Clinical Safety and Tolerability of Bifidobacterium bifidum BBi32: An 8‐Week Randomized, Double‐Blind, Placebo‐Controlled Trial With Genomic and In Vitro Corroboration

Abstract

Bifidobacterium bifidum BBi32, isolated from a healthy infant, underwent a multi‐tiered safety assessment to evaluate its genetic features, in vitro properties, and effects on gut microbiota and host biomarkers. Whole‐genome sequencing (WGS) and functional annotation were performed alongside in vitro assays assessing acid and bile tolerance, mucin degradation, hemolysis, Caco‐2 cytotoxicity, and antibiotic susceptibility. Acute oral toxicity was tested in mice. A randomized, double‐blind, placebo‐controlled clinical trial ( n = 40, 8 weeks) evaluated tolerability and exploratory endpoints, including hematology, liver and renal function, LL‐37 levels, gastrointestinal symptom scores, and 16S rRNA‐based microbiome profiling. The BBi32 genome comprised a 2.2 Mbp circular chromosome with 99.99% average nucleotide identity to the type strain, no plasmids, and no acquired antimicrobial resistance or virulence genes. Functional categories were enriched for ABC transporters, purine metabolism, and defense mechanisms. BBi32 demonstrated tolerance to acid and bile, lacked mucin‐degrading, or hemolytic activity, showed no cytotoxicity to Caco‐2 cells, and was susceptible to most antibiotics. Acute toxicity test yielded an LD 50 > 2 × 10 10 CFU/kg with no adverse effects. In the clinical trial, daily BBi32 administration (3 × 10 10 CFU) was well tolerated, with no hematological or hepatic abnormalities. Compared with placebo, BBi32 reduced uric acid, urea, and creatinine levels, increased LL‐37, and improved gastrointestinal symptom scores. Microbiome analysis revealed higher alpha diversity, distinct community clustering, enrichment of Romboutsia , and predicted functional shifts toward amino acid biosynthesis and peptidase activity. Genomic, in vitro, toxicological, and clinical data collectively indicate that BBi32 meets key safety criteria and favorably modulates host and microbiome biomarkers, supporting its probiotic potential.