Co-expression of Nisin Z and Leucocin C as a Basis for Effective Protection Against Listeria monocytogenes in Pasteurized Milk

Abstract

Nisin, an important bacteriocin from <i>Lactococcus lactis</i> subsp., is primarily active against various Gram-positive bacteria. Leucocin C, produced by <i>Leuconostoc carnosum</i> 4010, is a class IIa bacteriocin used to inhibit the growth of <i>Listeria monocytogenes.</i> Because two bacteriocins have different modes of action, the combined use of them could be a potential strategy for effective inhibition of foodborne pathogens. In this study, <i>L. lactis</i> N8-r-lecCI (N8 harboring <i>lecCI</i> gene) coexpressing nisin-leucocin C was constructed based on the food-grade carrier <i>L. lactis</i> N8. Production of both bacteriocins was stably maintained. Antimicrobial measurements showed that the recombinant strain is effectively against <i>Listeria monocytogenes</i> and <i>Staphylococcus aureus</i> and moderately against <i>Salmonella enterica</i> serovar Enteritidis and <i>Escherichia coli</i> because of its stronger antibacterial activity than the parental strain, this result first demonstrated that the co-expression of nisin and leucocin C results in highly efficient antimicrobial activity. The checkerboard assay showed that the antibacterial activity of <i>L. lactis</i> N8-r-lecCI supernatant was enhanced in the presence of low concentration of EDTA. Analysis of the scanning electron microscope image showed the biggest cellular morphology change in <i>L. monocytogenes</i> treated with a mixture of EDTA and <i>L. lactis</i> N8-r-lecCI supernatant. The practical effect was verified in pasteurized milk through time-kill assay. The <i>L. lactis</i> N8-r-lecCI strain expressing both nisin and leucocin C has a promising application prospect in pasteurized milk processing and preservation because of its strong antibacterial activity.