Characterization of anti-listeria bacteriocins isolated from shellfish: Potential antimicrobials to control non-fermented seafood

摘要

This work had as main objectives to characterize two bacteriocins produced by lactic acid bacteria (LAB)previously isolated from non-fermented seafood, in order to evaluate their potential as new food protectiveagents. The two bacteriocinogenic isolates were identified by Polymerase Chain Reaction (PCR) using genusandspecies-specific primers, and confirmed by 16S rDNA sequencing, as Enterococcus faecium and Pediococcuspentosaceus. The antimicrobial spectrum of each strain included several indicator microorganisms, some ofthem also isolated from seafood. Growth of Listeria innocua, L. monocytogenes, Staphylococcus aureus, Bacilluscereus and other LAB species were inhibited, although no inhibition of Gram-negative microorganisms wasobserved. Proteolytic, but not lipolytic or glycolytic enzymes, completely inactivated the antimicrobial effect ofboth cell-free supernatants confirming the proteinaceous nature of the inhibitors. The antimicrobial activitywas maintained after treatment with NaCl, SDS, Triton X-100, Tween 20, Tween 80 and EDTA after 2 h or 5 h ofexposure and both bacteriocins were stable over a wide range of pH and temperatures. Production of bacteriocinby E. faecium (bacALP7)was detected initially at exponential phase and reached a maximum activity of25,600 AU/ml in the early stationary phase, whereas bacteriocin production by P. pentosaceus ALP57(bacALP57) reached the maximum at exponential phase with 12,800 AU/ml. The bacteriocins did not kill L.monocytogenes ESB54 nor L. innocua 2030c however, cellular growth was reduced. The partially purifiedbacteriocins, bacALP7 and bacALP57, were below 6.5 kDa in size as determined by Tricine-SDS gel electrophoresis.E. faecium and P. pentosaceus contained DNA fragments corresponding in size to those recorded forenterocin B and pediocin PA-1, respectively. Sequencing of the fragments from both bacteriocins confirmed thehomology. To our knowledge, for the first time two LAB producing bacteriocins similar to pediocin PA-1 andenterocin B, were isolated from non-fermented shellfish. The adaptation of the cultures to seafood matricesmay be advantageous in terms of application as a biopreservation strategy for reduction of L. monocytogeneslevels in seafood products.