Characterization and Evaluation of Probiotic Potentials In Vitro of Bacillus Strains Isolated From Traditional Thai Natto

Traditional Thai natto is one source of Bacillus spp. Due to their spore-forming ability, Bacillus spp. are considered promising probiotic candidates capable of surviving harsh environmental condition. This study aimed to genetically identify and characterize Bacillus strains isolated from traditional Thai natto and evaluate their probiotic potential, including survival ability under various acidic pH levels, bile salt concentrations, and electrolyte monogastric system model (EMSM). Antibacterial activity and safety assessments including hemolytic activity and antibiotic susceptibility were also evaluated. More than 10 colonies were found in the samples. Only 3 isolates were selected based on Bacillus morphology. The isolates displayed biochemical features consistent with Bacillus and were identified with 16S rRNA sequencing, showing high sequence similarity to: B. subtilis CMI 7 (99.86%), B. subtilis CMI 9 (99.72%), and B. cereus CMI 10 (98.91%). All isolates exhibited key probiotic traits and showed statistically significant differences in survival rates under acidic pH (over 62.35±4.50% at 4 h; and over 51.61±5.08% at 24 h), bile salt concentrations (over 93.60±2.42% at 4 h; over 82.23±3.90% at 8 h; and over 39.65±3.02% at 24 h), and EMSM conditions (over 73.08±3.18% at 4 h). Isolates CMI 7 and CMI 9 exhibited antagonistic activity against S. aureus ATCC 9144 and strain DMST 2933 and E. coli ATCC 25923, demonstrating non-hemolytic and antibiotic-sensitive features. In contrast, isolate CMI 10 lacked antagonistic activity, exhibited β-hemolysis, and showed resistance to several antibiotics, representing a significant safety concern. This study suggests that B. subtilis CMI 7 and CMI 9 are probiotic candidates with safety profiles. Further study should be conducted to explore other biological properties of these Bacillus spp.