Impact of Clostridium butyricum HADIG-CB003 dietary supplementation on the gut microbiota of Kunming mice

Clostridium butyricum is a key anaerobic probiotic due to its ability to produce butyric acid and form spores. In this study, four strains were isolated from pig feces using tryptose sulfite cycloserine (TSC) plates and specific primers sequentially. The optimal strain, C. butyricum HADIG-CB003, identified via gas chromatography and enzyme tests, produced 1.54 g/L of butyric acid, with cellulase and amylase activities of 20.88 ± 1.27 U/mL and 16.04 ± 0.46 U/mL, respectively. In reinforced clostridial medium, it produced 2.48 L of gas per liter, with hydrogen making up 59.68% (1.48 L). The adhesion rate to porcine intestinal epithelial cells (IEC-J2) was 9.16% in vitro. Twelve 3-week-old male Kunming mice were split into two groups and fed a basal diet with or without 1 × 10¹⁰ CFU (colony-forming unit)/kg HADIG-CB003 for 2 weeks. The HADIG-CB003 diet did not significantly affect body weight or the feed-to-gain ratio but reduced harmful bacteria like Salmonella enterica and increased beneficial gut bacteria, elevating the Firmicutes/Bacteroidetes ratio (p < 0.05). Moreover, the HADIG-CB003 diet significantly increased fecal butyric acid levels and decreased microbial species richness (Chao1 index) in the cecal contents of the mice. The study shows that a TSC double-layer plate, PCR screening, and 16S rRNA sequencing effectively screen C. butyricum in pig feces. Additionally, adding HADIG-CB003 to Kunming mice diets significantly changed their gut microbiota in 2 weeks.