Milk-clotting protease enzyme producing bacteria from soils in Ethiopia

Abstract

Rennet (acidic proteases) are used to be traditionally extracted from animals as commercial sources of milk clotting (MC) enzymes for cheese production. However, the ever increasing demand of these enzymes necessitates search for alternative sources from fungi and bacteria. In this study, 49 bacterial strains were screened for milk-clotting (MCA) and protease (PA) activities in vitro using plate assay technique and further studied under Solid-state fermentation (SSF) and submerged fermentation (SmF). The result showed that 14 (29%) bacterial strains produced MCA on Skim milk agar forming clear zone diameter between 5.25 mm and 21 mm. Most of these MC bacterial strains were identified into the genus Bacillus; showing 100% sequence similarity with Bacillus tequilensis KCTC 13622, Bacillus subtilis subsp. subtilis NCBI 3610, Bacillus paramycoides NH24A2, Bacillus siamensis KCTC 13613. All of the strains induced MCA on SmF, and only 11 (79%) of the strains produced the enzyme under SSF. The strains produced enzymes on SmF ranging from 133.33 U/mL to 480 U/mL with MCA: PA ratio of 0.1–0.36. The MCA significantly increased during culture profiling upon partially optimized conditions with a maximum MCA production of 2533 U/mL and MCA/PA ratio of 14.05 recorded from Bacillus subtilis SMDFS 2B, which is a remarkable characteristic in the selection of commercially important rennet enzyme. Thus, Bacillus subtilis SMDFS 2B, together with B. siamensis 29B, can be further studied for enzyme purification under different optimization conditions to fully realize their potential for rennet enzyme production.

Keywords/phrases: Bacillus, Milk-clotting activity, Milk-clotting protease, Submerged fermentation