Membrane disruption and DNA binding of Staphylococcus aureus cell induced by a novel antimicrobial peptide produced by Lactobacillus paracasei subsp. tolerans FX-6

Previously, we have isolated a novel bacteriocin, peptide F1 from Tibetan Kefir, and demonstrated its superior antimicrobial activity. However, its antimicrobial mechanism is still undefined. This study was aimed to elucidate the antimicrobial mechanism of peptide F1 against Staphylococcus aureus. The antimicrobial effects of peptide F1 were characterized by the following methods: chemical assay to quantify cytoplasmic β-galactosidase leakage, atomic absorption spectrometry to measure the released potassium ions, transmission electron microscopy to visualize the cellular morphological changes, and electrophoresis analysis and atomic force microscopy together to exam the DNA binding activity. Our results revealed that peptide F1 exerted its bactericidal effects by damaging bacterial cell membranes and by binding to the genomic DNA in the cytoplasm, which both led to rapid cell death.     

An antimicrobial peptide screened from casein hydrolyzate by Saccharomyces cerevisiae cell membrane affinity method

A Saccharomyces cerevisiae cell membrane affinity screening method was developed to separate targeted antimicrobial peptides from the pepsin hydrolyzate of bovine casein. S. cerevisiae cell membranes were first immobilized on the surface of the silica gel to construct an affinity binding medium. A membrane-binding fraction was successfully screened by comparing the RP-HPLC fingerprint chromatograms of the hydrolyzate before and after adsorption with the adsorption medium. The amino acid sequence of the peptide was identified as LRLKKYKVPQL with the use of a matrix-assisted laser desorption/ionisation quadrupole time-of-flight tandem mass spectrometer. The sequence corresponded to amino acid residues 99–109 of bovine α_S1-casein. The results indicated that it is feasible to target screen antimicrobial peptides from protein hydrolyzate using S. cerevisiae cell membranes. The influences of thermal treatment, pH, ions, and enzymes on the activity of the purified peptide were also determined. The activity of the peptide was relatively thermally stable and was pH dependent. It retained more than 90% of its activity in the presence of 15% Na⁺, K⁺ and pepsin. Trypsin, proteinase K, divalent cation Mg²⁺ and Ca²⁺ reduced the activity to different extents. The peptide also showed antibacterial effectiveness in fresh pear juice. These observations provide further information on the application of protein-derived antimicrobial peptides in food systems.     

Impact of microfluidization or ultrasound processing on the antimicrobial activity against Escherichia coli of lemongrass oil-loaded nanoemulsions

The effect of the ultrasonication and microfluidization processing conditions on the antimicrobial activity of lemongrass oil–alginate nanoemulsions was studied. Sonication led to less effective nanoemulsions against Escherichia coli, being the loss of antimicrobial potential dependent on the amplitude and treatment time applied. Namely, nanoemulsions sonicated at 100 μm for 180 s almost completely lost their bactericidal action, leading to 0.3 log-reductions of E. coli population after 30 min of contact time. On the contrary, nanoemulsions processed by microfluidization exhibited an enhanced antimicrobial activity, which was proportional to the number of cycles through the microfluidization chamber. In fact, whereas the coarse emulsion reduced the E. coli population up to 0.66, 2.25 and 5.85 log-units after 5, 15 and 30 min of contact time, the microfluidized nanoemulsions (10 cycles, 150 MPa) achieved 1.37, 5.29 and 7.07 log-reductions. Therefore, nanoemulsions with an improved functionally could be obtained by microfluidization, whereas ultrasounds seem to have a deleterious impact on the antimicrobial activity of lemongrass essential oil.     

Isolation,identification and antimicrobial resistance of Cronobacter spp. isolated from various foods in China

Cronobacter spp. are important foodborne pathogens that can cause severe diseases such as meningitis, sepsis, and necrotizing enterocolitis in neonates. In this study, 195 food samples, including cereals, cereal products, powdered infant formula (PIF), infant food formula, herbs, spices, vegetables, and fruits, were analyzed for the presence of Cronobacter spp. by culture-based method. The presumptive isolates were further confirmed by targeting the 16S rDNA gene using PCR. Out of 195 samples, 13 samples (6.7%) were positive for Cronobacter species. 12 of 85 cereal and cereal products (14.1%), and 1 of 22 herbs and spices (4.5%) were contaminated. In contrast, no Cronobacter was detected in commercial powdered infant formula, infant food formula, vegetables, or fruits. Alignment of 16S rRNA gene sequences showed that 13 isolates was most closely related to the genus Cronobacter. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis revealed that Cronobacter sakazakii was the only Cronobacter species isolated from various food samples. The antimicrobial susceptibility of 13 Cronobacter isolates was determined by the standard disk diffusion method. All isolated strains, except one resistant to ampicillin, were sensitive or displayed intermediate susceptibility to the 10 antimicrobial agents investigated. No multiple drug resistance was observed.     

Inhibitory effect of Cinnamomum cassia oil on non-O157 Shiga toxin-producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) have caused numerous foodborne outbreaks. Compared with the most well-known STEC E. coli O157:H7, importance of non-O157 STEC has been underestimated and they have gained far less attention till increasing outbreaks recently. Using natural plant materials as antimicrobial agents is a heated area. Therefore in this study, Cinnamomum cassia, a widely used spice in cuisine, was tested for its antibacterial efficacy on CDC “top six” non-O157 STECs including O26, O45, O103, O111, O121, O145. Gas chromatography-mass spectrometry analysis showed that the major component of C. cassia oil was cinnamaldehyde (59.96%). The disk diffusion assay indicated that 20 μL 4% (v/v) C. cassia oil per disk resulted in inhibition zones of 15.0 mm, 18.5 mm, 15.7 mm, 19.3 mm, 18.8 mm, and 25.3 mm for O26:H11, O45:NM, O103:H2, O111:H2, O121:H19, and O145:NT, respectively. Minimum inhibitory concentration for all tested non-O157 STECs were 0.025% (v/v). Minimum bactericidal concentration was strain dependent, which was 0.05% (v/v) for O26:H11, O121:H19, O145:NT, while 0.1% (v/v) for O45:NM, O103:H2 and O111:H2. Growth kinetics showed that at the low inoculation of approximate 2.5 × 10⁵ CFU/mL, C. cassia oil at the concentration of 0.01875% (v/v) completely inhibited the growth of O26:H11 and O145:NT for at least 24 h, and increased the duration of lag phase of O45:NM, O103:H2, O111:H2, O121:H19 by18, 12, 6, and 16 h, respectively. Including 0.025% (v/v) C. cassia oil completely inhibited the growth of all tested non-O157 STECs for at least 24 h. At high inoculation of 5 × 10⁶ CFU/mL, inhibition effect of C. cassia oil decreased. Death curve showed that including as low as 0.05% (v/v) C. cassia oil could kill non-O157 STECs. 0.1% (v/v) C. cassia oil showed bactericidal effects on all tested non-O157 STECs within 15 min. C. cassia oil at the concentration of 0.15% (v/v) killed all O26:H11, O121:H19 and O145:NT within 30 min, while O45:NM, O103:H2 and O111:H2 at 120, 60, and 60 min, respectively. In conclusion, C. cassia oil can effectively inhibit the growth of non-O157 STECs at concentration as low as 0.025% (v/v). Our data suggest that C. cassia oil has the potential to be used as a natural antibacterial agent in food industry.     

Novel bacteriocin produced by Lactobacillus alimentarius FM-MM4 from a traditional Chinese fermented meat Nanx Wudl: Purification,identification and antimicrobial characteristics

The present study aims to purify and characterize a novel bacteriocin (designated as lactocin MM₄) produced by Lactobacillus alimentarius FM-MM₄, which was isolated from Nanx Wudl, a traditional Chinese fermented meat. Lactocin MM₄ was extracted by ethyl acetate and purified through cation exchange chromatography and semi-preparative high-performance liquid chromatography (RP-HPLC). As revealed by LC-MS/MS, lactocin MM₄ has a molecular mass of 1104.58 Da with the N-terminal sequence QGVGPLGQGHR, demonstrating low homology with previously reported class II bacteriocins. Lactocin MM₄ exhibited broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative food-borne pathogens, as well as several yeasts. It was considerably thermo-stable with 84.7% residual antimicrobial activity after exposure to121 °C for 15 min, and it was highly stable at acidic pH (2–5). Proteases, such as trypsin, protease K, papain and pepsin, could completely inactivate the bacteriocin, while other enzymes, such as lipase and amylase, had no effect on its antimicrobial activity. These results indicate the potential of lactocin MM₄ for application in food preservation.     

Evaluation of a novel antimicrobial solution and its potential for control Escherichia coli O157:H7, non-O157:H7 shiga toxin-producing E. coli,Salmonella spp., and Listeria monocytogenes on beef

The goal of this study was to evaluate the efficacy of a novel antimicrobial solution made with chitosan, lauric arginate ester, and organic acids on Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, and non-O157 shiga toxin-producing E. coli cocktails and to test its potential to be used as a marinade for raw beef. Fresh beef top round steaks were surface-inoculated with the pathogen cocktails at approximately 2.5 or 4.5 Log CFU/cm², marinated with the antimicrobial solution (AMS), and then stored at 4 °C for 6, 24, and 48 h. Three commercially available marinades were used for comparison. Results revealed that AMS had the most antimicrobial effect regardless of the type or inoculation level of pathogens (P < 0.05). After 6 h, the AMS marination reduced all pathogens to levels below the limit of detection (<1 Log CFU/cm²), resulting in a 3.5 Log CFU/cm² reduction. When AMS was diluted with autoclaved distilled water by 5 times (AMS 1:5) or 10 times (AMS 1:10), its antimicrobial efficacy was impacted by marination time, the inoculated pathogens, and the inoculation levels. This study demonstrates that the developed antimicrobial solution has a great potential to be used during marination by consumers to ensure better food safety.     

Improving the antimicrobial activity of d-limonene using a novel organogel-based nanoemulsion

The purpose of this research was to develop a novel antimicrobial delivery system by encapsulating d-limonene into an organogel-based nanoemulsion and investigating its antimicrobial activity. The d-limonene organogel-based nanoemulsion was prepared by high pressure homogenization method. The surfactant concentration had a major impact on the droplets' formation and distribution. At the optimal condition (10% w/w Tween 80, 100 Mpa, and 10 Cycles) the smallest droplet size (d ≈ 36 nm) could be obtained, which has shown a narrow structure and good stability. Results from the antimicrobial activity have shown the encapsulation of d-limonene (4% w/w) into the organogel-based nanoemulsion contributed to the increase of its antimicrobial activity. In addition, the mechanism of d-limonene organogel-based nanoemulsion against the tested microorganisms was studied by the electronic microscope observation and the cell constituent release. This research would have an important implication for the design of more efficient antimicrobial systems for food preservation and production.     

Evaluation of two antimicrobial packaging films against Escherichia coli O157:H7 strains in vitro and during storage of a Spanish ripened sheep cheese (Zamorano)

The antimicrobial activity of two packaging films (polypropylene – PP- and polyethylene terephthalate – PET-) coated with different concentrations (0, 4, 6 and 8%) of essential oil from Origanum vulgare (OR) and Ethyl Lauroyl Arginate HCl (LAE) was tested against two Escherichia coli O157:H7 strains using in vitro systems and a raw milk sheep cheese model (Zamorano). The influence of the antimicrobials on the sensory attributes of cheese was also evaluated. For both strains, the MBC (minimum bactericidal concentration) values were identical to their respective MIC (minimum inhibitory concentration) values and lower for LAE (25 mg/l) than for OR (200–400 mg/l). PP and PET films coated with OR were tested by a vapour phase assay and the Japanese Standard method (JIS Z 2801:2000). Both films coated with LAE were tested by an agar diffusion method. Overall, in vitro tests were effective against both strains. The inhibitory activity depended on the active compound concentration, the target strain and the packaging material, PET being more effective than PP. For inoculated cheese slices, OR and LAE PP films did not effectively decrease E. coli O157:H7 counts after 7-days cold storage. PET films incorporating 6 and 8% of OR and LAE significantly (p < 0.05) decreased the numbers of both strains and also did 4% for the reference and wild strain depending on the antimicrobial. LAE PP, OR PET and LAE PET did not significantly (p > 0.05) affect sensorial characteristics of Zamorano cheese. Packaging with PET films coated with ≥6% LAE concentrations might be useful in reducing E. coli O157:H7 numbers in sheep cheese.     

Inhibitory effect of nerol against Aspergillus niger on grapes through a membrane lesion mechanism

The antifungal activity of Nerol (NEL) was evaluated against Aspergillus niger, a known cause of grape spoilage, and possible modes of action were explored. The antifungal efficacy of NEL against A. niger has been proven in a dose-dependent manner through in vitro (mycelial growth and spore germination) and in vivo (grapes) tests. An obvious increase in the membrane permeability was detected through the determination of extracellular pH and conductivity. The damage in the cell membrane was measured by monitoring the influx of Propidium iodide (PI) using flow cytometry and was further verified through the inhibition of ergosterol synthesis in the cell membrane. NEL induced morphological changes, and the integrity of the A. niger fungal cells decreased with increasing NEL concentrations. A dose-dependent decrease in ergosterol production was also seen in A. niger cell membranes upon exposure to NEL. The mechanism-of-action study revealed that NEL activated a membrane-active mechanism that inhibits ergosterol synthesis and consequently compromised the membrane integrity of A. niger. As a result, the membrane permeability changes and causes cell death. The membrane lesion mechanism makes NEL a natural alternative to commercial fungicide for control of stored grape spoilage.     

The antimicrobial effects and synergistic antibacterial mechanism of the combination of ε-Polylysine and nisin against Bacillus subtilis

The aim of the present study was to evaluate the combined effects of ε-Polylysine (ε-PL) and nisin and investigate the synergistic action of these compounds against Bacillus subtilis (B. subtilis).The combination of ε-PL and nisin showed synergistic anti-microbial activity against Escherichia coli (E. coli), B. subtilis and Staphylococcus aureus (S. aureus). SEM and TEM microscopy revealed that combined treatment with ε-PL and nisin synergistically damaged the morphology of tested bacterial cells. Propidium iodide (PI) infiltration experiments indicated that combined treatment with ε-PL and nisin synergistically enhanced the permeability of the bacterial cell membrane, likely reflecting the inhibition of both Na⁺K⁺- and Ca⁺⁺ Mg⁺⁺-ATPase activities through these compounds. The fluorescence spectrum showed an interaction between ε-PL and DNA, but not between nisin and DNA. The mode of ε-PL in binding with DNA was similar to that of ethidium bromide (EB). These results indicated that the uptake of ε-PL into cells was promoted through nisin, and subsequently, ε-PL interacted with the intracellular DNA achieving a synergistic effect.     

Prevalence,antimicrobial resistance profiling and genetic diversity of Campylobacter jejuni and Campylobacter coli isolated from broilers at slaughter in China

A total of 651 samples from broiler cecal samples, carcasses, carcass parts collected at the slaughterhouse level in Sichuan Province of China were examined for the occurrence of Campylobacter jejuni and Campylobacter coli. After confirmed by species-specific multiplex PCR, the recovered isolates were examined for resistance to antimicrobials using an agar dilution method and investigated for the mutation of gyrA, tetO gene and V domain of 23S rRNA as well as the presence of class 1 integron and the associated gene cassettes. In addition, the genotype relatedness of the isolates was determined by pulsed-field gel electrophoresis (PFGE) profiling. The prevalence of Campylobacter was 56.1% in cecal samples, 31.0% in carcasses and 17.0% in carcass parts, respectively. Among them, C. jejuni accounted for 24.6% and C. coli occupied 20.0% of the samples. The strains of C. jejuni were most frequently resistant to ciprofloxacin (88.1%), followed by resistance to tetracycline (79.4%) and levofloxacin (78.1%). Most of the C. coli isolates were resistant to ciprofloxacin (100%), tetracycline (98.5%), levofloxacin (98.5%), clindamycin (98.5%) and erythromycin (93.9%). Antimicrobial resistance profiling showed that 93.7% of campylobacters were multidrug resistant (MDR) strains. Moreover, class 1 integrons were detected in 98.6% of MDR campylobacters, among which 98.7% were positive for C. jejuni and 98.5% for C. coli. Three kinds of gene cassettes-associated amplicons were identified and the amplicons profile of 1000–750–500–250 bp was the predominant pattern linked to the aminoglycoside resistance gene of aadA2. The presence of mutation in gyrA, tetO and 23S rRNA between C. jejuni and C. coli varied from 89.7% to 97.3%, 96.6% to 94.1%, and 95.0% to 96.7%, respectively. Finally, the results of PFGE indicated that, 33 PFGE profiles were generated among 78 isolates of C. jejuni and the similarity level ranged from 42.1% to 99.1%. By contrast, 15 PFGE patterns were produced among 68 C. coli isolates sharing a similarity level of 54.0%–99.7%. Comparison of the PFGE and antimicrobial resistance profiles of the isolates reflected the high genetic diversity of Campylobacter tested. A poor correlation among the antimicrobial resistance patterns, resistance determinants and PFGE genotypes was observed in C. jejuni. Our study showed that there were several points of cross-contamination during broiler slaughter, and a high diversity of PFGE types in the Campylobacter isolates with high resistances to ciprofloxacin and tetracycline.     

Antimicrobial effects of modified chitosan based coating containing nanoemulsion of essential oils,modified atmosphere packaging and gamma irradiation against Escherichia coli O157:H7 and Salmonella Typhimurium on green beans

The antibacterial activity of modified chitosan-based coatings containing nanoemulsion of essential oils (EOs), gamma irradiation, modified atmosphere packaging (MAP), alone or in combinations, against Escherichia coli O157:H7 and Salmonella Typhimurium was evaluated on inoculated green bean samples. Firstly, four different nanoemulsions, made of carvacrol, mandarin, bergamot and lemon Eos, respectively, were compared in terms of minimum inhibitory concentration (MIC) against the two bacteria evaluated in vitro using the micro-broth dilution method. Carvacrol nanoemulsion resulted to be the most effective antibacterial agent and was therefore selected to be incorporated into modified chitosan (MC) to form a bioactive coating. Secondly, the radiosensitivity of E. coli and S. Typhimurium to gamma irradiation was evaluated on inoculated green beans after coating deposition and MAP. Results showed that, without MAP, MC-based coating containing carvacrol nanoemulsion significantly increased the radiosensitization of E. coli and S. Typhimurium by 1.32-fold and 1.30-fold, respectively. Remarkably, the use of bioactive coating under MAP caused a synergistic effect with an increase in radiosensitivity by 1.80-fold and 1.89-fold for E. coli and S. Typhimurium, respectively. Thirdly, the antibacterial effects of the antimicrobial coating, gamma irradiation, MAP alone and their combinations were evaluated against these two bacteria during a 13-days storage of green beans at 4 °C. Bioactive coating deposition or gamma irradiation treatment resulted effective in controlling the growth of the two bacteria during the entire shelf-life. Moreover, it was also found that the combined treatment of antimicrobial coating, gamma irradiation and MAP caused the reduction of microbial population to undetectable levels during the whole storage period for E. coli and from day 7 to the end of storage for S. Typhimurium. The obtained results can be interested to food companies aiming to ensure the food safety with a prolonged shelf life.     

Prevalence and characterization of Escherichia coli O157:H7 from samples along the production line in Chinese beef-processing plants

This work investigated the prevalence of Escherichia coli O157:H7 and the antibiotic susceptibility, genetic diversity and virulence genes of isolated strains from four beef slaughter plants in China. A total of 510 samples (feces, hide, carcasses and raw meat) were tested for E. coli O157:H7 using enrichment, immunomagnetic separation, and plating on selective media. The prevalence of E. coli O157:H7 in the feces, hide, pre-evisceration carcass, post-evisceration carcass, post-washing carcass, chilled carcass, and raw meat samples was 1.43%, 1.43%, 0%, 0%, 0%, 1.25%, and 0%, respectively. Multiplex PCR assays were used for serotype confirmation and virulence gene detection. stx₂, eaeA and EHEC-hlyA genes were present in all six of the strains, and the stx₁ gene was not present. Fluorescence amplified fragment length polymorphism (FAFLP) was used to determine the genetic diversity of E. coli O157:H7 and revealed a high similarity between the strains isolated from feces and those isolated from carcasses. None of the isolated strains were found to be resistant to sixteen commonly used antimicrobial agents. The results of this study indicate that although E. coli O157:H7 contamination in the Chinese beef industry is sporadic and not as common as reported in other counties, all of the isolates contained three major virulence genes, presenting a high risk of disease for humans. The current research provides baseline information on E. coli O157:H7 prevalence and character profiles in Chinese beef-processing plants that can be used for future studies.     

Preparation of low- and medium-molecular weight chitosan nanoparticles and their antimicrobial evaluation against a panel of microorganisms,including cheese-derived cultures

This study employed the technique of ionic gelation in the manufacture of low- and medium-molecular weight chitosan nanoparticles. Nanoparticles were characterised (size, size distribution, surface charge and morphology) and their antimicrobial activity assessed against cheese-derived cultures, as well as a select panel of Gram-positive and Gram-negative microorganisms. Antimicrobial activity was determined by the minimum inhibition concentration via the micro dilution method using 96-well microplates. Synthesised particles were small-sized, with a moderate size distribution and positive zeta potential. Generated nanoparticles exhibited successful solubility in both water and acetic acid. Acidic nanosuspensions demonstrated greater microbial reduction than water-based nanoparticles, with no difference in activity observed between molecular weights. Cheese-derived cultures were effectively controlled, and Gram-negative species were more susceptible than Gram-positive species to the action of nanoparticles in acetic acid. Nanoparticles suspended in an acidic-based medium show promise as antimicrobial agents, particularly for use with cheese products.     

Assessment of sanitation efficacy against Escherichia coli O157:H7 by rapid measurement of intracellular oxidative stress,membrane damage or glucose active uptake

To reduce cross contamination with foodborne pathogens during washing procedures, sodium hypochlorite (NaOCl) and hydrogen peroxide (H₂O₂) are widely used in the fresh produce industry. To ensure food safety and minimize excessive use of sanitizers and water during sanitation, it is critical to develop a rapid method to assess sanitation efficacy. This study examines the potential for employing oxidative stress, membrane damage and glucose uptake measurements to assess the antimicrobial efficacy of NaOCl (0–70.4 ppm) and H₂O₂ (0–1.6% v/v) toward Escherichia coli O157:H7. The relative amount of intracellular reactive oxygen species (ROS) was measured using Aminophenyl fluorescein (APF) and 2′,7′- dichlorodihydrofluorescein diacetate (H₂DCFDA) for cells treated with NaOCl and H₂O₂ respectively. Results from these ROS sensitive probes revealed a limited correlation between these oxidative stress measurements and inactivation of bacteria measured using the plate counting method. Sanitation experiments, conducted with contaminated pre-cut lettuce leaves in water, were also carried out. Measurement of the bacterial membrane integrity was assessed using the membrane permeable probe propidium iodide (PI) and by evaluating effects on active transport by the glucose transport system using 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG). The results demonstrate that the relative increase in membrane permeability using PI correlated with NaOCl, but did not correlate with H₂O₂ induced reduction of bacterial survival quantified by the plate counting method. In contrast to the other approaches, monitoring the loss of glucose transport system function by measuring the uptake of 2-NBDG displayed strong correlation with the reduction of bacterial survival for both NaOCl and H₂O₂ treatment. Overall, the study demonstrated potential of glucose uptake measurements with 2-NBDG to serve as surrogate method for the traditional plate counting, which is the current gold standard used by the Food and Drugs Administration, to provide a faster analysis of sanitation processes.     

Purification,characterization and bactericidal mechanism of a broad spectrum bacteriocin with antimicrobial activity against multidrug-resistant strains produced by Lactobacillus coryniformis XN8

The multidrug-resistant strains of food spoilage bacteria and foodborne pathogenic bacteria in food are badly in need of being controlled by effective bio-preservatives. In this study, the XN8 strain was isolated from Jiangshui and identified as Lactobacillus coryniformis according to 16S rRNA gene sequence. One of the bacteriocins produced by XN8 was purified by ammonium sulfate precipitation and a series of chromatographic column, and designated as lactocin XN8-A (LXA). The molecular mass of LXA was 3100.0242 Da by MALDI-TOF MS. The LXA showed good heat, pH and storage stabilities. However, it was sensitive to proteases. The LXA was found to have a broad antimicrobial spectrum on both Gram-positive and Gram-negative bacteria including multidrug-resistant strains and Listeria monocytogenes. Its minimum inhibitory concentration (MIC) for both Escherichia coli and Staphylococcus aureus was 6.85 μg/mL. The LXA had a bactericidal mode without cell lysis by the growth curve and time-kill assay. The results of electron microscope showed that the LXA destroyed membrane permeability and induced pore-formation of target cells. Furthermore, the LXA induced cell cycle arrest at both G1 and G2/M phase by cell cycle analysis. This research suggests that the LXA has promising potential as bio-preservative in food industry.     

Development of a controlling method for Escherichia coli O157:H7 and Salmonella spp. in fresh market beef by using polylysine and modified atmosphere packaging

Escherichia coli O157:H7 and Salmonella spp. often contaminate fresh beef. In Japan, an E coli outbreak caused by raw beef made 181 people ill and 5 individuals dead in 2011. Responding to this outbreak, an effective sterilization method for fresh beef is expected to be developed. In this study, ε-polylysine combined with CO₂-packaging method was examined for controlling these pathogens in fresh beef. At an incubation temperature of 4 °C, approximately 4.3 log and 2.4 log reduction in bacterial numbers were observed after 7-day incubation for E. coli O157:H7 and Salmonella, respectively, in ε-polylysine-added beef. When effectiveness of CO₂-packaging combined with ε-polylysine was investigated, CO₂ did not have additional inhibiting effect on bacterial growth compared to only-ε-polylysine-treated samples when incubated at 4 °C. However, effectiveness of CO₂ was observed when incubated at 10 °C where approximately 2.9 log and 4.4 log reduction in E. coli cell numbers were observed in only-ε-polylysine-treated samples and polylysine- and CO₂-treated group, respectively, and approximately 1.7 log and 3.5 log reduction in Salmonella cell numbers were observed in only-ε-polylysine-treated samples and polylysine and CO₂-treated group, respectively. This study confirmed that ε-polylysine or ε-polylysine combined with CO₂ packaging are effective in preventing foodborne diseases caused by raw beef.     

Addition of phytochemical-rich plant extracts mitigate the antimicrobial activity of essential oil/wine mixtures against Escherichia coli O157:H7 but not against Salmonella enterica

Marinades for preparing raw meats for cooking are frequently made of wine and herbs. We simulated several formulations of potential antimicrobial marinades with these components and other food compatible/food derived extracts. Red wine formulations containing essential oils from oregano or thyme, or their primary active components carvacrol and thymol, respectively, and a mixture of plant extract powders from phytochemical-rich apple skin, green tea, and olive, were evaluated for inhibitory activity against the foodborne pathogens Escherichia coli O157:H7 and Salmonella enterica. Red wine alone exhibited low activity, as did the plant extract suspended in the wine. Surprisingly, the high activity of oregano or thyme essential oils in red wine was reduced in E. coli, but not in Salmonella, by addition of the plant extract. This study shows that essential oils in red wine can be an effective antimicrobial in food, however the possibility exists that phytochemicals, added to the treatment solution or natively present in the food itself, could adversely impact the antimicrobial activity and should be addressed with future studies.     

Inactivation by 405 ± 5 nm light emitting diode on Escherichia coli O157:H7, Salmonella Typhimurium,and Shigella sonnei under refrigerated condition might be due to the loss of membrane integrity

The objective of this study was to evaluate the antibacterial effect of 405 ± 5 nm light emitting diode (LED) on Escherichia coli O157:H7, Salmonella Typhimurium and Shigella sonnei. Its antibacterial mechanism was also investigated by determining the permeability of bacterial membrane and DNA degradation. Bacterial strains in phosphate-buffered saline were exposed to 405 ± 5 nm LED to a final dose of 486 J/cm² (7.5 h) at 4 °C. The inactivation curves were fitted by Weibull model to compare the sensitivities of pathogens to the LED illumination by calculating the decimal reduction times (t_R). The bacterial sensitivity to bile salts and NaCl by LED illumination was also determined. LIVE/DEAD^® BacLight™ staining as well as comet assay and DNA ladder analysis were carried out to determine the bacterial membrane integrity and DNA degradation, respectively. Results showed that LED illumination inactivated 1.0, 2.0, and 0.8 log CFU/ml for E. coli O157:H7, S. Typhimurium, and S. sonnei for 7.5 h, respectively. The comparison of t_R values demonstrated that S. Typhimurium was found to be the most (P < 0.05) susceptible strain to LED illumination. Regardless of the bacterial strain, the sensitivity of illuminated bacterial cells to bile salts and NaCl considerably increased compared to non-illuminated controls. Furthermore, LIVE/DEAD^® assay clearly showed that LED illumination resulted in loss of bacterial membrane permeability. On the other hand, no DNA degradation was observed by both comet assay and DNA ladder analysis. Therefore, these results suggest that the antibacterial effect of 405 ± 5 nm LED might be partly attributed to the physical damage to bacterial cell membrane. This study proposes that 405 ± 5 nm LED under refrigerated conditions may be effective to control the pathogens on foods.