1a. Objectives (from AD-416):
1. Develop and evaluate novel antagonists (e.g. Bacteriovorax, Bdellovibrio and non-pectolytic Pseudomonas) for biological-based intervention strategies, and identify means of combining pathogen microbial ecology with effective chemical and physical interventions. 2. Develop and optimize chemical decontamination interventions (e.g. novel sanitizer formulations and advanced gas-phase antimicrobial treatments), making use of pathogen microbial ecology information generated under Objective 1. 3. Develop nonthermal technologies (e.g. cold plasma, high-intensity monochromatic light and irradiation) as effective, waterless physical treatments, and establish protocols for combination treatments with interventions developed in the first two objectives.
1b. Approach (from AD-416):
A holistic approach to the development of new effective intervention technologies will be followed. In an iterative process of technology development, increased knowledge of pathogen ecology on produce surfaces will be used to optimize biological, chemical and physical control strategies; combinatorial use of intervention technologies will be examined for additive and synergistic effects while maintaining product quality. More rapid and successful commercialization will be fostered by determining the equipment and infrastructure required for large-scale implementation, as well as economic costs and benefits expected from the use of the new technologies. A key aspect of this effort to facilitate commercialization will be collaboration and information sharing with industry, including direct contact with potential end-users of technologies. Stakeholders will be updated on research goals and objectives of the project, and input will be sought from them to identify key problems to be solved. By proactively fostering these interactions in conjunction with site visits, annual scientific meetings, industry trade shows and similar venues, there will be opportunities in the early, mid-phase and in late stages of research will allow for the practical needs of industry to be addressed as the research is formulated and conducted.
3. Progress Report:
Objective 1. Sophorolipid (SL-p) is an environmentally friendly antimicrobial for use on produce. We evaluated different concentrations of SL-p, conventional sanitizer, and combinations of SL-p and sanitizer for inactivation of Listeria monocytogenes, Salmonella enterica, and Escherichia coli O157:H7 on grape tomatoes. Temperatures and storage time of SL-p application affected pathogen populations. Among the three pathogens, Listeria was the most sensitive to SL-p treatment in vitro, as reductions of 5 logs were recorded at 0.12% SL-p, with reductions of Salmonella and E. coli at 2-5%. Inactivation was similarly dose-dependent on grape tomatoes but was lower overall vs. in-vitro assays. In contrast, sanitizer and combinations of SL-p plus sanitizer showed greater than 5 log pathogen reductions. Combining SL-p with sanitizer or other antimicrobials will greatly reduce foodborne pathogen populations and enhance post-harvest produce safety. Post-harvest produce safety is improved with other combined treatments. We evaluated gamma radiation (0.5 kGy), sanitizer (100%), and competitive biocontrol microbes (cocktail Pseudomonas fluorescens, ~ 6 logs) on the survival and reduction of L. monocytogenes on post-harvest tomato and carrot. Produce storage temperatures (5-25C) and times (0-7days), with single, two or three treatment combinations were investigated for pathogen reductions. Overall, treatment efficacies on pathogen reductions for both commodities were sanitizer > radiation > biocontrol, with combination treatments generally more effective (up to 5.0 log reduction) than single treatments. Objective 2. Chlorine dioxide (ClO2) has demonstrated broad antimicrobial efficacy. Powdered chlorine dioxide precursors (sodium chlorite and citric acid) were able to reduce Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica by >99.9999% in 45 minutes on glass surfaces. Reductions were enhanced at refrigeration temperature, suggesting the importance of solubility of a gaseous disinfectant over simple reactivity. Salmonella on blueberries was reduced by 99.9 to >99.999% using a commercially available ClO2 gas generator under a variety of conditions. ClO2 was applied to mung bean seeds under tumbling and static conditions. Static gassing conditions producing higher reductions. Under optimized conditions, ClO2 reduced Salmonella on mung bean seeds by >99.9%. Salmonella was reduced on the resulting sprouted seeds by >99.999%, while maintaining a 94.9 to 98.5% sprout viability. Another seed treatment, hot water immersion at 60 and 80 °C for <3 minutes, successfully reduced heat-resistant S. enterica Senftenberg by >99.99%. Pathogen inactivation was not hampered by the seed hulling process. Overall, this suggests ClO2 and hot water treatments can effectively reduce Salmonella on seeds used for sprouting with little loss of sprout viability. Objective 3. Cold plasma is a novel food processing technology being developed to inactivate foodborne pathogens on fresh produce. An emerging area of research involves the use of cold plasma for treatment of plant seeds as a non-chemical antimicrobial process and to increase seed germination and overall crop yield. Corn and wheat seeds were treated with a cold plasma jet in a tumbling chamber for up to 5 minutes. Total aerobic plate counts were reduced by 60% log on corn, 50% log on wheat. Romaine lettuce seeds were treated with cold plasma for much shorter times of 15, 30 and 45 s. During a germination period of 6 days, shoot length was greatest for seeds treated with cold plasma for 45 s (p<0.01) while root length was greatest for seeds treated for 30 s (p<0.01). Germination and production rates were greater than the control at 30 s. Cold plasma is therefore shown to inactivate microorganisms on seeds, and to promote seed growth and germination. Although cold plasma does not rely on thermal inactivation for antimicrobial efficacy, the most energetic forms of plasma (e.g. plasma jets) can introduce heat onto food contact surfaces, with the thermal effect greatest in areas of restricted airflow. Vortex tube cooling (VTC) and conventional air jet cooling (CAJC) were evaluated for plasma treatment of a moving conveyor belt, an open-topped polyvinyl vessel, or a narrow-necked polyvinyl vessel. In the closed container (restricted airflow), the entirety of the plasma jet’s energy was captured, raising the internal temperature to 139.9 C. VTC-replacement reduced this to 112.3 C. Side jets were more effective, with CAJC and VTC reducing temperatures to 94.7 C and 77.8 C, respectively. In the open container (partial airflow), plasma alone raised temperatures to 117.5 C. VTC-replacement lowered this to 92.8 C. Again, side jets were more effective, with CAJC and VTC reducing temperatures to 51.2 C and 37.5 C, respectively. On the conveyor belt, with full airflow, the 180s plasma jet treatment raised belt temperature to 41.2 C. VTC-replacement reduced this to 38.9 C. Consistent with other applications, side jets were more effective, with CAJC and VTC reducing temperatures to 34.8 C and 28.0 C, respectively. VTC is a simple, robust, effective means of lowering impingement temperatures of plasma jets on a variety of contact surfaces. The mechanical simplicity, small footprint, and non-electrical nature of VTC recommend it for applications coupled with cold plasma in line systems and remote/field implementations of plasma-based disinfection systems.
1. Chlorine dioxide: in the package and on demand. To be most effective as an antimicrobial, chlorine dioxide needs to be generated in the produce package and on demand. ARS researchers in Wyndmoor, Pennsylvania worked with collaborators to develop an innovative and practical chlorine dioxide self-generating biobased package label. Using pectin and gelatin layers embedded with citric acid and sodium chlorite, respectively, these labels can generate and release chlorine dioxide at different concentrations and release rates simply by varying how the layers are put together. Changing the number of layers, their thickness, composition, or surface area, the presence or absence of barrier layers, etc., can allow customization for different commercial applications. The demonstrated technical feasibility of manufacturing these biobased labels indicated that they can enhance the microbiological safety and shelf life of packaged food.
2. Hot water makes for safer sprouting seeds. Mung bean sprouts have been implicated with numerous salmonellosis outbreaks. ARS researchers in Wyndmoor, Pennsylvania developed a hot water treatment that is capable of reducing Salmonella populations on mung bean sprouting seeds by 99.999%. Treating inoculated seeds using hot water at 80C with or without 0.2% Stepanol and 20 ppm chlorine for 90 and 120 seconds, respectively, completely inactivated pathogens on seeds, immediately after treatment and after 5 days of incubation at 21C and 75% humidity. Since this is a significant improvement over conventional treatments such as 120,000 ppm chlorine rinses or electrolyzed water for 24 h, this hot water process would be an improved, “green” method to reduce the risks of Salmonella on sprouts.
3. Reducing Listeria monocytogenes on cantaloupe with hot water. Listeria monocytogenes contamination of cantaloupes has resulted in some of the deadliest foodborne illness in recent years. ARS researchers in Wyndmoor, Pennsylvania developed direct-from-field surface pasteurization treatments for whole cantaloupe which can reduce L. monocytogenes by 99.999% and reduce total bacterial counts by at least 99.9%. An optimized treatment of 3.75 min in 80°C water is ready for commercialization.
4. Safer fresh-cut cantaloupes without chemical sanitizers. Fresh-cut cantaloupes have been implicated in numerous foodborne outbreaks of salmonellosis. ARS researchers in Wyndmoor, Pennsylvania collaborated with the University of Puerto Rico scientists to improve safety of fresh-cut cantaloupes. All fresh-cut samples prepared from hot water-treated cantaloupes were negative for the pathogen throughout the entire storage sampling, while those treated with chlorine dioxide- and chlorine-treated cantaloupe pieces had detectable pathogen levels in storage. This hot water process for whole melons can yield safer, higher-quality cut pieces than conventional chemical treatments. This hot water process has been commercialized.
5. Rapid detection of pathogenic bacteria on mung bean seeds. The more quickly pathogens can be detected and identified on foods, the safer our food supply will be. ARS researchers in Wyndmoor, Pennsylvania extracted certain cellular components (secondary metabolites) from Salmonella, L. monocytogenes or E. coli O157:H7, and identified them using advanced chemical analysis techniques (matrix assisted laser desorption ionization mass spectrometry coupled with tandem time of flight mass analysis). Certain distinct peaks were identified that can be related with proteins produced by the individual strains of these pathogens. This kind of chemical analysis of pathogenic strains is 300-800% faster than conventional microbial growth identification methods. Faster identification will allow food processers and packagers to more efficiently recall compromised products, thereby protecting the American consumer from foodborne human pathogens.
6. Cold plasma: cooling without coolant. Although cold plasma does not rely on thermal inactivation for antimicrobial efficacy, the most energetic forms of plasma (e.g. plasma jets) can introduce heat onto food contact surfaces, with the thermal effect greatest in areas of restricted airflow. ARS scientists in Wyndmoor, Pennsylvania combined vortex tube cooling (VTC) with plasma jets to provide spot-chilling without coolants, pumps, refrigerators, or electricity. The combination of VTC injectors mitigated temperature buildup of plasma in closed and open containers (the most challenging applications) by almost 70C, far surpassing conventional air-cooling methods. On conveyor belts, VTC mitigated temperature effects of plasma treatment almost entirely. VTC is a simple, robust, effective means of lowering impingement temperatures of plasma jets on a variety of contact surfaces. The mechanical simplicity, small footprint, and non-electrical nature of VTC recommend it for applications coupled with cold plasma in line systems and remote/field implementations of plasma-based disinfection systems.
7. New sanitizer strategies to protect produce. Sophorolipid (SL-p) is an environmentally friendly antimicrobial to inactivate pathogens on produce. ARS scientists in Wyndmoor, Pennsylvania used SL-p, alone and in combination with conventional sanitizers, to inactivate Listeria monocytogenes, Salmonella enterica, and Escherichia coli O157:H7 on grape tomatoes. SL-p alone was very effective, but SL-p plus sanitizer yielded greater than 99.999% pathogen reductions. Sophorolipids are biodegradable and meet GRAS (“generally regarded as safe”) standards. Combining SL-p with sanitizer or other antimicrobials will greatly reduce foodborne pathogen populations and enhance post-harvest produce safety.
8. Combined controls keep carrots clean from contamination. To control foodborne pathogens on post-harvest produce, ARS scientists in Wyndmoor, Pennsylvania evaluated gamma radiation (0.5 kGy), sanitizer (100%), and competitive biocontrol microbes (Pseudomonas fluorescens) for their ability to reduce Listeria monocytogenes on post-harvest tomato and carrot. Produce storage temperatures (5-25C) and times (0-7days), with single, two or three treatment combinations were investigated for pathogen reductions. Overall, treatment efficacies on pathogen reductions for both commodities were sanitizer > radiation > biocontrol, with combination treatments generally more effective (up to 5.0 log reduction) than single treatments. This integrated control strategy holds promise as a means to reduce the risks of L. monocytogenes on fresh produce.
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