Comparative Evaluation of Spores and Vegetative Forms of Bacillus subtilis and Bacillus licheniformis on Probiotic Functionality In Vitro and In Vivo

The probiotic effects of Bacillus are strain-specific and dependent on both spore and vegetative forms, but the distinct contributions of these forms to probiotic functionality are not well understood. This study aimed to evaluate and compare the impacts of vegetative forms and spores of Bacillus subtilis and Bacillus licheniformis on probiotic functions in vitro and in vivo. We systematically assessed the anaerobic metabolic capabilities and the potential to enhance the intestinal barrier function of four Bacillus strains, leading to the selection of Bacillus subtilis X22 and Bacillus licheniformis N-3 for detailed investigation. Utilizing in vitro fermentation with murine fecal microbiota, we observed that the spores form of Bacillus licheniformis N-3 noticeably positively regulated the gut microbiota under anaerobic conditions. Concurrently, both spore and vegetative forms of Bacillus licheniformis N-3 and Bacillus subtilis X22 demonstrated the ability to prevent pathogen adhesion, reduce inflammation, combat oxidative stress, and promote cellular autophagy to reduce apoptosis in response to enterotoxigenic Escherichia coli (ETEC) infection in the IPEC-J2 cell model. As a facultative anaerobe, Bacillus licheniformis N-3 exhibited a tendency toward superior regulatory capacity in enhancing the anti-infective activity of IPEC-J2 cells in vitro. In the pathogens challenge mouse model, B. licheniformis N-3 effectively preserved the integrity of jejunal tissue and enhanced the expression of glycoproteins in goblet cells. Moreover, B. licheniformis N-3 strengthened the epithelial barrier by increasing the levels of Occludin and Claudin-1 in the jejunum, thus promoting overall intestinal health. This research offers new insights into strain selection and the life cycle utilization of Bacillus probiotics.