Targeted Screening of Fiber Degrading Bacteria with Probiotic Function in Herbivore Feces

Cellulolytic bacteria with probiotic functions play a crucial role in promoting the intestinal health in herbivores. In this study, we aimed to correlate the 16S rRNA gene amplicon sequencing and fiber-degrading enzyme activity data from six different herbivore feces samples. By utilizing the separation and screening steps of probiotics, we targeted and screened high-efficiency fiber-degrading bacteria with probiotic functions. The animals included Maiwa Yak (MY), Holstein cow (CC), Tibetan sheep (TS), Southern Sichuan black goat (SG), Sichuan white rex rabbit (CR), and New Zealand white rabbit (ZR). The results showed that the enzymes associated with fiber degradation were higher in goat and sheep feces compared to cattle and rabbit's feces. Correlation analysis revealed that Bacillus and Fibrobacter were positively correlated with five types of fiber-degrading related enzymes. Notably, the relative abundance of Bacillus in the feces of Tibetan sheep was significantly higher than that of other five herbivores. A strain TS5 with good cellulose decomposition ability from the feces of Tibetan sheep by Congored staining, filter paper decomposition test, and enzyme activity determination was isolated. The strain was identified as Bacillus velezensis by biological characteristics, biochemical analysis, and 16S rRNA gene sequencing. To test the probiotic properties of Bacillus velezensis TS5, we evaluated its tolerance to acid and bile salt, production of digestive enzymes, antioxidants, antibacterial activity, and adhesion ability. The results showed that the strain had good tolerance to pH 2.0 and 0.3% bile salts, as well as good potential to produce cellulase, protease, amylase, and lipase. This strain also had good antioxidant capacity and the ability to antagonistic Staphylococcus aureus BJ216, Salmonella SC06, Enterotoxigenic Escherichia coli CVCC196, and Escherichia coli ATCC25922. More importantly, the strain had good self-aggregation and Caco-2 cell adhesion rate. In addition, we tested the safety of Bacillus velezensis TS5 by hemolysis test, antimicrobial susceptibility test, and acute toxicity test in mice. The results showed that the strain had no hemolytic phenotype, did not develop resistance to 19 commonly used antibiotics, had no cytotoxicity to Caco-2, and did not have acute toxic harm to mice. In summary, this study targeted isolated and screened a strain of Bacillus velezensis TS5 with high fiber-degrading ability and probiotic potency. This strain can be used as a potential probiotic for feeding microbial preparations for ruminants.