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United States Department of Agriculture

Agricultural Research Service

2008 Annual Report

1a.Objectives (from AD-416)
Develop more effective means for decontaminating organic and conventionally grown fresh and minimally processed fruits and vegetables including sprout seed containing human pathogens to ensure food safety and security by assessing the efficacy of new and/or improved intervention technologies. Determine effectiveness of treatment combinations (multiple hurdle approach). Assess factors that might limit treatment efficacy. Transfer effective decontamination technology to the produce industry in order to reduce the risk of foodborne illness.

1b.Approach (from AD-416)
A variety of chemical, physical and biological intervention technologies will be evaluated. Physical and chemical treatments include the use of hot water pasteurization, ultrasound, gaseous chlorine dioxide, cold plasma, hydrogen peroxide vapor, and ionizing radiation alone or in combination. Conduct studies on the use of single or multiple isolates of antagonistic bacteria for inhibiting the outgrowth of bacterial human pathogens on sprouting seed and on sprouts postharvest. Determine the mode of action of effective antagonists. Scale up studies of effective interventions from laboratory scale to pilot plant scale. Investigate changes in composition and structure of indigenous microbial communities in relation to shelf life and hygienic quality of produce while in storage. Study the formation of biofilms by pathogens alone or in combination with native microflora on the surface of selected produce. Evaluate the effects of the various interventions on sensory and nutritional quality attributes, yield, physiology, and shelf-life to ensure acceptable quality of treated foods.

3.Progress Report
There has been significant progress made on the project 1935-41420-011-00D, Intervention Technologies for Enhancing the Safety and Security of Fresh and Minimally Processed Produce and Solid Plant-Derived Foods.

A beneficial bacterium, Pseudomonas fluorescens 2-79 (pf 2-79), was discovered to be particularly effective in inhibiting the growth of pathogens such as Salmonella and E. coli O157:H7 that survived initial physical or chemical treatments. The bacteria also stopped the growth of two common spoilage bacteria and reduced the development of soft rot. Because it can grow at refrigeration temperatures, it is especially useful for control of pathogens on fresh produce that are often stored under refrigeration.

Two versions of cold plasma generating systems have been developed and tested to reduce the population of Escherichia coli O157:H7 and Salmonella Stanley on apples and almonds. Reductions ranged from 99.87 – 99.98% (2.9 to 3.7 log cfu/ml) for S. Stanley, and 99.96 – 99.97% (3.4 - 3.6 log cfu/ml) for E. coli O157:H7 on apples after 2 min treatment.

Ionizing irradiation enhanced microbial safety of fresh and fresh-cut produce. However allowing time for biofilm formation reduced the efficacy of irradiation in eliminating pathogens from leafy vegetables. Regarding the quality of irradiated fresh produce, most fresh-cut vegetables of thirteen common fresh-cut vegetables could tolerate 1 kGy radiation without deterioration in quality. At 1 kGy, no detectable amount of furan (a possible carcinogen) was produced from the vegetables. Numerous inquiries about produce irradiation have been received, resulting in hundreds of TV, radio, internet and newspaper articles featuring ARS irradiation research.

A biosafety level two pilot plant was established to provide researchers an ability to conduct pilot-plant scale decontamination studies of produce and equipment using human pathogens instead of surrogates. Pilot-scale washing treatments using 76C hot water for 3 min of cantaloupe resulted in more than 99.999% (5 log) reduction of Salmonella Poona. Large scale chlorine dioxide gas treatment of the un-inoculated melons yielded more than 99% (2 log) reductions in normal microflora. Shelf-life of whole melons and fresh-cut fruit prepared from the treated whole melons was extended by at least one month, and sensory qualities of fresh-cut fruit were maintained and in some instances were enhanced.

The progress made over last 12 months addressed the NP 108 2006-2010 Action Plan components 1.2.4 Processing Intervention Strategies by developing, improving and validating new chemical, biological and physical pathogen intervention strategies, and 1.2.3 Production and Processing Ecology by defining pathogen ecology including the importance of biofilm formation and the interaction of human pathogens with native microorganisms on produce and their effects on decontamination efficacy.

1. Cold plasma kills human pathogens on produce. The produce industry has a critical need for an effective antimicrobial treatment (a “kill step”) suitable for application to fresh and fresh-cut fruits and vegetables. Cold plasma is a novel food processing technology that uses energetic gases to kill pathogens on fragile surfaces. Cold plasma generated in a prototype gliding arc was applied to outbreak strains of Escherichia coli O157:H7 and Salmonella Stanley inoculated on the surfaces of golden delicious apples. Maximum reductions were obtained after 180 seconds of treatment, and ranged from 99.87 – 99.98% (2.9 to 3.7 log cfu/ml) for S. Stanley, and 99.96 – 99.97% (3.4 - 3.6 log cfu/ml) for E. coli O157:H7. The maximum temperature of any plasma treated apple was 50.8C (28C above ambient), indicating that antimicrobial effects were not the result of heat. Using a more advanced form of the arc discharge, studies to optimize the plasma chemistry have shown that a nitrogen feed gas increases UV-light production by 80% over that of an air-based feed gas. The advances made in this emerging technological field will give processors new cold plasma-based processing interventions to kill human pathogens on produce, thereby making these foods safer. NP 108 Food Safety 2006 – 2010 Action Plan Component(s) 1.2.4 Processing Intervention Strategies.

2. Biological control of human pathogens and spoilage bacteria on produce. Conventional washing and sanitization treatments can not completely eliminate human pathogens from produce. Used in combination with these conventional chemical treatments, a biological-based intervention may be used to effectively inhibit the re-growth of surviving pathogens. Using bell pepper disks as a model system, plant-associated bacterial antagonists were evaluated for their potential as biological agents for control of foodborne pathogens and spoilage bacteria. A strain of Pseudomonas fluorescens (designated Pf 2-79) was identified as the most effective among the bacterial antagonists investigated. Treatment with 100-fold higher number of Pf 2-79 than the pathogen reduces growth of pathogens by 99.9% (3 logs). Pf 2-79 is also effective in suppressing the growth of cold-tolerant pathogens such as Listeria monocytogenes and Yersinia enterocolitica and spoilage bacteria such as Pseudomonas marginalis. Application of Pf 2-79 and other bacterial antagonists shows promise as a new tool to restrict the proliferation of human pathogens and spoilage bacteria on produce. NP 108 Food Safety 2006 – 2010 Action Plan Component(s) 1.2.3 Production and Processing Ecology and 1.2.4 Processing Intervention Strategies.

3. Biofilms on lettuce influence efficacy of irradiation. Contamination of leafy green vegetables with Escherichia coli O157:H7 is a source of ongoing concern for consumers. E. coli O157:H7 in biofilms are relatively resistant to chemical treatments, but little is known about the response of leaf surface biofilms to irradiation. Leaves of Romaine lettuce and baby spinach were dip inoculated in a cocktail of three strains of E. coli O157:H7 and stored to allow biofilms to form. Chlorine washes were generally only moderately effective, and resulted in maximal reductions of 94.99% (1.3 log cfu/g) for baby spinach and 98.42% (1.8 log cfu/g) for Romaine. For both types of leaves, allowing time for biofilm formation reduced the efficacy of irradiation, up to 48h post-inoculation. From 0h of storage, D10 values (the dose required for a 1 log reduction) increased from 0.19 kGy to a maximum of 0.40-0.43 kGy for Romaine and 0.52-0.54 kGy for spinach. These results indicate that the biofilm habitat can reduce the efficacy of irradiation in eliminating pathogens from leafy vegetables. This information will allow processors to design better, more effective ways to use irradiation to improve the safety of leafy vegetables. NP 108 Food Safety 2006–2010 Action Plan Component(s) 1.2.3 Production and Processing Ecology and 1.2.4 Processing Intervention Strategies.

4. Radiation sensitivity of fresh vegetables. The industry is in need of a “kill” step to ensure microbial safety of fresh produce and gain the confidence of consumers. Many studies have demonstrated that a dose of 1 kGy radiation can achieve at least 99.999% (5 log) reduction of E. coli O157:H7 surface inoculated on fresh produce. Whether fresh-cut produce can tolerate the dose is unclear. Quality of thirteen common fresh-cut vegetables (Iceberg, Romaine, red and green leaf lettuce, spinach, tomato, cilantro, parsley, green onion, carrot, broccoli, red cabbage, and celery) irradiated at 1 kGy was investigated. The appearance, texture, and aroma of most of the 13 common fresh-cut vegetables were not negatively affected by the 1 kGy radiation. The appearance and aroma of many irradiated vegetables were better than that of the untreated samples after 14 days storage, due to the reduction of decay and browning. However, vitamin C content was reduced by irradiation in a few vegetables. Most fresh-cut vegetables could tolerate 1 kGy radiation without deterioration in quality. At low doses, no detectable amount of furan (a possible carcinogen) was produced from irradiation of the vegetables. This information is critical to the real-world application and implementation of irradiation as a food safety intervention. NP 108 Food Safety 2006-2010 Action Plan Component(s) 1.2.4 Processing Intervention Technologies.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of New CRADAS1
Number of the New MTAs (providing only)2
Number of Non-Peer Reviewed Presentations and Proceedings2
Number of Newspaper Articles and Other Presentations for Non-Science Audiences2

Review Publications
Fan, X., Sokorai, K.J. 2007. Effects of ionizing radiation on quality of frozen corn and peas. Journal of Food Protection. 70(8):1901-1908.

Liao, C. 2008. Growth of salmonella on sprouting alfalfa seeds as affected by the inoculum size, native microbial load, and Pseudomonas fluorescens 2-79. Letters in Applied Microbiology. 46:232-236.

Niemira, B.A. 2007. Relative Efficacy of Sodium Hypochlorite Wash Versus Irradiation to Inactivate Escherichia coli O157:H7 Internalized in Leaves of Romaine Lettuce and Baby Spinach. Journal of Food Protection. 70(11):2526-2532.

Sites, J.E., Annous, B.A., Walker, P.N., Burke, A.M. 2008. Development and Validation of a Pilot Scale Enhanced Biosafety Level Two Containment for Performance Evaluation of Produce Disinfection Technologies. Applied Biosafety. 13(1):30-44.

Fan, X., Annous, B.A., Beaulieu, J.C., Sites, J.E. 2008. Effect of hot water surface pasteurization of whole fruit on shelf life and quality of fresh-cut cantaloupes. Journal of Food Science. 73(3):M91-M98.

Fan, X., Sokorai, K.J. 2008. Effect of ionizing radiation on furan formation in fresh-cut fruits and vegetables. Journal of Food Science. 73(2):C79-C83.

Last Modified: 10/9/2015
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