 224231.
Malone, D.M. Growth patterns of Saccharomyces cer- 294. Wang, H.; Seki, M.; Furusaki, S. Mathematical model for
evisiae microcolonies in alginate and carrageenan gel analysis of mass transfer for immobilized cells in lactic
particles: eect of physical and chemical properties of gels. acid fermentation. Biotechnol. Prog. 1995, 11, 558564.
Enzyme Microb. Technol. 1996, 18, 366372. 295. Monbouquete, H.G.; Sayles, G.D.; Ollis, D.F. Immobi-
276. Stabler, C.; Wilks, K.; Sambanis, A.; Constantinidis, I. lized cell biocatalyst activation and pseudo-steady-state
The eects of alginate composition on encapsulated hTC3 behavior: model and experiment. Biotechnol. Bioeng.
cells. Biomaterials 2001, 22, 13011310. 1990, 39, 609629.
277. Kiy, T.; Tiedtke, A. Lysosomal enzymes produced by 296. Wijels, R.H.; de Gooijer, C.D.; Schepers, A.W.; Beuling,
immobilized Tetrahymena thermophila. Appl. Microbiol. E.E.; Mallee, L.R.; Tramper, J. Dynamic modeling of
Biotechnol. 1991, 35, 1418. immobilized Nitrosomonas europaea: implementation of
278. Bringi, V.; Shuler, M.L. A framework for understanding diusional limitation over expanding microcolonies.
the eects of immobilization on plant cells: dierentiation Enzyme Microb. Technol. 1995, 17, 462471.
of tracheary elements in tobacco In Physiology of 297. Laca, A.; Quiros, C.; Garc a, L.A.; D az, M. Modelling
Immobilized Cells; De Bont, J.A.M., Visser, J., Mattias- and description of internal proles in immobilized cell
son, B., Tramper, J., Eds.; Elsevier Science Publishers: systems. Biochem. Eng. J. 1998, 1, 225232.
Amsterdam, 1989; 161172. 298. Quiros, C.; Garc a, L.A.; Diaz, M. The evolution of the
279. Zayed, G. Evaluation of N2-xation eciency of Azoto- structure of calcium alginate beads and cell leakage during
bacter in alginate-encapsulated and free cell systems. protease production. Process Biochem. 1996, 21, 813822.
Egypt. J. Microbiol. 2000, 34, 4555. 299. dos Santos, V.A.P.M.; Marchal, L.M.; Tramper, J.;
280. Holcberg, I.B.; Margalith, P. Alcohol fermentation by Wijels, R.H. Modeling and evaluation of an integral re-
immobilized yeast at high sugar concentrations. Eur. J. moval system with microorganisms co-immobilized in
Appl. Microbiol. Biotechnol. 1981, 13, 133140. double-layer gel beads. Biotechnol. Prog. 1996, 12, 240248.
281. Buzas, Z.; Dallmann, K.; Szajani, B. Inuence of pH on 300. Laca, A.; Garc a, L.A.; D az, M. Analysis and description
the growth and ethanol production of free and immobi- of the evolution of alginate immobilized cells systems. J.
lized Saccharomyces cerevisiae cells. Biotechnol. Bioeng. Biotechnol. 2000, 80, 203215.
1989, 34, 882884. 301. Wijels, R.H.; de Gooijer, C.D.; Kortekaas, S.; Tramper,
282. Fortin, C.; Vuillemard, J.C. Eect of immobilization in J. Growth and substrate consumption of Nitrobactor agilis
calcium alginate beads on regulation of protease produc- cells immobilized in carrageenan: Part 2. Model evalua-
tion by Myxococus xanthus cells. In Physiology of tion. Biotechnol. Bioeng. 1991, 38, 232249.
40
Hydrothermal Degradation and Fractionation of Saccharides
and Polysaccharides
Ortwin Bobleter
University of Innsbruck, Innsbruck, Austria
I. INTRODUCTION which may grow to 10 billion people in this century, would

need 1.8 billion tons of foodstus per yearan amount not
Plant biomass, this many splendored thing, has nurtured easy to supply.
mankind since the birth of Homo sapiens, whose stan- Table 3 gives the content of water, carbohydrates, fat,
dard of living and health increased dramatically when he and protein [6] for several important foodstus, as well as
was able to use re by burning plant matter approxi- their yields in tons per hectare and year.
mately 500,000 years ago. The application of wooden The average harvest yields in 1999 are taken from the
hand tools introduced the next development phase, agri- FAO yearbook [7]. However, it must be considered that
culture (fA.D. 5000). This led to a steadily increasing these values have an enormous range both with regard to
supply of foodstus and still no nal limiting barriers can the year for these harvests and also the climate where the
be seen. harvests were yielded. In Austria, the wheat productivity
Wood became a very interesting product for building [9] between 1950 and 1999 grew from 2 to nearly 5 tons
houses, ships, and many other gadgets. With the invention ha1 year1. But in 1999 in Europe, the dierences [7] were
of paper (A.D. 105), writing obtained a new dimension and still between 2.1 (Spain) and 7.3 tons ha1 year1 (Nether-
introduced the communication age, which like all other lands). However, more important is that high harvest
progresses is not completely free of problems. results are only obtained by intensive fertilization and the
With the depletion of the world oil reserves in the 21st use of herbicides. Through these methods, agricultural
century [1], plant biomass will again become the main land and groundwater can be severely contaminated so
resource for the production of transport fuel, organic that new agricultural policies in many parts of the world
chemicals, and plastic materials. are required.
According to Whittaker and Likens [2] and Larcher From Tables 2 and 3, it can be estimated how much
[3], there is, with 117.5 billion tons per year, a large growth land is needed to produce, annually, 1.27 109 tons
of plant biomass on the continents of Earth (Table 1). High carbohydrates, 0.23 109 tons fat, and 0.32 109 tons
mean annual yields are produced in tropical rain forests, protein to feed the future world population of 10 billion. In
other woods, savannas, and steppes (22.0, 11.6, and 9.1 Table 4, two scenarios are drawn to reach this target
tons ha1 year1). Agricultural plantations with 6.5 tons approximately. Scenario I would need 14 108 ha, which
ha1 year1 take only fourth place. The question arises: Is is just about the total agricultural crop land of all con-
there enough land to cultivate food for the growing world tinents on Earth. The land requirement of this scenario is
population? The main nutrients (Table 2) are carbohy- mainly because of the uneconomical production of protein
drates in the form of saccharides and polysaccharides, fat, over the meat chain. In reality, the situation is even worse
and protein, which sum up to a daily consumption of because in a large number of countries, where hunger
approximately 500 g, this being equal to an energy content prevails, the harvest yields are much below the world
of 10,000 J per person. Roughage (f10 wt.%), fruits, average. In addition, roughage, fruit, and so forth are not
vitamins, and mineral compounds have not been taken yet taken into account and, therefore, the solution of
into consideration. Nevertheless, the world population, scenario I is not satisfactory.
893
894 Bobleter
Table 1 Annual Net Primary Production of Plant Biomass as Dry Matter on the Continents
Net primary production
Area Range Mean Regional yield

Vegetation (106 km2 = 108 ha) (tons ha1 year1) (tons ha1 year1) (109 tons year1)
Tropical rain forest 17.0 1035 22.0 37.4

Other woods 31.5 425 11.6 36.5
Savannas, meadows, steppes, and tundra 22.0 0.120 9.1 20.0
Agricultural crops 14.0 140 6.5 9.1
Swamps, marshes, and inland waters 4.0 160 17.0 6.8
Dry scrub land 8.5 215 7.0 6.0
Deserts (shrub, dry and cold) 42.0 04 0.4 1.7
Total 149.0 7.8 117.5
Scenario II obtains carbohydrates mainly from pota- atmosphere. On a sunny day, only 0.90 kW m2 of this
toes and wheat, fat from sunowers, and protein from radiation energy reaches the ground at a medium degree of
yeast (e.g., bakers yeast). Meat and milk-like products can latitude. The position of the sun between day and night,
certainly be produced from yeast, at the same time reducing clouds and fogs reduce this value to f0.20 kW m2 as an
the danger of infection by foot and mouth as well as mad average annual value at 40j latitude (e.g., Philadelphia,
cow disease. In this scenario, the food requirements for the USA; Toledo, Spain; Ankara, Turkey; and Beijing, China)
whole population can be met by 8 108 ha, leaving enough for the large energy spectrum of 3002200 nm. This adds up
space for planting fruit and raising animals for those people to 6.3 GJ m2 year1 [10]. If all this energy could be used by
who still do not accept vegetarianism in the near future. plants, a frightening 370 kg m2 year1 growth would be
For the production of yeast, no land requirement is given expected. However, the complicated photosynthesis system
in Table 4. The reason for this is that the biomass residue uses mainly two energy maxima: 700 nm (Photosystem I)
of cereal crop harvests, with f20 108 tons year1, is and 680 nm (Photosystem II). Through the limited use of
already more than enough raw material for conversion to the radiation, the real maximum harvests lie much lower.
sugars, from which the necessary amount of yeast can be Only 50% of the light has the photosynthetically active
obtained. In addition, there are large reserves of wood spectral range (PAR = 400700 nm). Approximately 10%
residues or short-rotation forestry plants that can be used are lost through reection, transmission, or absorption in
for this purpose. inactive tissues. Another 30% are lost through side eects
(heat, uorescence, etc.) during assimilation. Photorespi-
ration and dark respiration take the larger part of the
A. Major Plant Polysaccharides remaining 10% [11]. One of the highest yields was obtained
1. Polysaccharide Growth with algae in a nutrient solution tank, 14.3 kg m2 year1,
corresponding to a dry-matter production of as little as
For their growth, plants need a certain amount of light and
3.9% of the sun energy inux [1214].
live only in a relatively limited range of temperature and
The temperatures preferred by plants are between 5jC
with a reasonable amount of water.
and 25jC. Only the oceans come close to these ideal
Radiation, Temperature, and Humidity temperature conditions. On the continents, strong daily
The light used by plants for photosynthesis is gener- or seasonal deviations are common. The frost-free regions
ously delivered by the sun. At midday, an energy inux of are situated around the equator and include the tropical
1.4 kW m2, the solar constant, is received at the outer rain forests. Close to the Tropic of Cancer, occasional
Table 2 Mean Values of Human Consumption of the Main Nutrients
Average consumption per person

Energy content Consumption by 1010
Nutrient (103 kJ kg1) Daily (kJ) Daily (g) Annual (kg) people/year (109 tons)
Carbohydrates 17.2 6,000 349 127.4 1.274

Fat 39.0 2,500 64 23.4 0.234
Protein 17.2 1,500 87 31.8 0.318
Total 10,000 500 182.6 1.826
Hydrothermal Degradation and Fractionation 895
Table 3 Content of Water, Carbohydrate, Fat, Protein, and Average World Harvest Yields of Eight Important Foodstus
Carbohydrates Fat Protein

Harvest Water
Foodstu (tons ha1 year1) (%) % tons ha1 year1 % tons ha1 year1 % tons ha1 year1
Potato 16.4 77.8 16.9 2.8 0.1 0.02 2.0 0.33

Wheat 2.7 12.8 72.9 2.0 1.8 0.05 10.9 0.29
Rice, paddy 3.8 13.1 76.3 2.9 2.2 0.08 7.2 0.27
Maize 4.3 12.5 73.9 3.2 3.8 0.16 8.5 0.37
Sunower seed 1.2 6.6 18.6 0.2 49.0 0.59 22.5 0.27
Soybeans 2.1 8.5 28.3 0.6 18.3 0.38 34.3 0.72
Meat 0.7 56 25 0.18 16 0.11
Yeast, bakers 73.0 6.7 1.2 16.7
Note: Meat includes poultry, beef, and pork in the relation of 30:30:40 wt.%, which corresponds approximately to the present world production.
It is assumed that poultry and pigs are fed on maize, and cattle extensively through grazing (1.5, 1.06, and 0.4 tons meat per hectare and year,
respectively).
Source: Refs. 68.
frosts occur, which make life hard for plants, especially as the formation of carbohydrates is connected with the
high absolute maximum temperatures of up to 58jC are production of oxygen, O2. In a four-step reaction, the an-
measured (California, Mexico, and Libya). Some of the tennae pigments funnel the absorbed photons to the reac-
unproductive deserts also lie in this region. Around 40j tion center (P680), which becomes excited and gives an
latitude, cold winter regions are situated. The mean low electron to a pheophitin molecule. From there, the electron
temperatures are between 10jC and 40jC, to which is passed to the quinon, QA, and nally to the quinon
plants have to adjust, but their productivity considerably QB. After a second electron absorption, the quinon QB
drops compared to the region of the rain forests [15]. moves freely until it produces an adenosine triphosphate.
Approximately 12% of the land surface have less than With this energy-rich compound, the assimilation of CO2
250 mm of rain per year, making agriculture very unsatis- can begin.
factory [16]. Roughly, an equivalent area has more humid- In the CalvinBenson cycle, the pentose phosphate,
ity but still a decit of rain. Under these conditions, the ribulose-1,5-bis phosphate (RuBP), works rst as CO2
potential evaporation is higher than the actual rainfall. acceptor, whereby an unstable six-carbon molecule is
Large plant harvests are only reached when an amount of formed. In a very fast reaction, this molecule is disinte-
1000 mm and more precipitation (including snow in winter) grated into 3-phosphorus glycerinic acids (PGA). This
is furnished (Fig. 1). reaction product contains three carbon atoms, which gives
Photosynthesis the expression of a C3 path for this type of CO2 assimila-
tion. PGA is reduced by ATP and NADPH2 to glycerine-
During evolution, plants had to invent a most inge-
aldehyde-phosphate (GAP), which belongs to a pool, from
nious way to reduce nCO2 molecules to (CH2O)n, the
which higher carbohydrates (sugars, starch, hemicellulose,
carbohydrates. By the overall formula,
and cellulose, etc.) are formed.
The synthesis pathway from C3 carbohydrates to
nCO2 nH2 O ! CH2 On nO2 1 higher saccharides seems an energy-saving way. This will
Table 4 Two Scenarios of the Use of Land for the Production of Food, per Year, for 10 Billion People
Scenario I Scenario II
Land use Carbohydrate Fat Protein Land use Carbohydrate Fat Protein
Foodstu (108 ha) (108 tons) (108 tons) (108 tons) (108 ha) (108 tons) (108 tons) (108 tons)
Potato 3 8.4 0.06 0.99

Wheat 2 4.0 0.10 0.66 1 2.0 0.05 0.29
Rice 1 2.9 0.08 0.27 0.5 1.5 0.04 0.14
Maize 2 6.4 0.32 0.74
Sunower 3.5 0.7 2.07 0.95
Soybeans 1 0.6 0.38 0.72
Meat 8 1.44 0.88
Yeast 2.2 0.40 5.57
Total 14 13.9 2.3 3.3 8 14.8 2.6 7.9
896 Bobleter
must be considered. Other organic mass products

(e.g., plastic materials and bers) will have to be
manufactured from plant biomass.
These requirements can be fullled by three polysaccha-
rides, the main plant constituents: starch, cellulose, and
hemicellulose (polyoses). Starch is only briey described
because hydrothermal treatment of this compound is usu-
ally not necessary and its cost as a large-scale commodity is
still relatively high.
Starch
Starch serves as a polysaccharide energy reserve in
many plants. It is formed only by a-D-glucose units and
therefore belongs to the glucans. In amylose, the glucose
is linked chainwise in the (14)-position. The repeating unit
is h-maltose. It is important that h-maltose crystallizes
with one water molecule, whereas h-cellobiose, the repeat-
ing unit of cellulose, crystallizes without water. Obviously,
Figure 1 Net primary production of plant biomass (kg m2 the intermolecular H bonds of the latter already indicate
year1) versus precipitation (mm). (From Ref. 16.) the ability to form higher structures as manifested in
cellulose. In contrast to this, starch remains amorphous
when it is completely dry. The two compounds of starch,
amylose (frequently 2025%) and the strongly branched
be mentioned again in the chapter on the disintegration of amylopectin, are responsible for its hydrophilic nature.
C6 sugars, whereby several C3 reaction products are The nonbrous structure of starch indicates a higher
formed. energy content, easier penetration of water, and with it, a
The HatchSlackKurtschak pathway diers from the much facilitated hydrolytic attack by enzymes, for exam-
CalvinBenson cycle. In this case, it is not C3 compounds ple. This is also the reason why pretreatment or fraction-
that are formed, but an oxaloacetate, a C4 molecule. The ation of starch is usually not necessary.
xation of CO2 by phosphoenol pyruvate (PEP) and the Starch yields, taken as total carbohydrates in grain
PEP carboxylase are very ecient. Much lower CO2 con- and potatoes, lie on an average in the world between 2.0
centrations can be used as in the C3 path and the photo- and 3.2 tons ha1 year1 (Table 3). These values are
respiration is practically absent. Therefore plants following relatively low, so that starch most probably cannot com-
this C4 path in their assimilation usually have much higher pete with over 20 tons ha1 year1 of biomass growth,
biomass yields than C3 plants. which can be obtained without diculty in warm and
moderate regions of the world. The situation is dierent
2. Polysaccharide Structures when starch can be directly used. At the end of the last
The large variety of monomeric sugars enables the forma- century, approximately 13 million tons were produced
tion of an enormous number of polymeric species. Of these, worldwide to obtain glues, carriers, emulsiers, humidity
only a limited selection can be considered for technical buers, and so forth [17].
application. In this chapter, a further reduction to very few Cellulose
polysaccharides is made, for which the main reasons are as Cellulose is the most important glucan. The repeating
follows: unit of this homopolymer saccharide, the cellulose, is
The overproduction of food in the industrialized cellobiose, which consists of two glucose molecules (Fig.
countries suggests that new uses for agricultural 2a). The macromolecular chain containing only a-D-glu-
land, outside the usual food and feed line, should be cose is formed by h-(14)-glycosidic links (Fig. 2b). For
found. For this purpose, the main plant polysac- detailed structural studies, see also the contributions of
charides (cellulose and hemicellulose) are a prom- Kajiwara and Miyamoto [18], Zugenmaier [19], Ogawa and
ising resource. Yui [20], and Tvaroska and Taravel [21] and the relevant
The latter problem can only be solved by the mass articles in this book.
production of plant materials (e.g., short-rotation Cellulose is produced annually by plants on the con-
forestry, fast-growing annual plants) and/or the tinents in the enormous amount of f50 109 tons.
use of agricultural waste products (e.g., straw). Compared with this gure, the yearly (1998) crude oil
Petrochemically derived car fuel must be substi- delivery (3.5 109 tons), iron and steel manufacture (1.4
tuted by biofuels (e.g., ethanol) in this century. In 109 tons), and paper and cardboard production (0.29
developing countries, this alternative fuel should be 109 tons) are very modest amounts. In most plants, cellu-
produced immediately. lose contributes up to 50% of the total dry-matter content.
In the future, because of the increasing demand for The glucose molecules in the cellulose (Fig. 2c) can
paper, environmentally advantageous processes rotate; therefore, the OH groups can form H bridges both
Figure 2 Stereochemical formula of cellobiose and cellulose. (a) Cellobiose; (b) segment of cellulose; (c) two sections of cellulose
chains and their intermolecular and intramolecular bonds.
within its own molecule (intramolecular) and also with the sugars such as xylose (xylans or pentosans), mannose
neighboring chain (intermolecular). This leads to a very (mannans), or galactose (galactans). All hemicelluloses
rigid structure responsible for the surprising stability of the have side chains or groups like a-D-4-O-methyl glucuronic
plants. In Fig. 3, three possible forms of cellulose micro- acid, galactose, or arabinose and acetyl units. In Table 5,
brils are shown [22]. A low degree of crystallinity is given the sugar units of the hemicelluloses and their abundance in
in Fig. 3a and a high degree of crystallinity in Fig. 3b. Fig- hardwood and softwood are given.
ure 3c shows the cellulose molecules in a folded congura- Xylans
tion. Kajiwara and Miyamoto [18] showed that the most
As shown in Fig. 4a, a hardwood xylan exhibits, with
probable conguration of cellulose is a rather extended
its a-D-O-methyl glucuronic acid and acetyl groups, a
chain structure. Especially the great number of intermo-
molecular construction that does not allow the formation
lecular H bridges indicates the dierence between starch
of tightly packed brils. The homopolymer backbone of
and cellulose. To degrade cellulose, water temperatures of
f250jC or strong acids are needed. The enzymatic attack xylose units is linked by h-(14)-glycosidic bonds. In soft-
woods, arabino-4-O-methylglucuronoxylans are usually
requires specic pretreatment methods; otherwise, the
found (Table 5).
saccharication yields are dramatically low.
Mannans
Hemicelluloses (Polyoses) In coniferous trees, xylans are present to a much lesser
Hemicelluloses [23] are not built as uniformly as degree than glucomannans (Table 5). A glucomannan from
cellulose. The main structures of the polyoses contain C5 softwoods is depicted in Fig. 4b. Sometimes the term
898 Bobleter
However, their function for the plant water balance has

not yet been suciently studied. But the ease of water
penetration explains that the degradation of hemicelluloses
at higher temperatures with water or at lower temperatures
with acids or enzymes is very much facilitated compared
with that of cellulose. This fact holds also when the
activation energy of the cleavage of the glycosidic bond is
not very dierent between cellulose and hemicelluloses.
HemicelluloseLignin Bonds
The high water solubility of many hemicelluloses
indicates that they are usually bound covalently to lignin.
Figure 5 shows dierent ligninhemicellulose linkages
according to Fengel and Wegener [27]. The arrows depict
the position of possible bond cleavages during thermal
treatment. The weaker linkages break rst, but little is
known about the strength of the hemicelluloselignin bind-
ings. In addition, it is also possible that linkages within the
lignin structure break. As Meshgini and Sarkanen [28]
proved with synthetic lignin compounds, a-aryl ethers
have, with 79 kJ/mol, a much lower activation energy for
hydrolysis than h-aryl ethers (118 kJ/mol). The latter
diverge only slightly from the cleavage energy of h-(14)-
Figure 3 Cellulose brillous structures: (a) low crystallinity;
glycosidic bonds.
(b) high crystallinity; (c) folded models. (From Ref. 22.)
3. Plant Polysaccharide Composition, Resources,
and Costs
mannan is used only for compounds that contain at least
Selected plant materials, whose polysaccharides show good
85% of mannose units [26]. However, in this case, hemi-
possibilities for future technical use, have an approximate
celluloses that have also a lower mannose content are
content of cellulose of 3350%, hemicellulose of 630%,
counted as mannans.
and lignin of 929%. In Table 6, the percentages of
Galactans cellulose, hemicellulose, lignin, and ash for several hard-
This type of hemicellulose usually occurs in woods woods, softwoods, grasses, and lignocellulosic waste mate-
only to the amount of 0.53%. But there are exceptions: in rials are listed. In many cases, a comparison of the values is
the heartwood of larches, 1025% may be represented by still rather dicult because dierent analytical methods are
this hemicellulose (Fig. 4c). used and the biomass material shows natural uctuation in
Generally speaking, the side chains of the hemicellu- its composition. The analyses of wood demonstrate a
loses allow free access of water to these compounds. reasonable sum of 8899% for cellulose, hemicellulose,
Table 5 Compounds,a Percentage, and Degree of Polymerization (DPb) of Hemicelluloses (Polyoses) of Deciduous and
Coniferous Trees
Deciduous trees Coniferous trees

Hemicellulose
(polyoses) Percentage DP Compounds Percentage DP Compounds
Xylans 2030 100200 Xylose (Xyl) 510 70130 Xylose (Xyl)

4-O-methyl-glucoronic 4-O-methyl-glucoronic
acid (MGA) acid (MGA)
Acetyl gr. (Ac) Acetyl gr. (Ac)
Mannans 35 6070 Mannose (Man) 2025 Mannose (Man)
Glucose (Glu) Glucose (Glu)
Galactose (Gal)
Acetyl gr. (Ac)
Galactans 0.52 Galactose (Gal) 0.53 200360 Galactose (Gal)
Arabinose (Ara) Arabinose (Ara)
Rhamnose (Rha)
a
Data from Ref. 24.
b
Data from Ref. 25.
Figure 4 Hemicellulose (polyose) structures: (a) segment of hardwood xylan; (b) segment of softwood glucomannan; (c)
segment of larch wood arabinogalactan.
lignin, and ash. Some grass analyses give signicantly lower In warm climatic zones with enough water, more
sum values. The wheat straw example resulted from a than 40 tons ha1 year1 biomass production can
round-robin test by 11 institutions [37] and yielded a mean be achieved.
sum value of 92.7%. If the water extract (7.5%) is added, In climatically less favorable regions, harvests of
the total sum is 100.2%. 1020 tons ha1 year1 are possible (e.g., poplar,
In Table 7, the biomass production of several plants eucalyptus).
are given. Certain characteristics can clearly be read from High-starch-containing crops (maize and potatoes)
this table: are of interest for the food and starch industries but
have relatively large labor, fertilizer, and herbicide
In temperate humid climatic zones, it is possible to requirements.
harvest over 20 tons ha1 year1 of dry plant Harvest residues (e.g., straw, maize stalks, and
biomass (e.g., maize, willow, and miscanthus). restwood) are promising raw materials because of
900 Bobleter
Figure 5 Ligninhemicellulose linkages. Possible bond cleavages are indicated by arrows.
their high cellulose and hemicellulose content as new agricultural projects are not yet suciently commer-
well as their low prices. In several cases (e.g., straw cialized and therefore planting and harvesting equipment,
from grain crops), approximately the same amount for example, are still too expensive. In some cases (e.g.,
as the harvest can be assumed as agricultural waste miscanthus in Table 8), the cost of seedlings is, at present,
material. relatively high; a considerable decline can be expected in
the future, as indicated by the wide range of costs for this
In this connection, some economic considerations are of
plant. As soon as the rst economic breakthrough is
interest. In Table 8, several wood species and grasses are
established, corresponding price drops can be expected.
listed, including their costs in o. It can be seen that it
should easily be possible to produce certain plants at 50 o
per ton dry mater (DM). As an energy resource, 2.5 tons 4. Thermodynamic Properties of Celluloses
would be needed to replace 1 ton of oil. This means that 125 and Hemicelluloses
o per 2.5 tons of biomass is cheaper than 157 o per ton of Thermodynamic data can help in the interpretation of
crude oil at a 20 o/barrel price, not to mention the price conditions, reaction behavior, strength, and so forth of
per ton for a rened oil product (heating oil) as given in the molecules concerned. The case of macromolecular cel-
Table 8. lulose is more complicated than that of sugar compounds.
If large mass applications for plant biomass can be This becomes clear when the formation (DHform) and the
introduced (e.g., ethanol as car fuel), then even farmers combustion enthalpies (DHcomb) obtained by a modeling
could gain a positive outlook into the future: with a revenue procedure [64] are compared with the experimental com-
of $50 per ton and a harvest of 20 tons ha1 year1, the bustion enthalpies (Table 9). The modeling combustion
latter would bring $1000 ha1 year1 compared to the enthalpies show a dierence of 180 kJ/kg between the
approximately $800 ha1 year1, which an average grain crystalline and the amorphous forms. This relatively large
farmer makes in the United States. It should also be kept in energy value explains the stability and the dicult hydro-
mind that this agricultural changeover to energy farming lytic reaction conditions regarding crystalline cellulose.
would be combined with a lesser use of fertilizers and The experimental values for cellulose, having a crys-
herbicides and, in addition, could give more free time to tallinity of 6971%, agree relatively well with the results of
the farmer. the modeling procedure as long as cotton is excluded. The
The present prices for energy farming products are highest dierences are F0.36% of the average, which is
often still too high. There are several reasons for this: These somewhat higher than the accuracy of the calorimetric
Table 6 Composition of Selected Hard Woods, Soft Woods, Grasses, and Lignocellulosic Waste
(a) Holocellulose
(b) Sum of
Cellulose Hemicellulose Lignin (c) columns
Plant Scientic name (%) (%) (%) Ash (%) a + b + c (%) Reference
Hard woods
Trembling aspen Populus tremuloides, 49.4 21.22 18.1 0.4 89.1 29
Mich. 50.8 18.81 18.4 0.6 88.6 30
48 23 17 88 31
European beech Fagus silvatica 49.1 22.01 23.8 0.3 95.2 29
European birch Betula verrucosa, Erh. 48.5 25.11 19.4 0.3 93.3 29
White willow Salix alba L. 49.6 26.71 22.7 0.3 99.3 29
Soft woods
Balsam r Abies balsamea (L.) 49.4 15.42 27.7 0.4 92.9 29
Mill.
Douglas r Pseudotsuga menziesii 42.0 23.5 27.8 0.4 93.7 32
Mirg.
Austrian pine Pinus nigra, var. 49.5 11.01 27.2 0.2 87.9 29
Arnold
White spruce Picea glauca (Moench) 42.0 26.5 28.6 0.4 97.5 32
Voss
Grasses and lignocellulosic waste

Alfalfa (stalks) 48.5 6.5 16.6 70.6 33
Bagasse 33.4 30.04 18.9 2.4 84.7 34
40.6 32.2 15.0 87.8 31
Miscanthus 49.7 19.5 21.9 3.1 94.2 35
Giant reed Arunda donax 32.9 28.5 21.3 6.1 88.8 36
Sudan grass (stalks) 44.1 21.3 9.1 74.5 33
Wheat straw 42.1 26.14 19.8 2.5 92.7 37
Newspaper 43.23 16,7 24.6 4.2 88.7 38
Determination of the compounds; indices: 1 = pentosan; 2 = polyose; 3 = cellulose as glucan; 4 = hemicellulose as xylan.
measurements (0.050.1%). The cotton samples are obvi- Skrabal expressed these experimental results also in the
ously not comparable with pure cellulose and the theoret- form of an equation that still holds today:
ical value of xylan considerably diers from that of the
experimental ones. dC
kw ka H kb OH :C 2
dt
In this equation, [C], [H+], and [OH] are the concentra-

II. HYDROLYSIS AND DEGRADATION OF tions of the ether or ester, the H+ and OH ions, re-
SACCHARIDES AND POLYSACCHARIDES spectively. The constants kw, ka, and kb stand for the
A. Acidic and Alkaline Hydrolysis pH-independent, acid- and base-catalyzed reactions, re-
spectively. The pH-independent reaction constant, kw, can
Hydrolysis is one of the major degradation reactions of also be seen as the determining factor of a water-catalyzed
oligosaccharides and polysaccharides. Thereby, the glyco- reaction. At least in the horizontal region of curves b, d and
sidic bond between the sugar units is cleaved. Early in the e, the k values would be much lower if the reactions were
last century, Austrian chemists [65,66] analyzed the hydrol- only catalyzed by H+ and OH ions. Obviously, the
ysis of ethers and esters very thoroughly. An interesting horizontal parts of the curves, including the line c, are
result is given in Fig. 6. The reaction constant, k, can be determined by the action of water and are, therefore, water
pH-independent or show a fairly good linear dependence catalyzed, as pointed out by Skrabal [66].
when log k is plotted versus the pH. Several cases were In Fig. 7, three possible reaction paths of cellobiose
found where mixed behavior occurs; for example, with hydrolysis are demonstrated [67,68]. On the left side of this
increasing pH, the log k values decrease, then become pH- gure acid hydrolysis is shown, whereby the formation of a
independent and increase again at a higher pH range. conjugated acid, IIa, introduces the glycosidic bond cleav-
902 Bobleter
Table 7 Annual Production of Dry Matter of Selected Plants
Maximum harvest Typical harvest

Plant (tons ha1 year1) (tons ha1 year1) Harvest index Country References
C4 grasses
Sugar cane 6080 53 0.85 (M) USA, Fl 3941
Maize 2040 15.522.6 0.40.5 (S) A 39, 42
Tropical grasses 3080 1527 0.85 (M) USA 39, 43
Napier grass 45 1645 USA 44
Miscanthus 944 444 Europe 4547
C3 grasses
Wheat 1030 1525 0.250.45 (S) D 39
Rice 2050 0.40.55 (S) 39
Leguminosae and root crops

Potatoes 20 7.0 0.820.86 (R) D 7, 39, 48
Sugar beet 2030 0.450.67 (R) 39
Soybeans 1030 030.35 (S) 39
Forest plants
Pinus radiata 46 0.66 (W) 39
Spruce 22 0.61 (W) 39
Birch 13 27 0.70 (W) SF 39, 49
Energy farming
Willow 50 6.340 0.6 (W) S,CDN 5053
Poplar hybrids 3440 1525.3 0.5 (W) US, IRL, CDN 39, 54, 55
Eucalyptus grandis 41 717.4 Hawaii 39, 56
Bougainvillea 80 80 India 57
Harvest index: S = seeds and grains, M = mass above ground, R = roots, W = wood without leaves and small branches. A = Austria, CDN =
Canada, D = Germany, Fl = Florida, IRL = Ireland, S = Sweden, SF = Finland, US and USA = United States.
age and leads to the two glucose units. In the middle part adsorption (IIc). Water and the glycosidic bond split
of this gure, alkaline hydrolysis is depicted. The OH simultaneously (IIIc) and so form two glucoses again.
attack at the anomeric carbon atom, IIb, renders the Obviously, half of the cellobiose molecule follows the
cleavage of the O bridge (IIIb) and again yields the two acidic cleavage pattern in step IIIc and the other half the
glucose units. The hydrothermal or aquasolv cleavage on alkaline. As will be shown later, this hypothesis is also
the right side of the gure is rst characterized by the H2O supported by experiments.
Table 8 Examples of Reported Costs of Plant Biomass
Energy value Cost per Cost per

Plants and fuel (MJ/kg) ton (o) GJ (o) Remarks (country/year) Reference
Willow 14.2 30 2.1 70% DM (IRL/1986) 53

Eucalyptus 19.8 30 1.5 Max yield 35.6 Mg/(ha year) (USA/1989) 58
(19.7) 45 2.3 (Hawaii/1994) 59
Slash pine 19.7 40 2.0 (USA/1989) 58
Switchgrass (17.6) 1020 0.61.2 (USA/1991) Weather dependent 59
Softwood (19.7) 60 3.4 (S/2001) 60
Hardwood 19.7 52 2.6 (USA/1986) 61
Tall grass 17.6 32 1.8 (USA/1992) 43
Miscanthus 17.8 3682 1.94.4 Seedling costs! (D/1991) 62
Straw (17.6) av. 43 2.4 (GB/1976) 63
Heating oil 44.2 378 8.6 (A/2002)
The prices are taken from the literature cited and correspond to the year indicated. Foreign currencies are converted into o. The oil price is given
as reference. DM = Dry matter, IRL = Ireland, D = Germany, GB = Great Britain, S = Sweden and A = Austria.
Table 9 Thermodynamic Properties of Celluloses and Xylan
Modeling
DHform
298 DHcomb
298 DHcomb
298 Experimental
Polymer Chain fragment (kJ/unit) (kJ/unit) (kJ/kg) Phase or sample DHcomb (kJ/kg)
Cellulose 952.1 2,840.7 17,522 Crystalline

922.8 2,870.1 17,702 Amorphous
Fibers 17,543
Ramia 17,517
Cottonb 17,208
Cotton cellulose 17,438
Wood cellulose 17,472
Xylan 726.8 2,384.4 18,062 17,836
The results of a modeling method are compared with experimental values.

a
Mean of three experimental values.
b
Mean of two experimental values.
Source: Ref. 64.
1. Acid Hydrolysis of Glucans The expressions in brackets are the concentration symbols
A detailed description of acid hydrolysis of cellulose and of the above compounds. Combining Eqs. (2) and (3) and
hemicellulose is given by Abatzoglou and Chornet [69] in using [C] for the concentration of all oligomeric saccharides
this book. In the following chapter, only those aspects are and polysaccharides concerned, but represented by glucan
discussed, which may have an inuence on hydrothermal monomers, it follows that
degradation of saccharides and polysaccharides. dC
It can be assumed that the hydrolysis of cellulose kw ka H kb OH :C k1 C 4
dt
occurs in the same way as the cleavage of the cellobiose For glucose, the integrated form is given by
molecule. According to Saeman and Grethlein [70,71], the
reaction is a pseudo-rst-order sequential process: k2
Glu C0 ek1 t ek2 t Glu0 ek2 t 5
k1 k1
k1 k2 High [Glumax] values can only be reached when k1 H k2. In
cellulose ! glucose ! decomposed glucose 3 Fig. 8, this situation is clearly demonstrated. Fagan et al.
Cell Glu Dec
[72] showed that with acid hydrolysis (1% acid concentra-
tion), a maximum glucose value of f50% C0 can be
achieved at 230240jC, indicating that k1 and k2 are
approximately equal. With 0.2% acid concentration, not
more than 20% maximum glucose formation of the orig-
inal cellulose is obtainable. This excellent work certainly
explains why industrial acid hydrolysis was abandoned in
the western countries. With a 50% glucose yield, biomass is
too expensive to economically deliver sugars. In addition,
the highest curve in Fig. 8 shows that only 6 sec after the
peak, the glucose concentration is already 10% lower. For
technical application, such limitations are unacceptable.
In several experiments, the biomass is soaked in an
acidic solution, especially before steam treatment. This pro-
cess is called steam treatment with catalyst. Figure 8
indicates also for these conditions that, at low acid concen-
trations and temperatures below 220jC, not too much of
the cellulose is hydrolyzed. Some exceptions are reported
[73], which are, however, dicult to explain. Table 10 shows
that the strengths of the inorganic and organic acids dier
very much. The pKa values lie between 6 (HCl) and 4.8
Figure 6 Hydrolysis of esters and ethers. Dependence of the (CH3COOH). The low dissociation behavior of acetic acid
reaction constants k on the pH. (From Ref. 66.) indicates why this acid is an unfavorable hydrolysis agent
904 Bobleter
Figure 7 Hydrolysis of cellobiose. Acid (H+ and index a), alkaline (OH and index b), and water catalyzed (H2O and index c).
[74] and behaves more like an organosolv medium. In the activation energies of cellobiose, xylobiose, starch,
addition, its ionization constant, Ka, decreases from 1.75 cellulose, and hemicelluloses are so close together that
to 1.63 when the temperature is raised from 20jC to 50jC. the kinetic temperature behavior of their hydrolysis should
Table 11 gives the reaction conditions and the activa- not dier very much. Only saccharose is obviously much
tion energies for acid hydrolysis for several saccharides easier to split than the aforementioned compounds.
and polysaccharides. During the years of the Soviet Union, several large
In Szejtlis compilation [67] of cellobiose, the highest acid hydrolysis plants were still in operation. Insiders
deviation of the activation energies from the mean were in reported that, most of the time, half the factories were at
the region of approximately 10%. It is also surprising that a standstill, mainly owing to corrosion problems.
built, which reduce the pH during the reaction. In addition,

the side reactions are more pronounced than in acid
hydrolysis. Relevant experiments with cellobiose [79] ex-
plain this situation quite well. In Fig. 9, the alkaline
treatment of cellobiose (Cbi) is shown. The alkaline con-
centration (0.1 N NaOH) is rather high and the cellobiose
concentration low (10 g/L). Therefore the consumption of
Cbi follows a rst-order reaction well, which is not the case
at lower NaOH concentrations. However, it is surprising
that the maximum amount of glucose is not more than
f20%. Under these conditions, the glucose is transformed
to fructose (Lobry de Bruyn-van Ekenstein rearrange-
ment), but even the sum of glucose and fructose hardly
exceeds 30% of the cellobiose consumption. These results
lead to a rather complicated reaction scheme, as shown in
Fig. 10. The strong conversion to degradation products
explains why only a low-monomer sugar yield can be
achieved. The consumption of NaOH in the above experi-
Figure 8 Predicted glucose yields by acid hydrolysis of ments indicate that, at the end of the reaction, approxi-
paper cellulose. (From Ref. 72.) mately half of the saccharides are converted to acids. To
calculate the reaction mechanism, lactic acid was taken as
the major acidic compound. Obviously, the end products
of alkaline hydrolysis of carbohydrates are organic acids.
This is also conrmed by de Bruijn et al. [82], who found
2. Acid Hydrolysis of Hemicelluloses that a 100% acid formation at pH 14 occurs with 50%
lactic and 30% C-6 saccharinic acid.
A pentosan prepared from beechwood (Fagus crenata) was
Very few reaction kinetic constants for alkaline hy-
hydrolyzed with H2SO4 [77]. Two reaction regions were
drolysis are found in literature, for which one reason is
observed with the same activation energies (E1 = 129.2 kJ/
experimental diculty. The experiments described in Fig. 9
mol) but dierent frequency factors (A1 = 2.56 1015 and
lead to an approximate activation energy of E = 120 kJ/
2.57 1014). The acid hydrolysis [78] of xylans from wheat,
mol, which, again, is within the limits of the acid and
rye, barley, and rice showed that L-arabinose is cleaved
hydrothermal bond cleavage [64].
more quickly than xylose, and D-xyloseD-glucuronic acid
The astonishing fact remains that, with 0.1 N NaOH at
is further delayed in the reaction, which indicates that
70jC, more than 90% of the cellobiose is transformed after
dierent reaction mechanisms occur during the hydrolysis.
8 min, but cellulose can be mercerized [83] with 18% NaOH
This is probably also one of the reasons why the range of
at 100jC for 1 hr with little attack to this macromolecule.
activation energies (106.2159.7 kJ/mol) is very wide com-
Obviously, under these conditions, NaOH cannot break
pared with other measurements (Table 11). The inuence
the H bridges and even stabilizes the structure of micro-
of the crystallinity because of the H bonds explains the 60
brils, which is indicated by the gain of crystallinity of the
80 times faster hydrolysis reaction rates of xylans than of
cellulose bers after this treatment.
cellulose [67].
Experiments with supercritical ammonia [84] were also
The compound of xylose analogous to cellobiose is
carried out, but an ammonia concentration of approxi-
xylobiose (4-O-h-D-xylopyranosylD-xylopyranose). Its
mately 30% of the reactor volume and relatively high
activation energy for acid hydrolysis [67,76] is 137 kJ/
temperatures (150jC) were needed.
mol, which is again relatively close to the cleavage energy
of the glucans (Table 11).
B. The Organosolv Process
3. Alkaline Hydrolysis In 1931, a wood pretreatment process, which used a
Alkaline hydrolysis is also approximately proportional to mixture of an organic solvent with water, was proposed
the OH concentration. However, the measurements are by Kleinert and Tayenthal [85]. Frequently alcohols (e.g.,
much more dicult to carry out because organic acids are ethanol or methanol) were employed. At this point the
Table 10 pKa Values of Inorganic and Organic Acids
Acid HCl H2SO4 HNO3 CF3COOH H3PO4 HCOOH CH3COOH
pKa 6 3 1.32 0.23 1.96 3.7 4.8

906 Bobleter
Table 11 Reaction Conditions and Activation Energies of Acidic, Alkaline, and Hydrothermal Degradation of Saccharides and
Polysaccharides
Saccharides and Activation energy

polysaccharides Temperature (jC) Catalyst (kJ/mol) Remarks (reference)
Acid hydrolysis
Cellobiose 1899.5 HCl, H2SO4 125.4 Mean of 13 experiments after
H3PO4 Szejtli [67]
160220 H2SO4 133 [75]
Saccharose HCl, H2SO4 105.9 Mean of three experiments [67]
Xylobiose 6080 H2SO4 136.9 [67]
137 [76]
Starch 18100.9 HCl, H2SO4 122.5 Mean of two experiments [67]
Cellulose 0100 HCl, H2SO4 127.5 Mean of 14 experiments [67]
H3PO4
Hemicellulose H2SO4 129.2 [77]
106.2159.7 [78]
Alkaline hydrolysis
Cellobiose 6080 NaOH f120 [79]
Hydrothermal degradation
Cellobiose 180249 H2O 136.0 [80]
Cellulose (cotton) 215274 H2O 129.1 [81]
question arises: How does the added alcohol inuence the rst covered by a patent in 1968 [89] whereby in the
reaction behavior? There was the hope that the organic temperature range of 200260jC, up to 50% of the biomass
medium will increase the solubility of the lignin. This hope were dissolved during a 10-min reaction time. At the same
was only fullled to a very minor degree, and in certain time, also the expression hydrothermal was chosen in the
cases this attempted eect does not occur at all, as will be publication on hydrothermal degradation of glucose
shown later. In Table 12, dierent organic media applied in [90]. In the meantime, steam treatment of LCM became
the organosolv processes are listed. With the exception of an intensively investigated eld. Lora and Wayman [91]
methanol and ethanol, all other solvents are expensive, so published their paper (1978) on delignication of hard-
that a commercial application would only be advisable if woods and called the process autohydrolysis. Steam
lignin solubility were strongly increased, which is obviously pretreatment or steam extraction was introduced in 1981
not the case. Phenol and cresol are not acceptable in large- by Puls et al. [92], whereby the steam treatment was
scale technology because they cause health problems. followed by a cold water washing cycle. Since 1983, the
Acetic acid is taken here as an organosolv constituent, expression hydrothermolysis is also in use [93]. Conner
which, as mentioned earlier, is more realistic than its (1984) called his process water prehydrolysis [94]. In
inclusion among the acid-catalyzing media. However, there 1986, Heitz et al. [95] described their experiment by aque-
is also another disadvantage to using organic acids, espe- ous liquefaction or extraction. Hydrothermal pretreat-
cially acetic acid, for hydrolysis: At the applied temper- ment was also chosen in 1987 by Overend and Chornet
atures (e.g., 180220jC), these acids are reactive and are [96]. In 1994 the useful expression aquasolv was intro-
able to form esters with the saccharides. Under these duced by Antal [97].
conditions, quite considerable losses of acids [74] and the All these expressions are easy to understand and
formation of cellulose and hemicellulose esters occur. mostly self-explanatory. Nevertheless, a stronger denition
In the case of the technical application of the organo- of this working eld would be advisable. Therefore in
solv process, the necessary explosion-proof installations accordance with Overend, Chornet, Antal, and Garrote
are a severe nancial burden. et al. [31], it is proposed to use hydrothermal treatment or
pretreatment as general title that includes both subelds
steam treatment and aquasolv.
C. Hydrothermal Treatment (Steam
and Aquasolv Treatment) 1. Steam Treatment
The treatment of plant biomass (lignocellulosic material = Steam treatment can only be applied to solid biomass
LCM) with steam goes back to 1929 and 1932 when Mason material, such as wood chips, straw, etc. The evaluation
[87] and Babcock [88] introduced their steam explosion of the hydrothermal reaction behavior of water-soluble
process. Extraction treatment with hot liquid water was substances has to be carried out in hot liquid water. The
hemicellulose is already dissolved at 180jC. Between

220jC and 300jC, cellulose is also degraded. The dierence
between the cellulose and the wood curves is mainly
because the hydrothermal treatment of the plant matter
also dissolves a considerable part of the original lignin, but
the remaining part of the lignin retards, to a certain extent,
the hydrolysis of the wood cellulose. The isolated lignin
used in these experiments was obtained by HCI hydrolysis.
Therefore a certain change of the lignin structure must be
taken into account, but both curves, the lignin and the
wood degradation, correspond well in the temperature
region concerned.
The equipment used for this experiment [98] is
depicted in Fig. 12. The reaction vessel (RV) is lled with
biomass and the water vessel (WV) with an amount of
water that, when expanded during the temperature rise,
does not exceed the volume of the vessel. When the reaction
temperature is reached, the hot water is led through the
reaction vessel and the heat exchanger (HE). The ow rate
is adjusted by the valve (V2). At the same time, the
thermostat oil is pumped through the oil guide tube (G)
to allow a fast heat-up of the pressure vessel (F).
Low-Molecular Saccharides
To gain a better understanding of the process, it was
important to study the hydrothermal behavior of low-
molecular compounds. These have the advantage that most
of the degradation products can be quantitatively analyzed
and, in addition, the starting material (cellobiose) is soluble
in water.
For this purpose, cellobiose (Cbi) is a very suitable
Figure 9 Alkaline hydrolysis of cellobiose and the for- compound because it has the glucosidic bond and hydrol-
mation of D-glucose (Glu) and D-fructose (Fru). (a) ysis leads to two glucose (Glu) molecules. The reaction
Degradation of cellobiose (Cbi) with 0.1 N NaOH at 60jC, scheme can be written as
70jC, and 80jC. (b) Formation of glucose and fructose at
60jC and 80jC. The yields are given in percentage of the k1 k3 k2
Cbi ! M2 ! Glu ! Degradation products
initial cellobiose plotted versus the reaction time (min). The !
k4
plotted curves are results of the calculation. Degradation products
6
An intermediate, M2, is assumed, from which by the
large amount of results obtained by steam treatment reaction constant k3 glucose, and by k2 and k4, the degra-
experiments show that especially the separation (fraction- dation products are formed.
ation of LCM) of hemicellulose from the plant biomass Figure 13a shows the cellobiose consumption and Fig.
material was successfully carried out with this process. 13b shows the glucose formation and degradation [80].
Because fractionation is the main purpose of steam treat- Several features in these gures are remarkable: The reac-
ment, this eld will be described in Sec. III. tion rate for cellobiose at 180jC is still very low, but at
229jC, a 50% consumption is reached in a reaction time of
2. Aquasolv Treatment less than 2 min. In this case, k1Hk2, which can be seen from
This process uses only water at the chosen reaction tem- the dierent steepness of the curves in Fig. 13b. What is the
perature (e.g., 180220jC). In the rst work in 1968, it was reason for introducing the pathway with the reaction
already shown [89] that, at the above-mentioned temper- constant k4 in Eq. (6)? If the curves in Fig. 13a and b are
atures, hemicellulose and part of the lignin can be degrad- compared, it is evident that more cellobiose is degraded
ed, as can cellulose at higher temperatures. In Fig. 11, the than the equivalent amount of glucose formed. In addition,
solid spruce wood residues as dry matter after a 10-min the [Glumax]/[C] relation of the 249jC curve gives a k1/k2 =
ow-through treatment at the indicated temperatures are 1.5, whereas the mathematical treatment of Eq. (5) for this
given [98,99]. On the ordinate, the percentage of lignin, reaction results in a k1/k2 = 7.7. All these arguments lead
cellulose, and hemicellulose, according to Table 8, are also to the conclusion that a side reaction with the constant k4
shown. For comparison, the degradation of pure cellulose has an important inuence. [Glumax] under hydrothermal
and lignin is given. It is obvious that a large part of the conditions is much higher than in alkaline hydrolysis
908 Bobleter
Figure 10 Reaction scheme of the alkaline degradation of cellobiose.
(Fig. 9b) but lies in the middle between the alkaline and work [80] with glucose, it was shown that alkaline and
acid hydrolyses. This is again an indication that hydro- acidic solutions yield a dierent product spectrum, and
thermolysis follows partly the acidic and partly the alka- hydrothermolysis is related to both of them. Hydroxy-
line reaction path. The activation energy for the k1 path methylfurfural and furfural do not appear in alkaline
was found to be 136.0 kJ/mol, very close to the acid and treatment, whereas methyl glyoxal and dihydroxyaceton
alkaline hydrolyses. are relatively high yield reaction products only in aquasolv
A further method for the comparison of alkaline, treatment (Fig. 14).
acidic, and hydrothermal treatment is the analytical eval- A similar picture is obtained when C-14-labeled glu-
uation of the reaction products of saccharides. In a relevant cose is hydrothermally treated [100]. At rst, methyl
Table 12 Selection of Organosolv Media and Corresponding Cooking Conditions
Solvent % in H2O Wood: liquor Cooking temperature (jC)
Methanol 50100 1:10 130220

Ethanol 4060 1:615 120240
Propanediol 50 Not specied 190
Butanol 3070 1:1015 120250
Glycol 20100 1:46 100205
Tetrahydrofurfuryl alcohol 50100 1:10 95205
Phenol 2050 1:815 80205
Cresol 2080 1:78 160190
Acetic acid 5095 1:48 110220
Acetic acid/ethyl acetate 2633/3349 Not specied 165170
Source: Ref. 86.
glyoxal, 4, is strongly formed, dihydroxyacetone, 5, and

hydroxymethylfurfural (HMF) to a lesser extent. In a 1.33-
min autoclave experiment at 240jC, a 96% recovery yield
of the three compounds mentioned, including the
unreacted glucose, and the minor components (fructose,
glycolaldehyde, and furfural) was obtained. As mentioned
earlier, the formation of C3 compounds during the aqua-
solv treatment of C6 sugars has its analogy in the photo-
synthesis of hexoses by C 3 compounds. In these
chromatograms, the acids were not determined. The oc-
currence of acetic, glycolic, formic, and lactic acids was
already analyzed by MacLaurin and Green [101] and these
compounds were also found in hydrothermal treatment of
xylose [102,103]. The acids are responsible for the partial
neutralization of the base in alkaline treatment and for the
decrease of the pH in hydrothemolysis.
The results described (i.e., that hydrothermolysis pro-
duces compounds equivalent to alkaline and acidic treat-
ment, and that a lowering of the pH occurs) were the cause
of many discussions concerning the eld to which hydro-
thermolysis belongs. According to the experiments [75]
given in Fig. 15, the answer to this question can be given.
Owing to the fact that hydrothermal hydrolysis of cellobi-
Figure 11 Dry-matter residue after an aquasolv, percola- ose shows no pH dependence in the region of pH 3 to about
tion treatment (10-min reaction time). o, residue of spruce pH 5, it is catalyzed by neither alkaline nor acid media and,
wood; +, residue of cellulose; *, residue of lignin. therefore, constitutes an individual process. This behavior
Figure 12 Aqusolv ow-through (percolation) laboratory equipment with 124-mL reactor volume. (a) Schematic diagram;
N2 = nitrogen ask, Th = oil thermostat, V1 and V2 = valves, WV = water vessel, RV = reaction vessel, and HE = heat
exchanger. (b) Reaction vessel: A = biomass container, B = water inlet tube, C = water outlet tube, D = cover, E = seal, F =
pressure vessel, G = guide tube for thermo-oil, H = thermo-oil inlet and outlet.
910 Bobleter
Figure 13 Hydrothermal degradation of cellobiose and formation of glucose in the temperature range 180249jC. (a)
Cellobiose degradation versus reaction time. (b) Formation of glucose versus reaction time. The plotted curves are calculated
according to Eq. (6).
Figure 14 Hydrothermal, alkaline, and acidic degradation of cellobiose including the main reaction products.
Figure 15 Acid hydrolysis and aquasolv treatment of

Figure 16 Aquasolv treatment of D-xylose and the for-
cellobiose. The reaction constant k1 as a function of the
mation of furfural. The yields are given in mol% at
initial pH at 200jC. (z) pH before the reaction and (5) pH
temperatures between 180jC and 220jC.
after 50% cellobiose consumption.
corresponds to curve b in Fig. 6 and is another example of slow, but at 274jC after only 12-min reaction time, 50% of
Skrabals early discoveries. The pH below 3.2 remains the cellulose is solubilized. The dierences between the
constant during the reaction, but the initial pHs at 3.5 hydrothermal degradation of cellulose and cellobiose are
and 4.7 decrease with increasing reaction time to 3.4 and signicant. Even if it is considered that, at a given temper-
3.7, respectively, after 50% consumption of the cellobiose. ature, the same amount of h-glycosidic bonds are broken in
This eect is due to the formation of acids as mentioned both compounds, the chances of recombination are cer-
earlier. The rst-order reaction, as shown in Fig. 13, does tainly much higher in the dense cellulosic structure than in
not change during the whole reaction time, which is the dissolved cellobiose. In addition, it needs several h-gly-
additional proof that the pH in this region does not cosidic splitting processes until water-soluble glucans are
inuence the reaction rates. obtained. Therefore it is not surprising that a comparison
Xylose between the stability of cellulose (Fig. 17) and cellobiose
(Fig. 13), taking their half-lives at the same temperature
As regards the low-molecular pentosans, few experi- (240jC), renders a relation of 89:0.8 min. Nevertheless, the
mental results are available. However, the hydrothermal activation energy with 129.1 kJ/mol is again close to that of
degradation products of pentoses are similar to those of cellobiose and acid hydrolysis (Table 11).
the hexoses. With a view to the technical application, an important
In Fig. 16, the hydrothermolysis [102] of D-xylose, its question is: How far the reduction of the DP during
consumption, and the formation of the main product, hydrothermolysis reduces the ber properties of the cellu-
furfural, is shown. After 26 min at 220jC, a relatively high
50% furfural yield is obtained. At this temperature, the
formation of acids (formic, glycolic, acetic, lactic, and
peruvic acid) reaches about 20% of the original xylose
[103], which is more than in acid hydrolysis, but much less
than in alkaline treatment. In addition, the pH behaves in a
fashion similar to that of the hexoses and drops to 3 fairly
soon. The activation energy for the xylose degradation was
found to be Ea = 119.4 kJ/mol and the frequency factor to
be 4.8 1011.
High-Molecular Saccharides
The cellooligomers, with a degree of polymerization
(DP) higher than 8, are considered to be insoluble in water
[104]. The hydrothermal attack of the solid cellulose (Cel) is
more complicated than can be expressed by a simple
equationEq. (3). Nevertheless, it is astonishing that the
experiments show a clear pseudo-rst-order reaction. In Figure 17 Degradation of cellulose by aquasolv treatment.
Fig. 17, the results of the hydrothermal treatment of cotton The residue as percentage of the original cellulose is given as
cellulose are given [81]. At 215jC, the reaction rate is very a function of time (min) and temperature (jC).
912 Bobleter
Figure 18 Change of the degree of polymerization (DP) during aquasolv treatment at 195jC. The DP of the cellulosic residue of
wheat straw was determined as a function of the reaction time.
lose. At moderate temperatures, the DP decrease is quite In Table 13, the yields of hydrothermally pretreated paper
acceptable [105]. In Fig. 18, the results of the hydrothermal dunnage according to Reynolds et al. [106] are given.
experiments with wheat straw at 195jC show that, after a Approximately 50% of the original matter is obtained as
1-h reaction time, the DP is reduced from 1500 to 1000. char and 3042% of the char are organic solubles. Twelve
Considering that this treatment in a technical installation to 17% are transformed into gaseous products and 16
should last for not more than 20 min, the DP decrease 17% into water-soluble compounds. A very important
would be only approximately 10%. Experiments carried
out with a dierent equipment gave a DP loss of 11%,
which is in good agreement with the expectations. There-
fore the hydrothermally treated straw cellulose would be
suitable for chemical ber production.
In acid hydrolysis of cellulose, the relation of k1/k2 in
Eq. (3) increases with increasing temperature and, there-
fore, the maximum glucose yields are obtained at high
temperatures. To a certain extent, this is also true for the
hydrothermal aquasolv process. If pure cellulose (e.g.,
cotton) is treated [81] at a relatively high temperature,
good glucose recoveries can be achieved. In a percolation
experiment at 295jC, cotton is used as original cellulose. In
Fig. 19, the dierent ow rates in the 11-mL reaction vessel
correspond to the indicated residence times of the liquid at
the chosen reaction temperature. The highest glucose yield
is obtained after f1.2 min and approaches 50% of the
original cellulose. Including the other analyzed compounds
(Cbi = cellobiose, HMF = hydroxymethylfurfural, Ahg
= anhydroglucose, Fru = fructose, and F = furfural), an
amount of 68.8% is recovered. An additional 19.7% of the
solubles were not determined compounds, only 2.4% are
solid residues and 9.1% remained as losses. Figure 19 High-temperature hydrothermal treatment
If cellulose were cheaply available, this would be an (aquasolv at 295jC) of cellulose and the formation of the
interesting process. There is, however, a severe drawback: reaction products. G = glucose, Cbi = cellobiose, HMF =
The cellulose has to be relatively pure; otherwise, the sugar hydroxymethylfurfural, Ahg = anhydroglucose, Fru =
yield drops considerably. fructose, F = furfural. The ow rate (mL/min) is plotted
By further increasing the reaction temperatures or as well as the corresponding residence time (min) of the water
times, interesting product distributions can be expected. in the reaction vessel.
Table 13 Hydrothermal Treatment of Paper Dunnage at High Temperatures
Char
Reaction conditions (jC) Gas Watera Water solubles Organic solubles Extracted Sum
275 12 16 17 21 34 55
310 17 18 17 20 28 48
320 16 18 16 15 35 50
Product distribution and yield in wt.% after 30-min reaction time.
a
Water was determined by balance.
Source: Ref. 106.
feature of this process is that the slurry obtained is pump- at least part of the coal formation from plant biomass is due
able and can therefore be transferred easily for further to a hydrothermal process.
treatment (e.g., gasication). This advantage becomes clear Another important process was initiated by Antal et
when it is remembered that waste paper cannot be pumped, al. [107,108] by recognizing that high-temperature hydro-
the viscosity being far too high. Even the burning of paper thermal treatment of wood led to much higher yields of
in conventional incinerators does not function well, be- charcoal than the conventional processes were able to
cause the outside carbonization of the paper bales can achieve.
isolate the inner part of the package so that some of the
used journals can still be read after the ring process. The Hemicelluloses (Polyoses)
authors showed that the hydrothermal treatment of paper If plant material is treated hydrothermally at approx-
dunnage leads to chars that are very similar to type III imately 200jC, the hemicelluloses are dissolved. In Fig.
(coaly) geological samples. This is a strong indication that 20a, a percolation (ow-through) experiment [93] using
Figure 20 Aquasolv elution proles of poplar and birch wood. (a) Populus tremoloides treated at 186jC in a percolation
experiment; (b) birch wood percolation experiments at = 233jC and + = 199jC. The concentration in the eluate is given in
grams per liter as dry matter. The curves were obtained according to Eq. (7) with k = 16.
914 Bobleter
aspen wood is shown. The hemicellulose is nearly quanti- must and will be improved if large-scale industrial use of
tatively brought into solution and about half of the original hemicelluloses is in view.
lignin as well. The form of the bell-shaped elution prole
has an analogy in chromatography. In both these elds, the
eluted concentration C(t) at a time t depends on the III. HYDROTHERMAL FRACTIONATION
maximum concentration, Cmax, and the time tmax where OF POLYSACCHARIDES
the maximum concentration occurs (k is a dimensionless
A major problem in the chemical and technological use of
constant):
! the large plant resources is the robustness of the combined
ktmax t2 cellulose, lignin, and hemicellulose structure, which is
Ct Cmax exp 7 dicult to separate. To date, paper manufacturers usually
tmax t
use strong chemicals (e.g., sulte) to solubilize lignin.
In Fig. 20b, two birch wood elution experiments are plotted Environmentally favorable fractionation processes would
according to Eq. (7). Cmax and tmax are taken from the be desirable to obtain biomass fractions that can be trans-
experiments. The coincidence of the theoretical curve and formed more easily into the desired products with high
the measured points is fairly good, especially if it is yields. The separation of hemicellulose with a part of the
considered that the experiments were performed at dier- lignin from the original plant matter can result in a strongly
ent temperatures and that two dierent equipments (10-mL opened structure so that large molecules can easily pene-
and 50-L reactor volumes) were used. The constant k is 16 trate the material. In this way, enzymes, such as cellulases,
in both cases. guarantee high saccharication yields.
Additional work is needed to characterize the hemi-
cellulose eluted during hydrothermal treatment. An initial A. Low-Temperature Fractionation
comparison of eight dierent hemicelluloses is given in
Table 14 [109]. The rst four (bagasse, wheat straw, rice In this case, temperatures of about 100jC are meant.
straw, and miscanthus) were obtained by hydrothermal
percolation, the dissolved matter precipitated with ethanol, 1. Acidic and Alkaline Treatment
and the precipitate hydrolyzed with triuoroacetic acid and Acidic hydrolysis in this temperature region has not gained
then analyzed by capillary zone electrophoresis. The com- technical importance. Only for analytical purposes are
mercial xylan products (birch wood, Lenzing hemicellu- highly concentrated mineral acids and triuoroacetic acid
lose, and oat spelts) were directly hydrolyzed and analyzed in use [110]. Thereby, the polysaccharides are hydrolyzed
as before. and mainly monomeric sugars are obtained; the lignin
The recovery yield in these experiments is relatively remains as solid residue.
low and lies between 65.4% and 85.8%. The reason for this At low alkaline concentrations (e.g., 1% NaOH) and
can be found in several directions: The glucuronic and room temperature, only insignicant parts of hardwood
galacturonic acids are dicult to hydrolyze and were found and straw [111,112] are dissolved, but enzymatic hydrolysis
only in rice straw and miscanthus. The acetyl groups were is clearly enhanced. With higher NaOH concentrations
hydrolyzed to acetic acid and therefore not detected. Also, (e.g., 5%), f68% of the pentoses, including some cellulose
evaporable acids and furfural were not determined, be- and lignin, can be brought into solution [113]. Alkaline
cause of their high volatility. The analytical procedures treatment can make plant matter more digestible for the
Table 14 Determination of Hemicellulose Monosaccharides in Percent of the Initial Weight
(a) (c) (e) (f) (g) (h)

Source of Wheat (b) Rice (d) Birchwood Hemicellulose Oat spells Oat spells
hemicellulose straw Bagasse straw Miscanthus (Roth) (Lenzing) (Sigma) (Roth)
Xylose 55.36 42.16 47.60 55.31 61.67 76.16 77.84 70.26

Glucose 15.62 14.92 11.76 7.23 6.08 0.82 0.99 0.81
Arabinose 5.80 4.10 4.25 4.01 7.93 n.d.a 6.95 6.39
Galactose 4.69 4.10 4.07 2.04 1.58 n.d. n.d. n.d.
Fucose n.d. n.d. 0.44 0.12 0.65 n.d. n.d. n.d.
Galacturonic acid n.d. n.d. 0.70 0.85 n.d. n.d. n.d. n.d.
Glucuronic acid n.d. n.d. 0.87 n.d. n.d. n.d. n.d. n.d.
Total sugars 81.47 65.36 68.87 69.23 77.93 76.98 85.79 77.46
The polyoses a, b, c, and d were prepared by hydrothermal treatment and c, f, g, and h obtained commercially. The analyses were carried out by
capillary zone electrophoresis after acid hydrolysis of the preparation.
a
n.d. = not detected.
Source: Ref. 109.
cattle rumen. But the breakthrough in this eld, which was extraction step considerably increases the process costs.
expected some years ago, did not occur. This certainly was one of the reasons why, in technical
organosolv application, the hemicellulose was not recov-
2. Hydrothermal Fractionation ered fast enough, thereby undergoing severe degradation.
At low temperatures, cellulose, hemicellulose, and lignin In the Kehlheim plant (Germany), the alkaline organosolv
are still very stable, but at f120jC, tannins are dissolved process led to a complete loss of the hemicellulose.
by the use of water alone. For this purpose, tannin-rich
materials (e.g., barks) are quite suitable [114]. 3. Hydrothermal Treatment (Steam and Aquasolv
Treatment)
B. Medium-Temperature Fractionation Steam Treatment
In many laboratories, steam is in common use and can
Here temperatures in the region of 200jC are applied. If the
be furnished by simple steam generators. Thereby, opera-
temperature of water is raised from ambient to 200jC, the
tion temperature determines the steam pressure and boiling
increase of volume becomes severe and must be taken into
occurs until the equilibrium pressure (e.g., in the connected
account, especially when working with autoclaves. Also,
reaction vessel) brings boiling to a stop. In Table 15, steam
the saturation steam pressure of f2 MPa requires rela-
pressure values at temperatures between 100jC and 300jC
tively thick container and tube walls. Safety valves are a
are given. If dry plant biomass (e.g., wheat straw at 20jC) is
necessity for all ow-through and recirculation equip-
treated with 220jC steam, then the steam is cooled until the
ments. Graphs and tables (e.g., Table 15) should be on
biomass also reaches the reaction temperature of 220jC. In
view in every laboratory where work with such installations
a supposed 10-L reaction vessel containing a load of 1 kg of
is performed.
straw (consistency of 10%), the straw (with an assumed
and temperature-corrected specic heat of f2 kJ kg1
1. Acid and Alkaline Treatment
jC1) would need 400 kJ to bring the biomass over this
As already shown in Fig. 8, the temperature for acidic 200jC gap. The evaporation or condensation energy at this
saccharication of the biomass was strongly raised to temperature is 1856 kJ/kg; therefore 0.216 kg of steam will
obtain better sugar yields. For the isolation of hemicellu- be condensed and most of this liquid will be absorbed by
loses, the use of acids has practically no advantage. the plant material. If the biomass has a certain humidity,
In contrast to this, alkalis show a high dissolution which is usually the case, or the reactor walls are at rst
power for hemicelluloses, and alkaline treatment is an below the steam temperature, then additional condensa-
established method for the isolation of hemicelluloses. tion occurs.
However, this process is not applied on a large scale, partly Under these circumstances, it becomes clear why it is
because of the necessary elimination of alkalis after the dicult to work in the eld of steam explosion or steam
hemicellulose extraction. extraction with a dened amount of condensed water on
the biomass. Some experiments were carried out where
2. Organosolv Treatment practically no water condensation occurred. For this pur-
As already mentioned earlier, this process is very similar to pose, Schwald et al. [115] used a tube-like reactor [116]
the hydrothermal one. In the next subsection (e.g., Fig. 27), containing a light grid basket for the biomass. If conden-
it will be shown that similar amounts of hemicellulose and sation occurred on the wall, it was collected at the bottom
lignin can be dissolved in both processes. The necessary and could be drained o, which usually was not necessary.
separation of the organic solvent after the organosolv The high temperature (e.g., 240jC) applied probably pro-
Table 15 Properties of Water and Steam at Temperatures Between 100jC and 300jC
Vapor pressure
Volume Evaporation Heat content
Temperature (jC) (MPa) (bar) (L/kg) enthalpy (kJ/kg) (enthalpy) (kJ/kg)
100 0.10 1.00 1.044 2257 418.8

120 0.20 1.96 1.061 2202 503.4
140 0.36 3.57 1.080 2144 588.7
160 0.62 6.10 1.102 2081 675.1
180 1.01 9.89 1.127 2013 762.7
200 1.56 15.34 1.157 1938 852.0
220 2.43 23.78 1.190 1856 943.2
240 3.37 33.03 1.229 1765 1037.0
260 4.72 46.30 1.275 1661 1133.9
280 6.46 63.29 1.332 1544
300 8.65 84.78 1.404 1406
916 Bobleter
important is that not more than 23.5% of the used aspen

wood can be dissolved in water. This means that only
hemicellulose and no lignin is brought into solution when
pure steam treatment without condensation is applied.
A similar situation with very little steam condensation
is shown in Fig. 22. Chornet and Overend [117] called this
process Thermo-Mechanical Vapor (TMV) and used the
term severity [118] to characterize the reaction condi-
tions. Thereby, the reaction ordinate R0, is given by
T 100
R0 t exp 8
14:74
In this equation, the reaction time (t) is measured in minutes

and the reaction temperature (T ) in degrees Celsius. For the
determination of the actual reaction time and temperature,
an experimental grid is needed. In Table 16, such a grid is
given, whereby for the rst ve temperatures, a reaction
Figure 21 Steam treatment and fractionation of aspen time of 4 min is assumed. The temperatures corresponding
wood. The solid residue, the water-insoluble fraction (SHA- to this reaction time are also plotted in Fig. 22. It is obvious
WI) together with the water-soluble fraction (WS) give the that with increasing severity, more hemicellulose can be
recovery yield (RY). (From Ref. 115.) dissolved. At higher log R0 values, very little lignin is
dissolved, but the biomass losses become noticeable.
Equation (8) is an approximation to a rst-order
duced slightly superheated steam, which helped to avoid reaction, assuming that the yield is proportional to the
condensation. The results are demonstrated in Fig. 21. In reaction time (t) and that a temperature increase of 10jC
the rst 50 sec, the amount of biomass dissolved in water doubles the yield, which is the case when the activation
(WS) after the reaction and the remaining water-insoluble energy is f118 kJ/mol at 200jC.
part (SHA-WI) sum up very closely to a 100% recovery An example with high steam condensation is given in
yield (RY). At longer reaction times, degradation occurs Fig. 23. Puls et al. [119] called this process steam extrac-
mainly in the water-soluble part, the hemicellulose. More tion. After the steaming process, the biomass is extracted
Figure 22 Steam treatment and fractionation of Populus deltoides. Fractions as percentage of the initial wood. The severity is
calculated according to Eq. (8). For a reaction time of 4 min, the corresponding reaction temperatures (jC) are also plotted.
Table 16 Experimental Grid for Evaluating the Severity According to Eq. (8)
Reaction time,
Temperature, T (jC) t (min) T 100 (T 100)/(14.75) R0 Log R0
180 4 80 5.424 907 2.96

190 4 90 6.1017 1,786 3.25
200 4 100 6.7797 3,519 3.55
210 4 110 7.4576 6,932 3.84
220 4 120 8.1356 13,655 4.14
226 2 126 8.5424 10,255 4.01
with water. It is obvious that much higher amounts (e.g., It can be seen from Fig. 26 [121] that the 200jC region
40% of the initial material after 20-min reaction time) can gives good hydrothermal fractionation of poplar wood
be extracted compared to the experiments discussed before, biomass. Two dierent ow rates (3 and 9 mL/min) were
where practically no condensation occurred. In this case, used at 180jC and 200jC. These ow rates correspond to
most of the hemicellulose and a considerable part of the 2- and 0.67-min residence time, respectively, of the
the lignin are dissolved. Therefore steam extraction carried eluting water in the 6-mL reaction vessel. The recovery
out with the Hamburger equipment comes very close yield is between 96% and 99%, of which about 4050% are
to aquasolv. found in the solution and determined as dry matter (DM);
Aquasolv Treatment 4858% of the original biomass (DM) remained in the solid
residue. These residues contained 6979% cellulose, 12
The earliest experiments in Fig. 11 [98,99] already
20% lignin, some minor quantities of hemicellulose (4
showed that most of the hemicellulose can be dissolved
6%), and extractives (47%). Comparing the composition
with water at f200jC. Several ow-through equipments
of the original poplar wood (Fig. 27, column e) with the
were designed and built. Special endeavors were made to
hydrothermal residue, it is evident that nearly all hemicel-
achieve a fast rise in temperature of the owing water and a
lulose and approximately half of the lignin is dissolved. By
constant reaction temperature during the treatment, which
this treatment also, 58% of the cellulose is brought into
eorts were not easily accomplished with laboratory devi-
solution, obviously the less-crystalline part.
ces. In Fig. 24, an apparatus [99,120] is shown where the
This relatively simple fractionation process has the
pressure mantle of the reaction vessel was furnished with an
following advantages:
electrical heating coil to improve the temperature behavior
of the system. Practically no loss of biomass occurs.
In Fig. 25, hydrothermal elution proles of rice straw The eluate or extract contains most of the hemi-
in the temperature region of 165278jC are depicted [120]. cellulose, which can easily be further treated (e.g.,
As can be seen from this gure, the fractionation starts at by enzymatic saccharication).
approximately 180jC, reaches a relatively high separation The residue incorporates the cellulose and approx-
power at 200jC, and shows from 200jC to 250jC a slightly imately half of the original lignin.
increasing reaction prole. The larger part of the dissolved The cellulosic residue can be delignied or enzy-
biomass appears in the euent within 1015 min. matically saccharied well with yields of over 90%.
Figure 23 Steam treatment (steam extraction) of wheat straw; reaction temperature is 187jC and the reaction time is given in
minutes.
918 Bobleter
Figure 24 Aquasolv laboratory equipment; schematic diagram.
It is of interest how the aquasolv treatment compares with solv. Contrary to this, the hydrothermal treatment dis-
organosolv. In Fig. 27, both these treatments were ana- solves more biomass matter when wheat straw is used (Fig.
lyzed and the percentage of the remaining residue plotted 27b). More important is that the dierence between both
[122]. Several features are characteristic and can be seen in processes is not very great. These results show, as stated
Fig. 27a: At 205jC, 12% more biomass (poplar wood) than earlier, that large additional investments for the organo-
at 190jC is dissolved by the organosolv process; in this solv process will not pay if the fractionation behavior
region, the residue amount drops fairly steeply. Aquasolv shows no or little advantage compared to hydrothermol-
treatment has a more gradual decline from 190jC to ysis. Chemically related plants, such as hardwoods and
240jC. In the region 195235jC, the dissolving power of straw, exhibit similar fractionation behavior. In Fig. 28,
organosolv is somewhat stronger than in the case of aqua- poplar wood, wheat straw, birch wood, and rice straw are
Figure 25 Elution proles from aquasolv treatment of rice straw. Reaction temperature between 165jC and 278jC. The
biomass concentration is given as dry matter (DM) in mg/mL and the elution time in minutes.
Figure 26 Aquasolv fractionation of poplar wood. The yields as dry matter (DM) are given for two dierent ow rates (3 and 9
mL/min) and temperatures of 180jC and 200jC. (a) The water-soluble and the residue dry matter are given. (b) The composition
of the raw material and the residues as percentage of the original dry matter biomass as extractives, lignin, hemicellulose, and
cellulose are also given.
compared [46,120,122]. Between 65% and 40% remain in tion, as was determined by a subsequent acid hydrolysis.
the solid residue after aquasolv treatment at 170240jC. As mentioned earlier, in this case also, about half of the
The research group of Antal obtained corresponding lignin was solubilized. Small amounts of the cellulose are
results [97], as presented in Fig. 29. Some 4060% of the also dissolved.
10 plant species were solubilized in this aquasolv process. In addition to earlier autoclave experiments, many
The hemicellulose is nearly completely brought into solu- research projects were carried out with direct ow-through
Figure 27 Aquasolv (hydrothermal, HT = .) and organosolv (OS = 5) treatment of poplar wood and wheat straw. The solid
residues are given as dry matter in percentage of the initial biomass and the reaction temperature (jC). (a) Poplar wood; (b)
wheat straw. The organosolv mixture was water/methanol = 1:1 (v/v).
920 Bobleter
a ow-through or percolation experiment in a 10-L reactor is

shown [123]. For the wheat straw used, which was cut into
35-cm pieces, a maximum concentration of f9 g/L is
obtained. But even the withdrawal of the euent liquid in a
narrow time span of between 15 and 35 min would give
only an average concentration of f6 g/L. With denser
material (e.g., bagasse), the consistency and, with it, the
eluted concentrations are approximately doubled, which is
still not satisfactory.
In view of these results, it was necessary to analyze
how long a recirculation of the extracting liquid can be
carried out before the losses, mainly of hemicellulose,
become too severe. Figure 31 shows [124,125], such a
recirculation experiment at 200jC. As in the previous
example, the extraction begins when f180jC is reached.
When the reaction temperature (200jC) is held, the max-
imum concentration in the solution, with f29 g/L, exceeds
Figure 28 Solid residue (dry matter in percentage of the
by far the maximum concentration in the percolation
initial raw material) after aquasolv treatment of poplar
experiment (9 g/L), but around the peak of the DM curve,
wood, wheat straw, birch wood, and rice straw in the
temperature range between 170jC and 240jC.
severe degradation of the dissolved biomass begins. As
indicated in Fig. 31, nearly all dissolved matter is precip-
itated or reacts with the residue within 30 min. Prolonged
treatment in the recirculation mode therefore leads to
equipments. The latter have the advantage that the mech- dramatic losses of biomass in the liquid.
anism of the process can be better evaluated. However, for A combined process, rst recirculation and then direct
a technical application, there would be a serious disadvan- ow-through, avoids the disadvantages of both processes
tage because large volumes of water are needed, which is mentioned earlier and is shown in Fig. 32a [125]. Again,
connected with a high-energy consumption and a low using wheat straw, the maximum concentration is, with 36
biomass concentration in the eluted liquid. In Fig. 30, such g/L, much higher than in the percolation experiment. The
Figure 29 Aquasolv treatment of hardwoods and grasses. Recovery of hemicellulose sugars, solubilized cellulose, and lignin as
well as the percentage of the solubilization of the original sample.
Figure 30 Elution prole of a aquasolv percolation experiment, using wheat straw as biomass. The temperature (jC), the eluted
biomass as dry matter (DM), and the pH are plotted versus the reaction time (min).
replacement of one reactor volume with fresh water can doned in favor of vacuum drying. Frequently, in the
yield this relatively highly concentrated solution. Fig. 32b steaming process, the inside of the wood boards showed
gives an example of the combined process with bagasse as severe formation of cracks. This eect can be explained as
biomass [125]. The maximum concentration of f50 g/L is follows: The steam rst condenses on the cooler surface of
relatively high. Forty-eight percent of the original biomass the wood, thereby dissolving some of the hemicellulose
was dissolved and the pH decreased to 3.13. This process and a fraction of the lignin. When the steaming process
solves several problems inherent in the other procedures: continues, the outer layer of the wood is depleted of
Only one reactor volume is needed to replace the concen- hemicellulose and the wood structure can collapse in this
trated extraction solution. If a large part of the heat content region, whereby the water transport is strongly inhibited.
of the solution is regained by the use of a heat exchanger, As soon as the temperature of the wood approaches that
then the energy cost of the process should be modest. At the of the steam, the internal pressure is so high that cracking
same time, nearly 100% of the biomass is recovered, either can occur.
in solution or as residue. These facts are also veried by a simple experiment: A
piece of straw that is still wet after an aquasolv treatment,
whereby approximately 50% of its original weight was
4. Comparison of Steam Versus Aquasov Treatment extracted, shows a very fragile structure. After drying, such
Steam-drying of wood materials was a very common pretreated straw demonstrates greater strength than the
practice in Europe for many years. Because of unsatisfac- original untreated straw. It should also be mentioned that
tory results, this process was almost completely aban- materials with low consistency, e.g. straw, reduce their
Figure 31 Prole of a aquasolv recirculation experiment, using wheat straw as biomass. The temperature (jC), the eluted
biomass as dry matter (DM), and the pH are plotted versus the reaction time (min).
922 Bobleter
Figure 32 Prole of a combined aquasolv recirculation and percolation experiment. The temperature (jC), the eluted biomass
as dry matter (DM), and the pH are plotted versus the reaction time (min). (a) Biomass is wheat straw; (b) biomass is bagasse.
volume by nearly 50% during the aquasolv treatment. The straw, is chosen. This inuence is not so severe when
individual straw particle shows little change in its dimen- biomass materials with higher consistency, e.g., bagasse,
sions in this process. This indicates that half of its volume is wood chips, etc. are used. In addition, a careful energy
lled with water when enough water and pressure is recuperation has to be applied in the aquasolv processes,
available during the hydrothermal process and the cooling e.g., through the use of heat exchangers.
down period. A relatively large number of steam treatment experi-
The cases discussed above indicate the reasons for ments were carried out using a catalyst. Thereby, usually
some of the widely diering results obtained by steam the plant biomass was soaked in a mineral acid, alkali, or
treatment under almost identical conditions. With relative- carbonate solution and then the steam added. For com-
ly dry steam treatment, only hemicellulose but no lignin parison, Table 17 gives a selection of relevant results. Some
can be extracted [115], but with ample condensation, features are characteristic: Good hemicellulose recoveries
practically the same results are obtained as with the aqua- (70% to near theoretical values) are achieved. Also, the
solv process [119]. sugar and the ethanol yields (74% to near theoretical
An important dierence between both these processes values) are satisfactory. As disadvantages, the corrosion
is the greater use of energy in the aquasov process and waste water problems remain.
compared with steam treatment. Kazi et al [126] pub- Table 18 gives the result of pure steam treatment. In
lished a diagram (Fig. 33) showing the inuence of the most cases, a series of experiments were carried out but
consistency and temperature on the consumption of only optimal values are given in this table. After the steam
steam. At a given temperature (e.g., 220jC) and a con- treatment, approximately 1454% of the original biomass
sistency of 40%, only 1.4 kg steam per kilogram of were solubilized (overall dissolution). The hemicellulose
lignocellulosic biomass is required but 4.4 kg steam is recovery varies considerably between 32% and 88%. The
needed when a low 10% consistency, characteristic for ethanol yield with 67% to over 90% is relatively high.
Figure 33 Comparison of steam consumptions at dierent biomass consistencies and temperatures.
The aquasolv treatment is presented in Table 19. The For several years, Germany was much engaged [167] in
hemicellulose recovery with more than 80% and the etha- the development of the organosolv process, called the
nol yields with 80% and more than 90% are quite satis- MD-organocell process, The two-stage process was
factory. carried out rst with a 50% methanolwater mixture at
Of special interest are the experiments of Antal et al. 195jC and 4 MPa. To improve the delignication in a
[128,145,160] whereby the same equipment and the same second step, NaOH was added.
analytical methods were used to test the above-mentioned The 350-ton/day plant in Kehlheim, Germany, was in
processes. The results are given in Tables 1719. In all three operation for only 1 year, until 1993. Financial losses
processes, the hemicellulose recovery from poplar wood prevented the continuation of its operation.
are between 70% and 88% and the ethanol yields between
70% and 97%. Similar results were achieved with bagasse 2. Steam Explosion Process Equipment
and partly with corn ber. Figure 34 shows the correspond- Mason introduced the steam explosion process and
ing equipment [128] for the comparison of aquasolv and obtained a patent [87] in 1929. He was interested in a
steam treatment. debration process to obtain material for particle-board
Aquasolv shows somewhat higher ethanol yields than production. In his equipment, the wood chips were expelled
steam treatment but the winner of this race has to be by a quickly opening valve (steam explosion). In 1932,
found using reliable economical evaluations. Carefully Babcock [88] patented a similar device called the masonite
upgraded experiments would be a valuable help for such gun. The saturated steam led to water condensation on
investigations. the biomass, as mentioned in the patent. The target of this
work was the production of fermentable sugars.
C. Fractionation Process Equipment The question of how far this process is able to de-
brate, debrillate, or introduce rupture of the bers in the
1. Organosolv Equipment biomass material was a much discussed matter. In the
Not much later than Masons steam explosion, Kleinert meantime, opinion is increasing that the envisaged eects
and Tayenthal [85] in 1931 patented their organosolv occur only to a very minor extent [115]. A further devel-
process in which wood was treated with an alcoholwater opment was incorporated in the Canadian patent [168],
mixture. The process is called the Kleinert organosolv called the IOTECH Process. The nely chopped wood
process [164]. It is claimed [165] that, with softwood as was treated with steam at 230jC and 3.5 MPa and then
raw material, pulps can be obtained with 12.815% lignin explosively released from the reactor (ash hydrolysis).
(kappa numbers of 85100) and hardwood pulps should Important results of this work were that the hemicellulose
have lower lignin contents. Denser hardwoods like euca- could be extracted afterward with water and that the lignin
lyptus, alder, or oak can be more dicult to treat, and coalesced in the form of spheres. Small lignin spheres f1
separation of the hardwood species may be required. Am in diameter were detected, showing a glass transition
The ALCELLR pulping process introduced by temperature of 120jC [169]. It is understandable that this
Katzen [166] brought changes concerning mainly the ow process increases the accessibility of the large cellulase
of the organosolv liquid. enzyme molecules and thereby also the saccharication
924
Table 17 Steam Treatment with Catalyst
Reaction parameters Yields (%)
Temperature Time Overall Hemicellulose Total

Process target Raw material Catalyst (jC) (min) dissolution recovery sugars Ethanol Remarks Reference
Ethanol Softwood thinn. H2SO4 180 + 210 4 + 1.5 >90 55a 49a two-step process 127
Poplar wood 175 10 70 92a 128
Douglas r 215 2.3 33 80a 129
Bagasse SO2 205 10 31 72b 79b 130
Softwood 190 + 190 2 + 10 69a 80b 131
Douglas r 8090 90c 132
Douglas r 195 4.5 65 87a 133
Spruce 215 5 68b 134
Bagasse 215 3 37 84a 135
Softwood 215 5 88b 136
Sugars Softwood SO2 215 3 87a 137
Douglas r 215 2.4 50 16 94a 138
Corncob H2SO4 150 30 26 Xylooligosaccharide 139
Spruce SO2 210 2.5 32 92 140
Fractionation Bagasse SO2 118222 4.159 141
Softwood 195 5 23 70b 142
a
Percent of original glucan.
b
Percent of theory.
c
After sulfuric acid posthydrolysis.
Bobleter
Table 18 Steam Treatment

a
Temperature Pressure Time Severity Overall Hemicellulose
Process target Raw material (jC) (MPa) (min) (log R0) dissolution recovery Sugars Ethanol Remark Reference
Ethanol Rice straw 3.5 2 30 >90 86 143

Corn ber 215 2 37 40 90b 144
Hydrothermal Degradation and Fractionation
Bagasse 205 10 28 64 130

Bagasse 220 2 18 48 85c 145
Poplar wood 220 2 54 88 70b 128
Wheat straw 217 3 81 146
Softwood 210 4 60 50c Particle size 147
Aspen wood 214 6 85 148
Bagasse 216 4.0 25 67 149
Corn stalks, straw 220 2 80 90c 150
Cotton gin 3.56 83 151
Sugars Bagasse 1.6 5 66 152
Sugars, C-ber Hardwood 3 1.96 25 90 40% yield of C-ber 153
Fractionation Oak wood 4.0 72 154
Bagasse 190 10 34 155
Bagasse 195 4 33 70 156
Pulp Wheat straw 3.8 41.3 55 157
Particle board >190 Reduced swelling 158
Rice husks 210 3 3.7 14 32 159
a
Severity according to Eq. (8).
b
Percent of theory.
c
Based on residual cellulose.
925
926 Bobleter
Table 19 Aquasolv Processes
Temperature Time Severity Overall Cooking Hemicellulose

Process target Raw material (jC) (min) (log R0) dissolution yield recovery Ethanol Reference
Ethanol Bagasse 220 2 36 90 80a 145

Poplar wood 220 2 85 97b 128
Corn ber 215 2 54 82 86 144
Fractionation Bagasse 230 2 41 100 160
Corn stalks 4.25 51 96 161
Poplar wood 200 Percc 48 50 98 121
Wheat straw 210 52 122
200 42 58 123
Aspen wood 220 2 26 79 162
230 2 47 88 163
Pulp Wheat straw 192 Percc 40 60 125
Miscanthus 202 40 60 125
a
Based on residual cellulose.
b
Percent of original glucan.
c
Percolation experiment.
yield. In the same way, the nutritive value as feed for steam explosion process with strong vapor condensation.
ruminants was nearly doubled. If no steam condensation takes place, then the process is
A continuous process [170] was patented in 1981 in the called TMV (thermomechanical vapor). The inuence of
United States and called the STAKE Process. With the the sharing devices in the equipment was still regarded to be
application of an additional high inert-gas pressure and relatively high.
special bars in the reactor exit nozzle, an increase of the At SIRO, Melbourne, Australia, nonwood biomass
sharing eect on the biomass particles was envisaged [171]. was used as raw material. The equipment for this steam
Chornet and Overend [117] introduced the expression explosion work was called Siropulper [172,173]. In
thermomechanical aqueous/vapor (TMAV) for the practically all laboratory experiments described, the bio-
mass material was fed batchwise. A typical device is shown
in Fig. 35. Most biomass particles and papers are materials
that are very dicult to deliver by pumps. Therefore
continuously fed equipments are in little use so far. New
ecient feeding devices could bring a much-needed ad-
vance to this eld. Brown [174] patented a new device,
containing a coaxial reciprocating feeder. Also, the Ger-
man rm Putzmeister has been engaged in this eld for
several years.
3. Aquasolv Process Equipment

Low-Temperature Treatment
Low-temperature extractions are normally carried out
at temperatures up to 120jC. At this temperature, the
water pressure is only 0.2 MPa and therefore simple
reaction vessels can be employed. In several cases, water
alcohol mixtures are used, whereby slightly dierent pres-
sure conditions have to be taken into account.
Medium-Temperature Treatment
Figure 34 Diagram of an apparatus for aquasolv and steam
treatment. From the feed water tank (T1), the water is led to A schematic diagram of an equipment with a 10-L
the boiler (T2) and from there hot water or steam reaches the reaction volume is given in Fig. 36 [125]. The three process
reactor (R1). Biomass can be positioned over the condensed modes, direct ow-through (percolation), recirculation,
water. Product tank = T3. Product reservoir = T4. Internal and the combined process, can be carried out with this
thermocuple = T. (From Ref. 128.) device. For a technical application, two dierent pilot
Figure 35 Steam explosion laboratory unit; schematic diagram.
plants are proposed. For discontinuous operation, mainly half of the reactor volume. This higher consistency could
in countries with low labor costs, the equipment in Fig. 37a lead to higher euent concentrations. A device, which
is depicted. The reaction vessel is lled with the biomass, compensates this volume reduction, was invented by Kim
the air is expelled by steam, and then water at f200jC is et al. [183] (BSFT = bed-shrinking ow-through).
introduced. After a reaction time with recirculation of f15
min, the liquid is replaced by fresh water (f200jC), which
is again recirculated for about 10 min. This second water
IV. PRODUCT SPECTRUM FOR
lling takes up only minor amounts of biomass and it can
be used again after an intermediate storage for the next HYDROTHERMALLY TREATED
biomass lling. In Fig. 37b, a continuous equipment is POLYSACCHARIDES
designed, whereby the biomass is led through two temper- Plant biomass not only produces foodstus and construc-
ature zones in the reaction vessel. The extract of the lower tion materials but can also easily substitute practically all
part with a small biomass content can be used to treat the organic chemicals manufactured at present from petro-
biomass in the upper region. In this way, large biomass chemicals by the oil industry.
contents in the eluted liquid and low energy consumption
are achieved.
In designing hydrothermal fractionation equipment, A. Products from Low-Temperature Treatment
the characteristic behavior of plant biomass in aqueous
media must be taken into account. Especially grasses (e.g., As already mentioned, tannins [114] can be produced from
wheat straw) expand somewhat during the treatment, tannin-rich sources (e.g., barks) by hydrothermal treat-
which makes the biomass transport dicult. Straw can be ment at approximately 120jC. With organosolv mixtures,
lled into a reactor vessel to a consistency of approximately several chemicals, such as triglycerides of linoleic and
10% (dry weight per volume). However, during the reac- palmitic acid, vegetable oils, etc. [175], and pharmaceu-
tion, it settles down and does not require much more than ticals can be obtained.
928 Bobleter
Figure 36 Schematic diagram of an aquasolv equipment with a 10-L biomass reaction vessel (BM). The water can be led in a
direct ow-through (percolation) over the heat exchangers (HE1, HE2, and HE4) or in a recirculation mode (pump P2). In the
latter case, samples can be taken over the heat exchanger (HE3).
B. Products from Medium-Temperature By reduction, xylose is transformed to xylit, a sugar

Treatment that does not cause caries. A very important dehydration
product of xylose is furfural. Up to now, furfural produc-
In Fig. 38, an abbreviated scheme of products, which can tion has been carried out mainly by acidic distillation of
result after hydrothermal treatment, is shown [46]. As corn cobs. Figure 20 shows that also by hydrothermal
mentioned earlier, at reaction temperatures between treatment of xylose, relatively high yields of furfural can
180jC and 230jC, the aquasolv solution contains the be obtained [177] (e.g., 46 mol% at 220jC). This is ap-
hemicellulose and about half of the original lignin. proximately the same as that achieved with 0. 1 MH2SO4 at
1. Hemicellulose Solution 190jC. After elimination of the lignin from this solution
This solution can easily be fermented to methane with high (e.g., by extraction), a white hemicellulose powder is pro-
yields. Therefore a biogas plant could eciently use the duced. This powder can be used as a biodegradable plastic
liquid part of the hydrothermal process and furnish the component (e.g., in the form of polyurethans).
methane gas for heating or other caloric purposes.
The enzymatic saccharication renders mainly pen-
toses such as xylose. The enzymes can be directly added to 2. Cellulosic Residue
these solutions; the dissolved lignins do not have much The cellulosic residue still contains a fairly high amount of
inuence on the reaction. In Fig. 39, the xylose yields [176] lignin (e.g., 16%). Nevertheless, this type of pulp can be
of a birch wood hydrothermal solution are shown. The dry- used without further treatment for simple papers, like
matter content of the fractions in this percolation experi- cardboards. Tensile strengths of 3.35 km were reached
ment and the yields for three dierent enzymes are given in when wheat straw served as the raw material [123].
grams per liter. The results using the Roth enzyme come Further delignication can be carried out with NaOH at
relatively close to theory; the other sugars, acids and lignin, approximately 170jC, whereby about two-thirds of the
ll most of the gap between the dry matter in solution and lignin are removed [178]. The remaining lignin can be
the xylose curves. extracted by elementary chlorine-free methods (e.g., with
The pentoses can be fermented very eciently into ClO2 and/or H2O2). Papers with high whiteness are ob-
single-cell proteins, and also to butanol, acetone, and tainable in this way. The question if pure cellulose can also
ethanol. be produced by this method can be answered positively.
Figure 37 Proposed hydrothermal equipments. RV = reaction vessel, HE = heat exchangers, P = pumps. (a) Discontinuous
plant. The reaction vessel is lled for each treatment. The eluted liquid of a second treatment can be stored in the water storage
tank (WS) and used for a new biomass lling. (b) The continuous plant obtains the biomass by a biomass feeder (BF). The
reaction vessel (RV) contains two reaction stages. The slightly biomass-loaded liquid of stage 11 can be used again in stage 1.
The new solvent medium, methyl-morpholine-N-oxide that, thanks to increasing industrialization, the growth of
(MMNO), is a promising cellulose solvent [179]. Several the world population will come to a standstill at the end of
experiments [125] with MMNO gave a white cellulose this century and reach its maximum with 10 billion people.
powder with a good molecular distribution, even when Furthermore, the market for synthetic and, especially,
hydrothermally treated and bleached wheat straw was technical cellulose bers is growing, whereas that for wool
used as raw material. remains more or less constant. Owing to the above-men-
An interesting question arises in this context: How do tioned reasons, the cotton ber market is expected to
technical cellulosic bers, prepared, for example, with decrease considerably soon. The serious situation
MMNO, compare with natural cotton bers? In the year concerning the oil reserves is clearly shown by Deeyes
1999, a world cotton production of 18.4 million tons was [1] and reproduced in Fig. 40b.
reached. The harvest yield spreads from 110 to over 1000 Even when the large oil reserves of 2.1 trillion barrels
kg/ha, and an average of 540 kg/ha can be taken. Com- are assumed, we will pass the nal maximum in the next few
pared with this value, 1020 tons ha1 and more (Table 9) years. Are we prepared for these economical changes?
are easily obtainable, either as waste material (e.g., straw) Hydrothermally pretreated biomass is easily enzymat-
or as energy plants (e.g., miscanthus). If cellulose comprises ically hydrolyzed. The H bridges in the cellulosic residue
approximately 40% of the latter values, the yield is, are obviously cleaved so well that the large enzyme mole-
nevertheless, 48 tons ha1. It is hard to understand why cules can penetrate the structure without diculty and
cotton plantations (and doubtful defoliation methods) are produce glucose. With hardwoods and straw, it is possible
still in use, when, on an equivalent area, 715 times more to come close to theoretical yields.
cellulose can be grown even in temperate zones. In Fig. 40a, The enzymatically produced glucose and xylose can be
the ber consumption and the world population are given. fermented in several ways to products of technical interest.
The values from 1900 to 1995 are taken from literature The ve important fermentation pathways [181] produce
[180] and the values from the present day to 2100 are an lactic acid, isopropanol, 2,3-butenediol, ethanol, and n-
expected assumption. For this purpose, it was presumed butanol. From these chemicals, many valuable solvents or
930 Bobleter
Figure 38 Product spectrum obtainable after hydrothermal treatment of plant biomass.
Figure 39 Enzymatic saccharication of hydrothermally obtained eluates from birch wood. Three dierent hemicellulases were
directly applied to the eluted fractions. The concentration of xylose (in g/L) is given for each fraction.
Figure 40 (a) Fiber consumption and world population gures between 1900 and 2100 are given. Between 1900 and 1995, the
values are from literature; after 1995, the trend is projected. (b) Annual production of world oil (circles). The Gaussian curves
correspond to the total eventual oil recovery of 1.8 and 2.1 trillion barrels.
intermediates can be obtained: ethylene, ethylenoxide, 1. Economy

glycerin, propyleneoxide, and so forth. Using the mono- A reasonable price for plant raw material is a precondition
meric compounds also, many important high polymers can for an economic success of this process. As already men-
be produced: polyacrylic acid, polypropylene, polystyrene, tioned in Sec. I (Table 8), there is a good chance that in the
polyethylene, polyoxyethylene, and PVC. future, waste materials (e.g., waste paper and straw) as well
Ethanol deserves special attention because it may as short-rotation forestry products (e.g., poplar wood and
become the favorite future substitute for gasoline very willow) and energy plantations (e.g., miscanthus) will be
soon. Developing countries with few resources in foreign able to compete with crude oil and furnish a new plant
currency should be the rst to use this technology. The biomass industry.
industrial regions could benet from the ethanol fuel Mass products such as paper, bers, and alcohols as
technology through an additional agricultural use of land, fuel are of special interest. Simple papers like wrapping or
increase in technological know-how, and reduced energy brown paper can be obtained from straw [123] by hydro-
dependence on foreign countries. thermolysis without any further chemical treatment. Cer-
tain paper products obtained in this way will most
probably be the rst to reach the economic limit.
C. Products from High-Temperature Treatment Fiber production will change from cotton to chemi-
As mentioned earlier, the hydrothermal treatment of plant cally manufactured cellulose. The dierence between the
biomass at temperatures between 275jC and 320jC deliv- cellulose yield of a cotton eld and that of fast-growing
ers pumpable sludges. This advantage can be used to plants of the same area (e.g., maize or miscanthus) is so
deliver the biomass into gasication stations [182], thereby enormous that the chemical pathway will succeed. As soon
performing the Boudouar reaction, C + CO2 = 2CO, and as glucose can be obtained suciently cheaply from hy-
the watergas reaction, C + H2O = H2 + CO. These gases drothermally treated plants by enzymatic saccharication,
can be transformed to several other chemicals, such as the fermentation to lactic acid, isopropanol, 2,3 butane-
methanol or ammonia. Hydrothermally activated pyroly- diol, and n-butanol can be carried out without diculty.
sis of biomass [97] renders high yields of charcoal, and is a Ethanol fermentation holds a key position because of its
much improved process. good chances for wide application.
Special products like citric acid depend more or less
only on the sugar costs; therefore, glucose from hydrother-
D. Economic and Environmental Aspects mally treated plants will be in a good position in the
future. High yields of furfural can by obtained from hemi-
Economy and environmental acceptance will be the most celluloses; but furfural, as by-product in hydrothermal
important parameters for the breakthrough of the hydro- treatment, can have an adverse eect on anaerobic fermen-
thermal treatment and its follow-up products. tation [184].
932 Bobleter
The economic eect of a new biomass industry could 14. Neori, A.; Cohen, I.; Gordin, H. Ulva lactuca biolters for
help to change the unemployment trend in industrial- marine shpond euents. Bot. Mar. 1991, 34, 483.
15. Martyn, D. Climates of the World; Elsevier: Amsterdam,
ized countries.
1992; p. 34.
16. Lieth, H. Modelling the primary productivity of the world.
2. Environmental Considerations In Primary Productivity of the Biosphere; Lieth, H., Whit-
Short-rotation forestry and energy plants usually need very taker, R.R., Eds.; Springer: Berlin, 1975; p. 237.
few fertilizers and insecticides, which could improve the 17. Pfannemuller, B. Starke. In Polysaccharide; Burchard, W.,
Ed.; Springer-Verlag: Berlin, 1985; p. 25.
ground water considerably in many parts of the world. 18. Kajiwara; Miyamoto, T. Progress in structural character-
The use of pure water by hydrothermolysis (steam isation of functional polysaccharides. In Polysaccharides;
treatment and aquasolv) is an environmental bonus com- Dumitriu, S., Ed.; Marcel Dekker: New York, 1998; p. 1.
pared with conventional processes such as the sulte 19. Zugenmaier, P. Supramolecular structure of polysaccar-
treatment in the paper industry. The proposed cellulose ides. In Polysaccharides; Dumitriu, S., Ed.; Marcel Dekker:
ber production with MMNO would make the defoliation New York, 1998; p. 57.
20. Ogawa, K.; Yui, T. X-ray diraction study of polysacchar-
practice in cotton elds unnecessary. ides. In Polysaccharides; Dumitriu, S., Ed.; Marcel Dekker:
The use of biomass ethanol as car fuel would only give New York, 1998; p. 101.
o the same amount of CO2 as the plants assimilated for 21. Tvaroska, I.; Taravel, F.R. Computer modeling of poly-
their growth. Therefore hydrothermal pretreatment is saccharide-polysacharide interactions. In Polysaccharides;
connected with numerous positive environmental aspects. Dumitriu, S., Ed.; Marcel Dekker: New York, 1998; p. 173.
22. Fengel, D.; Wegener, G. WoodChemistry, Ultrastruc-
ture, Reactions; W. de Gruyter: Berlin, 1989; p. 97.
ACKNOWLEDGMENTS 23. Popa, V.I.; Spiridon, I. Hemicelluloses: Structure and
properties. In Polysaccharides; Dumitriu, S., Ed.; Marcel
My thanks to the research groups of the professors M. J. Dekker: New York, 1998; p. 297.
Antal Jr., B. Hahn Hagerdahl, G. Liden, J. N. Saddler, 24. Fengel, D.; Wegener, G. Polyosen und lignin-polysacchar-
and G. Zacchi for their generous supply of literature; Dr. id-komplexe. In Polysaccharide; Buchard, W., Ed.; Spring-
er-Verlag: Berlin, 1985; p. 45.
Burtscher for his help in computer work; and my wife, 25. Fengel, D.; Wegener, G. WoodChemistry, Ultrastruc-
Lucy, for her courageous endeavor to improve the English ture, Reactions; W. de Gruyter: Berlin, 1989; pp. 109, 111,
translation of this work. 115, 124, 45.
26. Franz, G. Struktur und biologische funktion von poly-
sacchariden. In Polysaccharide; Burchard, W., Ed.; Spring-
REFERENCES er-Verlag: Berlin, 1985; p. 1.
27. Fengel, D.; Wegener, G. WoodChemistry, Ultrastruc-
1. Deeyes, K.S. Hubberts PeakThe Impending World Oil ture, Reactions; W. de Gruyter: Berlin, 1989; p. 172.
Shortage; Princeton Univ. Press: Princeton, 2001; p. 147. 28. Meshgini, M.; Sarkanen, V. Syntheses and kinetics of acid
2. Whittaker, R.H.; Likens, G.E. The biosphere and man. In catalyzed hydrolyses of some a-aryl ether lignin model
Primary Productivity of the Biosphere; Lieth, H., Whittaker, compounds. Holzforschung 1989, 43, 239.
R.H., Eds.; Springer-Verlag: Berlin, 1975; p. 305. 29. Fengel, D.; Wegener, G. WoodChemistry, Ultrastruc-
3. Larcher, W. Physiological Plant Ecology; Springer-Verlag: ture, Reactions; W. de Gruyter: Berlin, 1989; pp. 5658.
Berlin, 1995; p. 152. 30. Chum, H.; Jonson, D.K.; Blake, S.; Baker, J.; Grohmann,
4. Encyclopaedia Britannica; W. Benton Publ.: Chicago, 1968; K.; Sarkanen, K.V.; Wallace, K.; Schroeder, H. Organo-
Vol. 16, p. 803. solv pretreatment for enzymatic hydrolysis of poplar. 1.
5. Farbiges Gr. Volkslexikon, Bibliograph. Institute, F. Spie- Enzymatic hydrolysis of cellulosic residues. Biotechnol.
gel Buch: Ulm, 1981; Vol. 3, p. 581. Bioeng. 1988, 31, 643.
6. Souci, S.W.; Fachmann, W.; Kraut, H. Food Composition 31. Garrote, G.; Dominguez, H.; Parajo, J.C. Hydrothermal
and Nutrition Tables 1981/82; Scherz, H., Kloos, G., Eds.; processing of lignocellulosic materials. Holz Roh- Werkst.
Wiss. Verlags G.: Stuttgart, 1981; pp. 290, 338, 486, 660, 1999, 57, 191.
666, 676, 804, 914, 1302. 32. Ladish, M.R.; Lin, K.W; Voloch, M.; Tsao, G.T. Process
7. FAO Yearbook Production, Vol. 53, 1999 (FAO Statistical consideration in the enzymatic hydrolysis of biomass.
Series No. 125); Food and Agricultural Organization of Enzyme Microbiol. Technol. 1982, 5, 82.
the United Nations: Rome, 2000; pp. 68, 70, 72, 77, 79, 33. Dale, B.E. Biomass rening: Protein and ethanol from
106, 111. alfalfa. Ind. Eng. Chem. Prod Res. Dev. 1983, 22, 466.
8. Haiger, A. Zucht. In Naturgemasse Viehwirtschaft; Haiger, 34. Esterbauer, H.; Jungschaer, G.; Schurz, J. Enzymatische
A., Storhaas, R., Bartussek, H., Eds.; Stuttgart: Ulmer, Hydrolyse von Cellulose. Holzforschung 1981, 35, 129.
1988; p. 37. 35. Kubikova, J. Hydrothermale Vorbehandlung von pan-
9. Krausmann, F. Erneuerbare Rohstoe in Osterreich- zlicher Biomasse fur die Zellsto- und Papierherstellung,
Chancen und Risken; BM Finanzen-Druck: Vienna, 1993. Ph.D. thesis, University of Innsbruck, 1994; p. 210.
10. Larcher, W. Physiological Plant Ecology; Springer-Verlag: 36. Shatokov, A.A.; Quilho, T.; Pereira, H. Giant reed-Arunda
Berlin, 1995; p. 33. donax. TAPPI 2001, 84, 96.
11. Beadle, C.L.; Long, S.P. PhotosynthesisIs it limiting to 37. Bonn, G.; Bobleter, O. Wheat Straw Analysis, Round-
biomass production? Biomass 1985, 8, 119. Robin Test. unpublished.
12. Kelly, G.J.; Latzko, E. Photosynthesis. Carbon metabo- 38. Steiner, W.; Steinmuller, H.; Esterbauer, H.; Laerty,
lism: On land and sea. Prog. Bot. 1984, 46, 68. R.M. Lignocellulosic residuesA source for fermentable
13. Moeller, H.W.; Grin, G.; Lee, V. In Aquatic biomass sugars: Availability and composition of raw materials,
production on sand, using sea water spray. Annual Confer- pretreatment and enzymatic saccharication. Biotechnol.
ence on Energy from Biomass and Wastes, June 1982. Biotech. 1978, 3, 6.
39. Larcher, W. Physiological Plant Ecology; Springer-Verlag: 61. Perlack, R.D.; Ranney, J.W.; Barron, W.F.; Cushman,
Berlin, 1995; p. 150. J.H.; Trimble, J.L. Short-rotation intensive culture for the
40. Coombs, J.; Khan, R.; Righelato, R.L.; Vlitos, A.J. Future production of energy feedstocks in the USA: A review of
sources of organic raw matter. In Carbohydrate as Renew- experimental results and remaining obstacles to commerci-
able Feedstock in Future Source of Organic Raw Materials; alization. Biomass 1986, 9, 145.
St-Pierre, L.E., Brown, G.R., Eds.; Pergamon Press: 62. Breuninger, E. Heute Weizen, morgen Elefantengras, Top.
Oxford, 1980; p. 533. Agrar 1991, 3, 30.
41. Gascho, G.J.; Shih, S.F. Cultural methods to increase 63. Morris, J.; Radley, R.W.; Smith, D.L.O.; Plom, A. Straw
sucrose and energy yields of sugarcane. Agron. J. 1981, 73, supply in the U.K. with particular reference to industrial
999. usage. Agric. Prog. 1976, 51, 77.
42. Results of Field Experiments, Report 1993, Landeslandwirt- 64. Tarchevsky, A.; Marchenko, G.N. Cellulose: Biosynthesis
schaftskammer fur Tirol: Innsbruck, 1994; p. 12. and Structure; Springer-Verlag: Berlin, 1991; pp. 211213.
43. Mislevy, P.; Fluck, R.C. Harvesting operations and 65. Wegscheider, R. Uber die Verseifung von Karbon- und
energetics of tall grasses for biomass energy production: Sulfonsaureestern. Phys. Chem. 1902, 41, 51.
A case study. Biomass Bioenergy 1992, 3, 381. 66. Skrabal, A. Uber ein sehr allgemeines Zeitgesetz der chem.
44. Sladden, S.E.; Bransby, D.I.; Aiken, G.E.; Prine, G.M. Kinetic und seine Deutung. Die Hydrolyse Geschwindig-
Biomass yield and composition, winter survival of tall keit der Organooxyde. Z. Elektrochem. 1927, 33, 322.
grasses in Alabama. Biomass Bioenergy 1993, 1, 123. 67. Szejtli, J. Saurehydrolyse glycosidischer Bindungen; Fach-
45. Kordsachia, O.; Baum, N.; Patt, R. Elefantengras-ein buchverlag: Leipzig, 1976; 344, 362.
potentieller Rohsto fur die Zellsto- und Papierindustrie. 68. Philipp, B.; Jacopian, V.; Loth, F.; Hirte, W.; Schulz, G.
Das Papier 1992, 46, 257. Inuence of cellulose physical structure on thermohydro-
46. Bobleter, O. Hydrothermal degradation of polymers lytic, hydrolytic and enzymatic degradation of cellulose. In
derived from plants. Prog. Polym. Sci. 1994, 19, 799. Hydrolysis of Cellulose: Mechanisms of Enzymatic and Acid
47. Schwarz, H. Miscanthus sinensis, Giganteus production Catalysis; Brown, R.D., Jurasek, I., Jurasek, L., Eds.;
on several sites in Austria. Biomass Bioenergy 1993, 5, Adv. Chem. Ser. 181, 1979; American Chemical Society:
413. Washington, DC, 1979; p. 127.
48. FAO Year Book Production, Vol. 48, 1994 (FAO Statistic 69. Abatzoglou, N.; Chornet, E. Acid hydrolysis of hemi-
No. 125); Food and Agriculture Organisation of the celluloses and cellulose: Theories and applications. In
United Nations: Rome, 1995; p. 90. Polysaccharides; Dumitriu, S., Ed.; Marcel Dekker: New
49. Ferm, A. Birch production and utilization for energy. York, 1998; p. 1007.
Biomass Bioenergy 1993, 4, 391. 70. Saeman, J. Kinetics of wood saccharication hydrolysis of
50. Willebrand, E.; Ledin, S.; Verwijst, T. Potential and cellulose and decomposition of sugars in dilute acids at high
economics of forestry options for carbon sequestration in temperatures. J. Ind. Eng. Chem. 1945, 37, 43.
India. Biomass Bioenergy 1993, 4, 323. 71. Grethlein, H.E. Chemical break down of cellulosic
51. Labrecque, M.; Teodorescu, T.I.; Coglistro, A.; Daigle, S. materials. J. Appl. Chem. Biotechnol. 1978, 28, 296.
Growth patterns and biomass productivity of two salix 72. Fagan, R.D.; Grethlein, H.E.; Converse, A.O.; Porteous,
species grown under short-rotation intensive culture in A. Kinetics of the acid hydrolysis of cellulose found in
southern Quebec. Biomass Bioenergy 1993, 4, 419. paper refuse. Environ. Sci. Technol. 1971, 5, 545.
52. White, E.H.; Gambles, R.L.; Zsua, L. Experiments with 73. Kim, J.S.; Lee, Y.Y.; Torget, R.W. Cellulose hydrolysis
willow as a wood biomass species. In Energy from Biomass under extremely low sulfuric acid and high-temperature
and Wastes XII; Klass, D.L., Ed.; Institute of Gas conditions. Appl. Biochem. Biotechnol. 2001, 91, 331.
Technology: Chicago, 1989; p. 125. 74. Prutsch, W. Untersuchung des chemischen Aufschlusses
53. McElroy, G.H.; Dawson, W.M. Biomass from short- panzlicher Biomasse unter hydrothermalen Bedingungen,
rotation coppice willow on marginal land. Biomass 1986, Ph.D. thesis, University of Innsbruck, 1989; pp. 200201.
10, 225. 75. Bobleter, O.; Schwald, W.; Concin, R.; Binder, H. Hy-
54. DeBell, D.S.; Clendenen, G.W.; Zasada, J.C. Growing drolysis of cellobiose in dilute sulfuric acid and under hy-
populus biomass: Comparison of woodgrass versus wider- drothermal conditions. J Carbohydr. Chem. 1986, 5, 387.
spaced short-rotation systems. Biomass Bioenergy 1993, 4, 76. Roy, N.; Timell, T.E. The acid hydrolysis of glycosides, IX:
305. Hydrolysis of two aldotrionronic acids derived from a-(4-
55. Heilman, P.E.; Fuguang, X. Inuence of nitrogen on O-methylglucurono) xylan, X: Hydrolysis of 2-O-(4-O-
growth and productivity of short rotation Populus tricho- methyl-a-D-glucopyranosyluronic), XI: Eect of pH on the
carpa and Populus deltoides hybrides. Can. J. For. Res. hydrolysis of acid disaccharides. Carbohydr. Res. 1968, 6,
1993, 23, 1863. 488.
56. Liu, W.; Merriam, R.A.; Phillips, V.D.; Singh, D. 77. Kobayashi, T.; Sakai, Y. Wood saccharication process
Estimating short-rotation Eucalyptus saligna production with strong H2SO4 (IV) hydrolysis rate of pentosan in dil.
in Hawaii: An integrated yields and economic model. H2SO4. Mokuzoitoka Shingikai Hokuku 5: 1(1956). Chem.
Bioresour. Technol. 1993, 45, 167. Abstr. 1960, 54, 6121.
57. Giridhar, G.; Deval, K.; Maheswari, R.C.; Vasudevan, P. 78. Dudkin, M.S.; Santowa, N.G.; Tatarkina, A.V. Hemi-
A source of energy. Biomass 1985, 7, 241. celluloses of cereals and leguminous plants. J. Angew.
58. Rockwood, D.L.; Dippon, D.R. Biological and economic Chem. 1965, 38, 173.
potentials of Eucalyptus grandis and Slash pine as biomass 79. Bonn, G.; Binder, H.; Leonhard, H.; Bobleter, O. The
energy crops. Biomass 1989, 20, 155. alkaline degradation of cellobiose to glucose and fructose.
59. Liang, T.; Khan, M.A.; Phillips, V.D.; Liu, W. Hawaii Monatsh. Chem. 1985, 116, 961.
natural resource information system: A tool for biomass 80. Bobleter, O.; Bonn, G. The hydrothermolysis of cellobiose
production management. Biomass Bioenergy 1994, 6, and its reaction product D-glucose. Carbohydr. Res. 1983,
431. 124, 185.
60. Sladden, S.E.; Brausby, D.I.; Aiken, G.E. Biomass yield, 81. Schwald, W.; Bobleter, O. Hydrothermolysis of cellulose
composition and production costs for eight switch grass under static and dynamic conditions at high temperatures.
varieties in Alabama. Biomass Bioenergy 1991, 1, 119. J. Carbohydr. Chem. 1989, 8, 565.
934 Bobleter
82. De Bruijn, J.M.; Kieboom, A.P.G.; van Bekkum, H.; von organosolv degradation of lignocellulosic biomass. Frese-
der Poel, P. Sugars as phosphate substitute in detergents. nius Z. Anal. Chem. 1988, 331, 46.
XIIth International Carbohydrate Symposium Abstracts; 104. Bonn, G.; Pecina, R.; Burtscher, E.; Bobleter, O. Sepa-
Vonk Publ.: Utrecht, 1984; p. 189. ration of wood degradation products by high-perform-
83. Dan, D.C.; Jaeopian, V.; Kasulke, K.; Philipp, B. Analysis ance liquid chromatography. J. Chromatogr. 1984, 287,
of the eect of the chemical and physical structure of 215.
substrates on the enzymatic degradation of cellulose. Acta 105. Zemann, A.; Bobleter, O.; Prutsch, W. Hydrothermoly-
Polym. 1980, 31, 388. sisA pretreatment for pulp production. In Ligno-
84. Garrett, E.R.; Young, J.F. Alkaline transformations Cellulosics, Science, Technology, Development and Use;
among glucose, fructose and mannose. J. Org. Chem. Kennedy, J.F., Phillips, G.O., Williams, P.A., Eds.; New
1970, 35, 3502. York: Ellis Horwood, 1992; p. 213.
85. Kleinert, T.N.; Tayenthal, K. Separation of cellulose and 106. Reynolds, J.G.; Burnham, A.K.; Wallman, H. Reactivity
incrustings, U.S. Patent No. 1,856,567, 1931. of paper residues produced by hydrothermal pretreatment
86. Johansson, A.; Aaltonen, O.; Ilinen, P. Organosolv process for municipal solid wastes. Energy Fuels 1997, 11,
pulpingMethods and pulp properties. Biomass 1987, 98.
13, 45. 107. Antal, M.J.; Mok, W.S.L.; Varhegyi, G.; Szetzely, T.
87. Mason, W.H. Cellulose or papermaking appl. U.S. Patent Review of methods for improving the yield of charcoal
No. 1,655,618, 1929. from biomass. Energy Fuels 1990, 4, 221.
88. Babcock, L.W. Fermentable sugars from wood, U.S. Pat- 108. Richard, J.R.; Antal, M.J. Thermogravimetric studies of
ent No. 1,825,464, 1932. charcoal formation from cellulose at elevated pressures. In
89. Bobleter, O.; Pape, G. Verfahren zum Abbau von Holz, Advances in Thermo-Chemical Biomass Conversion; Bridge-
Rinde und anderen Panzenmaterialien, Austrian Patent water, A.V., Ed.; Blackie Academic and Professional:
No. 263661, 25.7.1968. London, 1994; p. 784.
90. Bobleter, O.; Pape, G. Der hydrothermale Abbau von 109. Huber, Ch.; Grill, B.; Oefner, P.; Bobleter, O. Capillary
Glucose. Monatsh. Chem. 1968, 99, 1560. electrophoretic determination of the component mono-
91. Lora, J.H.; Wayman, M. Delignication of hardwoods by saccharides in hemicelluloses. Fresenius J. Anal. Chem.
autohydrolysis and extraction. TAPPI J. 1978, 61, 47. 1994, 348, 825.
92. Puls, J.; Dietrichs, H.H.; Sinner, M. Hydrolysis of hemi- 110. Fengel, D.; Wegener, O.; Heizmann, A.; Przyklenk, H.
celluloses by immobilized enzymes. In Energy from Analysis of wood and cellulose by total hydrolysis with
Biomass; Palz, W., Chartier, P., Hall, D.O., Eds.; Applied triuoroacetic acid. Holzforschung 1977, 31, 65.
Science Publ.: London, 1981; p. 348. 111. Mietz, M.; Berg, F.; Jacopian, V.; Philipp, B.; Sauer, I.
93. Bonn; Concin, R.; Bobleter, O. HydrothermolysisA new Scientic and technological aspects of mechanochemical
process for the utilization of biomass. Wood Sci. Technol. straw digestion. Acta Biotechnol. 1987, 7, 135.
1983, 17, 195. 112. Paul, D.; Jacopian, V.; Philipp, B. Morphological changes
94. Conner, A.H. Kinetic modeling of hardwood prehydrol- in sugar cane bagasse by mild alkali treatment. Cellul.
ysis. Part I: Xylan removal by water prehydrolysis. Wood Chem. Technol. 1986, 20, 357.
Fiber Sci. 1984, 16, 268. 113. Pekarovicova`, A.; Luza`kova`, V.; Reiser, V.; Pekarovic, J.
95. Heitz, M.; Carrasco, F.; Rubio, M.; Chauvette, G.; Enhancement of wheat straw enzymatic hydrolyzability by
Chornet, B.; Jaulin, L.; Overend, R.P. Generalized low temperature pretreatments. Holzforschung 1989, 43,
correlations for the aqueous liquefaction of lignocellulo- 65.
sics. Can. J. Chem. Eng. 1986, 64, 647. 114. Bonn, G. Tannin Extraktion. unpublished.
96. Overend, R.P.; Chornet, B. Fractionation of lignocellulo- 115. Schwald, W.; Brownell, H.H.; Saddler, J.N. Enzymatic
sics, by steam-aqueous pretreatments. Philos. Trans. R. hydrolyses of steam treated aspen wood: Inuence of
Soc. Lond., A. 1987, 321, 523. partial hemicellulose and lignin removal prior to pretreat-
97. Mok, W.S-L.; Antal, M.J. Jr. Biomass fractionation by hot ment. In Steam Explosion Techniques; Focher, B., Marzetti,
compressed liquid water. In Advances in Thermochemical A., Crescenzi, V., Eds.; Gordon and Breach Science Publ.:
Biomass Conversion; Bridgewater, A.V., Ed.; Blackie Philadelphia, 1991; p. 307.
Academic and Professional: London, 1994; p. 1572. 116. Brownell, H.H.; Saddler, J.N. Steam pretreatment of
98. Pape, G. Untersuchungen uber den hydrothermalen Ab- lignocellulosic material for enhanced enzymatic hydrolysis.
bau von Holzsubstanzen und Herstellung von Ionenaus- Biotechnol. Bioeng. 1987, 29, 228.
tauschern auf Holzbasis, Ph.D. thesis, University of 117. Chornet, E.; Overend, R.P. Liquid fuels from lignocellu-
Vienna, 1965. losics. In Steam Explosion Techniques; Focher, B., Mar-
99. Bobleter, O.; Binder, H.; Concin, R. Hydrothermolyse von zetti, A., Crescenzi, V., Eds.; Gordon and Breach Science
Panzenmaterialien. Chem. Rundsch. 1979, 32 (44), 1. Publ.: Philadelphia, 1991; p. 21.
100. Bonn, G.; Bobleter, O. Determination of the hydrothermal 118. Overend, R.P.; Chornet, E. Fractionation of lignocellulo-
degradation products of D-(U-C14) glucose and D-(U-C14) sics, by steam-aqueous pretreatments. Philos. Trans. R.
fructose by TLC. J. Radioanal. Chem. 1983, 79, 171. Soc. Lond., A. 1987, 321, 523.
101. MacLaurin, D.J.; Green, J.W. Carbohydrates in alkaline 119. Puls, J.; Dietrichs, H.H.; Sinner, M. Hydrolysis of hemi-
systems. II. Kinetics of the transformations and degrada- celluloses by immobilized enzymes. In Energy from
tion reactions of cellobiose, and 4-O-h-D-glucopyranosyl- Biomass; Palz, W., Chartier, P., Hall, D.O., Eds.; Applied
D-mannose in 1 N sodium hydroxide at 22jC. Can. J. Science Publ.: London, 1981; p. 348.
Chem. 1969, 47, 3957. 120. Liamsakul, W.; Zemann, A.; Bobleter, O. Hydrothermal
102. Oefner, P.J.; Lanziner, A.H.; Bonn, G.; Bobleter, O. pretreatment of rice straw. In Advances in Thermochemical
Quantitative studies on furfural and organic acid formation Biomass Conversion; Bridgewater, A.V., Ed.; Blackie
during hydrothermal, acidic and alkaline degradation of D- Academic and Professional: London, 1994; p. 1545.
xylose. Monatsh. Chem. 1992, 123, 547. 121. Bobleter, O.; Binder, H.; Concin, R.; Burtscher, E. The
103. Bonn, G.; Oefner, P.J.; Bobleter, O. Analytical determi- conversion of biomass to fuel raw material by hydro-
nation of organic acids formed during hydrothermal and thermal treatment. In Energy from Biomass; PaIz, W.,
Chartier, P., Hall, D.O., Eds.; Applied Science Publ.: chips. Can conditions for optimum hemicellulose recov-
London, 1981; p. 544. ery still provide adequate access for ecient enzyma-
122. Bonn, G.; Hormeyer, H.F.; Bobleter, O. Hydrothermal and tion hydrolysis? Appl. Biochem. Biotechnol. 2000, 84,
organosolv pretreatments of poplar wood and wheat straw 693.
for saccharication by a Trichoderma viride cellulase. 139. Yang, R.; Xu, S.; Wang, Z. Steaming extraction of corncob
Wood Sci. Technol. 1987, 21, 179. xylan for production of xylooligosaccharide. Wuxi Qing-
123. Kubikova, J.; Zemann, A.; Krkoska, P.; Bobleter, O. gong Daxua Xuebao 1998, 17, 50.
Hydrothermal pretreatment of wheat straw for the 140. Schwald, W.; Smaridge, T.; Breuil, C.; Saddler, J.N. The
production of pulp and paper. TAPPI 1996, 79, 163. inuence of SO2 impregnation and fractionation on
124. Kubikova, J. Hydrothermale Vorbehandlung von pan- product recovery and enzymic hydrolysis of steam-treated
zlicher Biomasse fur die Zellsto- und Papierherstellung, sprucewood. In Enzyme Systems for Lignocellulose Degra-
Ph.D. thesis, University of Innsbruck, 1994, p. 209. dation; Coughlan, M.P., Ed.; Elsevier Appl. Sci.: London,
125. Kubikova, J.; Lu, Ping; Zemann, A.; Krkoska, P.; 1989; p. 231.
Bobleter, O. Aquasolv pretreatment of plant materials 141. Mattos, L.R.; Teixeira da Silva, F. In Steam explosion of
for the production of cellulose and paper. Cellul. Chem. sugar cane bagasse catalysed by H2SO4: Eect of temper-
Technol. 2000, 34, 151. ature, time and acid content on sugarcane bagasse solubilisa-
126. Kazi, K.M.F.; Jollez, P.; Chornet, E. Preimpregnation: An tion. Brasilian Symposium on the Chemistry of Lignins and
important step for biomass rening processes. Biomass Other Wood Components, Proceedings, 6th, Guaratingue-
Bioenergy 1998, 15, 125. ta, Brazil; Fac. de Eng. Quim. de Lorena: Lorena, Brazil,
127. Nguyen, Q.A.; Tucker, M.P.; Keller, F.A.; Beaty, D.A.; 2001; p. 407.
Conners, K.M.; Eddy, F.P. Dilute acid hydrolysis of soft 142. Dieterich, J.B.; Mathias, A.L.; Ramos, L.P. In Acid-
woods. Appl. Biochem. Biotechnol. 1999, 77, 133. catalyzed steam explosion of Pinus taeda chips. I. Character-
128. Allen, S.G.; Schulman, D.; Lichwa, J.; Antal, M.J. Jr. A ization of water-soluble fraction. Brasilian Symposium on
comparison of aqueous and dilute-acid single-temperature the Chemistry of Lignins and Other Wood Components,
pretreatment of yellow poplar sawdust. Ind. Eng. Chem. Proceedings, 6th, Guaratingueta, Brazil; Fac. de Eng.
Res. 2001, 40, 2352. Quim. de Lorena: Lorena, Brazil, 2001; p. 189.
129. Schell, D.; Nguyen, Q.; Tucker, M.; Boynton, B. Pretreat- 143. Nakamura, Y.; Sawada, T.; Inoue, E. Enhanced ethanol
ment of softwood by acid-catalyzed steam explosion production from enzymatically treated steam-exploded rice
followed by alkali extraction. Appl. Biochem. Biotechnol. straw using extractive fermentation. J. Chem. Technol.
1998, 70, 17. Biotechnol. 2001, 76, 879.
130. Martin, C.; Galbe, M.; Nilvebrant, N-O.; Jonsson, L.J. 144. Allen, S.G.; Schulman, D.; Lichwa, J.; Antal, M.J. Jr.;
Comparison of the fermentability of enzymatic hydro- Laser, M.; Lynd, L.R. A comparison between hot liquid
lysates of sugar cane bagasse pretreated by steam explosion water and steam fractionation of corn ber. Ind. Eng.
using dierent impregnating agents. Appl. Biochem. Chem. Res. 2001, 40, 2934.
Biotechnol. 2002, 98, 699. 145. Laser, M.; Schulman, D.; Allen, S.G.; Lichwa, J.; Antal,
131. Soderstrom, J.; Pilcher, L.; Galbe, M.; Zacchi, G. Two-step M.J.; Lynd, L.R. A comparison of liquid hot water and
steam pretreatment of softwood with SO2 impregnation for steam pretreatments of sugar cane bagasse for bioconver-
ethanol Production. Appl. Biochem. Biotechnol. 2002, 98, sion to ethanol. Bioresour. Technol. 2002, 81, 33.
5. 146. Alfani, F.; Gallifuoco, A.; Saporosi, A.; Spera, A.;
132. Shevchenko, S.M.; Chang, K.; Robinson, J.; Saddler, J.N. Cantarella, M. Comparison of SHF and SFF processes
Optimization of monosaccharide recovery by post-hydrol- for the bioconversion of steam-exploded wheat straw. J.
ysis of water-soluble hemicellulose component after steam Ind. Microbiol. Biotechnol. 2000, 25, 184.
explosion of softwood chips. Bioresour. Technol. 2000, 72, 147. Ballesteros, I.; Oliva, J.M.; Navarro, A.A.; Gonzalez, A.;
207. Carrasco, J.; Ballesteros, M. Eect of chip size on steam
133. Wu, M.M.; Chang, K.; Gregg, D.J.; Boussaid, A.; Beatson, explosion pretreatment of softwood. Appl. Biochem.
R.B.; Saddler, J.N. Optimization of steam explosion to Biotechnol. 2000, 84, 97.
enhance hemicellulose recovery and enzymatic hydrolysis 148. De Bari, I.; Viola, E.; Barisano, D.; Cardinale, M.; Nanna,
of cellulose in softwoods. Appl. Biochem. Biotechnol. 1999, F.; Zimbardi, F.; Cardinale, G.; Braccio, G. Ethanol
77, 47. production at ask and pilot scale from concentrated
134. Stenberg, K.; Bollok, M.; Reczey, K.; Galbe, M.; Zacchi, slurries of steam-exploded aspen. Ind. Eng. Chem. Res.
G. Eect of substrate and cellulase concentration on 2002, 41, 1745.
simultaneous saccharication and fermentation of steam- 149. Kaar, W.B.; Gutierrez, C.V.; Kinishita, C.M. Steam
pretreated softwood for ethanol production. Biotechnol. treated rice industry residues as an attractive feedstock
Bioeng. 2000, 68, 204. for the wood based particle board industry in Italy.
135. Stenberg, K.; Galbe, M.; Zacchi, G. The inuence of lactic Biomass Bioenergy 1998, 14, 255.
acid formation on the simultaneous saccharication and 150. Belkacemi, K.; Turcotte, G.; Savoie, P. Aqueous/steam-
fermentation (SSF) of softwood to rthanol. Enzyme fractionated agricultural residues as substrates for ethanol
Microb. Technol. 2000, 26, 71. production. Ind. Eng. Chem. Res. 2002, 41, 173.
136. Alkasrawi, M.; Galbe, M.; Zacchi, G. Recirculation of 151. Jeoh, T.; Agblevor, F.A. Characterization and fermenta-
process streams in fuel ethanol production from softwood tion of steam exploded cotton gin waste. Biomass Bioenergy
based on simultaneous saccharication and fermentation. 2001, 21, 109.
Appl. Biochem. Biotechnol. 2002, 98, 849. 152. Ye, H.; Li, J.; Chen, B.; Ouyang, P. Steam explosion pre-
137. Tengborg, Ch.; Galbe, M.; Zacchi, G. Inuence of enzyme treatment for recycling bagasse. Huagong Shikan 2000,
loading and physical parameters on the enzymatic hydrol- 14, 12.
ysis of steam-pretreatment. Biotechnol. Prog. 2001, 17, 153. Shimizu, K.; Sudo, K.; Ono, H.; Ishihara, M.; Fujii, T.;
110. Hishiyama, S. Integrated process for total utilization of
138. Boussaid, A.; Esteghlalian, A.R.; Gregg, D.J.; Lee, K.H.; wood components by steam-explosion pretreatment. Bio-
Saddler, J.N. Steam pretreatment of Douglas-r wood mass Bioenergy 1998, 14, 195.
936 Bobleter
154. Ibrahim, M.; Glaser, W.G. Steam-assisted biomass 167. Pappens, R. PPI March 1990, 74.
fractionation. Part III: a quantitative evaluation of the 168. Delong, E.A. Making lignin separable from cellulose and
clean fractionation concept. Bioresour. Technol. 1999, hemicellulose in lignocellulose material, Canadian Patent
70, 181. No. 1,096,374, 1981.
155. Cunha, H.C.M.;; Silva, F.T. In Characterization of 169. Marchessault, R.H.; Malhotra, S.L.; Jones, A.Y.; Petrovic,
carbohydrates present in hydrolysate obtained from sugar- A. The wood explosion process: Characterization and uses
cane bagasse pretreated by steam explosion. Brasilian of lignin/cellulose products. In Wood and Agricultural
Symposium on the Chemistry of Lignins and Other Wood Residues; Soltes, E.J., Ed.; Academic Press: New York,
Components, Proceedings, 6th, Guaratingueta, Brazil; 1983, p. 401.
Fac. de Eng. Quim. de Lorena: Lorena, Brazil, 2001; p. 221. 170. Bender, R.H. U.S. Patent No. 4,136,207, 1979.
156. Galvao, M.L.; Ramos, L.P.; Echterho, M.F.; Fontana, 171. Manners, H.; Rowney, J.E. Recovery of cellulose bers
J.D. In Autohydrolysis and acid-catalyzed steam explosion from a composite lm, U.S. Patent No. 4,163,687, 1979.
of sugarcane bagasse. Brasilian Symposium on the Chem- 172. Manners, H.; Yuritta, J.P.; Menz, D.J. Explosion pulping
istry of Lignins and Other Wood Components, Proceed- of annual and fast growing plants. TAPPI 1981, 64, 93.
ings, 6th, Guaratingueta, Brazil; Fac. de Eng. Quim. de 173. Dekker, R.F.H.; Wallis, A.F.A. Autohydrolysisexplosion
Lorena: Lorena, Brazil, 2001; p. 200. as pretreatment for the enzymatic saccharication of
157. Montane, D.; Farriol, X.; Salvado, J.; Jollez, P.; Chornet, sunower seed hulls. Biotechnol. Lett. 1983, 5, 311.
E. Application of steam explosion to the fractionation and 174. Brown, B.B. U.S. Patent No. 4,211,164, 1980.
rapid vapor-phase alkaline pulping of wheat straw. 175. Antal, M.; Micko, M.M. Balsam poplar wood quality
Biomass Bioenergy 1998, 14, 261. parameters. Determination of chemical and physical
158. Sekino, N.; Inoue, M.; Irle, M.; Adcock, T. The mecha- properties. Holzforschung 1990, 42, 51.
nisms behind the improved dimensional stability of 176. Watch, E.; Zemann, A.; Schirmer, F.; Bonn, G.; Bobleter,
particleboards made from steam-pretreated particles. O. Enzymatic saccharication of hemicellulose obtained
Holzforschung 1999, 53, 435. from hydrothermally pretreated sugar cane bagasse and
159. Gerardi, V.; Minelli, F.; Vaggiano, D. Steam treated rice beech bark. Bioresour. Technol. 1992, 39, 173.
industry residues as an alternative feedstock for the wood 177. Oefner, P.J.; Lanziner, A.H.; Bonn, G.; Bobleter, O.
based particleboard industry in Italy. Biomass Bioenergy Quantitative studies on furfural and organic acid formation
1998, 14, 295. during hydrothermal, acidic and alkaline degradation of D-
160. Allen, S.G.; Kam, L.C.; Zemann, A.J.; Antal, M.J. Jr. xylose. Monatsh. Chem. 1992, 123, 547.
Fractionation of sugar cane with hot, compressed, liquid 178. Bobleter, O.; Grif, M.; Huber, Ch. Verfahren zur Hydro-
water. Ind. Eng. Chem. Res. 1996, 35, 2709. lyse von Panzenmaterialien, Austrian Patent No. 398 990
161. Rubio, M.; Tortosa, J.F.; Quesada, J.; Go`mez, D. B, 1995.
Fractionation of lignocellulosics. Solubilization of corn 179. Bocher, A.M.; Petropavlovsky, G.A.; Kallistof, O.V.
stalk hemicelluloses by autohydrolysis in aqueous medium. Structure formation in solutions and lms of cellulose
Biomass Bioenergy 1998, 15, 483. and its derivatives as determined by elastic light scattering
162. Bouchard, J.; Nguyen, T.S.; Chornet, E.; Overend, R.P. data. Cellul. Chem. Technol. 1993, 27, 137.
Analytical methodology for biomass pretreatment. Part 2: 180. Partnertre fur eine Textile Zukunft, Chemie, July/August
Characterisation of ltrates and cumulative distribution as 1996; p. 7.
a function of treatment severity. Bioresour. Technol. 1991, 181. Hanselmann, K.W. Experientia 1982, 38, 176.
36, 121. 182. Milligan, J.B.; Evans, G.D.; Bridgwater, A.V. Results from
163. Mok, W.S.-L.; Antal, M.J. Jr. Uncatalyzed solovolysis of a transparent open-core downdraft gasier. In Advances in
whole biomass hemicellulose by compressed liquid water. Thermochemical Biomass Conversion; Bridgwater, A.V.,
Ind. Eng. Chem. Res. 1992, 31, 1157. Ed.; Blackie Academic and Professional: London, 1994;
164. Kleinert, T.N. Mechanism of wood pulping, U.S. Patent p. 175.
No. 3,585,104, 1971. 183. Kim, J.S.; Li, Y.Y.; Torget, R.W. Cellulose hydrolysis
165. Aziz, S.; Sarkanen, K. Organosolv pulpingA review. under extremely low sulfuric acid and high-temperature
TAPPI 1989, 72, 169. conditions. Appl. Biochem. Biotechnol. 2001, 91, 331.
166. Katzen, R. Distillation method and apparatus for making 184. Horvath, I.S.; Taherzadeh, M.J.; Niklasson, C. Eects of
motor fuel grade anhydrous ethanol. Chemtech 1981, 3 furfural on anaerobic continuous cultivation of Saccha-
(11), 186. romyces cerevisiae. Biotechnol. Bioeng. 2001, 75, 540.
41
Cellulosic Biomass-Derived Products
Chembiotech Laboratories, Birmingham, United Kingdom
I. INTRODUCTION The term biomass is used to describe materials of

biological origin, which are either purposely grown, or
The key strategies for an energy policy are safety of supply, arise as by-products, residues, or wastes from a process or
low implementation costs, and environmental acceptabil- application. Although it is used mainly in reference to
ity. The rst of these has always been the most important materials of plant origin (phytomass), it also includes
since energy is the lifeblood of a modern industrial society. bones, proteins, lipids, and other biological components.
Acute population growth (expected to exceed 10 billion by Historically, renewable biomass resources, in the form
2050) is expected to contribute to the already growing of wood (and farm residues/wastes), were the principal
demand for energy in developed and developing countries. sources of fuel and construction materials throughout
A mere eight countries have >80% of all world crude oil the world, and this is still the case in many rural areas
reserves, six have >70% of all natural gas reserves, and (particularly in developing countries) because it is often
eight have >85% of all coal reserves. Countries such as readily available and needs little capital or technological
India, China, and Indonesia, which represent nearly half investment. Until the start of the 20th century, biomass
the worlds population, are therefore actively involved in and coal were the major sources of fuel, materials, and
developing and using renewable energy as the only means chemicals. Then nonrenewable resources such as oil and
of sustaining their rapidly growing energy requirements. natural gas (and coal) became the major fuel sources, as
More than half of Asia, Africa, and Latin America import well as the major sources of chemicals and plastic materi-
over 50% of their commercial energy. Most of these als. Signicant developments were also made in modern
countries export crops that fetch low prots and import construction material production, from metallic, mineral-
energy at high prices, which is not good for their econo- based (glass, ceramics), or chemical/synthetic sources.
mies [1]. These factors, along with intensive land use for food crops
Growing worldwide environmental awareness and in the developed world, resulted in a dramatic reduction in
concerns (regarding issues such as greenhouse gas emis- biomass utilization for the production of derived materials
sions, global warming, natural resource depletion) with and products.
continued use of conventional fossil and nuclear fuels will Interest in biomass utilization is growing as a result of
mean that future energy production will have to be as greater awareness of economic changes and political fac-
clean as technically possible and economically viable, tors associated with fossil fuels, and increasing environ-
which opens the door for renewable energy sources [2]. mental awareness, which has led to eorts to introduce
Considerable achievements and rapid progress is being more stringent global environmental controls. This will
made in areas of hydropower, biomass conversion, geo- shift the responsibility of dealing with waste/pollution
thermal energy, solar thermal technology, wind energy problems to the waste producers, thereby connecting prod-
conversion, and the increasing usage of photovoltaics. uct biocompatibility to production costs. Purpose-grown
Recent technological developments have also improved biomass is still little used in the developed world due to its
the cost-eectiveness of such renewable resources, making low physical and energy density (high moisture content),
their economic prospects look increasingly attractive. It relatively rapid biodegradation during storage, and cost
has been predicted that by 2030, 1520% of our prime (growing, harvesting, transporting, storing, processing, and
energy will be met by renewable energy resources [1,3,4]. using biomass all consume energy). Such factors will con-
937
tinue to discourage its purpose-grown use unless political material that consists of a reinforcement of cellulose
initiatives are taken and the necessary nancial incentives microbrils (5055% w/w) in a cementing matrix of hemi-
are given [5]. cellulose (1525% w/w) and lignin (2030% w/w) [1517].
Utilization of waste biomass, arising as by-products The components lignin, cellulose, and hemicellulose
from other processes or by recycling previously used (Fig. 1) are generally present in the ratio 1:2:1; however,
materials, is far more economically viable since conversion this varies depending on the species of origin. Tradition-
costs simply have to be exceeded by nonuse disposal costs ally, wood has been used as building material and in the
plus revenue gained from selling conversion end products. paper industry, or simply as a fuel by burning, but its future
Such renewed interest in biomass utilization (especially applications lie in areas of mass production of fuels and
solid biowaste) has stimulated research into the utilization chemical feedstocks by the large-scale microbial biocon-
of biomass feedstocks as an alternative source of energy version of lignocellulosic materials [1820]. Because of
and chemical production, which will simultaneously ad- their abundance, lignin, cellulose, and hemicellulose are
dress issues of renewable energy and environmental pollu- the basic chemical feedstocks derived from biomass and
tion [614]. great eorts are made to improve their separation.
Cellulose is largely crystalline, and consists of linear
chains of (1!4)-linked h-D-glucopyranosyl units (Fig. 1).
II. CELLULOSIC BIOMASS SOURCES These chains are packed in layers, held together by van der
AND COMPOSITION Waals forces with intramolecular and intermolecular hy-
drogen bonding [2123]. The majority of industrially uti-
Plant-based biomass resources include wood, agricultural lized cellulose currently nds its application in the pulp and
crops and residues, grasses, and components from these paper industry. Large amounts of cellulose currently nd
sources. Wood is by far the largest source of biomate- no application and are treated as waste, being burned or
rial. Wood tissues are built up from cells, most of which rotted naturally. However, as with other long-chain, nat-
are brous. The wood bers are themselves a composite ural polysaccharides, starch and chitin, the potential of
Figure 1 Chemical structures of (a) lignin, (b) cellulose, and (c) hemicelluloses.
Cellulosic Biomass-Derived Products 939
cellulose as a source material for higher value materials is cellulosic materials can be processed in a number of ways
now being fully realized. using chemical, biochemical, or thermochemical conver-
Various cellulosic raw materials are available that sion methodologies (Fig. 3). Several strategies are poten-
deserve scrutiny as potential source materials for chemical tially applicable or are being applied to cellulosics
and fuel production. Wood and woody plants as well as modication and utilization on an industrial scale.
waste products such as cardboard, sawdust, bagasse,
groundnut shells, jute sticks, spent lemongrass, cashew
and rubber tree wood, cotton linters, cotton waste, etc., A. Chemical Modification (Acid/Alkali
all potential renewable resources for the generation of Hydrolysis and Derivatization)
fermentable sugars, can also be utilized [24]. Among the
The most common method of converting cellulosics to
woody species, poplar and eucalyptus are good sources of
useful products is a straightforward hydrolysis to compo-
lignocellulose material suitable for energy production.
nent monosaccharides. The choices for this procedure are
Sweet sorghum produces about 30% sugar and 70%
either acidic/alkaline hydrolysis (or enzymic hydrolysis).
bagasse, a lignocellulose raw material, and can potentially
The advantages of acidic/alkaline hydrolysis stem from
produce 3050 tonnes of dry matter per hectare [25].
their nonspecicity (also the cause of their major draw-
backs), meaning that all of the cellulosic sample can be
III. CELLULOSIC BIOMASS CONVERSION hydrolyzed to glucose using a single application of acid/
TECHNOLOGY alkali [34]. The obvious major drawback is that anything in
the sample other than cellulose is also likely to react with
Cellulose is generally produced by pulping: acidic or the acid/alkali, a situation that can lead to the formation of
alkaline treatment results in the hydrolysis of hemicellulose harmful by-products. Treatment of lignocellulosic biomass
and the decomposition of lignin, so free cellulose bers can with dilute sulfuric acid is primarily used as a means of
be extracted from the medium [26,27]. Lignin and hemi- hemicellulose hydrolysis and a pretreatment for enzymatic
cellulose cannot be obtained in their polymeric forms by hydrolysis of cellulose [35]. Treatment of various celluloses
the common wood unlocking processes, but the develop- in supercritical water has also been used to produce glucose
ment of steam explosion and implosion techniques (Fig. 2) and its derivatives [36].
permits separation of cellulose, oligomeric hemicellulose, A multitude of detailed information is available on the
and lignin monomers in one process [2833]. In principle, chemical (acid and alkali) degradation of cellulose [3743].
Figure 2 Separation of lignin, cellulose, and hemicellulose by steam explosion.

Figure 3 Potential conversion routes for cellulosic materials.
The acid-induced depolymerization of polysaccharides (from pentoses) and 5-hydroxymethylfurfural (from hexo-
(hydrolysis of glycosidic linkages) is presented in Fig. 4. ses) (Fig. 5) [44].
Clearly, if sucient hydrolysis occurs then monosaccha- Cellulose glycosidic linkages are relatively alkali stable
rides will ultimately be produced; however, further acid below 170jC; however, degradation does occur via a
treatment results in the degradation/dehydration of the dierent mechanism, namely, an endwise degradation
released monosaccharides producing 2-furfuraldehyde known as peeling or unzipping. This involves the conver-
Figure 4 Acid-catalyzed hydrolysis of glycosidic linkages.

Figure 5 Acidic dehydration of monosaccharides.
Figure 6 Alkaline hydrolysis of cellulose.

Treatment with dilute mineral acids hydrolyzes some

glycosidic bonds (a similar eect is achievable using mild
enzymic modication). Products from these conversions
could be used when high-solid, low-viscosity pastes are
desired, e.g., in textile yarns, fabric nishing, gypsum
board manufacture, paper manufacture, and in gum
confectionery. The hydrolysis of sorghum straw using
hydrochloric acid has been investigated to produce xy-
lose-rich hydrolysates that can be bioconverted into xyli-
tol [45].
The reactivity of the three hydroxyl groups at the C2,
C3, and C6 positions of the D-glucosyl units of cellulose
oer a variety of possibilities for making useful deriva-
tives. In most cases, the desired properties are achieved by
producing a partially substituted derivative. Thus, the
Figure 7 Cellulose esterication using acids, anhydrides, or homopolymer is transformed into a copolymer of up to
acid chlorides. eight monomers randomly distributed along the polymer
chain. The description and characterization of polymers
of such complexity have been challenging problems of
long-standing interest [46].
The lack of solubility of native cellulose in water and
sion of the reducing end of the cellulose molecule releasing most organic solvent systems constitutes a major obstacle
D-glucoisosaccharinic acids via a h-alkoxycarbonyl elimi- for its utilization in many industrial applications. Chemical
nation of the rest of the cellulose chain (Fig. 6) [43]. This modication of cellulose was therefore initially performed
generates a new deprotonated reducing end group that to overcome this insolubility. An enormous number of cel-
undergoes further alkaline degradation, and so on. If this lulose derivatives have been synthesized and characterized
erosion of cellulose molecules from their reducing ends over the years [47]. The main classes of commercially pro-
were to continue unchecked, the whole of the cellulosic ma- duced cellulose derivatives are cellulose esters and cellu-
terial would eventually break down; however, a competing lose ethers. Esterication is often performed using organic
reaction, namely, h-hydroxycarbonyl elimination, occurs acids, anhydrides, or acid chlorides [48,156]. A general re-
at a slower rate producing terminal D-glucometasaccha- action scheme is provided in Fig. 7. The two major meth-
rinic acid end groups (Fig. 6). At temperatures >170jC, odologies utilized for the production of cellulose ethers,
random alkaline scission (hydrolysis of cellulose glycosidic namely, Williamson etherication and alkaline-catalyzed
linkages) occurs, generating new reducing end groups that oxalkylation [49], are presented in Fig. 8. Industrial modi-
can undergo peeling [34]. cation processes normally take place in heterogeneous
Figure 8 Cellulose etherication: (a) alkali treatment, (b) Williamson etherication, and (c) oxalkylation.
systems; that is, both the cellulose substrate and the pro- tinues to be the relatively slow rates of hydrolysis and
duced derivative are present as solids, either as dry matter the high cost of the enzymes [56]. Research eorts are
or suspended in the reaction medium. Thorough mixing/ therefore being directed toward understanding and mani-
stirring is often required to ensure uniform swelling, alkali pulating cellulase systems to achieve greater cellulase ac-
distribution, and production of a homogeneous product tivity, through optimizing cellulase mixtures and protein
(with respect to random distribution of added function- engineering of cellulases [71]. Optimal combination of cel-
ality). Nonrandom derivatization can result in poor prod- lulases should lead to increased rates and extents of hydrol-
uct solubility due to underivatized regions in the nal ysis since synergistic mechanisms are observed in several
product. Information on the applications of specic deriv- cellulase systems. The fementability of municipal waste
atives and products is provided in Section IV.A of this hemicelluloses (from teak, banana stalks, and sugarcane
chapter. bagasse) in the presence of Clostridium sp. have been
evaluated [72].
B. Biochemical Modification (Enzymatic Monosaccharides produced by cellulose degradation
Hydrolysis, Biodegradation, and Fermentation) can be treated with a variety of other enzymes leading to a
whole host of potential products, such as glucose and
Considerable research and development has been directed fructose syrups. Fructose syrups can be produced by
toward understanding and commercializing the enzymatic treatment of glucose with glucose isomerase [73]. The
hydrolysis of wood and cellulose, ranging from applied commercial production of fructose syrups was rst started
work on bioreactors to fundamental focusing on the in Japan. Such syrups are currently of great research
detailed molecular mechanisms of biological degradation interest, particularly in the development of fat substitutes
[5059]. However, cellulose requires an enzyme complex, and in the development of replacements for sucrose in
which acts in a synergistic manner to be totally hydrolyzed foods and beverages. These syrups could also be used as
to D-glucose. The reactivity of cellulose in the enzymatic is for animal feeds or as a chemical feedstock for enzyme/
hydrolysis depends on its physicochemical and structural fermentation processes, or they could be converted by
parameters [60,61]. Physicochemical parameters of cellu- other methodologies outlined in this chapter to produce
lose change during the course of enzymatic hydrolysis, and other more rened, higher-value chemicals.
this aects the kinetics of the process. An important Anaerobic digestion is an important phase of sewage
structural feature that aects the rate of enzymatic hydro- treatment in the developed world, and sewage gas (impure
lysis of cellulose bers is the degree of crystallinity of the methane) is often used for electricity production. Anaero-
cellulose. Cellulolytic enzymes are capable of degrading the bic digestion is now being applied increasingly to process
more readily accessible amorphous portion of cellulose, biowastes, particularly from farms and food factories [74].
but are unable to attack the less accessible crystalline In developing countries, especially India and China, many
portions. Studies on cellulose pretreatment have provided small digesters exist, and the biogas from them is com-
considerable insights into the inuence of crystallinity monly used for heating and sometimes for power genera-
and specic surface area on the rate and extent of hydro- tion. Large amounts of impure methane also arise from
lysis [56]. biological decay of domestic and commercial wastes that
As mentioned previously, the most common method are landlled. Landll gas is being utilized on a growing
of converting cellulosics to useful products is a straight- scale, and electricity production is an increasingly popular
forward hydrolysis to component monosaccharides. The method for exploiting the energy in that gas that would
advantage of carrying out this procedure enzymically using otherwise be wasted. It also provides a safe way of reducing
either a single enzyme, or a cocktail of cellulose degrading the danger of explosions and pollution by landll gas.
enzymes, is that the conditions are specic and nonde- Denmark has led the way in the development of bio-
structive (unlike acid/alkaline hydrolysis), and hence the gas plants and has over 20 large centralized gas plants
product can be tailored to suit the requirements of the [75]. They are an important part in the Danish energy
customer. Additionally the milder conditions are far less policy of reducing CO2 emissions by 20% by 2005, and
likely to result in the formation of harmful by-products. utilize admixed industrial organic wastes and manure
The group of enzymes that act on cellulose are collec- as substrate.
tively named cellulases. They are produced by a number of
bacteria and fungi, the most widely studied coming from
Trichoderma and Clostridium species [6265]. One of the C. Thermochemical Modification (Gasification/
most ecient decomposers of wood and other natural Combustion/Pyrolysis)
lignocellulose is white rot fungi, which performs enzymatic
hydrolysis of cellulose and oxidative breakdown of lignin Biomass is already being used, without preliminary con-
[6669]. version, as a fuel for direct combustion to raise steam and
Commercial preparations of cellulases are becoming hence to generate electricity. Wood, agricultural and for-
more widely available on an industrial scale, and many estry wastes, and domestic and commercial wastes can be
have been evaluated for their suitability for conversion of cited as examples of fuels utilized in this way. Direct
biomass [70]. However, the major drawback to commer- combustion is well suited to the use of biomass having
cialization of enzymatic cellulose hydrolysis processes con- fairly low contents of moisture and ash. The design of the
grate and boiler determines the completeness of combus- small-scale technologies (below 1 MW). Such systems
tion and eciency of heat transfer. Improved designs aim require good insulation since the poorer the insulation,
to improve eciency and to decrease noxious emissions. the more the reactor behaves like a stove, and the more CO2
Other improvements include methods for using waste heat and H2O are produced at the expense of reduced outputs of
to partially dry the feedstock. Many dierent kinds of CO and H2. Biomass is fed continuously so that the bed
biomass can be handled, and the combustion performance level in the gasier remains within certain limits. Producer
of eucalypt Eucalyptus nitens and aluminosilicate-rich bark gas is continuously sucked o at the bottom below a grate,
residue have been compared [76]. usually by the engine that is fuelled by this gas. Continuous
Gasication is the thermochemical conversion of or- removal of producer gas causes air to be continuously
ganic material into useful gases. Biomass can be reacted sucked into the bed (Fig. 9). Around the air entrances
with oxygen to produce mainly carbon monoxide and rapid, exothermic, partial combustion occurs at temper-
hydrogen, which can be used as a fuel or a chemical atures >1000jC, causing char deposition below the air inlet
feedstock. In principle, the technology is not much dierent (5). Gasication takes place below the air inlet [endother-
from combustion, in which organic material also reacts mic reactions (6) and (7)] [7881].
with oxygen, although in combustion the main products
are carbon dioxide and water. Whether a process results in 2C6 H10 O5 O2 ! 11C CO2 10H2 O 5
gasication or combustion depends on the air to fuel ratio.
Biomass (in the form of anhydrocellulose, C6H10O5) can C CO2 ! 2CO 6
undergo combustion (1) or gasication (2). In the combus-
tion reaction, much more heat is given o (17.5 MJ/kg) C H2 O ! CO H2 7
than in the gasication reaction (1.85 MJ/kg). Whereas
If wood is the feedstock, the heat produced in the
combustion converts a chemical feedstock into heat, gas-
combustion zone is used to dry it in the zone above the air
ication converts a solid carrier of chemical energy into a
inlet and to drive the endothermic gasication reactions.
gaseous carrier. The gas obtained from gasication can be
The wetter the wood and the greater the heat loss, the less
combusted in a second step resulting in the liberation of the
heat remains for gasication, and hence the lower the
chemical energy of the gas in the form of heat [reactions, (3)
heating value of the gas produced. The moisture content
and (4)].
of the wood should be <25%. This technology has found
2C6 H10 O5 12O2 ! 12CO2 application on a scale of a few megawatts, or less, making it
1 economically feasible only in developing countries, where
10H2 O combustion these reactors are found in signicant numbers.
2C6 H10 O5 O2 ! 12CO 10H2 (gasication) 2
2CO O2 ! 2CO2 3
2H2 O2 ! 2H2 O 4
With respect to production of electricity via biomass
conversion, gasication followed by combustion is pre-
ferred to direct combustion for several reasons. A gas has
signicantly better burning properties than a solid, there-
fore process control is easier, hardly any excess of air is
necessary, simple burners can be used, and there are no
particulate emissions and less gaseous pollutants. In addi-
tion, the gas obtained from gasication can be directly
converted to shaft power by combustion in a gas turbine,
gas, or duel-fuel diesel engine [77]. This avoids the less
ecient indirect steam cycle that is generally used with the
combustion of solids to transfer the heat from the com-
bustion gases via a steam turbine into shaft power to drive
the electricity generator. Therefore, electricity plants based
on gasication have higher overall eciencies than those
based on combustion, particularly at the relatively low
capacities relevant in biomass conversion technology (typ-
ically below 100 MWe). In these circumstances, even taking
into account the capital and running costs of a gasication
plant, the economics of electricity production of biomass
via gasication are believed to be more favorable than
those of systems using direct combustion.
Downdraft xed and moving bed gasication, using
air as the gasifying agent, are the most widely applied Figure 9 Overview of downdraft gasication.
In the developed world, electricity from biomass plete absence of oxidizing agent, or with such a limited
plants typically will have capacities in the range of 10 supply that gasication does not occur to an appreciable
100 MWe. For such capacities, conventional uidized beds, extent. Slow pyrolysis at low temperatures and long reac-
and circulating uidized beds are the most attractive tion times maximizes charcoal yields at about 30% w/w,
options [82]. In a circulating uid bed, small particles of comprising about 50% of the energy content of the feed-
biomass (typically a few millimeters in diameter) are con- stock. Flash pyrolysis at relatively low temperatures (typ-
tinuously fed to the gasier by a screw feeder. Air or oxygen ically 500jC, but not more than 750jC) and at very high
is fed as the gasifying agent at the bottom. The bed tem- reaction rates and short residence times of typically less
perature (typically 820jC) and the gas composition are than 1 sec, maximizes liquid yields at up to 80% w/w. Flash
highly inuenced by the oxygen/fuel ratio and fuel mois- pyrolysis at relatively high temperatures (above 700jC)
ture content. Fluidized beds are operated under pressure, maximizes gas yields at up to 80% w/w. Conventional
typically between 10 and 30 bar. Research has been car- pyrolysis at moderate temperatures of <600jC and mod-
ried out into more advanced processes, e.g., oxygen and erate reaction rates gives approximately equal propor-
steam gasication. Other specic types of gasier reactors tions of gas, liquid, and solid products [81,84]. The options
include updraft xed-bed, bubbling uid bed, entrained for biomass to bioenergy conversions are summarized in
ow, and twin uid bed reactors [78,80,81]. Straw is a Fig. 10.
potential substrate for use in a 25-MW power-generation A comprehensive survey of fast pyrolysis processes has
plant [83]. been published that describes all the pyrolysis processes for
Pyrolysis is the technology for the conversion of bio- liquids production that have been built and tested in the
mass into gas, liquid, and charthe relative proportions last 1015 years [85]. These include bubbling uid bed,
depending on the pyrolysis method and reaction param- circulating uid and transported beds, ablative pyrolysis,
eters. Fast or ash pyrolysis is used to maximize either entrained ow, rotating cone, and vacuum pyrolysis reac-
gas or liquid products according to the temperature em- tors. Pyrolysis is an economically viable route for power
ployed. Pyrolysis is thermal degradation, either in the com- generation that deserves critical attention [86,87].
Figure 10 Biomass to bioenergy conversion routes.

The liquid product of pyrolysis is a very complex sweep gas ow rate, and particle size on the yield and
mixture of oxygenated hydrocarbons, the composition of composition of solid product char [95]. A wide range of
which is determined intrinsically by the rate of reaction and thermal processes have been evaluated utilizing a variety of
product quenching, and extrinsically by the feed composi- biomass substrates [96].
tion. The complexity arises from the relatively uncontrolled
degradation of lignin, producing a broad spectrum of
phenolics and similar types of compounds that interreact. IV. DERIVED PRODUCTS AND APPLICATIONS
The liquid is referred to as oil, bio-oil or bio-crude-oil. It A. Cellulosic Derivatives (Ethers/Esters)
can be upgraded to liquid hydrocarbon fuels. There are two
types of liquids produced by pyrolysis of biomass, primary Cellulose derivatives are among the oldest industrially
bio-oil from ash pyrolysis processes and secondary oil or exploited substances. Derivatives such as nitrocellulose
tar from conventional or slow pyrolysis processes, as well (guncotton, collodium), cellulose ethers (e.g., methylcellu-
as by-products from gasication. Most attention is focused lose and carboxymethylcellulose), cellulose esters (e.g.,
on ash pyrolysis liquids because of their much higher cellulose acetate and mixed organic cellulose esters) have
yields and superior properties. been produced on an industrial scale for many years [97].
Oils produced by the pyrolysis of biomass are attrac- A variety of cellulosic ethers has been synthesized and
tive in environmental terms when compared to fuels de- have broad-ranging industrial applications [98100]. For
rived from petroleum, especially because their combustion example, they can be used in paint formulations and have
does not increase the carbon dioxide content of the atmo- found a number of specic applications in the ceramic
sphere, and because of their low sulfur content. However, materials industry [101,102]. Specic ether derivatives of
the crude oil products of pyrolysis from biomass have industrial importance include carboxymethylcellulose
several limitations. They have a high oxygen content (CMC), cyanoethylcellulose (CEC), ethylcellulose (EC),
(around 30% w/w), and they are unstable: solidication methylcellulose (MC), hydroxyethylcellulose (HEC), hy-
occurs when they are heated to 100 to 200jC, so they droxypropylcellulose (HPC), and mixed ethers such as
cannot be fractionated by distillation. They are not com- hydroxypropylmethylcellulose (HPMC), carboxymethyl-
pletely volatile and are poorly miscible with hydrocarbon hydroxyethylcellulose (CMHEC), and hydroxyethylmeth-
solvents. They contain dissolved water (up to 30% w/w). ylcellulose (HEMC) [103]. An overview of commercially
They are relatively viscous and are often acidic, thus produced cellulose ethers is provided in Table 1 [104].
presenting corrosion problems. Carboxymethylation of cellulose produces a range of
Liquid fuels have been produced from beech wood cold water soluble products, having high viscosity and
(Fagus orientalis), hazelnut shell (Corylus avellana), olive polyelectrolyte behavior (due to its anionic nature). CMC
husk wastes, cotton cocoon shell, and tobacco stalk by is used as a thickener in paints, wallpaper adhesives, oil well
pyrolysis and catalytic liquefaction [8893]. Steam pyroly- drilling muds, etc., and has shown potential in pharmaceu-
sis of nut shells and fruit stones and seeds has been used to tical applications (as a tablet disintegrant) and in deter-
produce gas and tar products, the latter of which have been gents (as an antiredeposition agent). HEC and HPC are
used to obtain carbon adsorbents [94]. Carbonization produced using ethylene and propylene oxides, respective-
experiments have also been performed on sugarcane ba- ly. HEC is a nonionic polymer that is compatible with a
gasse in a static, xed bed reactor to determine the eect of wide range of other water-soluble polymers. Solutions of
process variables such as temperature, heating rate, inert HEC exhibit strong pseudoplastic ow particularly at
Table 1 Commercially Produced Cellulose Ethers

Ether derivative Derivatizing reagent Solubility DS
Sodium carboxymethyl- Chloroacetic acid Water 0.51.3

cellulose (NaCMC)
Methylcellulose (MC) Chloromethane Water 1.52.4
Hydroxypropyl Chloromethane + Water 1.52.0
methylcellulose (HPMC) propylene oxide
Hydroxyethyl Chloromethane + Water 1.52.0
methylcellulose (HEMC) ethylene oxide
Hydroxyethylcellulose (HEC) Ethylene oxide Water 1.32.5
Ethylcellulose (EC) Ethyl chloride Organic solvents 2.32.6
Ethylmethylcellulose (EMC) Ethyl chloride + Water 1.01.3
chloromethane
Cyanoethylcellulose (CEC) Acrylonitrile Organic solvents 0.52.0
Benzylcellulose Benzyl chloride Organic solvents 1.82.0
higher molecular weights and solution concentrations. HPC sulfate, and nitrate. Cellulose nitrate is used in plastics,
is more hydrophobic than HEC due to the presence of the lacquers, coatings, and explosives [105].
methyl group side chain. This is responsible for its good
solubility in polar organic solvents and its insolubility in water B. Fermentation Products (Alcohols, Organic
above 45jC. In solid form HPC is thermoplastic and can be Acids, and Others)
extruded or injection molded. HEC is used in coatings, ce-
ments and plasters, thickeners, emulsifying agents, antifog- The industrial utilization of sugars in hydrolysates and
ging agents, oil-well fracturing and drilling uids, fabric and sulte spent liquors is based mostly on fermentation [106].
paper nishes, wetting solutions, binding and dispersing Ethanol is the most common fermentation product of D-
agents, lms, inks, corrosion and scale inhibitors, toothpaste, glucose and hexose sugars, but it can also be produced from
lubricants, sealants, adhesives, etc. HPC is used in manufac- xylose by certain microorganisms [107]. The interest in
turing injection-molded articles, pharmaceutical and personal biotechnical ethanol production has increased dramati-
hygiene articles, controlled-release tablet formulations, lms, cally in the search for more economic production of fuels,
textile warp sizes, occulants, wetting agents, thickeners, alcoholic beverages, etc. [57,108113]. Ethanol made from
binders, absorbents, etc. [103]. lignocellulosic biomass sources provides unique environ-
MC and EC are produced by reacting the corre- mental, economic, and strategic benets. Advances in pre-
sponding alkyl halides with alkali cellulose (Fig. 8). MC treatment by acid-catalyzed hemicellulose hydrolysis and
undergoes reversible gel formation in aqueous solution on enzymes for cellulose breakdown coupled with develop-
heating; many of its applications are based on its gelling ment of genetically engineered bacteria that ferment all ve
ability, and it is approved for food use. EC is thermoplastic, sugars in biomass to ethanol at high yields have been the
has considerable strength and exibility compared to other key to reducing costs [114]. Through continued advances
cellulose derivatives, and is used in lms, lacquers, adhe- in accessing the cellulose and hemicellulose fractions, the
sives, binders, and hot melts. CEC is prepared by the cost of biomass ethanol can be reduced to the point at
reaction of acrylonitrile with alkali cellulose. Low-DS which it is competitive as a pure fuel without subsidies (and
(0.30.5) CEC is resistant to mold and microbial attack not only viewed as potentially competitive for blending
and has been used in textiles [103]. with gasoline).
A variety of cellulose esters has been produced indus- Fermentation of cellulose-derived single sugars using a
trially. Cellulose acetate (CA) is recognized as the most commercial strain of yeast that can tolerate a high ethanol
important organic ester of cellulose due to its extensive concentration (ca. 40% yields are producible) has vast
applications in bers, plastics and coatings. CA is pro- potential. In the future fuel gasohols, as they are known,
duced by reacting high-purity cellulose with acetic anhy- are likely to be in great demand. Many governments (e.g.,
dride, using acetic acid as the solvent and sulfuric acid as United States and Brazil) currently provide tax incentives
catalyst. The applications of such derivatives rely on for gasohol production. It is likely that with diminishing
solubility in certain solvent systems, which is often linked fossil fuel reserves, other governments will take similar
to DS. Table 2 shows the DS, solubility, and associated action. Fermentation of biomass for the production of
applications of CA [9]. Other commercialized aliphatic alcohol for vehicle fuel is an important industry in Brazil,
cellulose esters include cellulose formate, propionate, bu- Zimbabwe and the United StatesBrazil from sugarcane,
tyrate, and mixed esters (e.g., acetatepropionate, acetate the United States from maize.
butyrate, etc). Low-viscosity cellulose propionate has been An example of a hydrolytic process for ethanol pro-
used for the production of lms with low odor, high duction from wood chips by fermentation of sugars is
melting point, and excellent surface hardness. Other sig- shown in Fig. 11. Alcohol has been produced from grain,
nicant cellulose esters include carbamates, sulfonates, molasses, whey, brushwood, other cellulosics, and a variety
of waste materials [111,115]. Pineapple peel waste has also
been evaluated as a substrate for ethanol production [116].
Very ecient mutants of Trichoderma reesei have been
developed and used in biofuel production [69,117,118].
Municipal solid waste, sewage sludge, industrial bio-
Table 2 Cellulose Acetate Applications as a Function of DS
sludge, manure, and agricultural residues have been con-
and Solubility
verted to a mixture of alcohols (predominantly 2-propanol,
DS Solubility Applications but also higher alcohols up to 7-tridecanol) using the
MixAlco process, which involves fermentation using a
1.81.9 Waterpropanolchloroform Films mixed culture of microorganisms to produce carboxylic
2.22.3 Acetone Lacquers acids, which are converted to carboxylate salts, concen-
2.32.4 Acetone Acetate silk trated, dried, thermally converted to ketones, and nally
2.52.6 Acetone Films, plastics
hydrogenated to alcohols [119].
2.82.9 Methylene chlorideethanol Plastics
Xylitol has been produced by bioconversion of xylose-
2.93.0 Methylene chloride Movie lm,
containing hydrolysates (obtained mainly from eucalyptus
wire insulation
wood) using Debaryomyces hansenii yeast strains [120].
Figure 11 Overview of the production of ethanol by fermentation of sugars obtained from wood chips by acid hydrolysis.
Glycerol has been produced by fermentation of cane sugar into account, nearly all metabolites from biomass can be
molasses and wheat milling residues (akalona) using Sac- considered not just as biochemicals but as chiral and
charomyces cerevisiae [121]. multifunctional speciality chemicals, which may serve as
Enzymic and microbial processes can lead to the convenient starting materials in organic syntheses. The
formation of several organic acids [122]. Of these, conver- demand for bioderived synthons will further increase in
sions of cellulose-derived sugars to citric and gluconic acids the future, when chemists become more aware of the
are by far the most commercially viable. Citric acid (and its potentials of enzyme-catalyzed reactions in organic syn-
salts) has properties as natural acidulants, as acid and pH thesis [124]. An indication of some of the compounds that
buers, and in the formation of metal complexes. Some can be produced by fermentation of the glucose obtained
80% of citric acid is used in the food industry where it acts by hydrolysis of cellulosic materials is shown in Figs. 12
as a pH stabilizer, a chelating agent, a avor enhancer, or as and 13.
a synergist with antioxidants. Of the remaining 20% of Growth factors, vitamins, antibiotics, and nucleotides
citric acid, 10% is used in pharmaceutical or cosmetic are all potentially producible using current fermentation
industries for drug delivery, avoring, and as an ingredient technology. With regard to antibiotics, oxytetracycline,
in anticoagulant solutions. The remaining 10% is utilized
in the industrial sector where it acts in metal cleaning, metal
deposition, ion sequestering, and as a cement additive
where it lengthens set time and reduces water requirements.
Gluconic acid and its sodium salt are used as seques-
tering agents to prevent soap lm formation in alkaline
cleaning formations for glassware and food processing
equipment. Sodium gluconate is used for the cleaning,
derusting, and paint stripping of metals. It is also added
to concrete where it lengthens set time and enhances
strength. Glucono-y-lactone is used in drying, textile print-
ing, and in baking powder. Calcium gluconate has found
success in the treatment of calcium deciency in both
humans and animals. It may also be possible to produce
organic acids at commercially viable levels from lower-
purity syrups.
Applied organic chemistry is shifting more and more
toward the synthesis of biologically active, multifunction-
al, and chiral compounds [123,124]. Taking such progress Figure 12 Compounds obtained from glucose fermentation.
Figure 13 Examples of the synthetic methodologies that can be used to convert cellulosic-derived glucose into other useful
products.
streptomycin, and cephalosporins are currently prepared nuclease from Penicillium citrinum. Production by fermen-
from noncarbohydrate materials, but technical expertise tation utilizes mutants of Brevibacterium ammoniagenesis.
exists for their production from cellulose-derived carbohy- Solid-state fermentation (SSF) using sugarcane ba-
drates if this decision is warranted economically. The gasse as the substrate has been used for the production of
preferred substrates for these processes are high dextrose protein-enriched feed, enzymes, amino acids, organic
equivalent cellulose hydrolysates (DE95) because of acids, and other compounds of pharmaceutical impor-
their lower viscosity and higher productivity than lower tance [126]. SSF closely resembles the natural way of life
DE samples. of lamentous fungi, and has been used to produce
Vitamin B12 is produced commercially by the fermen- biofuels, enzymes, animal feeds, biofertilisers, biopesti-
tation of glucose using Pseudomonas or Propionibacterium cides, biopromoters, and secondary metabolites [127].
strains. Mutants of the former are preferred for yield Vegetable wastes have been used as substrates in a range
reasons. Some 30% of Vitamin B2 currently produced is of SSF processes for the production of a wide range
from fermentation. Vitamin C is produced from glucose. of higher-value products, including avors and bione
Glucose is reduced to sorbitol, sorbitol is transformed to chemicals [128].
L-sorbose, and then L-sorbose is chemically transformed to
Vitamin C. An alternative route is chemical transformation C. Recycled Paper and Biodegradable Products
of 2-ketogulonic acid obtained from glucose by direct
fermentation [125]. Antibiotic synthesis (Antisomycin Most processed cellulose is used for the production of
and Pentenomycin) from cellulose breakdown products is cellulose pulp and paper. However, trees specically grown
also potentially viable. and harvested for this purpose are not the only sources of
The production of nucleotides and their derivatives by such cellulosic material, since cellulosic bers suitable for
microbial means using cellulose breakdown products as a the production of a wide range of paper grades can come
substrate shows vast potential. 5V-Inosine monophosphate from many agro-based resources and waste biomass
(IMP) and 5V-guanosine monophosphate (GMP) exhibit [129]. Cellulose pulp (and paper) are produced from many
strong avor-enhancing properties. IMP and GMP are sources, such as bagasse, bamboo, cotton, eucalyptus, ax,
produced by the enzymic hydrolysis of RNA using a hemp, jute/sisal, kenaf, straw, etc. [130].
Used scrap paper is a valuable resource and is a and synthetic polymers have been on the market for some
primary component of biowaste, with cellulose being its time [142]. Wood bers have been utilized in the produc-
dominant structural biopolymer. Such paper materials can tion of a number of thermoplastic composite materials
be recycled [131], or can be treated with cellulose hydro- [143]. However, in most of these cases, the biopolymers are
lyzing enzymes to bioconvert their cellulose component just lling materials. The properties of the new materials
into sugars such as glucose, which can be utilized as feed- are more or less those of the synthetic polymer, somewhat
stock for the synthesis of biodegradable products [109,132]. changed and often weakened by the presence of the ller. In
Dierent paper materials exhibit nonsimilar susceptibil- many cases, the attempted microbial degradation of the
ities toward cellulase preparations and consequently dif- biological components is hampered because it is isolated
ferent sugar releasing and bioconversion eciency patterns from the environment by the plastic layer. Some other
are observed with dierent grades and quantities of paper cellulose derivatives produce liquid crystal polymers,
[133,134]. which are of interest as speciality products in high-per-
Biopolymers can serve as promising raw materials for formance engineering plastics [144].
new biodegradable plastics [135]. As with starch, cellulose
is currently being put forward as a source material for use D. Membranes
in biodegradable materials:
Cellulose and its derivatives (such as ethers and esters)
1. Lactic acid, produced from fermentation of cel- possess excellent lm forming properties [105]. For this
lulose, has found use in the production of poly- reason, they have found utilization in membrane formation
mers. Currently, their use is limited to the medical science since the initiation of that eld of research [145].
eld, but improvements could lead to more wide- Ultraltration membranes for example are cast from cel-
spread use. lulose nitrate. Other cellulose membranes have found
2. Granular cellulose has been used as ller in various utilization in packaging (e.g., cellophane), food, farming,
plastics, having utilization potential in both pack- chemical industries, and in medicine [146,147].
aging and containers. Gelatinized (repasted)
cellulose has been shown to form a continuous
phase with the synthetic polymer as opposed to
being only ller. This discovery could lead to many V. CONCLUSIONS
commercially protable products.
The vast potential of cellulosic materials as a source of
3. Foams (for use in packing, re extinguishers, beer
high-quality, high-value products is only now being real-
froths, etc.) can be produced from both cellulose
ized. In a world that is becoming only too aware of its ever
and starch [136].
depleting stock of natural resources the waste of such
4. Grafting of cellulose onto a synthetic polymer has
valuable natural materials is viewed by the general public
been performed [137]. These cellulose graft co-
as nothing short of environmental suicide. However, de-
polymers have only yet been used as super-
spite all the advances in biotechnology, technical possibil-
absorbants, although they are under investigation
ities do not guarantee economic feasibility. Switching from
as potentially biodegradable (or semibiodegrad-
a petrol-based industry to a biomass-based one requires
able) plastics.
much extensive research, development and, not least, the
It should be noted that biodegradability begins at modication of existing or the construction of new plants.
100% with pure cellulose, and decreases as nondigestible A huge number of petrochemically derived compounds can
materials are blended or compounded with that cellulose. be produced from biomass routes (Fig. 14).
Therefore, some biodegradable plastics are often only Most of the obvious advantages of biomass utilization
partially degraded biologically. Most biopolymers do not cannot be directly converted into prot by manufacturers.
provide properties that have come to be expected from a A decrease in ecological damage, although for the benet
useful plastic material. They are neither thermostable nor of the community at large, is hardly benecial to the
elastic and, therefore, are dicult to process. They are short-term prot goals of many companies. Therefore the
sometimes even water soluble and are usually too unstable production of biobased chemicals is generally of little
for long-term applications (hence their interest in the eld commercial attraction, the majority of the chemical indus-
of biodegradable plastics). However, by slight chemical tries having adopted a wait-and-see policy. This leaves
derivatization, some biopolymers develop very interesting the burden of fundamental research and development into
and useful properties, e.g., the acetylation of cellulose, methods that can enhance the viability of commercial
which leads to the synthetic ber rayon [138]. treatment of biobased materials on an industrial scale in
As mentioned above, one approach to the utilization the hands of the academic scientic community. On a
of biopolymers in plastics is the combination of synthetic planet with diminishing resources the success of this global
petrol-based materials with natural polysaccharides project is a challenge that we can ill aord to lose.
[139,140]. A low-density board has been developed, con- Many alternative energies are seeing an increasing
sisting of wood pulp, glass ber, and polyethylene [141]. market share as their technologies and implementations
Some moldable woodplastic composites from wood meals improve, and their costs decline. However, this reects
Figure 14 Chemical products from petrochemical and biomass routes.

improvements in the characteristics of the technologies in 11. Polman, K. Review and analysis of renewable feedstocks
question. They are not guaranteed a market, since fossil- for the production of commodity chemicals. Appl.
Biochem. Biotechnol. 1994, 4546, 709722.
fuel-based industries will continue to dominate for the
12. Overend, R.P., Chornet, E., Eds. Biomass: a growth
foreseeable future, particularly in the developed world. opportunity in green energy and value-added products.
Their greatest potential for dramatic impart therefore lies Elsevier: Oxford, 1999.
in the Third World, where they can have a signicant 13. Pari, L. Energy production from biomass: The case of Italy.
impact on energy/fuel supply by reducing the need for Renew. Energy 2001, 22, 2130.
high-cost energy importation [148]. 14. Kruse, A.; Gawlik, A. Biomass conversion in water at 330
Environmental issues should ultimately be the driving 410 degrees C and 3050 MPa. Identication of key
compounds for indicating dierent chemical reaction
force for the successful development of economically viable pathways. Ind. Eng. Chem. Res. 2003, 42 (2), 267279.
uses of renewable energy sources. Concerns regarding 15. Krassig, D.H. Structure of cellulose and its relation to
dramatic fossil fuel depletion and the potential political properties of cellulose bers. In Cellulose and Its Deriva-
instability of many oil-producing countries have been tives: Chemistry, Biochemistry and Applications; Kennedy,
continuously raised since the 1970s and most predictions J.F., Phillips, G.O., Williams, P.A., Eds.; Ellis Horwood:
regarding petroleum supply, future oil process, resource Chichester, 1985; 326.
16. Betts, W.B.; Dart, R.K.; Ball, A.S.; Pedlar, S.L. Biosyn-
scarcity, etc., have proven to be extremely inaccurate [149]. thesis and structure of lignocellulose. In Biodegradation:
Public awareness and understanding of biomass utili- Natural and Synthetic Materials; Betts, W.B., Ed.;
zation is much poorer than other areas of bioenergy Springer-Verlag: Berlin, 1991; 139155.
production (such as wind, solar and tidal energy utiliza- 17. Saka, S. Structure and chemical composition of wood as a
tion). More emphasis needs to be placed on education of natural composite material. In Recent Research on Wood
the public in such technologies and education of young and Wood-based Materials, Current Japanese Materials
Research; Shiraishi, N., Kajita, H., Norimoto, M., Eds.;
people at school and university level so that future gen-
Elsevier Applied Science: London, 1993; Vol. 11, 120.
erations can rise to the challenge of renewable resource 18. Bisaria, V.S. Bioprocessing of agro-residues to glucose and
utilization for sustainable development [150155]. chemicals. In Bioconversion of Waste Materials to Industrial
Products; Martin, A.M., Ed.; Elsevier: London, 1991; 187
223.
REFERENCES 19. Chahal, S. Lignocellulosic wastes: Biological conversion.
In Bioconversion of Waste Materials to Industrial Products;
1. Sayigh, A. Renewable energyThe way forward. Appl. Martin, A.M., Ed.; Elsevier: London, 1991; 373400.
Energy 1999, 64, 1530. 20. Kennedy, J.F.; Melo, E.H.M. Bioconversions of cellu-
2. Doerell, P.E. All future energy will have to be clean. loseA major source of material for the biochemical
Appl. Energy 1999, 64, 7988. industry. Br. Polym. J. 1990, 23, 193198.
3. Kaygusuz, K. Renewable energy: Power for a sustainable 21. Tarchevsky, I.A.; Marchenko, G.N. Cellulose: Biosyn-
future. Energy Explor. Exploit. 2001, 19 (6), 603626. thesis and Structure. Springer-Verlag: Berlin, 1991.
4. Kaygusuz, K. Renewable energy sources: The key to a 22. Krassig, H.A. Cellulose: Structure, Accessibility and
better future. Energy Sources 2002, 24 (8), 787799. Reactivity. Gordon & Breach: Yverdon, 1993.
5. Ackermann, T.; Andersson, G.; Soder, L. Overview of 23. Kroon-Batenburg, L.M.J.; Kroon, J. The crystal and
government and market driven programs for the promo- molecular structures of cellulose. Carbohydr. Eur. 1995,
tion of renewable power generation. Renew. Energy 2001, 12, 1519.
22, 197204. 24. Lipinsky, E.S. Chemicals from biomassPetrochemical
6. Meshitsuka, G. Utilisation of wood and cellulose for substitution options. Science 1981, 212, 14651471.
chemicals and energy. In Wood and Cellulosic Chemistry; 25. Gosse, G.; Mitchell, P. Identication of suitable crops,
Hon, D.N.-S., Shiraishi, K., Eds.; Marcel Dekker: New production and harvesting. In Electricity from Biomass;
York, 1991; 9771013. Grassi, G., Trebbi, G., Pike, D.C., Eds.; CPL Press: New-
7. Narayan, R. Biomass (renewable) resources for production bury, 1992; 27.
of materials, chemicals and fuels. In Emerging Technologies 26. Bowen, I.J.; Hsu, J.C.L. Overview of emerging technolo-
for Materials and Chemicals from Biomass: ACS Sympo- gies in pulping and bleaching. Tappi J. 1990, 73, 205217.
sium Series 476; Rowell, R.M., Schultz, T.P., Narayan, R. 27. Phillips, G.O.; Meadows, J. Wood and cellulose science
Eds.; American Chemical Society: Washington, DC, 1992; and technology in Europe. In Wood Processing and
110. Utilisation; Kennedy, J.F., Phillips, G.O., Williams, P.A.,
8. Grohmann, K.; Wyman, C.E.; Himmel, M.E. Potential for Eds.; Ellis Horwood: Chichester, 1989; 328.
fuels from biomass and wastes. In Emerging Technologies 28. Shimizu, K.; Sudo, K.; Ono, H.; Fujii, T. Total utilisation
for Materials and Chemicals from Biomass: ACS Sympo- of wood components by steam explosion pretreatment. In
sium Series 476; Rowell, R.M., Schultz, T.P., Narayan, R., Wood Processing and Utilisation; Kennedy, J.F., Phillips,
Eds.; American Chemical Society: Washington, DC, 1992; G.O., Williams, P.A., Eds.; Ellis Horwood: Chichester,
354392. 1989; 407412.
9. Blazej, A.; Kos k, M. Phytomass: A Raw Material for 29. Tanaka, M.; Matsuno, R.; Converse, A.O. n-Butylamine
Chemistry and Biotechnology; Ellis Horwood: Chichester, and acidsteam implosion pretreatment of rice-straw and
1993. hardwood: Eects on substrate structure and enzymatic
10. Blazej, A.; Kos k, M. Environmentally acceptable con- hydrolysis. Enzyme Microb. Technol. 1990, 12, 190195.
version technology for the biochemical utilisation of 30. Meadows, J.; Vignon, M.R.; Phillips, G.O.; Rinaudo, M.
lignocellulosics. In Cellulosics: Pulp, Fibre and Environ- The applicability of the steam explosion process to achieve
mental Aspects; Kennedy, J.F., Phillips, G.O., Williams, separation of the major polymeric components of spruce
P.A., Eds.; Ellis Horwood: Chichester, 1993; 355364. softwood. In Cellulose Sources and Exploitation: Industrial
Utilisation, Biotechnology, and Physico-chemical Proper- tion and acetalization of cellulose in LiCl/DMAC. In
ties; Kennedy, J.F., Phillips, G.O., Williams, P.A., Eds.; Cellulose, Structural and Functional Aspects; Kennedy,
Ellis Horwood: Chichester, 1990; 443450. J.F., Phillips, G.O., Williams, P.A., Eds.; Ellis Horwood:
31. Focher, B., Marzetti, A., Crescenzi, V., Eds. Steam Chichester, 1989; 219224.
Explosion Techniques: Fundamentals and Industrial 49. Mondt, J.L. The use of cellulose derivatives in the paint and
Applications. Gordon & Breach: Reading, 1991. building industries. In Cellulose Sources and Exploitation;
32. Grethlein, H.E.; Converse, A.O. Common aspects of acid Kennedy, J.F., Phillips, G.O., Williams, P.A., Eds.; Ellis
prehydrolysis and steam explosion for pretreating wood. In Horwood: Chichester, 1990; 269278.
Enzymatic Hydrolysis of Cellulose; Walker, L.P., Wilson, 50. White, A.R. Visualisation of cellulases and cellulose
D.B., Eds.; Elsevier: London, 1991; 7782. degradation. In Cellulose and Other Natural Polymer
33. Kokta, B.V.; Ahmed, A.; Garceau, J.J.; Chen, R. Progress Systems: Biogenesis, Structure and Degradation; Brown,
of steam explosion pulping; an overview. In Ligno- R.M., Ed.; Plenum Press: New York, 1982; 489509.
cellulosics: Science, Technology, Development and Use; 51. Sagar, B.F. Mechanism of cellulase action. In Cellulose and
Kennedy, J.F., Phillips, G.O.,Williams, P.A., Eds.; Ellis Its Derivatives: Chemistry, Biochemistry and Applications;
Horwood: Chichester, 1992; 171212. Kennedy, J.F., Phillips, G.O., Williams, P.A., Eds.; Ellis
34. Nevell, T.P. Degradation of cellulose by acids, alkalis and Horwood: Chichester, 1985; 199210.
mechanical means. In Cellulose Chemistry and Its Applica- 52. Wood, T.M. Aspects of the biochemistry of cellulose
tions; Nevell, T.P., Zeronian, S.H., Eds.; Ellis Horwood: degradation. In Cellulose and Its Derivatives, Chemistry,
Chichester, 1985; 223242. Biochemistry and Applications; Kennedy, J.F. Phillips,
35. Lee, Y.Y.; Iyer, P.; Torget, R.W. Dilute-acid hydrolysis of G.O., Williams, P.A., Eds.; Ellis Horwood: Chichester,
lignocellulosic biomass. In Recent Progress in Bioconver- 1985; 173188.
sion of Lignocellulosics; Tsao, G.T., Ed.; Advances in 53. Finch, P.; Roberts, J.C. Enzymatic degradation of cellu-
Biochemical Engineering/Biotechnology. Springer-Verlag: lose. In Cellulose Chemistry and Its Applications; Nevell,
Berlin, 1999; Vol. 65, 93115. T.P., Zeronian, S.H., Eds.; Ellis Horwood: Chichester,
36. Saka, S.; Ueno, T. Chemical conversion of various 1985; 312343.
celluloses to glucose and its derivatives in supercritical 54. Jeries, T.W. Physical, chemical and biochemical consid-
water. Cellulose 1999, 6 (3), 177191. erations in the biological degradation of wood. In Wood
37. Philipp, B. Degradation of celluloseMechanisms and and CellulosicsIndustrial Utilisation, Biotechnology,
applications. Pure Appl. Chem. 1984, 56 (3), 391402. Structure and Properties; Kennedy, J.F., Phillips, G.O.,
38. Blazej, A.; Kos k, M.; Spilda, I. The degradation of Williams, P.A., Eds.; Ellis Horwood: Chichester, 1987;
cellulose by thermal, chemical and biochemical techniques. 213230.
In Cellulose Sources and Exploitation; Kennedy, J.F., 55. Puls, J.; Wood, T.M. The degradation pattern of cellulose
Phillips, G.O., Williams, P.A., Eds.; Ellis Horwood: by extracellular cellulases of aerobic and anaerobic micro-
Chichester, 1990; 385396. organisms. In Enzymatic Hydrolysis of Cellulose; Walker,
39. Blazej, A.; Kos k, M. Degradation reactions of cellulose L.P., Wilson, D.B., Eds.; Elsevier: London, 1991; 1520.
and lignocellulose. In Cellulose and Its Derivatives: 56. Walker, L.P.; Wilson, D.B. Enzymatic hydrolysis of cellu-
Chemistry, Biochemistry and Applications; Kennedy, J.F., lose: An overview. In Enzymatic Hydrolysis of Cellulose;
Phillips, G.O., Williams, P.A., Eds.; Ellis Horwood: Walker, L.P., Wilson, D.B., Eds.; Elsevier: London, 1991;
Chichester, 1985; 97117. 314.
40. Lai, Y.-Z. Chemical degradation. In Wood and Cellulosic 57. Coombs, J., Grassi, G., Eds. Cellulose Hydrolysis and
Chemistry; Hon, D.N.-S., Shiraishi, N., Eds.; Marcel Fermentation. Newbury: CPL Press, 1992.
Dekker: New York, 1991; 455523. 58. Elnasser, N.H.A.; Elshafei, H.A. Enzymatic hydrolysis of
41. Redfern, P.D. Alkaline Degradation of Carbohydrates. cellulose and related materials by streptomyces species.
Ph.D. thesis, University of Birmingham, UK, 1996. Microb. Res. 1994, 149 (2), 151156.
42. Klemm, D.; Philipp, B.; Heinze, T.; Heinze, U.; Wagen- 59. Beguin, P.; Aubert, J.P. The biological degradation of
knecht, W. Degradation of cellulose. In Comprehensive cellulose. FEMS Microbiol. Rev. 1994, 13 (1), 2528.
Cellulose Chemistry; Fundamentals and Analytical Meth- 60. Lee, Y.-H.; Fan, L.T. Kinetic studies of enzymatic-
ods; Wiley-VCH: Weinheim, 1998; Vol. 1, 83129. hydrolysis of insoluble celluloseAnalysis of the initial
43. Knill, C.J.; Kennedy, J.F. Degradation of cellulose under rates. Biotechnol. Bioeng. 1982, 24, 23832406.
alkaline conditions. Carbohydr. Polym. 2003, 51 (3), 281 61. Lee, S.B.; Shin, H.S.; Ryu, D.D.Y.; Mandels, M. Adsorp-
300. tion of cellulase on celluloseEect of physico-chemical
44. Alen, R.; Sjostrom, E. Degradative conversion of cellulose- properties of cellulose on adsorption and rate of hydrolysis.
containing materials into useful products. In Cellulose Biotechnol. Bioeng. 1982, 24, 21372153.
Chemistry and Its Applications; Nevell, T.P., Zeronian, 62. Linko, M.; Poutanen, K.; Viikari, L. New developments
S.H., Eds.; Ellis Horwood: Chichester, 1985; 530544. in the application of enzymes for biomass processing.
45. Herrera, A.; Tellez-Luis, S.J.; Ram rez, J.A.; Vazquez, M. In Enzyme Systems for Lignocellulose Degradation;
Production of xylose from sorghum straw using hydro- Coughlan, M.P., Ed.; Elsevier: London, 1989; 331346.
chloric acid. J. Cereal Sci. 2003, 37, 267274. 63. Esterbauer, H.; Steiner, W.; Labudova, I.; Herrman, A.;
46. Gelman, R.A. Characterisation of cellulose derivatives: Hayn, M. Production of Trichoderma cellulase in labo-
Distribution of substituent groups along the chain. In ratory and pilot scale. In Enzymatic Hydrolysis of Cellulose;
Cellulose and Its Derivatives, Chemistry, Biochemistry and Walker, L.P., Wilson, D.B., Eds.; Elsevier: London, 1991;
Applications; Kennedy, J.F., Phillips, G.O., Williams, P.A., 5166.
Eds.; Ellis Horwood: Chichester, 1985; 293300. 64. Goyal, A.; Ghosh, B.; Eveleigh, D. Characteristics of
47. Heinze, T.J., Glasser, W.G., Eds. Cellulose Derivatives fungal cellulases. In Enzymatic Hydrolysis of Cellulose;
Modication, Characterization, and Nanostructures. ACS Walker, L.P., Wilson, D.B., Eds.; Elsevier: London, 1991;
Symposium Series 668. Washington, DC: American 3750.
Chemical Society, 1998. 65. Wilson, D.B.; Irwin, D.C. Genetics and properties of
48. Ikeda, I.; Kurata, S.; Suzuki, K. Homogeneous esterica- cellulose. In Recent Progress in Bioconversion of Lignocel-
lulosics; Tsao, G.T., Ed.; Advances in Biochemical Engi- Blanco, F.; Alkorta, I.; Garbisu, C. Straw quality for its
neering/Biotechnology. Springer-Verlag: Berlin, 1999; Vol. combustion in a straw-red power plant. Biomass Bio-
65, 121. energy 2001, 21, 249258.
66. Hatakka, A.I.; Lundell, T.K.; Mohammadi, O.K.; Tervil- 84. Bridgwater, A.V. Conversion into fuels: Biomass pyrolysis
la-Wilo, A.L.M. Catalytic activities of lignin-degrading liquids production. In Electricity from Biomass; Grassi, G.
enzymes from the white-rot fungus Phlebia radiata: Lignin Trebbi, G., Pike, D.C., Eds.; CPL Press: Newbury, 1992;
model compound studies. In Biotechnology in Pulp and 1122.
Paper ManufactureApplications and Fundamental Inves- 85. Bridgwater, A.V.; Peacocke, G.V.C. Fast pyrolysis pro-
tigations; Kent Kirk, T., Chang, H.-M., Eds.; Butterworth- cesses for biomass. Sustain. Renew. Energy Rev. 1999, 4
Heinemann: Boston, 1990; 413420. (1), 173.
67. Shimada, M. Biochemical mechanisms for the biodegra- 86. Ganesh, A.; Banerjee, R. Biomass pyrolysis for power
dation of wood. In Recent Research on Wood and Wood- generationA potential technology. Renew. Energy 2001,
based Materials, Current Japanese Materials Research; 22, 914.
Shiraishi, N., Kajita, H., Norimoto, M., Eds.; Elsevier: 87. Jurado, F.; Cano, A.; Carpio, J. Modelling of combined
London, 1993;Vol. 11, 207222. cycle power plants using biomass. Renew. Energy 2003, 28,
68. Highley, T.L.; Dashek, W.V. Biotechnology in the study of 743753.
brown- and white-rot decay. In Forest Products Biotech- 88. Rustamov, V.R.; Abdullayev, K.M.; Samedov, E.A.
nology; Bruce, A., Palfreyman, J.W., Eds.; Taylor & Biomass conversion to liquid fuel by two-stage thermo-
Francis: London, 1998; 1536. chemical cycle. Energy Convers. Manag. 1998, 39 (9), 869
69. Harman, G.E., Kubicek, C.P., Eds. Trichoderma and 875.
Gliocladium. Enzymes, Biological Control and Commercial 89. Demirbas, A. Yields of oil products from thermochemical
Applications. Taylor & Francis: London, 1998; Vol. 2. biomass conversion processes. Energy Convers. Manag.
70. Nieves, R.A.; Ehrman, C.I.; Adney, W.S.; Elander, R.T.; 1998, 39 (7), 685690.
Himmel, M.E. Survey and analysis of commercial cellulase 90. Demirbas , A. Conversion of biomass using glycerin to
preparations suitable for biomass conversion to ethanol. liquid fuel for blending gasoline as alternative engine fuel.
World J. Microbiol. Biotechnol. 1998, 14 (2), 301304. Energy Convers. Manag. 2001, 42, 13571378.
71. Wilson, D.B.; Walker, L.P. Engineering cellulases. In 91. Demirbas , A. Biomass resource facilities and biomass
Enzymatic Hydrolysis of Cellulose; Walker, L.P., Wilson, conversion processing for fuels and chemicals. Energy
D.B., Eds.; Elsevier: London, 1991; 97100. Convers. Manag. 2001, 41, 17411748.
72. Murty, M.V.S.; Chandra, T.S. Fermentability of hemi- 92. Demirbas , A.; Caglar, A.; Akdeniz, F.; Gullu, D. Con-
celluloses extracted from municipal waste and commercial version of olive husk to liquid fuel by pyrolysis and
xylans by Clostridium sp. Appl. Microbiol. Biotechnol. catalytic liquefaction. Energy Sources 2000, 22 (7), 631
1997, 47 (3), 212217. 639.
73. Olsen, H.S. Enzymatic production of glucose syrups. In 93. Caglar, A.; Demirbas, A. Conversion of cotton cocoon shell
Handbook of Starch Hydrolysis Products and Their to liquid products by pyrolysis. Energy Convers. Manag.
Derivatives; Kearsley, M.W., Dziedzic, S.Z., Eds.; Blackie 2000, 41, 17491756.
Academic & Professional: Glasgow, 1995; 2664. 94. Savova, D.; Apak, E.; Ekinci, E.; Yardim, F.; Petrov, N.;
74. Chynoweth, D.P.; Owens, J.M.; Legrand, R. Renewable Budinova, T.; Razvigorova, M.; Minkova, V. Biomass
methane from anaerobic digestion of biomass. Renew. conversion to carbon adsorbents and gas. Biomass
Energy 2001, 22, 18. Bioenergy 2001, 21, 133142.
75. Mng, H.; Lund, H.; Hvelplund, F. Biogas plants in 95. Katyal, S.; Thambimuthu, K.; Valix, M. Carbonisation of
Denmark: Technological and economic developments. bagasse in a xed bed reactor: Inuence of process
Appl. Energy 1999, 64, 195206. variables on char yield and characteristics. Renew. Energy
76. Dare, P.; Giord, J.; Hooper, R.J.; Clemens, A.H.; 2003, 28, 713725.
Damiano, L.F.; Gong, D.; Matheson, T.W. Combustion 96. Hogan, E., Robert, J., Grassi, G., Bridgwater, A.V., Eds.;
performance of biomass residue and purpose grown Biomass Thermal Processing; CPL Press: Newbury, 1992.
species. Biomass Bioenergy 2001, 21, 277287. 97. Engelhardt, J. Sources, industrial derivatives and commer-
77. Witton, J.J.; Noordally, E.; Przybylski, J.M. Clean cial application of cellulose. Carbohydr. Eur. 1995, 12, 5
catalytic combustion of low heat value fuels from gas- 14.
ication processes. Chem. Eng. J. 2003, 91, 115121. 98. Felcht, U.-H. Cellulose ethersSynthesis, application and
78. Beenackers, A.A.C.M. Conversion into fuels: Gasication. analytical aspects. In Cellulose and Its Derivatives, Chem-
In Electricity from Biomass; Grassi, G., Trebbi, G., Pike, istry, Biochemistry and Applications; Kennedy, J.F., Phil-
D.C., Eds.; CPL Press: Newbury, 1992; 811. lips, G.O., Williams, P.A., Eds.; Ellis Horwood: Chichester,
79. Walker, M.; Jackson, G.; Peacocke, G.V.C. Small scale 1985; 273284.
biomass gasication: Development of a gas cleaning system 99. Reuben, J. Description and analysis of cellulose ethers. In
for power generation. In Progress in Thermochemical Cellulose, Structure Modication and Hydrolysis; Young,
Biomass Conversion; Bridgwater, A.V., Ed.; Blackwell R.A., Rowell, R.M., Eds.; Wiley: Chichester, 1986; 149
Scientic Publications, 2001: Oxford, 441451. 157.
80. Belgiorno, V.; De Feo, G.; Della Rocca, C.; Napoli, 100. Dahlgren, L. Cellulose ethersProperties and applica-
R.M.A. Waste Manage. 2003, 23, 115. tions. In Wood and CellulosicsIndustrial utilisation,
81. Bridgwater, A.V. Renewable fuels and chemicals by biotechnology, structure and properties; Kennedy, J.F., Phil-
thermal processing of biomass. Chem. Eng. J. 2003, 91, lips, G.O., Williams, P.A., Eds.; Ellis Horwood: Chichester,
87102. 1987; 427440.
82. Chen, G.; Andries, J.; Splietho, H. Biomass conversion 101. Vaughan, F. The use of cellulose ethers in ceramic tile
into fuel gas using circulating uidised bed technology: The adhesives. In Cellulose and Its Derivatives, Chemistry, Bio-
concept improvement and modelling discussion. Renew. chemistry and Applications; Kennedy, J.F., Phillips, G.O.,
Energy 2003, 28, 985994. Williams, P.A., Eds.; Ellis Horwood: Chichester, 1985;
83. Allica, J.H.; Mitre, A.J.; Bustamante, J.A.G.; Itoiz, C.; 311318.
102. Burneld, K.; Petersen, B.; Nellessen, B.; Schweizer, D. Use 119. Holtzapple, M.T.; Davison, R.R.; Ross, M.K.; Aldrett-
of cellulose ethers for rheological control in advanced Lee, S.; Nagwani, M.; Lee, C.M.; Lee, C.; Adelson, S.;
ceramics. In Cellulose, Structural and Functional Aspects; Kaar, W.; Gaskin, D.; Shirage, H.; Chang, N.S.; Chang,
Kennedy, J.F., Phillips, G.O., Williams, P.A., Eds.; Ellis V.S.; Loescher, M.E. Biomass conversion to mixed alcohol
Horwood: Chichester, 1989; 479485. fuels using the MixAlco process. Appl. Biochem. Bio-
103. Nicholson, M.D.; Merritt, F.M. Cellulose ethers. In Cellu- technol. 1999, 77-9, 609631.
lose Chemistry and Its Applications; Nevell, T.P., Zeronian, 120. Diz, J.; Cruz, J.M.; Dominguez, H.; Parajo, J.C. Xylitol
S.H., Eds.; Ellis Horwood: Chichester, 1985; 363383. production from eucalyptus wood hydrolysates in low-cost
104. Modied Cellulosics; Rowell, R.M., Ed.; Academic Press: fermentation media. Food Technol. Biotechnol. 2002, 40
New York, 1978. (3), 191197.
105. Wadsworth, L.C.; Daponte, D. Cellulose esters. In Cel- 121. Mostafa, N.A.; Magdy, Y.H. Utilization of molasses and
lulose Chemistry and Its Applications; Nevell, T.P., Zero- akalona hydrolysate for continuous glycerol production in
nian, S.H., Eds.; Ellis Horwood: Chichester, 1985; 344362. a packed bed bioreactor. Energy Convers. Manag. 1998, 39
106. Linko, M. Fermentation of cellulose feedstocks. In Wood (7), 671677.
and CellulosicsIndustrial Utilisation, Biotechnology, 122. Tsao, G.T.; Cao, N.J.; Du, J.; Gong, C.S. Production of
Structure and Properties; Kennedy, J.F., Phillips, G.O., multifunctional organic acids from renewable resources. In
Williams, P.A., Eds.; Chichester: Ellis Horwood, 1987; Recent Progress in Bioconversion of Lignocellulosics; Tsao,
231242. G.T., Ed.; Advances in Biochemical Engineering/Biotech-
107. Jeries, T.W.; Alexander, M.A. Production of ethanol nology. Springer-Verlag: Berlin, 1999; Vol. 65, 243280.
from xylose by Candida shehatae grown under continuous 123. Quadbeck-Seeger, H.J. Chemistry for the futureState of
or fed-batch conditions. In Biotechnology in Pulp and Paper the art and perspectives. Angew. Chem. Int. Ed. 1990, 29,
ManufactureApplications and Fundamental Investiga- 11771188.
tions; Kent Kirk, T., Chang, H.-M., Eds.; Butterwoth- 124. Ribbons, D.W.; Taylor, S.J.C.; Evans, C.T.; Thomas, S.D.;
Heinemann: Boston, 1990; 311321. Rossiter, J.T.; Widdowson, D.A. Biodegradation yield
108. Ladisch, M.R.; Svarczkopf, J.A. Ethanol production and novel intermediates for chemical synthesisStereospecic
the cost of fermentable sugars from biomass. In Enzymatic chiral synthon preparation: A review. Adv. Appl. Bio-
Hydrolysis of Cellulose; Walker, L.P., Wilson, D.B., Eds.; technol. Ser. 1990, 4, 213245.
Elsevier: London, 1991; 5166. 125. Boudrant, J. Microbial processes for ascorbic acid biosyn-
109. Grohmann, K.; Himmel, M.E. Enzymes for fuels and thesis: A review. Enzyme Microb. Technol. 1990, 12, 322
chemical feedstocks. In Enzymes in Biomass Conversion; 329.
Leatham, G.F., Himmel, M.E., Eds.; ACS Symposium 126. Pandey, A.; Soccol, C.R. Bioconversion of biomass: A case
Series 476. American Chemical Society: Washington, DC, study of lingo-cellulosics bioconversions in solid state
1991; 211. fermentation. Braz. Arch. Biol. Technol. 1998, 41 (4), 379
110. Katzen, R.; Fowler, D.E. Ethanol from lignocellulosic 389.
wastes with utilisation of recombinant bacteria. Appl. 127. Tengerdy, R.P.; Szakacs, G. Bioconversion of lignocellu-
Biochem. Biotechnol. 1994, 45-46, 697707. lose in solid substrate fermentation. Biochem. Eng. J. 2003,
111. The Alcohol TextbookEthanol Production by Fermenta- 13, 169179.
tion and DistillationIncorporating the Alcohol Alphabet; 128. Laufenberg, G.; Kunz, B.; Nystroem, M. Transformation
Lyons, T.P., Kelsall, D.R., Murtagh, J.E., Eds.; Notting- of vegetable waste into value added products: (A) the
ham University Press: Nottingham, 1995. upgrading concept; (B) practical implementations. Bio-
112. Saddler, J.N.; Gregg, D.J. Ethanol production from forest resour. Technol. 2003, 87, 167198.
product wastes. In Forest Products Biotechnology; Bruce, 129. Rowell, R.M., Young, R.A., Rowell, J.K., Eds.; Paper and
A., Palfreyman, J.W., Eds.; Taylor & Francis: London, Composites from Agro-Based Resources; CRC Press: Boca
1998; 1536. Raton, 1997.
113. Gong, C.S.; Cao, N.J.; Du, J.; Tsao, G.T. Ethanol 130. PPI International Pulp and Paper Directory. Miller Free-
production from renewable resources. In Recent Progress man: San Francisco, 1991.
in Bioconversion of Lignocellulosics; Tsao, G.T., Ed.; 131. Paper RecyclingStrategies, Economics and Technology;
Advances in Biochemical Engineering/Biotechnology. Patrick, K.L., Ed.; Miller Freeman: San Francisco, 1991.
Springer-Verlag: Berlin, 1999; Vol. 65, 207241. 132. Muhlemann, H.M.; Bungay, H.R. Research perspectives
114. Wyman, C.E. Biomass ethanol: Technical progress, oppor- for bioconversion of scrap paper. In Recent Progress in
tunities, commercial challenges. Annu. Rev. Energy Bioconversion of Lignocellulosics; Tsao, G.T., Ed.; Advan-
Environ. 1999, 24, 189226. ces in Biochemical Engineering/Biotechnology. Springer-
115. Nazare Pereira, A. Availability and kinetics of hydrolysis Verlag: Berlin, 1999; Vol. 65, 193206.
and fermentation of brushwood biomass for ethanol prod- 133. Viikari, L.; Kantelinen, A.; Ratto, M.; Sundquist, J. In
uction. In Cellulose Hydrolysis and Fermentation; Coombs, Enzymes in Biomass Conversion; Leatham, G.F., Himmel,
J., Grassi, G., Eds.; CPL Press: Newbury, 1992; 214222. M.E., Eds.; ACS Symposium Series 476. American Chem-
116. Tewari, H.K.; Marwaha, S.S.; Rupal, K.; Kennedy, J.F. ical Society: Washington, DC, 1991; 1221.
Bio-utilization of pineapple waste for ethanol generation. 134. van Wyk, J.P.H. Biodevelopment of wastepaper as a
In Wood and CellulosicsIndustrial utilisation, biotechnol- resource of renewable energy: Inuence of enzyme concen-
ogy, structure and properties; Kennedy, J.F., Phillips, G.O., tration and paper amount on the bioconversion process.
Williams, P.A., Eds.; Ellis Horwood: Chichester, 1987; Energy Fuels 2002, 16 (5), 12771279.
251259. 135. Evans, J.D.; Sidkar, S.K. Biodegradable plastics: An idea
117. Du, S.J.B.; Murray, W.D. Bioconversion of forest whose time has come. Chemtech 1990, 20, 3842.
products industry waste cellulosics to fuel ethanol: A 136. Hirose, S.; Nakamura, K.; Hatekeyama, H.; Meadows, J.;
review. Bioresour. Technol. 1996, 55, 133. Williams, P.A.; Phillips, G.O. Preparation and mechanical
118. Himmel, M.E.; Ruth, M.F.; Wyman, C.E. Cellulase for properties of polyurethane foams from lignocellulose
commodity products from cellulosic biomass. Curr. Opin. dissolved in polyethylene glycol. In Cellulosics: Materials
Biotechnol. 1999, 10, 358364. for Selective Separations and Other Technologies; Kennedy,
J.F., Phillips, G.O., Williams, P.A., Eds.; Ellis Horwood: nologies; Kennedy, J.F. Phillips, G.O., Williams, P.A.,
Chichester, 1993; 317331. Eds.; Ellis Horwood: Chichester, 1993; 113118.
137. Narayan, R. Engineering of controlled cellulosesynthetic 146. Ikada, Y. Biomedical applications of cellulose membranes.
polymer graft copolymer structures. In Cellulosics Utiliza- In Cellulose, Structural and Functional Aspects; Kennedy,
tion, Research and Rewards in Cellulosics; Inagaki, H. J.F. Phillips, G.O., Williams, P.A., Eds.; Ellis Horwood:
Phillips, G.O., Eds.; Elsevier: London, 1989; 110118. Chichester, 1989; 447455.
138. Turbak, A. Rayon. In Encyclopaedia of Polymer Science 147. Kamide, K.; Iijima, K. Recent Advances in Cellulose
Engineering; Kraschwitz, J., Ed.; Wiley: New York, 1988; membranes. In Cellulosic Polymers, Blends and Composites;
4572. Gilbert, R.D., Ed.; Carl Hanser Verlag: Munich, 1994;
139. Prudhomme, R.E. Microcomposites (or blends) from 189206.
synthetic polymers and cellulose or cellulose derivatives. 148. Junginger, M.; Faaij, A.; van den Broek, R.; Koopmans,
In Wood Processing and Utilisation; Kennedy, J.F., Phillips, A.; Hulscher, W. Fuel supply strategies for large-scale bio-
G.O., Williams, P.A., Eds.; Ellis Horwood: Chichester, energy projects in developing countries. Electricity gen-
1989; 221228. eration from agricultural and forest residues in Northeast
140. Youngquist, J.; Myers, G.E.; Harten, T.M. Lignocellulo- Thailand. Biomass Bioenergy 2001, 21, 259275.
sicplastic composites from recycled materials. In Emerg- 149. Lynch, M.C. Oil scarcity, oil crises, and alternative
ing Technologies for Materials and Chemicals from Biomass; energiesDont be fooled again. Appl. Energy 1999, 64,
Rowell, R.M., Schultz, T.P., Narayan, R., Eds.; ACS Sym- 3153.
posium Series. Washington, DC: ACS, 1992; 476, 4256. 150. Bhattacharya, S.C. Renewable energy education at the
141. Kawaguchi, K.; Murase, T.; Iida, K. New wood bre university level. Renew. Energy 2001, 22, 9197.
plastic composite for thermomoulding. In Wood Proces- 151. Ho, G.; Dallas, S.; Anda, M.; Mathew, K. Renewable
sing and Utilisation; Kennedy, J.F. Phillips, G.O., Williams, energy in the context of environmentally sound technolo-
P.A., Eds.; Ellis Horwood: Chichester, 1989; 237242. giesTraining and research programmes at the Environ-
142. Takase, S.; Shiraishi, N.; Takahama, M. Studies on mental Technology Centre, Murdoch University. Renew.
composites from wood and polypropylins. In Wood Energy 2001, 22, 105112.
Processing and Utilisation; Kennedy, J.F. Phillips, G.O., 152. Jennings, P.; Lund, C. Renewable energy education for
Williams, P.A., Eds.; Ellis Horwood: Chichester, 1989; sustainable development. Renew. Energy 2001, 22, 113
243249. 118.
143. Kokta, B.V.; Raj, R.G.; Maldas, D. Use of wood bres in 153. Lund, C.P.; Jennings, P.J. The potential, practice and
thermoplastic composites. In Lignocellulosics: Science, challenges of tertiary renewable energy education on the
Technology, Development and Use; Kennedy, J.F., Phillips, World Wide Web. Renew. Energy 2001, 22, 119125.
G.O., Williams, P.A., Eds.; Ellis Horwood: Chichester, 154. OMara, K.L.; Jennings, P.J. Innovative renewable energy
1992; 747762. education using the World Wide Web. Renew. Energy
144. Gilbert, R.D. Cellulose and cellulose derivatives as liquid 2001, 22, 135141.
crystals. In Agricultural and Synthetic Polymers; Glass, E., 155. Sims, R.E.H. BioenergyA renewable carbon sink.
Swift, G., Eds.; ACS Symposium Series. American Renew. Energy 2001, 22, 3137.
Chemical Society: Washington, DC, 1990; 433, 259272. 156. Shimizu, Y.; Hayashi, J. Esterication of cellulose with
145. Hicke, H.-G.; Paul, D. Manufacturing and performance of organic acids. In Cellulose, Structural and Functional
membranes from cellulose and cellulose esters. In Cellulo- Aspects; Kennedy, J.F. Phillips, G.O., Williams, P.A.,
sics: Materials for Selective Separations and Other Tech- Eds.; Ellis Horwood: Chichester, 1989; 187194.
42
Bioethanol Production from Lignocellulosic Material
* Kristian B. R. Krogh, and Christophe Rocay
Lisbeth Olsson, Henning Jrgensen,*
Center for Microbial Biotechnology BioCentrum-DTU, kgs. Lyngby, Denmark
I. INTRODUCTION dependence on oil, and they were the onset for intensied
search for alternative energy sources. One alternative to oil
Presently, bioethanol production receives tremendous at- has been ethanol, produced from lignocellulosic material
tention, as bioethanol used as fuel in the transportation also named bioethanol [1]. There is an international aware-
sector is sustainable and carbon dioxide neutral in contrast ness of the increasing CO2 concentration in the atmosphere
to fossil fuels. Furthermore, being produced from ligno- and negotiations have led to the formulation of the Kyoto
cellulosic material, it can represent a domestic energy Protocol, in which many countries have committed them-
source that is renewable. Bioethanol production is getting selves to decrease their emission levels of greenhouse gases.
close to commercialization and presently large resources A very important reason for renewed interest in bioetha-
are put into solving the bottlenecks in the process. The use nol, as an alternative to fossil fuel, is the accompanying
of ethanol is forecasted to grow rapidly in the coming years, reduction of carbon dioxide emission into the atmosphere.
and the rst plant producing fuel ethanol from lignocellu- The CO2 concentration in the atmosphere has increased by
losic material is expected to be running in the near future. 23% from 1750 to 1984 [2] with an additional increase of
This chapter describes and evaluates the process for 8% from 1984 to 2001z. The combustion of fossil fuels and
fuel ethanol production. The raw material, hydrolysis, and bioethanol results in production of CO2. However, the net
fermentation are described in detail, and dierent possibil- CO2 release is much lower in the case of bioethanol because
ities to perform these process steps in various process of the xation of CO2 during photosynthesis in plants [3].
designs are discussed. The bottlenecks for the process and During the last 20 years, the production of CO2 from
possible improvements in the future are assessed. combustion of petroleum has increased by more than
10% (Fig. 1; data are from Ocial Energy Statistics from
A. Ethanol and Oil as Transportation Fuels: the U.S. Government).
History and Interests
Just before 1900, Henry Ford built his rst vehicle, the B. Ethanol as a Transportation Fuel
quadricycle, which used ethanol as fuel. He also produced The volumetric energy content of ethanol is around two
the Model T using ethanol as fuel. At this time, gasoline thirds of the volumetric energy content in gasoline, so the
emerged as a more favorable transportation fuel, and the mileage should be reduced by 33% using ethanol as fuel
interest for ethanol decreased. This view has been domi- instead of gasoline. However, combustion of ethanol has a
nant until the oil price suddenly escalated in 1973, and 15% higher eciency as a result of a combination of a high
again in 1979. These oil crises demonstrated the worlds heat of vaporization, a high octane number, and a high gas
* Present address: Department of Forest and Forest Products,

z
Forest & Landscape, The Royal Veterinary and Agricultural Concentration measured at Mauna Loa by Carbon Dioxide Re-
University. search Group Scripps Institution of Oceanography, University of
y
Present address: Centro de Recursos Microbiologicos, Biotechnol- California, California, U.S.A.

ogy Unit, Faculty of Sciences and Technology, New University of Energy Information Administration, U.S. Department of Energy
Lisbon, Caparica, Portugal http://www.eia.doe.gov/pub/international/iealf/tableh2.xls
957
958 Olsson et al.
sion is increased in blends from pure gasoline up to E50,

but with E85 the NOx emission is decreased by 20%
compared to pure gasoline [4,6]. The emission of reactive
aldehydes, in particular acetaldehyde and formaldehyde, is
increased when the blend ratio of ethanol to gasoline
increases. However, as a result of the general decrease in
emission of pollutants, the United States made the 1990
Clean Air Act Amendments, which mandated that oxy-
genated additives (methyl-tertiary-butyl ether, MTBE;
ethyl-tertiary-butyl ether, ETBE; or ethanol) should be
added to at least a level of 2% by weight of oxygen to
decrease urban pollution in areas with too high levels of
carbon monoxide [7]. Critical amounts of MTBE found in
Figure 1 Global emission of CO2 resulting from the com- ground waters resulted in reluctance to the use of MTBE in
bustion of petroleum. gasoline as it can be toxic to humans; in California, the use
of MTBE will be banned from 2004. In Brazil, during the
last three decades, almost all gasoline sold is oxygenated as
to volume change [4], so the mileage eciency for ethanol is E22, as a result of alcohol programs [1].
only reduced by 2025% in comparison to gasoline [1].
Today, some cars, the Flexible Fuel Vehicles, can run on C. Production of Ethanol
mixtures of ethanol and gasoline, with an ethanol volume
content up to 85% (E85), and all new cars with a catalyst The feedstock for bioethanol production can either be
can run on E10 [5]. Combining ethanol and gasoline plants grown especially for this purpose or dierent waste
decreases the emission of carbon monoxide, volatile or- streams, e.g., agriculture, paper, forestry, and municipal
ganic compounds, and hydrocarbons. However, NOx emis- solid waste. After a mechanical degradation, the hydrolysis
Figure 2 Schematic overview of the production of bioethanol from lignocellulosic material. Dierent possible process schemes
are depicted. SHFseparate hydrolysis and fermentation, SSFsimultaneous saccharication and fermentation, CBPcon-
solidated bioprocessing.
Bioethanol Production from Lignocellulosic Material 959
Table 1 Percent Dry Weight Composition of Lignocellulose from Plants

Feedstock Cellulose Hemicelluloses (main fraction) Lignin Reference
Hardwood 54 21 (xylan) 22 [11]
Softwood 52 17 (glucomannan) 28 [11]
Wheat straw 38 33 (xylan) 9 [12]
Rice straw 32 24 (xylan) 13 [13]
Switchgrass 43 36 (xylan) 22 [9]
of the cellulose and hemicelluloses can either be performed higher content of cellulose and lignin, which favors rigidity
in one physical and/or chemical step or by a pretreatment compared to straw that has a relatively high content of
followed by enzymatic hydrolysis (Fig. 2). The enzymatic hemicelluloses, and therefore has more exibility. The
route has three dierent congurations: (1) SHF, separate cellulose content of softwood and hardwood is the same,
hydrolysis and fermentation; (2) SSF, simultaneous sac- but softwood contains more lignin and less hemicelluloses
charication and fermentation; (3) CBP, consolidated bio- [11]. The hemicellulose composition is also dierent in
processing. A requisite process step for SHF and SSF is the various plants (Table 1).
production of cellulases and hemicellulases (either on-site The content of glucomannan in softwood reects upon
or produced by industries specializing in the production). the total amount of monomeric sugar residues in the way
that pine has a relatively high mannose content and a
relatively low xylose content (Table 2).
II. LIGNOCELLULOSIC MATERIAL In the last two decades, extensive eorts have been put
into investigating the possibilities of optimizing the pro-
Lignocellulose is a generic term describing the main conductivity and composition of dierent feedstocks. Today,
stituents in most plants, namely cellulose, hemicelluloses, the perspectives of using lignocellulosic material as a
and lignin. Cellulose is a glucose polysaccharide, hemi- carbon source for ethanol production have increased the
celluloses are polysaccharides with a backbone of dierent interest in optimizing the composition of the plants. The
hexoses (glucose, mannose, galactose) and pentoses (xylan, chemical composition of lignocellulose can be changed to
arabinose), and lignin is a complex network of dierent ease the degradation and to improve the ethanol yield and
phenyl propane units ( p-coumaryl alcohol, coniferyl alco- productivity. A selection of dierent hybrid poplar clones
hol, sinapyl alcohol). In general, cellulose gives rigidity; has resulted in a clone with a signicantly higher cellulose
however, to confer rigidity, e.g., to a stem, there is a need and lower lignin content [14]. The relative amount of each
for some other materials that can stick and glue the lignin monomer in a tobacco plant has been modied and
polysaccharides together. The hemicelluloses provide the result was a higher degradability [15]. Furthermore, Hu
stickiness and lignin acts as glue. et al. [16] have produced and characterized transgenic
aspen trees (genus Populus) with a substantial decrease in
A. Composition of Lignocellulosic Material lignin content. The lower lignin content was compensated
by an increase in cellulose content so that the total cellu-
Lignocellulosic material comes from a variety of plants, loselignin mass remained the same.
and the composition depends not only on the plant, but In 1992, the Department of Energy (DOE) initiated a
also on the growth conditions [8,9], on the part of the plant program (at Oak Ridge National Laboratory) in which
[10], and on the age at harvesting. The average composition switchgrass has been chosen as a model crop in a biofuel
of lignocellulose is 4045% cellulose, 2030% hemicellu- feedstock development program. In selection of clones
loses, and 1525% lignin (Table 1). In general, wood has a from breeding experiments, it has been possible to nd a
Table 2 Percent Dry Weight Monomeric Composition of Lignocellulose Samples From Plants Analyzed
at the National Renewable Energy Laboratory, USA
Feedstock Glucose Xylose Galactose Arabinose Mannose
Beech 42.9 20.8 1.5 0.9
Poplar 49.9 17.4 1.2 1.8 4.7
Pine 46.4 8.8 2.4 11.7
Wheat straw 38.2 20.4 0.9 2.8 0.3
Rice straw 34.2 24.5
Switchgrass 31.0 20.4 0.9 2.8 0.3
Data not available.
Source: Ref. 14.
960 Olsson et al.
clone with a high biomass yield and relatively high cellulose problems with corrosion compared to, e.g., HCl. The use of
content [17]. The choice of variety at the specic location lower acid concentrations (<1%) demands the use of high
and the cultural practice (harvest frequency, fertilization, temperatures (180230jC) to achieve acceptable reaction
and plant spacing) have been proved to be important rates for the hydrolysis of the cellulose and to obtain a high
parameters for the productivity [1820]. Almost all growth yield of glucose [24,28]. Commonly, the dilute acid hydro-
experiments with switchgrass has been conducted in the lysis is performed in a plug-ow reactor operated at
United States; however, some growth experiments have temperatures above 200jC, acid concentrations from
demonstrated that switchgrass can be successfully cultivat- 0.2% to 1.5% (w/w), and a residence time of a few minutes
ed in parts of Europe as well [21]. or less [24,28]. The glucose yield seldom exceeds 5560% of
the theoretical using this method [24,28,29]. Furthermore,
at the elevated temperatures, the pentoses from the hemi-
III. HYDROLYSIS OF LIGNOCELLULOSE celluloses, and to a lesser extent the hexoses, are rapidly
degraded (see Fig. 5). The formation of degradation prod-
Over the years, a number of dierent methods have been ucts can inhibit the fermentation (which is discussed in
proposed for hydrolysis of the lignocellulosic material. detail in Sec. VII.A) and therefore the dilute acid hydrolysis
Generally, two routes are employed to hydrolyze the is often divided into a two-step process. In the rst step,
lignocellulosic material. The rst route is the use of acid mainly the hemicelluloses are hydrolyzed at less-severe
hydrolysis and the second is the use of a pretreatment conditions with temperatures around 170190jC and acid
process before enzymatic hydrolysis (Fig. 2). In both cases, concentrations around 0.51.2% (w/w) [2932]. Thereaf-
there are several possible methods or operation modes and ter, the remaining solids are removed and treated in the
the choice of method has to be based on a number of second step at harsher conditionsat higher temperatures
considerations, e.g., which raw material will be used, the (200230jC) and higher acid concentration [up to 2.5% (w/
organism used for fermentation of the released sugars, and w)] [5,29,32]. This results in overall yields of 5060% for
economics. Common to all of these methods is that the glucose and up to 90% for hemicellulose sugars (xylose,
lignocellulosic material is rst mechanically chipped, arabinose, mannose, and galactose) of the theoretical
grinded, or milled to increase the surface area [22]. yields [25,32,33]. More advanced dilute acid processes em-
ploy the use of new reactor designs, e.g., a countercurrent
A. Acid Hydrolysis shrinking-bed reactor with three thermal stages (175jC,
225jC, and 235jC) using 0.07% (w/w) H2SO4, which has
The rst commercial-scale plant using concentrated acid been reported to result in glucose yields up to 83% and
was constructed in Germany in 1937 and was operated xylose yields well above 90% of the theoretical yields
during World War II [23]. The process was a modication [28,33]. Although the sugar yields are much improved in
of the Bergius process, and it was operated at 35jC for 1 hr the two- or three-stage process, some of the sugars are still
using 41% HCl [24]. Later, other processes have been based degraded into inhibitors. Furthermore, the process results
on using concentrated HF or H2SO4 in the hydrolysis, the in a low sugar concentration [normally less than 5% (w/w)
advantage of H2SO4 being its lower price compared to HCl [28]] in the exit. The more advanced technologies have also
and HF. The concentrated acid hydrolysis is performed at only been tested at bench or small scale.
low temperature (around 30jC) and it results in a high yield
of both hexoses and pentoses (8590% of the theoretical
[5,25]) and it only cause a limited production of degrada- B. Pretreatment of Lignocellulose
tion products from the sugars [24,26]. The use of large
amounts of acid necessitates ecient recovery of the acid. In the utilization of enzymes for the hydrolysis of cellulose,
This is one of the major drawbacks of the method, as the a pretreatment of the lignocellulosic material is necessary
recovery is an energy-demanding and expensive step [25]. to break up the barrier made by both lignin and hemi-
Furthermore, the corrosive nature of the acids requires the celluloses. Furthermore, the physical and/or chemical pre-
use of expensive alloys in the construction of pipes and heat treatment may increase the accessible surface area and
exchangers. These problems have been the main obstacle change the crystallinity of the cellulose, which results in
for making the process economically feasible [23], and there increased digestibility of the cellulose [34]. From this, it is
have been less focus on this process during the last 20 years. obvious that the dilute acid hydrolysis mentioned in the
However, in the United States, some companies in coop- previous section can also be regarded as a pretreatment
eration with the DOE and the National Renewable Energy process when performed under mild conditions, e.g., the
Laboratory (NREL) are trying to implement new technol- rst step in two-step dilute acid hydrolysis [26,30,35].
ogies for concentrated acid hydrolysis into commercial-
scale facilities [27]. 1. Steam Explosion
Dilute acid has also been successfully used for the A pretreatment process that has attracted much interest is
hydrolysis of lignocellulosic material on a commercial- steam explosion and it is a commonly used method to
scale since the beginning of the 1930s (the Scholler or pretreat various types of lignocellulosic material [22,36].
Madison percolation process [23,24]). The most generally For several years, equipment for large-scale steam explo-
used acid is H2SO4 because of its lower price and fewer sion of wood has been sold [37], but much research is still
Bioethanol Production from Lignocellulosic Material 961
being put into improving and optimizing the method for The conditions reported as optimal for the pretreat-
dierent types of lignocellulosic material. Steam explosion ment of a number of dierent substrates to obtain high
as a pretreatment method has been implemented in digestibility of the cellulose and maximum recovery of the
a number of pilot plants around the world [38]. It hemicellulose sugars are the use of 0.56% (w/w) H2SO4 or
can be performed with or without (autohydrolysis) an SO2 and with a severity factor from 3 to 4, corresponding to
acidic catalyst. temperatures in the range 175215jC for 210 min. The
Autohydrolysis total sugar yields (glucose and xylose) obtained under these
conditions after enzymatic hydrolysis of the cellulose are
In steam explosion (autohydrolysis), the lignocellu-
normally between 65% and 80% of the theoretical value
losic material is heated with high-pressure steam to tem-
[5,45,4750].
peratures from 160jC to 250jC for several seconds and up
To avoid formation of degradation products, improve
to 10 min [36]. Thereafter, the pressure is released and the
the recovery of sugars, and simultaneously make the
material undergoes an explosive decompression. The high
cellulose fraction more accessible to enzymatic hydrolysis,
temperatures promote the formation of organic acids from
two-step strategies for steam explosion have also been
acetyl groups present in the material, and these acids cause
tested. In the rst step, the steam explosion is performed
autohydrolysis [34]. In combination with the sudden
using a low severity factor (temperature below 180jC) to
change in pressure, the hemicelluloses are partially hydro-
solubilize the fraction of hemicelluloses, and the cellulose
lyzed and solubilized. The lignin is redistributed on the
fraction is then subjected to a second steam explosion step
cellulose and to some extent removed from the insoluble
at higher temperature (above 210jC).
material [26]. Although higher temperatures result in in-
creased removal of hemicelluloses and in improved digest- Lignin Removal After Steam Explosion
ibility of the cellulose fraction, the higher temperatures
The insoluble fraction obtained after steam explosion
promote sugar degradation. The eect of temperature and
consists of mainly cellulose and lignin and minor amounts
time on the hydrolysis and formation of degradation
of hemicelluloses. During the pretreatment, only a minor
products has been described by the severity factor log
amount of lignin (1015%) is removed [39,44,49]. The
(Ro) (or reaction ordinate Ro):
lignin forms a barrier that prevents the enzymes from
accessing the cellulose and the performance is also de-
Tr Tb
logRo log t exp creased because of nonspecic and irreversible adsorption
14:75
of the enzymes onto lignin [34,51]. Therefore research
in which t is the reaction time (min), Tr is the reaction groups have investigated the possibility to remove the
temperature (jC), and Tb is the reference temperature, lignin in a posttreatment step after the steam explosion.
which is set to 100jC [39]. The optimal conditions for The lignin can be extracted from the water-insoluble
maximum sugar yield are temperatures from 190jC to fraction by ethanol, methanol, acetone, and ammonium
220jC and residence times are from 2 to 10 min, hydroxide, but most often an alkaline washing step using
corresponding to a severity factor between 3.0 and 4.5 0.4% NaOH is employed because of its high eciency and
[36,3942]. Although steam explosion has successfully been lower costs [37]. The extraction can be further increased
used for pretreatment of hardwood and agricultural resi- by the addition of H2O2. A posttreatment with 1% H2O2
dues such as straw, it is not very eective for pretreatment at pH 11.5 for 45 min at 80jC removed 90% of the lignin
of softwood [36,39,43]. and signicantly improved the total sugar yield after en-
zymatic hydrolysis from 59% to 82%. Furthermore, the
Acid-Catalyzed Steam Explosion lower lignin content resulted in a better performance of
The addition of an acidic catalyst in steam explosion the enzymes, and the enzyme addition could be reduced
has been recognized as a way to increase the digestibility of sixfold [49].
the cellulose, improve the hydrolysis of hemicelluloses, and
decrease the production of degradation products from the
sugars [22,36,44]. Most commonly, the lignocellulosic ma- 2. Ammonia Freeze Explosion
terial is impregnated with either H2SO4 or SO2 before the The ammonia freeze explosion (AFEX) is performed in a
steam explosion. During steaming, SO2 is converted by way similar to steam explosion. The dried lignocellulosic
oxidation into H2SO4, which then acts as the actual material is impregnated with 12 kg of liquid ammonia per
catalyst [36]. The use of SO2 is advantageous compared kilogram of biomass and heated to around 5090jC under
to H2SO4 as the gas penetrates easier and faster into the pressure of 1020 atm. After 1530 min, the pressure is
material and SO2 does not result in as serious corrosion explosively released [35,52]. The pretreatment with ammo-
problems as H2SO4 [5]. The use of SO2 for hydrolysis of nia results in decrystallization of the cellulose and the
softwood has also been found to be advantageous to explosive release of the pressure causes a disruption of
H2SO4 as it results in the same sugar yields but produce the ber structure. These alterations in the structure of the
a hydrolysate with better fermentability [45]. In general, cellulose and lignin increase the accessible surface area and
SO2-catalyzed steam explosion is regarded as one of the result in enhanced enzymatic digestibility [52]. The method
most eective pretreatment techniques for softwood ma- does not signicantly hydrolyze the hemicelluloses and
terial [43,46]. does not remove the lignin. The method has proven to be
962 Olsson et al.
ecient for pretreatment of herbaceous crops and grasses which is also made more accessible to enzymatic hydrolysis
with low lignin content, but it is not as ecient for [8,53]. Addition of Na2CO3 to increase the initial pH
materials with high lignin content, e.g., softwood, as a results in a decreased formation of inhibitory compounds,
result of the insignicant removal of lignin from the e.g., furfural and hydroxymethylfurfural [12]. The use of
insoluble material [22,35,52]. However, the advantage of alkaline wet oxidation for the pretreatment of wheat straw
the method is the low formation of inhibitors. The costs of has been extensively investigated [12,54,55] and under
ammonia and environmental concerns necessitate the re- optimal conditions a hemicellulose yield of 70% (xylose
covery of the large amounts of ammonia used in the and arabinose) and 96

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Polysaccharides Structural Diversity and Functional Versality

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The rst edition was published as Polysaccharides: Structural Diversity and Functional Versatility, edited by Severian Dumitriu (Marcel

Library of Congress Cataloging-in-Publication Data

This book is printed on acid-free paper.

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PRINTED IN THE UNITED STATES OF AMERICA

Foreword Hans-Peter-Fink iii

1. Progress in Structural Characterization of Functional Polysaccharides 1

2. Conformations, Structures, and Morphologies of Celluloses 41

3. Hydrogen Bonds in Cellulose and Cellulose Derivatives 69

4. X-ray Diraction Study of Polysaccharides 99

5. Recent Developments in Spectroscopic and Chemical Characterization of Cellulose 123

7. Light Scattering from Polysaccharides 189

8. Advances in Characterization of Polysaccharides in Aqueous Solution and Gel State 237

11. Computer Modeling of PolysaccharidePolysaccharide Interactions 281

12. Interactions Between Polysaccharides and Polypeptides 305

13. Rheological Behavior of Polysaccharides Aqueous Systems 357

14. Stability and Degradation of Polysaccharides 395

16. Microbial Exopolysaccharides 431

17. OrderDisorder Conformational Transition of Xanthan Gum 459

18. Hemicelluloses: Structure and Properties 475

19. Chemical Modication of Hemicelluloses and Gums 491

20. Role of Acetyl Substitution in Hardwood Xylan 509

22. Characterization and Properties of Hyaluronic Acid (Hyaluronan) 535

23. Chemical Functionalization of Cellulose 551

24. The Physical Chemistry of Starch 591

25. Starch: Commercial Sources and Derived Products 605

26. StructureProperty Relationship in Chitosans 625

28. Pharmaceutical Applications of Chitosan and Derivatives 661

29. Macromolecular Complexes of Chitosan 679

30. Polysialic Acid: Structure and Properties 707

31. Brain Proteoglycans 729

32. Crystal Structures of Glycolipids 743

33. Synthetic and Natural Polysaccharides with Anticoagulant Properties 773

35. Enzymatic Synthesis of Heparan Sulfate 811

36. Polysaccharide-Based Hydrogels in Tissue Engineering 817

38. Medical Foods and Fructooligosaccharides 853

39. Immobilization of Cells in Polysaccharide Gels 867

40. Hydrothermal Degradation and Fractionation of Saccharides and Polysaccharides 893

41. Cellulosic Biomass-Derived Products 937

42. Bioethanol Production from Lignocellulosic Material 957

43. Hydrolysis of Cellulose and Hemicellulose 995

44. New Development in Cellulose Technology 1035

45. Polysaccharide Surfactants: Structure, Synthesis, and Surface-Active Properties 1055

46. Structures and Functionalities of Membranes from Polysaccharide Derivatives 1087

47. Electro-optical Properties of Cellulose Derivative Composites 1123

48. Blends and Composites Based on Cellulose Materials 1141

49. Preparation and Properties of Cellulosic Bicomponent Fibers 1179

Ann-Christine Albertsson Royal Institute of Technology, Stockholm, Sweden

P. L. Almeida EST/IPS, Setubal, Portugal and FCT/UNL, Caparica, Portugal

Eric H. Anderson Case Western Reserve University, Cleveland, Ohio, U.S.A.

Emmanouil S. Avgerinos National Technical University of Athens, Athens, Greece

Evaggeli Billa National Technical University of Athens, Athens, Greece

Ortwin Bobleter University of Innsbruck, Innsbruck, Austria

John W. Brady Cornell University, Ithaca, New York, U.S.A.

Walther Burchard Institute of Macromolecular Chemistry, University of Freiburg, Germany

Georgeta Cazacu Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania

JoMay Chow Abbott Laboratories, Columbus, Ohio, U.S.A.

Stanley (Bob) S. Davis University of Nottingham, Nottingham, United Kingdom

Stephen R. Decker National Renewable Energy Laboratory, Golden, Colorado, U.S.A.

Jean-Louis Doublier INRA-Laboratoire de Physico-Chimie des Macromolecules, Nantes, France

Severian Dumitriu Sherbrooke University, Sherbrooke, Quebec, Canada