Preclinical Safety and Toxicological Assessment of Vitamin B ₁₂ Producing Probiotic Limosilactobacillus reuteri NCDC 958

The development of functional dairy foods using probiotic cultures with additional nutritional benefits has gained considerable attention. Limosilactobacillus reuteri NCDC 958 (LRN958) has been identified as a probiotic strain with the ability to produce vitamin B 12 ; however, establishing its safety profile is essential for its successful application in food and human use. In this study, the preclinical safety of the vitamin B 12 ‐producing LRN958 strain was assessed using integrated in vitro and in vivo approaches. In vitro evaluation revealed that the strain lacked virulence‐associated genes, did not produce biogenic amines or degrade mucin, and was susceptible to recommended antibiotics. In vivo safety was examined through acute, sub‐acute, and sub‐chronic oral toxicity studies in mice. Administration of LRN958 caused no mortality or clinical signs of toxicity. Body weight, hematological and biochemical parameters, organ indices, and histopathology remained comparable to controls. No bacterial translocation was observed, and immune markers, including IgA, IgG, IL‐10, TGF‐β, IL‐6, and TNF‐α, which showed no significant differences, indicating maintained immune homeostasis. The no‐observed‐effect level (NOEL) was 10 10 CFU/mouse/day. Overall, the findings confirm that LRN958 is safe, non‐pathogenic, and supports its potential clinical use and application in probiotic and functional food formulations.