Casein, Fluorescein Isothiocyanate as a Substrate for Measuring Protease Production by Psychrotrophic Bacteria

Abstract

Fluorescein isothiocyanate-labelled casein was evaluated as a substrate for measuring bacterial protease activity in milk. Using protease from Bacillus amyloliquefaciens, effects of sub-strate type, pH, temperature and enzyme concentration were determined. The procedure was sensitive to .012 unit of enzyme activity. The procedure was then used as a method to detect protease production by psychrotrophic bacteria growing in pasteurized skim milk at 7°C for 5 d. Three psychrotrophic bacteria were added at three different initial populations. Bacterial population , protease activity and cumulative free amino groups were monitored daily. Bacterial populations were not correlated with either protease activity or cumulative free amino groups. Pro-tease activity and cumulative free amino groups were correlated (r=.46, p=.001). Both protease activity and cumulative free amino groups peaked as the microorganisms entered the late log phase (approximately 10 8 colony forming units per ml). This occurred after incubation for 2 d at 7°C in two replications and after 3 d in the third. Both values decreased with continued storage. The trend toward extended storage of raw milk at refrigerator temperatures before processing favors growth of psychrotrophic microorganisms. Attention has been fo-cused on extracellular proteases produced by these organisms , which can survive ultra high temperature (UHT) processing of milk (1,6,8,11). These enzymes may result in significant reduction in the shelf-life of these products by hydrolyzing the milk proteins. Gillis et al. (6) found that when sensory evaluation, standard plate count, psychrotrophic bacteria count, pro-teolytic bacteria count, Hull test and trinitrobenzene-sul-fonic acid test (TNBS) were used to monitor shelf-life of milk following UHT processing, only sensory evaluation and the two protease assays gave useful data. This and previous research (9) indicate that knowledge of initial protease activity in raw milk may be useful in predicting keeping ability of milk before UHT processing. Fairbairn and Law (5) reviewed production, properties, effects and control of proteases from psychrotrophic 'Current address: