Tony Z. Jin

Antibacterial Activity and Mechanism of Action of Zinc Oxide Nanoparticles against Campylobacter jejuni

ABSTRACT The antibacterial effect of zinc oxide (ZnO) nanoparticles on Campylobacter jejuni was investigated for inhibition and inactivation of cell growth. The results showed that C. jejuni was extremely sensitive to treatment with ZnO nanoparticles. The MIC of ZnO nanoparticles for C. jejuni was determined to be 0.05 to 0.025 mg/ml, which is 8- to 16-fold lower than that for Salmonella enterica serovar Enteritidis and Escherichia coli O157:H7 (0.4 mg/ml). The action of ZnO nanoparticles against C. jejuni was determined to be bactericidal, not bacteriostatic. Scanning electron microscopy examination revealed that the majority of the cells transformed from spiral shapes into coccoid forms after exposure to 0.5 mg/ml of ZnO nanoparticles for 16 h, which is consistent with the morphological changes of C. jejuni under other stress conditions. These coccoid cells were found by ethidium monoazide-quantitative PCR (EMA-qPCR) to have a certain level of membrane leakage. To address the molecular basis of ZnO nanoparticle action, a large set of genes involved in cell stress response, motility, pathogenesis, and toxin production were selected for a gene expression study. Reverse transcription-quantitative PCR (RT-qPCR) showed that in response to treatment with ZnO nanoparticles, the expression levels of two oxidative stress genes (katA and ahpC) and a general stress response gene (dnaK) were increased 52-, 7-, and 17-fold, respectively. These results suggest that the antibacterial mechanism of ZnO nanoparticles is most likely due to disruption of the cell membrane and oxidative stress in Campylobacter.

Antimicrobial efficacy of zinc oxide quantum dots against Listeria monocytogenes, Salmonella Enteritidis, and Escherichia coli O157:H7.

Zinc oxide quantum dots (ZnO QDs) are nanoparticles of purified powdered ZnO. These were evaluated for antimicrobial activity against Listeria monocytogenes, Salmonella Enteritidis, and Escherichia coli O157:H7. The ZnO QDs were utilized as a powder, bound in a polystyrene film (ZnO-PS), or suspended in a polyvinylprolidone gel (ZnO-PVP). Bacteria cultures were inoculated into culture media or liquid egg white (LEW) and incubated at 22 degrees C. The inhibitory efficacies of ZnO QDs against 3 pathogens were concentration dependent and also related to type of application. The ZnO-PVP (3.2 mg ZnO/mL) treatment resulted in 5.3 log reduction of L. monocytogenes and 6.0 log reduction of E. coli O157:H7 in growth media after 48 h incubation, as compared to the controls. Listeria cells in the LEW control increased from 3.8 to 7.2 log CFU/mL during 8 d incubation, while the cells in the samples treated with 1.12 and 0.28 mg ZnO/mL were reduced to 1.4 and 3.0 log CFU/mL, respectively. After 8 d incubation, the cell populations of Salmonella in LEW in the presence of 1.12 and 0.28 mg ZnO/mL were reduced by 6.1 and 4.1 log CFU/mL over that of controls, respectively. ZnO powder and ZnO-PVP showed significant antimicrobial activities against all 3 pathogens in growth media and LEW. ZnO-PVP coating had less inhibitory effect than the direct addition of ZnO-PVP. No antimicrobial activities of ZnO-PS film were observed. This study suggested that the application of ZnO nanoparticles in food systems may be effective at inhibiting certain pathogens.

Biodegradable Polylactic Acid Polymer with Nisin for Use in Antimicrobial Food Packaging

Biodegradable polylactic acid (PLA) polymer was evaluated for its application as a material for antimicrobial food packaging. PLA films were incorporated with nisin to for control of foodborne pathogens. Antimicrobial activity of PLA/nisin films against Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella Enteritidis were evaluated in culture media and liquid foods (orange juice and liquid egg white). Scanned electron micrograph and confocal laser microscopy revealed that nisin particles were evenly distributed in PLA polymer matrix on the surface and inside of the PLA/nisin films. PLA/nisin significantly inhibited growth of L. monocytogenes in culture medium and liquid egg white. The greatest inhibition occurred at 24 h when the cell counts of L. monocytogenes in the PLA/nisin samples were 4.5 log CFU/mL less than the controls. PLA/nisin reduced the cell population of E. coli O157:H7 in orange juice from 7.5 to 3.5 log at 72 h whereas the control remained at about 6 log CFU/mL. PLA/nisin treatment resulted in a 2 log reduction of S. Enteritidis in liquid egg white at 24 degrees C. After 21 d at 4 degrees C the S. Enteritidis population from PLA/nisin treated liquid egg white (3.5 log CFU/mL) was significantly less than the control (6.8 log CFU/mL). E. coli O157:H7 in orange juice was more sensitive to PLA/nisin treatments than in culture medium. The results of this research demonstrated the retention of nisin activity when incorporated into the PLA polymer and its antimicrobial effectiveness against foodborne pathogens. The combination of a biopolymer and natural bacteriocin has potential for use in antimicrobial food packaging.

Inactivation of Salmonella in liquid egg albumen by antimicrobial bottle coatings infused with allyl isothiocyanate, nisin and zinc oxide nanoparticles

Aims:  To develop an antimicrobial bottle coating effective at inhibiting the growth of Salmonella in liquid egg albumen (egg white) and reduce the risk of human Salmonellosis.

Inactivation of Salmonella on whole cantaloupe by application of an antimicrobial coating containing chitosan and allyl isothiocyanate.

This study investigated the antimicrobial effect of a chitosan coating+allyl isothiocyanate (AIT) and nisin against Salmonella on whole fresh cantaloupes. Cantaloupes were inoculated with a cocktail of three Salmonella strains and treated with chitosan, chitosan+AIT, chitosan+nisin, and chitosan+AIT+nisin coatings. With AIT concentrations increasing from 10 to 60 μl/ml, the antibacterial effects of coating treatments against Salmonella increased. Chitosan coatings with 60 μl/ml AIT (chitosan+60AIT) reduced more than 5 log₁₀ CFU/cm² of Salmonella. The addition of nisin to the chitosan-AIT coating synergistically increased the antibacterial effect; coatings with nisin (25 mg/ml or 25,000 IU/ml)+30 μl/ml AIT resulted in a 4.8 log₁₀ reduction of Salmonella. The chitosan+60AIT coating significantly (p<0.05) reduced populations of native bacteria on cantaloupes to ca. 2 log₁₀ CFU/cm² during the first 6 days and populations remained unchanged through day 14 at 10 °C. The same coating treatment completely inactivated mold and yeast on cantaloupe at day 1 and no regrowth occurred even up to 14 days of storage. Scanning electron microscopy revealed that cell membrane damage and leakage of intercellular components occurred as a result of the chitosan-AIT coating treatments. No visual changes in overall appearance and color of cantaloupe rind and flesh due to coating treatments were observed. These results indicate that the application of an antimicrobial coating may be an effective method for decontamination of cantaloupes.

Preparation of Antimicrobial Membranes: Coextrusion of Poly(lactic acid) and Nisaplin in the Presence of Plasticizers

Nisin is a naturally occurring antimicrobial polypeptide and is popularly used in the food and food-packaging industries. Nisin is deactivated at temperatures higher than 120 degrees C and, therefore, cannot be directly incorporated into poly(L-lactic acid) (PLA), a biomass-derived biodegradable polymer, by coextrusion because PLA melts at temperatures around 160 degrees C or above. However, PLA can remain in a melt state at temperatures below the T(m) in the presence of lactic acid or other plasticizers. In the present study, PLA was coextruded with lactic acid, or lactide, or glycerol triacetate at 160 degrees C. After the PLA was melted, the temperature of the barrels was reduced to 120 degrees C, and then Nisaplin, the commercial formulation of nisin, was added and the extrusion was continued. The resultant extrudates possess the capability to suppress the growth of the pathogenic bacterial Listeria monocytogenes , demonstrating a significant antimicrobial activity. The present study provides a simple method to produce PLA-based antimicrobial membranes. The method can also be used for the coextrusion of other heat-sensitive substances and thermoplastics with high melting temperature.

Radiation sensitization and postirradiation proliferation of Listeria monocytogenes on ready-to-eat deli meat in the presence of pectin-nisin films.

In this study, the ability of pectin-nisin films in combination with ionizing radiation to eliminate Listeria monocytogenes and inhibit its postirradiation proliferation was evaluated. Pectin films containing 0.025% nisin were made by extrusion. The surface of a ready-to-eat turkey meat sample was inoculated with L. monocytogenes at 10(6) CFU/cm2 and covered with a piece of pectin-nisin film. The samples were vacuum packaged and irradiated at 0, 1, and 2 kGy. The treated samples were stored at 10 degrees C and withdrawn at 0, 1, 2, 4, and 8 weeks for microbial analysis. Reductions in L. monocytogenes viability of 1.42, 1.56, 2.85, 3.78, and 5.36 log CFU/cm2 were achieved for the treatments of 1 kGy, pectin-nisin film, 2 kGy, 1 kGy plus pectin-nisin film, and 2 kGy plus pectin-nisin film, respectively. The greatest reduction (5.5 log CFU/cm2) was observed at 1 week for the 2 kGy plus pectin-nisin film treatment, suggesting that nisin was further released from the film to the surface of meat samples. Pectin-nisin films used in this study did not prevent but did significantly slow (P < 0.05) the proliferation of the L. monocytogenes cells that survived irradiation during 8 weeks of storage at 10 degrees C. These data indicate the potential use of pectin-nisin films alone or in combination with ionizing radiation for preventing listeriosis due to postprocessing contamination of ready-to-eat meat products.

Inactivation of Listeria monocytogenes in Skim Milk and Liquid Egg White by Antimicrobial Bottle Coating with Polylactic Acid and Nisin

This study was to develop an antimicrobial bottle coating method to reduce the risk of outbreaks of human listeriosis caused by contaminated liquid foods. Liquid egg white and skim milk were inoculated with Listeria monocytogenes Scott A and stored in glass jars that were coated with a mixture of polylactic acid (PLA) polymer and nisin. The efficacy of PLA per nisin coating in inactivating L. monocytogenes was investigated at 10 and 4 degrees C. The pathogen grew well in skim milk without PLA/nisin coating treatments, reaching 8 log CFU/mL after 10 d and then remained constant up to 42 d at 10 degrees C. The growth of Listeria at 4 degrees C was slower than that at 10 degrees C, taking 21 d to obtain 8 log CFU/mL. At both storage temperatures, the PLA coating with 250 mg nisin completely inactivated the cells of L. monocytogenes after 3 d and throughout the 42-d storage period. In liquid egg white, Listeria cells in control and PLA coating without nisin samples declined 1 log CFU/mL during the first 6 d at 10 degrees C and during 28 d at 4 degrees C, and then increased to 8 or 5.5 log CFU/mL. The treatment of PLA coating with 250 mg nisin rapidly reduced the cell numbers of Listeria in liquid egg white to undetectable levels after 1 d, then remained undetectable throughout the 48 d storage period at 10 degrees C and the 70 d storage period at 4 degrees C. These data suggested that the PLA/nisin coating treatments effectively inactivated the cells of L. monocytogenes in liquid egg white and skim milk samples at both 10 and 4 degrees C. This study demonstrated the commercial potential of applying the antimicrobial bottle coating method to milk, liquid eggs, and possibly other fluid products.

Natural surface coating to inactivate Salmonella enterica serovar Typhimurium and maintain quality of cherry tomatoes.

The objective of the present study was to investigate the effectiveness of zein-based coatings in reducing populations of Salmonella enterica serovar Typhimurium and preserving quality of cherry tomatoes. Tomatoes were inoculated with a cocktail of S. Typhimurium LT2 plus three attenuated strains on the smooth skin surface and stem scar area. The zein-based coatings with and without cinnamon (up to 20%) and mustard essential oil or a commercial wax formulation were applied onto tomatoes and the treated fruits were stored at 10 °C for up to 3 weeks. Populations of S. Typhimurium decreased with increased essential oil concentration and storage duration. S. Typhimurium populations on the smooth skin surface were reduced by 4.6 and 2.8 log colony forming units(CFU)/g by the zein coatings with 20% cinnamon and 20% mustard oil, respectively, 5h after coating. The same coating reduced populations of S. Typhimurium to levels below detection limit (1.0 log CFU/g) on the stem scar area of tomato during 7 days of storage at 10 °C. Salmonella populations were not reduced on fruit coated with the commercial wax. All of the coatings resulted in reduced weight loss compared with uncoated control. Compared with the control, loss of firmness and ascorbic acid during storage was prevented by all of the coatings except the zein coating with 20% mustard oil which enhanced softening. Color was not consistently affected by any of the coating treatments during 21 days of storage at 10°C. The results suggest that the zein-based coating containing cinnamon oil might be used to enhance microbial safety and quality of tomato.

Effects of antimicrobial coatings and cryogenic freezing on survival and growth of Listeria innocua on frozen ready-to-eat shrimp during thawing.

Foodborne pathogens such as Listeria monocytogenes could pose a health risk on frozen ready-to-eat (RTE) shrimp as the pathogen could grow following thawing. In this study, antimicrobial-coating treatments alone, or in combination with cryogenic freezing, were evaluated for their ability to inhibit the growth of Listeria innocua, a surrogate for L. monocytogenes, on RTE shrimp. Cooked RTE shrimp were inoculated with L. innocua at 3 population levels and treated with coating solutions consisting of chitosan, allyl isothiocyanate (AIT), or lauric arginate ester (LAE). The treated shrimp were then stored at -18 °C for 6 d before being thawed at 4, 10, or 22 °C for either 24 or 48 h. Results revealed that antimicrobial coatings achieved approximately 5.5 to 1 log CFU/g reduction of L. innocua on RTE shrimp after the treatments, depending on the inoculated population levels. The coating-treated shrimp samples had significantly (P < 0.05) less L. innocua than controls at each thawing temperature and time. Cryogenic freezing in combination with coating treatments did not achieve synergistic effects against L. innocua. Antimicrobial coatings can help to improve product safety by reducing Listeria on RTE shrimp.