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

## TL;DR

A clinical trial found that Bifidobacterium bifidum BBi32 is safe and may improve gut health and immune markers.

## Contribution

This study provides comprehensive safety and tolerability evidence for BBi32 through genomic, in vitro, and clinical assessments.

## Key findings

- BBi32 was well tolerated in a clinical trial with no adverse hematological or hepatic effects.
- BBi32 reduced uric acid, urea, and creatinine levels while increasing LL-37 and improving gastrointestinal symptoms.
- Microbiome analysis showed increased alpha diversity and enrichment of Romboutsia with BBi32.

## 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 LD50 > 2 × 1010 CFU/kg with no adverse effects. In the clinical trial, daily BBi32 administration (3 × 1010 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.

An 8‐week randomized, double‐blind, placebo‐controlled trial found BBi32 well tolerated with no hematologic or hepatic abnormalities. Versus placebo, BBi32 reduced uric acid/urea/creatinine, increased LL‐37, and lowered gastrointestinal symptom scores. 16S profiling showed higher alpha diversity, distinct clustering, and Romboutsia enrichment—supporting safety and a microbiome‐mediated benefit on metabolic and mucosal‐immune biomarkers.