Measurement of microbial protease activity using a pH-stat titration

The activity of purified and crude microbial proteases was measured by titration at pH 9 using an automatic pH-stat instrument. The ability of the pH-stat titration method and the trinitrobenzenesulfonic acid (TNBS) method to detect protease activity was compared. The pH-stat titration produced higher measurements of activity than the TNBS method when purified protease from Bacillus amyloliquefaciens was tested. Protease activity determination using the two methods resulted in a linear correlation of R2 = 0.985 and the same repeatability (C.V. = 2.6%). The pH-stat titration method was more sensitive than the TNBS method in measuring activity of the purified protease, and it indicated greater proteolytic activity than the TNBS method when culture filtrates from five Pseudomonas spp. which produced proteolytic enzyme with greater activity at pH 9 than pH 7.5 were tested. When culture filtrates from four Pseudomonas spp. with similar but low proteolytic activity at pH 9 and 7.5 were tested, the two methods produced similar results. For proteases with optimum activity at or above pH 9, the pH-stat titration method was simplier, faster, and more sensitive than the TNBS method in determining activity

Lipolytic enzymes and hydrolytic rancidity

Lipolysis, the enzymic hydrolysis of milk lipids to free fatty acids and partial glycerides, is a constant concern to the dairy industry because of the detrimental effcts it can have on the flvor and other properties of milk and milk products. However, free fatty acids also contribute to the desirable flavor of milk and milk products when present at low concentrations and, in some cheeses, when present at high concentrations. The enzymes responsible for the detrimental effects of lipolysis are of two main types: those indigenous to milk, and those of microbial origin. The major indigenous milk enzyme is lipoprotein lipase. It is active on the fat in natural milk fat globules only after their disruption by physical treatments or if certain blood serum lipoproteins are present. The major microbial lipases are produced by psychrotrophic bacteria. Many of these enzymes are heat stable and are particularly significant in stored products. Human milk differs from cows' milk in that it contains two lipases, a lipoprotein lipase and a bile salt-stimulated lipase. The ability of the latter to cause considerable hydrolysis of ingested milk lipids has important nutritional implications