Investigating the Neurotoxic Threat Associated with Bacillus pumilus Probiotic Strains

Background and Objective: Probiotics offer multiple benefits, including improved gut health, immunity, productivity, and reduced antibiotic resistance, contributing to food security and environmental health. Although Bacillus pumilus has been reported to exhibit both antibiotic and probiotic characteristics, its functional mechanisms remain poorly understood, and its hypothetical proteins are largely unexplored. This study aimed to assess the probiotic potential of Bacillus pumilus strain D5 and to evaluate the physicochemical properties, interleukin-2 (IL-2)-inducing capacity, and safety of its protein-coding hypothetical proteins. Materials and Methods: The genome of Bacillus pumilus strain D5 was analyzed using in silico approaches to predict probiotic traits. Protein-coding hypothetical proteins were characterized for physicochemical properties and IL-2-inducing potential. Computational safety assessments were conducted to identify possible virulence or toxin-related features. Descriptive and comparative analyses were performed to determine the strain’s probiotic potential and the functional relevance of its proteins. The top 25 IL-2-inducing peptides from each protein were analyzed using one-way ANOVA, and mean differences were determined by Duncan’s Multiple Range Test at a 0.05 significance level. Results: The strain exhibited probiotic potential in 59% of evaluated parameters. All peptides derived from the hypothetical proteins demonstrated IL-2-inducing capacity, indicating possible immunomodulatory effects. The proteins showed stability under harsh environmental conditions, supporting their adaptability. However, two proteins were identified as potential bacterial toxins, including one with neurotoxic properties, suggesting possible safety risks. Conclusion: This study provides new insights into the functional roles and safety profile of hypothetical proteins in Bacillus pumilus strain D5. While the strain exhibits promising probiotic characteristics, the identification of toxin-related proteins raises safety concerns that must be addressed before its application as a feed additive or therapeutic agent. Further experimental validation is recommended to ensure safe and sustainable use.