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Structure-forming processes in Ca2+-induced whey protein isolate cold gelation
PII : S0958-6946(98)00011-9
Int. Dairy Journal 7 (1997) 827—834 ( 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$19.00#0.00
Structure-forming Processes in Ca21-induced Whey Protein Isolate Cold Gelation Parichat Hongsprabhas and Shai Barbut* Department of Animal Science, University of Guelph, Guelph, Ont., Canada N1G 2W1 (Received 14 May 1997; accepted 4 February 1998) ABSTRACT The structure-forming process of Ca2`-induced whey protein isolate (WPI) gels was studied, at 24°C, in the presence of 10 and 120 mM CaCl . Pre-heating WPI suspensions (10% protein) at 90°C dramatically increased specific viscosity, but did not change the 2 number of accessible sulfhydryl groups, compared to pre-heating at 70°C. The most important factor governing the process appeared to be CaCl concentration, rather than the reactive sulfhydryl groups. At 10 mM CaCl , the increase in aggregate size and network 2 2 connectivity over time was achieved by clustering of adjacent aggregates. At 120 mM CaCl , the increase in aggregate size and 2 connectivity was by enlargement of the aggregates which formed connected paths and filled up interstitial spaces. ( 1998 Elsevier Science Ltd. All rights reserved
aggregation at pH 7 (Stading et al., 1992; Boye et al., 1998). b-lg gels formed at pH'6, with no salt added, are transparent due to the formation of small aggregates (Stading and Hermansson, 1991; Langton and Hermansson, 1992; Stading et al., 1992). Overall, the gelation mechanisms involved in mixed-protein systems (e.g. WPI) merit further investigation. In Ca2`-induced cold gelation of WPI, pre-heating is an essential step required to modify the proteins (e.g. expose reactive groups, open the structure) which is necessary for a later network formation (Barbut and Foegeding, 1993). The modified protein structures have been reported to range from dimers to polymers and are MWdependent (Wang and Damodaran, 1990; Zhu and Damodaran, 1994) maintained by disulfide bonds (Zhu and Damodaran, 1994). Hongsprabhas and Barbut (1996) showed that WPI suspensions subjected to a preheating step (70°C for 30 min), resulted in opaque gels, whereas higher pre-heating (90°C) formed translucent gels after CaCl addition by dialysis. The former is prob2 ably due to the creation of low MW aggregates, compared to the high MW aggregates formed at 90°C. Gels formed by pre-heating to 70°C exhibited lower water holding capacity (WHC) than those obtained by treating at 90°C; fracture properties were not affected by increasing calcium from 5 to 120 mM. However, it was not clear if the fine-strand gel structure resulting from pre-heating at 90°C was due to a slow rate of aggregation (i.e. limited diffusion of CaCl into the dialysis tube, caused by the 2 entanglement of large soluble aggregates formed at the high pre-heating temperature), or due to differences in the exposed reactive binding sites (e.g. sulfhydryl groups, carboxylic groups, etc.) on the surfaces of the aggregates. Calcium chloride concentration is likely to be the major determinant in the aggregation process. Hongsprabhas et al. (1998) have reported that at 410 mM CaCl , the mechanism is mainly governed by charge 2 dispersion while at higher CaCl , i.e. 530 mM, it 2 is mainly governed by Ca2` cross-linking. The latter
INTRODUCTION Globular protein gels can be prepared with varying physical properties (e.g. colour, texture, water holding capacity, etc.) by modifying pH and/or ionic strength (Doi, 1993). The differences in the physical characteristics are related to the type of gel formed. These structures have been divided into two distinct types, i.e. linear and random aggregation (Tombs, 1974). The mechanisms determining linear or random aggregation of a pure globular protein system (e.g. ovalbumin, bovine serum albumin or lysozyme) have been reviewed by Doi (1993). It has been suggested that at a pH distant from the isoelectric point (pI), low ionic strength would lead to linear aggregation, while high ionic strength would result in random aggregation. The former gives rise to a transparent gel while the latter to an opaque gel formation. It was proposed that at a pH slightly above or below the pI (6—7 for whey proteins) and/or intermediate ionic strength, linear and random aggregates are mixed together, or linear aggregates may be clumped together to form the structure. Both cases could result in gels ranging from translucent to opaque (Doi, 1993). However, the gelation mechanisms within this intermediate ionic strength or pH have not been fully elucidated. Whey protein isolate (WPI) represents a mixed-protein system composed of four major proteins, i.e. b-lactoglobulin (b-lg), a-lactalbumin (a-la), bovine serum albumin (BSA) and immunoglobulins (Ig’s). Although BSA exhibits linear aggregation at pH 7 and low NaCl concentration (Murata et al., 1993), it is present in WPI in a much lower concentration than b-lg (7—10% versus 67—75%; Morr and Ha, 1993). Thus, it is likely that the entire microstructure would be dominated by the b-lg microstructure, showing both linear and random-type
* Corresponding author. Tel.: 519 824 4120. Fax: 001 519 767 5730. E-mail: [email protected]. 827
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implies that aggregation, resulting in network formation, requires specific binding sites. The difference in the gelation mechanism, caused by different CaCl concen2 tration, could possibly be responsible for the difference in the network building process at the microscopic level. Thus, the objective of this study was to investigate the process of network formation of WPI suspensions subjected to different degrees of pre-heating (70 vs 90°C) at different CaCl concentration (10 vs 120 mM) over 2 time. Such information could provide a better insight into the mechanism(s) of cold-set gelation of globular protein.
were carried out using the Phast System Separation and the Control and Development Unit (Pharmacia LKB) according to the manufacturer’s instructions. Gels were stained with Phast Gel Blue R (Coomassie R350 Dye) after separation. Relative viscosity
MATERIALS AND METHODS
The viscosities of unheated and pre-heated WPI suspensions were determined by using a Cannon—Fensketype viscometer (Fisher Scientific Limited, Unionville, ON) at 24°C (n"2/treatment/trial) similar to the method described by Zhu and Damodaran (1994). The relative viscosity was calculated from the relation (Bradbury, 1970).
Two lots of WPI (BiPro, Davisco International, Inc., Le Sueur, MN) were used: lot A with a protein concentration 90.5% and lot B with 91.0% as determined by Macro-Kjeldahl (AOAC, 1984) using an N factor of 6.38. WPI suspensions (10% protein w/v) were prepared in double-distilled water and pH adjusted to 7.0 with 0.1 M HCl or NaOH. The suspensions were degassed and preheated at 70 or 90°C for 30 min, cooled to 24°C for 2 h and kept at 1°C for 16 h before characterization or dialysis against 10 or 120 mM CaCl solutions, unless 2 otherwise stated.
t !t 0 g" 4 (1) t 0 where t and t are the flow times for a protein suspension 4 0 and double distilled water, respectively. The experiment was carried out in two separate trials. Each trial consisted of the two lots of WPI. The effect of pre-heating temperature was analyzed by the ANOVA procedure (SAS, 1990). Differences among treatments were determined by using the least significance difference at P40.05.
Gelation
Accessible sulfhydryl (SH) group content
The pre-heated WPI suspensions were dialyzed against 10 or 120 mM CaCl at room temperature using 2 the method described by Barbut and Foegeding (1993). At each time interval, one dialysis tube (20.4 mm diameter) per treatment was removed from its dialyzing solution and cut open in the middle so gel thickness at the periphery could be determined with a micrometer. The experiment was carried out in two separate trials. Each trial consisted of the two lots of WPI. The effect of pre-heating temperature was analyzed by a one-way nonparametric procedure (SAS, 1990). Scores were ranked by the Kruskal—Wallis test (SAS, 1990) and values replaced by rank were further analyzed by the General Linear Model (GLM) procedure. Differences among treatments were determined using the least significance difference at P40.05.
The concentration of accessible SH groups was determined by the method of Shimada and Cheftel (1988, 1989) using an Ellman’s reagent. WPI suspensions (n"2/ treatment/trial), obtained after cooling and storing for 2 h or 40 h, were diluted to 2% protein (w/v) with double-distilled water. One milliliter of diluted protein suspension was further diluted with 8 mM Tris-glycine buffer (containing 0.086 M Tris-HCl, 0.09 M glycine and 0.004 M disodium EDTA) at pH 8 and treated with 1 mL of Ellman’s reagent [4 mg mL~1 5,5@-dithiobis-2-nitro benzoic acid (DTNB) in Tris-glycine buffer pH 8]. The absorbance at 412 nm of samples and a blank (i.e. 9 mM Tris-glycine buffer plus 1 mL Ellman’s reagent) was read 15 min after mixing (UV-visible Recording Spectrophotometer 260; Shimadzu, Kyoto). Accessible SH group concentration was calculated:
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) WPI suspensions (10% protein, w/v) were pre-heated at 70, 80 or 90°C for 30 or 60 min, cooled to room temperature and kept at 1°C overnight. The unheated and pre-heated protein suspensions were diluted to 2 mg mL~1 protein with a 10 mM Tris/HCl buffer containing 1 mM EDTA and 2.5% SDS. The samples and low MW standards (including Phosphorylase b with a MW of 94,000; Bovine serum albumin, 67,000; Ovalbumin, 43,000; Carbonic anhydrase, 30,000; Soybean trypsin inhibitor, 20,100; and a-lactalbumin, 14,400; all from Pharmacia LKB, Montreal, PQ) were heated at 100°C for 5 min, both in the presence and absence of b-mercaptoethanol (5%), cooled and centrifuged to remove suspended insoluble materials. One microliter of each WPI mixture and the standards were placed on a gradient polyacrylamide 8—25% SDS gel (Phast gel) with SDS buffer strips (Pharmacia LKB). Separations
73.53]Absorbance at 412 nm kM SH g~1 protein" . mg protein (2) Note that the assay did not include 8 M urea in Trisglycine buffer in order to avoid solubilization of protein aggregates, because the reactive groups on the surface of the aggregates were of interest. The experiment was carried out in two separate trials. Each trial consisted of two lots of WPI. The effect of pre-heating temperature was analyzed by ANOVA (SAS, 1990). Differences among treatments were determined by using the least significance difference at P40.05. Scanning and transmission electron microscopy (SEM, TEM) WPI suspensions were dialyzed against 10 and 120 mM CaCl at room temperature using the method 2
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Structure-forming processes of Ca2`-induced WPI gels
described by Barbut and Foegeding (1993). At each time interval, dialysis tubes were removed from the respective dialyzing solutions and cut open in the middle. The gel located at the periphery was cut into small cubes (1]1]2 mm) and prepared for SEM and TEM using the methods described in Hongsprabhas et al. (1998). Briefly, samples (1]1]2 mm) were fixed in 2% glutaraldehyde #1% paraformaldehyde in 0.1 M PIPES buffer at pH 7 for 6 h, rinsed, post-fixed in 1%OsO overnight and 4 dried in a graded series of ethanol. For SEM, the samples were critical point dried, sputter coated with 30 nm of gold/palladium and viewed at 15 kV (Hitachi S-570, Tokyo, Japan). For TEM, the samples were embedded in Epon, thinly sectioned (90 nm thick), stained with uranyl acetate and lead acetate, and viewed at 80 kV (Jeol, Montreal, PQ). At least four micrographs/treatment were taken from different locations. Suspensions that had not yet formed a gel were discarded. RESULTS AND DISCUSSION Statistical analysis indicated that there were no trial or lot effects (P'0.05), so data were combined and representative SDS-PAGE and electron micrographs are presented. Pre-heating temperature significantly (P40.05) affected both the thickness of the forming gels (Table 1) and specific viscosity (Table 2). Accessible SH-content was not affected by increasing temperature from 70 to 90°C (Table 3). Because the progress of gelation (i.e., moving from the periphery of the dialysis tube towards the center) was controlled by both CaCl gradient and the aggregation 2 rate, different time intervals were used to measure the progress of gelation in the 10 and 120 mM treatments (Table 1). At the beginning, the thickness of gels formed by pre-heating to 90°C was higher than that of suspensions pre-heated to 70°C. Over time, the difference in thickness decreased. The results suggest that the entanglement of protein aggregates, which possibly occurred to a higher degree at the 90°C treatment, did not have an effect on the rate of Ca2` diffusion into the dialysis tube. Rather, gelation was likely dependent on the type of aggregates formed during the pre-heating step (e.g. number of reactive groups exposed on the surface, MW, etc.). The pH differences of the gel systems to which 10 and 120 mM CaCl was added were insignificant (6.49 vs 6.37, 2 respectively) and therefore not believed to be a major factor in the differences observed. SDS-PAGE showed that under non-reducing conditions (without b-mercaptoethanol, Fig. 1A) the majority of the pre-heated proteins precipitated in the wells and did not migrate into the gel. The unheated control proteins migrated and showed three typical distinct bands (a-la, MW&14,000; b-lg, MW&18,000 and BSA, MW&66,000). The intensity of the monomeric bands decreased as pre-heating temperature increased, suggesting that these proteins were involved in polymerization. The profiles obtained are in agreement with that reported by Zhu and Damodaran (1994) and Monahan et al. (1995) who showed a formation of covalently bound polymers at the expense of b-lg, a-la and BSA when whey proteins were heated to 570°C at pH 7. Zhu and Damodaran (1994) reported that pre-heating 5% WPI at 70°C resulted in dimerization, while polymerization occurred at pre-heating to 90°C. However, the effect of
Table 1. Effect of Pre-heating Temperature on Progression of Gel Formation, Measured as Thickness, of Ca2`-induced Cold-set WPI Gels [CaCl ] (mM) 2
10
120
Time (h)
Thickness (mm)
2.0 4.0 6.0 7.0 8.0 9.0 0.5 1.0 1.5 2.0 2.5 3.0
70°C
90°C
0.87* 1.27) 3.05%& 4.40$ 6.44" 10.20! 1.655 2.993 3.771 4.95/ 6.527.57+
1.50' 2.37& 3.88% 5.14# 7.09" 10.20! 2.024 3.322 4.450 5.36. 6.397.20,
Means (n"4) followed by a different superscript within each salt treatment are significantly different at P40.05. Table 2. Effect of Pre-heating Temperature on Relative Viscosity at 24°C of WPI Suspensions (10% Protein w/v) Heat treatment
Relative viscosity
Unheated Pre-heated at 70°C Pre-heated at 90°C
0.9" 7.2" 92.7!
Means (n"8) followed by a different superscript are significantly different (P40.05).
Table 3. Effect of Pre-heating Temperature and Cold Storage Time on Accessible Sulfhydryl Group Content (kM SH g~1 Protein) of Pre-heated WPI Suspensions (10% Protein w/v) Heat treatment
Unheated Pre-heated at 70°C Pre-heated at 90°C
Cold Storage 2h
40 h
13.51# 27.98!" 28.22!
12.28# 25.26" 28.16!"
Means (n"8) followed by a different superscript are significantly different (P40.05).
pre-heating at 70°C in this experiment was different from that reported by Zhu and Damodaran (1994), probably due to differences in the methods used. In addition, they determined the degree of polymerization of the 5% WPI immediately after rapid cooling of the heated protein suspension, while in this study the SDS-PAGE analysis was performed 18 h after heating the 10% protein suspensions. Under reducing conditions (Fig. 1B), a noticeable band was observed between 30,000 and 43,000 and the precipitates disappeared. This suggests that polymerization of whey protein monomers could occur via disulfide bonds. It has been suggested that the band between 30,000 and 43,000 is a dimer of a-la and b-lg (Li-Chan, 1983; Zhu and Damodaran, 1994; Monahan et al., 1995). However, the former authors noted that an additional factor (apart
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P. Hongsprabhas, S. Barbut
Fig. 1. SDS-polyacrylamide gel electrophoresis of WPI suspensions before and after pre-heating assayed in the (A) absence and (B) presence of b-mercaptoethanol. Lane 1"low MW standards, lane 2"unheated WPI, lane 3"WPI pre-heated 70°C 30 min, lane 4"WPI pre-heated 70°C, 60 min, lane 5"WPI pre-heated 80°C, 30 min, lane 6"WPI pre-heated 80°C, 60 min, lane 7"WPI pre-heated 90°C, 30 min and lane 8"WPI pre-heated 90°C, 60 min.
Fig. 2. Scanning electron micrographs of WPI gels subjected to pre-heating at 70°C (a, c) or 90°C (b, d) after dialysis against 10 mM CaCl solutions for 3.5 (a, b) or 6.5 (c, d) h. Bar"2 km. 2
Structure-forming processes of Ca2`-induced WPI gels
from disulfide bonds) is involved in stabilizing the dimer since it was not reduced by b-mercaptoethanol. Since the presence of high-MW aggregates also occurred in protein suspensions pre-heated to 70°C, further testing was carried out to determine whether these aggregates are different from those formed at 90°C. The specific viscosity of protein suspensions pre-heated to 90°C was much greater than that of unheated suspensions (Table 2) or suspensions pre-heated to 70°C. This suggests that pre-heating to 90°C for 30 min caused polymerization to a higher degree than pre-heating to 70°C. Thus, it is suggested that the difference in the size of the aggregates determines the type of gel formed during the later calcium addition (cold gelation). Pre-heating resulted in unfolding of protein molecules and polymerization. These alterations could expose buried reactive groups, leading to differences in the textural properties of the gels (Shimada and Cheftel, 1988;
831
Jeyarajah and Allen, 1994; Monahan et al., 1995). At 10% protein, Shimada and Cheftel (1988) reported that heating WPI suspensions to 85°C for 45 min (pH 7.5) did not change the disulfide bond content and slightly reduced the total SH content compared to unheated proteins. Our results indicate that pre-heating caused an increase in accessible SH content (Table 3). However, Shimada and Cheftel (1988) determined their total SH content in the presence of 8 M urea and 0.5% SDS. In any case, increasing the pre-heating temperature from 70 to 90°C in our study did not significantly change the amount of accessible SH content (Table 3). A preliminary trial indicated that there was no significant difference in turbidity (measured at 400 nm) between pre-heated and unheated protein suspensions (i.e. diluted protein suspensions of 0.2% protein in 8 mM Tris-glycine buffer at pH 8; the same concentration used in our sulfhydryl assay) as both suspensions remained clear before adding the
Fig. 3. Transmission electron micrographs of WPI gels subjected to pre-heating at 70°C (a, c) or 90°C (b, d) after dialysis against 10 mM CaCl solutions for 3.5 (a, b) or 6.5 (c, d) h. Bar"0.2 km. 2
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P. Hongsprabhas, S. Barbut
Ellman’s reagent. Thus, the increase in accessible SH content, reported here, did not affect the turbidity of the solutions. Comparing the SH content of pre-heated protein suspensions stored for either 2 or 40 h at 1°C was also considered since gelation time in this system usually requires 16—40 hr to complete (Hongsprabhas and Barbut, 1996). The two storage periods were used to examine if additional polymerization, via sulfhydryl/disulfide interchange reaction, could occur. The results indicate that SH content was not significantly affected by the storage time. Thus, it is possible that the transformation of soluble aggregates into a gel network is primarily influenced by Ca2`-mediated reactions, rather than by sulfhydryl/disulfide interchange reaction per se. In addition, the difference in pre-heating temperature did not appear to be large enough to cause any change in the content of accessible SH groups. This is in agreement with work by Wang and Damodaran (1990) in which aggregate size was more important than the number of SH reactive groups in determining the gelation of globular proteins.
The microstructure of gels formed after pre-heating at 70°C and dialyzing against 10 and 120 mM CaCl was 2 more porous than those where 90°C was used (Figs 2—5). Pre-heating to 70°C resulted in gels with larger size aggregates/strands and smoother edges than those formed after pre-heating to 90°C, particularly after a longer dialysis period (3.5 vs 1.5 h; Figs 2 and 4). The aggregates/strands size in the 70°C treatment dialyzed against 120 mM CaCl for 3.5 h was in the range of 200—400 nm 2 (Fig. 4c), while the 90°C treatment resulted in 60—150 nm aggregates (Fig. 4d). This could explain the more opaque appearance of gels prepared with pre-heating to 70°C compared to 90°C, at both CaCl concentrations. This 2 also confirms previous results where optical measurements of gels produced after pre-heating to 70°C resulted in higher ¸-values (i.e. whiter gels) than those formed after pre-heating to 90°C (Hongsprabhas and Barbut, 1996). The reason for the higher ¸-values is that more light is reflected from the larger aggregates. To date, there has been no experimental explanation as to why the small-size soluble aggregates (i.e. formed at low
Fig. 4. Scanning electron micrographs of WPI gels subjected to pre-heating at 70°C (a, c) or 90°C (b, d) after dialysis against 120 mM CaCl solutions for 1.5 (a, b) or 3.5 (c, d) h. Bar"2 km. 2
Structure-forming processes of Ca2`-induced WPI gels
833
Fig. 5. Transmission electron micrographs of WPI gels subjected to pre-heating at 70°C (a, c) or 90°C (b, d) after dialysis against 120 mM CaCl solutions for 1.5 (a, b) or 3.5 (c, d) h. Bar"0.2 km. 2
pre-heating; 70°C) have a tendency to form larger-size insoluble aggregates in the gel network. The micrographs show that raising CaCl content 2 from 10 to 120 mM resulted in increasing aggregate size, at any given pre-heating temperature and time (Figs 2—5). Increasing gelation time resulted in increasing aggregate/strand size. However, the process involved seems to be different between gels prepared by dialyzing against 10 and 120 mM CaCl . At 10 mM CaCl , the network struc2 2 ture which was formed after 3.5 h (Fig. 2a and b) appeared to consist of evenly dispersed small pores (note: samples were removed from the periphery of the dialysis tubes). After 6.5 h, the aggregate and pore sizes increased (Fig. 2c and d). The use of TEM at a higher magnification revealed that aggregation and/or network formation is
taking place by clumping of adjacent aggregates into larger ones. Thus, the network formed appeared to be more porous over time while the aggregate/strand size was increasing. This pattern was observed in the WPI gels prepared with pre-heating at both 70 and 90°C. At 120 mM CaCl , the network formed at the early 2 stage (1.5 h) appeared to be loose and porous (Figs 4a and b). Over time, the structure seemed to become more dense (Fig. 4c and d). This is in contrast to the structures formed at 10 mM Ca2`. Transmission electron micrographs (Fig. 5) show that the aggregation and connectivity of the network resulted from enlarging the protein aggregates/strands to fill up the inner spaces. This resulted in the development of a less porous gel structure over time in WPI suspensions pre-heated at either 70 or 90°C.
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It should be noted that some of the TEM micrographs show black spots which are the result of osmium precipitation. This is believed to be due to osmium sequestering by the divalent calcium ions as discussed by ParnellClunies et al. (1986). Matsushita (1989) indicated that under a diffusionlimited aggregation (DLA) regime, aggregates would have a less compact structure than when formed under a reaction-limited aggregation (RLA) regime. The TEM micrographs illustrate that at 120 mM CaCl the gelation 2 is primarily controlled by RLA and the aggregates appear to have a more compact structure than at 10 mM CaCl . The latter is mainly controlled by a DLA . The 2 results are also in agreement with computer simulation results reported by Meakin (1988, 1989). He suggested that at the early stages of colloidal aggregation, the system consists of a large number of dispersed particles which move within the fluid and may come in contact with each other, stick and form a cluster. The small clusters formed in this way continue to move within the solution (Brownian motion) and collide with other clusters to form larger clusters. However, under the RLA regime, more collisions between clusters are required before they stick or combine to form larger clusters. Meakin (1988, 1989) assumed in his simulation procedure that all possible configurations have to be explored before the cluster—cluster addition process actually takes place. This resulted in using a smaller sticking probability in the RLA simulation compared to the aggregation DLA. This slow aggregation condition may have led to a more compact structure of RLA aggregates compared to those formed in the DLA regime. CONCLUSION The microstructure study confirms that the network formation processes are governed by CaCl concentration 2 in the cold set gelation. This is consistent with previous results (Hongsprabhas et al., 1998) indicating that varying CaCl levels results in different gelation mechanisms. 2 Overall, electrostatic forces were shown to be more important than covalent bonds in controlling the way gels are formed, even at a protein concentration as high as 10%. This study sheds more light on the gelation mechanism of a mixed-protein system in which linear aggregation is not dominant. The increase in aggregate size and network connectivity over time was observed to occur by clustering of adjacent aggregates at low CaCl level, and 2 by the growth of aggregates to fill up spaces within the network at high CaCl concentration. 2 REFERENCES AOAC (1984) Official Methods of Analysis, 14th edn. Assoc. of Official Analytical Chemists, Arlington, VA. Barbut, S. and Foegeding, E.A. (1993) Ca2`-induced gelation of pre-heated whey protein isolate. Journal of Food Science 58, 867—871. Boye, J. I., Ma, C.-Y., Ismail, A., Harwalkar, V. R. and Kalab, M. (1998) Molecular and microstructural studies on thermal denaturation and gelation of b-lactoglobulin A and B. Journal of Agricultural and Food Chemistry (submitted for publication).
Bradbury, J. H. (1970) Viscosity. In Physical Principles and ¹echniques of Protein Chemistry, Part B, ed. S. J. Leach. Academic Press, New York, pp. 99—145. Doi, E. (1993) Gels and gelling of globular proteins. ¹rends in Food Science ¹echnology 4, 1—5. Hongsprabhas, P. and Barbut, S. (1996) Ca2`-induced gelation of whey protein isolate: effect of pre-heating. Food Research International 29, 135—139. Hongsprabhas, P., Barbut, S. and Marangoni, A. G. (1998) Mechanisms of Ca2`-induced cold gelation of whey protein isolate (submitted for publication). Jeyarajah, S. and Allen, J. C. (1994) Calcium binding and saltinduced structural changes of native and preheated b-lactoglobulin. Journal of Agricultural and Food Chemistry 42, 80—85. Langton, M. and Hermansson, A.-M. (1992) Fine-stranded and particulate gels of b-lactoglobulin and whey protein at varying pH. Food Hydrocolloids 5, 523—539. Li-Chan, E. (1983) Heat-induced changes in the proteins of whey protein concentrates. Journal of Food Science 46, 47—56. Matsushita, M. (1989) Experimental observations of aggregations. In ¹he Fractal Approach to Heterogeneous Chemistry: Surfaces, Colloids, Polymer, ed. D. Avnir. Wiley, Chichester, pp. 161—180. Meakin, P. (1988) Fractal aggregates. Advances in Colloid Interface Science 28, 249—331. Meakin, P. (1989) Simulations of aggregation processes. In ¹he Fractal Approach to Heterogeneous Chemistry: Surfaces, Colloids, Polymer, ed. D. Avnir. Wiley, Chichester, pp. 131—160. Monahan, F., German, J. B. and Kinsella, J. E. (1995) Effect of pH and temperature on protein unfolding and thiol/disulfide interchange reactions during heat-induced gelation of whey proteins. Journal of Agricultural and Food Chemistry 43, 46—52. Morr, C. V. and Ha, E. Y. W. (1993) Whey protein concentrates and isolates: processing and functional properties. Critical Reviews in Food Science Nutrition 33, 431—467. Murata, M., Tani, F., Higasa, T., Kitabatake, N. and Doi, E. (1993) Heat-induced transparent gel formation of bovine serum albumin. Bioscience Biotechnology Biochemistry 57, 43—46. Parnell-Clunies, E. M., Kakuda, Y. and Humphrey, R. (1986) Electron-dense granules in yogurt. Characterization by Xray microanalysis. Food Microstructure 5, 295—302. SAS (1990) ºser’s Guide: Statistics. SAS Institute. Cary, NC. Shimada, K. and Cheftel, J. C. (1988) Texture characteristics, protein solubility, and sulfhydryl group/disulfide bond contents of heat-induced gels of whey protein isolate. Journal of Agricultural and Food Chemistry 36, 1018—1025. Shimada, K. and Cheftel, J. C. (1989) Sulfhydryl group/disulfide bond interchange reactions during heat-induced gelation of whey protein isolates. Journal of Agricultural and Food Chemistry 37, 161—168. Stading, M. and Hermansson, A.-M. (1991) Large deformation properties of b-lactoglobulin gel structures. Food Hydrocolloids 5, 339—352. Stading, M., Langton, M. and Hermansson, A.-M. (1992) Inhomogeneous fine-stranded b-lactoglobulin gels. Food Hydrocolloids 6, 455—470. Tombs, M. P. (1974) Gelation of globular proteins. Faraday Discussion Chemical Society 57, 158—164. Wang, C.-H. and Damodaran, S. (1990) Thermal gelation of globular proteins: weight-average molecular dependence of gel strength. Journal of Agricultural and Food Chemistry 38, 1157—1164. Zhu, H. and Damodaran, S. (1994) Heat-induced conformational changes in whey protein isolate and its relation to foaming properties. Journal of Agricultural and Food Chemistry 42, 846—855.
Int. Dairy Journal 7 (1997) 827—834 ( 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$19.00#0.00
Structure-forming Processes in Ca21-induced Whey Protein Isolate Cold Gelation Parichat Hongsprabhas and Shai Barbut* Department of Animal Science, University of Guelph, Guelph, Ont., Canada N1G 2W1 (Received 14 May 1997; accepted 4 February 1998) ABSTRACT The structure-forming process of Ca2`-induced whey protein isolate (WPI) gels was studied, at 24°C, in the presence of 10 and 120 mM CaCl . Pre-heating WPI suspensions (10% protein) at 90°C dramatically increased specific viscosity, but did not change the 2 number of accessible sulfhydryl groups, compared to pre-heating at 70°C. The most important factor governing the process appeared to be CaCl concentration, rather than the reactive sulfhydryl groups. At 10 mM CaCl , the increase in aggregate size and network 2 2 connectivity over time was achieved by clustering of adjacent aggregates. At 120 mM CaCl , the increase in aggregate size and 2 connectivity was by enlargement of the aggregates which formed connected paths and filled up interstitial spaces. ( 1998 Elsevier Science Ltd. All rights reserved
aggregation at pH 7 (Stading et al., 1992; Boye et al., 1998). b-lg gels formed at pH'6, with no salt added, are transparent due to the formation of small aggregates (Stading and Hermansson, 1991; Langton and Hermansson, 1992; Stading et al., 1992). Overall, the gelation mechanisms involved in mixed-protein systems (e.g. WPI) merit further investigation. In Ca2`-induced cold gelation of WPI, pre-heating is an essential step required to modify the proteins (e.g. expose reactive groups, open the structure) which is necessary for a later network formation (Barbut and Foegeding, 1993). The modified protein structures have been reported to range from dimers to polymers and are MWdependent (Wang and Damodaran, 1990; Zhu and Damodaran, 1994) maintained by disulfide bonds (Zhu and Damodaran, 1994). Hongsprabhas and Barbut (1996) showed that WPI suspensions subjected to a preheating step (70°C for 30 min), resulted in opaque gels, whereas higher pre-heating (90°C) formed translucent gels after CaCl addition by dialysis. The former is prob2 ably due to the creation of low MW aggregates, compared to the high MW aggregates formed at 90°C. Gels formed by pre-heating to 70°C exhibited lower water holding capacity (WHC) than those obtained by treating at 90°C; fracture properties were not affected by increasing calcium from 5 to 120 mM. However, it was not clear if the fine-strand gel structure resulting from pre-heating at 90°C was due to a slow rate of aggregation (i.e. limited diffusion of CaCl into the dialysis tube, caused by the 2 entanglement of large soluble aggregates formed at the high pre-heating temperature), or due to differences in the exposed reactive binding sites (e.g. sulfhydryl groups, carboxylic groups, etc.) on the surfaces of the aggregates. Calcium chloride concentration is likely to be the major determinant in the aggregation process. Hongsprabhas et al. (1998) have reported that at 410 mM CaCl , the mechanism is mainly governed by charge 2 dispersion while at higher CaCl , i.e. 530 mM, it 2 is mainly governed by Ca2` cross-linking. The latter
INTRODUCTION Globular protein gels can be prepared with varying physical properties (e.g. colour, texture, water holding capacity, etc.) by modifying pH and/or ionic strength (Doi, 1993). The differences in the physical characteristics are related to the type of gel formed. These structures have been divided into two distinct types, i.e. linear and random aggregation (Tombs, 1974). The mechanisms determining linear or random aggregation of a pure globular protein system (e.g. ovalbumin, bovine serum albumin or lysozyme) have been reviewed by Doi (1993). It has been suggested that at a pH distant from the isoelectric point (pI), low ionic strength would lead to linear aggregation, while high ionic strength would result in random aggregation. The former gives rise to a transparent gel while the latter to an opaque gel formation. It was proposed that at a pH slightly above or below the pI (6—7 for whey proteins) and/or intermediate ionic strength, linear and random aggregates are mixed together, or linear aggregates may be clumped together to form the structure. Both cases could result in gels ranging from translucent to opaque (Doi, 1993). However, the gelation mechanisms within this intermediate ionic strength or pH have not been fully elucidated. Whey protein isolate (WPI) represents a mixed-protein system composed of four major proteins, i.e. b-lactoglobulin (b-lg), a-lactalbumin (a-la), bovine serum albumin (BSA) and immunoglobulins (Ig’s). Although BSA exhibits linear aggregation at pH 7 and low NaCl concentration (Murata et al., 1993), it is present in WPI in a much lower concentration than b-lg (7—10% versus 67—75%; Morr and Ha, 1993). Thus, it is likely that the entire microstructure would be dominated by the b-lg microstructure, showing both linear and random-type
* Corresponding author. Tel.: 519 824 4120. Fax: 001 519 767 5730. E-mail: [email protected]. 827
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implies that aggregation, resulting in network formation, requires specific binding sites. The difference in the gelation mechanism, caused by different CaCl concen2 tration, could possibly be responsible for the difference in the network building process at the microscopic level. Thus, the objective of this study was to investigate the process of network formation of WPI suspensions subjected to different degrees of pre-heating (70 vs 90°C) at different CaCl concentration (10 vs 120 mM) over 2 time. Such information could provide a better insight into the mechanism(s) of cold-set gelation of globular protein.
were carried out using the Phast System Separation and the Control and Development Unit (Pharmacia LKB) according to the manufacturer’s instructions. Gels were stained with Phast Gel Blue R (Coomassie R350 Dye) after separation. Relative viscosity
MATERIALS AND METHODS
The viscosities of unheated and pre-heated WPI suspensions were determined by using a Cannon—Fensketype viscometer (Fisher Scientific Limited, Unionville, ON) at 24°C (n"2/treatment/trial) similar to the method described by Zhu and Damodaran (1994). The relative viscosity was calculated from the relation (Bradbury, 1970).
Two lots of WPI (BiPro, Davisco International, Inc., Le Sueur, MN) were used: lot A with a protein concentration 90.5% and lot B with 91.0% as determined by Macro-Kjeldahl (AOAC, 1984) using an N factor of 6.38. WPI suspensions (10% protein w/v) were prepared in double-distilled water and pH adjusted to 7.0 with 0.1 M HCl or NaOH. The suspensions were degassed and preheated at 70 or 90°C for 30 min, cooled to 24°C for 2 h and kept at 1°C for 16 h before characterization or dialysis against 10 or 120 mM CaCl solutions, unless 2 otherwise stated.
t !t 0 g" 4 (1) t 0 where t and t are the flow times for a protein suspension 4 0 and double distilled water, respectively. The experiment was carried out in two separate trials. Each trial consisted of the two lots of WPI. The effect of pre-heating temperature was analyzed by the ANOVA procedure (SAS, 1990). Differences among treatments were determined by using the least significance difference at P40.05.
Gelation
Accessible sulfhydryl (SH) group content
The pre-heated WPI suspensions were dialyzed against 10 or 120 mM CaCl at room temperature using 2 the method described by Barbut and Foegeding (1993). At each time interval, one dialysis tube (20.4 mm diameter) per treatment was removed from its dialyzing solution and cut open in the middle so gel thickness at the periphery could be determined with a micrometer. The experiment was carried out in two separate trials. Each trial consisted of the two lots of WPI. The effect of pre-heating temperature was analyzed by a one-way nonparametric procedure (SAS, 1990). Scores were ranked by the Kruskal—Wallis test (SAS, 1990) and values replaced by rank were further analyzed by the General Linear Model (GLM) procedure. Differences among treatments were determined using the least significance difference at P40.05.
The concentration of accessible SH groups was determined by the method of Shimada and Cheftel (1988, 1989) using an Ellman’s reagent. WPI suspensions (n"2/ treatment/trial), obtained after cooling and storing for 2 h or 40 h, were diluted to 2% protein (w/v) with double-distilled water. One milliliter of diluted protein suspension was further diluted with 8 mM Tris-glycine buffer (containing 0.086 M Tris-HCl, 0.09 M glycine and 0.004 M disodium EDTA) at pH 8 and treated with 1 mL of Ellman’s reagent [4 mg mL~1 5,5@-dithiobis-2-nitro benzoic acid (DTNB) in Tris-glycine buffer pH 8]. The absorbance at 412 nm of samples and a blank (i.e. 9 mM Tris-glycine buffer plus 1 mL Ellman’s reagent) was read 15 min after mixing (UV-visible Recording Spectrophotometer 260; Shimadzu, Kyoto). Accessible SH group concentration was calculated:
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) WPI suspensions (10% protein, w/v) were pre-heated at 70, 80 or 90°C for 30 or 60 min, cooled to room temperature and kept at 1°C overnight. The unheated and pre-heated protein suspensions were diluted to 2 mg mL~1 protein with a 10 mM Tris/HCl buffer containing 1 mM EDTA and 2.5% SDS. The samples and low MW standards (including Phosphorylase b with a MW of 94,000; Bovine serum albumin, 67,000; Ovalbumin, 43,000; Carbonic anhydrase, 30,000; Soybean trypsin inhibitor, 20,100; and a-lactalbumin, 14,400; all from Pharmacia LKB, Montreal, PQ) were heated at 100°C for 5 min, both in the presence and absence of b-mercaptoethanol (5%), cooled and centrifuged to remove suspended insoluble materials. One microliter of each WPI mixture and the standards were placed on a gradient polyacrylamide 8—25% SDS gel (Phast gel) with SDS buffer strips (Pharmacia LKB). Separations
73.53]Absorbance at 412 nm kM SH g~1 protein" . mg protein (2) Note that the assay did not include 8 M urea in Trisglycine buffer in order to avoid solubilization of protein aggregates, because the reactive groups on the surface of the aggregates were of interest. The experiment was carried out in two separate trials. Each trial consisted of two lots of WPI. The effect of pre-heating temperature was analyzed by ANOVA (SAS, 1990). Differences among treatments were determined by using the least significance difference at P40.05. Scanning and transmission electron microscopy (SEM, TEM) WPI suspensions were dialyzed against 10 and 120 mM CaCl at room temperature using the method 2
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Structure-forming processes of Ca2`-induced WPI gels
described by Barbut and Foegeding (1993). At each time interval, dialysis tubes were removed from the respective dialyzing solutions and cut open in the middle. The gel located at the periphery was cut into small cubes (1]1]2 mm) and prepared for SEM and TEM using the methods described in Hongsprabhas et al. (1998). Briefly, samples (1]1]2 mm) were fixed in 2% glutaraldehyde #1% paraformaldehyde in 0.1 M PIPES buffer at pH 7 for 6 h, rinsed, post-fixed in 1%OsO overnight and 4 dried in a graded series of ethanol. For SEM, the samples were critical point dried, sputter coated with 30 nm of gold/palladium and viewed at 15 kV (Hitachi S-570, Tokyo, Japan). For TEM, the samples were embedded in Epon, thinly sectioned (90 nm thick), stained with uranyl acetate and lead acetate, and viewed at 80 kV (Jeol, Montreal, PQ). At least four micrographs/treatment were taken from different locations. Suspensions that had not yet formed a gel were discarded. RESULTS AND DISCUSSION Statistical analysis indicated that there were no trial or lot effects (P'0.05), so data were combined and representative SDS-PAGE and electron micrographs are presented. Pre-heating temperature significantly (P40.05) affected both the thickness of the forming gels (Table 1) and specific viscosity (Table 2). Accessible SH-content was not affected by increasing temperature from 70 to 90°C (Table 3). Because the progress of gelation (i.e., moving from the periphery of the dialysis tube towards the center) was controlled by both CaCl gradient and the aggregation 2 rate, different time intervals were used to measure the progress of gelation in the 10 and 120 mM treatments (Table 1). At the beginning, the thickness of gels formed by pre-heating to 90°C was higher than that of suspensions pre-heated to 70°C. Over time, the difference in thickness decreased. The results suggest that the entanglement of protein aggregates, which possibly occurred to a higher degree at the 90°C treatment, did not have an effect on the rate of Ca2` diffusion into the dialysis tube. Rather, gelation was likely dependent on the type of aggregates formed during the pre-heating step (e.g. number of reactive groups exposed on the surface, MW, etc.). The pH differences of the gel systems to which 10 and 120 mM CaCl was added were insignificant (6.49 vs 6.37, 2 respectively) and therefore not believed to be a major factor in the differences observed. SDS-PAGE showed that under non-reducing conditions (without b-mercaptoethanol, Fig. 1A) the majority of the pre-heated proteins precipitated in the wells and did not migrate into the gel. The unheated control proteins migrated and showed three typical distinct bands (a-la, MW&14,000; b-lg, MW&18,000 and BSA, MW&66,000). The intensity of the monomeric bands decreased as pre-heating temperature increased, suggesting that these proteins were involved in polymerization. The profiles obtained are in agreement with that reported by Zhu and Damodaran (1994) and Monahan et al. (1995) who showed a formation of covalently bound polymers at the expense of b-lg, a-la and BSA when whey proteins were heated to 570°C at pH 7. Zhu and Damodaran (1994) reported that pre-heating 5% WPI at 70°C resulted in dimerization, while polymerization occurred at pre-heating to 90°C. However, the effect of
Table 1. Effect of Pre-heating Temperature on Progression of Gel Formation, Measured as Thickness, of Ca2`-induced Cold-set WPI Gels [CaCl ] (mM) 2
10
120
Time (h)
Thickness (mm)
2.0 4.0 6.0 7.0 8.0 9.0 0.5 1.0 1.5 2.0 2.5 3.0
70°C
90°C
0.87* 1.27) 3.05%& 4.40$ 6.44" 10.20! 1.655 2.993 3.771 4.95/ 6.527.57+
1.50' 2.37& 3.88% 5.14# 7.09" 10.20! 2.024 3.322 4.450 5.36. 6.397.20,
Means (n"4) followed by a different superscript within each salt treatment are significantly different at P40.05. Table 2. Effect of Pre-heating Temperature on Relative Viscosity at 24°C of WPI Suspensions (10% Protein w/v) Heat treatment
Relative viscosity
Unheated Pre-heated at 70°C Pre-heated at 90°C
0.9" 7.2" 92.7!
Means (n"8) followed by a different superscript are significantly different (P40.05).
Table 3. Effect of Pre-heating Temperature and Cold Storage Time on Accessible Sulfhydryl Group Content (kM SH g~1 Protein) of Pre-heated WPI Suspensions (10% Protein w/v) Heat treatment
Unheated Pre-heated at 70°C Pre-heated at 90°C
Cold Storage 2h
40 h
13.51# 27.98!" 28.22!
12.28# 25.26" 28.16!"
Means (n"8) followed by a different superscript are significantly different (P40.05).
pre-heating at 70°C in this experiment was different from that reported by Zhu and Damodaran (1994), probably due to differences in the methods used. In addition, they determined the degree of polymerization of the 5% WPI immediately after rapid cooling of the heated protein suspension, while in this study the SDS-PAGE analysis was performed 18 h after heating the 10% protein suspensions. Under reducing conditions (Fig. 1B), a noticeable band was observed between 30,000 and 43,000 and the precipitates disappeared. This suggests that polymerization of whey protein monomers could occur via disulfide bonds. It has been suggested that the band between 30,000 and 43,000 is a dimer of a-la and b-lg (Li-Chan, 1983; Zhu and Damodaran, 1994; Monahan et al., 1995). However, the former authors noted that an additional factor (apart
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Fig. 1. SDS-polyacrylamide gel electrophoresis of WPI suspensions before and after pre-heating assayed in the (A) absence and (B) presence of b-mercaptoethanol. Lane 1"low MW standards, lane 2"unheated WPI, lane 3"WPI pre-heated 70°C 30 min, lane 4"WPI pre-heated 70°C, 60 min, lane 5"WPI pre-heated 80°C, 30 min, lane 6"WPI pre-heated 80°C, 60 min, lane 7"WPI pre-heated 90°C, 30 min and lane 8"WPI pre-heated 90°C, 60 min.
Fig. 2. Scanning electron micrographs of WPI gels subjected to pre-heating at 70°C (a, c) or 90°C (b, d) after dialysis against 10 mM CaCl solutions for 3.5 (a, b) or 6.5 (c, d) h. Bar"2 km. 2
Structure-forming processes of Ca2`-induced WPI gels
from disulfide bonds) is involved in stabilizing the dimer since it was not reduced by b-mercaptoethanol. Since the presence of high-MW aggregates also occurred in protein suspensions pre-heated to 70°C, further testing was carried out to determine whether these aggregates are different from those formed at 90°C. The specific viscosity of protein suspensions pre-heated to 90°C was much greater than that of unheated suspensions (Table 2) or suspensions pre-heated to 70°C. This suggests that pre-heating to 90°C for 30 min caused polymerization to a higher degree than pre-heating to 70°C. Thus, it is suggested that the difference in the size of the aggregates determines the type of gel formed during the later calcium addition (cold gelation). Pre-heating resulted in unfolding of protein molecules and polymerization. These alterations could expose buried reactive groups, leading to differences in the textural properties of the gels (Shimada and Cheftel, 1988;
831
Jeyarajah and Allen, 1994; Monahan et al., 1995). At 10% protein, Shimada and Cheftel (1988) reported that heating WPI suspensions to 85°C for 45 min (pH 7.5) did not change the disulfide bond content and slightly reduced the total SH content compared to unheated proteins. Our results indicate that pre-heating caused an increase in accessible SH content (Table 3). However, Shimada and Cheftel (1988) determined their total SH content in the presence of 8 M urea and 0.5% SDS. In any case, increasing the pre-heating temperature from 70 to 90°C in our study did not significantly change the amount of accessible SH content (Table 3). A preliminary trial indicated that there was no significant difference in turbidity (measured at 400 nm) between pre-heated and unheated protein suspensions (i.e. diluted protein suspensions of 0.2% protein in 8 mM Tris-glycine buffer at pH 8; the same concentration used in our sulfhydryl assay) as both suspensions remained clear before adding the
Fig. 3. Transmission electron micrographs of WPI gels subjected to pre-heating at 70°C (a, c) or 90°C (b, d) after dialysis against 10 mM CaCl solutions for 3.5 (a, b) or 6.5 (c, d) h. Bar"0.2 km. 2
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P. Hongsprabhas, S. Barbut
Ellman’s reagent. Thus, the increase in accessible SH content, reported here, did not affect the turbidity of the solutions. Comparing the SH content of pre-heated protein suspensions stored for either 2 or 40 h at 1°C was also considered since gelation time in this system usually requires 16—40 hr to complete (Hongsprabhas and Barbut, 1996). The two storage periods were used to examine if additional polymerization, via sulfhydryl/disulfide interchange reaction, could occur. The results indicate that SH content was not significantly affected by the storage time. Thus, it is possible that the transformation of soluble aggregates into a gel network is primarily influenced by Ca2`-mediated reactions, rather than by sulfhydryl/disulfide interchange reaction per se. In addition, the difference in pre-heating temperature did not appear to be large enough to cause any change in the content of accessible SH groups. This is in agreement with work by Wang and Damodaran (1990) in which aggregate size was more important than the number of SH reactive groups in determining the gelation of globular proteins.
The microstructure of gels formed after pre-heating at 70°C and dialyzing against 10 and 120 mM CaCl was 2 more porous than those where 90°C was used (Figs 2—5). Pre-heating to 70°C resulted in gels with larger size aggregates/strands and smoother edges than those formed after pre-heating to 90°C, particularly after a longer dialysis period (3.5 vs 1.5 h; Figs 2 and 4). The aggregates/strands size in the 70°C treatment dialyzed against 120 mM CaCl for 3.5 h was in the range of 200—400 nm 2 (Fig. 4c), while the 90°C treatment resulted in 60—150 nm aggregates (Fig. 4d). This could explain the more opaque appearance of gels prepared with pre-heating to 70°C compared to 90°C, at both CaCl concentrations. This 2 also confirms previous results where optical measurements of gels produced after pre-heating to 70°C resulted in higher ¸-values (i.e. whiter gels) than those formed after pre-heating to 90°C (Hongsprabhas and Barbut, 1996). The reason for the higher ¸-values is that more light is reflected from the larger aggregates. To date, there has been no experimental explanation as to why the small-size soluble aggregates (i.e. formed at low
Fig. 4. Scanning electron micrographs of WPI gels subjected to pre-heating at 70°C (a, c) or 90°C (b, d) after dialysis against 120 mM CaCl solutions for 1.5 (a, b) or 3.5 (c, d) h. Bar"2 km. 2
Structure-forming processes of Ca2`-induced WPI gels
833
Fig. 5. Transmission electron micrographs of WPI gels subjected to pre-heating at 70°C (a, c) or 90°C (b, d) after dialysis against 120 mM CaCl solutions for 1.5 (a, b) or 3.5 (c, d) h. Bar"0.2 km. 2
pre-heating; 70°C) have a tendency to form larger-size insoluble aggregates in the gel network. The micrographs show that raising CaCl content 2 from 10 to 120 mM resulted in increasing aggregate size, at any given pre-heating temperature and time (Figs 2—5). Increasing gelation time resulted in increasing aggregate/strand size. However, the process involved seems to be different between gels prepared by dialyzing against 10 and 120 mM CaCl . At 10 mM CaCl , the network struc2 2 ture which was formed after 3.5 h (Fig. 2a and b) appeared to consist of evenly dispersed small pores (note: samples were removed from the periphery of the dialysis tubes). After 6.5 h, the aggregate and pore sizes increased (Fig. 2c and d). The use of TEM at a higher magnification revealed that aggregation and/or network formation is
taking place by clumping of adjacent aggregates into larger ones. Thus, the network formed appeared to be more porous over time while the aggregate/strand size was increasing. This pattern was observed in the WPI gels prepared with pre-heating at both 70 and 90°C. At 120 mM CaCl , the network formed at the early 2 stage (1.5 h) appeared to be loose and porous (Figs 4a and b). Over time, the structure seemed to become more dense (Fig. 4c and d). This is in contrast to the structures formed at 10 mM Ca2`. Transmission electron micrographs (Fig. 5) show that the aggregation and connectivity of the network resulted from enlarging the protein aggregates/strands to fill up the inner spaces. This resulted in the development of a less porous gel structure over time in WPI suspensions pre-heated at either 70 or 90°C.
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It should be noted that some of the TEM micrographs show black spots which are the result of osmium precipitation. This is believed to be due to osmium sequestering by the divalent calcium ions as discussed by ParnellClunies et al. (1986). Matsushita (1989) indicated that under a diffusionlimited aggregation (DLA) regime, aggregates would have a less compact structure than when formed under a reaction-limited aggregation (RLA) regime. The TEM micrographs illustrate that at 120 mM CaCl the gelation 2 is primarily controlled by RLA and the aggregates appear to have a more compact structure than at 10 mM CaCl . The latter is mainly controlled by a DLA . The 2 results are also in agreement with computer simulation results reported by Meakin (1988, 1989). He suggested that at the early stages of colloidal aggregation, the system consists of a large number of dispersed particles which move within the fluid and may come in contact with each other, stick and form a cluster. The small clusters formed in this way continue to move within the solution (Brownian motion) and collide with other clusters to form larger clusters. However, under the RLA regime, more collisions between clusters are required before they stick or combine to form larger clusters. Meakin (1988, 1989) assumed in his simulation procedure that all possible configurations have to be explored before the cluster—cluster addition process actually takes place. This resulted in using a smaller sticking probability in the RLA simulation compared to the aggregation DLA. This slow aggregation condition may have led to a more compact structure of RLA aggregates compared to those formed in the DLA regime. CONCLUSION The microstructure study confirms that the network formation processes are governed by CaCl concentration 2 in the cold set gelation. This is consistent with previous results (Hongsprabhas et al., 1998) indicating that varying CaCl levels results in different gelation mechanisms. 2 Overall, electrostatic forces were shown to be more important than covalent bonds in controlling the way gels are formed, even at a protein concentration as high as 10%. This study sheds more light on the gelation mechanism of a mixed-protein system in which linear aggregation is not dominant. The increase in aggregate size and network connectivity over time was observed to occur by clustering of adjacent aggregates at low CaCl level, and 2 by the growth of aggregates to fill up spaces within the network at high CaCl concentration. 2 REFERENCES AOAC (1984) Official Methods of Analysis, 14th edn. Assoc. of Official Analytical Chemists, Arlington, VA. Barbut, S. and Foegeding, E.A. (1993) Ca2`-induced gelation of pre-heated whey protein isolate. Journal of Food Science 58, 867—871. Boye, J. I., Ma, C.-Y., Ismail, A., Harwalkar, V. R. and Kalab, M. (1998) Molecular and microstructural studies on thermal denaturation and gelation of b-lactoglobulin A and B. Journal of Agricultural and Food Chemistry (submitted for publication).
Bradbury, J. H. (1970) Viscosity. In Physical Principles and ¹echniques of Protein Chemistry, Part B, ed. S. J. Leach. Academic Press, New York, pp. 99—145. Doi, E. (1993) Gels and gelling of globular proteins. ¹rends in Food Science ¹echnology 4, 1—5. Hongsprabhas, P. and Barbut, S. (1996) Ca2`-induced gelation of whey protein isolate: effect of pre-heating. Food Research International 29, 135—139. Hongsprabhas, P., Barbut, S. and Marangoni, A. G. (1998) Mechanisms of Ca2`-induced cold gelation of whey protein isolate (submitted for publication). Jeyarajah, S. and Allen, J. C. (1994) Calcium binding and saltinduced structural changes of native and preheated b-lactoglobulin. Journal of Agricultural and Food Chemistry 42, 80—85. Langton, M. and Hermansson, A.-M. (1992) Fine-stranded and particulate gels of b-lactoglobulin and whey protein at varying pH. Food Hydrocolloids 5, 523—539. Li-Chan, E. (1983) Heat-induced changes in the proteins of whey protein concentrates. Journal of Food Science 46, 47—56. Matsushita, M. (1989) Experimental observations of aggregations. In ¹he Fractal Approach to Heterogeneous Chemistry: Surfaces, Colloids, Polymer, ed. D. Avnir. Wiley, Chichester, pp. 161—180. Meakin, P. (1988) Fractal aggregates. Advances in Colloid Interface Science 28, 249—331. Meakin, P. (1989) Simulations of aggregation processes. In ¹he Fractal Approach to Heterogeneous Chemistry: Surfaces, Colloids, Polymer, ed. D. Avnir. Wiley, Chichester, pp. 131—160. Monahan, F., German, J. B. and Kinsella, J. E. (1995) Effect of pH and temperature on protein unfolding and thiol/disulfide interchange reactions during heat-induced gelation of whey proteins. Journal of Agricultural and Food Chemistry 43, 46—52. Morr, C. V. and Ha, E. Y. W. (1993) Whey protein concentrates and isolates: processing and functional properties. Critical Reviews in Food Science Nutrition 33, 431—467. Murata, M., Tani, F., Higasa, T., Kitabatake, N. and Doi, E. (1993) Heat-induced transparent gel formation of bovine serum albumin. Bioscience Biotechnology Biochemistry 57, 43—46. Parnell-Clunies, E. M., Kakuda, Y. and Humphrey, R. (1986) Electron-dense granules in yogurt. Characterization by Xray microanalysis. Food Microstructure 5, 295—302. SAS (1990) ºser’s Guide: Statistics. SAS Institute. Cary, NC. Shimada, K. and Cheftel, J. C. (1988) Texture characteristics, protein solubility, and sulfhydryl group/disulfide bond contents of heat-induced gels of whey protein isolate. Journal of Agricultural and Food Chemistry 36, 1018—1025. Shimada, K. and Cheftel, J. C. (1989) Sulfhydryl group/disulfide bond interchange reactions during heat-induced gelation of whey protein isolates. Journal of Agricultural and Food Chemistry 37, 161—168. Stading, M. and Hermansson, A.-M. (1991) Large deformation properties of b-lactoglobulin gel structures. Food Hydrocolloids 5, 339—352. Stading, M., Langton, M. and Hermansson, A.-M. (1992) Inhomogeneous fine-stranded b-lactoglobulin gels. Food Hydrocolloids 6, 455—470. Tombs, M. P. (1974) Gelation of globular proteins. Faraday Discussion Chemical Society 57, 158—164. Wang, C.-H. and Damodaran, S. (1990) Thermal gelation of globular proteins: weight-average molecular dependence of gel strength. Journal of Agricultural and Food Chemistry 38, 1157—1164. Zhu, H. and Damodaran, S. (1994) Heat-induced conformational changes in whey protein isolate and its relation to foaming properties. Journal of Agricultural and Food Chemistry 42, 846—855.