Scribd Logo
Upload

Welcome to Scribd!

  • Full Text 01

    Uploaded by

    thái học “hochaha”
    12 views88 pages

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Report this Document

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    Download as pdf
    12 views88 pages

    Full Text 01

    Uploaded by

    thái học “hochaha”

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
    Flag for inappropriate content
    Download as pdf
    of 88
    SIK-report 2000 No. 665 Pectins - structure and gel forming properties a literature review Caroline Léfgren This review is a part of a PhD work at the Department of Food Science, Chalmers University of Technology, Sweden Supervisors: Anne-Marie Hermansson and Pernilla Walkenstrém ISBN 91-7290-204-3 CONTENTS 2.1 Composition and structur 1 Main backbone... 2 Side chains and substituents... 3 Hairy and smooth regions 7 . peice 4 Distribution of methoxyl ester grOUPS. wou rear 9 Enzymatic degradation.. 2.2.1 Homogalacturonan acting enzymes 222 Rhamnogalacturonan acting enzymes. 2.3. Chemical reactions. 23.1 Acidic degradation. 23.2 Alkaline degradation 233 Ammonia treatment, 2.4 Extraetion methods... 24.1 Laboratory extraction with chemical 24.2 Extraction with enzymes.... 243° Industrial extraction 3. PHYSICAL PROPERTIES. 3.1 Molecular weight. 3.2 Molecular conformation 3.3. Flexibility of the pectin chain 3.4 Solubility 3.5 Polyelectrolyte behaviour. 3.6 Aggregation in solution.. 4 HM PECTIN GELATION AND RHEOLOGY... 4.1 ‘The mechanism of gelation of HM pectin . 4.1.1 Bonds involved in gel formation... enn : 20 4.1.2 A method for estimating size of the junetion zones Piatti DD 413 — Gelation rate.. ea o aa a 4.2 Rheological Properties of HM pectin gels. 42.1 Setting temperature and setting time. 422 — Gel strength... 5 423 Gel strength measurements 424 Structure development rate 425 Ageing process, 426 Mechanical spectr 42.7 Creep Compliance. 428 Stress relaxation 5 LM PECTIN GELATION AND RHEOLOGY. see 43 5.1 The mechanism of gelation of LM pectin... 43 S11 ‘The egg-box model ae 43 5.12 The Ca" interaction... i . « 44 Sal 5.1 1 2 3 Thermodynamic aspects, 4 Gelation of amidated pectins. 52 5.2.1 Effect of extraction method. 5.2.2 Riffect of pH. 5.23. Effect of Ca” concentration, 5.2.4 The effect of pectin concentration. Rieologleal properties of LM pectin gs. 5.25 The combined calcium-pectin concentration effect... . 54 5.2.6 Effect of soluble Solid8.erern nnn een uc 55 5.2.7 Effect of temperature a 56 52.8 Effect of degree of methylation. 58 529 Effect of ionic strength “58 5.2.10 Creep compliance and stress relaxation measurements 58 5.2.11 Frequency studies eet so 6 GELATION OF SUGAR BEET PECTIN BY OXIDATIVE CROSSLINKING 7 MIXTURES OF PECTIN AND OTHER POLYMERS. 1 72 13 14 18 16 4d 8 PECTIN IN CELL WALLS... 81 8.11 812 82 821 822 — The pectin matrix.. 823 Structural proteins. 83 831 9 REFERENCES., Pectin/alginate Pectin/gelati Pectin/galactomannan. Pectin/carrageenan.. Pectin/f-lactoglobulin Pectin/easein Pectin/bovine serum albumin Cell wall components... Middle lamella. Primary and secondary cell wail 72 Cell wall structur ans The cellulose-xyloglucan network 15 Cell wall degradat Enzymatic activity... 1 INTRODUCTION It is well known that when fruits are mixed with water and sugar and heated, a gel will be formed when the mixture is cooled. The French chemist Henry Braconnot was one of the first persons who realized that this phenomenon was related to a specific colloidal substance, extracted from fruit during processing. In the year 1825 he coined the name “peetin” from the Greek word which means “to congeal” (Lockwood, 1972, Pilnik 1990). Pectin is widely used as a gelling agent in jams and jellies. Beside the gelling properties of pectin, it can be used as stabilizer, thickener and to improve the mouth feel and texture of food products. Jams and jellies are products that traditionally contain a large amount of sugar. The pectin used for this type of product, is high-methoxyl (HM) pectin, since HM pectin forms a gel in the presence of sugar and acid, However, the increasing demand for low sugar products limits the use of HM pectin. Low-methoxyl (LM) pectins are useful as gelling agents, in low sugar products. LM pectins are less sensitive to the sugar concentration but, on the other hand, require calcium ions for gel formation. Pectin is a complex polysaccharide. It ocours naturally in the cell walls in most of the higher plants. It is an essential component in the initial cell growth and in the ripening process. The ‘major part of the pestin is located in the middle lamella and in the primary cell wall. Since pectin is located in different parts in the cell wall, the extraction process required varies. The extraction method used for the preparation of pectin affects the chemical composition to varying degrees. The possibility of using different sources for pectin extraction makes the compound even more diverse and complex. However, even though pectin ean be extracted from a variety of plant sources, commercial pectins are almost exclusively extracted from citrus peel and apple pomace. The industrial production of pectin is a by-product industry using waste materials especially from fruit juice production, The complexity of pectin and the diversity of the different fractions represent a challenge to researchers in food science. This literature review is part of a Ph D project concerning the structure and functionality of pectin mixtures. The main topics of interest are the mechanisms involved in the gel formation process, possible interactions between different pectin fractions, between pectin and other biopolymers, and between pectin and cellular bound pectin. The complexity of the pectin molecule is reflected in chapter 2 of this review. Many attempts have been made to determine the composition and chemical structure of pectin. Since so much work has been done in this specific field, it would require a separate review to cover it all Chapter 2 contains a summary of the findings in this area In chapter 3 physical properties such as molecular weight, molecular conformation and aggregation of pectin molecules in solution are reviewed, The purpose of chapter 3 is to give a background to the chapters concerning gelation mechanisms. Since HM pectin and LM pectin differ in gelation mechanisms and somewhat in the gel properties, they are treated in two separate chapters. The gelation and theology of HM pectin gels are treated in chapter 4 and the gelation and theology of LM pectin gels in chapter 5, Chapters 4 and 5 are the main chapters in this literature review, and the intention has been to give a complete description of the work that has been done in this area The research concerning the gelation mechanism of HM pectin has been focused on the formation of junction zones by hydrophobic interactions and hydrogen bondings. Early studies of HM pectin were concerned with setting temperature and setting time. These parameters affect the gel properties of the resulting gel and are certainly important process parameters. Much of the early work on HM pectin also dealt with various methods of measuring gel strength. The studies may be more useful to jelly manufacturers than from a scientific point of view since it is hard to correlate the different results. The various gel strength measuring methods are listed in chapter 4. More recent studies of HM pectin gels deal with the rheological properties in terms of the structure development rate, ageing processes and mechanical spectra, The proposed egg-box model, which involves calcium ions in the gelation mechanism of LM pectin, has been known for decades. However, the interaction between LM pectin chains and calcium ions has also been thoroughly studied even in the nineties. Interesting results have been found, for example, concerning the influence of methoxyl group distribution on the interaction between LM pectin and calcium. Several studies characterize the rheological properties of HM and LM pectin gels. These studies give useful information on the functionality of pectin gels. The gel network structures are explained by the different gelation mechanisms for HM and LM pectin. It should be interesting to relate the theological properties and the gelation mechanisms to microscopy studies on pectin gels. However, results from microscopy studies of pectin gels are not very numerous. A third gelation mechanism is proposed for pectin originating from sugar beets. Since this type of pectin does not fit either the HM pectin chapter or the LM pectin chapter, itis briefly treated in a separate chapter, no. 6, Combinations of pectin and other biopolymers may give rise to interesting gel properties, such as phase separation and synergistic effects. Earlier work on pectin/biopolymer mixtures is summarized in chapter 7. Mixtures of pectin and alginate are known to cause interesting synergistic effects. The mixture can form a gel under conditions where none of the polymers an form a gel alone. Pectin and gelatin mixtures also exhibit interesting behaviour. Depending on the methoxyl group distribution in the pectin molecule, one-phase or two-phase systems are obtained. Other mixtures such as pectin/galactomannan, and pectin/B- lactoglobulin have also been studied recently. Since HM pectins, LM pectins, amidated peetins, ete. exhibit different structures, mixtures of pectins could give interesting results However, no work has been found in the pectin/pectin mixture area. This literature review concludes with a brief introduction to the world of cell walls. Cell walls are complex and fascinating. Research is progressing in this area but there is no complete description yet. In order to understand the origin of pectin, chapter 8 gives a brief orientation about pectin in cell walls. 2 CHEMICAL STRUCTURE The chemical structure of pectin is very complex and depends on the source, location and method of extraction (Thakur ef al, 1997). The chain structure of pectin consists mainly of galactopyranosyluronic acid (galacturonic acid, GalA) units. Some of the carboxyl groups are present in methyl ester form. The degree of methyl esterfication (DM, degree of methylation) divides pectins into two sections. In high-methoxyl pectin (HM) more than 50 % of the carboxyl groups are in methyl ester form and in low-methoxyl pectin (LM) less than 50 % of the carboxyl groups are in methyl ester form, Pectic acids are polymers of galacturonic acid units without, or with a negligible amount of, methyl ester groups. Salts of pectic acids are called pectates (BeMiller 1986), Pectinic acids are polygalacturonic acids with a variable, but greater than negligible, content of methyl ester groups. Salts of pectinic acids are called pectinates, Protopectin is the native pectin fractions in cell walls, which can not be extracted without degrading the structure (Voragen et al 1995), The complex chemical structure of pectin has been extensively studied. An excellent review is given by Voragen et af (1995), and the following sections (2.1-2.2) follow that work to a large extent. 2.1 Composition and structure 2.1.1 Main backbone Pectin has a chain structure mainly consisting of o-(1,4)-linked D-galacturonic acid units interrupted by the insertion of (1,2)-linked L-rhamnopyranosyl (rhamnose) residues (Neukom et al 1980). See figures 2-1 and 2-2. The rhamnose content in pectin is typically 1-4 % (Voragen et al 1995), The backbone is often referred to as the homogalacturonan chain (Schols and Voragen 1996). Figure 2-1 Schematic illustration of the polygalacturonie acid structure of pectin with DM = 40 9% From Parbo, 1067 9 ba.o-n Figure 2-2 Schematic illustration of the rhamnose insertion in the pectic backbone. From Oakenfull in “The Chemistry and Technology of Pectin" 1991. 2.1.2. Side chains and substituents Pectins consist of various amounts of sugars attached as side chains that can be linked either to the rhamnose groups or to the galacturonic acid groups. The most common are D-galactose, L-arabinose and D-xylose. Less common sugars are D-glucose, D-mannose, L-fucose and D- glucuronic acid. The composition of sugars varies according to the source of pectin, which can be seen in table 2-1. ‘Apple’ Sugarbee™ Carrot Plum’ Poiato™ Yield 14 1 13.5 28.6 13.1 Gal, 58.0 54.9 54.7 B.0 8.6 Rha + Fue 3.0 3.2 38 15, 13 Ara 23.0 12.5 117 539 70 Xyl 1.0 02 0.2 04 04 Man 1.0 07 Ld 0.5 Gal 5.0 8.1 83 15.2 55 Gie 3.0 03 12 3.8 45 eatin extracted with hot water “Pectins extracted by 0.05 M NaOH at 4 °C after extraction with water, oxalate and dilute acid 74 %. The polymers in this group consisted of very small non-esterified blocks. Group C consisted of pectins with non-esterified blocks of varying size. The three groups could possibly be correlated to the properties of pectin (Daas ef al 1999). 2.2. Enzymatic degradation The intramolecular distribution of the structural elements in pectin can be elucidated during chemical or enzymatic degradation. Chemical degradation methods often have poor selectivity, whereas enzymatic degradation often gives a well-defined result (Schols and Voragen, 1996). Two groups of pectin-active enzymes can be distinguished, those that acts on the homogalacturonan chain and those that acts on the rhamnogalacturonan region (Daas et al 1999), 2.2.1 Homogalacturonan acting enzymes Pectin methylesterase (PE) de-esterifies HM pectin resulting in LM pectin and release of methanol. PE extracted from plants degrades blockwise, whereas degradation with fungal PE results in randomly distributed methoxyl ester groups (Daas et al, 1999). Pectin acetyl esterase splits off acetyl groups from the galacturonic acid backbone. The enzymatic action results in a calcium-reactive polymer that can form calcium pectate gels (Voragen er al, 1995). Polygalacturonase (PG), pectin lyase (PL), and pectate lyase (PAL) depolymerise the galacturonic acid backbone, PG acts by hydrolytic cleaving of the linkage between two non- esterified adjacent GalA residues. The PG enzyme exists as endo-PG and exo-PG. The endo form cleaves the pectin chain randomly, whereas the exo-form splits off monomers or dimers from the reducing end of the pectin chain. PL splits HM pectin by a fi-elimination mechanism. PAL, on the other hand, acts by f-elimination on non-esterified galacturonic acid residues. Endo-PAL cleaves the pectin chain randomly, whereas exo-PAL splits off unsaturated dimers from the non-reducing end of the pectin chain (Daas et al, 1999, Kravtchenko et al, 1993). Anger and Dongowski (1988) reported that amidated groups on the pectic backbone do not disturb the activity of PG splitting of neighbouring glycosidic linkages. 2.2.2. Rhamnogalacturonan acting enzymes Enzymes acting on the hairy region of pectin are schematically described in figure 2-4, Rharmnogalacturonan (RG) hydrolase acts by hydrolysis of the linkage between thamnose and galacturonic acid. RG lyase splits rhamnose and galacturonic acid by B-elimination. RG acetyl esterase acts by removing acetyl groups from the hairy part of the pectin molecule 10 (Schols et al, 1997). The action of RG rhamnohydrolase, RG galacturonohydrolase and B- galactosidase are illustrated in figure 2-4, @ os Be Ae r roman f Fegalectosdase RG GalA-ase Figure 2-4 The action of rhamnogalacturonan (RG) specific enzymes (galA rhamnose, gal = galactose, Ac = acetyl groups). From Schols etal, 1997. galacturonic acid, rha = 2.3 Chemical reactions 2.3.1 Acidic degradation Pectins are most stable at pH = 3-4. At lower pH, degradation of methoxyl, acetyl, and neutral sugar groups occurs. Low pH in combination with high temperature causes accelerated degradation reaction Acid hydrolysis can be used for selective degradation of pectins, since different glycosidic linkages are hydrolysed at different rates. Glycosidic linkages at C-1 of neutral sugars are easily hydrolysed, whereas the linkage between two galacturonic acid residues is highly resistant to acidic conditions. In an acidic environment, degradation of the sidechains consisting of neutral sugars, as well as degradation of the backbone at the rhamnose insertions, is expected. In contrast, polygalacturonan sequences will only be hydrolysed at sufficiently high temperatures (Kravtchenko et al, 1993, Thibault et ai, 1993, Voragen et al, 1995) 2.3.2. Alkaline degradation In an alkaline environment at low temperatures, deesterfication during saponification (base- promoted hydrolysis of ester groups) occurs. At neutral or alkaline pH values, the pectin chain is degraded by B-elimination. The degradation only takes place at glycosidic linkages next to esterified galacturonic acid units. When the deesterfication proceeds, the rate of degradation thus decreases (Kravichenko et al, 1993), Kravichenko ef ai, (1992d) found that increased temperature increased the B-elimination rate more than the competitive deesterfication rate. Increased pH favoured the deesterfication process more than the B-elimination, Thus, increased temperature and decreased pH improved the degradation of the polymer. At 115 °C and pH = 5.0, 38 % of the glycosidic linkages in the galacturonic backbone was cleaved within 24 h, For comparison, the same sample treated at 80 °C and pH 5.5 was only degraded 5.5 %. At temperatures above 60 °C, -elimination occurs even under slightly acidic conditions. When the temperature increases further, the fi-elimination rate is higher at pH > 5 than the acid degradation process at pH = 3 (Voragen er al, 1995). 23.3 Ammonia treatment When pectin is treated with ammonia in alcoholic suspensions, some of the ester groups are replaced with acid amide groups (Kim et al 1978a, Anger and Dongowski, 1988). The DAm varies with the temperature, ammonia concentration, reaction time and polarity of the medium (Kim ef al, 1978a, Reitsma et ai, 1986, Kratchanov et al, 1989). Some properties of amidated pectins obiained after different times of reaction at 5°C in an alcoholic suspension are shown in table 2-2. e(h)_| Free carboxyl groups % DM% DAm% 0 (initial) 24.6 154 2 29,7 32.6 37.7 4 29.9 248 458 6 30.1 212 © 48.7 20 30.5 121575 Table 2-2 Influence of tine on the amidation process at 5°C in alcoholic suspension (10 g pectin, 60 ml isopropanol, 40 ml ammonia 17 M). Values from Reitsma et al, 1986 and Racapé et al, 1987. Viscosity measurements and gel permeation chromatography indicated that some depolymerization occurred during the preparation of the amidated pectins. The highest amidated samples were only partly water-soluble and were possibly aggregated (Racapé et al, 1987). Because of the alkaline environment, two undesirable side reactions occur during amidation with ammonia: deesterfication and chain splitting by B-elimination (Reitsma ef al, 1986). A linear relationship between the increase in amide content and the decrease in esterfication was found by Kim et al, (1978a) for a HM citrus peetin treated with ammonia in isopropanol. Kratchanov ef al, (1989) found that the polarity of the solution and the acid-base quilibrium of the interaction of ammonia with water significantly affected the amidation and deesterfication reactions. An increased rate of amidation was found when ammonium chloride was added to the amidation reaction by Denev et al, 1996. Padival et al (1979) carried out deesterfication of lime peel pectin with ammonia 2.4 Extraction methods The extraction method used for the preparation of pectin affects the chemical composition of the sample to varying extent. Kravichenko ef al, (1992a) found that the composition of industrial pectin fractions very similar to those prepared under mild conditions. However, the commercial pectin contained less neutral sugars, The following sections contain brief descriptions of laboratory and industrial preparation methods, Sections 2.41. and 2.4.2 largely follow the review given by Voragen et al, (1995). 2.4.1 Laboratory extraction with chemicals Laboratory prepared pectin fractions usually begins with grinding of the plant material in warm ethanol or acetone, followed by washing with ethanol to inactivate enzymes and 2 remove alcohol-soluble solids. Another method to suppress the enzyme activity is by wet milling at low temperatures followed by addition of sodium deoxycholate (SDC) or dodecyl sulfate (SDS). Ralet et al, (1994) and Thibault e¢ al, (1996) reported that the use of extrusion- cooking of cell wall material substantially increased the yield in the following water extraction step. By sequential extraction treatments, pectin fractions are obtained from the purified cell wall material. In the first step, the cell wall material is treated with cold and/or hot water or buffer solutions such as acetate or phosphate buffers. Water-soluble pectin will be extracted in this step. Water extraction generally yields pectin with a high DM and high neutral sugar content. The second step includes extraction with chelating agents such as EDTA, CDTA, sodium hexa metaphosphate and ammonium oxalate. Chelator-soluble pectins (calcium bond pectins) are extracted in this step. The best chelating effect for EDTA, sodium hexa meta phosphate and ammonium oxalate is obtained at neutral pH values. Since degradation by -elimination can occur at pH > 5, nondegradative extraction with chelator agents has to be carried out at pH =4-5 and low temperature (Renard ef al, 1993). Chelator-extracted pectins are rather highly methoxylated but show a lower neutral sugar content than water-extracted pectins. In the third step, the cell wall residue is treated with hot diluted acid, The remaining pectin fraction from the second step is tightly bound in the cell wall as protopectin, and therefore a degradative method is needed for extraction. The acid extraction causes degradation of various types of glycosidic linkages in the pectin molecules. Acid-soluble pectins will be released in this step. In the fourth and last step, cold diluted sodium hydroxide (0.05 M) is used as extractant, Sodium hydroxide causes a deesterfication of the pectin fraction. In order to minimize degradation by B-elimination, the extraction is carried out at a low temperature, 2.4.2. Extraetion with enzymes Pectins can be extracted from cell wall materials by the use of pectolytic enzymes. The degradation products obtained are either oligogalacturonides or rhamnogalacturonides, rich in neutral sugars. Pectins of high molecular weight and high galacturonic acid content can be obtained with protopectinases (Sakai, 1993). Pectins can also be extracted by other enzymes such as endo-arabinase, endo-galactanase and glucanases (Thibault et al, 1988). 24.3 Industrial extraction The main sources of commercial production of pectin are dry apple pomace and wet or dry citrus peel. The two raw materials will produce pectin with slightly different properties. Apple pectin usually forms heavier and more viscous gels than citrus pectin. Citrus pectin shows a lighter colour than apple pectin, which can be preferable in some applications (May, 1990). Pectin is commercially extracted by treating the raw material with hot dilute mineral acid at pH ~ 2. The following step includes separation of the hot pectin extract from the solid residue. ‘The solid is separated on filter presses, and the extract is clarified by, for example, kiselguhr filtration. The separation is a complicated process, since the solid phase is soft and the liquid 13 phase is viscous after acid treatment. There has to be a compromise between efficient extraction and the separation of the phases. The production of liquid pectins is then followed by treatment with carbon to remove colour and with o-amylase to degrade starch residues in the fraction, Starch can otherwise precipitate from the liquid product. The clarified extract is then concentrated under vacuum, Production of powdered pectins can be accomplished by mixing of the liquid pectin fraction with isopropanol, ethanol or methanol. Pectins precipitates at alcohol concentrations >45 %, Heavy metals, acid, and other aleohol-soluble materials are then removed from the pectin precipitate by washing. An alternative method for production of powdered pectin, often used in the past, is the use of aluminium hydroxide. Pectin can coprecipitate in the presence of colloidal aluminium hydroxide. Pectins with DM > 70 % do not precipitate well with this process, and the overall yield is lower than with alcohol precipitation. The advantages of this process are that the extract does not have to be concentrated before precipitation and the impurities are more readily removed (May, 1989, 1990, Voragen er al, 1995). 3 PHYSICAL PROPERTIES 3.1 Molecular weight ‘The molecular weight of pectins can differ markedly, depending on the raw material source and the extraction method used. Difficulties with the measurements can also cause large variations in the molecular weight and the molecular weight distribution. The heterogeneity of the sample and aggregation of pectin in solution ofien result in a broad molecular weight distribution (Voragen ef al. 1995). Light scattering is commonly used to determine weight- average molecular weights (M,,) (Anger and Berth 1986, Hourdet and Muller 1987, Berth and Lexow 1991, Panchev er al 1988). Values on the number-average molecular weight (Mi, ) can be derived from membrane osmometry or end-group analysis (Fishman et al 1986). The molecular weight distribution of pectin is obtained by size exclusion chromatography, SEC (Rinaudo 1996, Harding er al 1991, Berth and Lexow 1991, Deckers ef al 1986). In order to avoid aggregation of the pectin molecules, the polysaccharide must be isolated as a sodium salt before analysis. Ultracentrifugation and filtration through hydrophilic membranes have also been used to eliminate the effect of aggregation (Rinaudo 1996). Rinaudo (1996) found molecular weight values from static light scattering to be overestimated compared to values obtained from SEC. Light scattering gave My = 70 000, but the SEC result showed that 85 % of the pectin molecules had only My, = 34 800. This effect was pointed out as aggregation of the pectin molecules. Low angle laser light scattering (LALLS ) photometry was used by Kontominas and Kokini (1990) to measure molecular weight and molecular weight distribution of apple pectin. The molecular weight obtained with LALLS, Mw = 5.34x10° agreed well with results obtained with SEC, which showed My = 5.33%10°, The sample was found to be highly polydisperse with a polydispersity (M,, /M, ) of 15.4. ‘The Mark-Houwink-relation has often been used to relate intrinsic viscosity data to molecular weights. The general equation is: Eq 3-1 where [n] is the intrinsic viscosity and M, the viscometric average molecular weight. K and a are parameters which may depend on the pectin properties such as DM and rhamnose content. (Rinaudo 1996). 3.2. Molecular conformation The molecular structure of pectin has been studied in order to understand the gelling mechanism of the polysaccharide, Rees and Wight (1971) suggested that pectin molecules solution are formed in a single helix conformation. Calculations based on H-NMR studies showed that multiple-helix formation is impossible for pectin. Rees and Wight also proposed that the “kinking” effect of rhamnose units in the backbone limits the length of the junction. zones in the gel. See figure 3-1 in O-O-O-0 9 bO+0+0 Figure 3-1 Schematic diagram illustrating how a rhamnose insertion (Rha) causes kinking of the polygalacturonie acid chain. From Oakenfull in “The Chemistry and Technology of Pectin”, 1991. Walkinshaw and Amott (1981) performed X-ray diffraction studies on dried pectinic acid (DM > 55 %) and calcium pectate fibres (DM < 30 %) in the solid state in order to explain the nature and stability of pectin gels. They assumed that the structure of dried pectin fibres and pectin gels is the same. Two different helical structures with three galacturonic acids as repeating unit, were identified. Isolated pectinic acid chains were suggested to form a parallel packing arrangement stabilised by hydrogen bonds between oxygen atoms in the same chain and oxygen atoms from adjacent chains. Hydrogen bonds between water molecules and the peetinic chain were also presumed to contribute to the stabilisation, Walkinshaw and Amott also pointed to another interchain association: a hydrophobic interaction between the methyl groups gives an important contribution to the stability of the structure. See figure 3-2. Figure 3-2 The structure of polygalacturonic acid with DM = 100 9 Parallel chains are packed in a hexagonal lattice. Methyl hydrogens are denoted with filled circles. Hydrogen bonds are shown by dotied lines. From Walkinshaw and Arnott, 1981 b. In contrast to Walkinshaw’s assumption that both dried pectin fibres and pectin gels had the same molecular structure, circular dichroism studies indicated that pectin undergoes a polymorphic phase transition when calcium pectate gels are dried (Morris ef al 1982). They suggested a 2, helix conformation for the gel and a 3, helix conformation for the solid state. The conclusion was based on results from circular dichroism on pectin (poly-D-galacturonate) in comparison with alginate (poly-L-guluronate), which is the near-mirror image of poly-D- galacturonate. Poly-L-guluronate has been extensively studied and shows the 2; helix conformation in both the solid and the gel state. Circular dichroism on poly-L-guluronate in both the gel and the solid state showed similar behaviour, however, for poly-D-galacturonate the circular dichroism results differ markedly between the gel and the solid state. Jarvis et al (1995) performed studies of solid calcium pectate and gels with "'C NMR spectrometry. The pectin concentration in the gel corresponded to the plant cell wall concentration. "C NMR spectrometry showed that the solid calcium pectate gel contained almost as much 2; as 3, helix structure. In the gel a significant proportion of 3; helix structure could be identified, ‘Thibault and Rinaudo (1986a) suggested that three different conformations exist for LM pectin. Potentiometry and circular dichroism measurements showed different binding properties of LM pectin in acidic, sodium and calcium form, respectively. The different binding behaviour of the three pectins was explained by different conformations of the samples. Buhl (1986) found two different types of gels formed by potassium-induced gelation of LM pectin. The two types, which occurred at different pH values and K*-concentrations, were related to conformational changes in the pectin structure. However, it can be concluded that the structure of pectin is a very complex system, not fully understood yet. 3.3, Flexibility of the pectin chain ‘The conformation of pectin molecules in solution is described in the literature in different terms. Rinaudo (1996) describes the pectin molecules in solution as worm-like chains. Harding et ai (1989, 1991) talks about an extended rod conformation, and Hourdet and Muller (1991) suggest an extended coil conformation comparable with the conformation of alginate chains. ‘The flexibility of the chains can be characterised with the flexibility parameter B, introduced by Smitsrod and Haug (1971). For pectins, B varies between 0.017 and 0.072. Compared to other polymers, pectin shows similar flexibility to that of alginate, less flexibility than amylose and much more flexible behaviour than xanthan. The flexibility is affected to a certain extent by the composition of the molecule. Thereby, the presence of rhamnose units is reported to increase the flexibility of the chain, but amide groups, on the other hand, induce larger stiffness of the polymer (Voragen e# al 1995). The stiffness of the chains is characterised by a persistence length (Lp). The larger the Lp of the chain, the larger the deviation from gaussian behaviour. ‘iscometric and light scattering measurements on citrus pectin with DM = 38 % were performed by Axelos ef al (1987). A high value of the L/Lp ratio was found, where L is the contour length of the polymer, which indicated a flexible coil behaviour of the polymer. A semi-rigid structure of pectin in solution was reported by Ousalem ef al (1993). Dynamic Light Scattering was used to provide information on the size of the individual pectin molecules and size and content of the aggregates. The Lp values were found to vary with the DM for citrus pectin in solution. Since it is claimed that the pectin conformation is independent of the DM, the changes in persistence length were related to changes in the polymer/solvent interaction (Axelos et al 1996). Light scattering studies of pectin solution with varying DM were performed by Plashchina et al (1985). Maximum chain flexibility was obtained for samples with DM between 43 and 58 %, Similar results were obtained by Axelos ef a/ (1989). Panchev et al (1988) reported maximum. conformational flexibility for pectin with DM = 57-58 %, Viscometric studies of pectin samples with different DM showed increased stiffness of the chains when the DM increased from 60 % to 73 % (Michel et al 1982). Cros et al (1996) reported Lp values for apple and citrus pectin solutions derived from intrinsic viscosity and small angle neutron scattering (SANS). The results were also compared with values calculated from molecular modelling studies. Persistence lengths derived from intrinsic viscosity ranged between 59 and 126 A, and those derived from SANS ranged between 45 and 75 A. The different results for the two techniques were explained as follows: intrinsic viscosity measurements reflect the weight average molecular weight of all the species in solution, including aggregates, whereas SANS measurements provide values for the isolated chains. Molecular modelling studies were also made of homogalacturonan ch: (polygalacturonic acid units) and rhamnogalacturonan chains (polygalacturonic acid units with some rhamnose units inserted). The hamnose insertions in the polygalacturonic acid chain did not show any significant effect on the polymer properties in solution by the molecular modelling programme. The calculated persistence length for the homogalacturonan chain (Lp = 135 A) was considerably higher than experimental values obtained with SANS (Lp = 45-75 A). 3.4 Solubility Pectins are generally soluble in water and insoluble in organic solvents. The solubility decreases with increased ionic strength. Pectic acids and pectinie acids, with a low DM, are only soluble as sodium or potassium salts. Sodium pectinates are more soluble than calcium pectinates. There is no saturation limit for pectins, but it is very difficult to obtain solutions with concentrations higher than 3-4 %. Pectin can also be dissolved in DMSO, formamide, dimethylformamide and warm glycerol (Voragen et al 1995). 3.5. Polyelectrolyte behaviour Pectins behave as polyelectrolytes, since they have carboxyl groups on the backbone, A fundamental parameter to describe the polyelectrolyte behaviour is the charge density (?.), which is related to the average distance between two charged groups of the chain, The charge density is defined as follows: a hs bDKT £q32 where «is the degree of dissociation, « the electron charge, b the length of the monomeric unit, D the dieleleetric constant of the solvent and kT the Botzmann term. The charge density depends on DM, the distribution of carboxyl groups and the degree of polymerisation (Rinaudo, 1996). Another important parameter, that characterises the polyelectrolyte behaviour of pectin is the pK,- value for the carboxyl group. The pK, value is related to the degree of dissociation («) for the carboxyl group in following way: 2K, = pH + Heald a) Fg 3-3 Variations in pK, as a function of the degree of dissociation were studied with potentiometry by Michel et al (1984). Salt-free solutions of HM pectin (DM = 73 %) varying in sucrose content were investigated. For solutions with a sucrose content below 20 %, the pK, variation as a function of a followed the Lifson-Katchalsky theory. Above a 20 % sucrose content, the theory was not applicable, which was explained by aggregation of the pectin molecules. Another effect of the presence of sucrose was found with viscometric studies, Increased sucrose content in the solutions resulted in increased stiffness of the pectin chains, 3.6. Aggregation in solution Aggregation phenomena of pectin chains in solution have been studied with light scattering techniques (Kravtchenko et al 1992b, Berth et al 1994a-c) and viscometry (Michel ef al 1985). HM pectin in solution forms aggregates when the ionic strength is sufficiently high (120.05), when pH < 3, and when the sucrose content is 55-58 % w/w (Michel ef al 1985). The aggregation was detected by a slight increase in intrinsic viscosity and a sharp increase in the Huggins coefficient (K’) when the “sol-pregel” transition occurred, Kravichenko et al (1992a) found that lemon pectin fractionated with SEC easily formed aggregates in the presence of calcium. The aggregates could be disrupted by shear forces, heating, or the presence of a chelating agent. Aggregation in the presence of calcium or copper ions has also been studied by Paoletti et al (1986). ‘The aggregation of pectin in solution is very sensitive to the purification treatment. Berth et al (1994) studied the tendency towards aggregation for HM citrus pectin in phosphate buffer solution. The sample that was only filtered before light scattering measurements showed increased apparent molecular weight with rising polymer concentration, which indicated aggregation of the polymers. Samples that were purified with ultracentrifugation showed no aggregation behaviour. 19 4 HM PECTIN GELATION AND RHEOLOGY 4.1 The mechanism of gelation of HM pectin The gelation behaviour of pectin depends on the degree of esterfication. High-methoxyl pectins with DM above 50 % form a three-dimensional network by crosslinks between the polymer chains, so-called junction zones. The junction zones consist of segments from two or more polymer molecules and are stabilised by a combination of hydrogen bonds and hydrophobic interactions. See figure 4-1. Gelation of HM peetin requires a pH below 3.5 and a cosolute, usually sugar, to reduce the water activity. Figure 4-1 Schematic illustration of a gel network with junction zones, From Oakenfull and Scott, 1985. 4.1.1 Bonds involved in gel formation Walkinshaw and Amott (1981) suggested that the junction zones in the three-dimensional network of HM peetin gels are held together by hydrogen bonds and hydrophobic interactions, See figure 3-2 above. The hydrophobic interactions are framed when water molecules are mixed with non-polar methoxy! groups in the polymer chain, Since water and the methoxyl groups are incompatible, the methoxyl groups are forced to coalesce and reduce the contact area with water. Oakenfull and Scott (1984) have further investigated the role of the hydrophobic interactions between the ester methyl groups. The hydrophobic interactions are affected by different cosolutes and by changes in temperature, When dioxane or a small amount of ethanol or t- buthanol replaces sucrose in a pectin gel, the rupture strength of the gel is affected. Oakenfull and Scott (1984) suggested that changes in rupture strength could be related to the hydrophobic interactions between pairs of CHs-groups. The free energy of hydrophobic interaction between a pair of CHs-groups, AG*n, has been calculated for a model system with ethanol as cosolute (Oakenfull, 1991) 20 oF fe g, - i e ets By 3 g 1 : See — ae ahs a 0 100 200° © 300 400 150200 250 300 «350 Ethanol (gia) Ethanol concentration (g/kg) hydrophobic interaction between two CH;-groups DM prepared from HM pectin in the presence of in a pectin model system at 25°C. From Oakenfiull, ethanol at 25°C. From Oakenfull and Scott, 1984 1991. AG? x and rupture strength for pectin-ethanol systems are shown in figure 4-2 and 4-3, respectively. The three pectins, with different degrees of esterfication, shown in figure 4-3 exhibit a maximum in gel strength, which coincides with maximal hydrophobic interaction shown in figure 4-2. The effect of temperature on the rupture strength indicates that both hydrophobic interactions and hydrogen bondings stabilise HM-pectin gels. The curve for a pectin gel is sigmoidal with a small peak in rupture strength observed at ~30°. In comparison, a x-carrageenan gel decreased in rupture strength with increasing temperature, See figure 4-4. ‘TEMPERATURE fc) Figure 4-4 Effect of temperature on the rupture strength (RS) of gels. Solid curve: 3,6 g/kg pectin gelled with S5idgikg sucrose. Broken curve: Ig/kg x-carrageenan. From Oakenfull and Scott, 1984. Oakenfull and Scott (1984) qualitatively explained the result for the pectin gel as a contribution of both hydrogen bondings and hydrophobic interactions, When the temperature is increased, the hydrogen bondings are weakened, whereas hydrophobic interactions become stronger with increased temperature. These competing effects could explain the complex behaviour of rupture strength as a function of temperature in pectin gels. NMR studies of the relaxation time of pectin solutions and gels can provide information on the gelation process, Broiso et al (1993) found that NMR results corresponded to rheological measurements of pectin gels prepared with the same cosolute. Their result supported the interpretation that the cosolutes affect the hydrophobic interactions between the methoxyl 21 groups, and that the formation of junction zones in HM pectin gelation is strongly dependent on the hydrophobic interactions. 4.1.2 A method for estimating size of the junction zones A method for estimating the size and thermodynamic stability of the junction zones in non- covalent cross-linked gels, such as pectin, has been derived by Oakenfull and Scott (1984b). ‘The method is based on the theory of rubber elasticity which gives the following relationship between shear modulus (G) and the weight concentration of the polymer (c): G=cRT/Me Eq 4-1 where R is the gas constant, T the absolute temperature and M. the number average molecular weight of active chains, i.e. chains joining cross-links, The “free ends” are not included in Mc. An expression for M. was derived by Oakenfull and Scott, where J is the number of junction zones per unit volume, M) is the number average of the junction zones, M is the number average of the polymer chains and Nyy is Avogadro's number. yy MME, (IM 1 panto Noe Ne » Eq 4-2 If equation (4-2) is substituted in equation (4-1), the expression for G will be AT Mia M MiJ|-e Fg 43 where [J] is the molar concentration of the junction zones, J/Ny.. The molar concentration [J] can be calculated from the law of mass action if itis assumed that formation of junetion zones is an equilibrium process. This is valid for very weak gels with well-dispersed junction zones (Oakenfull 1991). From the law of mass action it then follows that K, = IM ine~M LJ) }" Fq44 where Kj is the association constant and n is the number of segments from the different polymer molecules involved. A combination of equation (4-3) and (4-4) gives four unknown quantities (M, M,, Ky and n) when G and ¢ are known. It is possible to optimise the values of the four unknown quantities to fit experimental data on G and c. The method for estimating the properties of the junction zones is only valid with two limitations. Firstly, the polymer gels must be weak at polymer concentrations just above the gel threshold, since the gel network has to obey gaussian statistics i.e. the active polymer chains have to be flexible and free to adopt all possible conformations. Secondly, the formation of junction zones must be treated as an equilibrium process, according to the law of mass action. Measurements of shear modulus for different concentrations close to the gel threshold thus give information on (1) how many segments from polymer chains participate in each junction zone (n), (2) the size of the junction zones (M,), and (3) the thermodynamic driving force for formation of junction zones (AG;°=-RTinK;). Oakenfull and Scott (1984b) found out with this method that there are two cross-linking loci (n) participating in each junction zone. A strong relationship between the number average of the junction zones (M,), the thermodynamic stability (AG;°) and the degree of esterfication was suggested by Oakenfull and Scott (1984a). My and AG;° seem to be proportional to the square of the degree of esterfication. The relationship indicates how the hydrophobic interactions between different methoxyl groups emphasises the gel formation process. 2 4.1.3 Gelation rate ‘The rate of formation of junction zones, i.e. the gelation rate, was suggested by Oakenfull and Scott (1986) to be: v eke ed where n both is the number of polymer segments involved and the reaction order. The method described above of estimating junction zone size and the number n was strongly questioned by Ross-Murphy (1991). He claims that a more complex function is needed to describe the reaction kinetics. A log-log plot of the reciprocal gelation time versus the polymer concentration will not give a linear plot over the whole concentration range as in the case of Eq 4-5, Ata critical gelation Cy, the curve will diverge. Ross-Murphy’s model for predicting the gelation rate is based on following equation: fog ceeceeeres * C/G)" =D" aes. K is a proportionality constant, ty is the gelation time, n the kinetic order and p is a critical exponent, 0 K' > Mg’ >Na’ Effect of sugar concentration and different cosolutes Sugar is the most commonly used cosolute in pectin gels. The presence of cosolutes reduces the water activity and the interactions between pectin and water. It also influences the hydrophobic bondings presumably by disrupting the cage of water molecules surrounding the ester groups (May and Stainsby, 1985). The sugar content therefore affects the rheological properties and the setting temperature of the gel. When the sugar concentration increases, the setting temperature increases. In figure 4-13 the changes in 5 are shown for samples with different sugar contents. During cooling the system changes from mainly viscous to mainly elastic behaviour as the gelation proceeds, 29 8 3 Phase angled, «| 8 rr a ee) 2 Temperature, °C Figure 4-13 Changes in phase angle during cooling of a slow set pectin gel in the presence of 65, 70, and 75% soluble solids, analyzed by change in phase angle, 6. From Barford and Pedersen, 1990. When sucrose is replaced by other sugars, the hydrophobic effects vary, since these sugars interact with water in different ways. May and Stainsby found that the setting temperature of HM-peetin gels increased when sucrose was partly replaced by maltose, lactose or glucose syrup, respectively. When the whole amount of sucrose was replaced by maltose, the setting temperature rose above 95°C. Dextrose and fructose affected the setting temperature in the opposite way. When sucrose was partly replaced by dextrose and fructose, the setting temperature fell. 4.2.2 Gelstrength Internal gel strength is affected by several different factors such as type of pectin, molecular weight, pH, soluble solids content and gel-forming conditions. The gel strength of pectin can differ depending on the source and on the extraction method used (Crandall and Wicker, 1986). For HM pectins the gel strength increases with increased DM up to about 70 %. Any further increase in DM will result in a decrease in gel strength. (Axelos et al, 1991). The relation between gel strength, pH and DM is shown in figure 4-13. DEGREE OF METHYLATION 63 73 at GEL STRENGTH GEL pH Figure 4-14 Relationships between gel sirength, degree of methylation and pH. From Erlich, 1977. Increased DM moves the optimal pH to higher values, Barford and Pedersen (1990) found a similar relation between the content of soluble solids and pH on the internal gel strength. Increased soluble solid content increases the gel strength and the pH optimum. When the sugar content is increased, less acid is needed for gelation, 30 The molecular weight affects the rupture strength of pectin gels. The rupture strength increases continuously with increased molecular weight (Mitchell, 1980). The thermal treatment of the pectin before gelation is also an important factor in the final gel strength. If the pectin solutions are held for many hours at high temperatures, thermal breakdown of the molecules can occur (Barford and Pedersen 1990). The cooling rate, time of ageing, stage of acid addition, type of cosolute and the use of buffer salts also influences the gel strength (Crandall and Wieker, 1986, Oakenfull and Scott, 1984a), ‘Test methods that measure the rupture strength of a gel may not be related to the elastic ‘modulus of the gel. Therefore, measurements of rupture strength will not necessarily rank a series of gels in the same sequence as small deformation tests without rupture (Mitchell 1980). Berth and Dahme (1991) used the shear modulus Gp as a normalised rigidity parameter to evaluate the gel strength of high-methoxy! apple and citrus pectins. The pectins were fractionated on size with GPC (gel permeation chromatography) and the different fractions showed different behaviour. The results showed that raising the degree of polymerisation increased the shear modulus. An increased amount of branched molecules or a greater degree of branching was shown to reduce the shear modulus. 4.2.3 Gel strength measurements ‘The pectin gel strength can be measured with several different methods. Many methods developed for the jelly manufacturer's needs are not easily correlated to each other. The need for a standardised pectin gel strength measuring method was met in 1959 when the Institute for Food Technologist’s presented the SAG method, described in detail below (IFT Committee on Pectin Standardisation 1959). Gel strength measurements can briefly be divided into two types, non-destructive and destructive methods. A non-destructive test measures the clastic properties of the gel within the linear viscoelastic region. Destructive tests have the advantage of being close to the consumer’s perception of a jelly. On the other hand, large deformations are more difficult to interpret and are not reproducible to the same extent as non-destructive methods. The description of gel strength methods given below largely follows the review by Crandall and Wicker (1986). Non-destructive test methods In the SAG Method a mixture of a standardised amount of sugar is cooked with a test amount of pectin. The mixture is poured into glasses of well-defined dimensions extended with tape at the top. After 18 to 24 hours the gel is ready and the tape is removed from the top. A stretched wire then cuts off the part of the gel held by the tape. After that the gel is turned upside down on to a glass plate to rest on the base that was formed during cutting. The gel will sag under its own weight and the loss in height is measured with a special micrometer called a Ridgelimeter (IFT Committee on Pectin Standardisation 1959, Crandall and Wicker, 1986, Rolin and de Vries 1990). BAR and FIRA Jelly Tester: These instruments measure the apparent clastic modulus with a torque on a blade immersed in the gel. The torque is applied by water flowing into a bucket and the amount of water needed to obtain a 30° turn of the blade is measured. An improved version of the instrument uses an electric motor connected to a torsion wire with a known torsion moment instead of flowing water to turn the blade, 31 Concentric Cylinder Instruments consist of two corrugated cylinders, one inner and one outer. The pectin mixture is poured between the cylinders and will then set. A torsion wire twists the inner cylinder, and the extent of torsion caused in the gel is measured. These types of instruments can also be used for creep compliance and stress relaxation measurements, Parallel Plate Instruments: The sample is placed between two parallel plates. Parallel plates have been used for creep compliance measurements. In Oscillatory Testing a small oscillatory stress is applied to the sample. The phase angle of the responding strain is measured. Most gels show a phase difference of less than 90° between stress and strain. Beveridge and Timbers (1989) carried out small amplitude oscillatory testing with a “versatile food rheometer” at a constant cooling rate. The output amplitude was, followed during the progress of gelation. During cooling of the pectin/sucrose solution, the amplitude began to increase almost linearly at 70-80 °C. During reheating from 25 °C, the amplitude decreased until the temperature passed 70 °C. After 70 °C the amplitude started to increase again. See figure 4-15. Different pectin concentrations were investigated with the oscillatory testing ‘Temperature ('c) or Amplitude (g.cm) Time nin) Figure 4-15 Ampliude and temperature as a function of time of oscillatory input. The slow set pectin concentration is 1 % and pH 2.82. From Beveridge and Timbers, 1989, Destructive methods ‘The first destructive test method known is the Finger Test, A slice of the pectin gel is placed between the thumb and the forefinger and squeezed until it breaks. The test gel is compared to a reference gel. Trained analysts can detect differences of 5% between the test gel and the reference, Tarr-Baker Gelometer (Delaware Jelly Strength Tester): A force is applied to a pectin gel by a piston powered by compression air. The method is sensitive to uneven application of the pressure and “skin” formation on the surface of the gel. A modified version of the instrument can be used for several elastic and breaking tests on the same gel. Luers-Lochmuller Pektinometer measures the force required to pull a metal figure out after being cast inside a pectin gel. The pectin mixture is poured into a special container with the ‘metal figure inside. The container sides are corrugated to avoid slip. See figure 4-16. 32 Figure 4-16 The Luers and Lochmuller pektinometer. From Beach et al, 1986, ‘The Herbstreith-Pektinometer is an improvement on Luers-Lochmuller’s device. The Herbstreith Pektinometer uses a special designed clear, corrugated cup with a plastic self- centred figure. 100 gram pectin mixture is poured into the cup and held for 2 hours at 20°C, ‘The position of the cup is then locked and a hook is attached to the plastic figure. The force required for an electric motor to pull the plastic figure is measured in Pektinometer units. One advantage with the method is that the breaking strength is measured from the inside of the gel, which thereby eliminates any problem with skin formation on the gel surface (Beach e al, 1986). Instron: The Instron Universal Testing Machine can be used for compression and tension measurements of samples loaded in a cell. The force is measured when the upper part of the cell is moved a given distance or time, Fundamental characteristics of the gel such as brittleness, hardness and elasticity can be quantitatively measured and related to sensory properties such as chewiness and gumminess. Apparent gel strength and rupture strength were measured by Oakenfull and Scott (1984a) using an Instron, The Voland Stevens LFRA Texture Analyser (VSTA) measures the breaking strength of the gel. A plunger is applied to the gel at a constant speed and the stress versus deformation graph is recorded. A maximum corresponding to the point at which the elastic limit of the gel is exceeded appears during the measurement (Rolin and de Vries, 1990). ‘The Bloom Gelometer is another plunger method, often used for classification of gelatines. It measures the force required to press a plunger of specified dimensions 4 mm into the gel (Rolin and de Vries, 1990). The Boucher Electronic Jelly Tester is based on the same principle as the Bloom Gelometer. The force required is measured by an electronic instrument (Rolin and de Vries, 1990). The Penetrometer measures how deep a cone, which has been placed on the surface of a gel, can penetrate the gel at a given time period. The driving force is only the gravitational force (Rolin and de Vries, 1990). ‘The Chatillon consists of a stationary mechanical gauge attached to a test stand, The force- sensitive gauche contains a calibrated spring, which is activated by moving a test sample cup (Johnson and Breene, 1988). 3 Comparison between different methods Johnson and Breene (1988) compared the precision of the Pektinometer with that of the Chatillon and Instron for gel strength measurement. Two different modes were tested, the pull and the push mode. The Pektinometer and Instron showed similar precision; i.e. the variance in the gel strength measurements was small for both modes. The simpler instrument, Chatillon, showed higher variability in the gel strength measurements. However, the precision of the Chatillon seemed to be high enough for routine gel strength measurements, Beach et al (1986) compared five different pectins standardised to 150° SAG using different test methods. The internal strength and breaking strength were measured with VSTA. The five pectin sorts differed markedly in internal gel strength when the pectin concentration was about 0.5 %. According to the SAG test, all five pectins were of the same quality. Further investigations were made of other pectin samples. The breaking strength values measured with the Herbstreith Pektinometer differed from results with the VSTA. Once again, the pectin samples were standardised to 150° SAG and were supposed to have equal gel strength. ‘The VSTA was compared to the Tar-Baker gelometer for its suitability for measuring pectin gel break strength. The results from the two methods were highly correlated and showed a Tinear behaviour between gel break strength and pectin concentration (Angalet, 1986) Rao et al (1989) compared the VSTA with an Instron in order to examine the feasibility of determining texture parameters with the VSTA. The internal gel strength measured with VSTA was also compared to results from SAG and Pektinometer measurements. Depending on the plunger used with VSTA, high or low correlation with the Pektinometer was obtained. High correlation was obtained for the VSTA and the SAG Ridgelimeter. 4.2.4 Structure development rate The structure development (SD) rate during gelation is a usefull measure to provide information about the time required to complete gelation or to obtain certain gel strength. The SD rate can be defined either as dG'/dt, or as dn*/dt where n* is the complex viscosity: nt=G*/o mts © js the frequency and G* is the complex modulus consisting of an elastic par the storage ‘modulus (G’) and one viscous part, the loss modulus (G’’): a= GY +@F aa ‘The magnitudes of SD rates can be used to examine the effect of different process variables, such as temperature and polymer hydration (Lopes da Silva and Rao, 1995). Rao and Cooley (1993) measured the incipient SD rate and the SD rate after 22 days of storing on pectin/fructose gels, Both hydrated and unhydrated pectin were used. In the incipient SD rate measurement, dispersions containing 65 % fructose and 0.5-1.0 % rapid set HM-pectin (DM = 49%) were cooled for 2 hours. The samples were placed in a cone and plate system of a Carri- Med Rheometer. The SD rates were calculated from the time-n/* data recorded, Since pectin gels are weak, the derivatives were calculated after careful interpolation of the data. SD rates during the temperature sweep are shown in figure 4-17, 4 Rate (poise/min) ‘Temperature (°C) Figure 4-17 Structure development rates during a temperature sweep in 1 % pectin and 65 % fructose gel at pH = 2.68, during cooling at 20 °C/h. From Rao and Cooley, 1993, The negative and positive rates are suggested to correspond to breakage and reformation of bonds during the cooling process. As can be seen in figure 4-17, the SD rates were higher in the hydrated samples than in the unhydrated samples. In the long-term SD investigation, samples were tested regularly during the storage period. The SD rates were strongly dependent on the storage temperature and pH of the gels. The SD rates were, as expected, highest in the beginning of the period. SD rates have also been measured during gelation by Lopes da Silva et al (1995). An Arrhenius relationship was applied to describe the temperature dependence of the SD rates. The SD rates during ageing at different temperatures were studied by Lopes da Silva er af (1995). The samples were quenched to the different ageing temperatures. Figure 4-18 shows the SD rates as a function of ageing time at different temperatures. ous 0.0 00s acai 0.00 -005 | -0.05 0010" 1010 20104 3.010 Aro L210 1:3 10° time (5) Figure 4-18 Structure development rates during ageing of a 1% HM pectin 160% sucrase dispersion at pH = 3.0 and isothermal conditions. (A) 5°C, (@) 15°C, (0) 20°C, (») 30°C, (+) 50°C. From Lopes da Silva et al, 1995. 35 All structural modifications were assumed to occur isothermally, due to the rapid quenching of the samples, An initial increase in the SD rates was observed during the ageing process. ‘The largest increase occurred in the sample at 30°C. Both higher and lower temperatures showed lower initial SD rates. The rapid increase in SD rates corresponds to the rapid formation of junction zones between pectin chains, according to Lopes da Silva et al (1995). The increase was followed by a decrease in SD rates. The SD rates continued to decrease during the whole ageing period. This behaviour was suggested to correspond to slow reorganisation of the network. The effect of ageing temperature on the SD rates can be quantified by calculating the average SD rates, SDRav: f(spryar ee where t; and t corresponds to the time interval over which dG’/dt data are obtained (Rao and Cooley, 1994, Lopes da Silva et al, 1995). When SDR. values are plotted against temperature, a bell-shaped dependence of temperature will occur. Lopes da Silva et al (1995) found a SDRy, maximum at 30°C for a 1 % HM pectin/60 % sucrose sample at pH = 3.0. The same ageing temperature was found for maximum elastie modulus (G’). In a similar way to the SDR, the average viscosity, n*, is defined by Rao and Cooley (1994): nea mest Di where t; and tz are the time interval over which the complex viscosity data are obtained. The SD rate is very sensitive to changes in pectin concentration (Rao and Cooley, 1994). When the pectin concentration is increased, the SD rate increases. Lopes da Silva et al (1995) reported a high power dependence of the SD rate versus concentration; dG’/dt « c*!, Rao and Cooley compared the SD rates for gels with fructose and gels with glucose. The pectin/glucose gels showed higher SD rates than the pectin/fructose gels. Hydrogen bonds and hydrophobic interactions seemed to affect the glucose gels more than the fructose gels, since the SD rate varied substantially during cooling from 50°C to 10°C. 42.5 Ageing process The network structure of pectin gels is stabilised by hydrogen bonds and hydrophobic interactions. During ageing at different temperatures the different interactions will change, which gives a complex behaviour in G’ and G"’ (Lopes da Silva and Goncalves, 1994). 1 % HM-pectin/60) % sucrose systems were studied at different ageing temperatures, The storage modulus increased as a function of time for all studied temperatures; however, at 5°C only a small increase was observed. See figure 4-19. 36 sean 2500 ee 2000 awe = 1x0 isc 1000 toe 2 rc sc tine 0) Figure 4-19 Storage modulus as a function of ageing time for HM pectin/sucrose gels at pH = 3.0. ‘Measurements were performed at 0.5 Hz and 3 % strain. From Lopes da Silva and Goncalves, 1994. At the end of the ageing process the G’-value at $°C is much less than G’ for other temperatures. This could be an effect of the temperature dependence of the hydrophobic interactions. At low temperatures hydrophobic interactions are weak. As a consequence of stronger hydrophobic interactions at higher temperatures, G’ increases from 5°C to 30°C. At higher temperatures hydrogen bonds are weakened, which gives a weaker gel and lower G’ at 50°C. The effect of ageing has been studied on 0.5 % pectin / 65 % fructose gels at different pH by Rao and Cooley (1993). At pH = 2.7, the rheological properties G’, G”’ and n* increased during storage for 22 days at temperatures above 18°C. Below 18°C the rheological properties decreased with time. In contrast, at pH = 3.0 no changes in G’, G’” or y* occurred during storage. 4.2.6 Mechanical spectra The reciprocal of the angular frequency, «», defines the time scale during a dynamic measurement. The mechanical properties of a material can be qualitatively determined as a function of time by scanning the frequency. Lopes da Silva and Goncalves (1994) interrupted the time sweep experiments to perform frequency sweeps (mechanical spectra) on the pectin sucrose system described above. The purpose was to find a power law dependence for G’ and G” on the frequency («) with the same exponent (n) as a possible measure of the gel point: G@)x

    You might also like