Previous studies have shown that Bacillus subtilis natto affects rumen fermentation and rumen microbial community structure, which are limited to detect a few microbial abundances using traditional methods. However, the regulation of B. subtilis natto on rumen microorganisms and the mechanisms of microbiota that affect rumen fermentation is still unclear. This study explored the effects of live and autoclaved B. subtilis natto on ruminal microbial composition and diversity in vitro using 16S rRNA gene sequencing and the underlying mechanisms. Rumen fluid was collected, allocated to thirty-six bottles, and divided into three treatments: CTR, blank control group without B. subtilis natto; LBS, CTR with 109 cfu of live B. subtilis natto; and ABS, CTR with 109 cfu of autoclaved B. subtilis natto. The rumen fluid was collected after 0, 6, 12, and 24 h of fermentation, and pH, ammonia nitrogen (NH3-N), microbial protein (MCP), and volatile fatty acids (VFAs) were determined. The diversity and composition of rumen microbiota were assessed by 16S rRNA gene sequencing. The results revealed LBS affected the concentrations of NH3-N, MCP, and VFAs (p < 0.05), especially after 12 h, which might be attributed to changes in 18 genera. Whereas ABS only enhanced pH and NH3-N concentration compared with the CTR group (p < 0.05), which might be associated with changes in six genera. Supplementation with live B. subtilis natto improved ruminal NH3-N and propionate concentrations, indicating that live bacteria were better than autoclaved ones. This study advances our understanding of B. subtilis natto in promoting ruminal fermentation, providing a new perspective for the precise utilization of B. subtilis natto in dairy rations.
Keywords: 16S rRNA gene sequencing; Bacillus subtilis natto; rumen fermentation in vitro; rumen microbiota; volatile fatty acid.