Safety Assessment of Lacticaseibacillus rhamnosus OF44, a Previously Isolated Human-origin Probiotic Candidate.

Abstract

Lacticaseibacillus rhamnosus is one of the most extensively studied and well-characterized probiotic species. The strain L. rhamnosus OF44, originally isolated from a healthy female fecal sample, has shown particular promise for its ability to modulate the vaginal microenvironment through pH reduction and inflammation control. However, its comprehensive safety profile remains unclear. In this study, we systematically evaluated the safety of the previously identified strain L. rhamnosus OF44 through a series of standardized assessments. Genomic analysis (99.9% coverage) revealed the absence of virulence genes, while identifying four genes associated with resistance to chlorhexidine and copper ions; these have been demonstrated to be intrinsic to L. rhamnosus. Additionally, phenotypic analysis, including minimum inhibitory concentration (MIC) testing, reverse mutation testing, β-hemolysis assessment, biological amines and D-lactate production (0.571 ± 0.005 mM), and genetic stability testing, revealed no adverse traits. Finally, in vivo studies (acute oral toxicity at up to 3 × 10¹² CFU/kg, with LD₅₀ > 15.0 g/kg·BW) confirmed that L. rhamnosus OF44 exhibited no detectable acute oral toxicity and pathogenicity. These results indicate that L. rhamnosus OF44 has a favorable safety profile under the tested conditions, although further chronic toxicity and human studies are warranted.