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United States Department of Agriculture

Agricultural Research Service

2007 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
All subordinate projects are making good progress toward meeting their research goals and objectives. Progress is monitored through meetings, site visits and conference calls.

1935-41420-011-01R – Reimbursable Cooperative Agreement with University of California, Davis. This is a final report. Biofilm formation by various isolates of Salmonella on cantaloupe rind surfaces during storage at 10 or 22C was demonstrated using SEM. Results demonstrated a relationship between the ability of Salmonella to form biofilms on cantaloupe surfaces and the production of curli and cellulose. Cellulose deficient mutants were far less able to adhere to surfaces. Artificially inoculated cantaloupes were fumigated with chlorine dioxide for up to 6 h. We achieved more than 5 logs (99.999%) reduction in Salmonella populations. Chlorine dioxide gas treatment also increased the shelf life of the whole cantaloupe by reducing spoilage microorganism and did not have apparent adverse effects on the quality. This project was terminated on August 31, 2006.

1935-41420-011-03S – Specific Cooperative Agreement with Drexel University, Philadelphia, PA. A prototype Nonthermal plasma (NTP) system was developed for evaluation at ERRC. Experiments with the prototype system established that Escherichia coli O157:H7 on lab media was effectively killed by a 2 minute treatment. The kill was a result of active plasma deposition, not by thermal effects. For pathogens inoculated onto the surface of a golden delicious apple, a 3 minute NTP treatment reduced Salmonella by 99.8%, E. coli O157:H7 by 99.95% and Listeria monocytogenes by 98.7%.

1935-41420-011-04N – Non-funded Cooperative Agreement with Del Monte Fresh Produce Company. Hot water pasteurization for decontamination of cantaloupe melons using the ERRC designed and built pilot scale equipment was validated at Cooperator’s processing facility is underway.

1935-41420-011-05S – Specific Cooperative Agreement with Purdue University, West Lafayette, IN. Cells of two Listeria monocytogenes isolates were able to form biofilms on stainless steel chips at 4, 20 and 37C. Planktonic cells of Listeria monocytogenes were more sensitive to inactivation by aqueous sanitizing solutions than biofilm cells.

1935-41420-011-06R – Reimbursable Cooperative Agreement with University of California, Davis. Preliminary studies were conducted, and a Research Associate was recruited. Future research under this project may result in improved means of disinfecting contaminated leafy greens during packing or fresh-cut processing.

For a complete report on the progress of these subordinate projects, see the corresponding annual report.

Killing Internalized E. coli O157:H7 in Leafy Greens: Internalized human pathogens are protected from surface treatments such as chlorine-based washes. To test a more penetrating process (irradiation), E. coli O157:H7 was inoculated to the interior leaf tissues of baby spinach and five different types of lettuce (Romaine, Green leaf, Red leaf, Iceberg and Boston). Chlorine wash treatments applied to these leaves were minimally effective, with 600ppm treatment yielding only a 90% reduction. Depending on the type of leafy green examined, irradiation reduced pathogen populations by 99.9 to 99.999% at the highest dose tested (1.5kGy). Irradiation is shown to be effective in reducing risk associated with human pathogens on leafy greens, even when these pathogens are internalized. NP 108 Food Safety 2006 – 2010 Action Plan Component(s) 1.2.4 Processing Intervention Strategies.

Inactivation of Pathogens in Biofilms: When pathogens, such as E. coli O157:H7 and Listeria monocytogenes, form biofilms (closely knit clusters of cells), they become dramatically more resistant to conventional chemical sanitizing treatments. Atomic force microscopy shows that the number of Listeria cells that initially attach to the surface influences how quickly biofilms form and how many cells survive. Listeria cells in biofilms are killed by irradiation, but their sensitivity depends on the isolate and the temperature of biofilm formation. E. coli O157:H7 cells in biofilms are similarly killed by irradiation, but the efficacy of the process is influenced by isolate and the maturity of the biofilm. These results show that, although the biofilm habitat can protect pathogen cells from chemical sanitizers, irradiation can be a viable tool for eliminating these difficult to kill pathogens. NP 108 Food Safety 2006 – 2010 Action Plan Component(s) 1.2.3 Production and Processing Ecology.

Improved Sanitizers, Packaging and Processes to Kill Pathogens: New processes to improve the microbial safety and quality of fresh and fresh-cut produce are an industry priority. Advanced sanitizing processes such as hot water pasteurization and improved chemical treatments acidified calcium sulfate and acidified sodium chlorite, were shown to reduce the surface microbial load of treated cantaloupes, and to improve the keeping quality of cut fruit derived from those melons. Optimized plastic films, such as polyvinyl chloride, were shown to be superior to low density polyethylene for use with in-package, gas-phase chlorine dioxide treatments. Nonthermal plasma, a novel sanitizing technology, reduced Salmonella by 99.87%, E. coli O157:H7 by 99.95% and Listeria monocytogenes by 98.74% when applied to the surface of inoculated apples. These advanced food safety tools will serve to protect consumers from pathogens on fresh and fresh-cut produce. NP 108 Food Safety 2006 – 2010 Action Plan Component(s) 1.2.4 Processing Intervention Strategies.

Biological control of human pathogens on produce: The survival and growth of human pathogens on produce depends on interactions with other microflora. Tests with Pseudomonas fluorescens #2-79, a selected biocontrol organism, showed that this antagonist suppressed the growth of Salmonella on sprouting seeds by 99–99.9%. Several different bioactive cultures of native microflora, originally isolated from baby carrots, suppressed the growth of Salmonella, Yersinia enterocolitica, E. coli O157:H7, and Listeria monocytogenes both in carrot juice and on green bell pepper disks by 99-99.999%. These data indicate that biocontrol organisms, whether used via a single antagonistic strain or as a complex bioactive microflora application, can effective suppress human pathogens on produce. NP 108 Food Safety 2006 – 2010 Action Plan Component(s) 2.1.5 Biological Technologies.

Controlling surface microfeatures to reduce contamination: The small-scale surface features of foods and food contact surfaces can provide shelters for pathogens such as E. coli O157:H7, Salmonella and Listeria monocytogenes, protecting them from chemicals, antimicrobial agents and physical removal. In order to understand how pathogens evade sanitizing treatments in these physical structures, studies were conducted to determine the risk factors associated with cracks and voids in the inner seed coat of sprouting seeds, the gap spacings of adjacent cells in spinach leaves, and microscratches in stainless steel, a common food-contact surface. By eliminating these risks (e.g., through seed sorting, improved produce handling, and by use of polished steel), risks of persistent pathogen contamination were reduced. These advanced studies of surface topologies allow for the identification and elimination of risk factors in foods and food contact surfaces. NP 108 Food Safety 2006 – 2010 Action Plan Component(s) 1.2.3 Production and Processing Ecology.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of non-peer reviewed presentations and proceedings17
Number of newspaper articles and other presentations for non-science audiences7

Review Publications
Annous, B.A., Kozempel, M.F. 2005. Surface pasteurization with hot water and steam. In: Sapers, G.M., GORNEY, J.R., Yousef, A.E., editors. Microbiology of Fruits and Vegetables. CRC Press. p. 479-496.

Fan, X., Fett, W.F., Mitchell, B.W. 2007. Effects of negative air ions on escherichia coli atcc 25922 inoculated onto mung bean seed and apple fruit . Journal of Food Protection. 70(1):204-208.

Kim, H., Feng, H., Kushad, M., Fan, X. 2006. Effects of ultrasound, irradiation, and acidic electrolyzed water on germination of alfalfa and broccoli seeds and on escherichia coli o157:h7. Journal of Food Science. 71(6):M168-M173.

Liao, C. 2007. Inhibitory effect on foodborne pathogens by native microflora associated with fresh peeled baby carrots. Journal of Food Science. 72(4):M134-M138.

Niemira, B.A. 2007. Irradiation Sensitivity of Planktonic and Biofilm-Associated Escherichia coli O157:H7 Isolates is Influenced by Culture Conditions. Applied and Environmental Microbiology. 73(10): 3239-3244.

Last Modified: 11/28/2015
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