Bile Salt Hydrolase Activity in the Food-Derived Strain Levilactobacillus brevis M3R3: Genomic and Functional Characterization

In the present study, Levilactobacillus brevis M3R3, isolated from traditional Sicilian sourdough, was evaluated for BSH functionality through kinetic growth and bile salts (BSs) deconjugation using UHPLC/HR-MS. Genome sequencing and in silico analysis were performed to assess its safety and probiotic potential. Results showed that the M3R3 strain tolerated different concentrations of conjugated BSs, with dose-dependent growth inhibition. In particular, exposure to 1.0% taurocholic and taurodeoxycholic acids reduced M3R3 growth, indicating an adaptive stress response. When a mixed BSs solution was used to mimic intestinal conditions, M3R3 maintained its growth, despite an extended lag phase and a reduced final biomass, likely due to toxic BSs accumulation. UHPLC/HR-MS confirmed a complete deconjugation of glyco-conjugated BSs and a partial deconjugation of the tauro-conjugated BSs. Whole-genome sequencing (2.30 Mbp, 2375 CDSs) revealed no antimicrobial resistance or virulence genes and identified two bsh genes (bsh_2A and bsh_3A), both expressed under control and BS-exposed conditions. Phylogenetic analysis of L. brevis BSH proteins identified three BSH clusters (I, II, and III), and bsh2 and bsh3 genes present in M3R3 strains were grouped in clusters II and III, respectively. Overall, genomic and functional characterizations support that L. brevis M3R3 is a safe and BSH-active strain, highlighting its potential as a functional ingredient in health promoting applications.