Enhanced production of extracellular protease from Bacillus subtilis 9 in shake-flask fermentation

Microbial proteases, due to their enzymatic capabilities, have a significant impact on several industrial applications, including animal health and nutrition. Bacillus spp. are known for their ability to secrete extracellular proteases, which help improve nutrient digestion leading to greater animal growth performance and overall health. This study utilizes a previously isolated Bacillus subtilis 9 strain to produce extracellular protease in shake-flask fermentation using minimal media supplemented with peptone (PM), wheat bran (WB), extruded soybean (ESB), and soybean meal (SBM). The freeze-dried powder of cell-free supernatant showed maximum protease activity of 12,102 U/g when ESB-based media was used, followed by media supplemented with WB (9,171 U/g) and PM (8,383 U/g) after 48 hours of fermentation (37°C and 150 rpm). Replacing a regular flask with a baffled flask led to a 7-fold increase in protease activity (12,102 U/g vs. 89,755 U/g) under the same fermentation conditions. Attempts to optimize protease production were made by replacing ESB with SBM at varying percentages between 0.5% and 4%. This resulted in a 345% rise in protease activity after 48 hours of fermentation, with the most effective concentration being 1% SBM. Further optimization, specifically extending fermentation time (36–72 hours), yielded a 15% increase at 60 hours of fermentation while maintaining SBM concentration at 1%. After a four-hour reaction time, the analysis of freeze-dried, unpurified protease showed 3-fold less free amino acid nitrogen (FAN, ppm) compared to purified protease (1422 vs. 3833). Large-scale production and purification processes are sought to improve protease activity and efficacy, rendering production cost-effective. This study provides valuable insights into optimizing Bacillus subtilis 9 protease production, paving the way for its efficient utilization in supporting animal health.