Anaerobic Germination and Bacteriocin-like Inhibitory Substance (BLIS) Production by Bacillus subtilis BSC35 Enable Inhibition of Clostridioides difficile and Clostridium perfringens

Bacteriocin-like inhibitory substances (BLIS) from spore-forming probiotics are promising natural antimicrobials for food safety and gut microbial management. While BLIS produced by Bacillus subtilis BSC35 has previously been shown to inhibit Clostridium perfringens, the production and activity of BLIS from spore-initiated cultures remain unexplored. In this study, we first evaluated the antibacterial activity of a bacteriocin-like inhibitory substance (BLIS BSC35) produced by B. subtilis BSC35 against Clostridiodes difficile of multiple ribotypes. Cell-free supernatants (CFS) of BSC35 exhibited consistent and quantifiable inhibition zones (≥18 mm) in well diffusion assays and inhibited bacterial growth. Transmission electron microscopy revealed bactericidal effects, including disruption of cell walls, membrane separation, and leakage of cytoplasmic contents. Secondly, we show that B. subtilis BSC35 efficiently produced heat-resistant spores under both aerobic and anaerobic conditions. And finally, CFS from spore-derived cultures retained antibacterial activity against C. difficile and C. perfringens. These findings demonstrate that spore-initiated B. subtilis BSC35 produces stable BLIS with broad inhibitory activity against the two clostridial species, supporting its potential application as a probiotic or bioprotective culture in food matrices and gastrointestinal contexts.