Isolation of bacteriocinogenic strain of Lactococcus lactis subsp. lactis from rocket salad (Eruca sativa Mill.) and evidences of production of a variant of nisin with modification in the leader-peptide

Abstract

In this study we have isolated and identified strains of lactic acid bacteria (LAB) that are potential bacteriocin producers from rocket salad samples acquired in Sao Paulo, SP, Brazil. Using the PCR-ARDRA method, Lactococcus lactis subsp. lactis MK02R was identified and characterized as the only bacteriocinogenic among 12 strains initially isolated. The analysis of the antimicrobial peptide produced by MK02R revealed the presence of nisin variant, a well-known lantibiotic, and one of the most studied and important antimicrobial peptides known today. The bacteriocin MK02R showed heat stability for 1 h at 60 °C and 100 °C and it was inactivated by treatment with proteolytic enzymes. The antimicrobial activity did not change with pH adjustment between 2.0 and 9.0 and the production was stimulated by the addition of 0.5 and 1.0% cysteine in MRS broth at 37 °C. Bacteriocin MK02R inhibited the growth of Enterococcus faecium, Lactobacillus sakei, Lactobacillus delbrueckii, Lb. sakei subsp. sakei, Listeria innocua and Listeria monocytogenes from different serological groups. Low levels of adsorption of bacteriocin MK02R were detected in the cell surface of the producer cells suggesting that bacteriocin MK02R remains bound to the outer surface and that it is released when the pH of the environment increases. The chromatographic studies and the genetic tests using primers nisF and nisR related to specific genes of nisin production confirmed that the antimicrobial MK02R is a natural variant of nisin. The partial sequencing of the reconstructed protein showed that the peptide has an amino acid change in the sequence of the leader peptide compared with nisin A, Z, Q, U and F, but the structure of the mature is homologous to that of nisin F.