Probiotic potential of riboflavin-overproducing Bacillus subtilis ACU-I163MR and ACU-I11MR, isolated from fermented African locust beans

Abstract

Riboflavin (vitamin B₂) is a water-soluble compound that plays an important role in multiple cellular functions. This study evaluates the probiotic potential of riboflavin-overproducing <i>Bacillus subtilis</i> strains isolated from fermented African locust beans. After strain improvement, <i>B. subtilis</i> ACU-I11MR and ACU-I163MR were selected due to their higher riboflavin production (0.01905±0.0005 mg l^-1 to 0.0259±0.0077 mg l^-1 and 0.0195±0.0054 mg l^-1 to 0.0267±0.0013 mg l^-1, respectively). Their safety was confirmed through haemolytic assay, antibiotic susceptibility tests and the absence of gelatinase and biogenic amine activity. Probiotic potential was assessed via <i>in vitro</i> assays including resistance to low pH, bile salts, phenol, temperature and NaCl; auto-aggregation; cell hydrophobicity; biofilm formation; antibacterial activity; and enzyme and exopolysaccharide production. Both strains were non-haemolytic and negative for gelatinase and biogenic amine activity. They showed significant viability at pH 2 (survival 85.05; 87.09%), 1% bile salts (survival 88.82; 87.64%) and 0.5% phenol (survival 48.80; 59.52%), respectively. ACU-I11MR was susceptible to 9 out of 12 antibiotics, while ACU-I163MR was 100% susceptible. The strains demonstrated strong cell surface adhesion and auto-aggregation and inhibited several pathogenic bacteria. They produce amylase, protease and exopolysaccharide and thrive under various temperature and NaCl conditions. <i>B. subtilis</i> ACU-I163MR, showing superior probiotic potential, could be a promising candidate for developing riboflavin-enriched <i>Bacillus</i>-fermented functional foods.