Detection of Heat-Resistant Proteases Produced by Psychrotrophs in Refrigerated Milk

Proteases from four species of Pseudomonas were added to pasteurized milk at .18, 1.8, and 18 units of protease activity per milliliter followed by incubation at 5 C for 18 days. During this period milks were analyzed every 2 days for proteolysis by methods of Hull, ninhydrin, and absorption at 280 nm. Sensory analysis was done twice weekly for 18 days. There were no significant differences noted for protease activity when ninhydrin and absorption at 280 nm were used; however, only 18 units protease/ml were detected by the Hull test. Sensory evaluation correlated with the Hull test data. When 50 and 500 cells/ml were added to milk, a curvilinear increase in proteolysis was noted by 11 and 13 days, respectively, for three of the four Pseudomonas species as detected by the Hull method. Heat inactivation of the proteases at 62.8°C for 30 min was 0 to 73.1% and 10 to 36% as measured by the Hull and agar diffusion methods, respectively.