The breadth and depth of knowledge of gums and stabilisers has increased tremendously over the last two decades, with researchers in industry and academia collaborating to accelerate the growth. Gums and Stabilisers for the Food Industry 11 presents the latest research in the field of hydrocolloids used in food. Bringing together contributions from international experts, the first section of the book investigates the advances in structure determination and characterisation of hydrocolloids, including the use of capillary electrophoresis. Later sections deal with rheological aspects of hydrocolloids in solutions and gels; the application of hydrocolloids in real food systems; and the interfacial behaviour and gelation of proteins. A discussion of the influence of hydrocolloids on human health is also included. Researchers and other professionals in industry and academia, particularly those involved directly with food science, will welcome this title as a source of the very latest information.
About the Author
Peter A Williams is Professor of Polymer and Colloid Chemistry and Director of the Centre for Water Soluble Polymers at the North East Wales Institute. Has published over 170 scientific papers and edited over 30 books. He is Editor-in-Chief of the international journal Food Hydrocolloids. His research is in the area of physicochemical characterisation, solution properties and interfacial behaviour of both natural and synthetic polymers. Recent work has been involved with the determination of molecular mass distribution using flow field flow fractionation coupled to light scattering, rheological behaviour of polymer solutions and gels, associative and segregative interaction of polysaccharides, development of polysaccharide-protein complexes as novel emulsifiers.
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Gums and stabilisers for the Food Industry 11
By Peter A. Williams, Glyn O. Phillips
The Royal Society of ChemistryCopyright © 2002 The Royal Society of Chemistry
All rights reserved.
CAPILLARY ELECTROPHORESIS AS A TOOL FOR THE CHARACTERISATION OF PECTIN FINE STRUCTURE
M.A.K. Williams, G.M.C. Buffet and T.J. Foster
Unilever Research Colworth, Colworth House, Shambrook, MK44 1LQ, Bedford, UK
1.1 Pectin Fine Structure
While pectin is essentially a linear co-polymer of galacturonic acid and its methylesterified counterpart, it is arguably one of the most complex of the plant cell polysaccharides. This complexity, manifest in a host of possible fine structure variants, gives pectin its utility of function and, combined with its horde of accompanying enzymes in-vivo, its ability to respond to a myriad of environmentally triggered stimuli. More generally, any process capable of modifying pectin's molecular level polymeric characteristics, such as the molar mass, the degree and distribution of methylesterification, amidation and acetylation, and the content, length and distribution of neutral sugar side-chains, may yield a tangible change in its manifest macroscopic properties. The determination of such polymeric structure attributes forms an extensive and exciting field of research of which only a small part can be covered in this article. In particular, the work reported here is focused on the characterisation of homogalactan regions and, more specifically, on the elucidation of the distribution of methylesterified residues.
1.2 Conventional Characterisation of Methylesterification
1.2.1 Average Degree of Methylesterification. At present the classical titration method'is still the official method used for the determination of the average degree of methylesterification (DE) of a pectin sample. Traditional alternatives involve the liberation of methanol by de-esterifying enzymes or by acid or alkali treatments, and its subsequent quantification by chromatography, while more recently methods have been described using infra-red spectroscopy.
1.2.2 Intermolecular Distribution of Methylesterification. Previously reported chromatographic methods for obtaining the intermolecular DE distribution (the variation in DE between different molecules in the same sample) involve binding pectin onto an ion exchange column under conditions of low ionic strength and subsequent elution of the pectin with a buffer, the ionic strength of which is gradually increased.' Fractions are collected and classically the pectin mass in the column eluates has been determined by a colorimetric assay with meta-hydroxy diphenyl (MHDP) while the DE has been inferred from the elution time." However, it should be noted that the ion-exchange chromatography (IEC) method is not without its problems. Pectins with blockwise intramolecular DE distributions are found to elute from the column in an irregular manner, which is potentially a serious problem, as the intramolecular charge distribution will be polydisperse to some degree even in a sample in which all chains possess the same average charge per residue. Furthermore, the binding of pectin to the column will be a function of its degree of polymerisation since the equilibrium constant for the binding of a polyanionic molecule to a polycationic IEC stationary phase depends on electrostatic interactions summed over all groups.
In an attempt to correct for such problems a method has been described in which eluates from IEC have been analysed by size exclusion chromatography (SEC) using a dual detection system monitoring refractive index and conductivity. In this method the DE of the eluates is actually measured, using a calibration linking the refractive index / conductivity ratio to DE, developed using pectin standards, rather than inferred from the IEC elution time. However, the eluates from IEC may still be polydisperse with respect to DE and it should be noted therefore that the DE distribution derived using these average values is still an approximation to some degree.
1.2.3 Intramolecular Distribution of Methylesterification. In general there are two approaches to the determination of intramolecular DE distribution (the spatial distribution of methylesterified residues along the pectic backbone). These are i) to attempt to measure the distribution directly by recording a property of the residues that is sensitive to the local residue type environment, and ii) to fragment the chain according to rules that depend upon the residue type environment, and analyse the fragments generated. Broadly speaking NMR methods have been applied that follow the first methodology,' while many enzymatic and chemical methods' have been described that utilise the second approach. Recent advances in this area include the separation of methylesterified enzyme digest fragments using high performance anion exchange chromatography (HPAEC) at pH 5, and the use of tandem mass spectrometry, which is able to locate the position of methylesterified residues within the surviving enzyme-resistant fragments.
The question of how to exploit the rich vein of experimental data that is now being obtained, in order to derive fine structure descriptors that efficiently encode the maximum structural information and offer useful substrate discrimination, drives a further interesting area of the work. Recently the "degree of blockiness" of a pectin substrate has been defined using the results from endo-polygalacturonase (endo-PG) digests, based on the liberation of unesterified mono-, di-, tri- and tetra-mers of galacturonic acid. Furthermore, using the proportion of unesterified mono-, di- and tri-galacturonic acid released in combination with the percentage of the total substrate galacturonic acid that is liberated in an unesterified form, and the ratio of the sums of peak areas obtained for methylesterified and unesterified fragments, sequence similarities have been represented by distance trees using computational techniques commonly exploited in the analysis of DNA and proteins. These approaches have been used to compare pectins with various average DE values and differing intramolecular distributions (generated by random or blockwise de-esterification) and clearly demonstrate a useful discriminatory ability.
Complementary approaches focus on minimising the set of pectin fine structures that are consistent with available digest information, in order to gain the most realistic representation of the polymeric backbone. This philosophy is essentially one of chain reconstruction. Practically, this type of work involves the computer simulation of test substrates and their digestion, with subsequent comparison of digest characteristics with those measured experimentally. To date this has been attempted simply with the aim of the reconstruction of the measured molecular weight distribution. However, recent attempts have been made at building more specific models of enzyme action that could, in principle, be used in such a fashion. An obvious problem here is the practical issue of computational time. To construct, and subsequently digest according to specific enzyme rules, all (statistically) possible fine structures that exist for a pectin molecule of some 500 residues in length is simply impractical. However, as more is understood about pectin biosynthesis it is possible that by limiting the set of trial pectin substrates to those that are biosynthetically likely such an approach may become more realistic.
1.3 Capillary Electrophoresis
While capillary electrophoresis (CE) has been routinely used to perform efficient separations of proteins and nucleic acids for a number of years," applications to the analysis of carbohydrates have taken by comparison longer to develop owing in part to a lack of charge or a chromophore for many members of this class of biomolecule. A number of techniques that circumvent this difficulty have now been successfully applied including complexation, for example of sugars with anionic borate and plant starches with iodine / iodide, derivatization with UV absorbing or fluorescent labels and the use of indirect UV detection at high pH. For large carbohydrate polymers derivatization methods are of limited scope as tagging can only be carried out at reducing sites, typically the end group, imparting only one label per molecule. However, unlike many other natural polysaccharides, pectins possess both charge and a UV chromophore, the carboxylate group, making CE an attractive analytical tool for the study of these carbohydrates.
In fact, one of us has previously reported that CE can be successfully used in order to measure the degree of methylesterification of a pectin sample, since there is a linear relationship between the electrophoretic mobility and the average charge per residue. While many other methods perform this feat equally well, an advantage of the electrophoretic method is its inherent separation quality. A symmetrical scaling of charge and hydrodynamic friction coefficient with degree of polymerisation (DP) means that each polymer chain, regardless of its DP, will elute according to its average charge density. Each migration time, therefore, marks species with a unique DE and peak shapes thus reflect the intermolecular DE distribution of the sample. This aspect of the CE methodology has also been investigated previously and while some differences were found between the detailed shapes of intermolecular DE distributions obtained by CE when compared with the results of a more conventional IEC-SEC methodology, average DE values, and moreover the spans of the extracted distributions, showed excellent agreement between techniques.
The aims of this article are two-fold. Firstly, further evidence that the CE peak shape is a useful measurement of the intermolecular DE distribution is presented, and secondly, it is demonstrated that the exact same CE methodology previously reported as a useful tool in the measurement of average DE values and intermolecular distributions, also offers an attractive alternative for the measurement of endo-PG digest patterns.
2. MATERIALS AND METHODS
2.1 Sample Preparation and Enzyme Digests
2.1.1 Substrates. All pectin samples were lemon peel pectins, supplied by Copenhagen Pectin (www.cpkelco.com). Initial characterisation was carried out by the manufacturer using the titration method. The pectin samples were supplied as powder and solutions were prepared by heating in de-ionised water at 60 °C for 30 minutes. Polygalacturonic acid was purchased from Sigma (www.sigma-aldrich.com) and prepared similarly.
2.1.2 Enzyme Digests. A commercial sample of endo-polygalacturonase from Aspergillus Niger was obtained from Megazyme (www.megazyme.com). Typically 1.7 U was added to 2.5 mls of 0.25 % pectin solution and digests were carried out for 48 hours at 40 °C unless otherwise stated.
2.1.3 Standards. Mono-, di- and tri-galacturonic acid were purchased from Sigma and solutions were prepared by mixing known weights of sample and de-ionised water in order to obtain the desired w / w concentration.
2.1.4 Buffers. Phosphate buffer at pH 7.0 was used as a CE background electrolyte (BGE) and was prepared by titrating aqueous 50 mM NaH2PO4 with 1 M NaOH. Sodium acetate at pH 5.0 was used as a HPAEC eluent and was prepared by titrating 1 M NaOAc with 1M HOAc. All buffers were filtered through 0.2 µm filters (Whatman).
2.2 Anion Exchange Chromatography and Fraction Collection
2.2.1 HPAEC. High performance anion exchange chromatography (HPAEC) was carried out using a Dionex-300 equipped with a Carbopac PA1 column and a PA100 guard. The separation was carried out at pH 5 and post-column addition of NaOH allowed for the use of PED detection, as reported previously. Samples were eluted with a 65 minute linear gradient of 0.05 to 0.7 M sodium acetate using a flow rate of 0.5 ml min-1, which was typically made up to 0.8 ml min-1 with the NaOH.
2.2.2 Fraction Collection. Fractions were collected from HPAEC for subsequent use in capillary electrophoresis. These were diluted, post collection, in order to achieve a sample ionic strength of 50 mM prior to injection. In contrast to higher ionic strengths that caused significant peak broadening, this was found to give negligible perturbation to the CE electropherograms, and dilution was favoured over complex desalting procedures. In order to collect sufficient quantities of the relevant methylesterified oligomers so that they could be clearly observed, even after dilution, a partial digest of a 5 % pectin sample was carried out and injected from a 500 µ injection loop. While this lead to significant peak broadening compared with the experiment carried out at lower column loadings the chromatogram was still clearly recognisable and individual peaks of interest sufficiently resolved to allow fraction collection. 100 fractions of 0.25 ml were collected.
2.3 Capillary Electrophoresis
Experiments were conducted using an automated CE system (HP 3D), equipped with a diode array detector. Electrophoresis was carried out in a fused silica capillary of internal diameter 50 µm and a total length of 64.5 cm (56 cm from inlet to detector). The capillary incorporated an extended light-path detection window (150 µm) and was thermostarted at 25 °C. All new capillaries were conditioned by rinsing for 1 hour with 1 M NaOH, 1 hour with a 0.1 M NaOH solution, 1 hour with water and 2 hours with BGE. Between runs the capillary was washed for 10 minutes with BGE. Detection was carried out using UV absorbance at 191 nm with a bandwidth of 2 ran. Samples were loaded hydrodynamically (various injection times at 5000 Pa, typically giving injection volumes of the order of 10 nL), and electrophoresed across a potential difference of 30 kV. All experiments were carried out at normal polarity (inlet anodic) unless otherwise stated. Electrophoretic mobilities, μ, are related to the migration times of the injected samples relative to a neutral marker, t and t0 respectively, by the equation:
μ = μobs - μeo = (1L/V)(1/t - 1/t0)
where L is the total length of the capillary, l is the distance from the inlet to detector, V is the applied voltage, μobs the observed mobility and μobs is the mobility of the electroosmotic flow (EOF).
3. RESULTS AND DISCUSSION
Figure 1 shows typical electropherograms obtained from 20 s injections of 0.5 % w/w solutions of pectin samples of (a) 77.8 %, (b) 55.8 % and (c) 31.1 % DE. The absorbance dip at around 4 minutes indicates the neutral marker position. It can clearly be seen that the migration time increases with decreasing DE (increasing charge).
At pH 7.0 the galacturonic acid groups, pKa = 3-4, are fully charged, and although pectin is susceptible to base-catalysed P-elimination above pH 4.5, no problems were encountered during run times of less than 20 minutes, at 25 °C, in the CE capillary. All these anionic polysaccharides migrate after the neutral marker. The observed mobility μobs is the vector sum of μeo and μ (see equation 1) and since μ is negative and smaller in magnitude than μeo the anions having the most negative mobility have the smallest μobsand thus the longest migration times. Also shown is the result obtained following a consecutive 7 s injection of all three samples. It can clearly be seen that the individual peak shapes are maintained in the mixed sample and, as reported previously, the peak widths are invariant to injection length, demonstrating that peak variance arising from the length of the injection plug is minimal. Owing to a linear relationship between electrophoretic mobility and average charge density, data such as that shown in figure 1 can be used to construct a simple transform that maps the migration time axis onto degree of methylesterification. In order to do this the electrophoretic mobilities of the peak maxima are taken to correspond to species with the sample average degree of methylesterification as determined by titration. It has previously been shown that following such a calibration, based on the simultaneous injection of three resolvable pectin standards, this approach is capable of extracting the average DE value of further pectin samples to within [+ or -] 2% (which is comparable with the uncertainties in the titration method). Furthermore, peak shapes transformed in this manner have been found to give intermolecular DE distributions whose spans agree well with those obtained by more conventional IEC-SEC methods.
Excerpted from Gums and stabilisers for the Food Industry 11 by Peter A. Williams, Glyn O. Phillips. Copyright © 2002 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents
Market Overview; Structure, Characterization and Interactions; Rheological Aspects; Hydrocolloids in Real Food Systems; Interfacial Behaviour and Gelation of Proteins; New Materials; Hydrocolloids and Health; Subject Index.