The investigation was undertaken to examine the extraction method of cheese lipase embedded firmly in the texture of ripened Cheddar chuese, and to clarify some properties of cheese lipase with a quantitative method which is used to determine weak activities. Micro-determination of free fatty acids liberated enabled us to determine the weak activity of the cheese lipase. Extraction of the lipase from the cheese should be performed from portions of cheese curd and fat separately. Amount of oleic

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... acid from triolein increased linearly with incubation time up to 16 hours. Optimum pH's of the cheese and the pancreatic lipases during lipolysis were found to be 7.0 and 8.0, respec tively. Relationship between substrate concentrations and lipase activity was expressed distinctly as Michaelis-Menten's plot, and estimation of Km value did indicate 0.09%, propriate period remarkably accelerated inactivation of the enzyme. Elution patterns by gel filtration with Sephadex G-200 of the concentrated enzyme solution showed the separation into 4 components and higher activities of lipase were found to be present in III fraction than the others. All proteins in the cheese lipase, the pancreatic lipase and III fraction obtained by gel filtration were separated on the polyacrylamide disc gels by electrophoresis. Lipase proteins on the gels were identified by lipolysis against synthetic substrates, naphthol-AS-nonanoate and acetate followed by staining of the gels with Fast blue BB salt. Formation of free fatty acids from milk fat during Cheddar cheese ripening is well known to be essential for the production of typical cheese flavor. A few reports1-3) have indicated that lipolysis in cheese resulted from the lipolytic action of cheese bacteria, in particular, lactic acid bacteria. It was also shown that starter bacteria and lactobacilli prevailing during the relatively late periods of cheese ripening enabled their lipases to play a role in formation of free fatty acids. These bacteria have the capacities to hydrolyze very weakly milk fats by their own lipases as reported recently4-6). At the present, the degradative enzymes of cheese bacteria participated in breakdown of milk protein and fat are considered to be divided into intracellular enzymes and those stripped from periplasmic regions by bacteriolysis. Extraction of the latter enzymes is expected to be difficult since those enzymes are firmly embedded in cheese textures. Therefore, it is important to confirm the procedure for the isolation of the hydrolytic enzymes set firmly in cheese. According to the finding with regard to lipase in Cheddar cheese7), activities of the enzyme extracted were very low at the starting of cheese ripening, but increased gradually with the rip-Jap. J. Zootech. Sci., 48, (12): 731-740.