Whole-Genome Sequence Analysis and Probiotic Characterization of 5-Methoxytryptophan-Producing Strain Lacticaseibacillus paracasei RM081

This study comprehensively examines the whole-genome sequence and probiotic potential of Lacticaseibacillus paracasei RM081, a strain originally isolated from raw bovine milk. Whole-genome sequencing and in silico analyses provided a robust molecular basis for its functional traits. The L. paracasei RM081 genome harbors an extensive repertoire of carbohydrate-active enzymes, suggesting strong prebiotic utilization capabilities. Crucially, genomic mining identified key genetic determinants for postbiotic synthesis, including the potential to synthesize the anti-inflammatory metabolite 5-methoxytryptophan (5-MTP). Moreover, comprehensive safety evaluations confirmed the absence of transferable antimicrobial resistance genes, virulence factors, biogenic amine-producing genes, and plasmids, indicating a secure genomic architecture without horizontal gene transfer risks. These genomic predictions were further substantiated by valid in vitro phenotypic models. The strain exhibited strong tolerance to gastric acid, maintaining high viability at pH 3.5 and 2.5 after 4 h, and survived well at 0.1% bile salt concentration. Furthermore, L. paracasei RM081 demonstrated robust cell surface properties, with a high auto-aggregation rate (85.0 ± 0.7%), hydrophobicity (71.5 ± 2.4%), and 78.0 ± 4.8% adhesion to Caco-2 intestinal epithelial cells, supporting its potential for colonization. Regarding antioxidant capacity, the cell-free supernatant displayed the highest DPPH scavenging activity (37%), indicating the active secretion of antioxidative metabolites. Collectively, these findings establish L. paracasei RM081 as a highly promising, safe probiotic and postbiotic candidate with verified colonization potential and functional capabilities.