1a. Objectives (from AD-416):
1. Develop and evaluate novel antagonists (e.g. Bacteriovorax, Bdellovibrio and non-pectolytic Pseudomonas) for biological-based intervention strategies, and identify means of combining pathogen microbial ecology with effective chemical and physical interventions. 2. Develop and optimize chemical decontamination interventions (e.g. novel sanitizer formulations and advanced gas-phase antimicrobial treatments), making use of pathogen microbial ecology information generated under Objective 1. 3. Develop nonthermal technologies (e.g. cold plasma, high-intensity monochromatic light and irradiation) as effective, waterless physical treatments, and establish protocols for combination treatments with interventions developed in the first two objectives.
1b. Approach (from AD-416):
A holistic approach to the development of new effective intervention technologies will be followed. In an iterative process of technology development, increased knowledge of pathogen ecology on produce surfaces will be used to optimize biological, chemical and physical control strategies; combinatorial use of intervention technologies will be examined for additive and synergistic effects while maintaining product quality. More rapid and successful commercialization will be fostered by determining the equipment and infrastructure required for large-scale implementation, as well as economic costs and benefits expected from the use of the new technologies. A key aspect of this effort to facilitate commercialization will be collaboration and information sharing with industry, including direct contact with potential end-users of technologies. Stakeholders will be updated on research goals and objectives of the project, and input will be sought from them to identify key problems to be solved. By proactively fostering these interactions in conjunction with site visits, annual scientific meetings, industry trade shows and similar venues, there will be opportunities in the early, mid-phase and in late stages of research will allow for the practical needs of industry to be addressed as the research is formulated and conducted.
3. Progress Report:
Progress was made on all objectives, all of which fall under NP108 –Food Safety, Component 1 Food-borne Contaminants: Problem Statement 1, Population Systems; and Problem Statement 4, Intervention and Control Strategies. Objective 1. The ability of biosurfactant sophorolipids (palmitic acid, oleic acid and stearic acids) to inactivate Listeria monocytogenes, Salmonella enterica, and Escherichia coli O157:H7 was determined. Using in-vitro assays, bacterial suspensions were inoculated in plate wells and sophorolipids were added at different concentrations. Following storage at 5C and 25C for either 30 min or 24 hours, bacterial pathogens were recovered by dilution plating on TSA agar. At 5C (30 min), pathogens were reduced by 68-98% across three L. monocytogenes strains. The reductions of L. monocytogenes varied, but significantly increased as the concentrations of sophorolipids increased. At 25C (30 minutes), average pathogen reductions ranged from 68-99.3%. At 25C (24 hours), inactivation of L. monocytogenes by palmitic acid (0.02-0.12%) in-vitro were significantly greater and ranged from 99.99-99.9999%. At 5C, lower reductions of 76-98% were attained. Inactivation was lower overall for Salmonella and E. coli O157:H7 strains than for the strains of L. monocytogenes. At 1-2% palmitic acid for 24 hours at 25C, mean reductions of S. enterica and E. coli O157:H7 ranged from 98-99.99% and 87-99.99%, respectively. In-vitro pathogen reductions by oleic and stearic acids were similar. On post-harvest tomato and strawberry, stem scars and produce surfaces were inoculated with pathogenic strains, followed by the application of sophorolipid. The after treatment, the pathogens were recovered on selective media. Palmitic acid applied on produce at the rate of 0.12%, resulted in pathogen reductions of 50-84% on tomato and 62-93% on strawberry. Application of a commercial sanitizer (“Lovit”) reduced pathogens by >99.999% on grape tomato by >99.9% logs on strawberry. Combinations of palmitic acid plus sanitizer reduced pathogens by 99.999% on grape tomato and by 99.2% on strawberry. Objective 2. Hot water treatments were optimized for sprouting seeds and fresh produce. Treatment of inoculated mung bean seeds in hot water at 80C with or without 0.2% Stepanol and 20 ppm chlorine for 90 and 120 seconds, respectively, resulted in complete inactivation of pathogenic Salmonella. Also, sprouts obtained from both optimized treatments of seeds showed no pathogen following 5 days of incubation at 21C and 75% humidity. Treatments did not affect the seed germination. This is a marked improvement over industry practice of soaking the inoculated seeds with 1,000 ppm chlorine or electrolyzed water for 24 hours, which resulted in 90% reduction in Salmonella populations. Applied to cantaloupe, we determined that an optimized treatment of 3.75 min in 80 degrees C water reduced L. monocytogenes by 99.999%, and reduced total bacterial counts by at least 99.9%. Objective 3: The efficacy of cold plasma, a novel antimicrobial process, was determined under varying conditions of feed gas humidity, composition and treatment conformation. In one study, Romaine lettuce was inoculated with Escherichia coli O157:H7, Salmonella, Listeria monocytogenes, and Tulane virus (TV) on Romaine lettuce. Treatment parameters included moisture vaporization, modified atmospheric packaging (MAP), and post-treatment storage. Inoculated leafy greens were packaged in either a Petri dish or a Nylon/polyethylene pouch with and without moisture vaporization. Additionally, a subset of pouch-packaged leaves was flushed with oxygen at 5% or 10% (balance of the gas was nitrogen). All of the packaged lettuce samples were treated with cold plasma at 47.6 kV for 5 min and then analyzed either immediately or following post-treatment storage for 24 h at 4C to assess the inhibition of microorganisms. Cold plasma inhibited E. coli O157:H7, Salmonella, L. monocytogenes, and TV by 92%, 60%, 90%, 95%, respectively. The inhibition of the bacteria was not significantly affected by the type of lettuce packaging or moisture vaporization (p > 0.05) but a reduced-oxygen MAP gas composition reduced the inhibition rates of E. coli O157:H7 and TV. L. monocytogenes continued to decline by an additional 75% in post-treatment cold storage. Both rigid and flexible conventional plastic packages appear to be suitable for the in-package decontamination of lettuce with this cold plasma system. In related studies, grape tomatoes were inoculated with Salmonella, packaged in a polyethylene terephthalate (PET) commercial clamshell container and treated with cold plasma at 35 kV at 1.1 kHz for 3 min. Cold plasma reduced Salmonella by approximately 90%, irrespectively of the size of container, the number of grape tomatoes, or the position of the tomato in the container (P > 0.05). Rolling during treatment significantly increased the Salmonella reduction rates to 99.9% and reduced all aerobic microorganisms by 95-97%. Growth of Salmonella, total aerobes, and yeast and molds on cold plasma-treated grape tomatoes was effectively prevented during storage for 21 days at 10C. Cold plasma did not influence the tomato color, firmness, weight loss, pH, total soluble solid content, and lycopene concentration at either 10C or 25C storage. These results demonstrated the potential for cold plasma as a post-packaging process to for packaged bulk tomatoes. To expand this work to study mixed salads, two different inoculation methods were used. Either cherry tomatoes or romaine lettuce were inoculated with a Salmonella cocktail and placed into a commercial PET plastic container and thoroughly mixed together with its non-inoculated counterpart. Mixed salads were allowed to dry in a biosafety cabinet for 1 h. Samples were treated with cold plasma inside plastic containers for 3 min. When lettuce was the inoculated counterpart, the kill level of Salmonella was significantly greater on tomatoes (83% reduction) compared to lettuce (55% reduction). When lettuce was the inoculated counterpart, Salmonella was significantly reduced on mixed salad only (49% reduction). Cold plasma can kill Salmonella in a pre-packaged, mixed salad, with efficacy dependent on the nature of contamination, direction of transfer, and on the surface topography of the contaminated commodity. Additional research was conducted on cold plasma inactivation of norovirus surrogates, on comparing vacuum-activated cold plasma vs. corona discharge cold plasma, combination treatments of cold plasma plus chemical sanitizers, and in applications of high-intensity monochromatic light as a surface sanitizing process. These results are developing cold plasma and related technologies as antimicrobial treatments for fresh and fresh-cut fruits and vegetables.
1. Reductions of pathogenic bacteria by natural antimicrobials. Natural products can improve the safety of post-harvest produce due to their antimicrobial properties and low toxicity. ARS Researchers at Wyndmoor, Pennsylvania investigated a class of antimicrobial compounds (“sophorolipids”) produced by bacteria for their ability to inactivate human pathogens in-vitro, on grape tomatoes and strawberries. Results showed that different concentrations of sophorolipids (such as palmitic acid) reduced the survival of pathogenic bacteria. Another pathogen, Listeria monocytogenes, was much more sensitive to antimicrobial than Salmonella enterica or Escherichia coli. The sophorolipds were more effective in solution trials than on fresh produce, but when applied in combination with a sanitizer, pathogens were reduced by more than 90% on tomatoes and strawberries. These novel antimicrobial compounds could see use as a “natural” treatment to improve the safety of fresh fruits and vegetables.
2. Soybean sprouts safety enhanced by gaseous chlorine dioxide and biocontrol bacteria. Since sprouts are often contaminated with human pathogens, ARS Researchers at Wyndmoor, Pennsylvania collaborated with the University of Puerto Rico scientists to improve sprout safety with gaseous chlorine dioxide and biocontrol (non-pathogenic) Pseudomonas microbes. This combination treatment reduced Salmonella Typhimurium on soybean sprouts by 99.7-99.9%, whereas the biocontrol microbe alone reduced Salmonella by only 60-92%. Produce treated with combinations of the two compounds showed further reductions of Salmonella levels on sprouts with little change in quality after 7 days of storage. The combined treatments, applied correctly, can enhance soybean sprout safety and protect the American consumer.
3. Hot water makes for safer sprouting seeds. Mung bean sprouts have been implicated with numerous salmonellosis outbreaks. ARS researchers at Wyndmoor, Pennsylvania developed a hot water treatment that is capable of reducing Salmonella populations on mung bean sprouting seeds by 99.9%. Treating sprouting seeds in hot water at 80C with or without chemical additives (0.2% Stepanol and 20 ppm chlorine) for 90 to 120 seconds completely inactivated pathogens on seeds, immediately after treatment and after 5 days of incubation at room temperature and 75% humidity. Since this is a significant improvement over conventional treatments such as 1,000 ppm chlorine rinses or electrolyzed water for 24 hours, this hot water process would be an improved, “green” method to reduce the risks of Salmonella on sprouts.
4. Reducing Listeria monocytogenes on cantaloupe with hot water. Listeria monocytogenes contamination of cantaloupes has resulted in some of the deadliest foodborne illness in recent years. ARS researchers at Wyndmoor, Pennsylvania developed direct-from-field surface pasteurization treatments for whole cantaloupe which can almost completely eliminate the pathogen from the surface of cantaloupe. A process of treating cantaloupes in 80C water for 4 minutes is ready for commercialization.
5. Safer fresh-cut cantaloupes without chemical sanitizers. Fresh-cut cantaloupes have been implicated in numerous foodborne outbreaks of salmonellosis. ARS researchers at Wyndmoor, Pennsylvania inoculated whole cantaloupes with Salmonella and compared 1) a 200 ppm, 20 min chlorine wash, 2) a 5 mg/L, 4.5 h chlorine dioxide gas fumigation, and 3) a 76C, 3 min hot water dip. Fresh-cut cantaloupes were prepared from treated whole cantaloupes and evaluated during refrigerated storage. All fresh-cut samples prepared from hot water-treated cantaloupes were negative for the pathogen throughout the entire storage sampling, while chlorine dioxide- and chlorine-treated cantaloupe pieces had detectable pathogen levels in storage. This hot water process for whole melons can yield safer, higher-quality cut pieces than conventional chemical treatments.
6. Rapid detection of pathogenic bacteria on mung bean seeds. The more quickly pathogens can be detected and identified on foods, the safer our food supply will be. ARS researchers at Wyndmoor, Pennsylvania extracted certain cellular components (secondary metabolites) from Salmonella, L. monocytogenes or E. coli O157:H7, and identified them using advanced chemical analysis techniques (matrix assisted laser desorption ionization mass spectrometry coupled with tandem time of flight mass analysis). Certain distinct peaks were identified that can be related with proteins produced by the individual strains of these pathogens. This kind of chemical analysis of pathogenic strains is 300-800% faster than conventional microbial growth identification methods. Faster identification will allow food processer and packagers to more efficiently recall compromised products, thereby protecting the American consumer from foodborne human pathogens.
7. Packaged lettuce made safer with cold plasma. Cold plasma is a promising new treatment for the inactivation of foodborne pathogens. ARS researchers at Wyndmoor, Pennsylvania inoculated Romaine lettuce with different pathogens under a variety of packaging conditions. Cold plasma inhibited E. coli O157:H7, Salmonella, L. monocytogenes, and Tulane virus by 92%, 60%, 90%, 95%, respectively, irrespective of packaging atmosphere humidity content. L. monocytogenes continued to decline in post-treatment cold storage, suggesting that cold plasma causes injury which leads to later cell death of the pathogen. Since both rigid and flexible conventional plastic packages appear to be suitable for the cold plasma decontamination of lettuce, this technology could be used to reduce foodborne illnesses associated with leafy greens.
8. Cold plasma improves safety of packaged tomatoes. Cold plasma is a promising new treatment for the inactivation of foodborne pathogens. ARS Researchers at Wyndmoor, Pennsylvania inoculated grape tomatoes with Salmonella and packaged them in a polyethylene terephthalate (PET) commercial clamshell container. A cold plasma treatment of 3 minutes eliminated spoilage yeasts and molds, and reduced the Salmonella by up to 99.95%. This improvement in safety and storability was achieved with no adverse effect on tomato color, firmness, water content, pH, total soluble solids or lycopene concentration. This cold plasma technology could be used to improve the safety and shelf-life of packaged tomatoes and other fresh and fresh-cut fruits and vegetables.
9. Cold plasma enhances chemical sanitizer efficacy. Enhanced antimicrobial performance is possible with the combination of conventional chemical sanitizers with novel nonthermal processes such as cold plasma. ARS researchers at Wyndmoor, Pennsylvania inoculated Listeria monocytogenes onto the surfaces and into the difficult-to-sanitize calyx areas (“blossom end”) of Granny Smith apples, then treated with 1) an antimicrobial solution, 2), cold plasma or 3) both treatments, in sequence. Alone, the chemical sanitizer and cold plasma reduced L. monocytogenes by 99% and 99.6%, respectively; together, the combination treatment reduced L. monocytogenes by greater than 99.99%, even in the calyx area. Optimized combinations of chemical sanitizers and cold plasma provide new, effective tools to significantly reduce human pathogens on fruits and vegetables, and improve food safety for the American consumer.
10. Human norovirus surrogate is inactivated by cold plasma. Viruses, including human norovirus, are currently the leading cause of foodborne outbreaks. ARS researchers at Wyndmoor, Pennsylvania used cold plasma applied to blueberries to inactivate two widely used surrogates for human norovirus: Tulane virus and murine norovirus. A treatment of 45 seconds reduce Tulane virus by 96%, with 99.97%, reduction after 120 seconds. Murine norovirus was reduced by 68% after only 15 seconds, and was eliminated (>99.999% reduction) after only 90 seconds of cold plasma treatment. Since cold plasma is a nonthermal process, these results show that with further optimization, cold plasma may be used by food processors as a new tool to reduce human norovirus on improve the safety of fresh and fresh-cut fruits and vegetables, including fragile produce such as berries.
Min, S., Roh, S., Boyd, G., Sites, J.E., Uknalis, J., Fan, X., Niemira, B.A. 2017. Inactivation of Escherichia coli 0157:H7 and aerobic microorganisms in Romaine lettuce packaged in a commercial polyethylene terephthalate container using atmospheric cold plasma. Journal of Food Protection. 80(1):35-43.
Rodriguez, A., Olanya, O.M., Annous, B.A., Cassidy, J.M., Orellana, L., Niemira, B.A. 2017. Survival of Salmonella Typhimurium on soybean sprouts after treatment with gaseous chlorine dioxide and biocontrol Pseudomonas bacteria. Food Science and Biotechnology. 26:513-520.
Min, S.C., Roh, S., Niemira, B.A., Boyd, G., Sites, J.E., Uknalis, J., Fan, X. 2017. In-package inhibition of E.coli 0157:H7 on bulk romaine lettuce using cold plasma. Food Microbiology. 65:1-6.
Ukuku, D.O., Geveke, D.J., Chau, L.I., Bigley, A., Niemira, B.A. 2017. Appearance and overall acceptability of fresh-cut cantaloupe pieces from whole melon treated with wet steam process. LWT - Food Science and Technology. doi: 10.1016/j.lwt.2017.04.033.
Jin, Z.T., Huang, M., Niemira, B.A., Cheng, L. 2016. Shelf life extension of fresh ginseng roots using sanitizer washing, edible antimicrobial coating and modified atmosphere packaging. International Journal of Food Science and Technology. doi: 10.1111/ijfs.13201.
Min, S., Roh, S., Niemira, B.A., Sites, J.E., Boyd, G., Lacombe, A. 2016. Dielectric barrier discharge atmospheric cold plasma inhibits Escherichia coli 0157:H7, Salmonella, Listeria monocytogenes, and Tulane virus in Romaine lettuce. International Journal of Food Microbiology. 237:114–120.
Ukuku, D.O., Geveke, D.J., Chau, L.I., Niemira, B.A. 2016. Microbial safety and overall quality of cantaloupe fresh-cut pieces prepared from whole fruit after wet steam treatment. International Journal of Food Microbiology. doi: 10.1016/j.ijfoodmicro.2016.05.019.
Ukuku, D.O., Mukhopadhyay, S., Geveke, D.J., Olanya, O.M., Niemira, B.A. 2016. Minimal thermal treatments for reducing bacterial population on cantaloupe rind surfaces and transfer to fresh-cut pieces. Journal of Food Protection. doi: 10.4315/0362-028X.JFP-16-046.
Lacombe, A.C., Beard, A., Hwang, C., Hill, D., Fan, X., Huang, L., Yoo, B.K., Niemira, B.A., Gurtler, J., Wu, V.C. 2016. Inactivation of Toxoplasma gondii on blueberries using low dose irradiation without affecting quality. Food Control. 73(2017):981-985.
Jin, Z.T., Huang, M., Niemira, B.A., Cheng, L. 2016. Microbial reduction and sensory quality preservation of fresh ginseng roots using nonthermal processing and antimicrobial packaging. Journal of Food Processing and Preservation. doi: 10.1111/jfpp.12871.
Lacombe, A.C., Niemira, B.A., Gurtler, J., Sites, J.E., Boyd, G., Kingsley, D.H., Li, X., Chen, H. 2017. Nonthermal inactivation of norovirus surrogates on blueberries using atmospheric cold plasma. Food Microbiology. 63:1-5.
He, Z., Zhang, M., Zhao, A., Olanya, O.M., Larkin, R.P., Honeycutt, C.W. 2016. Quantity and nature of water-extractable organic matter from sandy loam soils with potato cropping managements. Agricultural and Environmental Letters. 1:160023-160029.
Ojwang, D.J., Nyankanga, R.O., Imungi, J., Olanya, O.M., Ukuku, D.O. 2016. Cultivar preference and sensory evaluation of vegetable pigeon pea (Cajanus cajan) in Eastern Kenya. Food Security Journal. 8:757-767.
Larkin, R.P., Halloran, J.M., Honeycutt, C.W., Griffin, T.S., Olanya, O.M., He, Z. 2016. Cumulative and residual effects of different potato cropping system management strategies on soilborne diseases and soil microbial communities over time. Plant Pathology. 66:437-449. doi: 10.1111/ppa.12584.