Bacillus genus is a widespread heterogeneous group of Gram-positive, facultative anaerobic, endospore-forming bacteria occurring in many types of foods. Bacillus has been reported to produce more than 45 antimicrobial molecules: some of them are of clinical value, whereas others have been assayed in vitro against food-borne microorganisms. In this work, the antibacterial activity of the cell-free concentrate supernatant of Bacillus subtilis TR50, a strain isolated from the natural microflora of an Italian traditional cured sausage, was tested by the disc diffusion assay against B. cereus DSM4312, B. cereus DSM4313, Enterococcus faecalis V583, Listeria monocytogenes DSM20600, Staphylococcus aureus NCTC8325, Aeromonas hydrophila DSM30187, Escherichia coli ATCC35401, Helicobacter pylori ATCC43504, Salmonella enterica ATCC13311, Yersinia enterocolitica DSM4780 and 17 Bacillus spp. strains isolated from traditional and industrial cured sausages. All pathogens, except E. faecalis, resulted inhibited by the cell-free concentrated TR50 supernatant. The average size of the inhibition halos on the bacterial lawns of the Bacillus species assayed decreased in the following order: B. pumilus > B. licheniformis > B. thuringensis, whereas no antimicrobial activity was detected against B. amyloliquefaciens and the other B. subtilis strains. The cell-free concentrate TR50 supernatant was extracted with ethyl acetate and the resultant organic fraction proved to retain antimicrobial activity. The same fraction was both spotted on silica thin layer chromatography (TLC) plates and loaded on Tricine SDS-PAGE gel; both TLC plates and the Tricine SDS-PAGE gel were overlaid with the Isosensitest soft-agar inoculated with Bacillus cereus DSM 4312 or B. cereus 4313. The halos on the bacterial lawn agar resulted correlated with the retention factor (Rf) of the spots, that were subsequently scraped from TLC and extracted again with methanol; among the spots (Rf 0.50-0.88), only the compounds with Rf ranging from 0.49 to 0.58 and from 0.69 to 0.77 retained antimicrobial activity. Among bands revolved by Tricine SDS-PAGE, one with an apparent molecular weight of about 2.5 kDa, displayed inhibitory activity against the same indicator strains. Finally, the organic fraction further purified by reverse phase (RP)-HPLC showed that only 20% of the TR50 specific peaks were retained; some of them fell in the chromatographic region common to commercial iturins. These preliminary results highlight the antibacterial potential of B. subtilis TR50 against widespread meat-borne pathogens that could be exploited in the sausage curing process. Further analyses are in progress to identify active compounds even though results of analytical techniques suggest the presence of lipopeptides responsible antimicrobial activity.
Partial purification of antimicrobial compounds produced by Bacillus subtilis TR50 isolated from a cured sausage
Quintieri L;Caputo L;Morea M;Baruzzi F
2012
Abstract
Bacillus genus is a widespread heterogeneous group of Gram-positive, facultative anaerobic, endospore-forming bacteria occurring in many types of foods. Bacillus has been reported to produce more than 45 antimicrobial molecules: some of them are of clinical value, whereas others have been assayed in vitro against food-borne microorganisms. In this work, the antibacterial activity of the cell-free concentrate supernatant of Bacillus subtilis TR50, a strain isolated from the natural microflora of an Italian traditional cured sausage, was tested by the disc diffusion assay against B. cereus DSM4312, B. cereus DSM4313, Enterococcus faecalis V583, Listeria monocytogenes DSM20600, Staphylococcus aureus NCTC8325, Aeromonas hydrophila DSM30187, Escherichia coli ATCC35401, Helicobacter pylori ATCC43504, Salmonella enterica ATCC13311, Yersinia enterocolitica DSM4780 and 17 Bacillus spp. strains isolated from traditional and industrial cured sausages. All pathogens, except E. faecalis, resulted inhibited by the cell-free concentrated TR50 supernatant. The average size of the inhibition halos on the bacterial lawns of the Bacillus species assayed decreased in the following order: B. pumilus > B. licheniformis > B. thuringensis, whereas no antimicrobial activity was detected against B. amyloliquefaciens and the other B. subtilis strains. The cell-free concentrate TR50 supernatant was extracted with ethyl acetate and the resultant organic fraction proved to retain antimicrobial activity. The same fraction was both spotted on silica thin layer chromatography (TLC) plates and loaded on Tricine SDS-PAGE gel; both TLC plates and the Tricine SDS-PAGE gel were overlaid with the Isosensitest soft-agar inoculated with Bacillus cereus DSM 4312 or B. cereus 4313. The halos on the bacterial lawn agar resulted correlated with the retention factor (Rf) of the spots, that were subsequently scraped from TLC and extracted again with methanol; among the spots (Rf 0.50-0.88), only the compounds with Rf ranging from 0.49 to 0.58 and from 0.69 to 0.77 retained antimicrobial activity. Among bands revolved by Tricine SDS-PAGE, one with an apparent molecular weight of about 2.5 kDa, displayed inhibitory activity against the same indicator strains. Finally, the organic fraction further purified by reverse phase (RP)-HPLC showed that only 20% of the TR50 specific peaks were retained; some of them fell in the chromatographic region common to commercial iturins. These preliminary results highlight the antibacterial potential of B. subtilis TR50 against widespread meat-borne pathogens that could be exploited in the sausage curing process. Further analyses are in progress to identify active compounds even though results of analytical techniques suggest the presence of lipopeptides responsible antimicrobial activity.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
