Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Trends in Food Science & Technology 17 (2006) 97–104 Review Pectin: new insights into an old polymer are starting to gel William G.T Willatsa*, J. Paul Knoxb and Jørn Dalgaard Mikkelsenc a & The Department of Plant Physiology, The University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark. (Tel.: C45 35322132; fax: C45 35322128; e-mail: [email protected]) b Centre for Plant Sciences. University of Leeds, Leeds LS2 9JT, U.K. c Danisco Biotechnology, Langebrogade 1, DK-1001 Copenhagen K, Denmark Pectin is a high value functional food ingredient widely used as a gelling agent and stabilizer. It is also an abundant, ubiquitous and multifunctional component of the cell walls of all land plants. Food scientists and plant scientists therefore share a common goal to better understand the structure and functionalities of pectic polymers at the molecular level. The basic properties of pectin have been known for nearly 200 years, but recently there has been tremendous progress in our understanding of the very complex fine structure of pectic polymers and pectinolytic enzymes. This has been made possible by synergies between plant and food research and by the application of a range of state-of-the-art techniques including enzymatic fingerprinting, mass spectrometry, NMR, molecular modelling, and monoclonal antibodies. With this increased knowledge comes the prospect of novel applications. Producers are beginning to develop a new generation of sophisticated designer pectins with specific functionalities. Moreover, the ability to manipulate pectin in planta would have a major impact on fruit and vegetable quality and processing, as well as on pectin production. * Corresponding author. 0924-2244/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tifs.2005.10.008 Introduction Pectin is all around us. It is a major component of the cell walls of all land plants and in a normal western diet around 4–5 g of pectin are consumed each day (Pilnik, 1990). Extracted pectin is widely used as functional food ingredient and it (or its EU code, E440) is listed among the ingredients of innumerable food products. Worldwide annual consumption is estimated at around 45 million kilograms, with a global market value of at least 400 million Euros (Savary, Hotchkiss, Fishman, Cameron, & Shatters, 2003). The gelling properties of pectin, that are well known to home jam makers and industrial producers alike, were first described by Hennri Braconnot in 1825 (Braconnot, 1825). In his excellent review, W. Pilnik describes how Braconnot coined the name pectin, derived from the Greek phctos ‘pektikos’ meaning to congeal or solidify. Braconnot also predicted that it would have important functions in all plants and have many applications in the art of the ‘confiseur’ (Braconnot, 1825). On all points he was quite correct and the study of this remarkable macromolecule has been pursued vigorously by both plant and food scientists ever since. In the food industry pectin is known primarily as a gelling agent and is widely used in the production of jams and jellies, fruit juice, confectionary products and bakery fillings (May, 1997; Rolin & De Vries, 1990). The other major use of pectin is for the stabilisation of acidified milk drinks and yogurts. In all application areas the fine structure of pectin profoundly affects its functionality. This is reflected in the fact that although most plant tissues contain pectin, commercial production is based almost entirely on just a few sources that have the required properties (Thakur, Singh, & Handa, 1997). Currently, citrus peel and apple pomace are the major sources of extracted pectin whilst other potentially valuable sources remain largely unused because of certain undesirable structural properties. This close relationship between structure and function has motivated research into a better understanding of pectin structure with the eventual aim of refining the production process. It is envisaged that new ‘designer pectins’ with bespoke functionalities may be produced. This can be achieved to some extent by the rational modification of extracted pectin by chemical or enzymatic treatment. A more ambitious goal is to modify pectin structure within plants before extraction—thus the manufacture of 98 W.G.T. Willats et al. / Trends in Food Science & Technology 17 (2006) 97–104 commercial pectin may start with the manipulation of plant genes rather than with fruit pulp. Several reviews have described the structure, processing and applications of pectin (Pilnik, 1990; May, 1997; Rolin & De Vries, 1990). The purpose of this article is to highlight some of the most recent advances in pectin research from both plant and food science, and to discuss the consequences for pectin production and application. New insights into pectin structure The term ‘pectin’ is a somewhat misleading since it rather implies one molecule. In fact pectin describes a family of oligosaccharides and polysaccharides that have common features, but are extremely diverse in their fine structures (Ridley, O’Neill & Mohnen, 2001). However, all pectins are rich in galacturonic acid (GalA), and the FAO and EU stipulate that ‘pectin’ must consist of at least 65% GalA. Three major pectic polysaccharides are recognised, all containing GalA to a greater or lesser extent. Homogalacturonan (HG) is a linear polymer consisting of 1,4-linked a-D-GalA, whilst rhamnogalacturonan I (RGI) consists of the repeating disaccharide [/4)-a-D-GalA(1/2)-a-L-Rha-(1/] to which a variety of different glycan chains (principally arabinan and galactan) are attached to the Rha residues. The confusingly named rhamnogalacturonan II (RGII) has a backbone of HG rather than RG, with complex side chains attached to the to the GalA residues (Ridley et al., 2001; Willats, McCartney, Mackie & Knox, 2001a). Until recently it was accepted that rhamnogalacturonan and homogalacturonan domains constitute the ‘backbone’ of pectic polymers as shown in Fig. 1(A). However, an alterative structure has recently been proposed in which HG is a long side chain of RGI (Fig. 1(B)) (Vincken et al., 2003). One thing that is not disputed is that pectins are an extremely complex and structurally diverse group of polymers. The fine structures of pectins can be extremely heterogeneous between plants, between tissues, and even within a single cell wall. The chain lengths of the various domains can vary considerably, and the sugar composition of RGI can also be highly heterogeneous. In contrast, RGII is thought to have a highly conserved structure. Moreover, GalA in HG may be both methyl-esterified and acetylated. Both the degree of methylesterification (DE) and degree of acetylation (DA) have a profound impact on functional properties and pectins are traditionally categorised as high-ester or low-ester with DEs of O50% an !50% respectively (Rinaldo, 1996; Voragen, Pilnik, Thibault, Axelos & Renard, 1995). A pectin gel is formed when portions of HG are crossedlinked to form a three dimensional crystalline network in which water and solutes are trapped (Fig. 2(B)). Various factors determine gelling properties including temperature, A Homogalacturonan Rhamnogalacturonan II Rhamnogalacturonan I B Acetyl ester Methyl ester Galacturonic acid (GalA) Rhamnose (Rha) Apiose (Api) Fucose (Fuc) Aceric acid (AceA) Galactose (Gal) Arabinose (Ara) Xylose (Xyl) Glucuronic acid (GlcA) Deoxylyxoheptulopyranosylaric acid (Dha) Ketodeoxymannooctulopyranosylonic acid (KDO) Fig. 1. The basic structure of pectin. Schematic representations of the conventional (A) and recently proposed alternative (B) structures of pectin. It is important to note that the polymers shown here are intended only to illustrate the some of the major domains found in most pectins rather than definitive structures. W.G.T. Willats et al. / Trends in Food Science & Technology 17 (2006) 97–104 Fig. 2. Structure function relationships of pectin gels. (A) Citrus fruits are one of the most important sources of commercial pectin, and shown here are some of the commercial products, jams, jellies, fruit gums and acidified milk drinks manufactured by Danisco A/S using pectin extracted from limes. (B) Pectin gels are formed when HG domains are joined at junction zones, forming a crystalline network that traps water and other molecules. (C) An extensive series of pectins was produced by de-methylesterification of a high-ester sample (E81) using PMEs from orange (P-series), Aspergillus niger (F-series) and base (NaOH, B-series). This figure only shows a subset of the samples produced and described in Limberg et al., 2002. Underlined numbers indicate DE. Fungal PMEs generally have non-blockwise action patterns, producing HG with dispersed arrangement of methyl-esters along the HG chain. In contrast, plants PMEs usually have a blockwise action pattern, producing long stretches (or ‘blocks’) of un-esterified HG. Treatment with base produces a more random distribution of methyl-esters. The critical effect of methylester distribution pattern is shown by compression testing of lime pectin/calcium gels. (D) Two gels with similar degrees, but different patterns of methyl-esterification were produced from pectin samples P41 and F43. (E) Both gels were compressed to yield point but failed in very different ways. Gels made from P41 were brittle and failed due to cracks (not visible) running vertically through the gel. In contrast, F43 gels deformed in a more plastic fashion. 99 pectin type, DE, DA, pH, sugar and other solutes, and calcium. In high-ester pectins the junction zones are formed by the cross-linking of HG by hydrogen bridges and hydrophobic forces between methoxyl groups, both promoted by high sugar concentration and low pH. In low-ester pectins junction zones are formed by calcium cross-linking between free carboxyl groups. It is increasingly apparent that the pattern of methyl-esterification is also critical in determining rheological properties and new molecular tools allow us to study methylation patterns in ever more detail (Section 3). In a recent study, a series of lime pectin samples was produced from a single highly methyl-esterified mother pectin sample with a DE of 81% (E81). Fungal and plant pectin methyl-esterases (PMEs) were then used to de-methyl-esterify E81 and to generate a population of pectin samples with a range of DEs and patterns of methyl-esterification (Willats et al., 2001b; Limberg, Korner, Buchholt, Christensen, Roepstorff, & Mikkelsen, 2000; Korner, Limberg, Christensen, Mikkelsen & Roepstorff, 1999) (Fig. 2(C)). This series of well defined samples has provided a wealth of information about the functional implications of methylation patterns. One example is illustrated in Fig. 2(D) in which two lime pectin samples with almost the same DE, but different methyl-ester distribution patterns were subjected to compression testing and found to have dramatically different rheological properties. Plants can teach us a lot about the modulation of pectin structure, and the fine tuning of HG domains in particular. In most plant cells, pectin is initially synthesised in a highly methyl-esterified form but subsequently de-methyl-esterified in muro by a battery of PMEs in order to achieve required functionalities within individual cell walls. The importance of this process is illustrated by the model plant species Arabidopsis thaliana which has at least 50 genes encoding PMEs with diverse action pattern activities (Willats et al., 2001b. Catoire, Pierron, Morvan, Hervé du Penhoat & Goldberg, 1998). Molecular tools for pectin research There are many well established methods of assessing the performance of pectins in food matrices. Several recent advances in pectin analysis now make it possible to relate these findings in great detail to pectin structure. Monoclonal antibodies (mAbs) are widely used for the analysis of pectin in plant science because they allow defined structural domains to be precisely localised in the context of intact cell wall architecture. Such antibodies are now also being used for the analysis of food matrices. As with plant cell walls, their unique feature is that they can provide information about interactions between pectin and other food components in situ. Frozen sample preparation techniques and sub-zero microscopy even allow liquid and W.G.T. Willats et al. / Trends in Food Science & Technology 17 (2006) 97–104 100 LM6 A LM8 LM5 LM7 JIM7 JIM5 2F 4 ~30 GalA Ca2+ Ca2+ Ca2+ PAM1 LM8 Anti-xylogalacturonan LM6 Anti-arabinan LM5 Anti-galactan LM7 Anti-homogalacturonan JIM5 Anti-homogalacturonan JIM7 Anti-homogalacturonan 2F4 Anti-homogalacturonan PAM1 Anti-homogalacturonan 5) Willats, W.G.T. et al. (2000) Planta 218, 673-681 Willats et al. (1998) Carbohydrate Res. 308(1–2),149-152 Jones, L. et al. (1997) Plant Physiol. 113, 1405–1412 Willatset al. (2001) J. Biol. Chem. 276(22), 19404–19413 Clausenet al. (2003) Carbohydrateres. 338(17), 1797–1800 Clausenet al. (2003) Carbohydrateres., 338(17), 1797–1800 Liners et al. (1989) Plant Physiol. 91, 1419–1424 Willats et al. (1999) Plant J. 18(1), 57-65 GA (L PA ti-X an an ti-H G( G( ti-H an M8 M1 7) JIM n( cta ala ti-g an ) ) LM B DE (%): 81 66 41 Lime 16 58 Pectin 31 series 19 64 15 0 (1 4)- -D-gal(4) - BSA Xylogalacturonan Galactan Fig. 3. Anti-pectin antibodies. (A) Antibodies (mAbs) have now been produced against many of the structural domains found in pectic polymers and the names, epitopes and references of some widely used mAbs are shown. (B) Pectin microarrays combine high-throughput robotic spotting technology with the specificity of antibodies. In this example, a series of lime pectins and pectin oligomers were spotted onto polystyrene slides. The slides were probed with anti-pectin antibodies which were then detected using a fluorescent secondary antibody (Clausen, Willats & Knox, 2003). semi-liquid systems to be sections and probed with mAbs. In recent years the number of anti-pectin antibodies has increased significantly and mAbs with specificities for numerous side chain and backbone domains are now available (Fig. 3(A)) (Willats, McCartney & Knox, 2003). Of particular relevance for the food industry are a set of antibodies that bind to HG domains. LM7, JIM7 and JIM5 all bind to partially methyl-esterified HG, but have different specificities with respect to degree and pattern of methylesterification. In contrast, the epitope recognized by PAM1 consists of long stretches of un-esterified HG whilst 2F4 binds specifically to HG that is crossed linked via calcium (Willats et al., 2001a. Liners, Letesson, Didembourg & Van Cutsem, 1989). The epitope structures of JIM5, JIM7, LM7 and PAM1 have been characterised in detail over recent years, including by the use of a series of chemically synthesised partially methyl-esterified oligogalacturonides (Clausen, Willats & Knox, 2003). Even high throughput microarray technologies are now being applied to pectin research. Microarrays of pectic polymers have been created using novel activated polymer slides to which diverse polysaccharides can be immobilised without prior derivitisation (Willats, Rasmussen, Kristensen, Mikkelsen & Knox, 2000) (Fig. 3(B)). The slides were developed for anti-pectin antibody characterisation, but are now also being used for the high throughput analysis of the interactions between pectin and other food matrix components such as caseins. Enzymatic fingerprinting is a powerful technology that W.G.T. Willats et al. / Trends in Food Science & Technology 17 (2006) 97–104 A E81 F76 F69 F5 8 101 F3 1 F43 20 mAU 15 10 5 0 –5 4 5 6 7 8 9 10 11 12 Minutes B PC3 1.0 Bi-plot P60 P70 P65 P46 DE P73 Gal Rha P53 Transpo rt Ca P76 Ar a P41 B71 Activi ty Ca 0.5 0 –0.5 PGA F11 %GalA in alcohol Insoluble frac tion B43 %Dry mat ter %GalA B15 pH in 1% so lu ti on E81 F31 Gyration radio B64 F76 F43 F58 F69 Conduc tivi ty Charge dens ity Visco sit y Gal A MW mo l/g MW Dalto n B34 PC2 –1.0 –0.8 –0.6 –0.4 –0.2 0 0.2 0.4 0.6 0.8 1.0 Fig. 4. State of art tools for pectin analysis. (A) High performance capillary electrophoresis of a series of model lime pectins (described in 2C) The mobility of the pectin samples was strongly correlated to DE the width of the peak indicates the heterogeneity of the sample molecule. (B) Chemometrics is an important new tool for the analysis of pectin using multivariate principles. A series of model pectins (black circles, see also Fig. 2(C)) were subjected to a variety of different analyses (red diamonds). Principle component analysis (PCA) was used to evaluate the relationship between the functional and physical properties. involves the enzymatic digestion of pectin using enzymes with well defined cleavage specificities, and the subsequent detailed analysis of the resulting oligomers using chromatography and/or mass spectrometry (MS) (Limberg, Korner, Buchholt, Christensen, Roepstorff & Mikkelsen, 2000. Korner et al., 1999. Korner, Limberg, Mikkelsen & Roepstorff, 1998) (Fig. 2(C)). Furthermore, analysis of GalA oligomer fragments using electron spray ionisation MS it is possible to define the methyl-esterification patterns of each oligomer. Another promising novel technology of pectin analysis is capillary electrophoresis, as shown in Fig. 4(A), this is an effective method for separating population of pectins with different methylation states (Goubet, Ström, Dupree & William, 2005). These ‘pectinomics’ techniques add to our understanding of pectin structure, but some may in the future have more direct roles in pectin production, for example for ‘in-line’ analysis or for assessing the properties of starting pulps. As with all ’omics technologies it is sometimes difficult to extract the most relevant information. Chemometrics is a powerful and novel approach for combining and analysing large amounts of data, and is now being applied to pectin research. (Cerna et al., 2003). We have analysed more than 20 model lime pectin (see also Fig. 2(C)) and commercial pectin samples using a variety of methods including fourrier-transformed infrared spectroscopy (FT-IR), FT-RAMAN and near infrared resonance, enzymatic fingerprinting, Ion-Trap-MS and MALDI-TOF, capilary electrophorhesis, and a range of chemical composition analyses. A number of functional assays of the same pectins were also performed such as gel forming properties with calcium, viscosity, ion transport parameters and calcium activity coefficients, conductivity, and stabilization of proteins at low pH. As shown in Fig. 4(B), these data were combined using principal component analysis (PCA). This kind of analysis is proving useful in relating knowledge at the molecular level to a functional properties. Pectin biosynthesis and modification—towards in planta manipulation Advances in our understanding of pectin biosynthesis and modification are of importance to the food industry for W.G.T. Willats et al. / Trends in Food Science & Technology 17 (2006) 97–104 102 two reasons. First, native pectin in plant cell walls plays as central role in the ripening, texture, and storage qualities of fruits and vegetables. Second, a fuller understanding of the molecular basis of biosynthesis and modification may allow us to influence pectin quality and functionalities in planta even before extraction begins. Several studies have demonstrated that polygalacturonase (PG) and PME activity can be reduced using an antisense approach (Brummell & Harpster, 2001). In one study, tomato fruits were produced with reduced expression of both a PG gene (LePG) and a gene encoding an expansin cell wall structural protein (LeExp1). These fruits were found to be significantly firmer during ripening and were less susceptible to deterioration during long term storage (Kalamaki, Harpster, Palys, Labavitch, Reid & Brummell, 2003). It is also possible to transgenically manipulate the side chains of pectin. Expression of an RGI degrading lyase in potato resulted in significantly reduced levels of arabinan and galactan in the tubers which also had altered biophysical properties (Oomen et al., 2002. Ulvskov et al., 2004). In principle, pectin structure could also be modulated by manipulation of the glycosyl transferase (GT) enzymes responsible for joining together the monosaccharides in pectic polymers. However, the identification of GTencoding genes has proved to be extremely difficult. Based on our current understanding of the structure of the pectic matrix, at least 53 GTs must be involved in its construction, but only two have so far been identified at the genetic level, a putative HG synthase (Qua1) and a putative glucuronosyltransferase involved in RGII synthesis (AtGut1) (7, Mohnen, 1999. Bouton et al., 2002. Iwai, Masaoka, Ishii & Satoh, 2002). The identification and characterization of the genes involved in the assembly of all three cell wall polymer systems therefore remains a major challenge for plant science. This area of research also has practical relevance for pectin producers. An example is illustrated by the recent ‘EuroPectin’ European Framework V programme aimed at improving sugar beet pectin by gene manipulation. Sugar beet pulp is potentially an abundant and low cost source of pectin, but is rarely utilised because its high DA adversely affects functionality. However, identification and characterisation of plant acetyl-transferases and esterases may in the future lead to production of pectin with improved functionality from transgenic sugar beet. Pectin and health The health effects of pectin are receiving increasing interest. It is generally accepted that a high fibre diet is beneficial to health and pectin is an important soluble fibre component of fruits and vegetables. There is clear evidence that pectin can lower cholesterol levels, serum glucose levels and may also have anti-cancer activities (Yamada, A B Casein Pectin C Apple pectin Citrus pectin 1 40 Citrus pectin 2 30 Citrus pectin 3 20 10 E 12 % Sediment % Sediment D 50 Citrus pectin Experimental 10 8 6 4 2 0 0 0 0.1 0.2 0.3 0.4 Pectin concentration (%) 0.5 0 0.05 0.1 0.15 0.2 Pectin concentration (%) Fig. 5. Pectin and milk stabilistion. (A) The stabilization of acidified milk drinks is one of then most important applications of commercial pectin and the sedimentation that occurs without pectin is clearly seen. (B) Sedimentation occurs due to the aggregation of casein. (C) When pectin is added, it is thought that HG domains electrostatically binds to casein particles, thereby preventing clumping and sedimentation. The degree and pattern of methyl esterification are important in determining the effectiveness of pectins for stabilization. (D) The activity of a set of standard pectins from apple and citrus is shown, where citrus pectin 3 is the most effective at stabilizing drinking yoghurt. (E) A novel experimental pectin product was selected by testing a range of lime pectin samples with different degrees and patterns of methyl-esterification. It was found that PMEs that produced pectin with long un-esterified blocks that were most effective at preventing sedimentation at low concentrations. W.G.T. Willats et al. / Trends in Food Science & Technology 17 (2006) 97–104 103 1996. Behall & Reiser, 1986). Pectin and pectic oligosaccharides have been shown to induce apoptosis in human colonic adenocarenoma cells (Olano-Martin, Rimbach, Gibson & Rastall, 2003). Most studies have involved relatively crude pectin preparations containing a large number of different structural domains. It has therefore been impossible to causally relate specific health related activities to defined molecular structures. However, new techniques for pectin analyses, some of which are described above, are likely to make this possible soon. Importantly, advances in chromatographic separation techniques and synthetic chemistry allow high purity pectic domains to be generated for use in animal studies and in in vitro cell cultures. Progress is being made and most evidence indicates that the complex side chains of pectin are important with regard to anti-cancer activities and other bioactive properties (Yamada, Kiyohara & Matsumoto, 2003). So far pectin producers have been hesitant about promoting the potential neutraceutical effects of their products. However, if convincing evidence of the health activities of defined pectic domains is demonstrated then this may change. with the aim for producing a pectin with the greatest stabilisation capacity at the lowest concentration. Lime pectin with long contiguous stretches of un-esterfied GalA residues was significantly more effective at preventing sediment than a range of other pectins (Fig. 5). Pectinolytic enzymes The removal of pectin is just as important for some food applications as is the addition of pectin for others. The most important classes of industrial pectinolytic enzymes are PMEs, endo- and exo-PGs and pectin lyases. These enzymes are used to improve juice yield when pressing and are also employed to regulate the degree of haze and cloudiness. Pectinases are also used during oil extraction and in coffee and tea production where they are used to remove the mucilage coat from coffee beans and to accelerate tea fermentation (Kashyap, Vohra, Chopra & Tewari, 2001). Many industrial pectinases are from microbial sources and there have been major recent advances in our understanding of their structures and activities. For example, crystal structures have been obtained for plant and microbial PMEs and fungal PG (van Santen et al., 1999. Jenkins, Mayans, Smith, Worboys & Pickersgill, 2001. Johansson, El-Ahmad, Friemann, Jornvall, Markovic & Eklund, 2002). Site-directed mutagenesis has provided detailed insights into the molecular basis of their activity. The industrial importance of PMEs with novel modes of action is increasingly recognised. Increased knowledge of such activities can lead to more rational use and the production of pectins with tailor made functionalities (Savary et al., 2003). This is illustrated by the recent development of specialised pectin for acidified milk drink stabilization (Fig. 5). Pectins prevent sedimentation of milk proteins by the electrostatic binding of HG to casein. The details of this mechanism are not fully understood, but it is known that DE and methyl-ester pattern are important parameters. A variety of pectins that had been digested with PMEs from plant, bacterial and fungal sources were tested Acknowledgements Much of the work described here was performed during two EU Framework programmes (FP4, ERBIO4CT960685 and FP5; QLK3-1999/00089) and we thank all of the participants in these consortia. Additionally we thank Tove Christensen, Hans Christian Buchholt, Hanne Thorsø and Lars Norregaard for contributions to figures. Conclusion Food consumers expect that food should be convenient to prepare, tasty, safe, healthy and should have a good shelf life. Moreover, more and more consumers leave many aspects of the preparation of daily meals to the food industry. This creates an increasing demand for functional ingredients with superior properties in the production of foods—and ‘designer’ pectins are expected to play an important role in this future. Pectin producers now have the knowledge and technology to manipulate pectin at all stages of the production process and in the forthcoming years it is likely that pectin with new and improved functionalities will be produced. However, it will be important that these advances are carefully managed, and that pectin maintains its deserved reputation as a ‘natural’ product. References Behall, K., & Reiser, S. (1986). Effects of pectin on human metabolism. In M. L. Fishman, & J. J. Ren (Eds.), Chemistry and functions of pectins (pp. 248–265). Washington, DC: American Chemical Society. Bouton, S., Leboeuf, E., Mouille, G., Leydecker, M. T., Talbotec, J., Granier, F., et al. (2002). QUASIMODO1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion in Arabidopsis. Plant Cell, 14(10), 2577–2590. Braconnot, H. (1825). Annales de chimie et de physique-Annals of Chemistry and Physics, 28(2), 173–178. Brummell, D. A., & Harpster, M. H. (2001). Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants. Plant Molecular Biology, 47(1–2), 311–340. Catoire, L., Pierron, M., Morvan, C., Hervé du Penhoat, C., & Goldberg, R. (1998). Investigation of the action patterns of pectinmethylesterase isoforms through kinetic analyses and NMR spectroscopy. Implications in cell wall expansion. The Journal of Biological Chemistry, 273(50), 33150–33156. Cerna, M., Barros, A. S., Nunes, A., Rocha, S. M., Delgado, I., Copikova, J., et al. (2003). Use of FT-IR spectroscopy as a tool for the analysis of polysaccharide food additives. Carbohydrate Polymers, 37, 241–248. Clausen, M. H., Willats, W. G., & Knox, J. P. (2003). Synthetic methyl hexagalacturonate hapten inhibitors of anti- 104 W.G.T. Willats et al. / Trends in Food Science & Technology 17 (2006) 97–104 homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. Carbohydrate Research, 338(17), 1797–1800. Goubet, F., Ström, A., Dupree, P., & Williams, M. A. K. (2005). An investigation of pectin methyl-esterification patterns by two independent methods: Capillary electrophoresis and polysaccharide analysis using carbohydrate gel electrophoresis. Carbohydrate Research, 340, 1193–1199. Iwai, H., Masaoka, N., Ishii, T., & Satoh, S. (2002). A pectin glucuronyltransferase gene is essential for intercellular attachment in the plant meristem. Proceedings of the National Academy of Science USA, 99(25), 16319–16324. Jenkins, J., Mayans, O., Smith, D., Worboys, K., & Pickersgill, R. W. (2001). Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site. Journal of Molecular Biology, 305(4), 951–960. Johansson, K., El-Ahmad, M., Friemann, R., Jornvall, H., Markovic, O., & Eklund, H. (2002). Crystal structure of plant pectin methylesterase. FEBS Letters, 514(2–3), 243–249. Kalamaki, M. S., Harpster, M. H., Palys, J. M., Labavitch, J. M., Reid, D. S., & Brummell, D. A. (2003). Simultaneous transgenic suppression of LePG and LeExp1 influences rheological properties of juice and concentrates from a processing tomato variety. Journal of Agricultural and Food Chemistry, 51(25), 7456–7464. Kashyap, D. R., Vohra, P. K., Chopra, S., & Tewari, R. (2001). Applications of pectinases in the commercial sector. Bioresource Technology, 77(3), 215–227. Korner, R., Limberg, G., Christensen, T. M., Mikkelsen, J. D., & Roepstorff, P. (1999). Sequencing of partially methyl-esterified oligogalacturonates by tandem mass spectrometry and its use to determine pectinase specificities. Analytical Chemistry, 71(7), 1421–1427. Korner, R., Limberg, G., Mikkelsen, J. D., & Roepstorff, P. (1998). Characterization of enzymatic pectin digests by matrix-assisted laser desorption/ionization mass spectrometry. Journal of Mass Spectrometry, 33(9), 836–842. Limberg, G., Korner, R., Buchholt, H. C., Christensen, T. M., Roepstorff, P., & Mikkelsen, J. D. (2000). Quantification of the amount of galacturonic acid residues in blocksequences in pectin homogalacturonan by enzymatic fingerprinting with exoand endo-polygalacturonase II from Aspergillus niger. Carbohydrate Research, 327(3), 321–332. Liners, F., Letesson, J.-J., Didembourg, C., & Van Cutsem, P. (1989). Monoclonal antibodies against pectin. Recognition of a conformation induced by calcium. Plant Physiology, 91, 1419–1424. May, C. D. (1997). Pectins. In A. Imeson (Ed.), Thickening and gelling agents for food (pp. 124–152). London: Blackie Academic and Professional. Mohnen, D. (1999). Biosynthesis of pectins and galactomannans. In D. Barton, K. Nakanishi, & O. Meth-Cohn (Eds.), Comprehensive natural products chemistry (pp. 497–527). Oxford: Elsevier. Olano-Martin, E., Rimbach, G. H., Gibson, G. R., & Rastall, R. A. (2003). Pectin and pectic-oligosaccharides induce apoptosis in in vitro human colonic adenocarcinoma cells. Anticancer Research, 23(1A), 341–346. Oomen, R. J., Doeswijk-Voragen, C. H., Bush, M. S., Vincken, J. P., Borkhardt, B., van den Broek, L. A., et al. (2002). In muro fragmentation of the rhamnogalacturonan I backbone in potato (Solanum tuberosum L.) results in a reduction and altered location of the galactan and arabinan side-chains and abnormal periderm development. The Plant Journal, 30(4), 403–413. Pilnik, W. (1990). Pectin—a many splendoured thing. In G. O. Phillips, P. A. Williams, & D. J. Wedlock (Eds.), Gums and stabilizers for the food industry (pp. 313–326). Oxford: Oxford University Press. Ridley, B. L., O’Neill, M. A., & Mohnen, D. (2001). Pectins: Structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry, 57(6), 929–967. Rinaldo, M. (1996). Physicochemical properties of pectins in solution and in gel sates. In J. Visser, & A. G. J. Voragen (Eds.), Pectins and pectinases (pp. 21–33). London: Elsevier. Rolin, C., & De Vries, J. (1990). Pectin. In P. Harris (Ed.), Food gels (pp. 401–434). London: Elsevier. Savary, B. J., Hotchkiss, A. T., Fishman, M. L., Cameron, R. G., & Shatters, R. G. (2003). Development of a Valencia orange pectin methyl esterase for generating novel pectin products. In F. Voragen, H. Schols, & R. Visser (Eds.), Advances in pectin and pectinase research (pp. 345–361). The Netherlands: Kluwer Academic Publishers. Thakur, B. R., Singh, R. K., & Handa, A. K. (1997). Chemistry and uses of pectin—A review. Critical Reviews in Food Science and Nutrition, 37(1), 47–73. Ulvskov, P., Wium, H., Bruce, D., Jorgensen, B., Qvist, K. B., Skjot, M., et al. (2004). Biophysical consequences of remodeling the neutral side chains of rhamnogalacturonan I in tubers of transgenic potatoes. Planta, 220(4), 609–620. van Santen, Y., Benen, J. A., Schroter, K. H., Kalk, K. H., Armand, S., Visser, J., et al. (1999). 1.68-A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis. The Journal of Biological Chemistry, 274(43), 30474–30480. Vincken, J. P., Schols, H. A., Oomen, R. J., McCann, M. C., Ulvskov, P., Voragen, A. G., et al. (2003). If homogalacturonan were a side chain of rhamnogalacturonan I. Implications for cell wall architecture. Plant physiology, 132(4), 1781–1789. Voragen, A. G. J., Pilnik, W., Thibault, J. F., Axelos, M. A. V., & Renard, C. M. G. C. (1995). Pectins. In A. M. Stephen (Ed.), Food polysaccharides (pp. 287–339). New York: Marcel Dekker. Willats, W. G., McCartney, L., Mackie, W., & Knox, J. P. (2001). Pectin: Cell biology and prospects for functional analysis. Plant Molecular Biology, 47(1–2), 9–27. Willats, W. G., Orfila, C., Limberg, G., Buchholt, H. C., van Alebeek, G. J., Voragen, A. G., et al. (2001). Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion. The Journal of Biological Chemistry, 276(22), 19404–19413. Willats, W. G., Rasmussen, S. E., Kristensen, T., Mikkelsen, J. D., & Knox, J. P. (2000). Sugar-coated microarrays: A novel slide surface for the high-throughput analysis of glycans. Proteomics, 2(12), 1666–1671. Willats, W. G. T., McCartney, L., & Knox, J. P. (2003). Pectin cell biology: Complexity in context. In F. Voragen, H. Schols, & R. Visser (Eds.), Advances in pectin and pectinase research (pp. 147–157). Dordrecht: Kluwer Academic Publishers. Yamada, H. (1996). Contribution of pectins on health care. In J. Visser, & A. G. J. Voragen (Eds.), Pectins and pectinases (pp. 173– 190). Amsterdam: Elsevier. Yamada, H., Kiyohara, H., & Matsumoto, T. (2003). Recent studies on possible functions of bioactive pectins and pectic polysaccharides from medicinal herbs. In F. Voragen, H. Schols, & R. Visser (Eds.), Advances in pectin and pectinase research (pp. 481–490). Dordrecht: Kluwer Academic Publishers.