Microstructure and Rheological Behavior of Pure and Mixed Pectin

Aug 27, 2002 - Santanu Basu , U.S. Shivhare , T.V. Singh .... EFFECT OF PECTINOLYTIC AND AMYLOLYTIC ENZYMES ON APPLE JUICE TURBIDITY...
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Biomacromolecules 2002, 3, 1144-1153

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Microstructure and Rheological Behavior of Pure and Mixed Pectin Gels ¨ fgren, Pernilla Walkenstro ¨ m, and Anne-Marie Hermansson* Caroline Lo SIK, The Swedish Institute for Food and Biotechnology, PO Box 5401, 402 29 Go¨teborg, Sweden Received April 8, 2002; Revised Manuscript Received July 1, 2002

The microstructure and the rheological properties of pure HM (high methoxyl) and LM (low methoxyl) pectin gels and of mixed HM/LM pectin gels have been investigated. Gel formation of either the HM or LM pectin, or both, was initiated in the mixed gels by varying the sucrose and Ca2+ content. The microstructure was characterized by transmission electron microscopy, light microscopy, and confocal laser scanning microscopy. HM and LM pectin gels showed aggregated networks with large pores around 500 nm and network strands of similar character. Small differences could be found, such as a more inhomogeneous LM pectin network with shorter and more branched strands of flexible appearance. LM pectin also formed a weak gel in 60% sucrose in the absence of calcium. A highly inhomogeneous mixed gel structure was formed in the presence of 60% sucrose and Ca2+ ions, which showed large synergistic effects in rheological properties. Its formation was explained by the behavior of the corresponding pure gels. In the presence of 60% sucrose alone, a homogeneous, fine-stranded mixed network was formed, which showed weak synergistic effects. It is suggested that LM pectin interacts with HM pectin during gel formation, thereby hindering secondary aggregation leading to the aggregated networks observed for the pure gels. I. Introduction Pectin is a gelling biopolymer originating from plants. The polysaccharide is an essential component in both the initial cell growth and the ripening process. Industrial pectins are obtained by acid extraction, yielding a chain structure mainly consisting of R-(1-4)-D-galacturonic acid units interrupted by the insertion of few rhamnose residues. Neutral sugar units are attached to the backbone and concentrated in highly branched “hairy” regions.1 Some of the carboxyl groups in the polygalacturonic acid chain are present in methyl ester form. The degree of methylation (DM) divides pectin into two types. In high methoxyl (HM) pectin more than 50% of the carboxyl groups are methylated and in low methoxyl (LM) pectin less than 50% of the carboxyl groups are methylated. The degree of methylation is crucial for the gel formation of pectin. HM pectin gelation occurs in an acidic environment (pH ∼ 3) and in the presence of a cosolute, usually sugar, typically more than 60%. The cosolute reduces the water activity and the low pH reduces the ion dissociation, thereby enabling the formation of junction zones between the pectin chains. The network structure in HM pectin gels is based on hydrophobic interactions and hydrogen bonds.2,3 LM pectin gelation occurs in the presence of Ca2+ ions, both with and without sugar. The proposed mechanism for LM pectin gelation is based on the so-called egg-box model.4,5 It has also been reported that LM pectin gelation can occur in the absence of Ca2+ ions at decreased water activity and at pH values below 3, i.e., under conditions governing gel formation of HM pectin.6,7 The functional properties of pectin depend on the chemical and physical structure of the molecules. Important factors

are homogeneity in molecular weight and methyl ester distribution of the pectin molecule.8-11 Analyses of the chemical structure of pectin show the complexity of the polymer and have led to extensive studies.12-14 Less work is reported on the microstructure of pectin solutions and gels. No studies can be found focusing on the microstructure of pectin gels, but atomic force microscopy (AFM) has been used to investigate air-dried, dilute pectin solutions at a high magnification.15,16 The rheological properties of pectin gels are affected by several parameters such as sucrose content, pH, temperature conditions, and, in the case of LM pectin, calcium concentration. Evageliou et al.17 studied the gel formation of a laboratory preparation of a HM pectin with a DM of 70.3%. The study focused on the influence of pH, sugar type and annealing on HM pectin gels. At the start of cooling at 95 °C, 0.5% pectin samples (pH 3.0) with sugar content of 60% and above had already started to set. The high setting temperatures of HM pectin with high DM can make it difficult to catch the initial stages of gel formation. The calcium content strongly influences the rheological behavior of LM pectin gels.18,19 Increasing the calcium content increases G′, but at calcium levels that are too high, syneresis or precipitation of the pectin chains may occur.18-20 Durand and co-workers studied the rheological behavior of LM pectin samples during gelation at pH 7 at different temperatures. From studies at constant temperatures they found that the gel time increased roughly from 30 to 150 min with a temperature increase from 4 to 12 °C, when originally cooled from 65 °C. It was also interesting to note that the G′-G′′ crossover occurred at the same G′ value, independent of the temperature. This behavior suggests that

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the macroscopic rheology at the gel point is similar, independent of the time-temperature dependence.21 Pectin mixtures are widely used in the food industry. Pectin users often mix HM and LM pectin, and furthermore, in products such as jam, a mixture of added pectin and native pectin in the fruit is often present. Even so, the behavior of mixed pectin/pectin gels has not been a topic of previous studies and no information on their behavior exists. In contrast, mixed biopolymer systems, of which pectin is one component, have been extensively studied, such as pectin/ alginate,22-24 pectin/gelatin,25-28 and pectin/starch systems.29,30 In this study, the microstructure and the rheological properties of pure HM and LM pectin gels as well as of mixed pectin gels have been investigated. In the mixed gels, the individual gel formation mechanisms of HM and LM pectin have been alternated by varying the sucrose and calcium concentration. Mixtures in which both gel formation mechanisms have been active simultaneously have also been studied. The objective was to outline whether the individual gel formation conditions of HM and LM pectin result in differences in the gel structure and synergistic effects in the mixed gels. II. Materials and Methods A. Materials. The pectin samples, one HM and one LM pectin, were supplied by Danisco A/S, Denmark. The HM pectin was rapid set with a DM of 70.0%. The LM pectin had a DM of 33.5%. Unfortunately, no information is available on the de-esterification method used and, thus, on the distribution of the remaining methyl groups on the polygalacturonan backbone of the commercial LM-pectin chain. The HM pectin originated from citrus fruit and had a mean Mw (molecular weight) of 130 000. The mean Mw of the LM pectin was 85 000. B. Sample Preparation. Pure and mixed pectin gels of various concentrations and compositions were prepared at pH 3.0. The total pectin concentration of the gels was either 0.75 or 1.5% w/w. The conditions varying in the experimental design were the sucrose content (30% or 60%) and Ca2+-ion content (0.15% CaCl2‚2H2O). A method similar to that described by Lopes da Silva et al. was used for preparation of the gels.31 All blends were dissolved in 0.1 M citrate buffer (pH 3.0) under stirring for 2 h at room temperature. The samples were then heated to boiling point in an oil bath, temperature conditioned to 115 °C. For blends that contained no Ca2+ ions, the sugar was added to the heated blends under stirring. The blends containing Ca2+ ions were prepared by dissolving the CaCl2‚ 2H2O in a small amount of the citrate buffer. To avoid pregelation when the Ca2+ ions were added to blends containing both 60% sucrose and CaCl2, half the amount of sugar was dissolved in the calcium solution. The hot calcium solution was then slowly poured into the heated pectinsugar mixtures under vigorous stirring. The samples were heated to boiling point, and the weight was adjusted with citrate buffer. The mixtures were poured into cylindrical molds of stainless steel with an inner diameter of 30 mm or

directly applied to a preheated rheometer at 95 °C for gel formation tests. The samples in molds were cooled and allowed to stand at room temperature overnight. C. Microscopy. 1. Light Microscopy and Transmission Electron Microscopy. Small gel cubes, ∼1 × 1 × 1 mm, were carefully cut from the bulk gels and fixed in an aldehyde solution, based on citrate buffer, 2% glutaraldehyde, and 0.1% ruthenium red. Three different citrate buffers, all at pH 3.0, were used for fixation depending on the composition of the gel samples: (I) citrate buffer with 30% sucrose and 0.15% CaCl2‚2H2O; (II) citrate buffer with 50% sucrose and 0.15% CaCl2‚2H2O; (III) citrate buffer with 50% sucrose. The gel cubes were placed in the fixation solution for 2 h. The samples were rinsed twice in a buffer solution for 15 min. The samples were dehydrated in a graded ethanol series: 30, 50, 70, 90, 95, and 99%. The alcohol was then replaced by the acrylic resin LR White medium grade (Taab Laboratories, England) in a graded series with 33, 50, 67, and 100% LR White, with 1 h in each step except for 100% LR White, which was allowed to stand overnight. The polymerization of LR White was obtained at 60 °C for 12 h. Thin sections (70-80 nm) were cut with a diamond knife. The sections were transported onto Formvar-supported gold grids and stained with periodic acid, thiosemicarbazide, and silver proteinate, a method developed for polysaccharides.32,33 The samples were examined with a transmission electron microscope (LEO 906E, LEO Electron Microscopy Ltd., Cambridge, England) at 80 kV. Semithin sections (1.5 µm) for LM examination were cut with a glass knife and stained with Stevenel’s blue, a mixture of 2% potassium permanganate and 1.3% methylene blue.34 The samples were examined in a Nikon Microphot-FXA light microscope (Nikon Corp., Tokyo, Japan). 2. Confocal Laser Scanning Microscopy. Small gel slices, 1 mm thick, were stained with 0.1% Congo Red by soaking the gel piece in the staining solution. The structure was examined in a CLSM (confocal laser scanning microscope) (Leica TCS 4D, Heidelberg, Germany), equipped with an argon-krypton laser as the light source. The Congo Red was excited at a wavelength of 568 nm. The signal from the sample was detected with a long pass tritc filter at wavelengths above 590 nm. The image size was 512 × 512 pixels. D. Rheological Measurements. The rheological properties of the gels were analyzed by dynamic oscillatory measurements in a Bohlin VOR Rheometer (Bohlin Rheology, Chichester, U.K.). A frequency sweep indicated a minor frequency dependence in G′ and G′′, and a frequency of 1 Hz was used consistently during the measurements. The gel formation of the samples was investigated during cooling from 95 to 25 °C and a subsequent holding time at 25 °C. A couette measuring system was used with a cup 27 mm in inner diameter and a bob 25 mm in diameter. To avoid evaporation of the sample during measurements, the sample was covered with a thin layer of paraffin oil. The cooling rate was 3 °C/min, and measurements were made each minute using a strain of 1 × 10-3. The rheological properties of the gels set in molds were analyzed at 25 °C, using a parallel plate measuring system

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with antislip serrated surfaces and a diameter of 30 mm. The gels were cut into 5 mm thick slices, using a thin metal thread. The gel pieces were carefully placed on the lower plate, and a compression of 5% was applied to the gel piece by the upper plate. The samples were stabilized with an initial equilibrium time of 30 s before the oscillation started. During the measurements the strain 2.5 × 10-3 was used, which is within the linear viscoelastic region for the gels. Each experiment was conducted in three replicates. III. Results and Discussion Pure HM and LM pectin gels as well as mixed HM/LM pectin gels based on total pectin concentration of 0.75 and 1.5% were studied. The gels were composed of 60% sucrose with and without Ca2+ ions and of 30% sucrose with Ca2+ ions. As will be discussed later, strong synergism was noted for a mixed gel in the presence of both Ca2+ ions and 60% sucrose despite the fact that the pure LM pectin does not form a coherent gel under these conditions but rather a thick, grainy paste. It will also be demonstrated that a weak network of pure LM pectin is formed in 60% sucrose in the absence of Ca2+ ions, which is not self-supporting but has a bearing on the mixed gel characteristics. A. Properties of Pure HM and LM Pectin Gels. 1. Gel Microstructure. The gel network was characterized at different length scales by transmission electron microscopy (TEM). The lowest magnification was used to obtain information about the general nature of the network. Panels a and b of Figure 1 show that the 0.75% pectin gels are composed of surprisingly open networks with many pores well above 500 nm in size. We can also see that HM pectin forms a more homogeneous network than LM pectin. The LM pectin gel is composed of network regions with large pores as well as dense, compact regions with small pores. At higher magnifications, details of the network strands can be seen. Panels c-f of Figure 1 show that the strands are aggregated in bundles or loose aggregates and branch in an irregular manner. There is a tendency toward parallel alignment, which is somewhat higher for the HM than for the LM pectin investigated. The arrow in Figure 1e marks a loose aggregate of parallel strands. The LM pectin strands have a more flexible appearance than the HM pectin strands, which appear more stiff and straight. Furthermore, the LM pectin strands seem to be shorter and more branched than the HM pectin strands. Figure 2 shows TEM micrographs of a 1.5% HM pectin gel formed in the presence of 60% sucrose and calcium ions. The gel structure does not change in nature with increasing concentration, but as expected, the network is denser. At low magnification a homogeneous network structure is seen, as shown in Figure 2a. At higher magnification it can be seen that the network strands are composed of loose aggregates with a tendency toward parallel alignment rather than being individually distributed (see Figure 2b). Thus, the network strand characteristics are unaffected by the difference in concentration and by the presence of Ca2+ ions (compare Figure 2b with Figure 1c). Even if some differences can be seen between the pectin samples studied, their strand characteristics are of a similar

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nature. This is quite interesting, since two different gelation mechanisms are being proposed for HM and LM pectin. However, the existing models focus on interactions taking place at the molecular level and not on the overall network structure. The results shown in Figure 1 reveal similarity, and one may speculate that the conformation of the pectin in solution has a bearing on the possible modes of aggregation and network formation, even if the molecular interactions locking the structure in the aggregates differ due to the degree of methylation. The structure of pectin in dilute solutions has previously been investigated by AFM.15,16 The pectin was extracted from unripe tomato plant cell walls. Before microstructure analysis, the solutions of the pectin molecules were air-dried on a mica surface. It is interesting to note that the pectin aggregates shown by AFM are similar in shape to the network strands shown in Figure 1, even if they were not induced by sucrose or by calcium. The results show the coarse nature of the aggregated pectin network. The fact that 0.75-1.5% pectin gels form such an open structure with pores in the range of 500 nm indicates a high degree of aggregation. The length scales found in other biopolymer gels such as gelatin and carrageenan can be used for comparison. Gelatin gels are very homogeneous and dense with a pore size in the range of 10 nm, in the same concentration regimen.35-37 The gelatin strands are composed of renatured triple helices that do not aggregate.38 κ-Carrageenan, on the other hand, is known to be composed of aggregated helices. The network structure and the degree of aggregation can be varied by the ionic conditions. However, even aggregated potassium-induced κ-carrageenan gels have pores of finer dimensions than those observed in the pectin gels.39 Further work will be made to elucidate the kinetics during aggregation and gel formation of different pectin fractions. 2. Gel Rheology. The viscoelastic properties of the HM and LM pectin gels were investigated under the same conditions used for the microstructure evaluation. Figure 3 shows the storage modulus, G′, and the loss modulus, G′′, at 25 °C. From a comparison of the 0.75% HM gels, it can be seen that both G′ and G′′ are unaffected by the presence of Ca2+ ions, which is in agreement with the similarity in gel microstructure (see Figures 1 and 2). It can also be seen that the HM pectin gels are highly influenced by changes in concentration. The doubling in concentration from 0.75% to 1.5% increased the G′ 3-fold, from 1300 to 4300 Pa, and the G′′ 4-fold, from 70 to 300 Pa. The gel network became denser with increase in concentration, but the nature of the structure was unaffected by the increase in concentration from 0.75 to 1.5% (see Figures 1 and 2). Similar results with increased G′ as a consequence of increased concentration have previously been reported by Lopes da Silva et al.31 The 0.75% LM pectin gel has a G′ of around 600 Pa, which is half the G′ of the HM pectin gel at the same concentration (see Figure 3). The G′′ is around 30% lower for the LM pectin gel than that for the HM pectin gel. The difference in sucrose content as well as the network structure

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Figure 1. Network structures of pure pectin gels: (a, c, and e) 0.75% HM pectin in 60% sucrose; (b, d, and f) 0.75% LM pectin in 30% sucrose and 0.15% CaCl2‚2H2O.

and strand characteristics can explain the difference in strength between the HM and LM pectin gels investigated.

gel under conditions governing gel formation of HM pectin has also previously been reported in the literature.6,7

a. Network Formation of LM Pectin in the Absence of Ca2+ Ions. Generally, the LM pectin is not expected to form a gel in the absence of Ca2+ ions. A temperature sweep was conducted to confirm the fact that a weak LM pectin gel may form in the absence of Ca2+ ions. Figure 4 clearly shows that a weak network is formed in the presence of 60% sucrose without addition of Ca2+ ions. The gel point, measured as the G′-G′′ crossover, takes place at around 40 °C during cooling from 95 to 25 °C. The weak gel shows a G′ of 30 Pa and a G′′ of 20 Pa. The fact that LM pectin can form a

In contrast to the behavior of the LM pectin, the HM pectin did not form a gel in 30% sucrose under conditions favoring gel formation of LM pectin. Gilsenan et al.7 explained the unexpected gel formation of LM pectin as a result of the protonation of the carboxyl groups promoting conformational ordering and association by two different mechanisms: (i) suppression of electrostatic repulsion and (ii) allowing the carboxyl groups to act as hydrogen-bond donors. In this investigation, gel formation of the LM pectin was found also at pH values of 3.5 and 4.0 (no results shown). Since

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Figure 2. The network structure of a HM pectin gel: 1.5% HM pectin in 60% sucrose and 0.15% CaCl2‚2H2O.

Figure 3. G′ (a) and G′′ (b) of pure pectin gels of different compositions at 25 °C. Error bars represent standard deviation.

Figure 4. Gel formation as a function of temperature of LM pectin in the absence of Ca2+ ions: 0.75% LM pectin and 60% sucrose.

protonation of the carboxyl groups decreases with an increase in pH, the conformational ordering and association suggested by Gilsenan et al. is counteracted by the pH increase. More studies are needed to fully explain the gelling behavior of the LM pectin at low Ca2+ concentrations. B. Properties of Mixed HM/LM Pectin Gels. 1. Gel Microstructure. a. Mixed Gels Formed in the Absence of Ca2+ Ions. As shown in the previous section, pure HM and LM pectin gels are composed of open networks with the strands arranged in bundles or loose aggregates. Mixed

HM/LM pectin gels exhibit quite different characteristics, which depend on the prevailing gel formation conditions. A mixed gel of HM and LM pectin in the presence of 60% sucrose is shown in Figure 5. The given condition favors the gel formation of HM pectin. However, as demonstrated in Figure 4, a weak gel of LM pectin also occurs under these circumstances. The mixed network structure appears very dense and compact at the lower magnification (Figure 5a), and it is even difficult to observe any structure. A fine, threadlike structure is revealed at the higher magnification (Figure 5b). The strands form a homogeneous and fine network with small pores. The mixed gel structure is very different and far less aggregated than that of the pure HM and LM pectin gels (see Figures 1 and 2). The presence of a weak LM pectin network may function as a sterical hindrance and thus prevent aggregation of the HM pectin strands into the loose bundles of aggregates observed for the pure HM pectin gel. The results imply that the open, aggregated structure of the pure pectin gels may be a result of secondary aggregation, where a fine network is formed at the gel point followed by further aggregation and rearrangements. Secondary aggregation taking place after the gel point has previously been observed for gels of other biopolymers such as κ-carrageenan and β-lactoglobulin.39-41 The big differences between the state of aggregation in the

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Figure 5. The network structure of a mixed HM/LM pectin gel in the absence of Ca2+ ions: 0.75% HM pectin, 0.75% LM pectin, and 60% sucrose.

pure pectin gels and the mixed pectin gels in the absence of calcium illustrate the importance to elucidate the kinetics of gel formation at the supramolecular level. b. Mixed Gels Formed in the Presence of Ca2+ Ions. A totally different microstructure is found for a mixed gel formed in the presence of both 60% sucrose and Ca2+ ions. Under this condition, gel formation of HM pectin is favored by the sucrose content and gel formation of LM pectin is favored by the presence of calcium. TEM micrographs at five magnifications are shown in Figure 6. A highly inhomogeneous structure is revealed. The inhomogeneity is on a large scale that is not covered even by the lowest magnification used by TEM (Figure 6a). Instead, the micrographs show part of a phase-separated structure. At higher magnifications, as shown in Figure 6b-d, the network varies enormously in density. The gel is composed of dense network regions as well as loose, open network regions. The border between the dense and loose network regions appears rather sharp in the images shown in Figure 6b-d. Details of the dense and loose network areas are shown at a high magnification in panels e and f of Figure 6, respectively. The network strands composing the dense areas are shorter and more branched than those composing the loose areas, which appear stiffer and straighter. A comparison with the pure network strands shows that the network strands of the dense regions are more similar to the LM pectin strands (see Figure 1f), whereas the strands of the loose regions are more similar to the HM pectin strands (see Figure 1e). The presence of a phase-separated structure is clearly demonstrated from the LM and confocal laser scanning microscopy (CLSM) micrographs at low magnification shown in Figure 7. The images give better information on the length scale of the inhomogeneities in the structure, which seems to be on the millimeter or micrometer scale rather than on the nanometer scale. The structure shown by the light micrograph was prepared by the same procedure of fixation, dehydration, and plastic embedding as the samples prepared for TEM. The advantage of CLSM is the minimum of preparation necessary. The gel was investigated fully hydrated as a bulk sample, and only a fluorescent stain was

added. The CLSM therefore validates the information obtained by LM and TEM. The results show that the HM pectin forms a coherent gel (see Figure 3) in the presence of 60% sucrose and Ca2+ ions. The LM pectin does not form a gel at all, but rather a thick, grainy paste of hard gel particles. The HM pectin starts to gel after the sample is poured into the mold, i.e., at a temperature slightly below 95 °C, whereas the formation of the LM gel particles seems to take place directly in the beaker when the sucrose and Ca2+ ions are added at 95 °C. These observations indicate that the LM particles start to form slightly before the onset of network formation of the HM pectin. The results indicate that the dense network areas correspond to the gel particles formed by LM pectin and that the sparse network consists mainly of HM pectin. This would also be in agreement with the comparison made of the network strand characteristics of the mixed and pure gels discussed above. It is interesting to note that strongly aggregated LM pectin structures may occur at sufficiently high concentrations of calcium ions, which also previously has been shown for LM pectin/starch mixtures.29 2. Gel Rheology of Mixed Gels. The rheological behavior of the mixed gels varies to a large extent with the composition of the blend and resulting microstructure. Large synergistic effects or none at all is observed, depending on the complexity of the composition and, thus, on the gel formation mechanisms in favor. In the presence of 60% sucrose and Ca2+ ions, gel formation of both HM and LM pectin is favored. As previously shown, a very inhomogeneous phase-separated mixed gel is formed. Very strong synergistic rheological properties are obtained from this mixed gel structure. The gel has exceptionally high G′ and G′′ in comparison with the other mixed gels shown in Figure 8 and also in comparison with the pure gels shown in Figure 3. The behavior is not an effect of concentration, since a pure HM gel with the same total pectin concentration of 1.5% as the mixed gel (see Figure 3) shows lower G′ and G′′ than the inhomogeneous mixed gel. The same synergism is observed

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Figure 6. The network structure of a mixed HM/LM pectin gel in the presence of Ca2+ ions: 0.75% HM pectin, 0.75% LM pectin, 60% sucrose, and 0.15% CaCl2‚2H2O.

when comparing the pure and mixed pectin gel with a total concentration of 0.75%. A weak synergistic effect is also found for the dense finestranded mixed network formed in the presence of 60% sucrose (see Figure 5). The dense network shows a G′ of around 1700 Pa and a G′′ of 240 Pa (see Figure 8), while the corresponding pure 0.75% HM and LM pectin gels show G′ of 1300 and 30 Pa, respectively, and G′′ of around 70 and 20 Pa, respectively (see Figures 3 and 4). In this case, 1.5% HM pectin gel has a considerably higher G′ value (4300 Pa) than the 1.5% mixed gel. The mixed gel structure is

entirely different from that of the pure HM pectin gel, and it is therefore difficult to make a comparison between the structure and the rheological behavior of the gels. The aggregated HM pectin with stiffer strands gives rise to a higher storage modulus than the mixed fine stranded gel at the same concentration. On the other hand, we do not know to what extent the weak LM pectin gel participates and contributes to the network properties of the mixed gel. No synergistic effects were observed for mixed gels in the presence of Ca2+ ions and 30% sucrose. The composition favors the gel formation of the LM pectin only. The

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Figure 7. Images of a mixed HM/LM pectin gel in the presence of Ca2+ ions (0.75% HM pectin, 0.75% LM pectin, 60% sucrose, and 0.15% CaCl2‚2H2O): (a) LM image, dark areas correspond to the dense network regions; (b) CLSM image, light areas correspond to the dense network regions.

Figure 8. G′ and G′′ of mixed pectin gels of different compositions at 25 °C. Error bars represent standard deviation.

rheological properties of the mixed gel were similar to those of the pure LM pectin gel. The behavior indicates that the presence of HM pectin has a minor influence on the gel formation of the LM pectin. Figure 8 shows G′ and G′′ for the mixed gel based on 30% sucrose and Ca2+ ions. A comparison with pure LM pectin gel made under the same conditions shows that G′ is unaffected by the presence of HM pectin, whereas G′′ is higher for the mixed gel than for the pure LM pectin gel (see Figure 3). The enhanced G′′ indicates that the HM pectin increases the polymer concentration in the water-sucrose phase and thereby the viscosity. The HM pectin does not form a gel under these conditions, since the low sucrose content promotes solvent-pectin interactions rather than pectin-pectin interactions. The mixed gels show large differences, both in rheological behavior and microstructure, depending on the conditions chosen for gel formation. Two cases have been presented, where both HM and LM pectin are active in the gel formation. In the presence of both 60% sucrose and Ca2+ ions, a phase-separated structure is formed, which shows extremely large synergistic effects in both G′ and G′′. In contrast, a completely different structure is formed for the mixed gels in the presence of 60% sucrose and absence of Ca2+ ions. A dense, fine-stranded microstructure is formed,

in which the HM and LM pectin may be connected. A weak synergistic effect was also found for this mixture. Under conditions where only LM pectin formed a gel, no effect of HM pectin was detected in the storage modulus reflecting the network structure. Instead, the presence of HM pectin in the solute was reflected by an increase in the loss modulus. The results demonstrate the possibility to design pectin blends with specific rheological properties, not only by modifying the molecular properties of pectin fractions during production but also by mixing fractions and thereby tuning the gelforming behavior of the mixed systems.

IV. Conclusions This investigation presents for the first time the microstructure of pectin gels on several length scales. For the pure pectin gels, open networks were found, with large pores in the range of 500 nm and aggregated network strands. The nature of the HM and LM pectin networks is similar, even though small differences are found. The LM pectin network is more inhomogeneous than the HM pectin network. Furthermore, the LM pectin network strands appear more

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flexible and branched than the HM pectin strands, which appear stiffer and straighter. Both the microstructure and the rheological properties of the mixed gels were different from those of the pure gels. In the presence of both 60% sucrose and Ca2+ ions, the gel formation mechanisms of both HM and LM pectin are favored, and the resulting network is inhomogeneous, based on dense, compact network regions as well as more loose, sparse regions. The formation of the structure was interpreted in terms of the behavior of the pure samples under the same conditions. The inhomogeneous structure showed large synergistic effects in both G′ and G′′. In the presence of 60% sucrose without calcium addition, a mixed gel with a homogeneous fine-stranded network structure was formed. Under these conditions a weak LM pectin gel was formed, which took part in the gel formation of the mixed system and influenced the kinetics of gel formation and aggregation of the HM pectin strands. A weak synergistic effect in rheological behavior was found for this mixed gel structure. Acknowledgment. This work is part of the LiFT program (Future Technologies for Food Production), financed by the SSF (Swedish Foundation for Strategic Research). Siw Kidman is gratefully acknowledged for technical assistance with the microscopy work. Professor A. Voragen and Dr. H. Schols are thanked for fruitful discussions during preparation of the manuscript. References and Notes (1) Voragen A. G. J.; Pilnik, W.; Thibault, J.-F.; Axelos, M. A. V.; Renard, C. M. G. C. Pectins. In Food Polysaccharides and their Applications; Stephen, A. M., Ed.; Marcel Dekker: New York, 1995; pp 287-339. (2) Walkinshaw, M. D.; Arnott, S. Conformations and Interactions of pectins I & II. J. Mol. Biol. 1981, 153, 1055-1073, 1075-1085. (3) Oakenfull, D.; Scott, A. Hydrophobic interactions in the gelation of high methoxyl pectins. J. Food Sci. 1984, 49 (4), 1093-1109. (4) Grant, G. T.; Morris, E. R.; Rees, D. A.; Smith, P. J. C.; Thom, D. Biological interactions between polysaccharides and divalent cations: the egg-box model. FEBS Lett. 1973, 32 (1), 195-198. (5) Morris, E. R.; Powell, D. A.; Gidley, M. J.; Rees, D. A. Conformations and Interactions of Pectins I. Polymorphism between Gel and Solid States of Calcium Polygalacturonate. J. Mol. Biol. 1982, 155, 507-516. (6) Morris, E. R.; Gidley, M. J.; Murray, E. J.; Powell, D. A.; Rees, D. A. Characterization of pectin gelation under conditions of low water activity, by circular dichroism, competitive inhibition and mechanical properties. Int. J. Biol. Macromol. 1980, 2, 327-330. (7) Gilsenan, P. M.; Richardson, R. K.; Morris, E. R. Thermally reversible acid-induced gelation of low-methoxy pectin. Carbohydr. Polym. 2000, 41, 339-349. (8) Daas, P.; Meyer-Hansen, K.; Schols, H.; Ruiter, G. A.; Voragen, A. Investigation of the nonesterified galacturonic acid distribution in pectin with endopolygalacturonase. Carbohydr. Res. 1999, 318, 135145. (9) Daas, P.; Voragen, A.; Schols, H. Characterization of nonesterified galacturonic acid sequences in pectin with endopolygalacturonase. Carbohydr. Res. 2000, 326, 120-129. (10) Daas, P.; Boxma, B.; Hopman, A.; Voragen, A.; Schols, H. Nonesterified Galacturonic Acid Sequence Homology of Pectins. Biopolymers 2001, 58, 1-8. (11) Daas, P.; Voragen, A.; Schols, H. Study of the Methyl Ester Distribution in pectin with endo-Polygalacturonase and HighPerformance Size-Exclusion Chromatography. Biopolymers 2001, 58, 195-203. (12) Kravtchenko, T. P.; Voragen, A. G. J.; Pilnik, W. Analytical comparison of three industrial pectin preparations. Carbohydr. Polym. 1992, 18, 17-25.

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