Effects of Calcium, pH, and Blockiness on Kinetic Rheological

In Food polysaccharides and their applications; Stephen, A. M., Ed.; Marcel .... Rao, M. A.; Buren J. P, v.; Cooley, H. J. Rheological changes during ...
1 downloads 0 Views 562KB Size
Download PDF
Biomacromolecules 2005, 6, 646-652

646

Effects of Calcium, pH, and Blockiness on Kinetic Rheological Behavior and Microstructure of HM Pectin Gels Caroline Lo¨fgren,† Ste´ phanie Guillotin,‡ Hanne Evenbratt,† Henk Schols,‡ and Anne-Marie Hermansson†,* SIK, The Swedish Institute for Food and Biotechnology, P.O. Box 5401 SE-402 29 Go¨teborg, Sweden, and Agrotechnology and Food Sciences, Laboratory of Food Chemistry, Wageningen University, P.O. Box 8129 6700 EV Wageningen, The Netherlands Received July 1, 2004; Revised Manuscript Received November 3, 2004

The kinetic behavior during gel formation and the microstructure of 0.75% high methoxyl (HM) pectin gels in 60% sucrose have been investigated by oscillatory measurements and transmission electron microscopy for three comparable citrus pectin samples differing in their degree of blockiness (DB). Ca2+ addition at pH 3.0 resulted in faster gel formation and a lower storage modulus after 3 h for gels of the blockwise pectin A. For gels of the randomly esterified pectin B, the Ca2+ addition resulted in faster gel formation and a higher storage modulus at pH 3.0. At pH 3.5, both pectins A and B were reinforced by the addition of Ca2+. In the absence of Ca2+, the shortest gelation time was obtained for the sample with the highest DB. Microstructural characterization of the gel network, 4 and 20 h after gel preparation, showed no visible changes on a nanometer scale. The microstructure of pectins A and B without Ca2+ was similar, whereas the presence of Ca2+ in pectin A resulted in an inhomogeneous structure. I. Introduction Pectin is a natural polysaccharide, frequently used as a food additive due to its gel forming properties. The major constituent of pectin is R-(1-4) D-galacturonic acid units with some of the carboxyl groups present in methyl ester form. The polygalacturonic backbone is interrupted by the insertion of few rhamnose residues, and neutral sugar units are attached to the backbone and concentrated in highly branched “hairy regions”.1 Industrial pectins are usually extracted from citrus fruit and apples. The esterification patterns vary according to the extraction method used. Acidic deesterification causes random distribution of the methyl esters, whereas enzymatic degradation with plant pectinesterase results in blockwise distribution of the methyl esters.2,3 The degree of methyl esterification (DM) divides pectin into high methoxyl (HM) and low methoxyl (LM) pectin. HM pectin, with more than 50% of the carboxyl groups esterified, forms gels at pH < 3.5 and in the presence of more than 55% sugar or similar cosolute. Gel formation of LM pectin occurs in the presence of Ca2+ ions over a wide range of pH values both with and without sugar. The gel formation mechanism for HM pectin is based on hydrophobic interactions and hydrogen bonds.4 The hydrophobic interactions mainly occur between methyl esters at reduced water activity promoted by the cosolute. Hydrogen bonds are favored at low pH values due to suppression of electrostatic repulsion between pectin chains.5 The gel formation mechanism of LM pectin is based on the so-called egg box model, including * To whom correspondence should be addressed. E-mail: [email protected]. † SIK, The Swedish Institute for Food and Biotechnology. ‡ Wageningen University.

interactions between Ca2+ ions and carboxyl groups in the pectin backbone.6 The DM influences the functional properties of both HM and LM pectin, but in opposite ways. Increased DM results in more rapid gel formation for HM pectins but slower gel formation for LM pectins.3 The distribution of methyl esters also influences the functionality of pectin. Blockwisedistributed pectins show strong Ca2+-binding behavior and are often referred to as Ca2+-sensitive pectins.7,8 Yield stress measurements indicate greater interchain association for blockwise pectins than for random pectins in the presence of Ca2+ with DM ∼ 45%.9 Furthermore, Rolin reports that HM pectin with blockwise distribution of methyl esters form gels at a higher temperature than pectin with the same DM in a more uniform distribution.8 The kinetic behavior during gel formation of HM pectin at pH 3.0 in the presence of 60% sucrose has been studied by Lopes da Silva et al.10 The gel formation was strongly dependent on both concentration and temperature, and in addition, the rate of gelation close to the gel point was described as a second-order rate process. Aging experiments on HM pectin/sugar gels show a slight increase in the storage modulus, which is attributed to continuous reorganization of the gel network.11,12 HM pectin gels may be either reinforced or weakened by calcium addition,3,13 and furthermore, their setting temperature increases in the presence of calcium.13,14 Pectin mixtures are frequently used in the food industry to obtain products with desired texture and gelling characteristics. A previous study focused on the microstructure and the rheological properties of pectin mixtures under different gel formation conditions.15 The mixtures were also compared

10.1021/bm049619+ CCC: $30.25 © 2005 American Chemical Society Published on Web 01/27/2005

Biomacromolecules, Vol. 6, No. 2, 2005 647

Kinetic Behavior of HM Pectin Gels Table 1. Chemical Characteristics of the Pectins pectin

DM, %

DB, %

GalA content, %

A B C

74 72 82

16 5 13

82 74 78

neutral sugar content, % 7 12 6

with pure HM and LM pectin gels under the same conditions. Transmission electron microscopy revealed that the microstructures of pure and mixed pectin gels differed greatly. Pure HM and LM pectin gels were composed of open networks with pores in the range of 500 nm, whereas a mixed gel in the absence of calcium revealed a fine-stranded, homogeneous structure with much smaller pores. The results implied that the organization of the network structure of the gels depended largely on the kinetic properties. Large variations in kinetic behavior were also found for the different preparations, with rather slow gel formation for HM pectin and rapid gel formation for LM pectin. The mixed HM/LM pectin gel in the absence of calcium showed characteristics of both HM and LM pectin gelation.16 The microstructure of dried pectin solutions has previously been investigated by atomic force microscopy (AFM).17,18 More recently the microstructure of hydrated pectin gels was imaged by AFM, by scanning a thin layer of pectin adhered to a mica surface.19 The present work focuses on the kinetic behavior during gel formation of HM pectins having the same characteristics but differing in the internal distribution of the methyl esters. The influence of calcium was studied at pH 3.0 and 3.5 in the presence of 60% sucrose. The objective was to outline how calcium and pH affect both the kinetic behavior and the microstructure of HM pectin gels. II. Materials and Methods A. Materials. The pectin samples were produced by Degussa Texturant Systems, Redon, France. Three citrus pectin samples, A, B, and C, with about the same DM but differing in their degree of blockiness (DB) were investigated, see Table 1.20 Pectins A and B originate from the same lemon variety. The MW of the samples, determined with highperformance size exclusion chromatography (HPSEC), were ∼ 82 kDa for pectins A and C and ∼78 kDa for pectin B. B. Uronic Acid Content. Pectins (60 µg/mL) were boiled (1 h), cooled, and then saponified with sodium hydroxide (40 mM). The uronide content was determined by the automated colorimetric m-hydroxydiphenyl method.21-23 C. Degree of Blockiness. The pectins were digested with polygalacturonase of KluiVeromyces fragilis (PGkf) which needs 4 or more free GalA to act. Degradation products were analyzed by HPAEC at pH 5.20,24 Quantification of these oligomers is possible by integration from HPAEC pH 5 elution profiles. From these results, the degree of blockiness was determined25,26 (Table 1). A high DB value is indicative for the release of a high number of mono-, di-, and tri-GalA and thus for a blockwise distribution of nonesterified galacturonic acid residues in a pectin. D. Sample Preparation. The pectin concentration was 0.75% and sucrose concentration 60% for all samples. The

pectins were dissolved in 0.1 M citrate buffer at pH 3.0 or pH 3.5 and stirred at room temperature for 2 h. The samples were then heated to boiling point in an oil bath with a temperature of 115 °C. Sucrose was added to the samples, after which a preheated solution of CaCl2 in citrate buffer was added to the samples containing Ca2+. The samples were heated to boiling point, and the weight was adjusted with distilled water. To prepare them for microscopy, the samples were poured into cylindrical moulds of stainless steel and stored at 20 °C in a water bath. E. Rheological Measurements. The rheological properties during the gel formation were characterized by oscillatory measurements in a strain-controlled Bohlin VOR Rheometer (Bohlin Rheology, Chichester, U.K.) equipped with a concentric cylinder with a volume of 25 mL. The boiling samples were rapidly poured into the rheometer cup, temperature conditioned to 20 °C. The bob was lowered, and the surface of the sample was covered with paraffin oil to avoid evaporation. The temperature of 20 °C was obtained ∼7 min after the measurement started. The gel formation measurements were performed at a frequency of 1 Hz and a strain of 2 × 10-3 for 3 and 20 h. The rheological measurements were conducted in three replicates, except the 20 h measurements which were conducted in two replicates. F. Microscopy. Small gel cubes ∼1 × 1 × 1 mm were carefully cut from the bulk gels 4 and 20 h after gel preparation. The gels were fixed in an aldehyde solution, based on citrate buffer, 2% glutaraldehyde, and 0.1% ruthenium red. Two different citrate buffers, both at pH 3.0, were used for fixation depending on the composition of the gel samples: (I) citrate buffer with 50% sucrose and (II) citrate buffer with 50% sucrose and the same concentration of CaCl2‚H2O as in the gel. 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 with ethanol, and the acrylic resin was dehydrated with LR white (for details, see previous work).15 The polymerization of LR white was obtained at 60 °C for about 20 h. Thin sections (70-80 nm) were cut with a diamond knife. The sections were transferred to Formvar-supported gold grids and stained with periodic acid, thiosemicarbazide, and silver proteinate.27 The samples were examined with a transmission electron microscope (LEO 906E, LEO Electron Microscopy Ltd., Cambridge England) at 80 kV. Each microscopy preparation was conducted in two replicates and the conclusions of the microscopy results were based on a large number of TEM images. III. Results and Discussion A. Gel Formation and MicrostructuresEffect of Blockiness. 1. Gel Formation. Three comparable citrus pectins, A, B, and C, with DM in the range of 72-82% but differing in their degree of blockiness (DB) were investigated, see Table 1.20 The kinetic behavior during gel formation of A, B, and C with a pectin concentration of 0.75% and a sucrose concentration of 60% was followed at pH 3.0 for 20 h, see Figure 1. The results imply that changes in blockiness lead to variations in kinetic behavior. The most randomly

648

Biomacromolecules, Vol. 6, No. 2, 2005

Lo¨fgren et al.

Figure 1. Gel formation at pH 3.0 of (a) pectin A, (b) pectin B, and (c) pectin C. Gelation time vs degree of blockiness (d).

distributed pectin B exhibits a considerably lower storage modulus and also slower gel formation than the other samples. The shortest gelation time was obtained for pectin A, which is the most blockwise-distributed pectin. Pectins A and C show similar behavior, with a large increase in G′ during the first 4 h, followed by a small increase in G′ during the latter part of the measurement. After 20 h G′ is ∼1200 Pa for both pectins A and C. However, the pectins differ in gelation time i.e., the G′-G′′ crossover, which is 17 min for A and 29 min for C. G′′ passes through a local maximum for both A and C after about 50 and 65 min, respectively, which is often seen for gelling biopolymers.28,29 The behavior agrees well with the cure curves previously obtained at pH 3 for HM pectin at various concentrations in the presence of 60% sucrose.10,11 Pectin B behaves differently from A and C. The initial increase in G′ is smaller, and after 20 h G′ is about one-third of the values of A and C. Furthermore, the crossover occurs after 55 min for B. Figure 1d views the relation between gelation time and DB, which demonstrates that small differences in the chemical structure may lead to quite large variations in kinetic behavior. It is well-known that increased DM results in more rapid gel formation for HM pectins.1 High DM enables the formation of several hydrophobic interactions between methyl esters. One may speculate that when the methyl esters are concentrated into blocks the pectin will appear as a more rapidly set one compared to pectin with the same DM but with random distribution of methyl esters. 2. Microstructure. The G′ values of the three samples increases considerably over time, and the microstructures of pectins A and B were therefore studied on two different occasions during the gel formation. Gel pieces were fixed after 4 and 20 h, respectively. Figure 2, parts a and b, shows the microstructure of pectin B, 4 and 20 h after gel preparation. The gel structure does not change in nature, although the storage modulus increases from G′4h ∼ 190 Pa

Figure 2. Gel microstructure of pectins A and B at pH 3.0 after 4 and 20 h. (a) pectin B, 4 h; (b) pectin B, 20 h; (c) pectin A, 4 h; and (d) pectin A, 20 h.

to G′20h ∼ 440 Pa. Both images reveal rather homogeneous structures, even if some variations in the network density can be seen. The microstructure of pectin A appears similar to that of pectin B. Both pectin A and B reveal open network structures with strands arranged in loose aggregates which can be seen for pectin A at higher magnification in Figure 2c,d. In similarity with pectin B, no difference in the gel structure is observed between 4 h (Figure 2c) and 20 h (Figure 2d) although G′ increases from G′4h ∼ 800 Pa to G′20h ∼ 1100 Pa. The coarse network structures of pectins A and B agree well with the structure of commercial HM pectin (DM 70.0%, 60% sucrose, pH 3.0) shown previously.15

Kinetic Behavior of HM Pectin Gels

Biomacromolecules, Vol. 6, No. 2, 2005 649

Figure 3. Gel formation of pectins A and B in the presence of 0.15% CaCl2‚2H2O. (a) pectin A, pH 3.0; (b) pectin B, pH 3.0; (c) pectin A, pH 3.5; and (d) pectin B pH 3.5.

One may speculate that the increase in G′ between 4 and 20 h may be related to internal strengthening of the gel network by intermolecular forces such as hydrophobic interactions and hydrogen bonds, without any structural changes visible on a nanometer scale. However, a considerable increase in G′ also occurs during the initial 4 h of the gel formation process. During this period the basic network structure of the gel is expected to be developed, which then may be further reinforced by intramolecular forces. B. Kinetic Behavior and MicrostructuresEffect of Calcium. 1. Gel Formation at pH 3.0 and 3.5. The influence of Ca2+ addition on kinetic behavior was studied for pectins A and B. Both pectins show very rapid gel formation at pH 3.0 in the presence of Ca2+ with G′ > G′′ from the beginning of the measurements and with a sharp initial increase in G′ (Figure 3, parts a and b). For pectin A, G′ reaches a plateau after ∼1 h, whereas pectin B continues to increase slightly even after the first 2 h of the measurement. Both A and B exhibit more rapid gel formation with Ca2+ than without, and furthermore, the presence of Ca2+ also affects the final storage moduli of the pectins. From Figures 1a and 3a, it can be seen that the storage modulus of pectin A is higher in the absence of Ca2+ (G′20h ∼ 1050) than in the presence of Ca2+ (G′20h ∼ 650 Pa). The behavior of pectin A is also seen in pectin C and several other pectins with DM ranging from 67 to 74% (results not shown). In contrast, Figures 1b and 3b show that the storage modulus of pectin B is lower in the absence of Ca2+ (G′20h ∼ 440 Pa) than in the presence of Ca2+ (G′20h ∼ 700 Pa). The opposite behavior is observed for the loss modulus for pectin B, which is higher in the absence of Ca2+ (G′′20h ∼ 20 Pa) than in the presence of Ca2+ (G′′20h ∼ 5 Pa). The changes in G′ and G′′ indicate that gel formation of pectin B is favored by Ca2+ addition at pH 3.0, resulting in a stiffer and more elastic gel. Other kinetic effects occur at pH 3.5. In the absence of Ca2+, no gels are formed for pectins A and B (Figure 3,

parts c and d). At pH 3.0, the majority of the carboxylic groups are undissociated, which is a prerequisite for HM pectin gel formation with hydrogen bonds and hydrophobic interactions. At pH 3.5, the amount of dissociated carboxylic groups is increased, which seems to prevent HM pectin gelation in the absence of Ca2+. In the presence of Ca2+, weaker gels are formed at pH 3.5 than at pH 3.0. For pectin A, the Ca2+ addition leads to a slow, steady increase in G′, in contrast to the rapid increase in G′ at pH 3.0 (Figure 3c). Interestingly, after 20 h, G′ still increases and no constant value is reached. Pectin B forms a very weak gel at pH 3.5 with G′ ∼ G′′ (Figure 3d). One explanation might be that the presence of Ca2+ enables the unesterified sequences of the blockwise pectin A to participate in the gel formation, which results in a slow-set, rather weak gel. The randomly distributed pectin B consists of shorter unesterified sequences, which are less accessible for Ca2+-induced gel formation. The result is a system on the sol-gel threshold with G′ and G′′ < 20 Pa. Similar behavior as for A and B was reported by Rolin30 for 3% HM pectin samples with about the same DM (65-66%) but with low or high Ca2+ sensitivity. Gels were formed in the absence of sucrose at pH 3.1 for samples with high Ca2+ sensitivity (phase angle < 45°) but not for samples with low Ca2+ sensitivity (phase angle > 45°).30 2. Microstructure with and without Calcium. The large variations in kinetic behavior due to Ca2+ addition raised the question about microstructural variations among the gels. Gels of pectins A and B were prepared at pH 3.0 and fixed after 20 h. In the absence of Ca2+, the gel structure of pectins A and B is relatively similar, being composed of open, branched network structures (Figures 2b,d and 4a,b). The Ca2+ addition affects the microstructure of pectin A. In the presence of Ca2+, an inhomogeneous gel structure with large differences in the network density, containing both dense as well as sparse areas (Figure 4c), is revealed. The dense area is composed of a coherent network of aggregated strands

650

Biomacromolecules, Vol. 6, No. 2, 2005

Figure 4. Gel microstructure of pectins A and B at pH 3.0 after 20 h. (a) pectin A without Ca2+; (b) pectin B without Ca2+; (c) pectin A with 0.25% CaCl2‚2H2O, left, dense area; right, sparse area; and (d) pectin B with 0.25% CaCl2‚2H2O.

and small pores around 100 nm in diameter, whereas the sparse region consists of aggregated strands surrounded by pores up to around 500 nm in diameter. The inhomogeneous structure also found during the 4 h preparation (not shown) is probably an effect of the high Ca2+ sensitivity due to the blockwise methyl ester distribution in pectin A. It seems likely that when the Ca2+ solution is added to the sample dropwise rapid gel formation occurs in the vicinity of the droplets. The Ca2+ ions may contribute to the network formation, since they result in dense regions in the gel

Lo¨fgren et al.

structure. The inhomogeneous structure of pectin A in the presence of Ca2+ could also be a direct result of the very rapid kinetic behavior, which may prevent the pectin chains from forming a homogeneous gel network. In contrast to pectin A, the Ca2+ addition in pectin B does not affect the microstructure of the gels. Both in the absence and the presence of Ca2+, rather homogeneous gels are revealed for pectin B (Figure 4, parts b and d), which is interesting since large differences in kinetic behavior occur between the samples. However, the random distribution of methyl esters in pectin B results in a less Ca2+-sensitive pectin, which might be the reason for homogeneous gels both in the absence and the presence of Ca2+. Figure 4c,d show gels at a concentration of 0.25% CaCl2‚2H2O, but the same results were obtained at 0.15% CaCl2‚2H2O. 3. Kinetic Effects of Calcium Addition. Drastically different kinetic behavior is observed when Ca2+ is present in the gel formation. The presence of Ca2+ accelerates the gel formation for all preparations irrespective of Ca2+ concentration. The kinetic properties also exhibit large differences between pectins A and B, which may be attributed to the variations in Ca2+ sensitivity. Moreover, the pH increase from 3.0 to 3.5 strongly influences the kinetic behavior and the storage modulus of the samples, see Figure 5. For pectin A, the Ca2+ addition at pH 3.0 results in very rapid gel formation and also a complex behavior in the storage modulus (Figure 5a). The storage moduli of the Ca2+-containing samples reach a plateau within ∼1 h, whereas in the absence of Ca2+, the storage modulus increases continuously. The results imply that after 1 h the storage moduli for the Ca2+-containing samples are higher than for the sample without Ca2+, but after 3 h the opposite result is obtained, which is quite interesting since it illustrates the influence of the kinetic aspects on the gel properties. For pectin B at pH 3.0, the

Figure 5. Effect of Ca2+ addition in G′ for pectins A and B at pH 3.0 and 3.5. (a) pectin A, pH 3.0; (b) pectin B, pH 3.0; (c) pectin A, pH 3.5; and (d) pectin B, pH 3.5. The dotted lines in figures c and d indicate that no gelation occurred (G′ < G′′) during the measurement.

Kinetic Behavior of HM Pectin Gels

effect of Ca2+ addition is more straightforward; the Ca2+containing samples exhibit a higher storage modulus than the sample without Ca2+ throughout the measurements (Figure 5b). The kinetic behavior during gel formation also varies considerably with the amount of added Ca2+. Increased Ca2+ concentration results in a more rapid increase in the storage moduli for both pectins A and B. Very rapid gel formation may also be attributed to inhomogeneous gels, which is indicated in Figure 5a by the local maximum in the storage modulus for pectin A with 0.25% CaCl2‚2H2O. The result agrees well with the microstructure of pectin A with Ca2+ shown in Figure 4c, which reveals both dense and sparse areas. However, observations during the experimental work indicate clear, homogeneous gels on the macroscopic level both in the absence and the presence of Ca2+. In comparison, the addition of Ca2+ does not alter the nature of the microstructure of pectin B (Figures 4b,d). The large increase in the storage modulus during the initial part of the gel formation of pectin A with Ca2+ may be a consequence of Ca2+-supported bindings of unesterified sequences in the pectin backbone. One may speculate that an initial structure based on Ca2+ bindings to unesterified blocks may lock the structure. Thus, the possibilities for further strengthening of the gel network by hydrophobic interactions and hydrogen bonds are diminished. It is interesting to note that a higher storage modulus is obtained for pectin B in the presence of 0.25% CaCl2‚2H2O than for pectin A with the same amount of Ca2+, at pH 3.0. The rather slow gel formation seen for pectin B in the absence of Ca2+ could be an advantage in the presence of Ca2+, since the gel formation occurs steadily and thereby enables Ca2+ ions to support the gel structure based on the HM pectin mechanism. At pH 3.5, the Ca2+ addition results in a reinforced storage modulus for both pectins A and B. The dotted lines in Figure 5, parts c and d, represent G′, where G′ < G′′ i.e., no gel formation occurred in these preparations. Although pectins A and B are highly esterified, Ca2+ ions are required to support the gel formation when pH is increased from 3.0 to 3.5. The storage modulus in pectin A with 0.25% CaCl2‚ 2H2O changes in nature when pH increases from 3.0 to 3.5. At pH 3.0 the initial increase in G′ is very rapid and G′ > G′′ at the very start of measurements. At pH 3.5, the storage modulus increases steadily and rather slowly during the measurement period as a result of more dissociated carboxylic groups. The results indicate that the pectin A-Ca2+ interaction is favored by the pH increase since the slow gel formation that occurs at pH 3.5 enables Ca2+-supported gelation in the blockwise pectin A. In contrast, the storage modulus of the random pectin B with 0.25% CaCl2‚2H2O does not change in nature when pH increases from 3.0 to 3.5. Both curves reveal a rapid kinetic behavior and reach a plateau during the measurement period. In addition, the pH increase also leads to weaker gels for both pectins A and B due to poorer gel conditions for the HM pectin mechanism. It is well-known that the distribution of methyl esters influences the Ca2+-binding properties of pectins.7,9 This work contributes to the understanding of the functional behavior of pectin gels by studying three HM pectins with

Biomacromolecules, Vol. 6, No. 2, 2005 651

different degree of blockiness (DB). Although pectin A and B differ on GalA content as well as on neutral sugar (NS) content, it is assumed that this will not really affect the gelling behavior observed. It cannot be completely ruled out that differences in GalA, NS content, and MW may cause differences in gelation, but it makes more sense that the more blockwise distribution of methyl esters in pectin A is responsible for the given gelling properties in a Ca2+ environment. In addition, it can be seen from the work by Guillotin20 et al. that although the total NS content is slightly different for the two pectins, the rhamnose content is similar (0.4% in w/w). Furthermore Perez31 et al. concluded from molecular modeling experiments that rhamnose insertions do not affect the three-dimensional structure of pectins. It is rather interesting that variations in DB result in differences in kinetic behavior of pectins A, B, and C at pH 3. The blockwise pectin A and the random pectin B also showed different behavior after Ca2+ addition at pH 3.0. The random pectin B was reinforced by the Ca2+ addition, whereas the blockwise pectin A showed a lower storage modulus in the presence of Ca2+ after ∼3 h. The result is comparable with the different effects of Ca2+ addition to HM pectin gels observed earlier.8,13 Rolin reports that the breaking strength of blockwise HM pectin gels may increase with the amount of Ca2+, but in other cases that the heterogeneity may lead to gels with poor breaking strength.8 Interestingly, the effect of Ca2+ addition on kinetic behavior varies greatly. All samples showed more rapid gel formation in the presence than in the absence of Ca2+. In comparison, higher setting temperatures were obtained when Ca2+ was present in HM pectin/sucrose gels than for HM pectin/sucrose gels in the absence of Ca2+.13,14 The complexity of the kinetic behavior is illustrated in Figure 5a by the effect of Ca2+ in pectin A. After about 1 h, the Ca2+-containing gels showed higher storage moduli than the gel without Ca2+, whereas after 3 h the opposite behavior was observed. The results illustrate the importance of good insight into the kinetic relations during gel formation when designing products with specific gel properties. IV. Conclusions The kinetic behavior during gel formation is a crucial aspect for the understanding of the functional properties of HM pectin gels. This investigation presents the kinetic behavior during gel formation and the microstructure of three comparable HM pectins, differing in their degree of blockiness (DB). We have shown that the DB influenced the gelation time in the absence of Ca2+; the shortest gelation time was obtained for the sample with the highest DB. Furthermore, the presence of Ca2+ strongly affected the kinetic behavior and the rheological properties of the gels. More rapid gel formation was achieved in the presence of Ca2+, but the variations in DB led to different results in the storage modulus and the microstructure of the gels. The Ca2+ addition to the blockwise pectin A resulted in a complex behavior in the storage modulus and also inhomogeneous microstructure at pH 3.0. The kinetic behavior of HM pectin gels was also strongly affected by the pH increase. At pH

652

Biomacromolecules, Vol. 6, No. 2, 2005

3.5, a completely different kinetic behavior for the blockwise pectin A was obtained. Microstructural characterization of the gel network in the absence of Ca2+ 4 and 20 h after the gel preparation showed no visible changes on a nanometer scale. The results indicate that the macromolecular aggregation of the pectin network occurs during the first 4 h, and the later G′ increase between 4 and 20 h can be related to internal strengthening without any visible effects on the gel microstructure. In addition, the microstructure was unaffected by the distribution of methyl esters in the absence of Ca2+ since both A and B revealed similar structures comprising open, branched gel networks. This investigation shows that two pectins differing in DB can respond completely different on Ca2+ addition and pH changes. In general, it can be concluded that small differences in the chemical composition have a strong bearing on the kinetic behavior and the functionality of pectin gels. Acknowledgment. Prof. Mats Stading is thanked for rheological advice during the work. This project is a part of the LiFT program (Future Technologies for Food Production), financed by the SSF (Swedish Foundation for Strategic Research). 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) Taylor, A. J. Intramolecular distribution of carboxyl groups in low methoxyl pectins -a review. Carbohydr. Polym. 1982, 2, 9-17. (3) Rolin, C.; De Vries, J. Pectin. In Food gels; Harris, P., Ed.; Elsevier Sci. Publ. Ltd: New York, 1990; pp 401-434. (4) Oakenfull, D.; Scott, A. Hydrophobic interaction in the gelation of high methoxyl pectins. J. Food Sci. 1984, 49 (4), 1093-1109. (5) 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. (6) Axelos, M. A. V.; Thibault, J.-F. The chemistry of low-methoxyl pectin gelation. In The chemistry and technology of pectin; Walter, R. H., Ed.; Academic Press: New York, 1991; pp 109-118. (7) Thibault, J. F.; Rinaudo, M. Chain association of pectic molecules during calcium-induced gelation. Biopolymers 1986, 25 (3), 455468. (8) Rolin, C. Commercial pectin preparations. In Pectins and their manipuation. Seymor, G. B., Knox, J. P., Eds.; Blackwell Publishing: Cambridge, MA, 2002; pp 222-241. (9) Powell, D. A.; Morris, E. R.; Gidley, M. J.; Rees, D. A. Conformations and interactions of pectins II. Influence of residue sequence on chain association in calcium pectate gels. J. Mol. Biol. 1982, 155, 517-531. (10) Lopes da Silva, J. A.; Goncalves, M. P.; Rao, M. A. Kinetics and thermal behaviour of the structure formation process in HMP/sucrose gelation. Int. J. Biol. Macromol. 1995, 17 (1), 25-32. (11) Lopes da Silva, J. A.; Goncalves, M. P. Rheological study into the aging process of high methoxyl pectin/sucrose aqueous gels. Carbohydr. Polym. 1994, 24 (4), 235-245. (12) Rao, M. A.; Buren J. P, v.; Cooley, H. J. Rheological changes during gelation of high-methoxyl pectin/fructose dispersions: effect of temperature and aging. J. Food Sci. 1993, 58 (1), 173-176.

Lo¨fgren et al. (13) Neidhart, S.; Hannak, C.; Gierschner, K. Investigations of the influence of various cations on the rheological properties of highesterified pectin gels. In Pectins and Pectinases. Visser, J., Voragen, A. G. J., Eds.; Elsevier Science: New York, 1996; pp 583-590. (14) El Nawawi, S. A.; Heikel, Y. A. Factors affecting gelation of highester citrus pectin. Process Biochem. 1997, 32 (5), 381-385. (15) Lo¨fgren, C.; Walkenstro¨m, P.; Hermansson, A.-M. Microstructure and rheological behavior of pure and mixed pectin gels. Biomacromolecules 2002, 3 (6), 1144-1153. (16) Lo¨fgren, C.; Hermansson, A.-M. Microstructure and kinetic behaviour of pure and mixed pectin gels. In Gums and Stabilisers for the Food Industry 12; Williams, P. A., Phillips, G. O., Eds.; The Royal Society of Chemistry: London, 2004; pp 153-159. (17) Round, A. N.; MacDougall, A. J.; Ring, S.; Morris, V. J. Unexpected branching in pectin observed by atomic force microscopoy. Carbohydr. Res. 1997, 303, 251-253. (18) Round, A. J.; Rigby, N. M.; MacDougall, A. J.; Ring, S. G.; Morris, V. J. Investigating the nature of branching in pectin by atomic force microscopy and carbohydrate analysis. Carbohydr. Res. 2001, 331, 337-342. (19) Fishman, M. L.; Cooke, P. H.; Coffin, D. R. Nanostructure of native pectin sugar acid gels visualized by atomic force microscopy. Biomacromolecules 2004, 5 (2), 334-341. (20) Guillotin, S. E.; Bakx, E. J.; Boulenguer, P.; Mazoyer, J.; Schols, H. A.; Voragen, A. G. J. Populations having different GalA blocks characteristics are present in commercial pectins which are chemically similar but have different gelling properties. Carbohydr. Polym. Submitted for publication. (21) Ahmed, A. E. R.; Labavitch, J. M. A simplified method for accurate determination of cell wall uronide content. J. Food Biochem. 1977, 1, 361-365. (22) Blumenkrantz, N.; Asboe-Hansen, G. New method for quantitative determination of uronic acids. Anal. Biochem. 1973, 12, 484-489. (23) Thibault, J.-F. Automatisation du dosage des substances pectiques par la methode au meta-hydroxydiphenyl. Lebensm.-Wiss. u.Technol. 1979, 12, 247-251. (24) Daas, P. J. H.; Arisz, P. W.; Schols, H. A.; De Ruiter, G. A.; Voragen, A. G. J. Analysis of partially methyl esterified galacturonic acid oligomers by high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight spectrometry. Anal. Biochem. 1998, 257, 195-202. (25) Daas, P. J. H.; Meyer-Hansen, K.; Schols, H. A.; De Ruiter, G. A.; Voragen, A. G. J. Investigation of the nonesterified galacturonic acid distribution in pectin with endopolygalacturonase. Carbohydr. Res. 1999, 318, 135-145. (26) Daas, P. J. H.; Voragen, A. G. J.; Schols, H. A. Characterization of nonesterified galacturonic acid sequences in pectin with endopolygalacturonase. Carbohydr. Res. 2000, 326, 120-129. (27) Thiery, J. P. Mise en e´vidence des polysaccharides sur coupes fines en microscopie e´lectronique. J. Microsc. 1967, 6, 987-1018. (28) Power, D J.; Rodd, A. B.; Paterson, L.; Boger, D. V. Gel transition studies on nonideal polymer networks using small amplitude oscillatory rheometry. J. Rheol. 1998, 42 (5), 1021-1037. (29) Stading, M.; Hermansson, A.-M. Viscoelastic behaviour of betalactoglobulin gel structures. Food Hydrocolloids 1990, 4 (2), 1-21. (30) Rolin, C. Calcium sensitivity of high ester citrus pectins. In Gums and Stabilisers for the Food Industry 7; Phillips, G. O., Williams, P. A., Wedlock, D. J., Eds.; IRL Press: Oxford, U.K., 1994; pp 413422. (31) Perez, S.; Mazeau, K.; Herve du Penhoat, C. The three-dimensional structures of the pectic polysaccharides. Plant Physiol. Biochem. 2000, 38 (1/2), 37-55.

BM049619+