Studies on the Microbiology of Fish and Shellfish with Emphasis on Bacteriocin-like Substances to control Listeria monocytogenes

Abstract

Seafood permits the transmission of many bacterial pathogens. In order to reconcile consumer demands with important safety standards, traditional means of regulating microbial spoilage and safety hazards in foods are combined with novel technologies. These include biological antimicrobial systems, such as the use of lactic acid bacteria (LAB) and/or their bacteriocins, such as Carnobacterium maltaromaticum CS526 and its bacteriocin piscicocin CS526. The aims of this study were to investigate the presence of Listeria monocytogenes in temperate seafood, namely fresh and smoked salmon, fresh and smoked haddock, and fresh mussels and oysters. Additionally, there was an aim to recover, characterise and use bacteriocin-like-substance to control Listeria monocytogenes in cold smoked haddock. Vibrio spp., Enterobacteriaceae representatives, total aerobic heterotrophic counts and Listeria monocytogenes were isolated from commercially prepared smoked and fresh Atlantic salmon, smoked and fresh haddock, live mussels and oysters using selective media and tryptone soya agar (TSA). Vibrio spp. occurred in high densities (>106 CFU gˉ1) in mussels and Enterobacteriaceae representatives were recorded at >106 CFU gˉ1 in fresh salmon. Total aerobic heterotrophic counts in fresh salmon, live mussels and oysters reached 107, > 107, and > 106 CFU gˉ1, respectively. Listeria monocytogenes was recorded at 5.0 x 104 CFU gˉ1 in mussels. In total sixty one bacterial isolates were recovered from the seafood examined. The results revealed 19 genera of bacteria, i.e. Acinetobacter, Aerococcus, Aeromonas, Bacillus, Brochothrix, Carnobacterium, Citrobacter, Corynebacterium, Enterobacter, Escherichia coli, Moraxella, Micrococcus, Pseudomonas, Psychrobacter, Serratia, Shewanella, Staphylococcus, Vibrio and Listeria. The prominent characteristics of fish spoilage isolates were demonstrated by the ability of the isolates to reduce trimethylamine oxide (TMAO) to trimethylamine, and to produce H₂S. Sh. baltica OS185, Aeromonas spp. HB-6, Sh. baltica, Sh. putrefaciens, A. hydrophila HX201006-3, A. salmonicida subsp. achromogenes, A. hydrophila, C. freundii, Enterobacter cloacae were strong producers of TMA and H₂S. The spoilage microorganisms were tested for potential pathogenicity. The result revealed that 6/15 of the spoilage microorganisms produced proteolytic, lecithinase, blood (β and α haemolysin) and elastinase activity, respectively, whereas 7/15 of the spoilage microorganisms showed lipolytic activity. Cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances of Carnobacterium maltaromaticum MMF-32 and KOPRI 25789 producing strains isolated from commercially prepared smoked salmon were investigated for their potential antimicrobial activity against potentially pathogenic and food spoilage microorganisms. Generally, a broad spectrum of activity was revealed against potentially pathogenic and food spoilage microorganisms in vitro. Cold-smoked haddock treated with bacteriocin producing C. maltaromaticum MMF-32, C. piscicola A9b bacˉ phenotype nonbacteriocin producing strain a mutant of C. piscicola A9b bac+, cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances was challenged with L. monocytogenes ATCC 19114 up to 103 CFU gˉ1, respectively. Samples were stored at 4 °C for 10 days. L. monocytogenes and total bacterial counts were determined along with changes in total volatile base nitrogen (TVBN) and biogenic amines production as well as texture, colour and odour. Although the study on anti-listerial effects of C. maltaromaticum MMF-32 was not successful, this organism did have a positive effect on retention of firmness and sensory perception in cold smoked haddock.