Heat Stability of Residual Milk Clotting Enzymes in Cheese Whey

Eliminating calcium chloride and replacing whole casein with k-casein eliminated clouding and sharpened diffusion boundaries in casein-agar gels used for the estimation of residual milk clotting enzymes in curd and whey. It also eliminated the need for a highly purified calcium-free agar. The substrate contained .54 percent k-casein, 3.6 percent sodium acetate, .73 percent bacto-agar and 95.13 percent water. The pH was adjusted to 5.9 with .lN hydrochloric acid. Proteases derived from Mucor pusillus var Lindt, Mucor Miehei and Endothia parasitica, as well as rennet, procine and bovine pepsins were used at recommended levels to set milk for Cheddar cheese manufacture. Whey samples from each lot were taken at draining and adjusted to pH 5.2, 5.6, 6.0, 6.2, 6.6 and 7.0 prior to being heated to 68.3C, 71.1C and 73.9C for .25, .5, 1, 5, and 10 minutes. Enzyme activities were assayed before and after heating. Mucor miehei protease was the most heat stable at all pH values followed by Mucor pusillus protease, rennet, bovine pepsin, E. parasitica protease and porcine pepsin. The heat stability of all enzymes except E. parasitica protease decreased with increasing pH, E. parasitica protease decreased with decreasing pH. All enzymes were inactivated at the minimum heat treatment at pH 7.0 except E. parasitica protease which was most stable at that pH. At pH 5.2 M. miehei protease persisted after a 10 minute treatment at 73.9C