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D.N. Sila, S. Van Buggenhout, T. Duvetter, I. Fraeye, A. De Roeck, A. Van Loey, and M. Hendrickx
ABSTRACT: Pectin represents a very heterogeneous biopolymer whose functionality remains largely puzzling. The link between the pectin fine structure and functional properties with relevance to plant growth and development, as well as food processing, is continually being explored. This review describes the current knowledge of pectin structurefunction relationships. Key mechanisms dictating pectin structurefunction relationships are discussed, including the polymer biosynthesis, cross-linking mechanisms, enzymatic and nonenzymatic conversion reactions, solubility properties, and more. Insight into the polymer structurefunction relationships is highlighted by examining traditional and advanced methodologies used in pectin research. The role of pectin in modulating the quality characteristics of plant-based foods is underlined while pin-pointing some of the main challenges and perspectives. An integrated approach using the pectin structurefunction relationship in the precision engineering of mechanical properties of tissue-based systems is proposed.
Introduction
Plant cell walls are complex polysaccharide matrices with diverse structural and physiological roles. In higher plants, the primary cell wall predominantly comprises water, cellulose, hemicellulose, and pectin, and to a lesser extent of structural glycoproteins, phenolic esters, ionically and covalently bound minerals, and enzymes. Assembly and interactions of these components remain elusive despite increasing interest and research efforts. In fruits, vegetables, their preprocessed intermediates and processed nal products, changes in functional properties (texture, rheological characteristics, juice extractability) are directly related to structural changes in the cell-wall polymers, in particular pectin and to a lesser extent changes in hemicellulose and cellulosic material. Pectin represents a very complex family of plant cell-wall polysaccharides that play a signicant role in inuencing plant
MS 20080858 Submitted 10/30/2008, Accepted 12/28/2008 . Authors are with Laboratory of Food Technology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Dept. of Microbial and Molecular Systems (M2S), Katholieke Univ. Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium. Direct inquiries to author Hendrickx (E-mail: marc.hendrickx@biw.kuleuven.be).
growth and development (Bacic and others 1988) and also in food science (Schols and Voragen 1996; Willats and others 2006). To be able to improve our understanding of pectin, a comprehensive insight into its structure is essential to exploit and engineer its biological and industrial functions (Voragen and others 1995; Daas and others 2001). A major hurdle is the heterogeneous nature of the polymer which depends on its botanical origin (Huisman 2000), plant development stage (Huyskens-Keil and others 2006; Van Linden and others 2008), and the exact localization in the cell wall (Libermans and others 1999) and in planta. To date, pectin is depicted as a triad component encompassing homogalacturonan (HG), rhamnogalacturonan-I (RGI), and rhamnogalacturonan-II (RGII) (Thibault and others 1993; Zhan and others 1998). Except for RGII, these galacturonans do not have a stable denite structure (Willats and others 2001; ONeill and others 2004). In general, the pectin backbone is viewed to be composed of both HG and the core of RGI, the latter being branched with neutral sugar side chains (arabinan and/or arabinogalactan I and/or II) (Schols and Voragen 1996; Coenen and others 2007). An alternative model indicates the RGI as the main pectin backbone whereas HG and RGII form the side chains (Vincken and others 2003). Evidence to substantiate and support either of the models is increasingly being sought for. RGI comprises a highly diverse population of spatially and developmentally
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The plant cell wall and its complex carbohydrate structure require intricate biochemical machinery for biosynthesis and assembly. Figuring out how cell-wall polysaccharides are made is the 1st step toward regulating their production in plants. Significant progress has been made toward identifying genes encoding proteins that are required for plant cell-wall polysaccharide biosynthesis. For pectin, the biosynthetic processes involved in the assembly of the polymer remain poorly understood. This is
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depends upon various cross-links between the macromolecules within a cell wall and the intercellular adhesion. Suggested pectin cross-links include ionic bridges, borate-diol esters, uronyl esters, hydrophobic interactions, and ferulic acid linkages. The amount and contribution of each type of cross-link to the coherence of the cell wall is not fully understood. The predominant anionic polymer in the primary walls of many plants and in the middle lamellae is homogalacturonan (HG). HG chains can condense by cross-linking with divalent ions, particularly Ca2+ , forming junction zones linking parallel or antiparallel chains (Powell and others 1980; Jarvis 1984). The carboxyl groups of 2 galacturonic acid molecules form negatively charged pockets that can accommodate calcium cations (Braccini and others 1999; Willats and others 2001). Other diverse cross-linking mechanisms are known including alkali-labile covalent bonds that join pectic chains with one another or with insoluble nonpectic polysaccharides. Dimers of RGII can be found cross-linked by diester bonds through a boron atom (Fleischer and others 1999; Ishii and others 1999; P erez and others 2003). Covalent ester bonds between uronic and hydroxyl groups of neighboring polysaccharides are proposed (MacKinnon and others 2002). More recently, other cooperative covalent and noncovalent associations have been hypothesized, particularly the oxidative cross-linking via endogenous ferulic acid (Norsker and others 2000; Waldron and others 2003; Ferreira and others 2007). Ferulates are major crosslinking agents in monocotyledons, but other hydrocinnamic links like sinapateferulate cross-products have also been discovered implicating sinapates in a similar role (Ralph and others 2004). The arabinan side chains of RGI may be oxidatively cross-linked through ferulic acid (Ishii 1997), while feruloylated galactans have been shown in sugar beets (Clausen and others 2004). Hydrophobic interactions between methoxyl groups and hydrogen bonds between undissociated carboxyl and secondary alcohol groups may also be involved in holding pectic polysaccharides within the plant cell wall (Oakenfull and Scott 1984). Pectin binds to other cell-wall polysaccharides as indicated by interactions with glucuronoarabinoxylans (Carpita 1989; Suzuki and others 2000) and xyloglucans (Hayashi and others 1987). More recently, in vitro and in muro studies have demonstrated interactions with cellulose microbrils (Zykwinska and others 2005, 2006, 2007). Proteinpectin interactions are known to occur in the plant cell wall. This is evidenced by the existence and identication of several structural proteins in the cell wall, predominantly the hydroxyproline-rich glycoproteins (HRGPs) (Showalter 1993). It is hypothesized that these proteins are cross-linked through peroxidase-mediated tyrosine dimerization, probably explaining proteincarbohydrate complexing (Qi and others 1995; Brady and others 1996; Oudgenoeg and others 2002). At the same time, there has been consistent speculation that HRGPs form ionic complexes with pectins (Showalter 1993; Kieliszewski and Lamport 1994; Sommer-Knudsen and others 1998). There is a possibility of an amide linkage between polyamines and either 1 or 2 pectic galacturonate residues (Lenucci and others 2005). A model indicating different pectin types based on the proposed molecular interactions and their extraction behaviors (Table 1) has been proposed (Chang and others 1993).
Pectin conversion reactions
Despite individual cell-wall polysaccharides having been characterized and their structures identied, knowledge on pectin pectin interactions and the interconnections with the other cell-wall polysaccharides and proteins is limited. This interaction
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Plant biochemists and food scientists are highly interested in pectin conversion mechanisms to understand physiological and biochemical changes including possible structurefunction tailoring. Pectin can be digested in planta by endogenous and/or exogenous (pathogenic) enzymes, as well as by postharvest and/or processing dependent nonenzymatic conversion reactions (Figure 1). Despite the possibility of many pectin conversion
Figure 1 --- Schematic presentation of possible pectin (only homogalacturonan) conversion reactions in plant-based foods and possible routes for tailoring quality parameters: PME = pectinmethylesterase, Ca+2 = calcium crosslinking, PG = polygalacturonase, PL = pectate lyase, T = temperature, OMe = methoxy-esters, R 1 /R 2 = initial/terminal fragment of the pectin polymer.
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pressure, and so on, play an important role (Jurnak and others 1996; Verlent and others 2004; Duvetter and others 2006; Sila and others 2007a). For elaborations on these aspects refer to Part I, the complementary publication by Duvetter and others. A complete breakdown of pectin could be achieved only if different types of enzymes are present in the correct proportion (Versari and others 1999). Despite the increasing knowledge and database on enzymatic pectin conversions, exciting and difcult challenges remain to be addressed to broaden the understanding on enzymatic pectin structural changes. Nonetheless, in planta and ex situ enzymatic manipulation of pectin can positively yield plant-based foods with specic functional properties. The success of this largely depends on tailored boosting of the activity and/or inactivation of enzymes depending of the desired need. Nonenzymatic pectin conversion. Pectin is very stable around pH 3.5, its pKa value. Nonetheless, a number of reactions are proposed for its nonenzymatic degradation. This review discusses nonenzymatic pectin conversion in the HG region only. First and most important is the base-catalyzed splitting of pectin chains via the -elimination reaction, a process that occurs in parallel with de-esterication and proceeds even when pectin is heated at neutral or weakly acidic pH (Keijbets and Pilnik 1974). Most plant-based foods have a pH above 4.5 and are processed at 80 C or higher making them very susceptible to the -elimination reaction (Figure 1) (Sila and others 2005). The 2nd mechanism leading to pectin degradation during thermal processing is acid hydrolysis (pH < 3.0). In acid conditions, pectin with low DM hydrolyzes faster (Krall and McFeeters 1998) and the reaction is boosted by the simultaneous chemical demethoxylation of the polymer under such conditions (Van Buren 1979). Acid hydrolysis is of less importance during regular food processing. Finally, pectin can be degraded through hydroxyl radical-mediated scission of the polymer (Schopfer and others 2002; Liszkay and others 2003), which is thought to predominate in postharvest fruit softening (Fry and others 2002). To date, no information is given on its impact on processed plant-based foods. During thermal processing, pectin undergoes preponderant changes in the degree of polymerization explained by the elimination degradation. This reaction is promoted by (1) high methoxy-ester content, (2) increasing temperature, (3) increasing pH, and (4) presence of monovalent salts, phytates, malates, citrates (Keijbets and Pilnik 1974), EDTA (Vu and others 2006), and so on. With relevance to the -elimination reaction, the DM is more important than the distribution of the methoxy-esters in pectin (Fraeye and others 2007). Demonstration case studies using carrot, apple, and citrus pectin indicate that by tailoring the methoxy-ester content of pectin, the rate of the -elimination reaction can be controlled (Jarvis and others 2003; Sila and others 2006a, 2006b; Vu and others 2006; Diaz and others 2007; Fraeye and others 2007). In carrot pectin isolates, sensitivity to the -elimination reaction is high in the water-soluble pectin (WSP) fraction as opposed to chelator (CSP)- and alkali (NSP)-soluble fractions (Sila and others 2006a). Thermal digestion of the WSP reveals a random depolymerization pattern marked by a nonhomogeneous distribution of polymers, whereas the CSP and NSP fractions show a limited and more homogeneous thermal depolymerization (Figure 2) (Sila and others 2005). The difference in thermal sensitivity between the pectin fractions was ascribed to differences in DM, the WSP fraction being highly methoxylated as opposed to the lowly/poorly methoxy-esteried CSP and NSP fractions (Sila and others 2005, 2006a; Vu and others 2006). Kinetic information on the -elimination reaction is important to elucidate and tailor the mechanical properties of processed plant-based foods. Depending on the type of process, the processing conditions, and exposure time, the -elimination
0.7 kDa
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Figure 2 --- Thermal (110 C) depolymerization pattern of (A) water-soluble (WSP) and (B) chelator-soluble (CSP) pectin fraction (pH 6.5) from carrots. Monogalacturonic acid which had a retention time of 14 min was used a reference cut-off point (Sila and others 2006a).
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DM = 62.7% DM = 53.0% DM = 45.7% DM = 36.7%
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Figure 3 --- Kinetics of the -elimination reaction of carrot WSP indicating (A) the inuence of temperature on nonpretreated carrot pectin samples (DM = 62.7%) and (B) the inuence of DM at a constant temperature (110 C). AIR = alcohol insoluble residues.
Vol. 8, 2009COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY 91
The structural characteristics of pectin and the distribution of hydrophilic and hydrophobic groups on the molecule determine the solubility properties of the polymer. Partial demethoxylation of pectin lowers its solubility in water (Hsu and others 1965). Generally, pectin solubility in water is increased by decreasing the polymer size and increasing the methoxyester groups; however, the pH, temperature, and the solutes concentration in the environment are important (Towel and Christensen 1959; Simpson and others 1984). Increased steric interactions and the presence of charged groups reduce pectin solubility in water (Karr 1976). In commercial pectin, water-soluble carrier materials such as ne-powdered sugar or D-glucose and other compounds that increase hydration and dispersibility characteristics, are used as solubilizing aids (Towel and Christensen 1959). Postharvest changes in pectin solubility properties of fruit and vegetable materials are a common phenomenon. Redgwell and others (1997) found that the water-soluble pectin content of plantbased foods increased with ripening. This is highly pronounced in
avocado as compared to apple and watermelon cultivars. Complementary changes in pectin fractions have been reported in tomatoes, avocado, and melon (Carrington and others 1993; Rose and others 1998; Wakayabashi and others 2000; Van Linden and others 2008), with the water-soluble pectin increasing at the expense of decreasing sodium carbonate-soluble fractions. In these cases, the chelator (CDTA)-soluble fraction showed little or no change, except in melons where the polyuronide increased with ripening. Knowledge on postharvest changes in pectin solubility properties in relation to structural transformations is increasingly being accumulated (Hallett and others 1992; Redgwell and others 1997; Popper and Fry 2005; Zykwinska and others 2005; Brummell 2006). Thermal processing-related changes in pectin solubility in plant-based foods are linked to the -elimination depolymerization of pectin (Sakai and others 1993; Siliha and Giershner 1995; Voragen and others 1995; Thakur and others 1997). Despite limited information, it is evident that heat causes a substantial change in pectin solubility (Plat and others 1988; Ben-Shalom and others 1992). An increase in water-soluble pectin with increasing thermal process severity is noticed (Nyman and others 1993; Greve and others 1994; Sila and others 2005, 2006a, 2006b). This increase is paralleled by decreasing alkali-soluble pectin (Ben-Shalom and others 1992; Siliha and others 1996; Sila and others 2006b). Interestingly, preheating results in decreased pectin solubilization in water when compared to non-preheated samples (Siliha and Giershner 1995; Siliha and others 1996). The effect is more pronounced with a high-pressure pretreatment as demonstrated for carrots (Sila and others 2006b). However, the pretreatment effect is reversed by subsequent thermal processing accompanied by a preponderant change in molar mass distribution (Sila and others 2006b). When a high-pressure treatment is used as the main process, the solubilization of carrot pectin in water and its degree of depolymerization is greatly reduced when compared to thermally processed ones (De Roeck and others 2008). Decreasing the moisture content of thermally processed carrots reduces the solubility of pectin in water suggesting that the water activity (a w ) of the system inuences the heatinduced changes in polymer solubility (Georget and others 1998). Two processes seem to dictate thermal pectin solubilization: (1) rst the polymer is depolymerized via the -elimination
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WSP CSP NSP
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36.7% 45.7% 53.0% 59.2% 62.7% 0% 20% 40% 60% 80% 100%
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Figure 4 --- Interconversion of carrot pectin fractions as illustrated by (A) changing the in situ degree of methoxylation of carrot tissues using pretreatment conditions and (B) thermally processing (100 C) nonpretreated samples with increasing time.
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The analysis of complex polysaccharides presents a major challenge. Knowledge on pectin has largely been obtained from chemical-enzymatic analyses combined occasionally with microscopic examination of extracts. To understand changes in mechanical properties of plant-based foods, isolation of cell-wall materials, preferably in the form of alcohol-insoluble residue (AIR), is necessary (McFeeters and Armstrong 1984). Use of AIR precludes undesirable changes due to enzymatic activity by coprecipitating proteins, reserve polysaccharides (starch), lipids, polyphenols, and so on, while removing most free sugars and pigments (Selvendran and Ryden 1990). In some specic cases, additional washing steps are employed: for example, in starchy materials, ball milling in 80% ethanol followed by use of aqueous 90% dimethylsulfoxide (DMSO) removes the bulk of starch from the cell-wall material (Selvendran and Ryden 1990). Use of aqueous sodium deoxycholate (SDC) or sodium dodecyl sulfate (SDS) suppresses endogenous enzymes activity and removes intracellular proteins whereas phenol-acetic acid water (PAW) removes lipids and pigments (Selvedran 1975; Selvendran and Ryden 1990). The main drawback of these procedures is the destruction of intact cells making it complicated to examine ripening or processing-dependent changes in structure. However, at the molecular level, insight into the evolution of cell-wall components can be achieved. Chemical-enzymatic analysis of pectin involves different extraction protocols for sequentially isolating and characterizing the polymer. The most common methods entail pectin fractionation based on the successive extractions with water, chelating agents such as cyclohexane diamine tetraacetate (CDTA), acid (hot dilute acids), and alkali (cold dilute alkali), or extractions with cell-wall-degrading enzymes and combined chemical and enzymatic methods (Weightman and others 1994; Grohmann and others 1995; Sila and others 2006a, 2006b; Coenen and others 2007). While these isolates may be enriched with particular types of molecules, they always contain mixtures of polymers, either coextracted by solvents or polymers linked together covalently (Brummell 2006). Examining individual polymers and analyzing the degree of heterogeneity within a sample represents an important contribution to the understanding of biological macromolecules and their functionality. The chemical-enzymatic analytical tool box for cataloguing pectin structural changes is diverse and gradually expanding, but there is still room for further rene-
Titrimetric
Spectrophotometric Spectrophotometric
Dische 1947 Blumenkrantz and Asboe-Hansen 1973 Scott 1979 Walter and others 1993 FlissetiCozzi and Carpita 1991 Anthon and Barrett 2008
Spectrophotometric
Spectrophotometric Spectrophotometric
Jones and Albersheim 1972 Ford 1982 Garleb and others 1991
HPAEC-PAD
Spectrophotometric
Spectrophotometric
An improvement of the former. Fast, simple, and reproducible Simple, sensitive, and reproducible, but all the methoxy-esters may not be removed by PME Samples need to be uniformly wetted for repeatable results Sample preparation easier than HPLC, methanol peaks distinct Large sample size needed and peak interference by carbonates Small sample size, short analysis time Results are pH dependent, constant calibration, and there might be interference from other components
Spectrophotometric
McFeeters and Armstrong 1984 Huisman and others 2004 Voragen and others 1986 Levigne and others 2002 Gnanasambandam and Proctor 2000
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MS is a powerful tool for the structural characterization of carbohydrates based on their molecular mass, as well as the mass of their respective fragments. Insight into the polymer molecular mass, constituent monosaccharides, sequence of the monosaccharides, linkage type, stereochemistry of the monosaccharide units, anomericity of the glycosidic bond, branching positions, type of branching, modifying groups, types of modifying groups, and the quantity, can be obtained. In pectin research, the polymer can rst be fragmented enzymatically or nonenzymatically into its substituent oligomers, followed by separation by new sensitive MS techniques such as MALDI-TOF (matrixassisted laser desorption ionization-time of ight), ESI-ITMS (electrospray ionization-ion trap mass spectrometry) and ESI-Q-TOF (electrospray ionization-quadrupole-time of ight). The ESI-ITMS technique has successfully been applied for the structural determination of partially methoxylated and acetylated oligogalacturonides released from pectins (Qu em ener and others 2003a, 2003b). In sugar beet pectin isolates, ESI-ITMS technique has permitted the precise localization of ferulic acid substituents on arabinan oligosaccharides (Levigne and others 2004a, 2004b; Qu em ener and Ralet 2004). The ne structure of complex pectinderived oligosaccharides has been elucidated by coupling MS with other techniques such as CE-IT-MS and CE-MSn (Coenen and others 2008). By combining this technique with other available pectin analysis methodologies, new pectin structures can be identied, which can further bolster the available knowledge.
Recent developments in microscopy have aided in abstracting more information about the plant cell wall and its constituents at varying scales (Table 3). By combining the subtractive methods with cytochemical staining techniques, visualization of pectin isolates can be achieved (Roland and Vian 1991; Jauneau and others 1998) providing more ex situ information on the galacturonic acid content, degree of methoxylation and acetylation, localization of esteried groups, the molar mass distribution patterns, neutral sugar prole, anionic sites, distribution of galacturonic acid residues, the position of their glycosidic linkages, and, when coupled with selective enzymatic extraction, they can identify regions of distinct composition in pectin fractions. Powerful imaging technologies like atomic force microscopy (AFM) are increasingly nding application in pectin isolate studies (Yang and others 2006). The limitation of ex situ methodologies is lack of understanding of the subtle variations and complexities occurring within individual cells and tissues. Structurefunction relations are difcult to elucidate in planta, because of the difculty in selectively characterizing the polymeric ne structure in situ or removing biopolymer material for ex situ analysis without modifying the ne structure, changing its location and/or interactions within the host biomaterial. An in-depth insight can be obtained when the current ex situ pectin characterization procedures are complemented by in situ conrmations. This can help to locate, map, and identify the functionally relevant pectin molecules targeting plant structure engineering. Currently, advances in imaging technology allow direct in planta visualization of the molecular architecture of cell walls and the modications that occur to the polymers during growth and development (McCann and others 2001). Combined with the use of probes, imaging techniques can provide intricate details on the pectin polymer structure unquestionably unraveling newer insights into structurefunction relations. Among the traditional in situ imaging techniques applied to study cell-wall changes are light microscopy (Van Buggenhout and others 2006a, 2006b; Sila and others 2007b), transmission electron microscopy (Iwai and others 1999; Wi and others 2005), scanning electron microscopy (Rouilly and others 2006), and so on, whereas advanced imaging techniques like confocal laser scanning microscopy (CLSM), Fourier transfer infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), and others, are increasingly being explored. The application and some of the advantages and disadvantages of the different microscopic techniques are outlined in Table 3. However, most of the published work in this area focuses on studying cell-wall changes during plant growth and development. Food scientists can benet enormously by exploiting the advantages of imaging technology combined with the use of probes in an attempt to explain and engineer processingdependent changes of plant-based foods. In this review, AFM and uorescence microscopy will be discussed in more detail as they present some of the most exciting recent insights in the area of pectin cellular and tissue localization. Atomic force microscopy. Atomic force microscopy (AFM) is one of the most powerful tools for determining the surface topography of native biomolecules at subnanometer resolution (Shao and others 1996). Atomic-scale resolution is achieved by scanning an object point by point while contouring it as a constant small force is being applied. The technique has found application in imaging plant cell-wall polysaccharides thus providing information on the polymer mass and contour length distribution (Ridout and others 1998), interpolymer interactions and associations with other biomolecules (Adams and others 2004), the degree of branching (Gunning and others 2000, 2003; Round and others 2001; Adams and others 2003, 2005; Ovodova and others 2006), and in some cases the micro-mechanical behavior
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Table 3 --- Main advantages and limitations of the different microscopy techniques used in pectin research. Technique Application When combined with general staining : in situ analysis of cell-wall changes like a failure mechanism (cell rupture) and cell-wall separation When combined with specic staining (immunolabeling): in situ analysis of pectin structure Main advantages - Simple and effective technique - Large scanning area - Ambient temperature and pressure conditions - Minimal sample preparation Main limitations - Low spatial resolution - Low contrast without staining
Light microscopy
- Optical sectioning/3D images When combined with immunogold-labeling: in situ analysis of pectin structure - High resolution (to 1 nm) - Wide range of magnications (<20 to >80000) - Rapid analysis - Optical sectioning/3D images
- Blurred pictures due to out-of-focus material in thick samples - Extensive sample preparation for sectioning - Expensive - Sample exposed to vacuum - Extensive sample preparation - Expensive - Sample must be sectioned
Transmission EM Scanning EM
Ex situ analysis of individual pectin molecules (molar mass distribution, nature of branching. . . ) and of pectin interactions
- No contact, no light source - Angstrom resolution - Images of individual atoms, molecules - Ambient temperature and pressure conditions - Minimal sample preparation - Qualitative and quantitative data - Nondestructive technique - Spatial resolution (10 m) - Spatial resolution (1 m)
Vibrational microspectroscopy
FT-IR microspectroscopy Raman microspectroscopy
In situ analysis of pectin in plant cell walls: chemical and conformational changes
- Spectral interpretation - Not frequently used in pectin research (up to the present) - No confocal depth analysis - Cannot be performed on uorescent samples - Expensive - Complex technique
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Table 4 --- Monoclonal antibodies (Mabs) for mapping the localization and distribution of pectin polymers in planta. Mabs Antigen/epitope Reference Willats and others 1999 Willats and others 2000 Willats and others 2000 Clausen and others 2003 Willats and others 2000 Clausen and others 2003 Liners and others 1989 Liners and others 1992 Willats and others 2001 Clausen and others 2003 Oomen and others 2002 Obro and others 2004 Willats and others 1998 Oomen and others 2002 Obro and others 2004 Willats and others 2000 Clausen and others 2004
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Homogalacturonans (HG) PAM1 Binds to long stretches of unesteried HG, and needs about 30 contiguous nonmethoxy-esteried GalA residues for binding JIM5 Recognizes partially methoxy-esteried (up to 40%) HG epitopes: unesteried residues adjacent to or anked by residues with methoxy-ester groups. Relative low DM JIM7 Recognizes partially methoxy-esteried (up to 80%) epitopes of HG: methoxy-esteried residues with adjacent or anking unesteried GalA/alternating methoxy-esteried GalA residues. Does not bind to unesteried HG. Relatively high DM 2F4 Recognizes the de-esteried HG domain of pectin in the dimeric association of pectic chains through calcium ions. Binds to HG with DM up to 40% LM7 Requires 4 unesteried Galacturonic residues between methoxy-ester groups. Can be used to detect PG/PL fragmentation products Rhamnogalacturonans (RG) LM5 Recognizes galactan side chains, (14)--D-galactan LM6 Recognizes arabinan side chains, (15)--D-arabinan Recognizes xylogalacturonan Recognizes feruloylated-(14)--D-galactan
LM8 LM9
arabinose residues making feruloyl groups linked to the inner part of the side-chains more accessible (Micard and Thibault 1999). Although both the extent and nature of in vitro formed cross-links clearly differ from the cross-links found in in vivo cross-linking, enzyme-treated sugar beet pectin has some potential for food applications. For example, sugar beet pectin added to diluted black currant juice, milk, and luncheon meat also forms a gel upon enzymatic treatment. Hereby, laccases seem to be more efcient than POD (Norsker and others 2000). These observations open the door for tailored sugar beet pectin to be used as a functional food ingredient. However, unwanted side effects of the enzyme treatment, like oxidation of anthocyanins and lipids, should be minimized or avoided before industrial implementation.
Plant-based food
Figure 5 --- An integrated approach toward intelligent engineering of the mechanical properties of plant-based foods.
Molecular scale: Pectin Functionally relevant parameters -Galacturonic acid content -Degree of methoxylation =DM -Pattern of methoxylation -Degree and pattern of acetylation -Degree of polymerization -Pectin solubility -Degree and type of branching -Kinetics of pectin degradation
Mesoscopic scale: Cell Key morphological parameters -Cell integrity intactness -Micromechanical properties -Percentage damaged cells -Cell wall area changes -Cell failure mechanism
Macroscopic scale: Tissue Key texture attributes -Hardness, toughness -Rate of texture decay -Final texture index
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structural features aiming at specic functionalities. Other possible benets of the covalent cross-linking mechanisms include facilitating the incorporation of high ber ingredients into foods and formulations of new functional ingredients. A broader perspective and newer applications can emerge once the pectin structurefunction relationship is extended to a broad spectrum of pectin sources. For instance, sugar beet pectin has recently been labeled as a potential emulsier in food systems. Citrus pectin has also been shown to produce stable emulsions (Leroux and others 2003); however, there is little/no detailed description of the pectin structureemulsifying property relationships. Strategies for controlling/improving the emulsifying power of such kinds of pectin should be established through product formulation and process engineering. As the understanding on processstructurefunction relationships of pectin advances, other novel food applications will emerge. Knowledge in the food sector can be extended to pharmaceutical and nutraceutical applications, further widening the pectin exploitation scope. For instance, very recently, chemists developed a material from pectin and other natural polymers that can be used in biomedical supplies, such as prosthetic devices and scaffolding for tissue repair (Liu and others 2007). Other novel discoveries include the use of pectin fragments from orange peel to promote health by increasing the growth of benecial probiotic bacteria in the large intestine (Eliaz and others 2006). Research to elucidate and engineer the new functions is still in the initial phase. To obtain a holistic picture of the causeeffect relationship, abstracting information at the molecular, mesoscopic, and macroscopic scale is imperative. A combination of ex situ and in situ analyses, complemented by improved in muro/in planta examination, coupled with a better understanding and control of the mechanisms inuencing pectin structurefunction relations is important for improving the quality of plant-based foods.
Acknowledgment
The authors thank the Research Council, the Interfaculty Board for Development Cooperation of KULeuven, and the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen) for their support. S. Van Buggenhout is a Postdoctoral Research Fellow of the Research Foundation Flanders (FWO).
References
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