Objective Limosilactobacillus reuteri is a facultative anaerobic lactic acid bacterium commonly found in the intestines of vertebrates and mammals. This bacterium possesses various probiotic functions, such as cholesterol reduction, inhibition of pathogenic bacteria growth, and enhancement of immune function, making the isolation of strains highly applicable to the food fermentation industry crucial. Method In this study, MRS-CaCO₃ medium was used to isolate and cultivate L. reuteri from mouse feces. Preliminary identification was conducted through morphological observation, Gram staining, and 16S rRNA sequence analysis. The identified strains were subjected to growth performance, stress resistance, and antibacterial efficacy tests. Whole-genome sequencing and annotation were performed to explore the probiotic mechanisms and safety of the strain at the genetic level. Result The isolated L. reuteri GF304 had no hemolytic activity and demonstrated promising growth performance. It showed strong tolerances to acid and bile salts, with high survival rates in artificial gastrointestinal fluids. Additionally, it effectively inhibited the growth of Escherichia coli and Staphylococcus aureus, indicating its probiotic properties. Whole-genome sequencing analysis revealed that the genome of L. reuteri GF304 contained no virulence or antibiotic resistance genes. Instead, it harbored stress resistance and probiotic genes related to heat shock tolerance, cold shock tolerance, acid resistance, bile salt tolerance, adhesion, antioxidant capacity, and organic acid synthesis. The most prevalent CAZy classifications for strain GF304 were GH and GT. Secondary metabolite analysis revealed that strain GF304 possesses Type Ⅲ Polyketide Synthase biosynthetic gene clusters and RiPP biosynthetic gene clusters. Conclusion Based on its secure genomic profile (absence of virulence and antibiotic resistance genes) and the abundance of probiotic-associated gene clusters, combined with its excellent growth, stress tolerance, and antibacterial properties, L. reuteri GF304 demonstrates significant potential for development as a probiotic preparation.
