Evaluation of a fluorimetric assay on the lipases from strains of milk psychrotrophic bacteria

Abstract

SummaryA fluorimetric assay, developed for the assay in milk of the lipase of the raw milk psychrotrophPseudomonas fluorescensAR11 (Stead, 1983), was evaluated in both buffer and in milk using cell-free lipase preparations from 11 strains of lipolytic psychrotrophic bacteria isolated from milk. Lipases of all strains were more active against 4-methylumbelliferyl nonanoate (4-MUN) than against 4-methylumbelliferyl oleate (4-MUO) in the buffer system but most were less active against 4-MUN than against 4-MUO in the milk assay system. In both systems, 4-MUO had a much lower rate of non-enzymic hydrolysis than did 4-MUN. Lipase activities measured by radial diffusion in tributyrin agar and trioctanoin agar reflected activities against 4-MUO and 4-MUN, except that lipase from a strain of coliform was relatively more reactive against the 4-MU esters. When compared with assay in the buffer system the most effectively activated lipase in the milk system was that from AR11; of those from other strains, 6 were 50–69% as effectively activated, 2 were about 17% and 2 were 7–8% activated. This difference in behaviour was related to the direct inhibition of lipases of some of the strains by the mixture of sodium taurocholate (NaTC) and cetyltrimethylammonium bromide (CTAB) needed in the milk assay system to dissociate lipase from milk protein. Increasing the proportion of NaTC to CTAB increased the activities of the more weakly activated lipases but decreased those of others. The assay in milk may therefore underestimate the lipase from certain strains of psychrotrophic bacteria.