Eight foodborne pathogenic and spoilage type gram-positive bacteria were evaluated for their spontaneous resistance frequencies to the peptide antimicrobial nisin. In brain heart infusion medium, spontaneous nisin resistance frequencies were in the range of 10−6 to 10−8 when exposed to nisin at concentrations 2 to 8 times the minimal inhibitory concentrations. A resistant mutant of Listeria monocytogenes Scott A (2000 U nisin per ml) was obtained by increasing stepwise exposure to nisin and was subsequently characterized. Nisin was not inactivated after exposure to mutant or parent cells growing in brain heart medium. Membrane fatty acid composition, phase transition temperature profiles (by differential scanning calorimetry), and specific growth rates of the resistant mutant and parent strain were compared. The resistant mutant had a higher phase transition temperature, higher percentage of straight-chain fatty acids, and a lower percentage of branched chain-fatty acids. The specific growth rate (k) of the resistant mutant was significantly decreased at the suboptimal temperature of 20°C where (k) was 40.9% of the parent strain k. In contrast, at 37°C, the mutant (k) was 87.6% of the parent (k). Collectively, these observations indicated that as a resistance response to nisin, fundamental changes occurred in bacterial membrane structure and function as opposed to a resistance response involving nisin degradation.

Složilová I., Purkrtová S., Kosová M., Mihulová M., Šviráková E., Demnerová K. (2014): Antilisterial activity of lactic acid bacteria against Listeria monocytogenes strains originating from different sources. Czech J. Food Sci., 32: 145-151. Eight individual bacteriocin-producing lactic acid bacteria (LAB) strains and three bacteriocin-non-producing cheese starter cultures were evaluated for their ability to inhibit the growth of six Listeria monocytogenes strains, originating from the guinea-pig lymph nodes, raw cow milk, and manufacturing dairy equipment. Results showed that either live cells or cell-free neutralised supernatant (CFNS) and/or heated CFNS of six individual LAB strains (Lcc. lactis subsp. lactis CCDM 416 and NIZO R5, Lbc. plantarum HV 11 and DC 1246, P. acidilactici HV 12, and Ent. mundtii CCM 1282) and one starter culture (DELVO-ADD ® 100-X DSF) were effective in the suppression of at least one listeria strain. Neither any individual LAB strain nor starter culture was antagonistic toward all studied L. monocytogenes strains, indicating diverse sensitivity/resistance among L. monocytogenes strains to antimicrobial compounds of LAB. The significant susceptibility of listerias isolated from raw milk and dairy equipment together with the strong antilisterial activity of DELVO-ADD ® 100-X DSF could be applied in dairy technology, where commonly used starter cultures could play both the biopreservative and fermentation role.

Lysozyme-chitosan composite films were developed for enhancing the antimicrobial properties of chitosan films. A 10% lysozyme solution was incorporated into 2% chitosan film-forming solution (FFS) at a ratio of 0%, 20%, 60%, and 100% (w lysozyme/w chitosan). Films were prepared by solvent evaporation. Lysozyme release from the film matrix, the antimicrobial activity of films against Escherichia coli and Streptococcus faecalis, and basic film properties were investigated. The lysozyme release proportionally increased with increasing initial concentration of lysozyme in the film matrix, and the amount of released lysozyme was in natural log relationship with time. The films with 60% lysozyme incorporation enhanced the inhibition efficacy of chitosan films against both S. faecalis and E. coli, where 3.8 log cycles reduction in S. faecalis and 2.7 log cycles reduction in E. coli were achieved. Water vapor permeability of the chitosan films was not affected by lysozyme incorporation, whereas the tensile strength and percent elongation values decreased with increased lysozyme concentration. Scanning electron microscopy images revealed that lysozyme was homogeneously distributed throughout the film matrix. This study demonstrated that enhanced antimicrobial activity of lysozyme-chitosan composite films can be achieved by incorporating lysozyme into chitosan, thus broadening their applications in ensuring food quality and safety.

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The recent FDA affirmation of nisin, an antimicrobial peptide, as a GRAS (generally recognized as safe) additive in pasteurized cheese spreads has renewed interest in its potential application in US dairy products. Fluid milks were prepared with varying concentrations of milk fat (0 to 12.9%) and of nisin (0 to 50 U/ml). Biological activity assays using a sensitive indicator microorganism in a well diffusion system indicated that initial nisin activity (50 U/ml) decreased by about 33% when it was added to skim milk and by more than 88% when added to milk containing 12.9% fat. Nisin activity decreased by ca. 50% in milk containing 1.29% fat. Milks containing 0, 10, or 50 U/ml of nisin and varying fat percentages were challenged with approximately log10 7 to 7.5 cfu/ml of log phase Listeria monocytogenes Scott A or Jalisco. At 2 h after inoculation, the viable count of L. monocytogenes Scott A decreased to log10 .30 cfu/ml in skim milk with 50 U/ml of nisin, decreased to log10 2.90 cfu/ml in skim milk with 10 U/ml of nisin, and increased slightly (log10 7.8 cfu/ml) in skim milk without nisin. In half-and-half (12.9% milk fat), nisin was far less effective in inhibiting Listeria with populations decreasing to log10 6.57 cfu/ml for 10 U/ml of nisin and log10 5.87 cfu/ml for 50 U/ml. Similar results were obtained with L. monocytogenes Jalisco. The nonionic emulsifier, Tween 80, partially counteracted decreases of nisin activity in milks, whereas the anionic emulsifier, lecithin, had no effect. Addition of Tween 80 significantly increased the activity of nisin against L. monocytogenes in milk regardless of fat content.

Plantaricin W (Plw) is a new two-peptide bacteriocin, from Lactobacillus plantarum, which inhibits a large number of Gram-positive bacteria. The two peptides, Plwα (comprising 29 residues) and Plwβ (comprising 32 residues), were isolated from the culture supernatants and characterized. The individual peptides had low antimicrobial activity but acted synergistically, and synergism was seen at all mixing ratios tested. The data indicate that the two peptides work in a 1 :1 ratio. Chemical analyses showed that both peptides are lantibiotics, but two unmodified cysteines and one serine residue were present in Plwα, and Plwβ contained one cysteine residue. The Plw structural genes were sequenced and shown to encode prepeptides with sequence similarities to two other two-peptide lantibiotics, namely staphylococcin C55 and lacticin 3147. The conserved residues are mainly serines, threonines and cysteines that can be involved in intramolecular thioether bond formation in the C-terminal parts of the molecules. This indicates that these bacteriocins are members of a new family of lantibiotics with common bridging patterns, and that the ring structures play an important functional role. Based on the data a structural model is presented in which each peptide has a central lanthionine and two overlapping thioether bridges close to their C-termini.

No abstract

This study investigated the antimicrobial activities of chitosan-lysozyme (CL) composite films and coatings against tested microorganisms inoculated onto the surface of Mozzarella cheese. CL film-forming solutions (FFS) with a pH of 4.4 to 4.5 were prepared by incorporating 0% or 60% lysozyme (per dry weight of chitosan) into chitosan FFS with or without a pH adjustment to 5.2. Sliced cheese was subjected to 3 CL package applications: film, lamination on a multilayer coextruded film, and coating. Cheese was inoculated with Listeria monocytogenes, Escherichia coli, or Pseudomonas fluorescens at 10(4) CFU/g, or with mold and yeast at 10(2) CFU/g. Inoculated cheese was individually vacuum packaged and stored at 10 degrees C for sampling at 1, 7, and 14 d for bacteria, and at 10, 20, and 30 d for fungi. Inoculated bacteria survived but failed to multiply in untreated cheese during storage. Treated cheese received 0.43- to 1.25-, 0.40- to 1.40-, and 0.32- to 1.35-log reductions in E. coli, P. fluorescens, and L. monocytogenes, respectively. Incorporation of 60% lysozyme in chitosan FFS showed greater antimicrobial effect than chitosan alone on P. fluorescens and L. monocytogenes. The pH adjustment only affected the antimicrobial activity on L. monocytogenes, with lower pH (unadjusted) showing greater antimicrobial effect than pH 5.2. Mold and yeast increased to 10(5) CFU/g in untreated cheese after 30 d storage. Growth of mold was completely inhibited in cheese packaged with CL films, while 0.24- to 1.90- and 0.06- to 0.50-log reductions in mold populations were observed in cheese packaged with CL-laminated films and coatings, respectively. All CL packaging applications resulted in 0.01- to 0.64-log reduction in yeast populations.