Pectins in Processed Foods: Structure-Function Relationships

Pectins in Processed Fruits and Vegetables: Part II Structure Function Relationships
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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Pectin structurefunction relationships . . .

regulated polymers. It is structurally characterized by a long sequence of alternating rhamnose and galacturonic acid residues (Voragen and others 1995). Acetylation is possible on the galacturonic acid residues in RGI; however, the presence of methoxyesters is questioned (Morra and others 2004). In the case of ax, a methoxylated RGI-like cell-wall fraction has been reported (Rihouey and others 1995). Some of the rhamnosyl residues may be decorated with arabinan and/or galactan side chains. HG is a polymer of unsubstituted galacturonic acid (GalA) residues of approximated minimum length of 72 to 100 GalA residues (Thibault and others 1993) which can be methoxy-esteried at C-6 and/or acetylated on O-2 and O-3 (Mohnen 1999; Vincken and others 2003). In some cases such as in pea seed coats, apple, watermelon fruit, carrot, and others, the galacturonic acids of HG are substituted at C-3 with xylose (Renard and others 1997), while in duck weeds (Lemna minor and Spirodela polyrrhiza), substitution with apiose at C-2 or C-3 occurs (Longland and others 1989). These structural features, and the related modications, characteristically dene the functional properties of HG in ways that are not exactly known. The functions of the complex pectin domain remain vague and much research effort is needed to unravel pectin structurefunction relations, in particular the role in planta. The main aim of this review is to comprehensively discuss the latest ndings on the question of pectin structurefunction relationships, including the evolution in the pectin-analytical tool box. ions, ionic strength, temperature, and so on) strongly inuence functionality in complex ways due to the diverse mechanisms of molecular association (Smit and Bryant 1968; Gilsenan and others 2000). The best known and the most exploited pectin structurefunction relationship is the involvement of HG in calcium-mediated gel formation, qualifying it as a thickening, stabilizing, gelling, and/or texturizing agent. Among the most common applications, pectin is used as an ingredient in jams, jellies, confectioneries, desserts, yogurts, and more, due to its unique interactions. Cross-linking of HG with Ca2+ is promoted by the presence of long nonesteried consecutive galacturonic acid chains and only small amounts of hairs. In potatoes, the proportion of the hairy regions is too high (Ryden and Selvendran 1990), while in sugar beets the polymer is highly acetylated and is of relatively small molar mass explaining the poor gelling ability of pectin from such sources (Renard and Thibault 1993). Gelation is also dictated by the amount and distribution of methoxy ester groups over the entire pectin molecule (Thibault and Rinaudo 1985; Kravtchenko and others 1992; Thibault and Ralet 2003; Capel and others 2005; Strom and others 2007). In the case of in situ changes in solid plant-based foods, Ca2+ cross-linking of demethoxylated pectin is exploited in texture engineering: low-DM pectin being associated with higher thermal resistance (Sila and others 2004; Vu and others 2004; Smout and others 2005). For more information on this refer to Part III, the complementary publication by Van Buggenhout and others. Improved cation exchange capacity of pectin is currently exploited in decontaminating industrial efuents containing metals (Dronnet and others 1998; Thibault and Ralet 2003) and in mopping up heavy metals (lead, mercury, arsenic, and other toxic metals) from human blood (PectaSol R Modied Citrus Pectin, EcoNugenics, Santa Rosa, Calif., U.S.A.). The hydration property of pectin makes it suitable for the development of ber-enriched products. Dietary pectin is health promoting as it reduces the total cholesterol level and also decreases low-density lipoprotein cholesterol in blood (Aprikian and others 2003; Ram rez and others 2007). In pharmaceutical industries, pectin efciently encapsulates many active drug components thus making it a good drug delivery system (Huguet and others 2006); and in some cases, oral administration of modied pectin is associated with treating cancer (Pienta and others 1995; Nangia-Makker and others 2002). Highly acetylated pectin, like sugar beet pectin, shows good emulsion properties (Leroux and others 2003; Drusch 2007). On the other hand, knowledge on the degradation of the pectin structure using pectinases increases the extraction yield of juices and concentrates, and it can also be used to tailor juice cloud stability and clarity (Mutlu and others 1999; Demir and others 2007; Wilinska and others 2008). Ferulic acid can be biotransformed into avoring compounds like vanillin through chemical pathways (Rosazza and others 2005). Many plant-based food functionalities can be improved, controlled, and engineered, but a combination of new techniques and integration of approaches holds the key to success in this eld.
Pectin structurefunction relations

The quality characteristics, specically textural and rheological properties, of many plant-based foods depend largely on the pectin content and composition in combination with the type of postharvest handling processes and/or (pre-)processing steps applied. Great progress has been made in the characterization of the ne structure of pectin; however, the interaction of its constituent components to systems with specic biological and industrial function is not fully understood. The suitability of pectin for any application is governed by its structural features such as molar mass, neutral sugar content, proportions of smooth and hairy regions, ferulic acid substitution, amount of methoxy and acetyl esters, and distribution of the ester groups on the polymer (Daas and others 1998; Braccini and others 1999). Any process capable of modifying the molecular parameters may lead to signicant changes in the functional properties. In addition, the unique characteristics of pectin allow an interestingly diverse interaction space where hydrogen bonding, hydrophobic interactions, polyelectrolytic behavior, specic ionic interactions, and even covalent coupling can all play a possible role in determining the functional properties of the holding matrix. Specic pectin structural components have been mapped to specic regions of the plant cell wall, strongly suggesting that they play certain biological functions. In dicotyledons, pectin is the main polysaccharide constituent of the middle lamella. It is linked with controlling cell-wall porosity, intercellular adhesion, and controlling the ionic environment of the cell wall (Carpita and Gibeaut 1993). Different kinds of cross-links are important for strengthening the cell wall, intercellular adhesion, and normal growth in vascular plants (ONeill and others 2004). More recent research is asking how inter- and intramolecular distributions of methyl esters affect pectin functionality in vivo (Gaffe and others 1997; Willats and others 2001). Pectin exhibits a range of functional properties that are important for food technological, nutraceutical, and pharmaceutical applications. This depends on its structural changes and the state transitions involved during processing or treatment (Godeck and others 2001). The relations between these parameters with the conditions of the system (pH, dissolved solids, specic metal
Mechanisms Inuencing Pectin Functionality

Pectin biosynthesis
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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partly due to the structural complexity of the pectin triads which imparts diverse physical and biochemical properties important for biological and industrial functions (Ridley and others 2001; Willats and others 2001; Bacic 2006). To really understand pectin functionality, identication and manipulation of the biosynthetic enzymes and corresponding genes are necessary. To date, research has focused on understanding and controlling the mechanisms that inuence pectin biosynthesis, its topology in membranes, tracing the combination of proteins/enzymes needed in forming its functional complexes, and its deposition into the wall. In vivo labeling and immunocytochemical analyses suggest that pectin is synthesized in the Golgi apparatus (Staehelin and Moore 1995; Goubet and Mohnen 1999; Abdel-Massih and others 2007). A study of pectic polysaccharide localization using antibodies suggests that HG and RGI are synthesized within different compartments of the Golgi apparatus (Zhang and Staehelin 1992; Sterling and others 2001) and are transported from the Golgi apparatus in vesicles that migrate to, and fuse with, the plasma membrane. The synthesized polysaccharides are then freed into extracellular space and deposited into the cell wall (Balu ska and others 2005). The number of enzymes involved in the process is largely dictated by pectin complexity. Functional genomics and mutant studies indicate that the biosynthetic enzymes required comprise glycosyltransferases and a number of decorating enzymes including methyltransferases, acetyltransferases, and feruloyltransferases (Ishikawa and others 2004). No direct evidence has yet been reported for the subcellular localization of the glycosyltransferases. Of the 412 putative glycosyltransferases identied in the completed genome sequence of Arabidopsis (Henrissat and others 2001), it is predicted that at least 53 different glycosyltransferases (GTs) are required for pectin synthesis (Ridley and others 2001). However, genes for only 2 putative pectin biosynthetic GTs have been identied (Bouton and others 2002; Iwai and others 2002), but none of the biosynthetic enzymes has been puried using the traditional biochemical purication techniques (Ridley and others 2001). There are at least 11 different monosaccharides present in pectins, and thus it is expected that at least 11 different nucleotide sugars are involved in pectin biosynthesis. To accommodate the need for such a diversity of nucleotide sugars, plants possess an extensive nucleotide sugar interconversion pathway for the de novo synthesis of those nucleotide sugars (Feingold and Avigad 1980). The relative abundance of pectin and its structural details differs among cell types and species, and because of the lack of direct biochemical evidence of the enzymes involved and their specic activities, pectin biosynthesis remains unclear. While questions remain regarding pectin biosynthesis, the identication of genes encoding the biosynthetic enzymes is needed to elucidate the structure and function of each enzyme and to determine how the enzymes work together to synthesize the structurally complex pectic polymer (Ridley and others 2001). Generation of the required nucleotide sugars and oligo/polysaccharides substrates and determining the activity of the related biosynthetic enzymes holds a major breakthrough in understanding pectin biosynthesis and functionality. A single analytical approach cannot fully resolve the pectin paradox. A concerted effort of genetic, molecular, biochemical, and chemical approaches is necessary to be able to wholly understand pectin phylogeny and biosynthesis. For more information on pectin biosynthesis refer to Mohnen (2008).
Pectin cross-linking mechanisms
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

reactions, only the well-known HG conversion reactions are indicated in Figure 1. Knowledge about pectin conversion can help to (1) identify and track the most important enzymatic and nonenzymatic reaction mechanisms leading to pectin structural changes, (2) identify the key factors regulating the pectin conversion reactions, (3) ngerprint pectin structural changes and the impact of such changes in relation to physical properties of the system, (4) provide information on the nature and characteristics of postconversion products, and (5) open opportunities to custom-tailor pectin for food and other applications. Knowledge on the (bio-)chemical basis of pectin modication/degradation in plant-based foods is still inadequate. However, some of the modication/degradative changes and the impact of such transformations on physicochemical properties can be anticipated ultimately giving room for manipulating desired functional properties. Enzymatic pectin conversion. A wide range of endogenous Table 1 --- Different types of pectin based on molecular in- and exogenous enzymes can synergistically modify and degrade pectin smooth and hairy regions. In the smooth region, the enteraction and extractability (Chang and others 1993). zymes involved can either be esterases or depolymerases (see Bonding molecular Part I, the complementary publication by Duvetter and others). Type interaction Extractions Pectinmethylesterases (PME) catalyze the specic demethoxylation of HG within plant cell walls, releasing methanol and S-type pectin Weak bonds/van der With cold water protons (and creating negatively charged carboxyl groups). The Waals force demethoxylated HG can (1) cross-link with divalent ions (such A-type pectin Ionic interactions With cold chelating agents as Ca2+ , Mg2+ ) forming supramolecular assemblies and/or gels, B-type pectins Intensive hydrogen With hot water important for engineering texture/rheological properties, (2) form bonds a substrate for pectin depolymerizing enzymes, associated with C-type pectins Hydrogen bonds and With hot chelating agents texture/viscosity loss, although it is useful in increasing juice exionic interactions traction yields and in controlling cloud stability (Figure 1). The P-type pectins Covalent bonds With dilute acid/alkali depolymerizing enzymes include hydrolases (polygalacturonases
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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= PG) and lyases (pectin lyase, pectate lyase = PL). The hydrolases cleave glycosidic bonds via acid/base-assisted catalysis, while the lyases fragment the polymers via a -elimination reaction mechanism resulting in the formation of a double bond between C-4 and C-5 at the newly formed nonreducing end. They can be endo- or exo-acting. Pectin lyases preferentially attack highly methoxylated pectin chains as opposed to pectate lyases. PL has previously been thought to be of exogenous origin in plants; however, the recent discovery of abundant PL-like sequences in many plant genomes refutes this (Rodriguez and others 2002; Owino and others 2005). In the hairy region, the -1,4 bonds in RGI are hydrolyzed by rhamnogalacturonan rhamnohydrolases and rhamnogalacturonan lyases, while the -1,2 linkage is cleaved by either rhamnogalacturonan galacturonohydrolases or rhamnogalacturonan hydrolases (Mutter and others 1998). The hairy region degrading enzymes can be endo- or exo-acting. To extend the range of pectin functionality, understanding enzymatic modication of pectin is pertinent. Detailed information can be obtained by using model pectin systems and pure enzymes. However, for the correct interpretation of the results, sufcient knowledge of the enzymes mode of action as well as in-depth information on the methoxy-ester and galacturonic acid content of the degradation products is essential (Daas and others 1999). The amount and distribution of methoxy-esters on pectin is dependent on the pectin source, the extraction procedures used, and the endogenous/exogenous enzymes present. Most plant PMEs act in a processive fashion catalyzing the demethoxylation of pectin linearly along the chain of the molecule, giving rise to blocks of free carboxyl groups. Contrary, treatment of pectin with fungal PMEs (Aspergillus japonica, Aspergillus niger, Aspergillus foetidus) causes a more random cleavage of esteried carboxyl groups (Markovic and Kohn 1984; Thibault and Rinaudo 1985). Some fungal PMEs, for example, Trichoderma reesei result in blockwise arrangement of free carboxyl groups in the pectin molecule (Ralet and others 2001). The total amount of methoxy-ester groups per galacturonic acid chain is expressed as the degree of methoxylation (DM). By determining the total amounts of nonmethoxy-esteried residues released after enzymatic degradation of the homogalacturonan, information on the occurrence and average size of the nonsubstituted regions can be obtained which can be related to important functional properties. To describe the distribution of methoxy-esters over the galacturonan backbone, the term degree of blockiness = DB is used. It expresses the total amount of nonesteried galacturonic acid liberated as the percentage of the total number of nonesteried galacturonic acid present in pectin (Daas and others 1999, 2000; Strom and Williams 2004). The more the PME-induced demethoxylation, the more the nonmethoxy-esteried galacturonic acid is liberated by endopolygalacturonase action (Guillotin and others 2005). For pectins having similar DM, the higher the DB the more blockwise the distribution of the methoxy-esters in the pectin. However, pectins having similar DM and DB values may still differ in the size of the blocks. This ambiguity can be resolved by a 2nd parameter, the proportion of mono-, di-, and trigalacturonic acid in the endo-PG digests. Long degradable blocks will lead to the release of high amounts of di- and trigalacturonic acid compared to monogalacturonic acid upon PG digestion (Daas and others 1999, 2000). Pectin populations having different galacturonic acid block characteristics are present in commercial pectins that are chemically similar but having different functionalities (Guillotin and others 2005; Winning and others 2007). The degree of blockiness reveals clear differences between pectins having the same DM and different functional behavior. The yield of enzymatic conversion depends on enzyme activity and substrate accessibility. However, extrinsic and processinginduced factors like pH, salt concentration, temperature,
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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

reaction can be followed coupled with ngerprinting the molar mass distribution patterns of the thermal digest fragments. Generally, the kinetics of the -elimination reaction are described following zero order with strong temperature and DM dependence (Figure 3). For every 10 C increase in temperature, the reaction rate increases by a factor of 2 to 3.5 (Albersheim and others 1960; Krall and McFeeters 1998; Sila and others 2006a). The temperature sensitivity of the -elimination reaction can adequately be described using the Arrhenius model. Estimated E a values are in the range of 80 to 129 kJ/mol (Krall and McFeeters 1998; Sila and others 2005; Fraeye and others 2007). More recently, the -elimination reaction has been linked to in situ interconversion of pectin fractions during pretreatment and subsequent processing (Sila and others 2006b). The rate and extent of the interconversion is strongly inuenced by the in situ sample DM. In carrots, the in situ demethoxylation of pectin by elevating PME activity using various pretreatment conditions resulted in the conversion of water-soluble pectin to insoluble
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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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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.
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pectin (CSP and NSP) (Figure 4A). On the contrary, subjecting the pretreated samples to thermal processing reversed the pretreatment effect, the insoluble pectin fractions being reconverted into water-soluble pectin (Figure 4B). Evidence for the interconversion of pectin fractions is observed by monitoring the changing galacturonic acid content, the molar mass distribution proles, and the neutral sugar content (Sila and others 2005, 2006b; De Roeck and others 2008). In the case of high-pressure pasteurization (80 C, 0.1 to 600 MPa), there is a limited conversion of the insoluble pectin fractions to become water soluble when compared to thermally processed samples (De Roeck and others 2008). In this perspective, the next research question then becomes, to what extent can the pectin change when high-pressure thermal sterilization (HPS) is employed?, or, in other words, to what extent can the additional processing variable pressure inuence the kinetics of the -elimination reaction? From this perspective, the impact of HPS on plant-based foods texture/rheology and the causeeffect relation can be evaluated and compared to that of existing technologies. In the next section, relations between the pectin fractions interconversion and pectin solubility are elaborated further.
Pectin solubility properties
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
A
WSP CSP
B
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WSP CSP NSP
Degree of methoxylation
36.7% 45.7% 53.0% 59.2% 62.7% 0% 20% 40% 60% 80% 100%
Processing time (min)
NSP
90 60 30 0 0% 20% 40% 60% 80% 100%
Pectin fraction
Pectin fraction
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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reaction making the molecules small enough so that they no longer bind to the cell-wall gel framework (Rees 1972) followed by (2) partial solubilization. This is probably due to the fragmentation of the hairy region (Plat and others 1988) and cleavage of the backbone. Consequently, the aggregation of pectin molecules is reduced and the slippage factor increases (Kunzek and others 1999). There is a probability that the association with other cellwall polymers is broken (Massiot and others 1992). During the cooking of potatoes, more branched than unbranched polymers are solubilized (Marle and others 1997). In carrots, a pronounced cleavage of glycosidic bonds occurs during canning (Nyman and others 1994). Increased solubilization of nonstarch polysaccharide occurs in peas after cooking (Periago and others 1996). The mechanisms controlling these changes remain vague. However, it is explicitly clear that pectin solubility is linked to the perceived macrostructural (morphological, physical) changes in thermally processed plant-based foods (see Part III, of the complementary publication by Van Buggenhout and others). ment and improvement (Table 2). Some of the main protocols and terminologies used to study pectin structure are discussed below. Determination of degree of methoxylation (DM). Pectin in plant cell walls is esteried with methanol to a certain extent. The degree of methoxylation is expressed as a percentage of the ratio of the moles of methanol in a sample divided by the moles of galacturonic acid (Table 2). Depending on the experimental assay used in determining the terms of the ratio, the DM of the sample can vary slightly due to differences in the biochemical or biophysical principles (Table 2). However, the results should remain largely indicative and reliable (Klein and others 1995) and cross-validation of methodologies is encouraged. Chemical procedures for the DM determination mostly require bulky samples and they are time-consuming, whereas some of the noninvasive techniques like FTIR and NMR may be limited due to artifacts and interferences. More standardized and improved protocols are needed to better unravel and understand the pectin DM-function relation. Determination of degree of blockiness (DB). One way to characterize differences in distribution of substituent groups in pectin, for example, methoxy and amide groups, is establishing its degree of blockiness (DB). Information regarding the intramolecular distribution of substituent groups can be quantied, thus allowing qualitative discrimination between pectins from different sources (Daas and others 1999, 2000; Guillotin and others 2007). As the pectin molecules are too large and heterogeneous to analyze as a whole, the polymer is usually degraded to smaller oligomers that can be identied and quantied. To determine the DB, pectins can be digested with an endo-polygalacturonase (endo-PG) obtained from Kluyveromyces fragilis and the degradation products can be separated and quantied using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) at pH 5 (Daas and others 1999, 2001). Detection of the oligomers is possible after post-column addition of sodium hydroxide. The amount of nonmethoxyl-esteried mono, di-, and trigalacturonic acid released by the enzyme, compared to the amount of nonmethoxyl-esteried galacturonic acid present in the sample, is used to express the DB. However, since high salt concentration is used to aid in eluting pectin oligomers by HPAEC, online mass detection is hindered. After desalting, fully and partially methoxylated oligogalacturonides can be identied by using matrix-assisted laser desorption/ionization time-ofight mass spectrometry (MALDITOF MS) (Guillotin and others 2006). More recently, capillary electrophoresis (CE) has successfully been adapted to determine the DB of several commercial pectins (Guillotin and others 2007). In combination with laser-induced uorescence (LIF) detection, CE allows an effective separation of differently substituted galacturonic acid-containing oligomers obtained at low pH. Here, mono- and oligosaccharides are derivatized with the uorescent 9-aminopyrene-1,4,6trisulfonate (APTS) and subjected to CE-LIF (Kabel and others 2006). The resolution is high and analysis time is short. Determination of average molar mass. To determine the molar mass (MM) and control the quality of the polysaccharide, highperformance size-exclusion chromatography (HPSEC) is widely employed. The fundamental metric of size for pectin in HPSEC is based on separation according to the hydrodynamic volume of the species. Commercially, many HPSEC columns with varying separation capabilities are available. The separated pectin segments can be detected by at least 1 detector, the signal of which must represent the concentration of the polymer. Bulk property detectors like refractive index (RI) detectors are common in pectin research (White and others 1999; Sila and others 2006a, 2006b; Zhang and others 2007; Van Linden and others 2008). The MM of the fragments can be estimated by calibrating with a polymer
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Methodological Approaches toward Understanding Pectin StructureFunction Relations

(Bio)-chemical analysis of pectin
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-

Table 2 --- Advances in the analytical tool box used for the chemical characterization of galacturonic acid and methanol content in pectin. Chemical methods used for analysis of galacturonic acid Method Galacturonic acid determination Modied decarboxylation of GalA by boiling in 19% HCl or 57% hydriodic acid, trapping the CO 2 in NaOH/Ba(OH) 2 followed by titration with HCl/H 2 SO 4 Acidalcohol treatment, titration of 1% of the solution in standard alkali. Saponication of methoxy-esters, neutralization of excess alkali, and titration of carboxyl groups CarbazoleH 2 SO 4 reaction m-HydroxyphenylH 2 SO 4 reaction 3,5-dimethylphenolH 2 SO 4 reaction or XylenolH 2 SO 4 reaction Combination of carbazole and m-hydroxyphenyl method Combined enzymatic and Colorimetric (arsenic-Nelson reagent reaction) Conversion of galaturonates to alditol acetate Trimethyl silyl derivatization TFA hydrolysis followed by derivatization Combined TFA-enzymatic hydrolysis Methanol determination Alkaline saponication (NaOH), oxidation by potassium permanganate, and condensation in pentadione Alkaline saponication (NaOH), oxidation by alcohol oxidase, and condensation in pentadione Enzymatic hydrolysis (PME), alcohol oxidase, purpald Alkaline saponication (1 M NaOH) Alkaline saponication (0.1 N NaOH) Saponication in isopropanol Pectin saponication, use of CuSO 4 as a precipitant Direct measurement of methoxy-esters Analytical technique Titrimetric Conductance Advantages/ Disadvantages Reliable, no neutral sugar interference Time consuming, large samples needed (250 mg) Simple, fast, and simultaneous determination of the GalA and methoxy-ester content Correction of acetyl groups needed requiring other methods Interference from nonuronide carbohydrates More specic for uronides and less sensitive to neutral sugars except at high concentrations High specicity for uronic acids Less sensitive to neutral sugar interference Neutral sugar interference eliminated but time-consuming Simple and selective for uronic acid over neutral sugars Poor recovery of galacturonic acid Poor recovery, time-consuming Superior to GLC methods, but incomplete derivatization results in low recovery Selective, sensitive, no need for derivatization Fast, simple, but requires long oxidation time Reference McCready and others 1946 Schultz 1965 man 1979 Theander and A
Titrimetric
Gee and others 1958
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
GLC GLC HPLC
Jones and Albersheim 1972 Ford 1982 Garleb and others 1991
HPAEC-PAD
Garna and others 2006
Spectrophotometric
Wood and Siddiqui 1971
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
Klavons and Bennett 1986
Spectrophotometric
Anthon and Barrett 2004
GC GC headspace analysis HPLC HPLC FTIR
McFeeters and Armstrong 1984 Huisman and others 2004 Voragen and others 1986 Levigne and others 2002 Gnanasambandam and Proctor 2000
Moles of methanol Degree of methoxylation = Moles 100. of galacturonic acid
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which has structure and hydrodynamic characteristics close to those of polygalacturonic acid, for example, pullulan (Sila and others 2006a, 2006b). The density detector, using the principle of a mechanical oscillator (Trathnigg and Jorde 1987), can be combined with the RI detector providing more information in the analysis of aliphatic polymers. The development of light-scattering detectors has further simplied molar mass distribution detection by the use of the so-called universal calibrations which allow the determination of the MM of any polysaccharide type based on its elution volume (Gomez and others 1997; Corredig and others 2000). By coupling RI detection of the eluent with light-scattering measurements, absolute molar mass values of the polymer can be obtained. Consequently, both the average MM and the distribution of the polymer can be estimated (Corredig and others 2000). Advanced MM-sensitive detectors like multiple angle light scattering (Nguyen and others 1998) and differential viscometers (Mendichi and Schieroni 1998) are increasingly becoming popular. Determination of -elimination reaction. Mostly, the elimination reaction is followed by monitoring the evolution of unsaturated galacturonides from a sample (Kravtchenko and others 1992; Ceci and Lazano 1998; Sila and others 2005; Vu and others 2006; Fraeye and others 2007). This is based on the detection of the olenic bond of the unsaturated ester by its UV absorption at 230 to 240 nm (Kravtchenko and others 1992; Ceci and Lazano 1998). Alternatively, the periodate-thiobarbituric acid test can be used (Rombouts and Pilnik 1972; Keijbets and others 1976), but changes in molar extinction coefcients of the degradation products, which varies with chain length, have to be taken into account. Fingerprinting the molar mass distribution patterns of the thermal digest fragments using size-exclusion chromatography can provide useful insights of the extent and mechanism of -elimination depolymerization (Deckers and others 1986; Sila and others 2006a, 2006b). In some cases, the decrease in viscosity of juices and pastes or texture loss in solid plant-based foods is used to indicate the -elimination reaction (Sila and others 2005; Anthon and others 2008).
Mass spectrometry (MS) technique Microscopic analysis of pectin
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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of tissues (Jurvelin and others 1996). AFM can directly image individual pectin molecules and polymers. Notable applications include the visualization of the structures of pectin molecules extracted from tomato and sugar beet cell-wall material (Kirby and others 2008), examining sodium carbonate-soluble (NSP) and chelate-soluble pectin (CSP) from unripe tomatoes (Round and others 1997, 2001), water-soluble pectin (WSP) from peaches (Yang and others 2005), the morphological arrangement of CSP in peach (Yang and others 2006), and it can be used to help tracing the mode of action of endogenous enzymes on pectin (Limberg and others 2000a, 2000b; Vetter and Kunzek 2003). The information obtained can be used to explain the heterogeneity of a polymer at the molecular level while complementing other established techniques. The possibility to directly observe pectin systems in their native form opens an exciting window to analyze their structural and functional properties at the submolecular level. Fluorescence microscopy. Fluorescent probes are increasingly being used in a wide range of biological materials. The evolution of uorescence microscopy has stimulated research on uorescent markers with dened excitation and emission spectra as specic labels of cellular functions. If the cellular component of interest is nonuorescent, specic staining or immunolabeling techniques are used. In the latter, both direct and indirect immunolabeling is possible. Indirect immunolabeling involves using commercially available secondary antibodies against the antibody binding to the target epitope, to which a variety of different uorescent probes can be attached, such as Texas red or Lissamine rhodamine. On the other hand, direct labeling of the target antibody with a uorescent probe is possible. Kits are commercially available for conjugating different uorescent probes, for example, Alexa uor dyes (molecular probes) to target antibody epitopes. Immunouorescence antibody assays allow precise and targeted localization of a compound in situ. In plant cell-wall research, monoclonal (Mab) and/or polyclonal (Pab) antibodies can be used. Mabs are highly specic to minute amounts of conformational or even sequential epitopes. Dened Mabs against cell-wall polysaccharides and the associated phenolic components have been very useful as markers of developmental state providing insight into developmental signals (McCabe and others 1997). An extensive library of Mabs with well dened specicities for both HG and the side chains of RGI is currently being exploited in plant science, and to a very limited extent in explaining processing-dependent changes (Table 4). For anti-HG Mabs, epitope specicity can be dened by the degree and pattern of esterication and the extent of calcium
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
Wide eld microscopy
Confocal laser scanning microscopy Electron 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
Atomic force microscopy
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)
- Extremely slow - Small scanning area - Expensive
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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cross-linking (Clausen and others 2003) allowing specic localization of pectin in planta and in foods (Arltoft and others 2007). For example, JIM5 has been successfully used for in situ localization of pectin in yogurt and model milk gels (Arltoft and others 2006). Consequently, pectin molecular heterogeneity can be evaluated in the context of individual cell-wall architecture and its modications during cell development and/or (pre-)processing (Willats and others 2001). Use of Mabs combined with pectinolytic enzymes provides a valuable tool for ngerprinting pectin structural changes. Combining confocal laser scanning microscopy (CLSM) and immunouorescence allows optimal use of each of their advantages, namely, spatial analysis (3-D imaging) and antibody sensitivity at near-theoretical resolution of antigenic targets on native tissue samples (Baschong and others 2001). To further extend the pectin research methodological tool box, antibodies against pectin-modifying enzymes and the related inhibitors need to be developed. In conjunction with other rapid and noninvasive approaches, antibody tagging may usefully be integrated with molecular genetic analysis to explain and complement subtractive pectin analysis. This would necessitate localization and identication of the spatial distribution of the functionally relevant pectin domains in plant-based foods creating room for targeted manipulation to suit application. In addition, if advanced biophysical methods that permit improved imaging at nanoscale can be introduced to study processingrelated changes, more information on how pectin is organized in planta can be obtained and how this is related to physical properties. Much is still needed for the comprehension of the multivariate behavior of pectin during growth, development, and (pre)-processing. change in subtle ways depending on the source of pectin and the environmental conditions. Links between the pectin structure and rheology have been indicated (Lofgren and others 2002, 2005, 2006; Donato and others 2005; Strom and others 2007) and it has been possible to establish relations between the polysaccharide microstructure and the rheology of products by employing the polysaccharides as a stabilizer (Arltoft and others 2006). The rheological properties of pectin gels containing apple and citrus pectin have been studied extensively (Chou and others 1991; da Silva and Gonc alves 1994). High-methoxy (HMP) pectins (DM > 50%) form gels mainly by hydrophobic interactions and hydrogen bonds at a pH 3.5 and in the presence of more than 55% sugar (Oakenfull and Scott 1984). Low-methoxy (LMP) pectins (DM < 50%) are often used in low-sugar products due to their gel-forming properties without or with only a small amount of sugar and in the presence of Ca2+ . Strong synergistic effects in the rheological properties of mixed HMP/LMP gel in the presence of Ca2+ and 60% sucrose at pH 3 have been reported (Lofgren and Hermansson 2004). The DM and the distribution of methoxy-esters are important for gelation. For HMP, the higher the DM and the more blockwise the distribution of methoxy-esters the faster the gelation process (Lofgren and others 2005). For instance, stronger gels can be prepared using pectin extracted from strawberries (DM 67%) when compared to raspberry (DM 62%) and blackberry (DM 57%) pectin (Haminiuk and others 2006). In the case of raspberry jam preparations, the higher the DM of the pectin used the higher the resulting jam texture parameters (Kopjar and others 2007). The setting time of the pectin gels also depends on the DM: for a given pH, the setting time increases with decreasing DM. Recently, it was demonstrated that the degree of blockiness (DB) inuences the gelation time in the absence of Ca2+ (Lofgren and others 2005). The gelation time is shorter for samples with a higher DB, that is, the distribution of nonesteried galacturonic acids is blockwise (Lofgren and others 2005). For LMP, the rate of gelation increases with decreasing DM. Gelation in LMP is relatively less dependent of the soluble solids content and the pH value of the product when compared to HMP. The pattern of methoxylation also inuences the sensitivity of LMP to Ca2+ : the more blockwise
Pectin as an Ingredient in Food Applications: Gelling Properties

Pectin is used as an ingredient in many food applications, such as jam, jellies, yogurt, and more. The polymer is known to be a principal component for the formation of gels. Rheological characteristics like viscosity or viscoelastic properties of systems
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

the distribution of free carboxyl groups the higher the Ca2+ sensitivity (Ralet and others 2001). However, random distribution of free carboxyl groups means more homogeneous distribution of interchain cross-linking and, consequently, stronger Ca2+ gels (depending on DM) as compared to blockwise distribution. Other factors like the calcium ion concentration, sugar type, buffer salts, temperature, and others, also play a role. Because of its peculiar gelling properties, LMP is commonly used in the production of low-calorie jams, jellies, preserves, and milk desserts (Thibault and Ralet 2003; Acosta and others 2008). Two types of intermolecular association govern the gelation process: (1) interchain condensation through calcium ions and (2) nonionic associations analogous to those observed in conventional high-methoxy pectin. The 2nd mechanism is becoming progressively more signicant as the pH is decreased (Gilsenan and others 2000). For LMP, it is hypothesized that the pectin chain conformation in a solution at low pH is predominantly 3-fold, with little conformational change on adoption of the ordered, intermolecular structure. At a high pH, the conformation is predominantly 2-fold, with only limited conversion to the 3fold (acid) form on cooling (Gilsenan and others 2000). Steady shear measurements on solutions of pectin reveal shear thickening, except at the lowest concentration where no shear thickening is found (Kjniksen and others 2005). It has been demonstrated that a weak oscillatory shear perturbation builds up multi-chain aggregates in dilute solutions, or an interconnected network in a semidilute concentration condition. These association structures are mainly stabilized by hydrogen bonds and the rapid buildup of energetic cross-links is governed by a cooperative zipping of aligned and stretched chains (Kjniksen and others 2005). In general, the rate of gel structure development and gel strength is inuenced by the type of pectin. By manipulating the properties of pectin gels, the most critical material properties in plant-based foods can be identied and thus can be tailored to meet specic desired functions. Currently, citrus peel and apple pomace, both by-products of the juice industry, are the major sources of extracted pectin. However, sugar beet pulp is potentially an abundant source of low-cost pectin. It is rarely utilized because its high degree of acetylation and relatively low molar mass in conjunction with the associated proteins adversely affects functionality. Sugar beet pectin does not produce a strong network to trap free water as much as citrus pectin does, and when added to food products like orange syrup and tomato ketchup it does not show good gelling properties. However, modication of sugar beet pectin can alter functionality desirably, namely, increasing the viscosity of pastes/purees (Mesbahi and others 2005). Moreover, the presence of feruloyl groups on the sugar beet pectin side-chains implies the possibility of a nontraditional gelation process. Oxidative enzymes can oxidize ferulic acid groups, and as a result of the radical-mediated process a covalent bond is formed between ferulic acid residues forming a hydrated network. Cross-linking using peroxidase (POD) requires the addition of hydrogen peroxide, the type and the amount of dehydrodiferulate formed being greatly affected by the hydrogen peroxide concentration (Baydoun and others 2004). Instead of using a POD-H 2 O 2 system for which the oxidizing agent is a substrate, and thus using undesirable food substances, a single enzyme, laccase, can be added to gel-feruloylated pectins (Micard and Thibault 1999). The gelling rate and mechanical properties of laccase-induced gels largely depend on the level of laccase activity. Gels formed at slow rates (by adding 67 nanokatal laccase/g pectin) are more rigid. During faster gelation, the molecules do not have time to become aligned and less covalent cross-linking occurs (Kuuva and others 2003). Laccase-induced gelation is favored by the addition of -arabinofuranosidase which aids in removing the peripheral
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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.
Pectin in Relation to the Mechanical Properties of Plant-Based Foods

Texture, rheology, and/or cloud stability of juices are key quality characteristics of many plant-based foods and are increasingly becoming selection criteria for consumers. It is known that plant structure plays an important role in determining the mechanical properties of many plant-based foods thus having triggered an unprecedented research into trying to elucidate the relation(s) between these aspects. Sensory characteristics (texture/rheology) are determined by the initial structure of the food material and the changes produced by physiological, preprocessing, and/or processing conditions. It is the goal of the food processing industry to maintain and/or modify the characteristics of the food matrix through the treatments that are applied. The effect of different processing technologies (traditional and novel) on plant tissue-based foods, namely, texture degradation in solid plant-based foods and rheological property transformations in pastes/purees has been reviewed (see Part III, the complementary publication by Van Buggenhout and others). Nevertheless, considering the empirical nature of most of the studies on the effect of (pre-)processing conditions on the mechanical properties of plant-based food products, correlations between pectin molecular changes in situ with micro- and macromechanical properties of the system are suggested (Figure 5). Such an approach can be used as an example for the systematic and targeted control of plant-based foods functional properties. However, it should be noted that each case study must be treated differently due to the diverse differences in pectin content and composition, differences in the endogenous enzyme contents, presence of some texture related proteins like expansins, and so on.
Pectin in Relation to Cloud Loss in Juices

Fruit juices and juice drinks are generally sold either as claried (apple juice) or cloudy products of variable density (orange juice). The mechanism why some of these extracts stay cloudy while others spontaneously clarify is not well understood. Cloud stability is a key visual quality attribute inuencing consumer acceptability of cloudy products (Versteeg and others 1980; Cameron and others 1998; Beveridge 2002). The cloud particles impart characteristic avor, color, and mouthfeel. It is hypothesized that clouds contain a positively charged protein core enveloped in a carbohydrate shell consisting of, among others, negatively charged pectin (Endo 1965; Yamasaki and others 1967). Depectination can partially degrade the polymer exposing the inner protein core leading to aggregation between polycations and polyanions thus resulting in colloid occulation (Beveridge 1997). Electrostatic repulsion of colloidal particles and other like-charged particles occurs (McClements 1999), which might be inuenced by pH. For juice cloud to remain suspended it must be of the appropriate specic gravity, particle size, and charge (Baker and Cameron

1999). Unfortunately, very little information is available on the nature of this electrosteric stabilization and how to control it. Cloud loss is a major defect associated with enzymatic precipitation of cloud in juices, a successive process starting with pectin demethoxylation by PME (Cameron and others 1998), followed by cross-linking of the deesteried pectin chains via cations forming aggregates of increased molar mass which then precipitate (Termote and others 1977). Knowledge of the critical point at which pectin becomes susceptible to cationic precipitation can be of major use in predicting juice cloud stability. However, this cannot be easily achieved since pectin is heterogeneous and plant PMEs work processively. Five technological approaches have been put forward/employed to tackle cloud loss: (1) degradation of pectin using depolymerizing enzymes like PG and/or PL which hinders PME-induced precipitation (Kropps and Pilnik 1974). The drawback can be excessive loss of viscosity for high-viscosity products. Alternatively, use of a combination of different enzymes, preferably the hairy region-degrading enzymes such as a galactanase, an arabinanase, a -galactosidase, a -xylosidase, and/or an -arabinosidase (Heldt-Hansen and Hyttel 2000). It may be desirable to reduce the degree of acetylation of the backbone, such as, by use of the enzyme rhamnogalacturonan acetyl esterase thus enhancing the activity of the debranching enzymes (Searle-van Leeuwen and others 1992); (2) use of PME inhibitors, utilizing either pectic acid hydrolysates which compete with pectin molecules as substrate for PME action (Termote and others 1977) or use of glycoproteic inhibitors like kiwi PMEI (Balestrieri and others 1990; Castaldo and others 1991); (3) thermal inactivation of PME taking care not to cause burnt avors (Laratta and others 1995; Collet and others 2005); (4) high-pressure mild-temperature inactivation of PME, so that nutritional and organoleptic properties are not adversely affected (Hendrickx and others 1998; Yen and Lin 1998; Nienaber and Shellhammer 2001a, 2001b; Balogh and others 2004; Guiavarch and others 2005; Baron and others 2006); and (5) use of synthetic and/or natural clouding agents, the latter being preferred (Espachs-Barroso and others 2003). Despite the current knowledge, much research effort is needed to rene the understanding on the mechanism of cloud stabilization, especially the role of pectin debranching enzymes and proteins.
Challenges and Future Perspectives

To ll in the knowledge gaps and provide formulators with key information about pectin functionality, attention must be paid to rene pectin structurefunction relations. First and foremost, an understanding is required of the pectin biosynthetic machinery by identifying and purifying most of the membranebound enzymes involved in the synthesis of the polymer, its methoxylation and acetylation, and the pool of genes involved in pectin biosynthesis. Such knowledge can be the basis for upgrading low-quality agricultural raw materials using genetic engineering and plant breeding to produce plants with specic pectin structural features meeting certain biological and industrial functions. By combining in planta pectin manipulation with new pectin extraction methods and down-stream processing, tailored pectins with well-controlled degrees and distribution of methoxylation/acetylation can be obtained further enlarging the pectin structurefunction window. Success in this eld is possible by employing 2 complementary approaches: (1) completing the
Plant-based food
Mechanistic insight into Structure-function relation
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
Identification of critical material properties (correlations and link) influencing functionality
Tailoring structural features to meet desired functionality
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identication, purication, and characterization of genes coding for the membrane-bound enzymes involved in pectin synthesis, its methoxylation, and acetylation; and (2) using Arabidopsis genetics for the discovery of new genes involved in pectin synthesis. A deeper insight on the enzymatic and nonenzymatic pectin conversion reactions is needed. Most of the published work on the enzymatic pectin conversion reactions has focused on PME and PG. However, other important pectin-related enzymes such as PL, xylogalacturonan hydrolases, rhamnogalacturonan hydrolases, and lyases have been described and more are being discovered. These enzymes have been suggested to play a role in plant development and pre- and postharvest ripening processes as well as in the case of microbial invasions of plant tissues. Despite the possible role of enzymes in changing the properties of the system, information on the stability and/or catalytic activity of these enzymes is currently missing. Boosting this knowledge would possibly form a basis for bio-mimetic approaches of fully exploiting the endogenous properties of plant-based raw materials in controlled pectin modication during processing and tailoring plant tissue structure of intermediates and nal (food) products. Special attention needs to be given to nonenzymatic pectin degradation mechanisms, particularly the demethoxylation and depolymerization processes and how they affect the polymer solubility properties. Furthermore, the phenomenon of pectin fraction interconversions needs to be more deeply explored. Use of model pectin systems to discriminate and identify the critical factors dictating functionality is encouraged, a process that can facilitate interfering with reactions in food systems in favor of the desired mechanical properties. As shown in Figure 1, pectin conversion reactions are important and many routes exist for tailoring the functional properties of the system. Pectin structure assignment and tracking the molecular changes of the complex polymer triads during growth, development, and food processing is a prerequisite for fully decoding the biological and industrial functions. The expanding chemical-enzymatic pectin analysis tool kit based on pectin extracts digestion remains inadequate. Advanced instrumental imaging techniques combined with the use of probes have helped to further understand the ex situ and in situ pectin heterogeneity, topography, localization, pectinpectin and pectinnonpectic components interactions. A more insightful information catalogue on the pectin structurefunction relationship can emerge when a complementary approach is employed. This calls for concerted efforts and synergies between different disciplines and players (molecular biology, biochemistry, biotechnology, food science, pharmacists, instrument manufacturers, to name the major ones) allowing pooling of knowledge and the technologies needed to better decipher pectin structure. The discovery of new pectin coupling mechanisms, like the oxidative cross-linking via endogenous ferulic acid, opens a new dimension in pectin structurefunction engineering. Ferulic acid is widely distributed throughout the plant kingdom (Ou and Kwok 2004). Ferulic acid gels are thermo-irreversible (Norsker and others 2000) and the acid is less affected by pH changes making it very important for foods subjected to more alkaline processing conditions (Ou and Kwok 2004). The pectin coupling process through ferulic acid is mechanistically different from that of calcium ions. Therefore, probably a synergistic effect might be obtained when combining Ca2+ infusion with exogenous ferulic acid impregnation, a mechanism that remains unexplored in gelation/texture/rheology engineering. More interestingly, peroxidase also mediates pectinprotein coupling through tyrosine dimerization in planta (Oudgenoeg and others 2002). It would be interesting to know to what extent the pectin-ferulic acid and/or tyrosine cross-links are affected when peroxidase activity is enhanced. This insight can lead to tailored manipulation of pectin
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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).
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Pectins in Processed Foods: Structure-Function Relationships

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Pectins in Processed Fruits and Vegetables: Part II Structure Function Relationships

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 8, 2009

2009 Institute of Food Technologists R

Pectin structurefunction relationships . . .

Pectin structurefunction relations

Mechanisms Inuencing Pectin Functionality

Vol. 8, 2009COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY

CRFSFS: Comprehensive Reviews in Food Science and Food Safety

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 8, 2009

Pectin structurefunction relationships . . .

CRFSFS: Comprehensive Reviews in Food Science and Food Safety

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 8, 2009

Pectin structurefunction relationships . . .

110C, 30 min 110C, 1 h 110C, 2 h 110C, 4 h

Unsaturated galacturonides (mM/ g AIR)

Unsaturated galacturonides (mM/ g AIR)

110C 100C 90C

CRFSFS: Comprehensive Reviews in Food Science and Food Safety

Processing time (min)

90 60 30 0 0% 20% 40% 60% 80% 100%

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 8, 2009

Pectin structurefunction relationships . . .

Methodological Approaches toward Understanding Pectin StructureFunction Relations

Vol. 8, 2009COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY

CRFSFS: Comprehensive Reviews in Food Science and Food Safety

Gee and others 1958

GLC GLC HPLC

Garna and others 2006

Wood and Siddiqui 1971

Klavons and Bennett 1986

Anthon and Barrett 2004

GC GC headspace analysis HPLC HPLC FTIR

Moles of methanol Degree of methoxylation = Moles 100. of galacturonic acid

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 8, 2009

Pectin structurefunction relationships . . .

Vol. 8, 2009COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY

CRFSFS: Comprehensive Reviews in Food Science and Food Safety

Wide eld microscopy

Confocal laser scanning microscopy Electron microscopy

Atomic force microscopy

- Extremely slow - Small scanning area - Expensive

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 8, 2009

Pectin structurefunction relationships . . .

Pectin as an Ingredient in Food Applications: Gelling Properties

Vol. 8, 2009COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY

CRFSFS: Comprehensive Reviews in Food Science and Food Safety

Pectin in Relation to the Mechanical Properties of Plant-Based Foods

Pectin in Relation to Cloud Loss in Juices

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 8, 2009

Pectin structurefunction relationships . . .

Challenges and Future Perspectives

Mechanistic insight into Structure-function relation

Identification of critical material properties (correlations and link) influencing functionality

Tailoring structural features to meet desired functionality

Vol. 8, 2009COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY

CRFSFS: Comprehensive Reviews in Food Science and Food Safety

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 8, 2009

Pectin structurefunction relationships . . .

Vol. 8, 2009COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY

CRFSFS: Comprehensive Reviews in Food Science and Food Safety

COMPREHENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETYVol. 8, 2009

Pectin structurefunction relationships . . .