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United States Department of Agriculture

Agricultural Research Service

Research Project: INTERVENTION TECHNOLOGIES FOR ENHANCING THE SAFETY AND SECURITY OF FRESH AND MINIMALLY PROCESSED PRODUCE AND SOLID PLANT-DERIVED FOODS

Location: Food Safety and Intervention Technologies

2010 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
The extent to which the biofilm habitat fosters recovery of irradiated E. coli O157:H7 was determined on leaf surfaces. Iceberg lettuce was inoculated with a cocktail of E. coli O157:H7 isolates and stored to promote biofilm formation. Contaminated leaf materials were irradiated at doses from 0.25-1.5 kGy, then sampled immediately or returned to cold storage to allow for potential recovery and regrowth. Irradiation effectively reduced the population of E. coli O157:H7 on lettuce leaves immediately after treatment. Pathogen population did not subsequently recover during refrigerated storage, indicating a durable reduction of total pathogen load. Therefore, the biofilm habitat does not facilitate post-irradiation recovery of the pathogen. Irradiation is an important potential intervention for fresh and fresh-cut fruits and vegetables; these results can be used to better establish best practices for incorporating irradiation into a lettuce/cut salad processing chain. To establish parameters for effective use of gas-phase chlorine dioxide (ClO2) on tomatoes, whole tomatoes were inoculated with Salmonella Poona RM 2350 to an approximate final concentration of 5 log CFU/gm, and stored at 4C for 24 h prior to treatment. Tomatoes were fumigated with ClO2 for up to 6 h in a closed chamber that was developed at ERRC, using two different technologies for generating ClO2. Following treatment, residual (non-injured and injured) populations of Salmonella Poona on tomatoes were enumerated using XLT-4 selective agar and TSA overlaid with XLT-4 agar media, respectively. There was a greater than 4.5 log CFU/g reduction in Salmonella Poona populations following ClO2 treatment for 6 h. Population reductions following ClO2 treatment were similar irrespective of the technology used to generate ClO2. The treatment helped increase the shelf life of the tomatoes by reducing the spoilage microorganism populations on the surface, and did not seem to have adverse effects on the quality of this commodity. The distribution, growth, and inactivation of representative groups of native microflora and of experimentally-inoculated Salmonella Saintpaul were determined on jalapeño peppers. Two genetically-modified strains of Salm. Saintpaul producing either green- or red-fluorescent protein were constructed and used in the study. Microbiological analyses showed that jalapeño peppers contained an average of 5.6 log units of total aerobic count and 3.5, 1.8, and 1.9 log units, respectively, of enterobacteriaceae, lactic acid bacteria, and yeast/mold per gram of tissue. Strains typical of Pseudomonas accounted for 8.3% of total aerobic count, and 0.2 % of which exhibited pectolytic activity. On inoculated peppers, a vast majority (> 90%) of Salm. Saintpaul was recovered from stem/calyx and only a small proportion recovered from fleshy pods. Growth of Salm. Saintpaul on peppers was indicated by an increase in the population of 3 log units after incubation of samples at 20°C for 48 hr. Fluorescent Salm. Saintpaul aggregates could be readily detected on stem/calyx using stereofluorescence imaging microscopy and scanning electron microscopy.


4.Accomplishments
1. Post-irradiation recovery of E. coli O157:H7 biofilms on lettuce: Pathogenic E. coli O157:H7 has previously been shown to increase in resistance to irradiation when allowed to form biofilm-like attachment to the surfaces of Romaine lettuce and spinach. No previous data existed on the extent to which the biofilm habitat fosters recovery of E. coli O157:H7 on leaf surfaces in cold storage subsequent to irradiation treatment. ARS researchers in Wyndmoor, PA inoculated Iceberg lettuce with a cocktail of E. coli O157:H7 isolates and stored to promote biofilm formation. Contaminated leaf materials were irradiated at doses from 0.25-1.5 kGy, then sampled immediately or returned to cold storage so as to evaluate potential recovery and regrowth. Irradiation effectively reduced the population of E. coli O157:H7 on lettuce leaves immediately after treatment. Pathogen population did not subsequently recover during refrigerated storage, indicating a durable reduction of total pathogen load. Therefore, the biofilm habitat does not facilitate post-irradiation recovery of the pathogen. When used in-line as a post-packaging treatment or off-site at a third-party facility, irradiation has the potential to be a practical antimicrobial intervention for bagged lettuce or cut salads. These results can be used to better establish best practices for incorporating irradiation into the processing chain.

2. Method to preserve the quality of irradiated whole-head Iceberg lettuce: Irradiation of Iceberg lettuce was recently approved by FDA to enhance microbial safety and to extend shelf-life at doses up to 4 kGy. However radiation tolerance of whole head lettuce is unclear. The present study by ARS researchers in Wyndmoor, PA was conducted to investigate the effect of irradiation on quality of head Iceberg lettuce and explore means to reduce irradiation-induced injury. In the first experiment, head lettuce were irradiated at 0, 0.5 and 1.0 kGy and stored at 4 °C for 14 days. Results showed that irradiation induced symptoms similar to russet spotting and other discolorations (pink ribs, rusty brown and vein brown) both in external leaves and internal leaves observed on 7 and 14 days at 4 ° C after irradiation. Irradiation at 0.5 and 1.0 kGy had no consistent effect on butt discoloration, decay or softening of lettuce. In subsequent experiments, the following treatments were used to reduce irradiation-induced injury: pretreatment with ~1 ppm 1-methylcyclopropene (1-MCP) before irradiation, modified atmosphere packages during and after irradiation, and oxygen exclusion during irradiation. Modified atmosphere packaging almost completely eliminated the injury caused by irradiation. Pretreatment with 1-MCP or irradiation in the absence of oxygen had little effect on the irradiation-induced tissue discoloration. Our results suggest that irradiation combined with modified atmosphere packaging could provide significant antimicrobial benefits without undue sensory impact in head lettuce.

3. Growth of Salmonella on jalapeño peppers: Consumption of Salmonella-contaminated jalapeño peppers was implicated in one of the largest foodborne illness outbreaks in the summer of 2008. The distribution, growth, and inactivation of representative groups of native microflora and of experimentally-inoculated Salmonella Saintpaul were determined on jalapeño peppers. Microbiological analyses by ARS researchers in Wyndmoor, PA showed that jalapeño peppers contained an average of close to 1 million bacteria, ~3000, ~60 and ~90 cells, respectively, of enterobacteriaceae, lactic acid bacteria, and yeast/mold per gram of tissue. On Salmonella inoculated peppers, > 90% of the pathogens was recovered from stem/calyx and only a small proportion recovered from fleshy pods. Growth of Salmonella on peppers was indicated by an increase in the population of 3 log units after incubation of samples at 20°C for 48 hr. Salmonella survived for at least 8 weeks on peppers stored at 4°C. Immersion of inoculated peppers in 200 ppm of sodium hypochlorite, acidified sodium chlorite, or peroxyacetic acid for 10 min could reduce Salmonella on stem/calyx by 30 to 50 cells and on flesh by 125 to 250 cells. These results highlight the potential for contamination of defined parts of this commodity, which will help processors and consumers to avoid cross-contamination.

4. Gaseous chlorine dioxide kills Salmonella on tomatoes: Tomatoes have been implicated in fourteen outbreaks of Salmonellosis in the U.S. since 1996. Gaseous chlorine dioxide may be capable of inactivating human pathogens attached to inaccessible sites within biofilm on the tomato surfaces. ARS researchers in Wyndmoor, PA inoculated tomatoes with Salmonella Poona RM 2350 to an approximate final concentration of 5 log CFU/g, and stored at 4°C for 24 h prior to treatment. Tomatoes were fumigated with chlorine dioxide for up to 6 h in a closed chamber. For both chlorine dioxide generating technologies tested, a 6h treatment resulted in greater than 4.5 log CFU/g reductions in Salmonella, even in inaccessible sites such as the stem scar area. The treatment helped increase the shelf life of the tomatoes by reducing the spoilage microorganism populations on the surface with no adverse effects on tomato quality. This technology will be of practical significance to growers, processors and retailers of fresh tomatoes.


Review Publications
Rajkowski, K.T., Ashurst, K. 2009. Use of 1 % peroxyacetic acid sanitizer in an air-mixing wash basin to remove bacterial pathogen from seeds. Foodborne Pathogens and Disease. 6(9):1041-1046.

Annous, B.A., Berrang. M., Burnett, S., Dykes, G., Frank, J., Graz, M., Karunasagar, I., Lupin, H., Matthews, K., Reilly, A., Sanderson, B., Zheng, Y. 2009. Use of Chlorine-Containing Disinfectants in Food Production and Food Processing. In: Benefits and risks of the use of chlorine-containing disinfectants in food production and food processing. Geneva, Switzerland. Food and Agriculture Organization of the United Nations and World Health Organization. p. 4-47.

Netramai, S., Rubino, M., Aurus, R., Annous, B.A. 2010. Effect of chlorine dioxide gas on physical, thermal, mechanical, and barrier properties of polymeric packaging materials. Journal of Applied Polymer Science. 115:1742-1750.

Fan, X., Sokorai, K.J., Liao, C., Cooke, P.H., Zhang, H.Q. 2009. Antibrowning and antimicrobial properties of sodium acid sulfate in apple slices. Journal of Food Science. 74(9):M485-M492.

Fan, X., Annous, B.A., Keskinen, L.A., Mattheis, J.P. 2009. Use of chemical sanitizers to reduce microbial populations and maintain quality of whole and fresh-cut cantaloupe. Journal of Food Protection. 72(12):2453-2460.

Annous, B.A., Smith, J.L., Fratamico, P.M., and Solomon, E.B. 2009. Biofilms in fresh fruit and vegetables. In: Fratamico, P.M., Annous, B.A., Gunther N.W. IV, editors. Biofilms in the food and beverage industries. 1st edition. Boca Raton, FL: Woodhead Publishing Limited and CRC Press LLC. p. 517-535.

Fan, X., Niemira, B.A., Doona, C.J., Feeherry, F.E., Gravani, R.B. 2009. Microbial Safety of Fresh Produce - Preface. In: Fan, X., Niemira, B.A., Doona, C.J., Feeherry, F.E., Gravani, R.B., editors. Microbial Safety of Fresh Produce. Ames, IA: Willey-Blackwell. p. XV-XVI.

Fan, X., Niemira, B.A., Doona, C.J., Feeherry, F.E., Gravani, R.B. 2009. Microbial safety of fresh produce. Ames, IA: Wiley-Blackwell. 446 p.

Netramai, S., Rubino, M., Aurus, R., Annous, B.A. 2009. Mass Transfer Study of Chlorine Dioxide Gas Through Polymeric Packaging Materials. Journal of Applied Polymer Science. 2929-2936.

Guan, W., Fan, X. 2010. Combination of sodium chlorite and calcium propionate reduces enzymatic browning and microbial population of fresh-cut ‘Granny Smith’ apples. Journal of Food Science. 75(2):M72-M77.

Niemira, B.A., Fan, X. 2009. Irradiation enhances quality and microbial safety of fresh and fresh-cut fruits and vegetables. In Fan, X., Niemira, B.A., Doona, C.J., Feeherry, F., Gravani, R.B. editors. Microbial Safety of Fresh Produce. Ames, IA: Blackwell. 10:191-204.

Niemira, B.A., Fan, X., Gravani, R., Doona, C., Feherry, F. 2009. Research Needs and Future Directions. In Fan, X., Niemira, B.A., Doona, C.J., Feeherry, F., Gravani, R.B. editors. Microbial Safety of Fresh Produce. Ames, IA: Blackwell. 23:421-426.

Niemira, B.A., Annous, B.A., Fan, X., Liao, C., Sites, J.E. 2009. Inactivation of Microbial Contaminants in Fresh Produce. In Al-Taher, F., Jackson, L.,DeVries, J., editors. Intentional and Unintentional Food Contaminants. Washington, DC: ACS Press. 12:183-206.

Last Modified: 7/31/2014
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