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.