Divergicin M35-Chitosan Film: Development and Characterization.

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.

The Impact of Chitosan-Divergicin Film on Growth of Listeria monocytogenes in Cold-Smoked Salmon.

The aim of this study was to evaluate the impact of chitosan film, with bacteriocin divergicin 35 incorporate, on growth of Listeria monocytogenes in Cold smoked salmon. The simples of Cold-smoked wild salmon were inoculated with L. monocytogenes and treated with chitosan (100 kDa, 94.7% de-acetylated) and divergicin M35 was stored for 3 weeks at 4-8°C. The compounds were applied to the fish flesh in the form of solution or dried film. The film reduced L. monocytogenes to below the detection limit (<50 cfu/g) and kept total counts below 104 cfu per g compared to 109 cfu per g in control samples while the effectiveness of the solution was very limited. The inhibitory activity of the film lasted for 3 weeks, while the solution had no effect on L. monocytogenes counts measured on day 14. The film provided a better preservation of fish color (redness) and firmness than others treatments, while the solution had little impact on these parameters. It kept the volatile basic nitrogen (17.5 mg N/100 g) below the control value 29.9 mg N/100 g. Divergicin-loaded chitosan film thus may represent an interesting alternative for the bio-preservation of cold-smoked fish.

Heat inactivation of hepatitis A virus and a norovirus surrogate in soft-shell clams (Mya arenaria).

The effectiveness of different thermal treatments for inactivating two viruses in clams was evaluated. Soft-shell clam digestive glands experimentally contaminated with hepatitis A virus (HAV) or murine norovirus (MNV) were heated for 90, 180, or 300 seconds at 85°C or 90°C in glass vials or plastic bags with 200 g of soft-shell clam meat. Inactivation was measured by plaque assay and real-time reverse-transcription (RT)-polymerase chain reaction assay. Measured inactivation was similar using both assays. The 90°C for 90 seconds treatment reduced MNV-1 titer by 3.33 log cycles and HAV by 2.66 log cycles. At 90°C for 180 seconds, both MNV-1 and HAV were completely inactivated (titer reduced by 5.47 log cycles) in glass vials. In the presence of clam meat as well, HAV inactivation was complete at 90°C for 180 seconds. In general, HAV was more resistant to heat treatment than MNV-1, suggesting that it would require a more severe treatment than human norovirus for inactivation in soft-shell clams. The results of the present study should contribute to the development of strategies for controlling the spread of enteric viral illness via shellfish.

Evidence of Antibacterial Activities in Peptide Fractions Originating from Snow Crab (Chionoecetes opilio) By-Products.

Antibacterial peptide fractions generated via proteolytic processing of snow crab by-products exhibited activity against Gram-negative and Gram-positive bacteria. Among the bacterial strains tested, peptide fractions demonstrated inhibitory activity against the Gram-negative bacteria such as Aeromonas caviae, Aeromonas hydrophila, Campylobacter jejuni, Listonella anguillarum, Morganella morganii, Shewanella putrefasciens, Vibrio parahaemolyticus and Vibrio vulnificus and against a few Gram-positive bacteria such as Listeria monocytogenes, Staphylococcus epidermidis and Streptococcus agalactiae. The principal bioactive peptide fraction was comprised mainly of proteins and minerals (74.3 and 15.5%, respectively). Lipids were not detected. The amino acid content revealed that arginine (4.6%), glutamic acid (5.3%) and tyrosine (4.8%) residues were represented in the highest composition in the antibacterial peptide fraction. The optimal inhibitory activity was observed at alkaline pH. The V. vulnificus strain, most sensitive to the peptide fraction, was used to develop purification methods. The most promising chromatography resins selected for purification, in order to isolate peptides of interest and to carry out their detailed biochemical characterization, were the SP-Sepharose™ Fast Flow cation exchanger and the Phenyl Sepharose™ High Performance hydrophobic interaction media. The partially purified antibacterial peptide fraction was analyzed for minimum inhibitory concentration (MIC) determination, and the value obtained was 25 µg ml(-1). Following mass spectrometry analysis, the active peptide fraction seems to be a complex of molecules comprised of several amino acids and other organic compounds. In addition, copper was the main metal found in the active peptide fraction. Results indicate the production of antibacterial molecules from crustacean by-products that support further applications for high-value bioproducts in several areas such as food and health.

Pelagic fish hydrolysates as peptones for bacterial culture media.

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.