Acute and sub-chronic oral toxicity evaluation and gut microbiota modulation of indigenous Lactobacillus helveticus strains from Indonesia in rats.

Probiotics are widely used to support gastrointestinal health, but their safety must be evaluated on a strain-specific basis. Lactobacillus helveticus DLBSA201 and DLBSA202 are indigenous strains with previously demonstrated probiotic potential in vitro. However, their in vivo safety profile has not been fully assessed. This study aimed to evaluate the acute and sub-chronic oral toxicity of these strains and their effects on selected gut microbial parameters in Wistar rats. In the acute toxicity study, rats received a single oral dose of L. helveticus DLBSA201 or DLBSA202 at 2.5 × 1010, 2.5 × 1011, or 5 × 1011 CFU/rat. In the sub-chronic study, rats were orally administered a 1:1 mixture of both strains daily for 90 days at doses of 1 × 109, 1 × 1010, or 1 × 1011 CFU/rat/day. Clinical observations, hematological and biochemical parameters, histopathology, bacterial translocation, bacterial enzyme activity, and fecal microbiological analyses were evaluated. No mortality or treatment-related adverse effects were observed, and the LD50 of both strains was estimated to be greater than 5 × 1011 CFU/rat. No significant treatment-related toxicological abnormalities were detected in the sub-chronic study, with a NOAEL of 1 × 1011 CFU/rat/day. Probiotic administration was also associated with reduced harmful bacterial enzyme activity and altered selected fecal bacterial populations. In conclusion, L. helveticus DLBSA201 and DLBSA202 were well tolerated in Wistar rats at the tested dose range and study duration, and warrant further investigation using comprehensive microbiome and clinical approaches.