THE ROLE OF MICROBIAL PROTEASE AND PHOSPHOLIPASE IN LIPOLYSIS OF RAW MILK (PSYCHOROTROPHS, PH-STAT MEASUREMENT

The effect of extracellular protease from Pseudomonas sp. and phospholipase from Bacillus cereus on lipolysis of washed cream at 37 and 7(DEGREES)C was studied. The preliminary work on the use of pH-stat titration for measuring the rate of lipolysis in presence of protease led to the development of a pH-stat titration method for measuring protease activity. This method is useful for measuring the activity of alkaline proteases with optimum activity at or above pH 9 in crude or purified enzyme preparations. The pH-stat titration method of proteolysis detection was compared with the trinitrobenzenesulfonic acid method. The activity measurements of Bacillus amyloliquefaciens purified protease using the two methods were highly correlated. The pH-stat titration method is simple, more rapid and more sensitive than the trinitrobenzenesulfonic acid method for measuring proteolysis. The pH-stat titration method gave higher protease activity measurements for Pseudomonas spp. culture filtrates which have protease more active at pH 9 than pH 7.5.;The pH-stat titration was used to measure the rate of lipolysis in the presence of protease. In this study, the pH-stat titration was not useful for measuring the rate of lipolysis in presence of phospholipase.;The rate of lipolysis of washed cream increased with the time of preincubation with protease at pH 9 and 37(DEGREES)C. Lipolysis rate and degree of proteolysis were highly correlated (R('2) = 0.89). Preincubation of washed cream with phospholipase (0.34 unit/ml) at pH 7.35 and 37(DEGREES)C did not affect the rate of lipolysis. Incubation of washed cream with lipase and protease, phospholipase or both protease and phospholipase at pH 6.65 and 37(DEGREES)C increased lipolysis significantly. The presence of protease resulted in a decrease in the rate of increase lipolysis. This could be due to lipase hydrolysis by the microbial protease. The increase in free fatty acid concentration due to the combined effect of the protease and phospholipase during the first 6 h of incubation was higher than the sum of the increases due to each enzyme. Protease and phospholipase inhibited lipolysis in washed cream incubated at pH 6.65 and 7(DEGREES)C. Lipolysis in washed cream incubated with only lipase at 7(DEGREES)C was higher than expected, possibly a result of the disruption of the milk fat globule membrane at low temperature.