Intestinal organoid screen reveals that Bacillus velezensis PGM541 promotes epithelial proliferation via its metabolite butyric acid.

BACKGROUND: Probiotics have been widely used for the regulation of intestinal health. Current screening methods for probiotics typically rely on animal or two-dimensional cell models. In this study, we employed intestinal organoids to identify a candidate probiotic strain. Furthermore, we investigated the potential mechanisms through which this strain and its active metabolites exert their effects, thereby evaluating the efficacy of this screening approach. RESULTS: Firstly, candidate probiotic strain PGM541 was identified from a porcine-derived Bacillus library by assessing organoid viability. Subsequently, to validate the organoid screening reliability, the potential mechanism of strain PGM541 on the intestinal epithelium was investigated; it was found to exhibit probiotic functions by regulating cell proliferation in both in vitro organoid and in vivo piglet models. Furthermore, organoid screening combined with metabolomic analysis identified butyric acid (BA) as the key bioactive metabolite responsible for driving epithelial proliferation. Whole-genome and transcriptomic analyses revealed the biosynthetic pathway of BA in strain PGM541. Importantly, BA receptor blockade experiments directly confirmed that BA enhances epithelial proliferation via interaction with the FFAR2 receptor, thereby validating its functional activity. Additionally, strain PGM541 exhibited protective effects against dextran sulfate sodium (DSS)-induced colitis, further validating the effectiveness of the intestinal organoid platform for probiotic screening. CONCLUSIONS: The probiotic strain PGM541, which was screened using intestinal organoids, promotes intestinal epithelial cell proliferation via its metabolite BA activating the FFAR2 receptor. These findings demonstrate that the intestinal organoid model serves as an effective platform for both preliminary probiotic screening and mechanistic investigation. Video Abstract.