Quantitative polyacrylamide gel electrophoresis has been used to measure residual native whey proteins remaining after heat treatment of skim-milk and cheese whey in a kinetic study. The denaturation of a-lactalbumin (a-la) appeared to be first order, but was probably a second-order reaction displaying pseudo firstorder kinetics. The denaturation of both yff-lactoglobulin A and B (/MgA and /MgB) followed second-order kinetics while that of serum albumin was more complex, and could equally well be described as first or second order. Equations are given relating log k x (in s-1 ) to temperature for a-la denaturation in skim-milk between 70 and 95 °C and between 100 and 150 °C. Similarly, equations relating log k 2 (in lg-1^-1 ) to temperature are given for /MgA in skim-milk between 100 and 150 °C, and for yff-lgB between 95 and 150 °C. The relative heat stability of /MgA and /MgB was found to vary.Below 95 °C /MgA appeared slightly more thermostable than /MgB in skim-milk, and the same was observed in cheese whey below 100 °C. Above these temperatures yff-lgB appeared more stable than /MgA. Denaturation of /MgB was only slightly more rapid in skim-milk than in whey at temperatures below 95 °C, but was significantly slower at higher temperatures.The recovery of residual protein from cheese whey is important to the dairy industry, and the denaturation of these whey proteins is therefore of considerable interest. However, the many studies which have been reported are often not directly comparable because of the variation in experimental technique and in the means used to express results. Kinetic data on rates of denaturation are rarely available, and then are not always in agreement. Lyster (1970), using immunochemical techniques, has measured the rates of denaturation of a-lactalbumin (a-la) and /Mactoglobulin (/?-lg) in heated milk, and found a-la to follow first-order kinetics, while /Mg followed second-order kinetics. Other workers have reported the denaturation of y#-lg as first order (Gough & Jenness, 1962). A rapid accurate measure of individual native whey proteins in complex mixtures is possible using polyacrylamide gel electrophoresis (PAGE) (Hillier, 1976;Darling & Butcher, 1976). This technique has been used for a comprehensive study of the kinetics of denaturation of the major proteins in milk and cheese whey over a wide range of temperatures.
SummaryA method is described for the quantitative estimation of a mixture of whey proteins by spectrophotometric scanning of the stained protein bands following polyacrylamide-gel electrophoresis. The incorporation of β-lactoglobulin A as an internal standard improves the accuracy of the technique. The method has been used to measure residual native whey proteins in milk after heating. Results are presented for whey protein denaturation in skim-milk after heat treatment at 130 and 140 °C for various periods of time.
Whey protein preparations isolated from cheese whey form stable gels when their aqueous solutions are heated at 80°C. The gels consist of polypeptide chains crosslinked by disulphide bonds. They dissolve if a sulphydryl (-SH) reagent is added after heating; their formation is retarded if compounds that react with -SH groups are added before heating. Opaque gels are formed from powders containing relatively large amounts of total -SH; clear gels form from those with low total -SH. Gels form faster on heating in Nz instead of air. Gels form more slowly at alkaline pH; it is suggested that this effect is caused by electrostatic repulsion between protein molecules with like charges.
Measurements of residual native protein remaining after heat treatment of cheese whey have been made using quantitative polyacrylamide gel electrophoresis. The results have been expressed in terms of kinetic constants. The effect of concentration was investigated up to 3 times the normal total solids content, dialysis treatments were used to study the effect of non-protein constituents, and the effect of pH was studied at pH 4, 6 and 9. The results indicated that increased total solids (TS) concentration slowed the denaturation of /9-lactoglobulin A and B (/?-lg A, yff-lg B) but hastened the denaturation of a-lactalbumin (a-la). However, increased lactose concentration slowed the denaturation of both a-la and /ff-lg, perhaps by preventing formation of heat-induced complexes. Increased Ca concentration, up to 0-4 mg/ml, tended to slow the denaturation of both proteins, but further increase in Ca up to 0-9 mg/ml produced little effect. The rate of denaturation of both a-la and /#-lg was slower at pH 4 than at pH 6 or 9, and was probably slowest at the isoelectric point. However, not all the changes associated with pH could be explained in terms of net molecular electrostatic charge. The genetic variants of '/?-lg showed different heat stabilities -below 90 °C /?-lgA was more stable than /MgB, but above 90 °C the situation was reversed at all TS concentrations, and pH 6. However, at pH 4 and 9, yff-lg A was less stable than /?-lgB over the entire temperature range at normal concentration.
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