Functional and Genomic Insights into Clostridium butyricum MCC0233: Probiotic Features and Safety Assessment

The growing limitations of conventional probiotics have driven interest in next-generation probiotics (NGPs), with Clostridium butyricum emerging as a promising candidate due to its spore-forming nature, metabolic adaptability, and therapeutic potential. This study presents a comprehensive characterization of C. butyricum MCC0233, focusing on its probiotic functionality, genomic attributes, and safety profile. In vitro assays revealed significant resilience of MCC0233 to gastrointestinal stress, retaining 20% viability after 2 h in simulated gastric fluid and 87.9% after 6 h in simulated intestinal fluid. MCC0233 demonstrated strong adhesion to intestinal epithelial cells, 143 ± 4 bacterial cells per 100 HT-29 cells and 241 ± 1 bacterial cells per 100 Caco-2 cells. The strain exhibited high antioxidant activity (82.91%) and significant cholesterol assimilation (67.02%), which is the first report of this property in C. butyricum. It also showed broad-spectrum antimicrobial activity and effective co-aggregation with various enteric pathogens. The whole-genome sequencing revealed a 4.62 Mbp genome with 4,151 protein-coding genes and a GC content of 28.8%. Functional annotation identified genes linked to probiotic traits, including those involved in butyrate production, biosynthesis of branched-chain and aromatic amino acids, and folate (vitamin B9) synthesis. Comparative genomics highlighted strain-specific features contributing to its functional potential. Importantly, both in vitro and in silico safety assessments confirmed the absence of transferable antimicrobial resistance genes, virulence factors, or plasmids. These findings establish C. butyricum MCC0233 as a robust, safe, and multifunctional next-generation probiotic candidate, with promising applications in gut health, metabolic regulation, and microbial therapeutics.