Milk-Clotting Protease From Bacillus stercorisNCCP-3139: A Potential Microbial Rennet for Cheese Production

Area of Science:

• Microbiology
• Enzymology
• Food Science

Background:

• Traditional cheese making relies on calf rennet, facing supply and ethical challenges.
• Microbial milk-clotting enzymes (MCEs) are sought as sustainable alternatives.
• Soil bacteria are a rich source of novel enzymes with industrial applications.

Purpose of the Study:

• To isolate and characterize a milk-clotting protease from a soil bacterium.
• To evaluate its potential as a rennet substitute in cheese production.
• To determine the enzyme's biochemical properties and optimal activity conditions.

Main Methods:

• Isolation and identification of *Bacillus stercoris* NCCP-3139 using morphological, biochemical, and 16S rRNA gene sequencing.
• Enzyme purification via ammonium sulfate precipitation, dialysis, ion-exchange, and gel filtration chromatography.
• Assessment of milk-clotting activity, casein hydrolysis, and enzyme characterization (pH, temperature, metal ion effects).

Main Results:

• A milk-clotting protease was purified from *B. stercoris*, exhibiting significant caseinolytic and milk-clotting activities.
• The enzyme showed optimal activity at 55°C and pH 9.0, with enhanced performance in sheep and buffalo milk.
• Purification significantly increased specific activity and the milk-clotting-to-proteolytic activity ratio, indicating high selectivity.

Conclusions:

• The purified *B. stercoris* protease demonstrates potent milk-clotting capabilities and high catalytic selectivity.
• This microbial enzyme is a viable and promising alternative to animal rennet for industrial cheese production.
• Further research can explore its application in various dairy processes.