A semiautomated reflectance colorimetric method for the determination of lipase activity in milk

Measurement of heat-stable lipase activity in dairy products relies on methods that are slow or that cannot be used in turbid solutions, which limits their industrial value. A need exists for a rapid, simple, informative assay to detect lipase activity in dairy products. In this study, we observed that hydrolysis of p-nitrophenyl esters, monitored by reflectance colorimetry, was linearly correlated to spectrophotometry (R(2) = 0.93) and release of titratable FFA (R(2) = 0.92 to 0.97), indicating that chromogenic substrates were useful in determining lipase activity. However, at the concentrations reported in milk, FFA inhibited p-nitrophenyl caprylate hydrolysis, which led to an underestimation of lipase activity in milk that had previously undergone lipolysis. Milk fat also significantly reduced hydrolysis of the chromogenic substrates tested but could be accounted for by a correction equation. To demonstrate the use of the assay, lipase activity in UHT skim milk inoculated with Pseudomonas fluorescens AFT36 was followed using reflectance colorimetry and tributyrin agar. Lipase activity increased as cell numbers increased during 106 h of incubation. Extracellular lipase activity was detected after 10 h of incubation using reflectance colorimetry, but 28 h were required using tributyrin agar. Reflectance colorimetry and chromogenic substrates allowed a rapid, sensitive, and meaningful detection of esterase and lipase activity in milk.