Purpose: To identify a Bacillus sp. isolate from pineapple pulp that produces a bacteriocin-like substance (BLIS) and to purify partially the antimicrobial peptide.
Methods: Bacillus sp. was isolated on MRS agar, cultured in MRS broth (24 h/30 °C) and, its activity against Listeria monocytogenes was tested by critical dilution assay (AU/ml). The neutralized filter-sterilized supernatant of the MRS culture was purified with Amberlite® XAD-16 resin (Sigma-Aldrich) using water, 20% ethanol and 70% isopropanol. Evaporated extract was applied on solid phase extraction column - SPE (C18, Varian) and eluted with water, 30% ethanol and 20, 40 and 70% isopropanol. The last fraction was selected, solvents were removed and the extract was analyzed by tricine SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Gel was revealed by silver staining and with Listeria monocytogenes (biological revelation). Possible hemolytic activity of extract was tested by streaking it on blood agar plate (37 °C/24 h).
Results: Isolate was named Bacillus cereus LFB – FIOCRUZ 1638 and the initial supernatant presented activity of 400 AU/ml. The antimicrobial activity of supernatant was stable at 75 °C/30 min, but 50% of activity was lost at 95 °C/30 min, while no activity remained after 121 °C/20 min (autoclave). Activity was detected also in supernatant when pH varied from 1.9 to 8.0 (10 °C for 24 h). The molecular weight of the BLIS was estimated by SDS-PAGE as 3.5 to 8.5kDa.
Significance: Despite negative association of Bacillus cereus with foods, purified bacteriocin preparations from Bacillus sp. may have technological advantages for food processing when compared to bacteriocins from lactic acid bacteria, due to large thermal and pH stability.