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Calorimetric and rheological behavior of cowpea protein plus starch (cowpea and corn) gels
Food Hydrocolloids
Vol. 11 no. 3 pp. 339-345, 1997
Calorimetric and rheological behavior of cowpea protein plus starch (cowpea and corn) gels Paul E.Okechukwu 1 and M.A.Raoz Department of Food Science and Technology, Cornell University-Geneva, New York, NY 14456-0462, USA IOn leave from the Federal Polytechnic, Oko, Nigeria 2To whom correspondence should be addressed
Abstract Thermal transition and gel formation in dispersions (10% solids) of cowpea protein, and in mixtures of the protein and corn starch and cowpea starch, were examined using a differential scanning calorimeter (DS C) and dynamic rheometry, respectively. In mixtures of the protein and corn starch, two DSC peaks due to starch gelatinization and protein denaturation were observed. A single peak observed during the heating of the protein/cowpea starch mixtures appeared to be an overlap of the starch and protein endotherm peaks. With an increase in the protein/starch ratio (R), the starch gelatinization onset and peak temperatures shifted to higher values, while endotherm enthalpy decreasedproportionally. G of the protein/starch gels increased with aging time at 20°C and attained plateau levels that decreased with increase in protein/starch ratio ( R ) . For protein/cowpea starch gels, G growth with time was adequately described by afirst-order kinetic model. In the early stages of structure development, afaster increase in G for the protein/cowpea starch mixture ensured higher levels of G after 1 h of aging compared with that for starch gel at the same starch concentration. In comparison to corn starch gels, there was no significant increase in G of gels ofprotein/corn starch.
Introduction Proteins and starches are important food components that are individually or collectively utilized to increase viscosity, stabilize dispersions, and provide texture and firmness. These functional attributes are based on the water-holding and gelation ability of the polymers during and after thermal transition. As the concentration of the polymers exceeds a threshold, gelation results from hyperentanglements as well as intermolecular associations and cross-links. Below this threshold, increased solution or dispersion viscosity and yield stress may be achieved. The gelation of globular proteins has been extensively studied by various techniques (1-5). Thermal gelation of proteins is widely accepted to involve the partial unfolding of protein molecular strands at temperatures higher than the transition onset, and subsequent aggregation, association and cross-linking to form a gel network. Thermal and rheological studies on the gelation of soybean 7S and lIS proteins by glucono-delta-lactone (GDL) (6) and by heat (7)
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have shown that the gelation rate is first order with GDL and increases with GDL concentration and heating temperature. Starch gels are composites in which intact and ruptured swollen granules are embedded in a continuous gel network. The continuous matrix may be composed of amylose or a mixture of amylose and amylopectin, depending on the type of starch, and pasting conditions (8-10). Amylose appears to be the major component of starch whose concentration in the continuous phase determines whether a heated starch dispersion would gel or not. The polymer is leached out as granule swelling progresses during heating at temperatures higher than the gelatinization onset. The critical concentration (Co) of amylose abo ve which a gel is formed is -0.8-1.1%, and appears to be independent of molecular weight and origin of the biopolymer (11-13). At relatively low starch concentrations , as filled gels, the mechanical properties of starch gels depend on those of the continuous matrix, the volume fraction of the granules, shape and
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deformability of the granules, and the granule-matrix interaction (8,14-16). The selectivity in interaction between proteins and starches was shown in dynamic rheological studies. Addition of gluten to 6.5-6.6% (w/w) dispersions of different starches at a 1%level calculated on a dry starch basis increased the G of wheat and rye starches, lowered that of maize starch, but did not affect the modulus of barley, triticale and potato starches (17). G was also found to increase with increasing amounts of gluten (1-4%) added to the wheat starch. Studies on starches with different amylose/amylopectin ratios suggest that gelatinization of amylopectin and not amylose favors a synergistic interaction with proteins leading to an increase in dispersion viscosity (18,19). The amylose/ amylopecin ratio, and type of protein and starch, are important factors influencing protein-starch interaction. Differences in the rheological behavior of gels from mixtures of fish protein and starch were attributed to the variation in the gelatinization temperatures of the starches in relation to the transition temperature of the protein. In such binary systems, the continuous gel network would be formed by the component with the lower transition temperature (2). Based on studies of starches and bovine serum albumin, Muhrbeck and Eliassen (20) showed that when a starch network is formed before the gel formation of the protein, the two structures supplement each other without any specific interaction, to form a complex system with two continuous networks. When the protein network is formed first, the diffusion and aggregation of the amylose molecules and the swelling of the granules are hindered. The starch cannot form a continuous network, but will instead act as a filler reinforcing the protein network. Because proteins and starches are thermodynamically different polymers, their joint presence may lead to phase separation, inversion or mutual interaction with significant consequences on texture (21). More information is required to enable gainful exploitation of the protein-starch interaction for the design and evaluation of texture in foods. The purpose of this study was to examine the interaction of cowpea protein with cowpea starch; a thermal procedure involving differential scanning calorimetry was used to probe the transition temperature, while rheological methods were used to examine the mechanical properties of gels formed by heating dispersions of cowpea and corn starch, cowpea protein, and their mixtures.
Materials and methods Unmodified corn starch used in this study was purchased from Sigma Chemical Co., St Louis, MO. Cowpea starch and protein extraction
Cowpea (Vigna unguiculata) seeds were soaked in tap water for 3 h and subsequently decorticated by manual abrasion. Following the methods of starch extraction described by Cheng-yii and Shin-ming (22) and Schoch and Maywald
(23), the cleaned cotyledons were blended to a slurry of fine particles with five parts by weight of 0.2% sodium sulfite solution in a Waring blender. The slurry settled for -2 h at ambient temperature, and was filtered through an ASTM No. 140 standard sieve. Prime starch was recovered as sediment from the filtrate after settling for 2 h. The starch was washed three times and vacuum dried at 35°C for -48 h. Using the method of Williams et al. (24), the amylose content of corn starch and cowpea starch was determined to be -20 and 30%, respectively. Protein was extracted from the decorticated cowpea cotyledons using the single-stage method of Molina et al. (25), freeze-dried, and stored in air-tight containers until experimentation. Slurry and gel preparation
Dispersions of starch and protein (4-12% solids) were made by thoroughly dispersing weighed amounts of the dry samples in distilled water in a 50 ml wide-bottom conical flask. The pH of the protein dispersion was adjusted to 7.0 with 2 N NaOH. Dispersions (10% solids, pH 7.0) of mixtures of starch and protein at various weight ratios were also prepared. The dispersions were allowed to hydrate for -6 h at 10°C before thermal analysis and gel preparation for rheometry. One concern in gel preparation was the settling of starch granules during heating of low-concentration starch dispersions (17,26,27), which was minimized by keeping the dispersion agitated. The other concern was loss of moisture at 85°C in an open geometry (17), such as the plate-cone geometry of a rheometer. Therefore, two different procedures were attempted as a necessary compromise between the aforementioned concerns and alteration of structure. (i) Thoroughly mixed dispersions (-20 g) were heated in stoppered conical flasks in a water bath for 15 min at 85°C, a technique similar to that of Lindahl and Eliasson (17). A thermocouple introduced through the cap of the flask was used to monitor the temperature of the dispersions. (ii) The heated dispersion at 85°C was quenched to 25°C in an ice bath and transferred to a 25°C water bath, and the resulting gel was aged for 1 h before rheological evaluation. Although the word gel is being used to describe the samples obtained after cooling, because of the relatively low solids content and the presence of starch granules, they could very well be called heated dispersions. Thermal analysis
A differential scanning calorimeter (DSC) (Model 2910, TA Instruments, New Castle, DE) was used to examine thermal transitions. Approximately 10-15 mg of a dispersion were placed in a DSC aluminum pan, hermetically sealed, and scanned over 30-150°C in the DSC at 5°C/min with a sealed empty pan as a reference. After heating, the sample pans were punctured and the pan contents dried at 105°C to constant weight and the true solids content of the sample was calculated.
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Figure 1 DSC thermogram of corn starch, cowpea protein and corn starch-protein mixtures at 10% solids and various protein! starch ratios (R).
Figure 2 DSC thermogram of cowpea starch, cowpea protein and cowpea starch-protein mixtures at 10% solids and various protein! starch ratios (R) .
Rheometry
The transition temperatures of the protein, shown to contain mostly 7S globulins (28-30), were higher than the corresponding gelatinization temperatures of corn starch and cowpea starch. At 10% solids, blends of the protein and corn starch produced a single endotherm peak which gradually broadened into a bimodal peak as the protein! starch ratio (R) increased (Fig. I). The thermogram appeared to be an overlap of two transition processes. The first peak, with T p increasing gradually from the starch gelatinization peak temperature to higher temperatures with increase in R, is attributed to starch gelatinization, while the second peak with T p close to the protein peak temperature is apparently related to protein denaturation. Mixtures of cowpea starch and protein showed only a single endotherm peak at various levels of R (Fig. 2). The absence of an emerging protein peak in the cowpea starch-protein mixtures may reflect the smaller difference in the peak temperatures of cowpea starch and protein (8°C) compared with that of corn starch and cowpea protein (12°C). Both To and T p of the single peak observed for the protein--cowpea starch mixtures increased proportionally with an increase in the protein content of the blends, while Tc quickly increased to the Tc of the protein (Fig. 3). A similar effect of the protein on the starch peak transition temperatures was observed in mixtures of corn starch and cowpea protein (Fig. 4). The transition enthalpies of the mixtures at 10% solids content decreased proportionally from the value of the starch to that of the protein with an increase in the protein/starch ratio. The trends in starch transition temperatures and enthalpy are indicative of hindrance to starch gelatinization similar to the presence of salt (31,32) and fish proteins (2). The trends also suggest the absence of any specific interaction between the starches and the protein before heat treatment.
Dynamic rheological tests were performed at 20°C on gel samples with a cone (6 em diameter, 2° angle) and plate geometry of a Carri-Med CSL2 100 rheometer (TA Instruments). The cone-plate geometry provides uniform shear and is well suited for heated starch dispersions. Samples of the gel aged for 1 h at 25°C were subjected to a frequency sweep at I% strain over 0.1-10 Hz in the linear viscoelastic range determined from torque sweepsat 5 Hz. In addition, samples of hot gels at 85°C were quenched rapidly to 20°C on the rheometer plate and subjected to a time sweep of 10 h at 1% strain and 1 Hz. Because the heated and unheated 10% cowpea protein dispersions were fluid in nature, it was felt that flow data would be useful. Therefore, shear stress-shear rate data were obtained on unsheared samples in both increasing and decreasing stress modes in the range 0.1-20 Pa in 5 min. Magn itudes of shear stress, 0, and shear rate, y, from the down curve of shear stress sweep were used in data analysis.
Results and discussion Differential scanning calorimetry
The average values of onset (To), peak (Tp ) and concluding (Tc) temperatures during gelatinization measured by DSC at solids content up to 15% were: 62, 68 and 73°C for corn starch, and 66, 72 and 78°C for cowpea starch, respectively. Transition enthalpies for the gelatinization of the starches were 10.8 J/g for corn starch and 8.5 J/g for cowpea starch. DSC thermograms (Figs I and 2) of cowpea protein isolate at 10% solids, pH - 7.0, revealed that the protein denatured over 72-86°C with a transition endotherm of 4.9 J/g that peaked at SO.2°C.
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Rheological data Starch gels Storage modulus (G) development as a function of aging time at 20 D C of cowpea starch gels of various concentrations showed a two-stage pattern (Fig. 5). The initial stage showed a concentration dependence and G increased rapidly with aging time. The second phase showed a slower increase in G and appeared to reach a limiting value. With increasing starch concentration, the second phase in G development became shorter and G approached its limiting value within a shorter time span. Changes in loss tangent (tan 0) with time were also similarly affected by starch concentration. At 5% starch concentration, tan 0 decreased from 0.25 to 0.1 and 0.05 after 2 and 10 h of aging at 20D C, respectively. At the
Figure 5 Effect of aging time on the gel strength of 5% (0) and 9% (. ) cowpea starch gels at 20°e. Insert shows the variation of tan 0 for the gels during aging.
higher concentration of 9% starch, tan 0 fell from 0.19 to 0.1 in <10 min and approached a limiting value of 0.03 after 10 h of aging. Similar trends were also observed for corn starch gels. The two-phase nature of G growth in starch gels has been attributed to the gelation of amylose in the continuous matrix during the early stages of aging and the retrogradation of amylopectin observed after long periods of aging (8,13,15,33). The decrease in the rate of amylose retrogradation with increase in starch concentration has also been attributed to the decrease in molecular mobility as a result of swellingand gelation. Values of G at 1 Hz of freshly prepared cowpea starch gels exhibited a power law increase with starch concentration (C). Over the range of 4--10% cowpea starch, G ex: (;3.7 (R2 = 0.964). For corn starch gels, over 6-12%, G ex: C4.8 (R2 = 0.984); however, over the range 7-12% starch, G increased linearly with starch concentration (G ex: 504.1 C, R2= 0.984), in agreement with Evans and Haisman (34). Protein dispersions andgels Unheated 10% solids cowpea protein dispersion, pH - 7.0, sheared at 20DC over 1-550 s-l, exhibited weak thixotropic behavior and followed the simple power law model. (1)
After heating at 85D C for 15 min, the dispersion exhibited higher viscosity and greater thixotropic response. Log-log plots of the descending segment of the shear stress versus shear rate cycle (not shown here) revealed that while the flow behavior index, n, decreased from 0.92 in the native state to 0.83 after heating, the consistency K increased from 20.2 to 107.4 mpa.s", Although the increase in thixotropy of the 10% solids dispersion after heating suggests the possibility of structure formation, its storage modulus was not detectable with our rheometer. Protein solutions (pH - 7.0) at
Cowpea protein-starch interaction
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G max =7.69(x s)3.35 R2 = 0.998 Figure 6 Gel strength development during the aging of cowpea protein solutions (pH - 7.0) at 20°C after heatingat 85°C for 15 min. Insert shows the decreasein tan 0 during aging. Percent protein: 13% 6 ,15% 0 , 20% D .
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concentrations ~ 13% formed gels on cooling after heating at 85°e (Fig. 6). The almost linear increase in G and the associated decrease in tan 8 over the entire 10 h aging period suggests a gradual development in the elastic characteristic of protein gels (35). Cowpea protein starch mixtures Figure 7 shows the G development in 10% solids gels from blends of cowpea protein and cowpea starch over a 10 h aging period at 20°e. In general, G increased from an initial value, Go, to a maximum plateau value, G max' that increased with the proportion of starch in the mixture. A slight decrease in G was observed during the later part of aging, probably due to weakening of starch granules. The rise in G
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The nearly equal values of the exponents for the variation of G with starch concentration in starch/protein mixtures (within the range of R < 1/9) and in cowpea starch gels emphasize the important contribution of starch to the structure of the former. G development in the protein-starch gels followed an essentially apparent first-order process and did not show either the dominant two-stage growth pattern seen in starch gels or the almost linear pattern observed in the cowpea protein gels. A combination of the development patterns in the protein and starch components is apparent in the protein-starch gel. It appears that at pH values close to 7.0, the cowpea protein solubilized and formed the continuous phase in which raw starch granules were dispersed. On heating, the granules absorbed water and as they swelled they exuded some amylose into the continuous matrix prior to the protein denaturation. It has been suggested (20) that the higher transition temperature of the protein compared with that of starch creates the environment for the leaching of amylose into the solution of the protein. It is likely that the co-existence of the protein and the amylose in the continuous phase may be responsible for the distinct kinetic pattern in the observed development of G in the protein-starch mixtures. For such a system, the protein/starch ratio (R) would reflect the protein/amylose ratio in the continuous matrix and may be considered to be an important parameter for assessing G of the mixture gels. Figure 8 shows the variation of G at 1 Hz of protein! starch gels (10% solids) from frequency sweeps on gels after 1 h of aging with starch fraction (x s) . Up to x, < 0.8 (R < 2/8), G of the mixed gels was higher than that of cowpea starch gel at the same concentration. For x, > 0.8, G of the starch gel was higher than G of the mixed gel. Figure 8 also contains G at 1 Hz from frequency sweeps on gels of cowpea protein and corn starch after 1 h of aging at 25°e. Addition of the cowpea protein to corn starch at high levels did not affect the magnitude of G of the gel and at low levels resulted in a slight decrease. A similar decrease in G was observed after the addition of 1% gluten to corn starch (17). Recognizing that G of 10% solids cowpea protein gels was
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too low to be measured, the shape of the modulus-starch fraction curves of the mixed gels containing either corn starch or cowpea starch suggests phase separation (21). The higher G values of the protein/cowpea starch gels at low levels of starch appear to be due to favorable kinetics during the early stages of G development (Fig. 9). For aging periods of <250 min in 5% solids gels, G of the mixed gel (5% solids and 5 parts protein and 5 parts starch) was more than that of the starch gel of equivalent solids content. At a higher starch and solids level (9%), G of the mixed gel (9% solids and 1 part protein and 9 parts starch) was lower throughout the aging period.
Conclusion At a heating rate of 5°C/min, at various protein/starch ratios, DSC data of cowpea protein with corn starch showed a bimodal DSC endotherm and a single peak with cowpea starch. With an increase in protein/starch ratio, transition onset and peak temperatures increased while endotherm enthalpy decreased proportionally. For gels made from protein and cowpea starch at protein/starch ratios <2/8, G was slightly higher than that of cowpea starch at the same starch fraction; however, no increase in G was observed in gels with corn starch. G of gels made from protein/starch mixtures increased with aging time and attained plateau values whose magnitudes decreased with increase in protein/starch ratio. For gels of protein/cowpea starch, the increase in G with time was adequately described by a first-order kinetic model. In the early stages of structure development, a faster increase in G for the mixture resulted in a higher levelof G compared with that for starch gel at the same starch level after 1 h of aging.
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Figure 9 Comparison of G development of cowpea starch gels with and without added cowpea protein (5% solids: all starch 0, 5/5 protein/starch A) (9% solids: all starch e, 1/9 protein/starch 0).
Acknowledgement Partial support for this work was provided by the USDA Regional Research Project, NC136.
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16. Eliasson ,A.e. (1986) 1. Texture Stud., 17,357-375. 17. Lindahl,L. and Eliasson,A.e. (1986) 1. Sci. Food Agric., 37, 1125-1132. 18. Chedid,L.L. and Kokini,IL.(1992) Cereal Chern., 69, 551-555. 19. Madeka,H . and Kokini,I.L. (1992) Cereal Chern., 69, 489-494 . 20. Muhrbeck,P. and Eliasson,A.e. (1991) 1. Texture Stud, 22,317-332. 21. Tolstoguzov,y.B. (1986) In Mitchell,IR. and Ledward,D.A. (eds), Functional Properties of Food Macromolecules. Elsevier Applied Science, London, pp. 385-415. 22. Cheng-yi.L. and Shin-ming.C, (1981) 1. Food Sci., 46, 78-82. 23. Schoch,T.S. and Maywald,E.e. (1968) Cereal Chern., 45, 564--568. 24. Williams.P'C, Kuzina,ED. and Hlynka,I. (1970) Cereal Chern., 47, 411-420. 25. Molina,M.R., Argueta,e.E. and Bressani,R. (1976) 1. Food Sci., 41,928-931. 26. Evans,I.D. and Lips,A. (1992) 1. Texture Stud. , 23, 69-86. 27. Okechukwu,P.E. and Rao,M.A. (1995) 1. Texture Stud., 26,501-516. 28. Hirano,H., Kagawa ,H. and Okubo,K. (1992) Phytoch emistry, 31, 731-735. 29. Wright ,D.I and Boulter,D. (1980) 1. Sci. Food Agric., 31, 1231-1241. 30. Khan ,M.R.I., Gatehouse,IA. and Boulter,D. (1980) 1. Exp. Bot. , 31, 1599-1611. 31. Rockland,L.B., Jones,ET. and Hahn,D.M. (1977) 1. Food Sci., 42, 1204-1210. 32. Lund,D. (1984) Crit. Rev. Food Sci. Nutr., 20,249-273. 33. Doublier,lL. and Llarnas.G, (1993) In Dickinson ,E. and Walstra,P. (eds), Food Colloids and Polymers; Stability and Mechanical Properties. The Royal Society of Chemistry, Cambridge, pp. 139-146. 34. Evans, I. 0. and Haisman,D.R . (1979) 1. Texture Stud., 10, 347-370. 35. Philips,L.G., Whitehead,D.M. and Kinsella,l (1994) Structure-function Properties of Food Proteins. Academic Press, New York. Received on December 19, 1995; accepted on April 12, 1996
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Calorimetric and rheological behavior of cowpea protein plus starch (cowpea and corn) gels Paul E.Okechukwu 1 and M.A.Raoz Department of Food Science and Technology, Cornell University-Geneva, New York, NY 14456-0462, USA IOn leave from the Federal Polytechnic, Oko, Nigeria 2To whom correspondence should be addressed
Abstract Thermal transition and gel formation in dispersions (10% solids) of cowpea protein, and in mixtures of the protein and corn starch and cowpea starch, were examined using a differential scanning calorimeter (DS C) and dynamic rheometry, respectively. In mixtures of the protein and corn starch, two DSC peaks due to starch gelatinization and protein denaturation were observed. A single peak observed during the heating of the protein/cowpea starch mixtures appeared to be an overlap of the starch and protein endotherm peaks. With an increase in the protein/starch ratio (R), the starch gelatinization onset and peak temperatures shifted to higher values, while endotherm enthalpy decreasedproportionally. G of the protein/starch gels increased with aging time at 20°C and attained plateau levels that decreased with increase in protein/starch ratio ( R ) . For protein/cowpea starch gels, G growth with time was adequately described by afirst-order kinetic model. In the early stages of structure development, afaster increase in G for the protein/cowpea starch mixture ensured higher levels of G after 1 h of aging compared with that for starch gel at the same starch concentration. In comparison to corn starch gels, there was no significant increase in G of gels ofprotein/corn starch.
Introduction Proteins and starches are important food components that are individually or collectively utilized to increase viscosity, stabilize dispersions, and provide texture and firmness. These functional attributes are based on the water-holding and gelation ability of the polymers during and after thermal transition. As the concentration of the polymers exceeds a threshold, gelation results from hyperentanglements as well as intermolecular associations and cross-links. Below this threshold, increased solution or dispersion viscosity and yield stress may be achieved. The gelation of globular proteins has been extensively studied by various techniques (1-5). Thermal gelation of proteins is widely accepted to involve the partial unfolding of protein molecular strands at temperatures higher than the transition onset, and subsequent aggregation, association and cross-linking to form a gel network. Thermal and rheological studies on the gelation of soybean 7S and lIS proteins by glucono-delta-lactone (GDL) (6) and by heat (7)
© Oxford University Press
have shown that the gelation rate is first order with GDL and increases with GDL concentration and heating temperature. Starch gels are composites in which intact and ruptured swollen granules are embedded in a continuous gel network. The continuous matrix may be composed of amylose or a mixture of amylose and amylopectin, depending on the type of starch, and pasting conditions (8-10). Amylose appears to be the major component of starch whose concentration in the continuous phase determines whether a heated starch dispersion would gel or not. The polymer is leached out as granule swelling progresses during heating at temperatures higher than the gelatinization onset. The critical concentration (Co) of amylose abo ve which a gel is formed is -0.8-1.1%, and appears to be independent of molecular weight and origin of the biopolymer (11-13). At relatively low starch concentrations , as filled gels, the mechanical properties of starch gels depend on those of the continuous matrix, the volume fraction of the granules, shape and
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deformability of the granules, and the granule-matrix interaction (8,14-16). The selectivity in interaction between proteins and starches was shown in dynamic rheological studies. Addition of gluten to 6.5-6.6% (w/w) dispersions of different starches at a 1%level calculated on a dry starch basis increased the G of wheat and rye starches, lowered that of maize starch, but did not affect the modulus of barley, triticale and potato starches (17). G was also found to increase with increasing amounts of gluten (1-4%) added to the wheat starch. Studies on starches with different amylose/amylopectin ratios suggest that gelatinization of amylopectin and not amylose favors a synergistic interaction with proteins leading to an increase in dispersion viscosity (18,19). The amylose/ amylopecin ratio, and type of protein and starch, are important factors influencing protein-starch interaction. Differences in the rheological behavior of gels from mixtures of fish protein and starch were attributed to the variation in the gelatinization temperatures of the starches in relation to the transition temperature of the protein. In such binary systems, the continuous gel network would be formed by the component with the lower transition temperature (2). Based on studies of starches and bovine serum albumin, Muhrbeck and Eliassen (20) showed that when a starch network is formed before the gel formation of the protein, the two structures supplement each other without any specific interaction, to form a complex system with two continuous networks. When the protein network is formed first, the diffusion and aggregation of the amylose molecules and the swelling of the granules are hindered. The starch cannot form a continuous network, but will instead act as a filler reinforcing the protein network. Because proteins and starches are thermodynamically different polymers, their joint presence may lead to phase separation, inversion or mutual interaction with significant consequences on texture (21). More information is required to enable gainful exploitation of the protein-starch interaction for the design and evaluation of texture in foods. The purpose of this study was to examine the interaction of cowpea protein with cowpea starch; a thermal procedure involving differential scanning calorimetry was used to probe the transition temperature, while rheological methods were used to examine the mechanical properties of gels formed by heating dispersions of cowpea and corn starch, cowpea protein, and their mixtures.
Materials and methods Unmodified corn starch used in this study was purchased from Sigma Chemical Co., St Louis, MO. Cowpea starch and protein extraction
Cowpea (Vigna unguiculata) seeds were soaked in tap water for 3 h and subsequently decorticated by manual abrasion. Following the methods of starch extraction described by Cheng-yii and Shin-ming (22) and Schoch and Maywald
(23), the cleaned cotyledons were blended to a slurry of fine particles with five parts by weight of 0.2% sodium sulfite solution in a Waring blender. The slurry settled for -2 h at ambient temperature, and was filtered through an ASTM No. 140 standard sieve. Prime starch was recovered as sediment from the filtrate after settling for 2 h. The starch was washed three times and vacuum dried at 35°C for -48 h. Using the method of Williams et al. (24), the amylose content of corn starch and cowpea starch was determined to be -20 and 30%, respectively. Protein was extracted from the decorticated cowpea cotyledons using the single-stage method of Molina et al. (25), freeze-dried, and stored in air-tight containers until experimentation. Slurry and gel preparation
Dispersions of starch and protein (4-12% solids) were made by thoroughly dispersing weighed amounts of the dry samples in distilled water in a 50 ml wide-bottom conical flask. The pH of the protein dispersion was adjusted to 7.0 with 2 N NaOH. Dispersions (10% solids, pH 7.0) of mixtures of starch and protein at various weight ratios were also prepared. The dispersions were allowed to hydrate for -6 h at 10°C before thermal analysis and gel preparation for rheometry. One concern in gel preparation was the settling of starch granules during heating of low-concentration starch dispersions (17,26,27), which was minimized by keeping the dispersion agitated. The other concern was loss of moisture at 85°C in an open geometry (17), such as the plate-cone geometry of a rheometer. Therefore, two different procedures were attempted as a necessary compromise between the aforementioned concerns and alteration of structure. (i) Thoroughly mixed dispersions (-20 g) were heated in stoppered conical flasks in a water bath for 15 min at 85°C, a technique similar to that of Lindahl and Eliasson (17). A thermocouple introduced through the cap of the flask was used to monitor the temperature of the dispersions. (ii) The heated dispersion at 85°C was quenched to 25°C in an ice bath and transferred to a 25°C water bath, and the resulting gel was aged for 1 h before rheological evaluation. Although the word gel is being used to describe the samples obtained after cooling, because of the relatively low solids content and the presence of starch granules, they could very well be called heated dispersions. Thermal analysis
A differential scanning calorimeter (DSC) (Model 2910, TA Instruments, New Castle, DE) was used to examine thermal transitions. Approximately 10-15 mg of a dispersion were placed in a DSC aluminum pan, hermetically sealed, and scanned over 30-150°C in the DSC at 5°C/min with a sealed empty pan as a reference. After heating, the sample pans were punctured and the pan contents dried at 105°C to constant weight and the true solids content of the sample was calculated.
341
Cowpea protein-starch interaction
cornstare
60
70
80
90
100
Temperature °C
60
70
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Temperature (oq
Figure 1 DSC thermogram of corn starch, cowpea protein and corn starch-protein mixtures at 10% solids and various protein! starch ratios (R).
Figure 2 DSC thermogram of cowpea starch, cowpea protein and cowpea starch-protein mixtures at 10% solids and various protein! starch ratios (R) .
Rheometry
The transition temperatures of the protein, shown to contain mostly 7S globulins (28-30), were higher than the corresponding gelatinization temperatures of corn starch and cowpea starch. At 10% solids, blends of the protein and corn starch produced a single endotherm peak which gradually broadened into a bimodal peak as the protein! starch ratio (R) increased (Fig. I). The thermogram appeared to be an overlap of two transition processes. The first peak, with T p increasing gradually from the starch gelatinization peak temperature to higher temperatures with increase in R, is attributed to starch gelatinization, while the second peak with T p close to the protein peak temperature is apparently related to protein denaturation. Mixtures of cowpea starch and protein showed only a single endotherm peak at various levels of R (Fig. 2). The absence of an emerging protein peak in the cowpea starch-protein mixtures may reflect the smaller difference in the peak temperatures of cowpea starch and protein (8°C) compared with that of corn starch and cowpea protein (12°C). Both To and T p of the single peak observed for the protein--cowpea starch mixtures increased proportionally with an increase in the protein content of the blends, while Tc quickly increased to the Tc of the protein (Fig. 3). A similar effect of the protein on the starch peak transition temperatures was observed in mixtures of corn starch and cowpea protein (Fig. 4). The transition enthalpies of the mixtures at 10% solids content decreased proportionally from the value of the starch to that of the protein with an increase in the protein/starch ratio. The trends in starch transition temperatures and enthalpy are indicative of hindrance to starch gelatinization similar to the presence of salt (31,32) and fish proteins (2). The trends also suggest the absence of any specific interaction between the starches and the protein before heat treatment.
Dynamic rheological tests were performed at 20°C on gel samples with a cone (6 em diameter, 2° angle) and plate geometry of a Carri-Med CSL2 100 rheometer (TA Instruments). The cone-plate geometry provides uniform shear and is well suited for heated starch dispersions. Samples of the gel aged for 1 h at 25°C were subjected to a frequency sweep at I% strain over 0.1-10 Hz in the linear viscoelastic range determined from torque sweepsat 5 Hz. In addition, samples of hot gels at 85°C were quenched rapidly to 20°C on the rheometer plate and subjected to a time sweep of 10 h at 1% strain and 1 Hz. Because the heated and unheated 10% cowpea protein dispersions were fluid in nature, it was felt that flow data would be useful. Therefore, shear stress-shear rate data were obtained on unsheared samples in both increasing and decreasing stress modes in the range 0.1-20 Pa in 5 min. Magn itudes of shear stress, 0, and shear rate, y, from the down curve of shear stress sweep were used in data analysis.
Results and discussion Differential scanning calorimetry
The average values of onset (To), peak (Tp ) and concluding (Tc) temperatures during gelatinization measured by DSC at solids content up to 15% were: 62, 68 and 73°C for corn starch, and 66, 72 and 78°C for cowpea starch, respectively. Transition enthalpies for the gelatinization of the starches were 10.8 J/g for corn starch and 8.5 J/g for cowpea starch. DSC thermograms (Figs I and 2) of cowpea protein isolate at 10% solids, pH - 7.0, revealed that the protein denatured over 72-86°C with a transition endotherm of 4.9 J/g that peaked at SO.2°C.
P. E. Okechukwuand M.A. Rao
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Figure 3 Effect of cowpea protein addition to the gelatinization onset (To, _), peak (Tp, . ) and concluding (Te, A) temperatures of cowpea starch in excess moisture.
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Rheological data Starch gels Storage modulus (G) development as a function of aging time at 20 D C of cowpea starch gels of various concentrations showed a two-stage pattern (Fig. 5). The initial stage showed a concentration dependence and G increased rapidly with aging time. The second phase showed a slower increase in G and appeared to reach a limiting value. With increasing starch concentration, the second phase in G development became shorter and G approached its limiting value within a shorter time span. Changes in loss tangent (tan 0) with time were also similarly affected by starch concentration. At 5% starch concentration, tan 0 decreased from 0.25 to 0.1 and 0.05 after 2 and 10 h of aging at 20D C, respectively. At the
Figure 5 Effect of aging time on the gel strength of 5% (0) and 9% (. ) cowpea starch gels at 20°e. Insert shows the variation of tan 0 for the gels during aging.
higher concentration of 9% starch, tan 0 fell from 0.19 to 0.1 in <10 min and approached a limiting value of 0.03 after 10 h of aging. Similar trends were also observed for corn starch gels. The two-phase nature of G growth in starch gels has been attributed to the gelation of amylose in the continuous matrix during the early stages of aging and the retrogradation of amylopectin observed after long periods of aging (8,13,15,33). The decrease in the rate of amylose retrogradation with increase in starch concentration has also been attributed to the decrease in molecular mobility as a result of swellingand gelation. Values of G at 1 Hz of freshly prepared cowpea starch gels exhibited a power law increase with starch concentration (C). Over the range of 4--10% cowpea starch, G ex: (;3.7 (R2 = 0.964). For corn starch gels, over 6-12%, G ex: C4.8 (R2 = 0.984); however, over the range 7-12% starch, G increased linearly with starch concentration (G ex: 504.1 C, R2= 0.984), in agreement with Evans and Haisman (34). Protein dispersions andgels Unheated 10% solids cowpea protein dispersion, pH - 7.0, sheared at 20DC over 1-550 s-l, exhibited weak thixotropic behavior and followed the simple power law model. (1)
After heating at 85D C for 15 min, the dispersion exhibited higher viscosity and greater thixotropic response. Log-log plots of the descending segment of the shear stress versus shear rate cycle (not shown here) revealed that while the flow behavior index, n, decreased from 0.92 in the native state to 0.83 after heating, the consistency K increased from 20.2 to 107.4 mpa.s", Although the increase in thixotropy of the 10% solids dispersion after heating suggests the possibility of structure formation, its storage modulus was not detectable with our rheometer. Protein solutions (pH - 7.0) at
Cowpea protein-starch interaction
343
up to the maximum value followed an apparent first-order rate process [equation (2)]:
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G max =7.69(x s)3.35 R2 = 0.998 Figure 6 Gel strength development during the aging of cowpea protein solutions (pH - 7.0) at 20°C after heatingat 85°C for 15 min. Insert shows the decreasein tan 0 during aging. Percent protein: 13% 6 ,15% 0 , 20% D .
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concentrations ~ 13% formed gels on cooling after heating at 85°e (Fig. 6). The almost linear increase in G and the associated decrease in tan 8 over the entire 10 h aging period suggests a gradual development in the elastic characteristic of protein gels (35). Cowpea protein starch mixtures Figure 7 shows the G development in 10% solids gels from blends of cowpea protein and cowpea starch over a 10 h aging period at 20°e. In general, G increased from an initial value, Go, to a maximum plateau value, G max' that increased with the proportion of starch in the mixture. A slight decrease in G was observed during the later part of aging, probably due to weakening of starch granules. The rise in G
(2)
(3)
The nearly equal values of the exponents for the variation of G with starch concentration in starch/protein mixtures (within the range of R < 1/9) and in cowpea starch gels emphasize the important contribution of starch to the structure of the former. G development in the protein-starch gels followed an essentially apparent first-order process and did not show either the dominant two-stage growth pattern seen in starch gels or the almost linear pattern observed in the cowpea protein gels. A combination of the development patterns in the protein and starch components is apparent in the protein-starch gel. It appears that at pH values close to 7.0, the cowpea protein solubilized and formed the continuous phase in which raw starch granules were dispersed. On heating, the granules absorbed water and as they swelled they exuded some amylose into the continuous matrix prior to the protein denaturation. It has been suggested (20) that the higher transition temperature of the protein compared with that of starch creates the environment for the leaching of amylose into the solution of the protein. It is likely that the co-existence of the protein and the amylose in the continuous phase may be responsible for the distinct kinetic pattern in the observed development of G in the protein-starch mixtures. For such a system, the protein/starch ratio (R) would reflect the protein/amylose ratio in the continuous matrix and may be considered to be an important parameter for assessing G of the mixture gels. Figure 8 shows the variation of G at 1 Hz of protein! starch gels (10% solids) from frequency sweeps on gels after 1 h of aging with starch fraction (x s) . Up to x, < 0.8 (R < 2/8), G of the mixed gels was higher than that of cowpea starch gel at the same concentration. For x, > 0.8, G of the starch gel was higher than G of the mixed gel. Figure 8 also contains G at 1 Hz from frequency sweeps on gels of cowpea protein and corn starch after 1 h of aging at 25°e. Addition of the cowpea protein to corn starch at high levels did not affect the magnitude of G of the gel and at low levels resulted in a slight decrease. A similar decrease in G was observed after the addition of 1% gluten to corn starch (17). Recognizing that G of 10% solids cowpea protein gels was
344
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Figure 8 Influence of starch concentration on the storage modulus at 1 Hz of com starch and cowpea starch gels in the presence of cowpea protein. Note that in order to avoid overlap with cowpea starch data, G values of protein/com starch and com starch gels were increased by 500 Pa. (Cowpea starch _, cowpea starch & protein A, com starch + 500 Pa e, com starch & cowpea protein + 500Pa+.)
too low to be measured, the shape of the modulus-starch fraction curves of the mixed gels containing either corn starch or cowpea starch suggests phase separation (21). The higher G values of the protein/cowpea starch gels at low levels of starch appear to be due to favorable kinetics during the early stages of G development (Fig. 9). For aging periods of <250 min in 5% solids gels, G of the mixed gel (5% solids and 5 parts protein and 5 parts starch) was more than that of the starch gel of equivalent solids content. At a higher starch and solids level (9%), G of the mixed gel (9% solids and 1 part protein and 9 parts starch) was lower throughout the aging period.
Conclusion At a heating rate of 5°C/min, at various protein/starch ratios, DSC data of cowpea protein with corn starch showed a bimodal DSC endotherm and a single peak with cowpea starch. With an increase in protein/starch ratio, transition onset and peak temperatures increased while endotherm enthalpy decreased proportionally. For gels made from protein and cowpea starch at protein/starch ratios <2/8, G was slightly higher than that of cowpea starch at the same starch fraction; however, no increase in G was observed in gels with corn starch. G of gels made from protein/starch mixtures increased with aging time and attained plateau values whose magnitudes decreased with increase in protein/starch ratio. For gels of protein/cowpea starch, the increase in G with time was adequately described by a first-order kinetic model. In the early stages of structure development, a faster increase in G for the mixture resulted in a higher levelof G compared with that for starch gel at the same starch level after 1 h of aging.
200
300
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500
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Aging time (min)
Figure 9 Comparison of G development of cowpea starch gels with and without added cowpea protein (5% solids: all starch 0, 5/5 protein/starch A) (9% solids: all starch e, 1/9 protein/starch 0).
Acknowledgement Partial support for this work was provided by the USDA Regional Research Project, NC136.
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Cowpea protein-starch interaction
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