Integrated Production, Genomic Safety, Quality, and Stability Characterization of a Human-Derived Limosilactobacillus reuteri ATA-LTC-Lr050600 Strain for Probiotic Applications

Probiotic quality and safety require strain-specific characterization supported by controlled production and comprehensive evaluation. This study aimed to integrate controlled fermentation and biomass production, downstream processing and lyophilization, microbiological quality control, batch reproducibility, whole-genome characterization, in vitro functional and safety-related assessment, real-time stability monitoring, and OECD-compliant in vivo safety evaluation of the human-derived Limosilactobacillus reuteri strain ATA-LTC-Lr050600. The strain was produced under controlled fermentation conditions, followed by harvesting, protective formulation, and lyophilization. Product quality was assessed by standardized microbiological testing, whole-genome sequencing, and in vitro assays evaluating acid and bile tolerance, adhesion to a fibroblast cell model, hemolytic activity, and cytotoxicity. Real-time stability was monitored during storage, and safety was further evaluated using OECD-compliant acute and repeated-dose oral toxicity studies. The production process generated a lyophilized preparation with consistent microbiological quality and reproducible manufacturing characteristics. Whole-genome sequencing confirmed strain identity and did not detect acquired antimicrobial resistance determinants. The strain tolerated simulated gastrointestinal conditions, adhered to the evaluated fibroblast cell model, and showed no detectable hemolytic activity or cytotoxicity under the experimental conditions. Product viability and microbiological quality were maintained throughout the monitored storage period. In the in vivo studies, no treatment-related adverse effects were identified, and the repeated-dose study established a no-observed-adverse-effect level of 1000 mg/kg/day under the conditions examined. Collectively, these integrated findings provide a basis for further strain-specific evaluation and support the continued development of L. reuteri ATA-LTC-Lr050600 as a well-characterized probiotic candidate under the conditions examined.