The cultures used were isolates of proteolytic Pseudomonas spp., P. fluorescens isolates 65 and 117 and P. fragi isolate 102 (Craven 1993). Theywere grown inUHTskim milk for 8 days at 4C with circular agitation at 150 rpm. The cell concentration was approximately 106 cells/mL. Following incubation, the culture was centrifuged at 3,000 g for 10 min at 4C. The supernatant was then filter-sterilized by passing through a 220-nm filter and used as the source of crude protease.
Cultures and Preparation of Crude Protease