Research and safety evaluation of the Bacillus subtilis Eramic 26 strain for application in probiotic production

Abstract

This study evaluated the safety of the Bacillus subtilis Eramic 26 strain through assessments of antibacterial activity, hemolytic potential, cytotoxicity, and virulence/antibiotic resistance gene analysis, following the Generally Recognised As Safe (GRAS) guidelines of the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA). Antibacterial activity was tested against five indicator bacterial strains: Escherichia coli, Salmonella typhimurium, Enterococcus faecium, Staphylococcus aureus, and Shigella flexneri. Cytotoxicity was evaluated using two cell lines: human HT-29 and Vero. The results showed that B. subtilis Eramic 26 did not inhibit the tested bacterial strains and did not exhibit cytotoxic effects in either the HT-29 or Vero cell line. Genomic analysis revealed the presence of antibiotic resistance genes such as fosB, satA, and ydhE/norM, as well as mutations in gyrA and gyrB; however, all were located on the chromosome and not associated with mobile genetic elements. The strain harboured bacteriocin-encoding genes, including bacilysin and subtilosin, which enhance competitive fitness. Overall, the findings indicate that B. subtilis Eramic 26 is a safe bacterial strain with strong potential for application in probiotic production.