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

Fluorescein isothiocyanate-labelled casein was evaluated as a substrate for measuring bacterial protease activity in milk. Using protease from Bacillus amyloliquefaciens , effects of substrate 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 10⁸ 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.