Enzymatic and Physical

Modifications of Starch
 Content
 Introduction of enzymes
 Enzymatic starch degradation: MW reduction to small sugars or
oligosaccharides
 Starch refining
 Cyclodextrin
 Maltooligosaccharides and Isomaltooligosaccharides
 Debranched starch for making resistant starch
 Enzymatic starch modifications: no or minor MW reduction, or MW
increase
 Modification by beta-amylase and maltogenic alpha-amylase
 Increased branching by starch branching enzymes
 Alpha-glucan chain extension by amylosucrase
 Physical starch modifications
 Hydrothermal treatment
 Irradiation and microwave
 High pressure processing

2
 Enzyme classification and Nomenclature
 Enzymes are classified by the type of reactions they catalyze
No. Class Type of reaction catalyzed
1 Oxidoreductases Transfer of electrons
2 Transferases Group transfer reactions
3 Hydrolases Hydrolysis reaction (group transfer to water)
4 Lyases Group addition to double bonds, or double bond formation
by group removals
5 Isomerases Group transfer within molecules to yield isomeric form
6 Ligases Formation of C-C, C-S, C-O, and C-N bonds among two
molecules coupled with ATP cleavage
 All enzymes have formal E.C. numbers and names. Most have trivial names
 ATP + D-glucose → ADP + D-glucose 6-phosphate (by hexokinase)

 E.C. number: E.C. 2 (transferase class).7 (phosphotransferase subclass).1

(hydroxyl group as acceptor).1 (D-glucose as acceptor)

3
Introduction of enzymes
 How Enzymes Work

 Enzyme catalyzed reaction

 Takes place within the confines of a pocket on the enzyme: active site
 Substrate: molecule bound in the active site and acted upon
 Enzymes affect reaction rate, not equilibrium
 Free energy, G
 Ground state: contribution to G by an average molecule
 Standard free energy change, G O (Biochemical standard energy
change, G’ O)
 At transition state, decay to S or P state is equally probable
 Activation energy, G‡
 Catalyst enhance reaction rates by lowering activation energies
 Formation of reaction intermediates

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Introduction of enzymes
 Catalysis by Enzyme

Reaction with no catalysis Enzyme-catalyzed reaction

Lehninger Principles of Biochemistry, Fourth Edition

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Introduction of enzymes
 Enzyme Kinetics
Enzyme kinetics
 To describe how the reaction rate changes in response to experimental
conditions; An approach to understand mechanism of enzyme catalysis
 Important concepts
 Reactions involving enzyme-substrate complex (ES)

 Pre-steady-state: during which ES concentration [ES] builds up

 Steady-state: [ES] remains approximately constant over time
 Initial rate (velocity), V0
 Maximum rate (velocity), Vmax
 Steady-state kinetics: Michaelis-Menten equation
Vmax [S]
V0 =
Km + [S]

6
Introduction of enzymes
 Enzyme Kinetics

Km

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Introduction of enzymes
 Enzyme Kinetics
Determination and interpretation of Michaelis-Menten equation

 Lineweaver-Burk equation is used to determine Km and Vmax

1 Km 1
= +
V0 Vmax[S] Vmax

Double reciprocal plot

Intercept

Lehninger Principles of Biochemistry, Fourth Edition

8
Introduction of enzymes
 Starch Refining

Enzymes used
 -Amylase hydrolyzes interior -1,4-glucosidic bonds
 -Amylase hydrolyzes -1,4-glucosidic bonds from non-reducing ends
and release maltose
 Glucoamylase (amyloglucosidase) hydrolyzes -1,4 and -1,6-
glucosidic bonds and release glucose
 Debranching enzymes (isoamylase and pullulanase) hydrolyze -1,6-
glucosidic bonds and release linear chains
Products
 Simple sugars: glucose, maltose, fructose, HFCS
 Syrup: DE (dextrose equivalent) >20
 Maltodextrin: DE <20

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Enzymatic starch degradation
 Starch Refining
Steam Starch slurry

Liquefaction -amylase

Products of different
Maltodextrin dextrose equivalent (DE)
Amyloglucosidase -amylase
Pullulanase Saccharification Pullulanase
Glucose Maltose syrup
syrup
Fermentation Isomerization Isomerase

Purification & High fructose corn

Ethanol crystallization syrup (HFCS)

Crystallized glucose

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Enzymatic starch degradation
 Cyclodextrin
Starch Liquefaction
Cyclodextrin
glucanotransferase
(CTGase)
Β-CD Enzyme conversion

Solvent extraction

α-, β-, or γ-CD based on

specific solvent used

α-CD β-CD γ-CD

Szejtli, Chem. Rev. 1998, 98, 1743-1753 11

Enzymatic starch degradation
 Maltooligosaccharides and Isomaltooligosaccharides

Amylases producing enriched maltotriose and maltotetraose

 AMT 1.2 L (Amano) is a maltotriose forming amylase
 Maltotriose was claimed to have the benefits
 Resistant to crystallization (humectant)
 Preventing starch retrogradation
Transglucosidase (TG) producing isomaltooligosaccharides
(prebiotic)
Β-amylase

TG
+
TG Panose
+ +
TG
+ + Isomaltose

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Enzymatic starch degradation
 Debranched Starch for Making Resistant Starch

Starch (except for high amylose starch)

Liquefaction

Debranching Pullulanase

Retrogradation

Drying

Resistant starch
Issues:
 To reduce the viscosity allowing for economic processing
 To increase the thermal stability of crystalline structure after retrogradation

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Enzymatic starch degradation
 Beta-amylase & Maltogenic α-amylase Retard Retrogradation

Change of relative PNMR solid

content (∆S’, %) measured
during 4°C storage for isolated
amylopectin after partial β-
amylolysis. ECL values are labeled
(Yao et al., J. Agric. Food Chem. 2003,
51, 4066-4071)

Maltogenic α-amylase:
 Degrades amylopectin & amylose to produce maltose & oligosaccharides
 Functions like an endo-enzyme
 Retard starch retrogradation by shortening external chains

Enzymatic starch modifications

 Some Enzymes for Bread Making

Fungal -amylase Acting on damaged starch, producing sugar,

increasing volume, flavor, and crust color

Acting mostly on amylopectin, reducing

Maltogenic -amylase external chain length, reducing
retrogradation, and extending shelf life

Xylanase, pentosanase, Partially hydrolyzing pentosan, reducing

Bread negative effect of insoluble pentosan, and
& hemicellulase
making increasing dough machinability & stability
and crumb structure & volume,

Dough conditioning: larger volume, more

Lipase uniform crumb structure, possibly forming
linkage between gliadin and glutenin

Resulting in stronger dough, mechanism not

Glucose oxidase
clear

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Enzymatic starch modifications
 Increased Branching by Starch Branching Enzymes (SBE)

OH
O O O O O O

Reaction
SBE
I
O
O
O O O H

OH
O O O O O O
+
OH
O O O O O

Reaction SBE
II
OH
O O O

O +
OH
O O O O O

Potentials:
 Reduce starch retrogradation by shortening external chain length
 Reduce digestibility by forming more branches, since α-1,6 linkages are much
less susceptible to glucoamylase than 1,4 linkages

Enzymatic starch modifications

 Alpha-glucan Chain Extension by Amylosucrase
 Sucrose + (α-1,4-D-glucosyl)(n) = D-fructose + (1,4-α-D-glucosyl)(n+1)
 Used to synthesize amylose with certain length

Low
concentration

High
concentration

Potocki-Veronese, G. et al, Biomacromolecules 2005, 5,1000

Enzymatic starch modifications
 Amylosucrase Forms Dendritic Nanoparticles

a. The surface chains of an initial glycogen particle (IGP) are extended by

amylosucrase to form linear glucan chains (LGC)
b. The chains are further elongated, forming a corona around the glycogen core
c. The elongated chains form double helical segments and crystallites, resulting in a
shrinkage of the corona and an increase in density and crystallinity

Putaux, J-L. et al. Biomacromolecules 2006, 7,1720.

Enzymatic starch modifications
 Hydrothermal Treatment

 Annealing: starch incubated in excess or intermediate water content (>40%)

at a temperature above the glass transition temperature but below the
gelatinization temperature
 Heat-moisture treatment: moisture level is low (<35%), temperature is above
the glass transition temperature and may reach up to 100oC
 Both lead to substantial change in starch properties: lower peak viscosity
and setbacks, greater swelling consistency
 Annealing leads to increased and narrower gelatinization temperature,
whereas heat-moisture treatment results in broader ones
 Annealing and heat-moisture treatment at low moisture content have no
impact on starch crystallinity, whereas heat-moisture treatment at higher
moisture content leads to partial gelatinization

Physical starch modifications

 Hydrothermal Treatment to Prepare Resistant Starch

High amylose
Annealing ~100oC
starch

Swelling Debranching Annealing Resistant starch

Annealing
Partial acid hydrolysis
Heat-moisture treatment

Effect of amylose chain length on RS

 DP<100: not long enough to form resistant crystallites
 DP100-260: RS increases to a maximum
 DP>300: too long, not easily reach the required alignment of chains
for resistant crystallites

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Physical starch modifications
 Irradiation
 Radiation: transmission of energy through space
 Electromagnetic waves (EM):

E = h = hc/ : frequency, c: speed of light, : wavelength

h:Planck's constant
Microwave (10-1 - 10-3m)

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Physical starch modifications
 Starch Modification by Irradiation

 Decrease of degree of polymerization of starch molecules

 Radiolytic end product is similar irrespective of the type of starch used
 Degradations of amylose and amylopectin lead to change of properties

Left: Starch pasting

results for rice samples
receiving different γ-ray
irradiation doses.

Yu & Wang, Food Research

International , 2007, 40: 297–303

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Physical starch modifications
 Microwave Heating

 By passing non-ionizing* microwave radiation at a frequency of 2.45 GHz (or

0.915 GHz for industrial oven) through food materials
 Water, fat, and other substances absorb energy by dielectric heating
 Many molecules (e.g. water) are electric dipoles, i.e. they have a positive
charge at one end and a negative charge at the other. They rotate to align
themselves with the alternating electric field of the microwaves
 This molecular movement creates heat
 Microwave heating is more efficient on liquid water than on fats and sugars
(which have less molecular dipole moment)

* Non-ionizing radiation: electromagnetic radiation that does not carry enough

energy per quantum to ionize atoms or molecules (i.e. to completely remove
an electron from an atom or molecule. Non-ionizing radiation is not mutagenic.

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Physical starch modifications
 Microwave Treated Starch
Microwave condition: 30% moisture, 60 min, 2.45 GHz, 0.5 W/g energy

Brabender viscosity curves for 8% solution of corn starch (left) and waxy corn starch (right)
Lewandowicz et al. Carbohydrate Polymers 2000, 42: 193–199

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Physical starch modifications
 Microwave Treated Starch
Corn starch 95 C, left: native, right: microwaved

Waxy corn starch 75oC, left: native, right: microwaved

Lewandowicz et al. Carbohydrate Polymers 2000, 42: 193–199

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Physical starch modifications
 High Pressure Processing (HPP)

 Also known as High Hydrostatic Pressure (HHP) processing

 Food material subjected to 100-900 MPa (commercially 400-700

MPa)

 Pressurization carried out in a pressure vessel containing a fluid

(e.g. water) as the pressure transmitting medium

 Pressure applied isostatically (equally applied in all directions)

 HPP processing system consists of pressure vessel,

pressurization system, temperature control, and product
handling device

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Physical starch modifications
 HPP Treated Starch

Bauer and Knorr. Journal of Food Engineering 2005, 68: 329–334

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Physical starch modifications