Kinetics of changes in plasmin activity and proteolysis on heating milk

Heat-induced inactivation of bovine plasmin, denaturation of β-lactoglobulin (β-lg), the interactions between both species and casein micelles and the subsequent net effect on proteolysis of β-casein was studied in a model system consisting of phosphocasein and β-lg in synthetic milk ultrafiltrate. The inactivation of plasmin and denaturation of β-lg were first order reactions, with the rate of inactivation of plasmin being greater than the rate of denaturation of β-lg. The predominant mechanism involved in the denaturation of plasmin in the temperature range 65-80°C was its interaction with β-lg (k^r at 60°C, 0.0526; E_a, 176 KJ/mol). At the point of complete inactivation of plasmin ~45% of the β-lg remained undenatured. Thermal inactivation of plasmin through other mechanisms was negligible. The association of β-lg with the casein micelles at 60°C had a rate constant of 3.71×10 ^-5 min^-1 and an E_a of 259 KJ/mol; thermal denaturation of β-lg was of much less importance, with a rate constant at 60°C of the order of 1×10^-10 min^-1 and an E _a of 250 KJ/mol. On denaturation of all β-lg in the system, a maximum of ~55% was associated with the casein micelles. The effect of heating on the subsequent hydrolysis of β-casein indicated that the level of plasmin activity was the most important factor affecting proteolysis, while the interaction of β-lg with the casein micelles had limited effect. Overall, thermal stability of plasmin in milk is very much dependent upon its interaction with β-lg.