Gums and Stabilisers for the Food Industry 15

By Peter A. Williams, Glyn O. Phillips

The Royal Society of Chemistry

Copyright © 2010 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-1-84755-199-3

Contents

Structure and Characterisation,
Technofunctionality,
Mixed Hydrocolloid Systems,
Food Applications,
Hydrocolloids and Health,
Hydrogels for Medical Applications,
Subject Index, 435,

CHAPTER 1

Structure and Characterisation

PECTIC OLIGOSACCHARIDES

1 INTRODUCTION

Pectin is a natural constituent of all terrestrial plants. It is one of the major plant cell wall components and probably the most complex macromolecule in nature since it can be composed of 17 different monosaccharides, some of them being esterified by methyl, acetyl or feruloyl groups. Degrading cell wall materials or extracted pectins by purified pectolytic enzymes showed that the different monosaccharides are not randomly distributed along the pectin macromolecule but are concentrated within different pectic structural domains. Over the years, many pectic structural domains have been identified and pectins are currently viewed as multiblock co-biopolymers containing: (i) homogalacturonan (HG), (ii) xylogalacturonan (XGA), (iii) rhamnogalacturonan I backbone (RG-I), encompassing arabinan, galactan and arabinogalactan I and II sidechains, and (iv) rhamnogalacturonan II (RG-II) (Figure l).

The proportion, fine structure and maybe length of each domain vary widely depending on the plant source and the extraction conditions applied. This impinges on the pectin physico-chemical and hydrodynamic properties and hence on the functionality in the specific cellular context as well as in the food application field. Although most plant tissues contain pectin, solely few plant sources are currently used for commercial pectin extraction and commercial pectin is produced almost exclusively from citrus peel or apple pomace.

Since pectins are extremely complex multiblock biopolymers, analysis on extracted pectin whole macromolecules is not sufficient to give an insight into the pectin fine structure. To reveal its structural characteristics, pectin is commonly degraded into oligosaccharides by enzymatic means. After fractionation of the degradation products, different sets of pectic oligosaccharides, which are in the analytical range of several analytical techniques, can be obtained. These pectic oligosaccharides can be used for structural analysis, to search for biological activities, or to generate specific antibodies.

2 METHOD AND RESULTS

2.1 Fine structure of homogalacturonan domains: methylation

Homogalacturonan (HG) is the simplest and the most abundant structural domain. It consists of a linear chain of around 100 (1,4)-linked α-D-galacturonic acid (GalA) units. GalA residues are partly methyl-esterified at C-6 and both the degree of methylesterification (DM, number of methylated GalA residues for 100 total GalA residues) and the distribution of methyl-esterified residues have a deep impact on pectin associative properties via calcium ions. It has been reported that 7-20 contiguous free GalA residues are required for calcium bridging. Pectins thereby exhibit stronger interaction with calcium when the DM is low and when methyl esters are blockwisely distributed.

In the past ten years, a lot of progress has been made in the understanding of methyl esters distribution due to:

(i) the generation of adequate pectic substrates

(ii) the increased availability of pure pectolytic enzymes for deesterification or hydrolysis purposes

(iii) the development of techniques capable of separating methyl-esterified oligogalacturonides

(iv) the development of mass spectrometry for oligosaccharides structural characterization.

Series of model pectins were generated by separately treating the same mother pectin with base, fungal pectin-methyl-esterase (f-PME) and plant-pectin-methyl-esterase (pPME). Chemical deesterification and deesterification by f-PME produce a random deesterification pattern while the action of p-PME is believed to follow a single chain mechanism deesterifying pectin in a blockwise fashion (Figure 2). Model pectins differing solely by their degree and pattern of methyl-esterification were thereby produced.

One important breakthrough was the development of HPAEC at low pH followed by PAD-detection for the analysis of partly methyl-esterified GalA oligomers. Different commercial pectins were hydrolyzed by a purified endo-polygalacturonase of Kluyveromyces fragiles. This enzyme degrades HG backbone only when more than 4 adjacent non-methylesterified GalA residues are present (Figure 3). Complex elution patterns were obtained by HPAEC analysis at pH 5 with a series of non-esterified GalA oligomers of dp 1 to 3 (very small amounts of dp4 were also detected for some samples) and various peaks attributed to partly methylated GalA oligomers on the basis of off-line Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (MALDITOF-MS) analyses. Quantification of non-esterified GalA oligomers by HPAEC at pH5 allowed the authors to introduce the concept of “degree of blockiness, DB” and, more recently, the concept of “absolute degree of blockiness, DBabs”, DB being the amount of unesterified monomers, dimers and trimers liberated by the enzyme related to the total amount of non-esterified GalA units in the pectin and DBabs the amount of unesterified monomers, dimers and trimers liberated by the enzyme related to the total amount of GalA units (including methylesterified ones) in the pectin (Figure 3). Limberg et at. used a slightly different fingerprinting approach using endo- and exopolygalacturonases from Aspergillus nigerP The combined use of these two enzymes allowed quantifying the amount of GalA units located in deesterified blocks (so-called blocksequence, BS, by the authors) by simply measuring the amount of GalA monomers released. Both approaches proved to be powerful in differentiating pectin samples of similar DM but different methylesterification patterns.

Partly methylated oligomers generated by enzymatic digestion of pectin can be efficiently separated by HPAEC at low pH and by capillary electrophoresis. The various methyl-esterified oligomers can be easily recognized on MALDI-TOF-MS spectra. The development of powerful analyzers with MS/MS or MS” capability further allowed the application of mass spectrometry techniques to reveal the exact location of the methyl esters within the GalA oligomers. Unfortunately, quantification of the partly methylated oligomers is not yet possible neither by MS, nor by HPAEC or CE due to the lack of adequate standards. Although this part of the information about methyl-ester distribution is still lacking, DBabs and BS (Figure 4) proved to be good indicators of pectin calcium sensitivity

2.2. Fine structure of homogalacturonan domains: acetylation

In some plant species such as sugar beet for example, GalA units in HG regions are partly acetyl-esterified at O-2 or O-3 on top of methyl-esterification at C-6. Acetylation of HG domains strongly alter pectins associative properties via calcium ions. On the other hand, acetylation has been related to the ability of sugar beet pectin to stabilize emulsions. To get a better understanding of the relationship between the acetylation of pectin and their associative or emulsifying properties, more information about the distribution of acetyl groups onto HG domains is needed. As a consequence, sugar beet pectin was degraded by purified pectolytic enzymes and degradation products were separated by anion-exchange chromatography (Figure 5).

Several fractions corresponding to oligomers of GalA were recovered (A-1 to A-7) (Figure 5) and analysed for their dp, DM and DAc. The quantative recovery with respect to both GalA and acetyl groups was calculated. Next the different fractions were analysed by ESI-IT-MSn. The electrospray ionisation followed by formation of fragment ions by collision-induced dissociation allows sequencing of oligosaccharides. Carbohydrates undergo two types of fragmentation, those related to cleavage of glycosidic bonds and those related to cross-ring cleavages. The carbohydrate fragmentation nomenclature was introduced by Domon and Costello. The negative ion mode provides the simplest spectra with, for oligogalacturonates, two types of glycosidic cleavage ions arising from the C- and Z-series and a sole specific cross-ring cleavage ion arising from the A-series. The complementary glycosidic cleavage ions of the C- and Z-series that were observed allowed sensitive sequencing of the different oligogalacturonates (Figure 6). Furthermore, the 0.2An cross-ring cleavage ions proved to be highly diagnostic ions permitting precise assignment of acetyl groups to O-2 or O-3 of oligogalacturonates (Figure 6).

The quantitative recovery with respect to both GalA and acetyl groups, the structural characterization by ESI-IT-MS of most of the oligogalacturonates and the known chain length of HG domains permitted depiction as a quantitative representation of the different HG-derived oligomers. So a list of oligogalacturonates sequences was obtained but the puzzle of in which order they were connected one to the other in the original sugar beet pectin had still to be solved. This was recently done by using different endo-polygalacturonases known to display variable tolerance towards methyl and acetyl groups. A blockwise distribution of acetyl groups onto HG domains with, on average, four zones of 7-15 contiguous non-acetylated GalA units was evidenced, intermolecular discrepancies being however most probable.

2.3 Fine structure of rhamnogalacturonan I domains

Another major pectic domain is rhamnogalacturonan I (RG-I). The RG-I backbone consists of [2)-α-L-Rhap-(l,4)-α-D-GalpA-1] repeats, the rhamnosyl residues being substituted, mainly at O-4, by variable arabinose- and/or galactose-containing side-chains. RG-I domains are highly acetylated. Although RG-I is a major pectic structural domain, not much is known about its function in the molecular architecture of pectin. Acid-extracted pectins used as food ingredients are indeed very rich in HG domains, which determine to a large extent the physical properties of pectic macromolecules. However, in the plant cell wall context, the fine structure of “hairy regions” (encompassing RG-I domains and neutral sugars side chains) and how it evolves during growth and development is very important to understand how cell wall properties are regulated in different cell types or at different developmental stages. Modified pectic “hairy regions” (MHR) isolated from apple juice produced by the liquefaction process were structurally characterised thanks to the isolation and purification of a rhamnogalacturonase, an enzyme able to cleave galactopyranosyluronic-rhamnopyranosyl linkages. The occurrence of pectic “hairy regions” in various plant cell wall materials and their degradability by rhamnogalacturonase was then assessed. Different sub-units were identified in MHR among which stubs of the backbone rich in long arabinan side chains that are not degraded by rhamnogalacturonase and subunits corresponding to the rhamnogalacturonase-degradable part of the molecule. NMR analyses allowed evidencing that rhamnogalacturonase oligomers consist of between 4 and 9 sugar units with a backbone of alternating Rha and GalA residues, partly substituted with galactose residues linked to C-4 of the Rha moiety. The arrangement of the different subunits and particularly the distribution of the rhamnogalacturonase-degradable parts over the pectin molecules seems rather diverse depending on the plant source and the extraction conditions. More recently, selective chemical β-eliminative degradation was successful in liberating RG-I fragments, which were previously not accessible.

The complexity of pectin structure, and especially of RG-I domains, provides a multiplicity of structural epitopes and much effort has been devoted to the generation of diverse antibodies with the aim of identifying the different pectin structures in cell and development contexts. Although antibodies against arabinan and (arabino)-galactan side-chains have been developed and are available, well-characterised antibodies against the RG-I backbone have not been described yet. With the aim of producing antibodies against RG-I backbone, we recently took advantage of seed mucilage. Mucilage is released from myxospermous seeds upon imbibition, and, in Arabidopsis thaliana, consists of a water-soluble outer layer and an adherent inner layer, the water-soluble outer layer consisting of unbranched RG-I. Water-soluble Arabidopsis thaliana seed mucilage was therefore degraded by a rhamnogalacturonase for the production of unbranched RG-I oligomers of various dp. Oligomers were separated by size-exclusion chromatography and fractions corresponding to dp 6-10 were pooled. Analysis by ESI-IT-MSn showed that oligomers had an alternating sequence of Rha and GalA units and always encompassed a GalA residue at the reducing end. These oligomers were used for neoglycoprotein preparation by reductive amination (Figure 7).

Several monoclonal antibodies were generated and characterised; they are currently used to detect RG-I in cell walls in a range of plant species at different developmental stages and will thereby help to understand the biosynthesis and the role of this peculiar pectic domain.

CONCLUSION

Structural analysis of pectin is a big challenge due to the complexity of this multi-block cobiopolymer. Revealing fine structure of cell-wall polysaccharides is crucial for understanding the fine tuning of polysaccharides in the cell wall context and how this fine tuning impacts on polymer interaction properties, cell wall architecture and hence cell wall mechanical properties.

CHAPTER 2

TAILORING PECTIN WITH SPECIFIC SHAPE, COMPOSITION AND ESTERIFICATION PATTERN

C. Rolin, I.B. Chrestensen, K.M. Hansen, J. Staunstrup, S. Sørenson

ABSTRACT

Citrus fruit is industrially utilised for co-producing fruit juice, aromatic oils, and peel for pectin production. Citrus peel is today by far the most important source of commercial pectin. Amongst the citrus fruits, orange is the more abundantly available source, and it is today used for a considerable amount of pectin. Anyway, since production of pectin from orange peel involves significant technological challenges, lime and lemon have a longer tradition in the pectin industry, and together they still account for the largest production volume. Commercial pectin from lime and lemon peel is characteristic by having a very high proportion of polymeric material that in analysis upon exhaustive hydrolysis yields galacturonic acid. The molecules are on average elongated having large [η]/Mw-roportion in comparison with pectin from at least some other studied sources.

The traditional pectin manufacturing process comprises incubation of the materials at elevated temperature and low pH in order to increase the yield of pectin and also reduce the pectin degree of methyl esterification (DM) to the level that suits commercial uses. The de-esterification with acid is believed to remove esterified methyl groups randomly, so that the substitution pattern approaches statistical randomness. In published literature it is sometimes postulated that the above traits, high galacturonan content and elongated shape, are a consequence of the massive acid use. Both traits are highly desirable for the traditional technofunctional uses for pectin, viz. gels and protein stabilisation, but the putative beneficial physiological effects that have been published for diverse, mostly noncommercial, pectin preparations may perhaps depend on other characteristics. Thus, it is interesting to study pectin samples of higher non-galacturonan content, or different shape, or different pattern of methyl ester substitution.

The use of enzymes for de-esterifying pectin has been commercially implemented in the pectin industry, and it is nowadays possible to produce citrus pectin of low DM without exposing the materials to excessive acidity. It has thereby become possible to assess to how large extent the typical traits of present-day commercial pectin are predetermined by the protopectin of the raw material, versus the alternative, that they are a consequence of manufacturing conditions. The present paper addresses this question as well as the differences in DM substitution pattern between acid de-esterified and enzyme de-esterified pectin.

1. INTRODUCTION

Pectin is a generic term for a diversity of complex materials that can be isolated from plants. When characterising these materials in scientific studies they are typically broken down in some limited and controlled way, and the thus treated materials are fractionated and characterised individually. In this way it has been established that pectin molecules contain characteristically different regions, for example homogalacturonan (HG) and at least two kinds of rhamnogalacturonans, RG-I and RG-II, which, in turn, possess galacturonan-containing regions and other regions that sometimes are referred to as “the neutral sugars”. The designation “neutral” is because these polysaccharides in contrast to the galacturonans do not possess (acidic) carboxyl groups.

(Continues…)Excerpted from Gums and Stabilisers for the Food Industry 15 by Peter A. Williams, Glyn O. Phillips. Copyright © 2010 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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