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Chitosan films loaded with bacteriocin were examined by FTIR spectroscopy, tested for color, puncture strength, water vapor permeability, and as antimicrobials of Listeria innocua HPB13. Divergicin M35, a bacteriocin produced by Carnobacterium divergens, was incorporated into films made with chitosan of molecular mass 2 kDa, 20 kDa, or 100 kDa and de-acetylated either 87% or 95%. Only 100 kDa chitosan yielded films that could be peeled and handled easily. The higher degree of de-acetylation increased the total color factor (ΔE) of bacteriocin-loaded films, their permeability, and puncture strength. Incorporation of divergicin M35 into the films increased amide I peak intensity but otherwise did not induce significant structural change. The FTIR spectra of divergicin M35 shed from the films did not differ from those of the original free bacteriocin, except in overall peak intensity. The release of active divergicin M35 from the film was faster into the buffer than into tryptic soy broth and peaked at 10-12 h in both cases. Chitosan 95% de-acetylated and loaded with divergicin M35 was the most active, producing a six-log drop in Listeria innocua HPB13 viable count within 24 h. These results suggest that the biocompatible and biodegradable films developed here have the potential for application as antimicrobials of Listeria spp. in foods, especially ready-to-eat, minimally processed products.

Cold-smoked salmon treated with divergicin M35-producing Carnobacterium divergens M35, C. divergens ATCC 35677 (a non-producer of bacteriocin), purified divergicin M35 or supernatants of C. divergens M35 culture in snow crab hepatopancreas (SCH) medium or MRS broth was challenged with Listeria monocytogenes (up to 10 3 CFU/g). Samples were stored at 4 °C for up to four weeks. L. monocytogenes , total bacterial and lactic acid bacterial counts were determined along with changes in total volatile base nitrogen (TVBN) and biogenic amine production as well as texture, color and odor. A 2.6 log CFU/g reduction in L. monocytogenes was obtained for up to 10 days of storage in samples treated with C. divergens M35. Purified divergicin M35 (50 μg/g), SCH supernatant or MRS supernatant brought reductions of 1 log CFU/g at the beginning of storage. However, the anti-listerial activity of the supernatants lasted for 15 days compared to 3 days for purified divergicin M35. Color and texture were affected little in samples containing C. divergens M35 compared to un-inoculated samples. TVBN and biogenic amine production, particularly tyramine, remained below the maximum acceptable level in fish appreciation. These results clearly show the potential of C. divergens M35 culture as well as divergicin M35 bio-ingredient for application to the inactivation of L. monocytogenes in ready-to-eat seafood.

The antimicrobial activities of the class IIa bacteriocin divergicin M35 and several types of chitosan against Listeria monocytogenes were quantified by agar diffusion, critical micro-dilution, and viable count and observed by electron microscopy. Antimicrobial activity of chitosan depended on its molecular mass (MM) and the pH. Three chitosans with MM values of 2, 20, and 100 kDa and 87.4% degree of deacetylation (DDA) were chosen for further study, based on high anti-listerial activity at pH 4.5. Electron microscopy suggested that the mechanism of anti-listerial activity also varied with the MM. Low-MM chitosan appeared to inhibit L. monocytogenes by affecting cell permeability and growth, whereas medium- and high-MM chitosan may form a barrier on the cell surface that prevents entry of nutrients. The minimum inhibitory concentrations (MICs) of 2, 20, and 100 kDa chitosan and divergicin M35 against a divergicin-resistant strain of L. monocytogenes (LSD 535) were 2.5, 2.5, 0.625, and 0.25 mg/mL, respectively. The combination of any of these 3 chitosans and divergicin M35 appeared to have an additive effect against L. monocytogenes, as determined by fractional inhibitory concentration (FIC) index. This study provides useful data for the development of chitosan films incorporating divergicin M35 for inhibiting L. monocytogenes in foods.

For several years in the Quebec fisheries’ industry,landings of pelagic fish have been calculated at over 4000 tons. These under-exploited species, rich in lipids and proteins, could be used in valuable new products. In the present study, hydrolysates of mackerel and herring were produced and utilized as sources of peptones in the formulation of new bacterial culture media. The molecular weight distribution analysis showed that molecules present in the hydrolysates were lower than 1300 Da for herring, and lower than 930 Da for mackerel. The formulated media were compared with reference media using 6 bacterial strains (3 lactic acid (LAB) and 3 non-lactic). The absorbance (OD) and carbohydrate measurements revealed that the formulated media possessed similar yields in comparison with the reference media. Finally, the inhibition of Listeria innocua by LAB bacteriocins was evaluated. Results obtained for Pediococcus acidilactici demonstrated high activities for each medium studied. Thus, the medium containing herring peptones generated the highest bacteriocin titre (32768 AU/mL), followed by both the medium containing mackerel peptones and the MRS7 medium (16384 AU/mL). Each medium containing the fish hydrolysates efficiently supported the growth of the bacterial strains. Pelagic fish peptones are promising as a novel bacterial culture media.

Carnobacterium divergens M35, isolated from a commercial sample of frozen smoked mussels, produces a new bacteriocin, divergicin M35, a class IIa bacteriocin. Divergicin M35 is sensitive to pronase-E, alpha-chymotrypsin and proteinase K, but not to trypsin and withstands thermal treatments up to 121 degrees C for 30 min. Divergicin M35 was extracted from the culture supernatant of C. divergens M35 using an SP-Sepharose cation-exchange column, desalted and purified on a C18 Sep-Pack column and further purified by reverse phase-high pressure liquid chromatography. This procedure allowed the recovery of 10% of the bacteriocin present in the culture supernatant with purity higher than 99%. Divergicin M35 had a molecular mass of 4518.75 Da as determined by mass spectrometry, a pI value of 8.3 and positive net charge (+3). The amino acid sequence of divergicin M35 was found to consist of 43 amino acid with four cysteine residues (Cys10, 15, 25, 43) and showed 80.5% homology with divercin V41 (80.5%) and 80.0% with bavaricin MN. Divergicin M35 showed powerful antilisterial activity, especially against Listeria monocytogenes and was also active against carnobacteria but not against strains of Lactococcus, Lactobacillus, Enterococcus, Bifidobacteria and Escherichia. Divergicin M35 production began in late exponential phase and reached a maximum activity of 65,000 AU/ml in early stationary phase. Initial broth pH, Tween 80 and acetate did not affect C. divergens M35 growth or divergicin production. This bacteriocin may be a potential tool for inhibiting L. monocytogenes in seafood products that do not usually undergo an adequate heat treatment.