Intervention Methods for Enahancing the Microbiological Safety of Fresh and Fresh-Cut Produce

NON-TECHNICAL SUMMARY: The number of foodborne illness outbreaks associated with the consumption of fresh and minimally processed fruits and vegetables have dramatically increased in the past thirty years. Traditional sanitizers such as chlorine, have poor efficacy when used for fruits and vegetables, and foodborne illness due to surviving pathogens may occur. Thermal inactivation of contaminating pathogenic and spoilage microorganisms on fresh and minimally produce is not feasible due to organoleptic changes. Identifying critical points during post harvest produce production will allow focused preventative steps to reduce pathogen contamination. As an additional level of protection from foodborne pathogens in fresh produce, effective pathogen decontamination methods will enhance the safety of produce for consumers.

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APPROACH: Obj. 1. To identify critical factors for produce during growing and processing, farm and processing plants will be sampled at various points (soil, water, food contact surfaces, slicers, and rinse waters) for the presence of generic E. coli, an indicator of fecal contamination, using Violet Red Bile Agar. Three farms and processing plants representing a range of production capacities will be sampled. The number of samples for each location, specific processing step and equipment locations will vary but a minimum of 3 per location up to as many as 10 samples per location depending on the initial findings. Each location sampling point represents the highest risk of pathogen introduction or potential multiplication of contaminating pathogens. Ten gram samples or 10 cm2 contact surfaces, and 100 ml water samples will be diluted or filtered as necessary and plated according to standard microbiological practices. Samples testing positive for E. coli will be sampled again for the presence of pathogenic E. coli using Chromagar and Salmonella using BSA or XLD Agar. <p>Obj. 2. In order to reproducibly assess the decontamination treatments, fresh lettuce (iceberg or romaine) will be obtained from a local retail grocery store. The lettuce (cut and whole leaf) will be spot inoculated with a cocktail of 5 E. coli O157:H7-GFP strains and a cocktail of 5 Salmonella-GFP strains at different inoculation levels (103 to 106/g). The lettuce will be immersed in the inoculum mixture for 10 minutes and then removed from the inoculum, spread over sterile cheese cloth and allowed to dry for 30 minutes in a biosafety cabinet. A representative sample will be taken to determine the initial pathogen levels for each treatment as described above. Decontamination Treatments: Varying levels of allyl isothiocynate (1-5 mg), dimethyl dicarbonate (250-1000 ppm) and pyruvate (10-150 mg) will be placed on sterile cotton discs and placed on the interior of a rigid polypropylene container (500 ml) and polyethylene bags (commercial produce bags) containing 100 g of inoculated produce. The samples will be placed at 40 degrees F and sampled every 2 days over the course of 14 days. As controls, inoculated produce with no treatments will be treated in the same manner as the treatment samples. To enumerate the GFP E. coli O157:H7 and Salmonella, 10 g samples of each treatment will be homogenized with 90 ml of sterile 0.1 percent peptone and further 10 fold dilutions will be made using sterile 9 ml 0.1 percent peptone water blanks. Appropriate dilutions will be surface plated onto TSA supplemented with 20 ug/ml isopropyl-b- D-thiogalactoside to allow for recovery of injured or stressed bacteria. The plates will then be incubated at 37 degrees C for 72 hours to allow for injured pathogens to resuscitate. Total plate counts using Plate Count Agar, for the produce samples will also be determined to establish the effect on the indigenous microflora. All experiments will be plated in duplicate for each. <p>Obj. 3. Once scientific data is generated from the research outlined in Obj. 1 and 2, appropriate extension materials will be developed targeting audiences on the farm and in processing plants.
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PROGRESS: 2006/10 TO 2007/09<BR>
OUTPUTS: The novel antifungal compound produced by a bacterial strain originally isolated from honey has been purified and chemically characterized as described in previous reports. The genetic elements coding for the antifungal protein have been characterized at the DNA level. The entire coding region for the preprotein has been sequenced and analyzed. From the analysis, the protein does not show any homology to other antifungal proteins and peptides from the iturin or surfactant groups. High DNA homology was observed to genes identified in bacterial genome sequences but the function had not been assigned to the putative proteins encoded by these genes. DNA sequencing and analysis of the upstream and downstream regions of the gene coding for the antifungal protein did not show any additional open reading frames. It is not known whether an immunity protein is required or present which is usually immediately downstream of the antimicrobial peptide or protein. In addition, transport and regulatory elements for the antifungal protein were not observed. It is likely that these essential proteins are located distal to the gene coding for the antifungal protein. Based on the DNA sequence, the preprotein has a leader or signal peptide that is cleaved in the active mature antifungal protein. The antifungal proteins exhibits a broad spectrum of activity that includes but is not limited to Penicillium, Byssochlamys and Aspergillus. The antifungal protein did not show activity against a limited range of yeast tested that included Saccharomyces and Candida. <BR>PARTICIPANTS: Randy W. Worobo (PI) John J. Churey (Research Support Specialist) Hyungjae Lee (postdoctoral researcher)
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IMPACT: 2006/10 TO 2007/09<BR>
The antifungal protein represents a novel antifungal protein that is active against both human clinical and foodborne pathogens. Existing antifungal proteins and peptides are limited due to cellular toxicity by pore formation on the cell membranes of eukaryotic cells. Due to the size of this antifungal protein and preliminary tests, cell toxicity due to cell membrane pore formation does not occur. There is a potential for this protein to be used as an orally administered antifungal agent for human therapy or as an antifungal compound for foods to prevent spoilage or protection against mycotoxin producing molds.