PROBIOTIC POTENTIAL OF RIBOFLAVIN-OVERPRODUCING Bacillus subtilis ACU-I163MR AND ACU-I11MR, ISOLATED FROM FERMENTED AFRICAN LOCUST BEANS

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 Bacillus subtilis strains isolated from fermented African locust beans. After strain improvement, Bacillus subtilis ACU-I11MR and ACU-I163MR were selected due to their higher riboflavin production (0.01905±0.0005 mg/L to 0.0259±0.0077 mg/L and 0.0195±0.0054 mg/L to 0.0267±0.0013 mg/L, 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 in vitro assays including resistance to low pH, bile salts, phenol, temperature, and NaCl; auto-aggregation; cell hydrophobicity; biofilm formation; antibacterial activity; enzyme and exopolysaccharides production. Both strains were non-haemolytic, 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 thrived under various temperature and NaCl conditions. Bacillus subtilis ACU-I163MR, showing superior probiotic potential, could be a promising candidate for developing riboflavin-enriched Bacillus-fermented functional foods.