Production, stability, gene sequencing and in situ anti-Listeria activity of mundticin KS expressed by three Enterococcus mundtii strains

Three enterococci (WFE3, WFE20 and WFE31) selected as presumptive bacteriocin producers were found to be active against Listeria monocytogenes. In this study, due to their potential industrial/food applications, the three bacterial isolates were extensively characterized. Identification was performed by means of a combined 16S rRNA gene sequencing and multiplex PCR approach, and was confirmed with the sequencing of a partial region of a protein-encoding gene, namely pheS. The three isolates belonged unequivocally to the species Enterococcus mundtii. The randomly amplified polymorphic DNA (RAPD) analysis recognized three distinct strains. The supernatants were mainly active against Listeria spp., but some lactic acid bacteria were also inhibited. The proteinaceous nature of the three supernatants was detected after treatment with proteinase K, protease B and trypsin. The bacteriocins were found to be heat resistant, stable in a large pH range and in presence of ethanol. The bacteriocins were not adsorbed onto the surface of the producer cells and their effect was bactericidal. The production of bacteriocins was higher at neutral pHs and temperatures in the range 30-37 °C. The active supernatants did not show cytotoxicity against human erythrocytes and the three strains were susceptible to the action of common antibiotics. The genetic characterization of the bacteriocin genes showed that all three strains produced mundticin KS. They produced it in five food model systems, sterilized by thermal treatment or filtration, prepared from fresh vegetables, cereals, cheeses, meats and fishes. The in situ anti-listerial activities of the strains WFE3, WFE20 and WFE31 were quantitatively different.