In Vitro probiotic assessment and optimization of enhanced spore production in submerged fermentation of Bacillus toyonensis OQ071612.

Certain species within the Bacillus genus, such as Bacillus toyonensis, are widely used as commercial probiotics; however, comprehensive screening and safety evaluation remain essential because some Bacillus species, including B. anthracis and B. cereus, are pathogenic and toxin-producing. In the present study, a soil-derived isolate, B. toyonensis OQ071612, exhibited strong inhibitory activity against multiple representative human enteropathogens and was therefore selected for detailed characterization. The strain demonstrated high tolerance to harsh gastrointestinal and processing-related stress conditions, including low pH, simulated gastric fluid, simulated intestinal fluid, bile salts, and elevated temperatures. In addition, B. toyonensis OQ071612 displayed several desirable probiotic attributes, such as antioxidant activity and bile salt hydrolase (BSH) activity, while showing no hemolytic activity and minimal cytotoxicity toward Caco-2 cells. Antibiotic susceptibility testing revealed sensitivity to vancomycin, levofloxacin, sulfamethoxazole, and doxycycline, with intermediate sensitivity to azithromycin and amoxicillin, indicating the absence of acquired antimicrobial resistance associated with mobile genetic elements. Response surface methodology (RSM) was employed to optimize biomass and spore production, achieving optimal conditions at pH 6.5, 40 °C, 150 rpm agitation, and 0.26 vvm aeration, resulting in 2.7-fold and 6.5-fold increases in biomass and spore yields, respectively. Furthermore, supplementation with B. toyonensis OQ071612 significantly enhanced body mass gain in rats, supporting its potential as a probiotic candidate for animal feed and suggesting possible relevance for future human applications. Collectively, these findings demonstrate that B. toyonensis OQ071612 possesses strong probiotic functionality, favorable preclinical safety characteristics, and promising industrial scalability, supporting its potential as a probiotic candidate for animal feed applications and providing a basis for future evaluation in human-related applications.