Characterization of bacteriocin, probiotic attributes and genomic information of Bacillus subtilis S1

This study investigated the probiotic potential of Bacillus subtilis strain S1, isolated from fermented chilli pickle. Genome sequencing revealed a genome size of 4.48 Mb, 42.6% GC content, and 4,969 protein-coding sequences, including genes linked to probiotic activity. In vitro assays confirmed that strain S1 displayed strong aggregation, antioxidant activity, hydrophobicity, and tolerance to acid, bile, and simulated gastrointestinal conditions. It showed antibiotic resistance within safe limits and passed hemolytic and fibrinolytic safety assessments. Notably, strain S1 remained stable at alkaline pH and in up to 6.0% saline. Functional characterization showed that the strain produces antimicrobial metabolites with strong inhibitory activity against a wide range of food-borne pathogens. The metabolite remained stable up to pH 8.0 and 50℃. Enzymatic treatment efficiently identified the substance as protein-based. Purification and mass spectrometry identified the metabolite as Subtilosin A, which demonstrated promising antibacterial activity against Listeria monocytogenes. FE-SEM analysis provided insight into the mechanism of action of Subtilosin A. B. subtilis S1 whole genome sequence analysis confirms its uniqueness in possessing three different bacteriocins, such as Subtilin, Subtilosin A, and Bacillibactin. Subtilosin A and previously reported sequence of Subtilosin A showed no genetic variations. These findings indicate that the isolated S1 strain possesses genetic traits associated with probiotic properties, as confirmed by experimental evidence. This study is novel for isolating, identifying, and sequencing bacteriocins from a traditional fermented chilli pickle, thereby reporting the first Bacillus spp. This represents a major advance in microbial biopreservation, enabling safer, germ-free foods.