<OL> <LI>To determine the nature of tomato contamination through the use of bioluminescent Salmonella enterica applied directly to the growing plant or fresh fruit<LI>To determine the effect of high pressure processing on Salmonella enterica populations and post-harvest ripening of whole green tomatoes<LI>To determine the efficacy of high pressure processing to control Salmonella enterica in peanut butter<LI>To survey fresh market farmers in Virginia, Georgia, and South Carolina and to survey managers of small farm markets in order to understand current practices and to guide research and Extension efforts. </OL>The primary outputs of this work are expected to be data that will be useful for improved application of high pressure technology, to improve understanding of tomato contamination, and to improve development of educational materials for farmers.
Non-Technical Summary: Due to food safety concerns regarding fresh and minimally processed fruits and vegetables and RTE foods, the food industry is searching for improved understanding of contamination mechanisms as well as processing/treatment options to ensure the safety of such products. Thermal processing, although effective for reduction of pathogenic and spoilage microorganisms, is not a viable option for minimally processed fruits and vegetables due to the undesirable effects of heat on fresh fruits and vegetable products and the high cost associated with such processes. Additionally, RTE products, like peanut butter, may require post-processing treatments where thermal processes may not be feasible. Therefore, the alternative processing methodology, high pressure processing, may provide an alternative to thermal treatment for the processing of produce and other RTE products. Additionally, improved understanding of how pathogenic bacteria interact with plants in the pre- and post-harvest environments may yield new directions for control of harmful microorganisms in the pre-harvest environment. This information, combined with better understanding of current production and handling practices of fruit and vegetable producers will yield robust extension programming to improve the safety of fresh produce. <P> Approach: TOMATO CONTAMINATION STUDY: Salmonella enterica will transformed, via plasmid, to carry the lux-cassette to produce bioluminescence. A loopful of each of the bioluminescent Salmonella serotypes will be used to inoculate 25 ml of Tryptic soy broth (with antibiotic) followed by incubation at 37C for 24 hrs. Serial dilutions will be performed to test bioluminescence of different concentrations (5 log and 9 log). In order to assess the survival and bioluminescence of Salmonella in soil, 10g of quartz and sand particles will be added to 50 ml of TSB (with antibiotic). The media will be inoculated with the bioluminescent Salmonella enterica and incubated at 37C for 24 hrs. The quartz particles will then be vacuum filtered to drain off excess media and dried for 24 hours. One ml of inoculated distilled water and 1g of soil and dust particles will be placed in the 96-well plates. After 30 days an image will be taken using the CCD camera. Finally, blossoms of growing tomato plants will be inoculated with bioluminescent Salmonella and when the fruit is harvested, the fruit will be subjected to CCD imaging. HIGH PRESSURE PROCESSING AND RIPENING OF TOMATOES: Salmonella enterica will be inoculated onto whole tomatoes (green unripened) via a vacuum method to achieve internalization of the pathogen into the fruit, then bagged in 1% CaCl solution. The bagged whole tomatoes will be subjected to pressures up to 550 MPa for 2 min at 17C (initial temperature). Following treatment, the whole tomato will be transferred to a fresh sterile bag and homogenized for 1 minute followed by plating. Additionally, inoculated samples that receive no HPP treatment will be analyzed microbiologically as described above. Un-inoculated and S. enterica inoculated un-ripened green tomatoes will be prepared and HPP treated as described above. Immediately following processing, tomatoes subjected to 125 ppm ethylene via a flow through gas system for up to 7 days. Tomatoes will then be evaluated for appropriate ripening as compared to non-pressurized fruit. HIGH PRESSURE PROCESSING OF PEANUT BUTTER: Salmonella enterica cultures will undergo successive 24-hour transfers into TSB at 37C prior to use. A commercial creamy-style and a commercial reduced-fat peanut butter will used for this study. A Salmonella suspension (1 ml) will be added to the peanut butter and mixed for 2 min. Inoculated peanut butter samples will be pressure treated at 350, 450 and 550 MPa. Sample will be plated directly after pressurization and after 24 hours. FARM SURVEY: Farm Markets Survey: Two surveys - One survey will target small to medium farms and the other will target small farm market managers. Survey participants will be asked about food safety practices and policies of their markets as well as characteristics of their vendors. Survey design and implementation will be conducted and data analyzed by the Virginia Tech Survey Center. Year 3 - A second wave of surveys designed to assess gains in knowledge will be conducted as described previously.