Purpose: The purpose of this study was to screen strains of B. subtilis subsp. subtilis (six isolates) and B. licheniformis (four isolates), representing the predominant Bacillus spp. during Bikalga (Hibiscus sabdariffa fermented seeds) fermentation for the production of antimicrobial substances and to characterize the antimicrobial compound(s) produced by a selected B. subtilis subsp. subtilis isolate.
Methods: Antimicrobial activity of the strains was determined by agar spot and well assay. Antimicrobial substance produced by the selected isolate was tested for sensitivity to heat, pH, enzymes and chemicals. Direct detection of antimicrobial activity was carried out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the inhibitory compounds were identified by PCR and Ultra High Performance Liquid Chromatography-Time of Flight Mass Spectrometry (UHPLC-TOFMS).
Results: Cell-free supernatants of three B. subtilis subsp. subtilis (G2, H4 and F1) showed the best activity being inhibitory to Listeria monocytogenes, Micrococcus luteus, Staphylococcus aureus and Bacillus cereus. The antimicrobial substance produced by B. subtilis subsp. subtilis H4 was further characterized showing sensitivity to protease, trypsin, α-amylase, lipase II and mercapto-ethanol. PCR detection of yiwB, sboA, spoX, albA and spaS, etnSgenes and genes coding for surfactins and plipastatins indicated a potential for subtilosin, subtilin and lipopeptide production, respectively. UHPLC-TOFMS analysis of the single band associated with antimicrobial activity in SDS-PAGE allowed identification of surfactin and a protein with a monoisotopic mass of 3346.59 Da, which is dissimilar in size to subtilosin and subtilin.
Significance: These results suggest that H4 in addition to surfactin, may produce a novel bacteriocin which may be relevant in food applications.