Effects of sucrose and sorbitol on the gel formation of a whey protein isolate

Food Hydrocolloids 16 (2002) 489±497

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Effects of sucrose and sorbitol on the gel formation of a whey protein isolate Stephan Dierckx*, Andre Huyghebaert Department of Food Technology and Nutrition, Ghent University, Coupure Links 653, B9000 Gent, Belgium Received 12 October 2001; revised 16 November 2001; accepted 10 December 2001

Abstract Thermal analysis by differential scanning calorimetry and dynamic rheology were used to evaluate possible effects of sucrose and sorbitol at various concentrations on the thermogelation of a 150 g kg 21 whey protein isolate solution at pH 6.0 and 8.5. At both pH values, addition of these solutes caused an increase in the thermal transition temperature of the protein denaturation/aggregation process Ttr, with a linear relationship between solute concentration (on a w/w basis) and Ttr. Dynamic oscillation tests showed that the gelation temperature Tgel was raised accordingly. The transition enthalpy DHtr was only affected slightly and non-speci®c (changes within 1 J g 21 protein). Calculation of the apparent activation energy of the gelsetting Eaapp based on the temperature dependence of the gelation time tgel, as well as the G p value, obtained after slow heating of the solutions, revealed effects of both sucrose and sorbitol at pH 6.0 but not at pH 8.5, especially at high concentrations of these solutes. This lead to the conclusion that the interactions between the polyhydric solutes and the whey proteins (mainly b-lactoglobulin) were of a very weak nature. Furthermore, based on differences in the gelation mechanism of whey proteins at pH 6.0 and 8.5, it was suggested that sucrose and sorbitol affect protein±protein interactions in gels through enhancement of hydrophobic interactions. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Gelation; Whey proteins; Polyhydric compounds; Thermal analysis; Dynamic rheology

1. Introduction Whey proteins are important ingredients in many food products, providing essential amino acids and exhibiting a broad range of functional properties such as gel formation, foaming, and emulsi®cation. Various whey protein products have been developed over the past decades, often with a speci®c application ®eld. One of such products is whey protein isolate (WPI), either produced by ion-exchange and subsequent ultra®ltration (UF) or micro®ltration and UF. According to the American Dairy Products Institute, the protein content should be at least 90% on an `as is' basis. Since there seems to be an increased interest in whey products with a high protein content, it can be expected that the share of WPI on the market will grow (Horton, 1998). Sucrose and sorbitol are naturally occurring polyhydric compounds and can be found in a wide variety of food products. It is well known that both sucrose and sorbitol have an effect on the denaturation of proteins. For instance, Jou and Harper (1996) reported an increased denaturation temperature Ttr of a whey protein concentrate in * Corresponding author. Tel.: 132-9-2646163; fax: 132-9-2646218. E-mail address: [email protected] (S. Dierckx).

the presence of sucrose while Boye, Ismael and Alli (1996b) reported the same effect for the single whey protein blactoglobulin. Harwalkar (1986) found a similar effect for sorbitol on the denaturation of b-lactoglobulin. This increased Ttr is thought to originate from a more unfavourable contact of the sugar or the polyalcohol with the denatured, unfolded form of the protein, compared to contacts with the native, folded form (Arakawa, Bhat, & Timasheff, 1990). The main effect for sugars seems to be related to their ability to increase the surface tension of water whereas for polyalcohols to their highly favourable contact with water (Arakawa et al., 1990; Timasheff, 1998). However, it is important to realize that the interactions between proteins and co-solvents like sugars and polyalcohols are weak and in general non-speci®c (Timasheff, 1993). Although the stabilizing effect on proteins of some mono- and disaccharides as well as a number of polyalcohols is well documented in literature, little is known about the actual importance of this behaviour with respect to protein gel formation. In the presented study, the gelation of a WPI in the presence of sorbitol or sucrose is evaluated. Such interactions may be important in food products, such as cakes or cookies, where the temperature range of thermal events like protein denaturation and starch gelatinisation

0268-005X/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0268-005 X(01)00 129-1

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should be in a speci®c range to obtain the desired texture for the ®nal product (Donovan, 1977). 2. Experimental 2.1. Material The protein preparation used is a commercial WPI produced by Besnier±Bridel Alimentaire (ReÂtiers, France), prepared from sweet or mixed whey by a-lactalbumin precipitation at acid pH, ion-exchange chromatography of the residual whey, followed by UF and spray-drying (Rialland & Barbier, 1988). Moisture content, analysed according to FIL26A (IDF, 1993b), was, on a product basis, (50.3 ^ 0.4) g kg 21 moisture, protein and non-protein nitrogen (IDF, 1993a) were (857 ^ 8) and (4.6 ^ 0.1) g kg 21, respectively. Lactose content, determined by GC, was (2.7 ^ 0.3) g kg 21 and the protein preparation contained (1.9 ^ 0.4) g kg 21 fat (IDF, 1987). Cations analysed and determined by ¯ame atomic absorption spectrometry were Ca 21 (1.625 ^ 0.006), K 1 (0.529 ^ 0.002), and Na 1 (1.208 ^ 0.005) g kg 21. The protein fraction was mainly comprised of b-lactoglobulin, as analysed by SDS-PAGE. According to the de®nitions set by the American Dairy Products Institute (Horton, 1998), this whey protein preparation should not be called `protein isolate' but rather `protein concentrate'. However, since it mainly consists of b-lactoglobulin it can be differentiated from most commercial available concentrates and therefore, the term WPI is used. d-Sorbitol and d-(1)-sucrose, both of analytical grade, were purchased from Acros Organics (Geel, Belgium). 2.2. Sample preparation An amount of WPI-powder to achieve a ®nal concentration of 150 g kg 21 was dispersed in distilled water and the dispersion was stirred for 1 h at room temperature. Sucrose or sorbitol were added to obtain a ®nal concentration of 125, 250, or 400 g kg 21, after centrifugation of the protein solution (15 min at 10,000g) to remove insoluble particles, and stirred until complete dissolution of the carbohydrate was obtained. The solution was kept over night at 4 8C. The pH of the solutions was adjusted to 6.0 or 8.5, using 0.01, 0.1 or 1.0 mol l 21 HCl or NaOH. The protein solutions were degassed under vacuum for 15 min prior to use. Since all solutions were used within 24 h after preparation, no antimicrobial agent was added. 2.3. Techniques For differential scanning calorimetry (DSC), about 15 mg of sample was hermetically sealed in coated aluminium pans. The thermogram was obtained with a DSC 2010 from TA-Instruments (Brussels, Belgium) and a sucrose or sorbitol solution, at the same concentration as used in the sample, was used as a reference. The temperature was

increased from 25 to 110 8C at a rate of 5 8C min 21. Three parameters are determined, based on the thermograms of all solutions, i.e. the transition temperature Ttr, the transition enthalpy DHtr, and the width at half-peak height T1/2. The temperature Ttr is determined as the temperature at maximum de¯ection of the baseline and is a good approximation for the protein denaturation temperature (Ma, Harwalkar, & Maurice, 1990). The value of DHtr is calculated based on the area under the transition peak, using a straight extension of the baseline. Rheological measurements were carried out on a Bohlin CVO50 (Bohlin Instruments Ltd, United Kingdom) controlled stress rheometer, equipped with a circulating water bath for temperature control (^0.1 8C), using a cone±plate geometry (diameter 40 mm, truncation angle 48, gap ®xed at 150 mm). For isothermal measurements, the sample was poured on the pre-heated bottom plate while the upper cone was lowered from a ®xed position, marked as time zero. Temperatures were chosen as such that gel formation occurred within 900 s after initiation of the test. The gelation temperature was determined by heating the sample between the cone/plate geometry at a ®xed heating rate of 2.5 8C min 21 from 25 8C till Ttr 1 2 8C. In the latter case, changes in the gap due to thermal expansion of the cone and plate were corrected by the software. The strain value was set at 0.025, which was within the linear viscoelastic region at 1 Hz as evaluated with preliminary tests. All analyses were done in triplicate, i.e. three separate solutions were prepared and each run once, and statistical analysis was done with SPSS 10.0 (SPSS Inc., Chicago), using tukey's test (a ˆ 0.05) for comparison of mean values.

3. Results and discussion 3.1. Thermal analysis The thermograms obtained for the WPI-solutions at pH 6.0 and 8.5, without the addition of sucrose or sorbitol, are shown in Fig. 1. The thermograms show one major endothermic peak. This peak can be attributed to the denaturation/aggregation of b-lactoglobulin. Heating puri®ed b-lactoglobulin (obtained from the Institut National de la Recherche Agronomique, France) con®rmed this ®nding. At pH 6.5, a slight shoulder on the low temperature end of the peak probably indicates the presence of some alactalbumin (Bernal & Jelen, 1985). Addition of 125, 250, or 400 g kg 21 sucrose or sorbitol to the protein solutions at pH 6.0 and 8.5 caused a shift of the endothermic peak to higher temperatures. This effect is shown in Fig. 2 as the increase of Ttr in function of increasing carbohydrate concentration. In general, Ttr values are higher at pH 6.0 than at 8.5, which corresponds to the lower thermal stability of b-lactoglobulin at alkaline pH values (Boye, Ismael, & Alli, 1996b). An increased thermal stability of whey proteins in the presence of carbohydrates has been reported in

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491

Fig. 1. Thermograms of 150 g kg 21 whey protein isolate solutions at pH 6 (Ð) and pH 8.5 (- - -) recorded at 5 8C min 21 (exotherm up).

literature by various authors (Harwalkar, 1986; Jou & Harper, 1996) and is attributed to preferential hydration of proteins in the presence of solutes, such as carbohydrates (Arakawa et al., 1990; Xie & Timasheff, 1997). However, in this study, concentration effects cannot be excluded since part of the water is replaced by either sucrose or sorbitol. Hence, a higher actual protein concentration is obtained, which is known to have an effect on the thermal stability of proteins (Relkin & Launay, 1990). Nevertheless, since the effect of sucrose is different from that of sorbitol at both pH values, especially at higher solute concentration (Fig. 2), it can be concluded that effects, other than an increased protein concentration, have to be taken into account. Results on the effects of carbohydrates on the denaturation enthalpy, reported in literature, are not unambiguous. Jou and Harper (1996) and Xie and Timasheff (1997) reported that the DHtr was generally independent of the solute concentration while Harwalkar (1986) found that this parameter changed without any speci®c pattern. Table 1 lists the transition enthalpies associated with the transition peak. It should be noticed that especially at 400 g kg 21 rather large standard deviations were obtained due to dif®culties in construction of the extended baseline. Although differences exist, they are in the order of magnitude of 1 J g 21 protein without a clear pattern. At pH 6.0, the width at half-peak height (T1/2) for the protein solution 5.8 8C, was not signi®cantly altered upon addition of sucrose or sorbitol. Boye, Alli and Ismael (1996a) observed the same effect for bovine serum albumin at pH < 6.8 and

suggested that sugars do not interfere with the cooperativity of unfolding. Sorbitol, however, did cause a signi®cant increase of the T1/2-value at pH 8.5 with a minimum value for T1/2 of 10.8 8C for the protein solution and a maximum of 13.6 8C in the presence of 400 g kg 21 sorbitol. In all cases, the values of T1/2 at pH 6.0 are signi®cantly lower than those at pH 8.5. This observation may be contributed to differences in the conformation of b-lactoglobulin. Between pH 6.5 and 8.0, this protein undergoes a reversible conformational transition (Tanford, Bunville, & Nozaki, 1959); furthermore, the thiol group becomes increasingly active at higher pH values (Shimada & Cheftel, 1989) affecting the aggregation mechanism (Roefs & de Kruif, 1994). This change in molecular structure may be a possible factor in the differences of the effect of carbohydrate addition at pH 6.0 versus at pH 8.5. 3.2. Isothermal gel formation For all solutions, isothermal gel formation curves were obtained similarly as those shown in Fig. 3 for the solutions without addition of carbohydrates. The gelation time tgel was taken as the time at which the complex modulus G p became larger than 1 Pa (threshold value). At higher temperatures tgel decreased, indicating that gel formation occurred more quickly at higher temperatures. No conclusion can be drawn about the values of the G p at equilibrium since it is known that for whey proteins this value is only obtained after prolonged holding times (order of hours) (Paulsson, Dejmek, & van Vliet, 1990; Stading & Hermansson, 1990) and in this

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Fig. 2. Variations of the transition temperature of the thermal denaturation, determined by DSC, of a 150 g kg 21 whey protein isolate solution at (a) pH 6.0 and (b) pH 8.5 as affected by the addition of sucrose (Ð) or sorbitol (- - -). The inner line represents the ®tted linear regression curve while the outer lines indicate the 95% con®dence area of this ®t.

research the isothermal gel formation was studied only for about 900 s at maximum. Tobitani and Ross-Murphy (1997) developed an elegant model to describe globular protein gel formation, assuming that gelation can be described as an nth-order irreversible reaction of junction formation whereby one junction is formed by n-fold sites on polymer particles. They found an inverse relationship between tgel and the reaction rate constant k. Hence, the effect of

temperature can be described by the Arrhenius model: 1 < e2Ea =RT tgel where Ea is an (apparent) activation energy (J mol 21), R the universal gas constant, and T the temperature (K). If the gelation time tgel is taken as the time at which G p . 1 Pa,

S. Dierckx, A. Huyghebaert / Food Hydrocolloids 16 (2002) 489±497 Table 1 Effect of various concentrations of sucrose or sorbitol on the thermal transition enthalpy DHtr (J g 21) of 150 g kg 21 whey protein isolate, determined by DSC at a scan rate of 5 8C min 21. abcValues with the same alphabetical superscript in one column are not signi®cantly different (a ˆ 0.05) within one carbohydrate group Polyhydric compound Concentration (g kg 21) pH 6.0

None Sucrose Sorbitol

125 250 400 125 250 400

pH 8.5

DHtr

SD DHtr

SD

13.1 ac 12.2 ab 13.1 a 11.2 b 13.5 c 13.8 c 12.6 c

0.3 9.1 ab 0.1 9.7 a 0.3 9.6 a 0.8 7.8 a 1.0 10.5 c 0.4 10.4 bc 1.0 10.2 bc

0.5 0.4 0.1 2.2 0.5 0.4 0.8

493

values for Ea are obtained as listed in Table 2. Although in general an R 2 $ 0.9 was obtained for the individual samples and the tests were done in a random order, quite large standard deviations can be noticed for certain experiments. This may be contributed to the fact that, especially at high temperatures, gelation proceeded very fast and the formation of air bubbles could also be observed, despite degassing of the solutions prior to the analysis. Nevertheless, substantial differences exist between the effect of sucrose and sorbitol at pH 6.0 compared to pH 8.5. At pH 8.5, neither sucrose nor sorbitol affect Ea signi®cantly whereas at pH 6.0, sorbitol causes an increase in Ea at all concentrations investigated and sucrose at 400 g kg 21, compared to the solution with no addition of sucrose or sorbitol. This observation may re¯ect differences in the gelation mechanism of whey proteins at both pH-values. At near-neutral pH, the gelation process is

Fig. 3. Evolution of the complex modulus at isothermal conditions of a 150 g kg 21 whey protein isolate solution at (a) pH 6.0 A: 75 8C, W: 77 8C, S: 79 8C, e: 81 8C, and (b) pH 8.5 A: 57.5 8C, W: 60.0 8C, S: 62.5 8C, e: 65.0 8C, £ : 67.5 8C. Frequency 1 Hz, strain 0.025.

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Table 2 Apparent activation energy Ea (kJ mol 21) of 150 g kg 21 whey protein isolate, as derived from the Arrhenius relationship between the temperature at isothermal gelation and the respective gelation time tgel, and the effect of various concentrations of sucrose or sorbitol. abcdData with the same superscript are not signi®cantly different (a ˆ 0.05) within one type of carbohydrate and one pH value Polyhydric compound

None Sucrose Sorbitol

Concentration (g kg 21)

125 250 400 125 250 400

pH 6.0

pH 8.5

Ea

SD

Ea

SD

28.9 a 36.8 a 45.0 ab 68.1 b 64.9 cd 54.4 c 71.6 d

5.7 9.8 6.1 14.4 4.3 6.1 6.2

16.7 ab 14.6 a 16.0 a 19.0 a 14.5 b 14.5 b 19.3 b

2.4 7.1 1.3 5.6 5.3 1.5 0.7

mainly dominated by hydrophobic and electrostatic interactions as well as hydrogen bonding (Oakenfull, Pearce, & Burley, 1997; Ross-Murphy, 1995) while at more alkaline pH disulphide bonds are more dominant (Damodaran, 1996; Shimada & Cheftel, 1989). Furthermore, it is interesting to note that Back, Oakenfull, and Smith (1979) already pointed out that sucrose and sorbitol may enhance hydrophobic interactions in protein±protein interactions while more recently Kamiyama, Sadahide, Nogusa, and Gekko (1999) suggested that polyols were able to stabilize the molten globule state of horse cytochrome C through enhancement of hydrophobic interactions. Hence, it is likely that at conditions where covalent interactions dominate protein aggregation the effect of carbohydrates is of less importance than at conditions where non-covalent interactions are at play. 3.3. Gelation curves Slow heating of the protein solutions resulted in the formation of a gel. This lead to an increase of G p and a decrease of the phase angle d near the gel temperature, typical for the denaturation and gel formation of globular proteins (Fig. 4). The shape of the curves is not visibly affected by the addition of sucrose or sorbitol. The curves, however, are shifted to higher temperatures, i.e. the gelation is delayed upon addition of these solutes. This is in agreement with the increased thermal transition temperatures observed during the DSC measurements. Taking the temperature at which G p reaches a threshold value of 1 Pa as the gelation temperature Tgel, then a nearly linear relationship can be found between this Tgel and the transition temperature Ttr, the latter obtained from the DSC thermograms as indicated earlier. This linear relationship is illustrated in Fig. 5. It is important to mention, however, that due to practical reasons the heating rates were different for the thermal, i.e. DSC, and rheological experiments. The heating rates were 5.0 and 2.5 8C min 21, respectively. Nevertheless, it can be concluded that the increase in Tgel is mainly due to the shift of the thermal transition temperature ( < denaturation temperature) to higher values upon addition of sucrose and sorbitol.

At pH 6.0, Tgel is slightly lower than Ttr while at pH 8.5 these values are very similar in the temperature range studied, probably indicating differences in the gelation mechanism and/or gelation kinetics. After the rapid initial decrease of d at the beginning of the gelation stage, the phase angle levels off to reach a quasi-equilibrium value. The values obtained for d after an isothermal period at 900 s at Ttr 1 2 8C are given in Table 3. Since G p is still increasing at this point, as can be derived from Fig. 4, it is clear that gel setting still continues. As indicated earlier, whey protein gels reach an equilibrium state, with respect to structure and texture, only after some hours at isothermal conditions (Paulsson et al., 1990; Stading & Hermansson, 1990). Hence, in this research only intermediate values, i.e. after 900 s at isothermal conditions, were obtained (Table 3). Values for the phase angle are higher at pH 6.0 than at 8.5, i.e. the gels are slightly less elastic. This is in accordance with their visual appearance. Gels at pH 6.0 are of a more particulate, opaque nature whereas the gels at alkaline pH are transparent and rubbery. Such pH-dependent changes in appearance are already well known and described by many authors, e.g. Langton and Hermansson (1992) and Stading, Langton, and Hermansson (1995). The phase angle is less strongly affected by the addition of carbohydrates, compared to the effect of pH. Only for a carbohydrate concentration of 400 g kg 21 and at pH 8.5, the value for the phase angle is higher than the value for the solution without carbohydrate addition. This means that a slightly more viscous gel is obtained (also the value at 250 g kg 21 sucrose at pH 6.0 seems to give an increase but one of the individual values was exceptionally high). A possible explanation may be that at such high solute concentration, the increased viscosity impairs protein± protein contact in addition to charge repulsions. A more pronounced effect can be seen on the complex modulus, although only at pH 6.0 and high carbohydrate concentration. Both sucrose and sorbitol cause an increase in G p at 400 g kg 21. There is no difference in effect between the solutes since the G p values, obtained for both polyhydric compounds at that concentration are not signi®cantly different. The fact that G p is only affected at high concentrations,

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495

Fig. 4. Evolution of complex modulus (B) and phase angle (A) of a 150 g kg 21 whey protein isolate solution at increasing temperature and subsequent holding period ( £ ) at (a) pH 6.0 and (b) pH 8.5. Frequency 1 Hz, strain 0.025.

is an illustration of the fact that interaction forces between the whey proteins and the polyhydric compounds are of a weak nature, as already outlined by Timasheff (1993, 1998). Furthermore, the difference in effect at pH 6.0 and 8.5 may be additional proof that polyhydric compounds may strengthen non-covalent protein±protein interaction, especially hydrophobic interactions. In this respect it is interesting to note the slight decrease in the G p (although not statistically at a ˆ 0.05) at 400 g kg 21 carbohydrate concentration and pH 8.5. 4. General conclusions Carbohydrates such as sucrose and sorbitol affect the

various stages during the gelation of whey proteins. Sucrose and sorbitol cause an increase in the thermal transition temperature and subsequently raise the temperature at which whey proteins are able to form a gel. Both solutes slightly affect the enthalpy of the thermal transition, depending on the pH, but no clear pattern is observed. At pH 8.5, sorbitol seems to in¯uence the protein conformation in such a way that protein unfolding becomes less cooperative. Since DSC only re¯ects the net thermal effects and does not give direct information about the protein's molecular structure, changes in this structure being either small or without causing changes in the thermal properties are not re¯ected. With respect to the gelation step, a clear effect of sucrose or sorbitol is only noticeable at pH 6.0, speci®cally at high concentrations. This was related to differences in the

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Fig. 5. Correlation between the transition temperature, determined by DSC at 5.0 8C min 21, and the gelation temperature, determined by dynamic rheology at 2.5 8C min 21, of a 150 g kg 21 whey protein isolate solution at (a) pH 6.0 and (b) pH 8.5 as affected by the addition of sucrose (A, Ð) or sorbitol (W, - - -). The various points indicate the independent replicates, the inner line represents the ®tted linear regression curve while the outer lines indicate the 95% con®dence area of this ®t. Table 3 Effect of various concentrations of sucrose and sorbitol on the complex modulus G p (kPa) and phase angle d (8) of 150 g kg 21 WPI-solutions after a heating step at 2.5 8C min 21 and an isothermal holding period of 900 s at Ttr 1 2 8C as obtained by dynamic rheology (g 0.025 and n 1 Hz). abcdValues with the same alphabetical superscript in one column are not signi®cantly different (a ˆ 0.05) within one carbohydrate group; § effect of carbohydrate type is not signi®cant at the concentration and pH indicated Polyhydric compound

Concentration (g kg 21)

pH 6.0 G

None Sucrose Sorbitol

125 250 400 125 250 400

p

14.2 ac 18.7 a§ 15.7 a§ 33.3 b§ 14.4 c§ 19.3 c§ 40.9 d§

pH 8.5 SD

d

SD

Gp

SD

d

SD

1.9 0.7 2.9 4.5 2.7 1.9 4.8

10.3 acd 11.0 ab§ 11.2 b§ 10.7 ab 10.4 d§ 10.5 d§ 10.0 c

0.1 0.3 0.6 0.3 0.1 0.2 0.0

14.5 ab 15.7 a§ 14.8 a 13.3 a§ 14.1 b§ 10.7 b 11.9 b§

1.2 1.8 1.0 2.4 2.2 0.4 3.0

8.5 ac 9.3 ab§ 9.0 ab§ 10.1 b§ 9.4 cd§ 9.5 cd§ 10.3 d§

0.6 0.2 0.4 0.5 0.4 0.5 0.2

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gelation mechanism of whey proteins at this pH, where noncovalent interactions predominate, compared to the more alkaline pH, where covalent disulphide linkages are more important. Hence, the increase in G p is attributed to enhancement of the hydrophobic protein±protein interactions in the presence of high concentrations of sucrose or sorbitol. Since the value of d is not altered to a large extent, the nature of the gel is not very much affected by the polyhydric solutes. Acknowledgements This research is part of a PhD-project subsidized by the Fund for Scienti®c ResearchÐFlanders (Belgium). The authors greatly acknowledge the practical assistance of Monique Jooris. References Arakawa, T., Bhat, R., & Timasheff, S. N. (1990). Why preferential hydration does not always stabilize the native structure of globular proteins. Biochemistry, 29, 1924±1931. Back, J. F., Oakenfull, D., & Smith, M. B. (1979). Increased thermal stability of proteins in the presence of sugars and polyols. Biochemistry, 18, 5191±5196. Bernal, V., & Jelen, P. (1985). Thermal stability of whey proteinsÐa calorimetric study. Journal of Dairy Science, 68, 2847±2852. Boye, J. I., Alli, I., & Ismail, A. A. (1996a). Interactions involved in the gelation of bovine serum albumin. Journal of Agricultural and Food Chemistry, 44, 996±1004. Boye, J. I., Ismael, A. A., & Alli, I. (1996b). Effects of physicochemical factors on the secondary structure of b-lactoglobulin. Journal of Dairy Research, 63, 97±109. Damodaran, S. (1996). Functional properties. In S. Nakai & H. W. Modler, Food proteins properties and characterization (pp. 167±234). New York: VCH Publishers. Donovan, J. W. (1977). A study of the baking process by differential scanning calorimetry. Journal of the Science of Food and Agriculture, 28, 571±578. Harwalkar, V. R. (1986). Effects of polyols upon thermal properties of blactoglobulin. Journal of Dairy Science, 69 (Suppl. 1), 84. Horton, B. (1998).The whey processing industryÐinto the 21st century. Proceedings of the Second International Whey Conference, Chicago 27±29 October 1997 (pp. 12±25). Brussels: International Dairy Federation. IDF (1987). Determination of fat content. FIL-IDF Standard 9C (7p). Brussels: International Dairy Federation. IDF (1993a). Determination of nitrogen content. FIL-IDF Provisional Standard 20B (12p). Brussels: International Dairy Federation. IDF (1993b). Determination of water content. FIL-IDF Provisional Standard 26A (2p). Brussels: International Dairy Federation.

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