Effect of temperature and pH on the interactions of whey proteins with casein micelles in skim milk

Food Research International,Vol. 29, No. 1, pp. 49-55, 1996 Copyright 0 1996 Canadian Institute of Food Science and Technology Published by Elsevier Science Ltd Printed in Great Britain 0963-9969/96$15.00+ .OO

ELSEVIER 0963-9969(95)00058-5

Effect of temperature and pH on the interactions of whey proteins with casein micelles in skim milk Milena Corredig & Douglas G. Dalgleish” Department of Food Science, University of Guelph, Guelph, Ontario, Canada i?IG 2Wl

Skim milk was heated at temperatures in the range 7559O”C, at pH values of 6.8, 6.2 and 5.8. The amounts of a-lactalbumin and p-lactoglobulin which interacted with the casein micelles during heat treatment were quantified by SDS-polyacrylamide gel electrophoresis of the micellar fractions isolated by ultracentrifugation. Both o-lactalbumin and ,&lactoglobulin appeared to interact similarly with casein micelles at temperatures up to 85°C. The amount of whey protein complexed with micelles increased with time, reaching plateau values that, at the highest temperatures, were comparable with the quantity present in the original skim milk. In general, faster reaction of the whey proteins with the micelles was found at lower pH and higher temperatures. The rates and extent of the reaction changed also when additional cr-lactalbumin and P-lactoglobulin isolates were added to milk before heating. The reaction between cl-lactatbumin and casein micelles depended to a relatively small extent upon environmental variations (pH and temperature), while /I-lactoglobulin interactions were more affected, so that a more complex behaviour may be attributed to the latter protein. Copyright 0 1996 Canadian Institute of Food Science and Technology Keywords:

milk, milk protein, casein micelle, heating, protein interactions.

bridges seem responsible for the maintenance of the reversible conformational change upon denaturation, in the presence of calcium (Swaisgood, 1992; Relkin et al., 1992). However, this thermal stability resulting from the high reversibility of the denaturation of a-la is not found when other proteins such as ,&lg and bovine serum albumin are present in the system (de Wit & Klarenbeek, 1984). Many studies have dealt with the kinetics of denaturation of p-lg and a-la in milk and whey and isolated solutions (Hillier & Lyster, 1979; Manji & Kakuda, 1986; Dannenberg & Kessler, 1988a), but agreement on the order of the reaction has not been achieved. In general, the rate and the extent of whey protein denaturation depends on environmental conditions (Hillier et al., 1979; Dannenberg & Kessler, 1988b); this is confirmed by the evidence that pH has a strong influence on the heat stability of milk (Singh & Creamer, 1992). However, it is often impossible to make direct comparisons between literature studies, because different analytical methods, or different means of expressing the results, have been employed. It is evident, in any case, that a

INTRODUCTION At temperatures higher than 7O”C, an extensive denaturation of whey proteins occurs (de Wit & Klarenbeek, 1984). By studying the loss of solubility at pH 4.6 of whey proteins, and by differential scanning calorimetry, it has been shown that immunoglobulins and serum albumin are the most easily denatured, ,&lactoglobulin (p-lg) is intermediate and a-lactalbumin (a-la) is the most heat stable (Ruegg & Moor, 1977). The free cysteine residue contained in the p-lg structure (Swaisgood, 1982) seems to be fundamental in the denaturation process, which involves several steps: dissociation of quaternary structure, changes in the conformation of the polypeptide chain and aggregation via disulphide bridging (Relkin & Launay, 1990). Differential scanning calorimetric studies show that a-la has a denaturation temperature of 64°C and high reversibility in solution (Ruegg & Moor, 1977; de Wit & Swinkels, 1980). The cysteine residues present in cy-la as four disulphide *To whom correspondence should be addressed. 49

50

M. Corredig, D. G. Dalgleish

close relation between serum protein denaturation and the formation of aggregates in the milk system exists (Dalgleish, 1990). Indeed, most of the literature on whey protein denaturation focuses the attention on residual native protein in solution after denaturation rather than on the complexes formed and the intermediates involved in the reaction (Hillier & Lyster, 1979; Manji & Kakuda, 1986; Dannenberg & Kessler, 1988a). The main complex formed during heat treatment of milk is believed to be the /?-lg/K-casein complex, the formation of which has profound implications in the renneting process (Hill, 1989). At low temperature, the reaction between /?-lg and n-casein seems to be driven mainly by hydrophobic interactions (Haque & Kinsella, 1988; Jang & Swaisgood, 1990), although after heat treatment the main characteristic of the complex is intermolecular disulphide bonding (Hill, 1989). This complex is also known as one of the major factors responsible for the heat stability-pH profile of milk (Singh & Creamer, 1992). The original ratio between fl-lg and K-casein is considered to be more important in heat stability of milk than the concentration of the two proteins per se (Sweetsur 8z White, 1974). However, the P-lg/K-casein complex is not the only one which is formed during heat treatment, since all of the cysteinecontaining proteins may play a role in the interactions (Dalgleish, 1990). Heat-induced intermolecular complexes may also form between o-la and ,&lg (Shalabi & Wheelock, 1976), and the presence of both whey proteins in the heated micelles has been described recently (Law el al., 1994). The present research aimed to quantify the amounts of a-la and p-lg complexed to the casein micelles, as a function of time, pH and heating temperature. These interactions were studied not in model systems but in skim milk.

MATERIALS

AND METHODS

Whole fresh milk was collected from the Elora Research Centre Dairy Herd of the University of Guelph. After addition of 0.02% sodium azide, the milk was skimmed at 8°C by centrifuging at 3000 g for 20 min in a JZMC Beckman preparative centrifuge, with JA-14 rotor (Beckman, Palo Alto, CA). Skim milk samples were either heat treated immediately or were first adjusted to pH 6.2 or 5.8 by slow addition of 1 M HCl with constant stirring. In some experiments, 2 mg/ml-’ of isolates of individual whey proteins were added to the skim milk before heating. The isolated fractions of a-la and p-lg were provided by Protose Separations Inc. (Teeswater, ON). Skim milk was heated in 15 ml aliquots in test tubes in a temperature controlled bath, at 75,80,85 and 90°C for times ranging from 1 to 80 min. A time of 105 s was necessary to reach the required temperature, and after

the treatment the milk was immediately cooled in an ice bath. Isolation of casein micelles Samples of heated skim milk were centrifuged with. a Beckman preparative ultracentrifuge (model L&70M, TI-70i rotor) at 60000 g for 40 min. Preliminary studies were performed to determine the optimum length of the run (i.e. which left the lowest amount possible of casein micelles in the serum phase). The supematant was decanted and the micellar pellet was resuspended in buffer at pH 7.0, containing 20 mM imidazole, 5 mM CaClz and 50 mM NaCl. This buffer was chosen to stabilize the micellar material and to observe it in its native state while washing away any uncomplexed whey proteins from the micellar pellet. After a second ultracentrifugation, under the same conditions, the micellar pellet was collected and drained on Whatman No. 1 filter paper (Whatman, Maidstone, UK). This technique is suitable for isolating micelles, but it will also cosediment any aggregates of whey proteins which may be formed independent of the casein. It is not possible to separate these two complexes from the heated milk. We can only follow other studies (Hillier & Lyster, 1979; Manji & Kakuda, 1986; Singh & Fox, 1987; Dalgleish, 1990) and assume that all of the whey protein lost from solution is complexed to casein. It is certainly the case for homogenized milk (Sharma & Dalgleish, 1993) that the whey protein is complexed with the casein. Determination of whey protein bound to casein micelks A weighed amount of the uhracentrifuged miceliar fraction (about 0.015 g) was suspended in 0.200 ml of buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0); 0.250 ml of a 20% solution of sodium dodecyl sulphate (SDS), 0.100 ml of a solution of bromophenol blue (0.01%) and 0.100 ml of 2-mercaptoethanol were then added. This mixture was stirred constantly for 5 min in a boiling water bath. Aliquots (1 ~1) of samples of denatured protein solution were loaded onto a 20% homogeneous SDS-gel and run in a rapid electrophoresis system (Phastsystem, Pharmacia-LKB, Baii d’Urfe, Quebec, Canada) at 15°C. The protein bands were stained using coomassie blue indicator. The gels were dried and scanned using an Ultrascan laser densitometer (Ultrascan XL, Pharmacia-LKB, Baie d’Urfe, Quebec, Canada), at a wavelength of 633 mn and the density of the protein bands was expressed as a series of peaks as a function of the distance from the loading point. The areas under the peaks were integrated and summed with the software provided with the Ultrascan equipment. The individual bands were identified by running, on the same gel, internal standards (buffer solutions of caseinate and whey protein isolate of known composition). An average of five independent

Interactions of whey proteins experiments was taken for each single condition of temperature, pH and protein concentration studied. The amounts of o-la and p-lg were related to the amount of K-casein in the micellar sample. Because staining may vary from gel to gel, the whey proteins were always measured relative to the K-casein in the same sample. Because the proteins had different staining densities, their apparent ratios from the scanner were corrected by comparison with standard curves, where samples of cr-la, p-lg and n-casein were analyzed by electrophoresis at various concentrations in a range of ratios whey protein/n-casein between 0.02 and 1.2 (mg/mg). Statistical analysis Results were analyzed statistically using the SAS package @AS, 1991). The general linear model procedure was used to analyze the variance of the factors that affected the outcome variables: pH, temperature, time and their interactions.

became faster, the amounts of a-la and /?-lg per rc-casein increased rapidly, and the maximum values were reached within 20 min of the start of the heat treatment (Fig. 1). At 9O”C, p-lg reacted with the micelles considerably faster than did o-la. The interactions of both a-la and &lg with casein micelles at 75°C were slower than at 85 and 90°C. The maximum values determined at 80, 85 and 90°C were slightly lower than the ratios of whey protein/K-casein determined in raw skim milk, but it was evident that most of the available a-la and p-lg were complexed with the casein micelles. It should be emphasized also that the sum of the molar ratios of o-la and ,0-lg to Kc-caseinwas approximately 1.5 mol of whey protein per mole of K-casein. This result clearly demonstrated that the reaction was not simply between single molecules of casein and whey protein. At pH 6.2, the reaction was followed for a shorter period of time because the heat-induced interactions were faster. As was found for milk at its native pH, increasing the heating time gave increasing ratios of o-la and /3-lg to r;-casein, and maximum values were reached

RESULTS quantify the amount of serum proteins in the original skim milk, and their ratio relative to the amount of K-casein present, SDS-polyacrylamide gel electrophoresis was performed on different dilutions of unheated milk, and the protein bands were quantified by laser scanning densitometry. The areas of the scanned bands of a-la and p-lg were proportional to the protein concentration in the sample. The analysis allowed the determination of a-la/E-casein and ,&lg/K-casein ratios in the skim milk, which were 0.44 f 0.06 mg/mg for o-la/ rc-casein and 1.O f 0.09 mg/mg for &lg/K-casein.

To

51

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Effect of different pH values At the natural pH of milk (6.75-6.8), the quantity of whey protein found complexed with casein micelles increased with the time and severity of heat treatment, as shown in Fig. 1. At 75°C slow increases in the amounts of a-la and @-lg bound to the casein micelles were measured, tending towards plateau values after 35-40 min of heating. The two whey proteins seemed to have similar rates of interaction with casein micelles at this temperature, although the maximum values were lower than those found at the other temperatures studied (i.e. neither protein showed complete binding to the casein micelles at 75°C). In general, at pH 6.8, the interactions of the two whey proteins with the casein micelles were similar at temperatures below 90°C. These data seemed in disagreement with previous findings, where differential scanning calorimetry techniques show a-la to be relatively stable, because of its reversible denaturation (Ruegg & Moor, 1977). When the temperature was increased to 80, 85 or 9O”C, the reactions

10

20

30

40

50

60

70

80

Heating time [min]

Fig. 1. Weight ratio of cr-lactalbumin/K-casein (A) and p-

lactoglobulin/K-easein (B) in the casein micelles as a function of time, for heat treatment at 75 (m). 85 (0) and 90°C (A). The analyses were carried out on casein micelles isolated by ultracentrifugation from skim milk at the native pH (6.8). Results are the average of five independent experiments. Lines are drawn for guidance only.

52

44. Corredig, D. G. Dalgleish

within a few minutes of treatment (results not shown). Temperature changes did not greatly influence the behaviour of a-la, however, since its kinetics were not different within experimental error for temperatures between 75-90°C. On the other hand, the interaction of p-lg at 90°C was faster than at 80 and 85°C. When skim milk was heated at pH 6.2, the average plateau values of o-la and ,&lg found in the casein micelles were 0.4 mg/ mg for o-la/K-casein and 1.0 mg/mg for P-lg/K-casein, comparable to the whey protein/K-casein ratios found in the original skim milk. In addition, at 90°C p-lg reacted much faster than a-la, reaching a plateau after only about 3-4 min of thermal treatment. At pH 5.8, the milks tended to be unstable and a visible coagulation of the skim milk samples occurred after few minutes of heat treatment, at all of the temperatures employed. Under these circumstances, measurement of the real ratios of the whey proteins to Ic-casein in the micelles became more difficult. The milk was heated until the interaction seemed to have reached its plateau. The possibility, especially in the case of long

exposure to high temperature, of an overestimation of the quantity of whey protein interacted with casein micelles, had to be considered, because of simple entrapping and coprecipitation of denatured whey proteins in the ultracentrifuged isolate. Generally, both a-la and /?-lg in the micelles, expressed as a ratio to micellar K-casein, increased their amount with time and temperature (Fig. 2). At 75°C neither a-la nor /3-lg reached what was assumed to be the plateau of the interaction, and a-la seemed to react faster than p-lg. At this temperature, the behaviour of p-lg was different from previous observations; the protein was not detected in the micelles, because an induction period occurred during the first 15 min of treatment. This result was in agreement with some anomalous behaviour of p-lg observed by Kella & Kinsella (1988). The phenomenon may be related to a change in the equilibria, at pH lower than 6.0, between the octamer-dimermonomer forms of the protein in solution. At 8o”C, the induction stage disappeared and only ,L?-lgseemed to reach a maximum of interaction with the casein

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5

10

15

20

25

30

Heating time [min]

Fig. 2. Weight ratio of a-lactalbumin/~-casein (A) and /3lactoglobulin/K-casein (B) in the casein micelles as a function of time, for heat treatment at 75°C (m), 80°C (0) and 85°C (A). The analyses were carried out on casein micelles isolated by ultracentrifugation from skim milk at pH 5.8. .Results are the average of five independent experiments. Lines are drawn for guidance only.

0.8

0

om .rn

0

10

20

30

40

50

60

Heating time [min]

Fig. 3. Weight ratio of o-la&albumin/n-casein (A) and /3lactoglobulin/K-casein (B) in the casein micelles as a function of time, for different temperatures and pH values: pH 6.2, 85°C (0); pH 6.2, 90°C (a): pH 6.8, 90°C (H); pH 5.8, 85°C (A). The analyses were carried out on casein micelles isolated by ultracentrifugation from skim milk after heating. Results are the average of five independent experiments.

Interactions of whey proteins micelles. At 85”C, the reaction of a-la was faster than at 75 and 80°C. This contrasted with the results at pH 6.2, where the reaction rates were similar at 80 and 85°C. Furthermore, no difference was found in the overall kinetic behaviour of a-la and p-lg at temperatures up to 85°C. In agreement with the previous cases, but with a more obvious difference, at pH 5.8, the interaction of ,&lg with casein micelles was more affected by temperature changes than the reaction of cr-la. The /3-lg/K-casein ratio showed different rates of change at all three temperatures studied. By comparing the kinetics of the binding of whey proteins with casein micelles at the same temperatures for different pH values, it became clear that a similar interaction behaviour occurred at pH 6.2 and 6.8 at every temperature up to 9O”C, while faster reactions occurred at pH 5.8. At this pH, the behaviour of a-la and /?-lg was in fact different at 85°C (Fig. 3). Effect of addition of individual wla and p-lg Results of either a-la are shown formed at

the or in pH

trials carried out by adding 2 mg/ml-’ of p-lg isolates to skim milk before heating Figs 4 and 5. The experiment was per6.8 and at 8O”C, and results were com-

=

pared with a control (skim milk at native pH, heated at the same temperature). These protein preparations contained only cr-la and p-lg, but were only 80% pure, as determined by electrophoresis; the p-lg contained 20% of a-la and vice versa. Figure 4 illustrates how the addition of 2 mg/ml-* of @lg isolate to skim milk affects the interaction behaviour of a-la and p-lg with the micelles. The a-la reaction at 80°C was faster in the presence of ,fNg (Fig. 4A), but the a-la/K-casein ratio reached the same plateau value as the control. The presence of additional p-lg greatly increased the rate of reaction of the total p-lg in the milk, but again the plateau value of @lg/ K-casein did not change. The increase of the concentration of ,&lg in the skim milk was from about 3.2 mg/ ml-’ of p-lg to 5.2 mg/ml-‘. Therefore, at 80°C and pH 6.8, the additional p-lg markedly influenced the kinetics of interaction of the whey proteins, without increasing the number of protein molecules bound to the micelles. In the same way, when 2 mg/ml-’ of a-la was added to skim milk and heat treatment at 80°C was performed (Fig. 5), there were changes in the reactions of both a-la and p-lg, compared with the control. In this case, not only was the interaction of cr-la with casein micelles

0.6

0.6 t 5i E 0.7 1

a

53

A

I

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0.5 1

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0

0

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Fig. 4. Effect of

10 20 30 40 50 60 70 60 Heating time [min]

addition of 2 mg/ml-’ of @lactoglobulin to

0

10 2030405060 70 60 Heating time [min]

skim milk on the weight ratio of cr-lactalbumin/n-casein (A) and &lactoglobulin/K-casein (B) as functions of time in heated skim milk; /?-lactoglobulin added (a), control (0). Heat

Fig. 5. Effect of addition of 2 mg/ml-’ of cY-lactalbumin to skim milk on the weight ratio of a-lactalbuminln-casein (A) and @lactoglobulin/n-casein (B) as functions of time in heated skim milk; with cY-lactalbumin added (0); control (0). Heat

treatment was performed at 8o”C, pH 6.8. Results are the

treatment was performed at 8o”C, pH 6.8. Results are the

average of five independent

experiments.

average of five independent experiments.

54

A4. Corredig, D. G. Dalgleish

faster, but the amount of protein bound to the micelles was much greater, presumably because of the increased amount available. The amount of o-la present in skim milk after the addition 2 mg/ml-i of protein isolate was comparable to the concentration of ,%lg (around 3.2 mg/ml-r) in the original skim milk. If the proteins interacted in proportion to their ratios, we would expect that the a-la/K-casein ratio in the heated micelles from the milk containing added a-la would be similar to the /3-lg/K-casein ratio in the micelles from the original milk, as was in fact observed. This was expressing the results on a weight basis; on a molar basis it implies more reaction of o-la with the casein micelles. The addition of 2 mg/ml-’ a-la also caused p-lg to interact faster with the micelles, and its kinetic curve was comparable to the one measured when isolated ,&lg was added to the milk (Fig. 4B). However, the plateau ratio of p-lg to n-casein was not altered with the addition of o-la. There was no evidence that the two proteins competed with one another.

DISCUSSION Environmental changes profoundly affected the interaction behaviour of both whey proteins, and their amount complexed with the casein micelles. Increasing the time of treatment gave more extensive reactions, and higher temperatures caused faster protein-protein interactions. In addition, a faster interaction with casein micelles generally occurred at the lowest pH values, as well as an increased amount of whey protein complexed. However, the p-lg seemed to be more dependent on pH and temperature changes than a-la. Analysis of variance showed that changing pH was not significant for a-la (P = 0.13), but was significant for the denaturation of p-lg (P < 0.01). The interaction kinetics of cr-la and p-lg tended to be similar at temperatures lower than 85-90°C; it was established by analysis of variance that temperature was a significant variable for both proteins (P < 0.001). In this study, the temperature range 85-90°C appeared to be rather critical for the reactions occurring during heat treatment. It is well known from a number of studies that changes in the slope of the Arrhenius plots occur around 90°C in spite of the wide disagreement on the order of the denaturation kinetics between authors (Hillier & Lyster, 1979; Dannenberg & Kessler, 1986; Manji & Kakuda, 1986). The change in the relative behaviour of the two whey proteins with casein micelles found in this research is fully in agreement with this critical change in the Arrhenius plot slope (i.e. in the whey protein denaturation kinetics). In particular, the divergence between the behaviour of the two proteins was observed at 9O”C, where the a-la reaction was considerably slower than @-lg (Dannenberg & Kessler, 1986). At this temperature, a change in the mechanism

of the interaction might have occurred. From the original ratio of the two whey proteins in skim milk, we can see that, at low temperature, a-la interacted more efficiently with casein micelles than did p-lg, because its initial concentration in the milk is lower, yet it reacts at the same rate. The plateau values reached by a-la and /I-lg and their molar ratio with K-casein, as determined in this research, showed that more than 1 mol of whey protein per mole of K-casein was present in the micelles isolated after treatment. It is believed that P-lg/n-casein is the main complex present in the micelles after heating (Hill, 1989), but our results showed that other interactions have to occur. It is known, for instance, that cr,z-casein forms complexes with ,@lg after thermal treatment of milk (Snoeren & van der Spek, 1977), and heat-induced a-la/P-lg complexes may occur as well (Shalabi & Wheelock, 1976). The addition of fresh /I-lg to skim milk did not influence the amount of a-la complexed with the micelles (P > 0.01). On the other hand, higher concentration of o-la in skim milk caused not only a faster reaction of a-la (P < O.Ol), but also a large and almost quantitative increase in the amount of the protein bound to the casein micelles. It seems, therefore, that the presence of smaller amounts of a-la bound to casein micelles in skim milk is simply a reflection of the lower concentration in the original skim milk. Increasing the quantity of /I-lg in skim milk influenced the kinetics of its interaction with the casein micelles, but not its maximum ratio with Ic-casein. The addition of a-la also increased the rate of interaction of p-lg as had the higher concentration of p-lg. This behaviour suggests that, in the reaction with p-lg, only a limited number of reactive sites on the casein micelles are involved. Because ,&lg seemed to be more affected by environmental conditions @H and temperature) than a-la, it is possible that the reaction of /3-lg is dependent on the surface characteristics of the casein micelles. In heated skim milk, both whey proteins were found in the complex with casein micelles in ratios comparable to those present in the original skim milk (i.e. most of the whey protein present in solution interacted with the micelles). During treatment at temperatures below 90°C a soluble complex between o-la and p-lg might be postulated as an intermediate of the reaction with the micelles. The presence of whey protein complexes prior to binding to the micelles would explain the catalytic effect of a higher concentration of cr-la on the reaction of p-lg with the micelles observed in Fig. 5B. The molar ratio whey protein/K-casein observed in the normal complexes was about 1.5, therefore a complex polymerization occurs between o-la, P-lg and K-casein; and cr,+asein might play a role in the complex formation as well, being the other cysteine-containing casein in skim milk.

55

Interactions of whey proteins ACKNOWLEDGEMENTS

This project was supported by the Ontario Dairy Council and the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors thank Protose Separations Inc., Teeswater, Ontario, for providing whey protein isolates.

Kella, D. & Kinsella, J. E. (1988). Enhanced thermodynamic stability of @lactoglobulin at low pH: a possible mechanism. Biochem. J., 255, 113-18. Law, A. J. R., Banks, J. M., Horne, D. S., Leaver, J. 8~ West, I. G. (1994). Denaturation of the whey proteins in heated milk and their incorporation into cheddar cheese. Milchwissenschaft, 49, 63-7.

Manji, B. & Kakuda, Y. (1986). Thermal denaturation of whey proteins in skim milk. Can. Inst. Food Sci. Technol. J., 19, 163-6.

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Dannenberg, F. & Kessler, H. G. (1986). Reaction kinetics of the denaturation of whey proteins. In Food Engineering and Process Applications, eds M. Le Maguer 8c P. J. Jelen, Elsevier Applied Science, London, pp. 33546. Dannenberg, F. & Kessler, H. G. (1988a). Reaction kinetics of the denaturation of whey proteins in milk. J. Food Sci., 53,25843.

Dannenberg, F. & Kessler, H. G. (1988b). Thermodynamic approach to kinetics of ,&lactoglobulin denaturation in heated skim milk and sweet whey. Milchwissenschaft, 43, 13942.

de Wit, J. N. & Klarenbeek, G. (1984). Effects of various heat treatments on structure and solubility of whey proteins. J. Dairy Sci., 67, 2701-10.

de Wit, J. N. & Swinkels, G. A. M. (1980). A differential scanning calorimetric study of the thermal denaturation of bovine /I-lactoglobulin. Thermal behaviour at temperatures up to 100°C. Biochim. Biophys. Acta., 624, 4(r 50.

Haque, Z. & Kinsella, J. E. (1988). Interaction between heated n-casein and fi-lactoglobulin: predominance of hydrophobic interactions in the initial stage of complex formation. J. Dairy Res., 55, 67-80. Hill, A. R. (1989). The ,&lactoglobulin-n-casein complex. Can. Inst. Food Sci. Technol. J., 22, 12&3.

Hillier, R. M. & Lyster, R. L. J. (1979). Whey protein denaturation in heated milk and cheese whey. J. Dairy Res., 46, 9>102.

Hillier, R. M., Lyster, R. L J. & Cheeseman, G. C. (1979). Thermal denaturation of a-lactalbumin and P-lactoglobulin in cheese-whey: effect of total solids concentration and pH. J. Dairy Res., 46, 103-11. Jang, H. D. & Swaisgood, H. E. (1990). Disulfide bond formation between thermally denatured P-lactoglobulin and n-casein micelles. J. Dairy Sci., 73, 9OW.

Relkin, P. & Launay, B. (1990). Concentration effects on the kinetics of /I-lactoglobulin heat denaturation: a differential scanning calorimetric study. Food Hydrocoll., 4, 1932.

Relkin, P., Eynard, L. & Launay, B. (1992). Thermodynamic parameters of ,&lactoglobulin and cr-lactalbumin. A DSC study of denaturation by heating. Thermochim. Acta., 204, 111-21.

Ruegg, M. & Moor, U. (1977). Calorimetric study of the thermal denaturation of whey proteins in simulated milk ultrafiltrate. J. Dairy Res., 44, 509-20. SAS (1991). SASSTAT User’s Guide, SAS Institute, Inc., Cary, NC. Shalabi, S. I. & Wheelock, J. V. (1976). The role of o-lactalbumin in the primary phase of chymosin action on heated milk. J. Dairy Res., 43, 331-5. Sharma, S. K. & Dalgleish, D. G. (1993). Interactions between milk serum proteins and synthetic Fat Globule Membrane during heating of homogenized whole milk. J. Agric. Food Chem., 41, 1407-12. Singh, H. & Creamer, L. K. (1992). Heat stability of milk. In Advanced Dairy Chemistry-Z. Proteins, ed. P. F. Fox. Elsevier, London, pp. 621-56. Singh, H. & Fox, P. F. (1987). Heat stability of milk: role of /I-lactoglobulin in the pH-dependent dissociation of micellar n-casein. J. Dairy Res., 54, 509-21. Snoeren, T. H. M. & van der Spek, C. A. (1977). The isolation of a heat induced complex from UHTST milk. Neth. Milk Dairy J., 31, 352-5.

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(Received 1 May 1995; accepted 14 July 1995)