Probiotic characterization of <i>Bacillus subtilis</i> Strain BS-Q and its cell-free-supernatant-mediated inhibition of <i>Clostridium perfringens</i>

This study characterized Bacillus subtilis (B. subtilis) BS-Q as a novel probiotic candidate against Clostridium perfringens (C. perfringens). Polyphasic analysis, including 16S rRNA gene sequencing (>99% similarity to reference B. subtilis strains), identified the isolate as BS-Q. It exhibited robust growth, and sporulation was observed. Its cell-free supernatant (CFS) demonstrated potent, broad-spectrum inhibition against C. perfringens types A, C, and G with a minimum inhibitory concentration (MIC) of 33.06 ± 0.53 μg/mL. The antibacterial activity was localized to the CFS and showed remarkable stability after exposure to harsh conditions: high temperature (100°C for 30 min), a broad pH range (3.0-11.0), and high salinity (up to 10% NaCl). Protease treatment markedly reduced the activity, confirming the proteinaceous nature of the antimicrobial compounds, which was supported by the presence of unique protein bands (25-35 kDa) in the CFS as revealed by SDS-PAGE. In conclusion, B. subtilis BS-Q produces highly stable, proteinaceous antimicrobials effective against C. perfringens. Its strong anti-clostridial activity and resilience position it as a promising probiotic and a potential antibiotic alternative for controlling necrotic enteritis in poultry.