Determination of Cheese Proteinase

Abstract

20 g. of Cheddar cheese is suspended in about 100 ml. of water with a power mixer, diluted to 200 ml., and homogenized several times. A 2.5% casein substrate suspension containing 0.02[image] Na citrate and 0.5% gum ghatti is prepd. in a power mixer. The substrate suspension is adjusted to pH 5, homogenized several times, and filtered to remove traces of undispersed casein. For the analysis, the cheese suspension and a substrate mixture consisting of 5 ml. of casein suspension, 1 ml. of 1 [image] acetate buffer at pH 5, 1 ml. of 0.015[image] cys-teine-HCl, and 0.2 ml. of toluol are warmed to 40[degree]C. 3 ml. of suitably diluted cheese suspension are added and the whole is incubated 5 hrs. at 40 [degree]C. Before and after incubation, the incubation mixture is homogenized and 1-ml. aliquots are removed for non-protein N analysis. One cheese proteinase unit is the amt. of enzyme liberating 1 [gamma] of non-protein N per 10.2 ml. of incubation mixture under the conditions described above. Casein hydrolysis is directly proportional to cheese proteinase when < 700 units are present. The Cheddar cheese proteinase system hydrolyzes casein most rapidly at pH 5 with a secondary optimum at pH 7-8. Only the pro-teolytic activity at pH 5 is important in ripening Cheddar cheese (pH 5-5.4). Proteolytic activity at pH 5 is enhanced in the presence of reducing agents, particularly cysteine.