1a. Objectives (from AD-416):
1. Develop new effective chemical and physical decontamination interventions for produce and/or improve the performance of current interventions such as gas-phase antimicrobials and cold plasma. Develop protocols for implementing interventions within a multi-step approach that improves decontamination efficacy, retains product quality and/or enhances the efficiency and practicality of the effective interventions. a. Develop and optimize gas-phase antimicrobial treatments and precision thermal treatments. b. Develop and optimize cold plasma and irradiation as non-thermal antimicrobial treatments. 2. Understand ecological factors that influence treatment decontamination efficacy, including interaction of human pathogens with native microorganisms and behavioral factors such as attachment, internalization and biofilm formation. Use this information to develop and evaluate biological-based intervention strategies for pathogen reduction while maintaining product quality. 3. Develop and evaluate process models, including economic analysis models, in order to identify barriers to commercialization and to facilitate technology transfer and commercial adoption of interventions and intervention combinations.
1b. Approach (from AD-416):
As part of this project, new and/or improved antimicrobial intervention technologies will be developed and optimized, focusing on chemical and non-thermal physical interventions. Physical and chemical treatments include the use of hot water pasteurization, gaseous chlorine dioxide, cold plasma, hydrogen peroxide vapor, and ionizing radiation alone or in combination. The microbial ecology of human pathogens on the surfaces of commodities, including attachment, biofilm formation and internalization, can alter the efficacy of the intervention. Research to better understand this aspect of pathogen biology, as well as interactions with native microflora including spoilage organisms, will be used in an iterative approach; this data will assist in the development and optimization of intervention strategies, including microbial antagonist-based biological controls. Initial studies will concentrate on high-risk produce commodities, such as leafy greens and tomatoes, and will also focus on additional products identified as contributing to foodborne illnesses. Intervention strategies will be examined for their effects on product quality and shelf-life. To facilitate industry implementation of promising treatments and treatment combinations, engineering process models and economic models will be developed to identify key barriers to commercialization during scale-up. This information will guide research efforts to address the most important aspects of successful implementation. Effective, cost-efficient intervention technologies will be transferred to industry to reduce the risk of produce-related outbreaks of foodborne illness.
3. Progress Report:
Microbial contamination of fresh produce is a continuing serious problem. Under Objective 1, we determined that time in refrigerated storage and modified atmosphere packaging influences the response of pathogens to irradiation. Tomato slices were packaged and stored for 24 or 48 hours between inoculation and treatment. Reduced oxygen generally resulted in higher doses being required to achieve the same reductions. Time required for refrigerated holding of processed tomatoes or shipment to an off-site irradiation service provider may therefore alter the efficacy of irradiation if reduced oxygen MAP is used, an important factor in protocol design. We increased the efficiency and throughput of a chemical-free sanitation system. Hot water surface pasteurization at 92 C (198 F) for 90 seconds reduced Salmonella on cantaloupe by more than 99.999%. An even faster version of the process is undergoing field trials. Using hot water at 71 C (160 F) for 45 seconds, an industry partner had the processing equipment built and installed at their facility. Shelf life for intact cantaloupe and fresh-cut pieces was increased, while total plate counts were reduced by at least 99.9% and coliforms were reduced to non-detectable levels. A CRADA is under development with the collaborating industry to optimize the treatment conditions under the commercial production conditions. Chlorine dioxide (ClO2) gas treatment (0.5 mg/l for 15 minutes) of mung bean seeds and sprouts resulted in excess of 99.999% reduction of inoculated Salmonella. On a small pilot scale, green and ripened tomatoes were inoculated with Salmonella and stored for 24 hours before gaseous ClO2 treatments (0.4 or 0.8 mg/l for up to 6 hours). Optimized treatments reduced Salmonella by 99.996% following up to 14 days of storage. The treatment increased the shelf life with no adverse effects on the texture and color qualities. This data suggest the feasibility of this process for enhancing the safety and shelf life of this commodity. As part of Objective 2, we evaluated a new selective medium for enumeration of E. coli and P. fluorescens (biocontrol microbe) from co-cultures on media and inoculated spinach and determined the efficacy of the biocontrol organism in controlling the pathogen. E. coli O157:H7 and biocontrol bacteria were inoculated on produce and stored for 24 and 48 hours at 5 to 30 C. The biocontrol reduced E. coli O157:H7 by 70 – 95%, with suppressive effects greatest at 15 C. Data suggest that various mechanisms contribute to biocontrol efficacy. Ongoing research is evaluating synergy of biocontrol in combination with various interventions. Overall, our research is on schedule for all our Objectives, and is seeing significant interest from our stakeholders.
1. Recovery of Escherichia coli O157:H7 and Pseudomonas fluorescens (biocontrol microbe) from spinach. Pre and post-harvest contamination of leafy greens by E. coli O157:H7 is an ongoing threat to consumers. Development of new bio-based means to control this pathogen is hampered by the technical difficulty of isolating effective control bacteria. ARS researchers at Wyndmoor, Pennsylvania developed a new selective assay procedure that will make this process faster, more accurate and more efficient. By using R & F chromogenic media, P. fluorescens and E. coli O157:H7 in co-cultures can be recovered, differentiated, and counted. This advance of technology has been validated for both culture media and inoculated spinach. The enhanced ability to isolate effective control bacteria will speed the development of biological controls that will protect consumers from E. coli O157:H7 and other pathogens on leafy greens and other commodities.
2. Reduction of Escherichia coli O157:H7 on spinach by Pseudomonas fluorescens. Food-borne outbreaks of E. coli O157:H7 are associated with consumption of contaminated leafy greens and vegetables. ARS researchers at Wyndmoor, Pennsylvania dip-inoculated spinach with biocontrol organism (P. fluorescens) for 5 minutes and then inoculated with E. coli O157:H7. The biocontrol suppression of E. coli O157:H7 populations ranged from 0.5-2.1 logs. By applying non-pathogenic biocontrol microbe on spinach at post-harvest, contamination of food-borne bacteria can be greatly reduced. To improve efficacy, this technique is being combined with other intervention technologies.
3. Irradiation makes tomatoes safer. Salmonella contamination of tomatoes is a recurrent food safety concern. Irradiation effectively inactivates this pathogen, but the interaction of time in refrigerated storage and modified atmosphere packaging may influence how it works. Salmonella-inoculated Roma tomatoes were packaged by ARS researchers at Wyndmoor, Pennsylvania under air and various reduced-oxygen atmospheres. Before irradiation, the packages were kept in refrigerated storage for 24 or 48 hours after inoculation, to simulate the potential time delay between packaging and irradiation treatment. Irradiation effectively reduced Salmonella, but the dose necessary for a given reduction varied significantly among the combinations of time and atmospheres. Reduced oxygen generally resulted in higher doses being required, with the highest dose required for tomatoes packaged in pure nitrogen. These results suggest that time required for refrigerated holding of processed Roma tomatoes or shipment to an off-site irradiation service provider may alter the efficacy of irradiation if reduced oxygen MAP is used. This information will be useful for designing irradiation protocols for real-world, commercial environments.
4. Safe cantaloupes: faster and cheaper. Cantaloupe melons have been implicated in at least seven outbreaks of Salmonella and one outbreak of Listeria monocytogenes, and resulting in more than 36 deaths since 1990. Previous research showed that a 3 minute treatment in hot water effectively eliminated Salmonella. The goal of the current research was to speed up the process to a commercially viable level. In this work, ARS researchers at Wyndmoor, Pennsylvania demonstrated that surface pasteurization in hot water at 92 degress C for 90 seconds gave a 99.999% inactivation of Salmonella, results in greater than that which can be obtained by chlorine-based and experimental sanitizer solutions. An even faster version of the process (71 degrees C (160 F) for 45 seconds) is undergoing field trials by an industry partner at a commercial cantaloupe processing facility. Total plate count was reduced by at least 99.9%, while total coliforms were reduced to non-detectable levels. This held true for the rind of the intact cantaloupe and for the fresh-cut product made from them. The reduction in microbial levels was translated into an increase in the shelf life of the whole and the fresh-cut cantaloupe. Surface pasteurization enhances the microbiological safety of cantaloupes at speeds that will better integrate with commercial throughput requirements. This means safer product while saving time and money. These findings will assist food industry and regulatory agencies in establishing processing guidelines to guard against pathogens, thereby decreasing the incidence of illness outbreaks. A CRADA is under development with the collaborating industry to optimize the treatment conditions under the commercial production conditions.
5. Chlorine dioxide: good for sprouts, good for tomatoes. A 15 minute chlorine dioxide gas (0.5 mg/l) treatment of mung bean seeds and sprouts reduced 99.999% of inoculated Salmonella. Using the same Eastern Regional Research Center-developed chlorine dioxide system, a large-scale treatment of green and ripened tomatoes was over 6 hours in a 10 cubic foot chamber. Inoculated tomatoes were treated then stored for up to 14 days. Salmonella was reduced by 99.996% over the course of the study. The treatment helped increase the shelf life of produce by reducing the spoilage microorganism populations on the surface. Also, this treatment has no adverse effects on the texture and color qualities other than bleached stem scar. This data suggest the feasibility of this process for enhancing the safety and shelf life of this commodity. The newly developed process by ARS researchers at Wyndmoor, Pennsylvania suggests the feasibility of using this treatment by the industry to enhance the safety of seeds, sprouts and tomatoes.
Olanya, O.M., Annous, B.A., Niemira, B.A., Ukuku, D.O., Sommers, C.H. 2012. Effects of media on recovery of Escherichia coli 0157:H7 and Pseudomonas fluorescens from spinach. Journal of Food Safety. 32:492-501.