Antilisterial Effect Research Articles

Development of Whey Protein Concentrate / Pullulan Composite Films Containing Bacteriophage A511: Functional Properties and Anti-Listerial Effects During Storage

Development of Whey Protein Concentrate / Pullulan Composite Films Containing Bacteriophage A511: Functional Properties and Anti-Listerial Effects During Storage

Encapsulation of Purified Pediocin of Pediococcus pentosaceus into Liposome Based Nanovesicles and its Antilisterial Effect.

To encapsulate a purified bacteriocin into a nanovesicles and check its antibacterial effect. Although the use of nano-encapsulated bacteriocins in food matrices is poorly reported, encapsulated nisin can reduce L. monocytogenes counts in whole and skimmed milk and in soft cheese. The present study deals with the extraction and purification of a bacteriocin from an isolated strain Pediococcus pentosaceus KC692718. A comparative study of the effect of free pediocin and liposome encapsulated pediocin against Listeria sp. was performed. The purification of the extracted cell free supernatant was subjected to ammonium sulphate precipitation, cation exchange chromatography followed by gel permeation chromatography. The bacteriocin activity and protein concentration were determined using Lowry's method. The characterization of the pure pediocin was done. Liposome like nanovesicle was constructed and the stability of the liposome encapsulated pediocin was checked. Finally, the antibacterial effect was comparatively studied of the free pediocin, liposome, and liposome encapsulated pediocin simultaneously. The pediocin of 3.6kDa was purified with a specific activity of 898.8. AU/mg. It remained stable from pH 2.0-8.0 was found to be moderately stable above 80°C and remain stable for one month when stored at -20°C. The encapsulated pediocin showed stability since it retained 50% of its initial activity. The encapsulated pediocin showed 89% of encapsulation efficiency. The encapsulated pediocin not only improved pediocin stability but also enhanced the controlled release of the antimicrobial substances, enough for inhibiting the foodborne pathogen L. monocytogenes.

Antilisterial Effect of a Natural Formulation Based on Citrus Extract in Ready-To-Eat Foods.

Controlling Listeria in food is a major challenge, especially because it can persist for years in food processing plants. The best option to control this pathogen is the implementation of effective cleaning and disinfection procedures that guarantee the safety and quality of the final products. In addition, consumer trends are changing, being more aware of the importance of food safety and demanding natural foods, minimally processed and free of chemical additives. For this reason, the current consumption model is focusing on the development of preservatives of natural origin, from plants or microorganisms. In sum, this study aimed to evaluate the antimicrobial effectiveness of a citrus extract formulation rich in flavonoids against several L. monocytogenes and L. innocua strains, using in vitro test (agar diffusion test, minimum bactericidal concentration (MBC), and time-kill curves) and challenge test in food trials (carne mechada, salami, fresh salmon, lettuce, brine, and mozzarella cheese). The results presented in this work show that citrus extract, at doses of 5 and 10%, had a relevant antimicrobial activity in vitro against the target strains tested. Besides this, citrus extract applied on the surface of food had a significant antilisterial activity, mainly in carne mechada and mozzarella cheese, with reductions of up to eight logarithmic units with respect to the control. These results suggest that citrus extract can be considered a promising tool to improve the hygienic quality of ready-to-eat foods.

Monitoring Growth Compatibility and Bacteriocin Gene Transcription of Adjunct and Starter Lactic Acid Bacterial Strains in Milk

When developing protective starter cultures for application in cheese technologies, monitoring growth interactions between starter and adjunct lactic acid bacterial (LAB) species and in situ expression of bacteriocin genes in the mixtures is crucial. This study first aimed to monitor the growth of mixed LAB strain populations during milk model fermentations by microbial counts and real-time quantitative PCR. The primary starter strains, Streptococcus thermophilus ST1 and costarter Lactococcus lactis subsp. cremoris M78, served as the basic starter composite coinoculated in all milk treatments. Adjunct bacteriocinogenic Enterococcus faecium strains KE82 and GL31 and the ripening Lactiplantibacillus plantarum H25 strain were added separately to the starter composite, resulting in four LAB combination treatments. The second aim was to quantify gene transcripts of nisin and enterocins B and A synthesized by strains M78, KE82, and GL31, respectively, by reverse transcription-real-time quantitative PCR and to detect the in situ antilisterial effects of the cocultures. Adjunct LAB strains showed growth compatibility with the starter, since all of them exhibited 2- to 3-log-unit increases in their population levels compared to their initial inoculation levels, with ST1 prevailing in all treatments. KE82 grew more competitively than GL31, whereas cocultures with KE82 displayed the strongest in situ antilisterial activity. Nisin gene expression levels were higher at the exponential phase of microbial growth in all treatments. Finally, the expression levels of nisin and enterocin A and B genes were interrelated, indicating an antagonistic activity.

Synergistic antimicrobial activities of plant essential oils against Listeria monocytogenes in organic tomato juice

The objective of this study was to identify combinations of plant essential oils (EOs) that have synergistic antilisterial effects in raw organic tomato juice. Among the EOs approved for addition to organic foods in the Republic of Korea, twenty-four EOs were screened for antilisterial activity using a well diffusion assay. Six EOs (cinnamon bark, lemon, lemongrass, oregano, peppermint, and thyme thymol) showed relatively strong inhibitory activities (diameter of zone of inhibition ≥ 35.0 mm) against Listeria monocytogenes. The minimal inhibitory concentrations (MICs) and minimal lethal concentrations (MLCs) of these EOs against L. monocytogenes in a laboratory medium were determined. Oregano and thyme thymol EOs had the lowest MIC (0.3125 μL/mL), and cinnamon bark, oregano, and thyme thymol EOs had the lowest MLC (0.6250 μL/mL). The synergistic antilisterial activities of combinations of EOs in a laboratory medium were determined using a modified checkerboard assay. Combinations of cinnamon bark and lemongrass EOs, cinnamon bark and thyme thymol EOs, and lemongrass and thyme thymol EOs showed synergistic antilisterial activities (fractional inhibitory concentration index = 0.5). A combination of cinnamon bark and thyme thymol EOs was tested for synergistic antilisterial activity in tomato juice incubated at 10 °C and 25 °C for up to 48 h. The number of L. monocytogenes in juice treated with combined EOs at a total concentration of 0.3125 μL/mL decreased significantly (P ≤ 0.05) compared to numbers in juice treated with 0.3125 μL/mL of either cinnamon bark EO or thyme thymol EO alone. These observations show the potential for using combinations of EOs for controlling L. monocytogenes in juice released from cut or bruised raw organic tomatoes intended for consumption in food service or home settings.

Application of Enterococcus faecium KE82, an Enterocin A-B-P–Producing Strain, as an Adjunct Culture Enhances Inactivation of Listeria monocytogenes during Traditional Protected Designation of Origin Galotyri Processing

The ability of the enterocin A-B-P-producing Enterococcus faecium KE82 adjunct strain to inactivate Listeria monocytogenes during protected designation of origin Galotyri processing was evaluated. Three trials were conducted with artisan cheeses made from traditionally "boiled" (85°C) ewe's milk. The milk was cooled at 42°C and divided in two treatments. A1 milk was inoculated with Streptococcus thermophilus ST1 and Lactococcus lactis subsp. cremoris M78, and A2 was inoculated with the basic starter ST1+M78 plus KE82 (step 1). All milks were fermented at 20 to 22°C for 24 h (step 2), and the curds were drained at 12°C for 72 h (step 3) and then salted with 1.5 to 1.8% salt to obtain the fresh Galotyri cheeses (step 4). These fresh cheeses were then ripened at 4°C for 30 days (step 5). Because artificial listerial contamination in the dairy plant was prohibited, samples of A1 and A2 cheese milk (200 mL) or curd (200 g) were collected after steps 1 through 5, inoculated with L. monocytogenes 10 (3 to 4 log CFU/mL or g), incubated at 37, 22, 12, and 4°C for predefined periods, and analyzed for microbial levels and pH. L. monocytogenes levels declined in all cheese curd portions contaminated after steps 2 through 5 (pH 4.36 to 4.84) when stored at 4 or 12°C for 15 days. The final net reductions in Listeria populations were 2.00-, 1.07-, 0.54-, and 0.61-log greater in the A2 than in the A1 curd portions after steps 2, 3, 4, and 5, respectively. In step 1, conducted to simulate the whole cheese milk fermentation process, L. monocytogenes levels declined by 1.47 log CFU/mL more in the A2 than in the A1 milk portions after 72 h at 22°C; however, slight growth (0.6 log CFU/mL) occurred during the first 6 h at 37°C. E. faecium KE82 was compatible with the starter culture and enhanced inactivation of L. monocytogenes during all steps of Galotyri cheese processing. The antilisterial effects of the combined acid and enterocin were the weakest in the fermenting milks, the strongest in the unsalted fermented curds, and declined again in the salted fresh cheeses.

Antimicrobial effect of nisin in processed cheese - Quantification of residual nisin by LC-MS/MS and development of new growth and growth boundary model for Listeria monocytogenes

This study tested the hypothesis that growth of Listeria monocytogenes in processed cheese with added nisin can be predicted from residual nisin A concentrations in the final product after processing. A LC-MS/MS method and a bioassay were studied to quantify residual nisin A concentrations and a growth and growth boundary model was developed to predict the antilisterial effect in processed cheese. 278 growth rates were determined in broth for 11 L. monocytogenes isolates and used to determine 13 minimum inhibitory concentration (MIC) values for nisin between pH 5.5 and 6.5. To supplement these data, 67 MIC-values at different pH-values were collected from the scientific literature. A MIC-term was developed to describe the effect of pH on nisin MIC-values. An available growth and growth boundary model (doi: https://doi.org/10.1016/j.fm.2019.103255) was expanded with the new MIC-term for nisin to predict growth in processed cheese. To generate data for model evaluation and further model development, challenge tests with a total of 45 growth curves, were performed using processed cheese. Cheeses were formulated with 11.2 or 12.0 ppm of nisin A and heat treated to obtain residual nisin A concentrations ranging from 0.56 to 5.28 ppm. Below 15 °C, nisin resulted in extended lag times. A global regression approach was used to fit all growth curves determined in challenge tests. This was obtained by combining the secondary growth and growth boundary model including the new term for the inhibiting effect of nisin on μmax with the primary logistic growth model with delay. This model appropriately described the growth inhibiting effect of residual nisin A and showed that relative lag times depended on storage temperatures. With residual nisin A concentrations, other product characteristics and storage temperature as input the new model correctly predicted all observed growth and no-growth responses for L. monocytogenes. This model can support development of nisin A containing recipes for processed cheese that prevent growth of L. monocytogenes. Residual nisin A concentrations in processed cheese were accurately quantified by the developed LC-MS/MS method with recoveries of 83 to 110% and limits of detection and quantification being 0.04 and 0.13 ppm, respectively. The tested bioassay was less precise and nisin A recoveries varied for 53% to 94%.

Antilisterial effects of lysozyme-nisin combination at temperature and pH ranges optimal for lysozyme activity: Test of key findings to inactivate Listeria in raw milk

This study aimed to determine antilisterial potential of lysozyme (LYS)-nisin (NIS) combination at temperatures and pHs optimal for LYS activity. Tests in buffers at pH 4.5 and 6.0 showed that heating at 50 or 60 °C combined with LYS-NIS caused higher Listeria innocua inactivation (- 6.2 to >6.6 log) than heating alone (- 0.05 to 5.5 log), or heating combined with LYS (- 4.34 to 6.0 log) or NIS (3.9 to >6.6). The antimicrobial performance of LYS-NIS in buffer at 50 °C was not pH-dependant (5.8–5.9 logs) while heating at 60 °C with LYS-NIS at pH 6.0 (>6.6 logs) caused higher Listeria reduction than that at pH 4.5 (5.7 logs). Heating at 50 °C for 45 min alone or in combination with LYS-NIS caused 0 and 5.5 logs Listeria reduction in milk, respectively. In contrast, Listeria inactivation in milk at 60 °C occurred mainly by heat (5.5 logs) with limited contribution of LYS and/or NIS. Milk heated at 50 °C maintained 73% of NIS and 63% of LYS activity. Application of LYS-NIS at 50 °C provides an opportunity to improve milk safety with less destruction of milk enzyme and microbial flora necessary to obtain desired ripening periods, and aroma and flavour in traditional cheeses.

Improved control of Listeria monocytogenes during storage of raw salmon by treatment with the fermentate Verdad N6 and nisin

Fresh Atlantic salmon (Salmo salar) represents a healthy, nutritious food with global distribution and increasing consumption and economic value. Contaminating Listeria monocytogenes in fresh salmon represents a health hazard to consumers, is linked to extensive product recalls and is a major challenge for salmon processors. Verdad N6, a commercially available buffered vinegar, was evaluated as a treatment for raw salmon fillets either alone or in combination with the antimicrobial peptide nisin, with regard to anti-listerial effects under processing and storage, and influence on sensory quality and background microbiota. Salmon fillets were surface contaminated with L. monocytogenes and immersed in solutions of Verdad N6 or treated with nisin or a combination of these two treatments. Levels of L. monocytogenes were determined during vacuum-pack refrigerated storage. The use of Verdad N6 resulted in increased lag times and substantially reduced growth of L. monocytogenes. The inhibitory effects were dependent on Verdad N6 levels, immersion time, and storage time and temperature. A 5 s immersion in 10% Verdad N6 solution at 4 °C reduced growth of L. monocytogenes from log 2.8 to log 1 after 12 days of storage. Nisin (0.2–1 ppm) had listericidal effects up to 1 log but did not inhibit regrowth when used alone. Appropriate combinations of Verdad N6 and nisin led to L. monocytogenes levels no higher after 12 days of storage than the initial levels. The inhibitory effects were markedly lower at 7 °C than at 4 °C. Salmon with Verdad N6 showed reduced levels of total counts during storage indicating a longer shelf-life, and a shift in the dominating bacteria with reduced and increased relative levels of Enterobacteriaceae and lactic acid bacteria, respectively. Sensory analyses of raw and cooked Verdad N6 treated a non-treated salmon resulted in small differences. In summary, Verdad N6 is an option for production of high-quality raw salmon with increased shelf-life and enhanced food safety through its Listeria inhibiting effects. The application of Verdad N6 in combination with nisin treatment can further reduce the listeria-risks of these products.

The Phage Holin HolGH15 Exhibits Potential As an Antibacterial Agent to Control Listeria monocytogenes.

Listeria monocytogenes is an important foodborne pathogen that is a serious threat to public health security, and new strategies to control this bacterium in food are needed. HolGH15, derived from Staphylococcus aureus phage GH15, has shown antibacterial activity against several bacterial species. In this work, the antilisterial behavior and effectiveness of HolGH15 are further studied. To elucidate its antimicrobial modes against L. monocytogenes, cell integrity and membrane permeabilization assays were performed. When treated with HolGH15, the release of 260-nm-absorbing materials of L. monocytogenes was rapidly increased. HolGH15 triggered a significant increase in fluorescence intensity by flow cytometry. In membrane permeabilization assays, the cytoplasmic β-galactosidase of L. monocytogenes treated with HolGH15 was released via an increase in the permeability of the membrane. HolGH15 caused changes in the structural properties of L. monocytogenes cells resulting in shrinkage, which evoked the release and removal of cellular contents and finally lead to cell death. Electron microscopy observations indicated that HolGH15 exhibited excellent bactericidal potency by permeabilizing the cell membrane, damaging membrane integrity, and inducing cellular content shrinkage or loss. Moreover, HolGH15 (at the final concentration of 240 μg/mL) reduced L. monocytogenes (at the initial concentration of 106 colony-forming unit/mL) to an undetectable level at 4°C. Collectively, HolGH15 has potential as a novel antimicrobial agent against L. monocytogenes in the manufacture and store of food by spraying or soaking, especially at refrigerated temperature.

Synergistic, bacteriostatic effect of propolis and glycerol against Listeria monocytogenes in chocolate milk under refrigerated storage.

Propolis ethanolic extracts, with or without glycerol, were added into pasteurized, non-fat chocolate milk, which was artificially contaminated with Listeria monocytogenes. The addition of propolis ethanolic extracts dissolved into glycerol led to a definite anti-listerial effect in milk stored at 4 ℃, with both propolis concentrations tested (2 or 4 mg of dry propolis ethanolic extract per milliliter of chocolate milk) leading to inhibition of L. monocytogenes growth throughout 20 days of storage. The combined addition of propolis ethanolic extracts with glycerol was also effective in significantly reducing the rate of growth of L. monocytogenes in chocolate milk stored under improper (10 ℃) refrigeration storage conditions (more than five-fold increase in the generation time of L. monocytogenes compared to control trials). Finally, the combined addition of a deodorized propolis ethanolic extract with glycerol resulted in a significant anti-listerial effect upon storage of contaminated milk at 4 ℃ (more than three-fold increase in the generation time of L. monocytogenes compared to controls) and in a smaller anti-listerial effect upon milk storage at 10 ℃ (two-fold increase in the generation time of the pathogen compared to controls). Of note, chocolate milk containing deodorized propolis ethanolic extract and glycerol received a positive consumer acceptability score on the nine-point hedonic scale (median acceptability score of "7"). Hence, propolis may possess a promising role as a natural anti-listerial preservative in dairy drinks.

Cell Growth Density and Nisin A Activity of the Indigenous Lactococcus lactis subsp. cremoris M78 Costarter Depend Strongly on Inoculation Levels of a Commercial Streptococcus thermophilus Starter in Milk: Practical Aspects for Traditional Greek Cheese Processors

Mixed thermophilic and mesophilic commercial starter cultures (CSCs), particularly those including Streptococcus thermophilus as a primary milk acidifier, have been found to reduce growth and counteract in situ nisin A (NisA+) antilisterial effects by the novel, indigenous Lactococcus lactis subsp. cremoris M78 costarter in traditional Graviera thermized milk cheese curds. Therefore, this model challenge study evaluated growth and in situ NisA+ activity of strain M78 in coculture with S. thermophilus ST1 singly in sterilized raw milk (SRM). Strain ST1, derived from a CSC for cheese, was challenged at two inoculation levels (5 and 7 log CFU/mL) in SRM against 6 and 3 log CFU/mL of strain M78 and Listeria monocytogenes, respectively. Pure cultures of each strain and cocultures of strain ST1 with the CSC L. lactis LL2, in replacement of strain M78, served as controls. At the high (7-log) inoculation level, the rapid, competitive growth (>9.3 log CFU/mL) of S. thermophilus ST1 reduced growth of both L. lactis by at least 10-fold; the industrial strain LL2 retained slightly higher relative population densities (7.4 to 9.1%) than the wild NisA+ strain M78 (3.8 to 5.6%) after 6 h at 37°C, followed by an additional 66 h of incubation at 22°C. In full contrast, at the low (5-log) inoculation level, S. thermophilus ST1 failed to predominate in SRM at 6 h; thus, the starter lactic acid bacteria populations were reversed in favor of L. lactis. Notably, strain M78 retained higher relative population densities (83.0 to 90.1%) than the CSC strain LL2 (80.3 to 85.2%) at 22°C. Moreover, at the 5-log ST1 level, the direct and deferred in situ NisA+ activities of strain M78 were at similar levels with its pure culture with L. monocytogenes in SRM, whereas at the 7-log ST1 level, the respective NisA+ effects were counteracted. Hence, 10- to 100-fold lowered inoculation levels of CSC S. thermophilus are required to enhance the performance of the M78 costarter in traditional Greek cheese technologies.

Preparation of phosphatidylcholine nanovesicles containing bacteriocin CAMT2 and their anti-listerial activity

A novel bacteriocin CAMT2, produced by Bacillus amyloliquefaciens ZJHD3-06, has potential as a natural biopreservative for the control of food-borne spoilage and pathogenic bacteria. To avoid interaction of CAMT2 with components of food that may adversely impact its antibacterial activity, CAMT2 was encapsulated into nanovesicles prepared from soybean phosphatidylcholine. The encapsulation of CAMT2 exhibited a limited impact on functional structure and crystallinity of bacteriocin CAMT2, but a high anti-listerial activity in agar, and increase its stability in food at refrigeration temperature (4 °C). The results also showed that both encapsulated and free CAMT2 had good anti-listerial effect in skim milk at refrigeration temperature. However, encapsulated CAMT2 performed better than free CAMT2 against Listeria in whole milk. These results showed that nano-encapsulation is an effective method of protecting bacteriocin from fat in milk and retaining its antimicrobial efficacy.

Elucidating antimicrobial mechanism of nisin and grape seed extract against Listeria monocytogenes in broth and on shrimp through NMR-based metabolomics approach

Nisin and grape seed extract (GSE) have been widely used as food preservatives; however, the mechanism against pathogens at molecular level has not been well elucidated. This work aimed to investigate their antimicrobial effect against Listeria monocytogenes and to elucidate the mechanism by NMR-based metabolomics. Nisin exhibited enhanced in vitro antilisterial effect when combined with GSE (4.49 log CFU/mL reduction). Marked change in cell membrane permeability was observed in the combination group using confocal laser scanning microscopy; this was verified by increased leakage of protein and nucleic acid. The underlying antimicrobial mechanism was revealed by NMR coupled with multivariate analysis. Significant decreases in threonine, cysteine, ATP, NADP, adenine were observed, whereas a few of metabolites such as lactic acid and γ-aminobutyric acid (GABA) increased after nisin-GSE treatment (P < 0.05). Pathway analysis further manifested that the nisin-GSE inhibited the survival of L. monocytogenes by blocking the TCA cycle, amino acid biosynthesis and energy-producing pathway. Lastly, nisin and GSE were applied to shrimp and binary combination showed remarkably antilisterial activity (1.79 log CFU/g reduction). GABA shunt and protein degradation from shrimp compensated the unbalanced glycolysis and amino acid metabolism by providing energy and carbon source for L. monocytogenes inoculated on shrimp. Thus, they were more tolerant to nisin and GSE stresses as compared to the broth-grown culture.

Antilisterial effect of juniper (Juniperus communis) and its mixed application with winter savory (Satureja montana) in beef protection

Juniperus communis essential oil and post-distillation waste were tested for their antibacterial potential against common food contaminants. Results of microdilution assay directed further study of Listeria monocytogenes. Checkerboard assay showed synergistic antilisterial effect of both substances with conventional antibiotics. By evaluating of their cytotoxicity on human colon cells, post-distillation waste was denoted as selective against L. monocytogenes, being able to interfere with its in vitro adhesion on colon cells. On the other hand, the food preservative potential of J. communis essential oil was studied and compared with the activity of Satureja montana essential oil. In a microdilution assay, both substances induced antilisterial effect against reference ATCC 19111 strain and three wild isolates. A checkerboard assay showed synergism against isolates. An in vitro time-kill assay was used to confirm the types of interaction, and to estimate curve MIC values. Finally, in situ antilisterial efficacies of the individual essential oils and their mixture on red wine-marinated beef, previously inoculated with ATCC 19111 strain or primoisolate from beef carcass, were determined. All treatments enhanced the antilisterial potential of wine marinade. In conclusion, derivatives of J. communis possess significant antilisterial potential both in in vitro and in situ conditions, so further research is advised.

Antilisterial Effect and Influence on Listeria monocytogenes Gene Expression of Enterocin or Enterococcus faecalis in Sliced Dry-Cured Ham Stored at 7°C

In this study, we focused on the effect of an enterocin or an Enterococcus faecalis strain added onto sliced dry-cured ham that was artificially inoculated with Listeria monocytogenes and stored at 7°C. The population of L. monocytogenes and the expression of five genes were monitored throughout the storage period. A persistent and a nonpersistent strain were tested, and both were influenced by the presence of the enterocin; both populations were reduced by more than 2 Log CFU/g after 14 days compared with the control, noninoculated ham. The presence of E. faecalis, a bacteriocin-producing lactic acid bacterium, had a less pronounced effect on the viable counts for both strains. Concerning gene expression, a common trend observed for both strains in the presence of enterocin was the down-regulation of genes tested after 30 min of storage at 7°C. For the remainder of the storage period, the expression fluctuated but was mostly reduced. Similarly, the presence of E. faecalis led to an overall down-regulation of genes. The effect on gene expression of both enterocin and E. faecalis was more pronounced on the nonpersistent L. monocytogenes strain. Although the potential of a bacteriocin and a bacteriocin-producing microorganism to control L. monocytogenes was confirmed, this study highlights that gene expression may be influenced and needs to be evaluated when considering such biopreservation interventions.

Combined Antilisterial Effects of Barberry Extract, Cinnamaldehyde and Nisin

Combined Antilisterial Effects of Barberry Extract, Cinnamaldehyde and Nisin

Sublethal Injury Caused to Listeria monocytogenes by Natural Plant Extracts: Case Study on Grape Seed Extract and Garlic Extract

Natural antimicrobials, such as grape seed extract (GSE) and garlic extract (GE), are often used as (a part of) novel food preservation technologies, especially due to their antilisterial effect. However, little is known on the extent of sublethal injury (SI) these extracts cause to Listeria monocytogenes, possibly leading to overestimated efficacies for such novel technologies. The influence of both extracts on the sublethal injury of L. monocytogenes strain LMG23775 was investigated, also using propidium iodide staining to investigate the nature of the injury. Minimum inhibitory concentrations were 500 mg gallic acid equivalents (GAE)/L and 7.5 μg allicin/mL for GSE and GE, respectively. These concentrations slowed down the growth of L. monocytogenes, while only causing a significant amount of SI for GSE. Pure extracts caused inactivation of the cells, with GSE being the most effective. Sublethal injury from pure GSE was mainly caused by membrane damage. In pure GE, a significant amount of SI, not caused by membrane damage, was also present, albeit less pronounced than in pure GSE. In conclusion, both extracts cause a significant amount of sublethal injury to L. monocytogenes, which is not taken into account in relevant studies investigating novel food preservation applications involving natural plant extracts.

Preliminary Evaluation of Anti-Listerial Bacteriocin-like Peptide Produced by Enterococcus lactis PMD74 Isolated from Ezine Cheese

Enterococcus lactis PMD74 is a novel strain with a notably high antimicrobial activity. The present study evaluated the anti-listerial effect of a bacteriocin-like peptide (BLIP) produced by E. lactis PMD74 isolated from Ezine cheese (PDO). The strain was screened for its antimicrobial activity against 22 indicator strains using both agar spot and well diffusion methods. We observed that the neutralized cell-free supernatant (CFS) of E. lactis PMD74 exhibited varying levels of antimicrobial activity against both closely and distantly related pathogenic strains, with the highest activity displayed against Listeria monocytogenes strains. Although thermostable and resistant to lysozyme treatment, BLIP could be completely inactivated by trypsin, proteinase K, and α-chymotrypsin treatments. BLIP production starts in the early exponential growth phase of E. lactis PMD74 (3 h incubation, 400 AU mL–1) and reaches its maximal production (6400 AU mL–1) at the end of the exponential growth phase. Moreover, it is stable in the pH range of 2.0 to 7.0. The treatment of cultures of L. monocytogenes ATCC 7644 and Escherichia coli ATCC 26922 with sterilized CFS exhibited bactericidal and bacteriostatic effects, respectively. Furthermore, co-inoculation of L. monocytogenes ATCC 7644 and E. lactis PMD74 in skim milk led to complete loss of viability of L. monocytogenes ATCC 7644. These findings suggest that BLIP produced by E. lactis PMD74 could serve as a promising food preservative agent owing to its bactericidal and bacteriostatic properties.

The Influence of Heat-Killed Enterococcus faecium BGPAS1-3 on the Tight Junction Protein Expression and Immune Function in Differentiated Caco-2 Cells Infected With Listeria monocytogenes ATCC 19111.

Listeria monocytogenes, the common foodborne pathogenic bacteria species, compromises the intestinal epithelial barrier, leading to development of the listeriosis, a severe disease especially among immunocompromised individuals. L. monocytogenes infection usually requires antibiotic treatment, however, excessive use of antibiotics promotes emergence of antibiotic resistance and the destruction of gut microbiota. Probiotics, including lactic acid bacteria (LAB), have been repeatedly proven as an alternative approach for the treatment of various infections. We have analyzed the potential of Enterococcus faecium BGPAS1-3, a dairy isolate exhibiting strong direct antilisterial effect, to modulate the response of differentiated Caco-2 intestinal epithelial cells to L. monocytogenes ATCC 19111 infection. We showed that the molecule with antilisterial effect is a bacterial cell-wall protein that is highly resistant to the high-temperature treatment. When we tested the antilisterial potential of heat-killed BGPAS1-3, we found that it could prevent tight junction disruption in differentiated Caco-2 monolayer infected with L. monocytogenes ATCC 19111, induce antilisterial host response mechanisms, and stimulate the production of protective TGF-β in intestinal epithelial cells. We also showed that the modulation of MyD88 dependent TLR2 and TLR4 pathways by BGPAS1-3 are involved in host response against L. monocytogenes ATCC 19111. Since heat-killed BGPAS1-3 possess strong antilisterial effects, such postbiotic could be used as a controllable and safe therapeutic.