Probiotic properties of Bacillus subtilis isolates from neonatal meconium

Neonatal meconium represents a relatively underexplored microbial source that may harbour microorganisms with probiotic potential, uniquely adapted for intestinal colonization and metabolic health. From 120 meconium samples, 167 spore-forming isolates were screened to identify strains exhibiting desirable probiotic functional and safety attributes. Three strains (ZBOE6, ZBRV8, and ZBTM2) were selected and identified as Bacillus subtilis through whole-genome sequencing. These strains showed non-haemolytic (α/γ types) and DNase negative phenotypes, indicating non-pathogenic profile, along with detectable bile salt hydrolase ( Bsh ) activity. The isolates exhibited tolerance to adverse stressors, including acidic conditions, bile salts, and simulated gastric and intestinal conditions. Isolates showed epithelial adhesion potential (up to 87.5% for ZBRV8) and auto-aggregation efficiencies ranging from 60 to 80%. In vitro assays using Caco-2 cell lines demonstrated anti-adhesive potential against E. coli through competitive, exclusion, and displacement mechanisms. The strains showed cholesterol assimilation potential greater than 60% and antioxidant activity up to 62.7% (DPPH scavenging) and 97.9% (hydroxyl radical scavenging). In vitro safety assessment revealed no biogenic amine production, minimal cytotoxicity (