Comprehensive genomic and phenotypic analysis of Limosilactobacillus reuteri ID-D01 reveals safety and probiotic potential

Limosilactobacillus reuteri ID-D01 , isolated from a healthy human, has demonstrated performance-enhancing and muscle fatigue recovery effects in previous studies. However, its genetic characteristics and safety have not yet been evaluated. This study aimed to analyze the genetic properties and assess the safety of L. reuteri ID-D01 as a probiotic based on international standards, including antibiotic susceptibility, virulence factors, hemolysis, metabolic characteristics, and cytotoxicity. Genomic analysis revealed that the genome of L. reuteri ID-D01 (2.14 Mb, GC content 38.7%) contained 3,903 pan-genome genes and 1,378 core-genome genes, as determined through comparative analysis with 16 L. reuteri strains. Safety assessment results showed intrinsic resistance to kanamycin, no acquired virulence factor genes, absence of β-hemolysis, and lack of the ability to produce toxic metabolites such as bile salt deconjugation and D-lactate. Additionally, no cytotoxicity was observed in gut epithelial cells. A single-dose acute oral toxicity study observed no significant adverse effects in rats, except for transient diarrhea on the day of administration. Furthermore, bioinformatic analysis identified functional genes in the L. reuteri ID-D01 strain associated with its adaptation to the human gastrointestinal environment. Thus, L. reuteri ID-D01 was considered safe and suitable for use as a probiotic strain in the functional food market. • Genetic characteristics and probiotic safety of L. reuteri ID-D01 were comprehensively analyzed • Whole-genome sequencing of L. reuteri ID-D01 identified 160 unique strain-specific genes • The strain exhibits intrinsic kanamycin resistance, with no acquired virulence factor genes • The acute oral toxicity study observed no significant adverse effects in rats • Genomic and safety profiles support the probiotic potential of L. reuteri ID-D01 for functional food applications