Progress 12/20/10 to 12/19/15
Outputs
Progress Report 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. 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. Over the course of this Project, a range of technologies have been developed and/or optimized for the control of human pathogens on fresh and fresh-cut fruits and vegetables. These include precision thermal treatments, chemical applications, biological controls, and nonthermal processing technologies. Based on optimized protocols, cost modeling for these determined how much these technologies would cost to use commercially. OBJECTIVE 1: HOT WATER (92C, 90s) reduced Salmonella (> 99.999%) on cantaloupe and tomatoes, with excellent quality retention (color, texture, ripening). A lower HEAT TREATMENT (65C) plus CHLORINE DIOXIDE (ClO2) gas (3.5 mg/L) reduced Salmonella by >99.999% on mungbean seeds with no impact on germination. Oregano oil (500 ppm) reduced dried-on Salmonella on lettuce by 95.0%, 97.8% or 99.5%. Very short treatments (20s) with COLD PLASMA (an energized gas) reduced Salmonella on tomatoes by 81%, reduced E. coli O157:H7 on almonds by 95%, & reduced chemically-resistant Salmonella biofilms by 99.3%. Treatments up to 60s reduced aerobic organisms on blueberries by 84-97% immediately after exposure & by 97-99% after 7d at 4C without significant quality loss. COLD PLASMA also reduced human norovirus surrogates Tulane Virus by 98% (45s) & Murine Norovirus by 90% (15s). Produce was stored up to 72h after inoculation (but before IRRADIATION) to simulate potential time delay between packaging & treatment. From 0h to 72h, D10 values (dose required for 90% reduction) increased from 0.28 kGy to 0.34 kGy (spinach) or from 0.30 kGy to 0.37 kGy (Romaine). Thus, time required for shipment to an off-site irradiator may alter efficacy. OBJECTIVE 2: The COMPETITIVE EXCLUSION MICROBE (CEM) Pseudomonas fluorescens reduced Salmonella by 50-99% on spinach. On tomatoes, the CEM P. chlororaphis reduced Salmonella by 99%. When CEM was followed by a 4 h gas-phase CHLORINE DIOXIDE treatment (146 ppm), Salmonella was reduced by 99.997%. E. coli O157:H7 Chromogenic Medium (RFCM) was shown to be an effective selective medium for CEM research & best suited for simultaneous recovery of E. coli O157:H7 & CEM. OBJECTIVE 3: PULSED ELECTRIC FIELDS, CEM, CHLORINE DIOXIDE, COLD PLASMA & other interventions were modeled. Costs for CEM range from $0.05-$0.95 per kg of tomato (small-scale) or $0.0041-$0.0075 per kg (large-scale). Variable costs (95%) vs. fixed costs (5%) indicates a limited projected ROI for small scale production. Approximately 91% of active CHLORINE DIOXIDE is lost to reactive surfaces with a high chemical demand, such as cardboard & wood. With plastic containers, consumption was drastically reduced. Per 1000 h operation, COLD PLASMA consumes ~90kW, worth ~$4500; feed gas consumption (300-2400 m^3/h) and composition were primary cost factors. A process flow analysis for PULSED ELECTRIC FIELDS for a strawberry puree mode showed that flavor & color were improved and E. coli was reduced by 99.99999% at only 52.5C, electric field strength of 24 kV/cm. Accomplishments 01 Packaged lettuce made safer with cold plasma. ARS researchers at Wyndmoor, Pennsylvania have developed �cold plasma�, a chemical-free means to kill the human pathogen Escherichia coli O157:H7 on Romaine lettuce leaves inside a commercial package. To better model commercial leafy greens packaging, and to determine how uniform the cold plasma treatment is, leaves were piled on top of each other inside a commercial �clamshell�-type package. After a 10 minute treatment, the process was shown to have reduced the E. coli O157:H7 pathogen by up to 93%, even for leaves in the middle of the pile. The treatment efficacy was uniform, and cause no changes in the surface morphology, leaf color, respiration rate, or weight of the samples, regardless of location in the stack of leaves. This cold plasma system effectively inhibited E. coli O157:H7 on bulk lettuce in clamshell containers in a uniform manner, without affecting the physical and biological properties and thus holds promise as a post-packaging process for fresh and fresh-cut fruits and vegetables. 02 Harmful parasite stopped by irradiation. ARS researchers at Wyndmoor, Pennsylvania have demonstrated that a low dose of food irradiation effectively inactivates the protozoan parasite Toxoplasma gondii, a contaminant of irrigation water and fresh edible produce. Current washing steps in produce processing may not be effective for eliminating T. gondii from at-risk varieties of produce. In this study, blueberries inoculated with T. gondii were treated with low doses of food irradiation, comparable to the doses currently used to inactivate insect pests. When the surviving T. gondii oocysts and excysted sporozoites were counted, it was determined that these low doses of irradiation reduced the pathogen by at least 99.99%. Berries were assessed for compression firmness, surface color, and total anthocyanins at these efficacious doses, and showed no significant changes in any of these quality factors. Low-dose irradiation is therefore shown to be a potential intervention measure for controlling T. gondii contamination on blueberries without affecting product quality. 03 Mood Indigo, killing Salmonella and Escherichia coli with blue light. ARS researchers in Wyndmoor, Pennsylvania are developing a food safety intervention that uses high-intensity blue light at a specific wavelength (405 nanometers) to kill such as Escherichia coli O157:H7, Salmonella, and their non-pathogenic surrogate bacteria on almonds. After inoculated almonds were treated with 405 nm light for 0, 1, 2, 4, 6, 8, and 10 min, the non-pathogenic surrogates were reduced by 89% - 99%. Pathogenic Salmonella was reduced by 80% while pathogenic E. coli O157:H7 was reduced by 99.7%. High-intensity blue light therefore has potential for development into an effective antimicrobial treatment for low-moisture foods, particularly for foods contaminated with E. coli O157:H7. 04 Putting a price tag on biological controls. ARS researchers in Wyndmoor, Pennsylvania have developed a formal cost model analysis for competitive exclusion microbes (CEM) for biocontrol of Salmonella enterica on tomatoes. The team found that the unit costs of CEM biocontrol range from $0.05-$0.95 per kg of tomato for the small-scale and $0.0041-$0.0075 per kg for the large CEM production models. Since total variable costs for CEM were 95% of total production costs, while fixed costs were only 5% of total costs, CEM is therefore best suited for large-scale application. Overall, the estimated total annual cost of CEM for control of Salmonella enterica on tomatoes ($0.0058 - 0.073 per kg) is greater than sodium hypochlorite (~$0.00046 per kg) or gaseous chlorine dioxide ($0.02 - $0.21 per kg). For high value produce, CEM may complement existing technologies if efficacy and delivery systems can be optimized and its effects on gut microflora and associated factors are evaluated.
Impacts
(N/A)
Publications
- Olanya, O.M., Sites, J.E., Hoshide, A.K. 2016. Cost modeling of pseudomonoas fluorescens and pseudomonoas chlororphis biocontrol for competitive exclusion of salmonella enterica on tomatoes. Biocontrol Science and Technology. 26:651-664.
- He, Z., Zhang, H., Olanya, O.M., Frantz, J.M., Larkin, R.P. 2012. Differences in modified Morgan phosphorus levels determined by colorimetric and inductively coupled plasma methods. Open Journal of Soil Science. 2(3):256-262.
- Olanya, O.M., Lakshman, D.K. 2015. Potential of predatory bacteria as biocontrol agents for foodborne and plant pathogens. Journal of Plant Pathology. 97:405-417.
- Olanya, O.M., Anwar, M., He, Z., Larkin, R.P., Honeycutt, C.W. 2016. Survival potential of Phytophthora infestans in relation to environmental factors and late blight occurrence. Journal of Plant Protection Research. 56:73-81.
- Ojwang, D.J., Nyankanga, R.O., Imungi, J., Olanya, O.M. 2016. Plant characteristics and growth parameters of vegetable pigeon pea cultivars. HortTechnology. 26:97-105.
- Ojwang, D.J., Nyankanga, R.O., Imungi, J., Olanya, O.M., Ukuku, D.O. 2016. Cultivar preference and sensory evaluation of vegetable pigeon pea (Cajanus cajan) in Eastern Kenya. Food Security Journal. 8:757-767.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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. Sub-surface growth of E. coli O157:H7 visualized with fluorescent markers. Produce safety research often involves the use of gfp (green fluorescent protein)-labeled bacterial cells for ease of visualizing attachment of cells to leaf tissues. However, these modified cells do not behave as the parent cells and leaf auto-fluorescence can interfere with localization and imaging. This study developed a non-genetically labeled fluorescent microorganism. An overnight culture of Escherichia coli O157:H7 was stained with SYTO9. The stained culture was suspended in sterile water and was stored at room temperature (RT; with or without light) or 4C (without light) for up to 7 d. Romaine lettuce leaves were dip inoculated using SYTO 9-stained cells and were stored for up to 5 d at 4 degrees C. Inoculated lettuce pieces were also observed before and after washing with 20 or 200 ppm chlorine solution. All stained cultures remained viable and fluorescent following 7 d of storage, with or without light, at RT or 4 degrees C. Colonization of romaine lettuce leaves by the SYTO9-stained E. coli O157:H7 cells was easily visualized via confocal microscopy and was further examined via scanning electron microscopy. Although viable cells were still present, fluorescence was inhibited following 20 or 200 ppm chlorine wash. The E. coli O157:H7 cells infiltrated leaf stomata and became incorporated within mixed culture biofilms within 24 h of inoculation. This easy method of staining, which does not require specialized equipment or training, allows for the observation of static bacterial population attachment to produce surfaces. Also, unlike gfp-labeled cells, auto fluorescence of lettuce did not interfere with observing SYTO9-dyed cells. Low-dose irradiation + gas-phase chlorine dioxide on leafy greens. Leafy greens continue to be a source of foodborne illness for consumers. Irradiation and gas-phase chlorine dioxide are each known to reduce human pathogens on leafy greens. However, the potential for combination treatments with these processes is unexplored. Here, iceberg lettuce was inoculated with a cocktail of pathogenic strains of E. coli O157:H7 and treated with factorial combinations of low-dose irradiation and gas-phase chlorine dioxide. Inoculated leaves were treated with 0.0 ppm (control), 0.1 ppm, or 0.4 ppm chlorine dioxide gas. Gas exposure time was 15 minutes. After gas treatment, leaves were bagged in a controlled environment to retain the active gas. They were then irradiated at 0.0 (control), 0.25, 0.5, 0.75 or 1.0 kGy. Both 0.1 and 0.4 ppm resulted in reductions of 1.80 - 1.96 log CFU/g (98.4 - 98.9%). Treatment with 0.75 kGy yielded a reduction from the untreated control of 5.95 log CFU/g (>99. 999%), while 1.0 kGy reduced the population to below detectable levels. For both 0.1 and 0.4 ppm, combining the two treatments reduced the population to below detectable levels at only 0.5 kGy. The two treatments are thus shown to compatibly work together to enhance inactivation of pathogens on the surface of iceberg lettuce. Controlling pathogens with friendly bacteria. We determined natural microflora associated with tomato and evaluated the inactivation of Salmonella serovars by Pseudomonas fluorescens and P. chlororaphis on tomatoes based on stem scar inoculations. The populations of lactic acid bacteria, Pseudomonas sp., aerobic mesophilic bacteria, yeasts and molds on unsanitized tomatoes were: 3.31-4.84, 3.93-4.77, 4.09-4.80 and 3.83-4. 67 log CFU/g of produce, respectively. When P. fluorescens were pre- emptively applied on tomatoes and then inoculated with Salmonella, the reductions of Salmonella montevideo ranged from 0.51-0.90 log CFU/g of produce. Bacterial (Salmonella montevideo) reductions attributed to the biocontrol activity (P. chlororaphis) ranged from 1.35 to 2.00 log CFU/g of produce and the S. typhimurium serovar treated with P. fluorescens was 0.46 to 1.07 log CFU/g of produce. The combined use of P. chlororaphis and gaseous chlorine dioxide led to significant reductions of S. montevideo and S. typhimurium is a novel technique that can be applied for control of foodborne pathogens. Process models to describe the interactions of these control mechanisms are under development. Potential for bio-control of food-borne pathogens with Bacteriovorax spp. and implications for food safety. Bacteriovorax spp. (Bvx) are delta proteobacteria adapted to marine ecosystems where salinity concentration range from 1-3%. Due to their predation of Gram-negative bacteria, Bvx may have great potential for biocontrol of food-borne pathogens on fruits and leafy greens. The goal of this research was to optimize the plaque assay for quantifying Bvx isolates. We determined the predation of E. coli O157:H7 (Ec) and formation of Bvx plaques by Bvx strains G3, S11, OR7, and OS1 on polypeptone peptone medium (PP20) amended with sterilized seawater (SW) in a double agar plaque assay. For plaque assays of Bvx, bottom and top PP20 agar layers were amended with: a) NaCl (bottom layer) +SW (top layer), b) SW (bottom)+NaCl (top), c) SW (bottom)+SW (top), and d) NaCl-MgCl-CaCl (bottom)+NaCl (top) and plaques were enumerated on Ec host cells. The effects of incubation temperatures (22, 26, 31, 37 deg C) on plaque development were assessed on lawns of Ec. Plaque forming units (PFU/ml) on PP20 amended with SW ranged from 0.56 x 10e6 (OR7) to 2.07 x 10e6 (isolate S11). PP20 amended with NaCl and divalent calcium and magnesium had the lowest mean number of plaques (0.73 x 10e6 PFU/ml), while SW+NaCl (1.22 x 10e6) had the greatest counts. Except for G3, storage temperatures varied with Bvx, as temperature optima for OR7 and S11 was 26 deg C, but were non-significant (P>0.05) for G3. These results suggest that food-borne pathogens and Bvx may be assessed on PP20 devoid of SW, which could otherwise provide a source of variability from one seawater collection to another. Storage temperatures and media amendment provide useful parameters for quantifying Bacteriovorax efficacy as a biocontrol agent. Cold plasma on blueberries. Cold plasma is a novel nonthermal technology, potentially useful in food processing settings. Berries were treated with atmospheric CP for 0, 15, 30, 45, 60, 90, or 120 s at a working distance of 7.5 cm with a mixture of 4 cubic feet/minute (cfm) of CP jet and 7 cfm of ambient air. Blueberries were sampled for total aerobic plate count (APC) and yeast/molds immediately after treatment and at 1, 2, and 7 days. Blueberries were also analyzed for compression firmness, surface color, and total anthocyanins immediately after each treatment. All treatments with CP significantly (P < 0.05) reduced APC after exposure, with reductions ranging from 0.8 to 1.6 log CFU/g and 1.5 to 2. 0 log CFU/g compared to the control after 1 and 7 days, respectively. Treatments longer than 60 s resulted in significant reductions in firmness, although it was demonstrated that collisions between the berries and the container contributed significantly to softening. A significant reduction in anthocyanins was observed after 90 s. The surface color measurements were significantly impacted after 120 s for the L* and a* values and 45 s for the b*values. CP can inactivate microorganisms on blueberries and could be optimized to improve the safety and quality of produce. Designing a Chlorine Dioxide Self-Releasing Package Label to Improve Fresh Produce Safety. This project was in collaboration with Rutgers University. A PhD student conducted the research under ARS researcher supervision at ERRC. The objective of this work was to evaluate the feasibility of an innovative and practical ClO2 self-releasing package label made of synthetic or biobased polymers. These labels, embedded with ClO2 precursors, have the ability to generate and release ClO2 in a controlled manner within the package in response to a controlled activation mechanism to improve the safety of packaged fresh produce. The package labels were manufactured using extrusion or solution casting. Synthetic labels were extruded with different thickness. Biobased labels were casted onto glass frame and dried. Gaseous ClO2 release was triggered by a controlled activation mechanism. Concentration of ClO2 released from labels was quantified over time using UV spectrophotometer at 360 nm. The antimicrobial effectiveness of both the synthetic and biobased labels was evaluated against the growth of Salmonella Montevideo G4639 on trypticase Soy Agar (TSA) plates and on inoculated mung bean seeds. The findings of both the release kinetics and microbial study are in good agreement and support the technical and practical feasibility of synthetic package labels in terms of label design and manufacture; ClO2 generation and release after controlled activation at levels within the effective concentration range reported in literature for inactivation of microorganisms on fresh produce; and antimicrobial effectiveness against Salmonella growth on TSA plates. Development of hot water treatment for enhancing safety of mungbean seeds. Sprouts provide good matrices for microbial localization and growth due to optimal conditions of temperature and humidity while sprouting. Thus purpose of this work was to enhance the safety of mungbean seeds prior to sprouting. Treatment of inoculated seeds in hot water at 80 and 90 deg C for 90 seconds resulted in complete inactivation of Salmonella cells. However, seeds treated at 90 deg C failed to germinate. Treatment of inoculated mungbean seeds at 80 deg C for 120 seconds resulted in complete inactivation of pathogenic cells on inoculated seeds. Also, sprouts obtained from the optimized treatment of seeds showed no pathogenic cells following 5 days of incubation at 21 deg C and 75% humidity. These results suggest that treatment of seeds at 80 deg C for 120 seconds would enhance the safety of mungbean sprouts. Accomplishments 01 Biocontrol organisms inactivate Salmonella on tomatoes. Tomatoes can be contaminated by Salmonella, presenting a risk to consumers. ARS researchers at Wyndmoor, Pennsylvania, recorded populations of lactic acid bacteria, Pseudomonas sp., aerobic mesophilic bacteria, yeasts and molds ranging from 3.31 to 4.80 and 2.79 to 3.37 log CFU/g on sanitized and unsanitized tomatoes. The researchers determined that P. chlororaphis and P. fluorescens suppressed S. montevideo by 0.74-0.95 and 0.51-0.90 log CFU/g, respectively. The reductions of another Salmonella strain, S. typhimurium, were 1.35-2.00 and 0.46-1.07 log CFU/ g of tomatoes, respectively. The biocontrol experiments demonstrate that Pseudomonas strains may be effective in reducing Salmonella, providing a biological-based means of improving the safety of fresh tomatoes for consumers. 02 Combining biocontrol approaches with gaseous chlorine dioxide for effective control of foodborne pathogens. Tomatoes and other fresh produce can be contaminated by Salmonella, presenting a risk to consumers. ARS researchers in Wyndmoor, Pennsylvania, combined biological controls with innovative chemical treatments in order to control Salmonella. A gaseous chlorine dioxide treatment (0.4 mg/L) for 2 or 4 hrs (90% R.H, 13C) in combination with P. chlororaphis resulted in significant reductions of Salmonella populations on tomato. Salmonella montevideo and S. Typhmurium populations were reduced to 0. 82 and <0.30 log CFU/g of produce relative to untreated controls which had 5.42 and 5.37 log CFU g-1 of produce, respectively. Reduction of Salmonella on tomatoes treated with biocontrol was 2 to 3 log CFU/g of produce. These results demonstrate that Pseudomonas strains, used in combination with innovative chemical treatments, effectively reducing Salmonella on tomatoes, providing a biological-based means of improving the safety of fresh produce for consumers. 03 Killing pathogens with blue light. Nuts, berries and other types of produce are difficult to treat with conventional sanitizers, yet can be contaminated with E. coli O157:H7, Salmonella and other pathogens. ARS researchers at Wyndmoor, Pennsylvania, investigated the disinfecting properties of high-intensity blue light, generated from an array of narrow-band 405 nm light emitting diodes (LED), against E. coli O157:H7, Salmonella, and their non-pathogenic surrogates on the surface of almonds. Almonds were inoculated with E. coli O157:H7 or pathogenic Salmonella, and, in separate studies, with the non-pathogenic surrogates E.coli K-12 and an avirulent strain of Salmonella typhimurium. After treatment with blue light for up to 600 seconds, the blue light yielded significant reduction of E. coli (1 log CFU/g at 480 seconds) and Salmonella (0.5 log CFU/g at 600 seconds). The nonpathogenic surrogates showed a similar response. This antimicrobial process is zero-contact, waterless, chemical-free and nonthermal, and is a promising technology for producers of low moisture foods. 04 Making berries safer with cold plasma. Nuts, berries and other types of produce can be contaminated with human pathogens, putting consumers at risk. Cold plasma is a novel nonthermal technology, potentially useful in food processing settings. ARS researchers at Wyndmoor, Pennsylvania, treated blueberries with atmospheric cold plasma for times up to 120 seconds at a working distance of 7.5 cm. Immediately after treatment and at 1, 2, and 7 days of storage, blueberries were sampled to establish 1) total microbial levels, 2) levels of yeasts and molds, and 3) quality factors such as compression firmness, surface color, and total anthocyanins (the compounds that make blueberries blue) . All treatments with cold plasma significantly (P < 0.05) reduced microbial load immediately after treatment and after time in storage. Reductions ranged from 0.8 to 1.6 log CFU/g and 1.5 to 2.0 log CFU/g compared to the control after 1 and 7 days, respectively. Cold plasma can inactivate microorganisms on blueberries at treatment times that do not impact color, texture or firmness. 05 Chlorine dioxide plus dry heat enhances safety of mungbean sprouts. Although nutritious, fresh sprouts provide plenty of tiny spaces for human pathogens to hide and multiply during sprout production. ARS researchers at Wyndmoor, Pennsylvania, evaluated the effectiveness of the combined dry heat and chlorine dioxide gas (3.5 mg/liter air) treatment with mechanical mixing (tumbling) to eliminate Salmonella on artificially inoculated mungbean seeds. Dry heat treatments (55, 60, or 70 degrees C) for up to 8 h reduced Salmonella populations in excess of 3 log CFU/gm. The use of tumbling while treating the seeds resulted in up to 1.6 log CFU/gm reduction in Salmonella populations as compared to no tumbling. Dry heat treatment at 65 degrees C for 18 h with tumbling resulted in a complete inactivation of Salmonella populations on seeds and with low inoculum levels as compared to high inoculum. The increased reductions in pathogenic populations on the seeds with the use of tumbling could be attributed to increased uniformity of heat transfer and exposure to chlorine dioxide gas. This combined treatment represent a viable process for enhancing the safety of fresh sprouts without compromising their germination or sprout quality.
Impacts
(N/A)
Publications
- Olanya, O.M., Niemira, B.A., Phillips, J.G. 2015. Effects of gamma irradiation on the survival of Pseudomonas fluorescens inoculated on romaine lettuce. LWT - Food Science and Technology. 62:55-61.
- Olanya, O.M., Taylor, J., Ukuku, D.O., Malik, N.S. 2014. Inactivation of Salmonella serovars by Pseudomonas chlororaphis and Pseudomonas fluorescens strains on tomatoes. Biocontrol Science and Technology. 25(4) :399-413. DOI:10.1080/09583157.2014.982513.
- Olanya, O.M., Annous, B.A., Taylor, J. 2015. Effects of Pseudomonas chlororaphis and gaseous chlorine dioxide on the survival of Salmonella enterica on tomatoes. International Journal of Food Science and Technology. 50:1102-1108.
- Prodduk, V., Annous, B.A., Liu, L.S., Yam, K.L. 2014. Evaluation of chlorine dioxide gas treatment to inactivate Salmonella enterica on mungbean sprouts. Journal of Food Protection. 77(11):1876-1881.
- Annous, B.A., Burke, A.M. 2015. Development of combined dry heat and chlorine dioxide gas treatment with mechanical mixing for inactivation of Salmonella enterica serovar Montevideo on mungbean seeds. Journal of Food Protection. 78(5):868-872.
- Nicholoson, A.M., Gurtler, J., Bailey, R., Niemira, B.A., Douds, D.D. 2015. Influence of mycorrhizal fungi on fate of E. coli 0157:H7 in soil and Salmonella in soil and internalization into romaine lettuce plants. International Journal of Food Microbiology. 192:95-102.
- Lacombe, A.C., Niemira, B.A., Gurtler, J., Fan, X., Sites, J.E., Boyd, G., Chen, H. 2015. Atmospheric cold plasma inactivation of Aerobic Microorganisms on blueberries and effects on quality attributes. Food Microbiology. 46:479-484.
- Niemira, B.A. 2014. Irradiation, microwave and alternative energy-based treatments for low water activity foods. In: M. Doyle,J. Kornacki and J. Gurtler (editors). Microbiological Safety of Low Water Foods and Spices, Springer, New York, NY. p. 389-401.
- Olanya, O.M., Larkin, R.P., Honeycutt, C.W. 2015. Incidence of Phytophthora infestans (Mont.) de Bary on potato and tomato in Maine, 2006- 2010. Journal of Plant Protection Research. 55:58-68.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. Microbial contamination of fresh produce is a continuing serious problem. Under Objective 1, we determined that chlorine dioxide gas treatments of inoculated tomatoes at 0.2, 0.4, and 0.8 mg/l air for 2, 4, and 8 h reduced Salmonella populations on tomato by at least 99.9%. Treatments included the use of a modified storage atmosphere (97% nitrogen, 3% oxygen) at 13�C and 90% relative humidity for the first 5 days after chlorine dioxide treatment, and then storage for an additional 7 days in a standard atmosphere at 21�C and 75% relative humidity to allow for ripening. Color and texture profiles of treated tomatoes were not significantly different from the control samples. These results indicate that chlorine dioxide gas phase treatments could enhance the microbiological safety of tomatoes and extended the shelf life without affecting the ripening process. Cold plasma is a relatively new nonthermal antimicrobial intervention. We tested cold plasma for its ability to inactivate Salmonella biofilms � tightly knit communities that resist chemical sanitizers. A three-strain Salmonella culture was grown to form adherent biofilms for 24, 48 or 72 hours on a test surface (glass slides). These were placed on a conveyor belt and passed at various line speeds to provide exposure times of 5, 10 or 15 s. The test plate was either 5 cm or 7.5 cm under a plasma jet emitter operating at one atmosphere using filtered air as the feed gas. The frequency of high voltage electricity was varied from 23 kHz to 48 kHz. At the closer spacing (5 cm), cold plasma reduced Salmonella biofilms by up to 97.3% (5 s), 98.5% (10 s) or 99.3% (15 s). Increasing the distance to 7.5 cm generally reduced the efficacy of the 15 s treatment, but had variable effects on the 5 and 10 s treatments. Variation of the high voltage electricity had a greater effect on 10 and 15 s treatments, particularly at the 7.5 cm spacing. For each combination of time, distance and frequency, Salmonella biofilms of 24, 48 and 72 hours growth responded consistently with each other. The results show that short treatments with cold plasma yielded up to a 99.3% reduction of a durable form of Salmonella contamination on a model food contact surface. This technology shows promise as a possible tool for rapid disinfection of materials associated with food processing. Small fruits such as berries represent a challenge for sanitization. Berries were treated with atmospheric cold plasma for 0, 15, 30, 45, 60, 90, or 120s at a working distance of 7.5 cm with a mixture of 4 cubic feet/minute (cfm) of cold plasma jet and 7 cfm of ambient air. Blueberries were sampled for total aerobic plate count (APC) and yeast/ molds immediately after treatment and at 1, 2, and 7 days. Blueberries were also analyzed for compression firmness, surface color, and total anthocyanins immediately after each treatment. All treatments with cold plasma significantly reduced APC after exposure, with plate counts 84.1 - 97.5% less than the untreated control after 1 day in storage and 96.8 � 99.0% less than the control after 7 days. Treatments longer than 60s resulted in significant reductions in firmness, although it was demonstrated that collisions between the berries and the container contributed significantly to softening. A significant reduction in anthocyanins was observed after 90s. The surface color measurements were significantly impacted after 120s for the L* and a* values and 45 s for the b* values. Cold plasma can inactivate microorganisms on blueberries and could be optimized to improve the safety and quality of produce. Foodborne outbreaks of Escherichia coli O157:H7 and other pathogenic bacteria have been associated with consumption of leafy vegetables. Under Objective 2, we determined the growth of the human pathogen Escherichia coli O157:H7 and the biocontrol organism Pseudomonas fluorescens (Pf 27-9) when grown together compared to their grown in isolated monocultures. We examined sterile distilled water (SDW), buffered peptone water (BPW) and trypticase soy broth (TSB) as interaction media. We also assessed the effects of temperatures (5, 10, 15, 20, 25, 35, and 37C) and storage time (0, 2, 4, 6, 24, and 48 hrs) on bacteria populations. The greatest inhibition of E. coli O157:H7 was seen in TSB at 20C, wherein the presence of the biocontrol organism resulted in a suppression of up to 99. 9%. At warmer temperatures, the suppressive effect was lessened. In general, the suppression of E. coli O157:H7 by Pf 2-79 varied among conditions of substrate availability, storage temperatures, and culturing time. These results suggest a role for biocontrol organisms such as Pf 27- 9 in the suppression of E. coli O157:H7 on stored produce. Under Objective 3, a Class II cost analysis was completed for chlorine dioxide treatment of tomatoes. A hypothetical commercial storage system incorporating the antimicrobial interventions tested would yield a treatment cost of approximately $0.17 per kg, with an optimized cost estimated to be $0.07 per kg. A key finding of the cost analysis was related to shipping containers, boxes and equipment. Approximately 91% of the active chlorine dioxide is lost to reactive surfaces with a high chemical demand, such as cardboard and wood. When reactive containers were replaced with nonreactive plastic containers in the cost modeling, the consumption of the chlorine dioxide gas was drastically reduced. Accomplishments 01 Chlorine dioxide reduces Salmonella on tomatoes. Microbial contamination of fresh produce is a continuing serious problem. ARS researchers at Wyndmoor, Pennsylvania showed that chlorine dioxide gas treatments reduced Salmonella on inoculated tomatoes by at least 99.9%. By multi-stage control of the storage atmosphere after treatment, antimicrobial efficacy went hand-in-hand with conventional ripening. Color and texture profiles of treated tomatoes were not significantly different from the controls. Chlorine dioxide gas can enhance microbiological safety and extended tomato shelf life. 02 Killing Salmonella biofilms with cold plasma. Biofilms can contaminate food contact surfaces, resisting conventional chemical sanitizers. ARS researchers at Wyndmoor, Pennsylvania grew durable biofilms from multiple strains of Salmonella culture and treated with short exposures (5 to 15 seconds) of cold plasma, an antimicrobial process. All of the short exposures to this new sanitizing technology reduced pathogens on these inert surfaces, with the greatest reduction of 99.3% after only 15 seconds of treatment. Short treatment times are essential for integration into economical, practical sanitizing protocols. This technology shows promise as a possible tool for rapid disinfection of materials associated with food processing. 03 Controlling E. coli O157:H7 with friendly bacteria. Foodborne outbreaks of Escherichia coli O157:H7 and other pathogenic bacteria have been associated with consumption of leafy vegetables. The biocontrol organism Pseudomonas fluorescens can suppress this pathogen. In studies conducted by ARS researchers at Wyndmoor, Pennsylvania, P. fluorescens suppressed the growth of E. coli O157:H7 by up to 99.9% when the two organisms were grown together in culture media at 20C. At warmer temperatures, the suppressive effect was lessened. In general, the suppression of E. coli O157:H7 by P. fluorescens varied among conditions of substrate availability, storage temperatures, and culturing time. These results suggest a role for biocontrol organisms in the suppression of E. coli O157:H7 on stored produce.
Impacts
(N/A)
Publications
- Larkin, R.P., Honeycutt, C.W., Olanya, O.M., Halloran, J.M., He, Z. 2012. Impacts of crop rotation and irrigation on soilborne diseases and soil microbial communities. In: He, Z., Larkin, R.P., Honeycutt, C.W., editors. Sustainable potato production: global case studies. Amsterdam, The Netherlands: Springer. p. 23-41.
- He, Z., Honeycutt, C., Olanya, O.M., Larkin, R.P., Halloran, J.M., Frantz, J. 2012. Comparison of soil phosphorus status and organic matter composition in potato fields with different crop rotation systems. In: He, Z., Larkin, R.P., Honeycutt, C.W., editors. Sustainable Potato Production: Global Case Studies. Amsterdam, The Netherlands: Springer. 61-79.
- Frantz, J., Larkin, R.P., Trusty, G.M., Honeycutt, C., He, Z., Olanya, O.M. , Halloran, J.M. 2012. Comparing modeled productivity to historical data in New England potato production systems. In: He, Z., Larkin, R.P., Honeycutt, C.W., editors. Sustainable Potato Production: Global Case Studies. Amsterdam, The Netherlands: Springer. p. 81-85.
- Halloran, J.M., Larkin, R.P., Defauw, S.L., Olanya, O.M., He, Z. 2013. Economic potential of compost amendment as an alternative to irrigation in Maine potato production systems. American Journal of Plant Sciences. 4:238- 245.
- Ukuku, D.O., Mukhopadhyay, S., Olanya, O.M. 2013. Effect of organic acid treatments on microbial safety and overall acceptability of fresh-cut melon cubes. International Journal of Food, Agriculture, and the Environment. 11(3&4):340-345.
- Niemira, B.A., Boyd, G., Sites, J.E. 2014. Cold plasma rapid decontamination of food contact surfaces contaminated with Salmonella biofilms. Journal of Food Science. 79(5):M917-M922.
- Niemira, B.A., Zhang, H.Q. 2014. Book Chapter. Advanced technologies for detection and elimination of bacterial pathogens. in K. Matthews, G. Sapers, C. Gerba (eds)The Produce Contamination Problem: Causes and Solutions, 2nd Edition, Elsevier.pp.433-450.
- Nyankanga, R.O., Kiplagat, K.W., Narla, R.D., Shibairo, S.I., Kabira, J.N., Landeo, J.A., Olanya, O.M. 2014. Effects of early and late harvest on agronomic performance and stability of late blight resistant (R-gene free) potato genotypes. Journal of Crop Science and Biotechnology. 17:89-96.
- Olanya, O.M., Ukuku, D.O., Niemira, B.A. 2014. Effects of temperature and storage time on resting populations of Escherichia coli 0157:H7 and Pseudomonas flurorescens in vitro. Food Control. 39:128-134.
- Olanya, O.M., Honeycutt, C., He, Z., Larkin, R.P., Halloran, J.M., Frantz, J. 2012. Early and late blight potential on Russet Burbank potato as affected by microclimate, cropping systems and irrigation management in North-eastern United States. In: He, Z., Larkin, R.P., Honeycutt, C.W., editors. Sustainable Potato Production: Global Case Studies. Amsterdam, The Netherlands: Springer. 43-60.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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.
Impacts
(N/A)
Publications
- 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.
- Annous, B.A., Burke, A.M., Sites, J.E., Phillips, J.G. 2013. Commercial thermal process for inactivating Salmonella Poona on surfaces of whole fresh cantaloupes. Journal of Food Protection. Volume 76(3):420-428.
- Olanya, O.M., Ukuku, D.O., Annous, B.A., Niemira, B.A., Sommers, C.H. 2013. Efficacy of Pseudomonas fluorescens for biocontrol of Escherichia coli 0157:H7 on spinach. International Journal of Food, Agriculture, and the Environment. 11:86-91.
- Guo, M., Yang, R., Antenucci, R., Mills, B., Cassidy, J.M., Scullen, O.J., Sites, J.E., Rajkowski, K.T., Sommers, C.H., Jin, Z.T. 2013. Inactivation of natural microflora and Listeria innocua on raw whole shrimp by ozonated water, antimicrobial coatings, and cryogenic freezing. Food Control. 34:24- 30.
- Kamleh, R., Jurdi, M., Annous, B.A. 2012. Management of microbial food safety in the Arab countries. Journal of Food Protection. Volume 75, No.11. , Pages-2082-2090.
- Niemira, B.A., Fan, X. 2012. Advances in processing technologies to preserve and enhance the safety of fresh and fresh-cut fruits and vegetables. Encyclopedia of Food Microbiology. Food Irradiation Research Technology, 2nd Edition.Wiley-Blackwell Publishing, Ames, IA.
- Niemira, B.A. 2012. Cold plasma as a food processing technology. Book Chapter. Encyclopedia of Agricultural, Food and Biological Engineering, DOI:10.1081/E-EAFE2-120048302.
- Niemira, B.A., Boyd, G. 2013. Influence of modified atmosphere and varying time in storage on the irradiation sensitivity of Salmonella on sliced roma tomatoes. Journal of Radiation Physics and Chemistry.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. Consumption of fresh fruits and vegetables is an important part of healthy diet and anti-obesity efforts. However, contamination of produce such as cantaloupe, tomato, sprouts, nuts and jalepeno peppers, is a continuing serious problem. Our research under Objective 2 seeks to determine how pathogens persist on produce. This information will help guide our efforts to inactivate them. On artificially inoculated peppers, ARS researchers in Wyndmoor, PA recovered > 90% of Salmonella from the stem and calyx and only a small proportion from the fleshy pods. We showed that Salmonella grew by 3 logs (over a thousand fold) on peppers after incubation at 20C for 48 hr and could survive for at least 8 weeks on peppers stored at 4C. Under Objective 1, we tested a variety of antimicrobial treatments, both chemical and physical. By immersing inoculated peppers in various antimicrobial solutions for 10 min, we were able to reduce Salmonella on stem/calyx by 96.8-98.0% and on flesh by 99. 2-99.6%. Using a 0.5 mg/l treatment of another type of chemical, chlorine dioxide, we reduced Salmonella on inoculated mung bean seeds and mung bean sprouts by 99.999%. This 15 minute treatment is commercially feasible and can enhance the safety of the sprouts. Although these intense levels of conventional chemical treatments show promise, there remains a critical need for novel, non-chemical antimicrobial interventions. Using an innovative new technology, cold plasma, we inactivated both Salmonella and Escherichia coli O157:H7 on dry almonds. Because the speed of the treatment is very important to processors, we used rapid treatments of 10 or 20 seconds, applied to the inoculated almonds at 2, 4 or 6 cm from the cold plasma emitter. The greatest inactivation we obtained was a 95.4% reduction of E. coli O157:H7. We found that air was generally a more effective cold plasma feed gas than nitrogen. Cold plasma therefore shows promise as a nonthermal treatment for tree nuts. Using a 45-second, direct-from-field treatment in 70C (160F) hot water, we eliminated coliforms and reduced total plate count on cantaloupe by at least 99.9%. This process can be integrated with existing commercial processing facilities & equipment. We�re collaborating with an industry partner to evaluate the quality, shelf- life, and sensory quality of fresh and fresh-cut cantaloupes treated with the ERRC hot water process. We also found that a longer hot water treatment of 3.5 minutes reduced Salmonella on fresh green tomatoes by up to 99.9999% without obvious visual injury and without changes to maturation. This promises to be a flexible, effective and economical antimicrobial process for intact fruits and vegetables. In our research with human norovirus, we found that high pressure processing (HPP) effectively disrupts the structure and function of the human norovirus capsid at pressure levels of 700-900MPa, for a holding time of 1 � 5 minutes. Since norovirus is a pathogen of concern for fresh foods, this information will help us design ways to use HPP so foods will be safer to consume. Overall, our research is on schedule for all our Objectives, and is seeing significant interest from our stakeholders. Accomplishments 01 Cold plasma inactivates pathogens on almonds. Contamination of raw nuts including almonds, is a food safety concern. ARS researchers at Wyndmoor Pennsylvania used rapid cold plasma treatments, 10 or 20 seconds, to inactivate both Salmonella and Escherichia coli O157:H7 from dry almonds Inoculated almonds were treated with at 2, 4 or 6 cm from the cold plasm emitter. The greatest effect observed was a 95.4% reduction of E. coli O157:H7. The interaction of treatment time with distance from plasma emitter head was complex, and isolate-dependent. In general, air was a more effective cold plasma feed gas than nitrogen. This treatment is waterless, contact-free and uses no antimicrobial chemicals. Short pulse of atmospheric pressure cold plasma therefore show promise as a nontherm treatment for tree nuts, a technological advance which will have applications in improving the safety of this commodity for producers and consumers. 02 Finding the �Danger Zone� on a jalape�o pepper. Consumption of Salmonel contaminated jalape�o peppers has been implicated in foodborne illness outbreaks. Working with artificially inoculated peppers, ARS researchers at Wyndmoor, Pennsylvania recovered > 90% of the introduced Salmonella from the stem/calyx and recovered only a small proportion from fleshy po Salmonella grew by 3 logs on peppers after incubation at 20C (68F) for hr and could survive for at least 8 weeks on peppers stored at 4C (40F). Immersion of inoculated peppers in 200 ppm of sodium hypochlorite, acidified sodium chlorite, or peroxyacetic acid for 10 min could reduce the number of Salmonella on stem/calyx by 96.8-98.0% and on flesh by 99. 99.6%. By clearly identifying where pathogens reside on peppers, processors will be better able to provide safer produce to consumers. 03 A simple treatment to eliminate Salmonella from tomatoes. Numerous Salmonella outbreaks have been associated with fresh tomatoes. ARS researchers at Wyndmoor, Pennsylvania dipped artificially contaminated green tomatoes in hot water for 3.5 minutes. This treatment at 70C (160F reduced Salmonella by up to 99.9999% without visible injury or changes t maturation. When combined with refrigerated storage, the treated tomatoe remained free of Salmonella. This chemical-free process of surface pasteurization holds promise for use in the fresh produce industry, and can enhance the microbiological safety of tomatoes. 04 Safer, cleaner cantaloupes in 45 seconds. Cantaloupes have been implicated in some of the deadliest foodborne illness outbreaks due to contamination with Salmonella and Listeria monocytogenes. A surface pasteurization process developed by ARS researchers at Wyndmoor, Pennsylvania is now being tested commercially for whole cantaloupe. A ve brief, direct-from-field treatment in 70C (160F) hot water reduces total plate count by at least 99.9% and total coliforms to non-detectable leve This 45 second process dovetails in existing commercial cantaloupe processing lines. Collaboration between ERRC and the industry partner is underway to evaluate the effect of this commercial process on microbial quality, shelf-life, and sensory quality of fresh and fresh-cut cantaloupes.
Impacts
(N/A)
Publications
- Niemira, B.A. 2012. Cold plasma decontamination of foods. Annual Review of Food Science and Technology. 3:125-142.
- Niemira, B.A., Boyd, G. 2012. Cold plasma reduction of Salmonella and Escherichia coli 0157:H7 on almonds using ambient pressure gases. Journal of Food Science. 77:M171-175.
- Gunduz, G.T., Niemira, B.A., Gonul, S.A., Karapinar, M. 2012. Antimicrobial activity of oregano oil on iceberg lettuce with different attachment conditions. Journal of Food Science. 77(7):412-415.
- Fangfei, L., Huang, P., Neetoo, H., Gurtler, J., Niemira, B.A., Chen, H., Jaing, X., Li, J. 2012. High pressure inactivation of human norovirus virus-like particles: evidence that the capsid of human norovirus is highly pressure resistant. Applied and Environmental Microbiology. DOI: 10. 1128/AEM.00532-12.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. The project has completed research in support of NP Component 1D (Pathogen Toxins and Chemical Contaminants-Intervention Strategies). Results of the research (described below) have addressed the Project Objectives. As a newly established Project, progress has been based on continuation of studies from the previous project (011), and establishment of new research. The impact of post-contamination storage time on the efficacy of irradiation was determined for leafy vegetables. Leaves of Romaine lettuce and baby spinach were dip inoculated in a cocktail of three strains of Salmonella. Leaves were stored at 4C to allow biofilms to form, then treated with either a sodium hypochlorite wash or increasing doses of irradiation. Chlorine washes yielded maximal reductions of 1.9 log cfu/g. From 0 h of storage, D10 (the dose required for a 90% reduction) increased from 0.28 kGy to a maximum of 0.34 kGy for spinach. For Romaine, D10 increased from 0.30 kGy at 0h to 0.37 kGy at 72 h. The biofilm habitat can reduce the efficacy of irradiation in eliminating pathogens from leafy vegetables. These results can be used to better establish best practices for incorporating irradiation into a lettuce/cut salad processing chain. Cold plasma was tested for ability to remove biofilms from food-contact surfaces. Salmonella and E. coli O157:H7 cultures were allowed to form adherent biofilms on glass. These were placed on a conveyor belt and passed at various line speeds under a plasma jet emitter. Optimized treatments of 15 seconds were able to reduce the most durable forms of E. coli O157:H7 biofilms by 3.03 log cfu/cm, and Salmonella biofilms by 2.12 log cfu/cm. Cold plasma shows promise as a rapid treatment for effectively inactivating persistent contamination on food contact surfaces. In collaboration with industry, two large scale field trials to evaluate chlorine dioxide (ClO2) were conducted. Trial data showed chlorine dioxide gas effectively penetrated fruit boxes and delivered a uniform treatment. Low dose gassing with ClO2 delayed mold development on whole pineapples and plantains, thereby increasing shelf life. Companion studies with pathogen-inoculated produce showed that ClO2 fumigation treatments reduced Salmonella by 4.5 log on tomato and 5 log on cantaloupe following 7 or 8 days of storage. The treatment also helped increase the shelf life. These data suggest the feasibility of ClO2 fumigation for enhancing the safety and shelf life of stored and shipped whole commodities. The research program on microbial ecology and biological control organisms was completely suspended after scientist died on October 17, 2010. Another scientist joined project 018 on July 18, 2011 and assumed the responsibilities for the microbial ecology research under the new plan. Accomplishments 01 Rapid treatment with cold plasma kills Salmonella and E. coli O157:H7. Cross-contamination of fresh produce from persistent pathogen reservoirs is a known risk factor in processing environments. Industry requires a waterless, zero-contact, chemical-free method for removing pathogens fro food-contact surfaces. Cold plasma was tested by ARS researchers at Wyndmoor, PA, for its ability to remove biofilms from food-contact surfaces. Salmonella and E. coli O157:H7 cultures were allowed to form adherent biofilms for 24, 48 or 72 hours on a test surface (glass slides These were placed on a conveyor belt and passed at various line speeds either 5cm or 7.5 cm under a plasma jet emitter. The frequency of cold plasma generation was varied from 23kHz to 48kHz. Optimized treatments o 5, 10 or 15 seconds were able to reduce the most durable forms of E. col O157:H7 biofilms by 94.4%, 99.7% and 99.9%, respectively. These rapid treatments reduced the most durable forms of Salmonella biofilms by 88.0 97.4% and 99.2% respectively. Cold plasma effectively inactivated persistent contamination on food contact surfaces associated with fruits and vegetables processing. 02 Chlorine dioxide kills Salmonella on green tomatoes and cantaloupe. Tomatoes and cantaloupes are recurrent sources of contamination for huma pathogens. Pilot scale ClO2 gas treatment of intact produce was conducte by ARS researchers in Wyndmoor, PA, at the ERRC. Green tomatoes and cantaloupe were inoculated to 10,000 cfu/g with Salmonella Montevideo or Poona, respectively, and stored at 4C or 12.5C for 24 h prior to treatme ClO2 treatments consisted of 6 h fumigations at 0.4 or 0.8 mg/l (tomato or 1.0 mg/l (cantaloupes). Tomatoes were stored at 18C, while cantaloupes were stored at 4C. Reductions of 99.997% on tomato and 99.99 on cantaloupe were seen following 7 or 8 days of storage, respectively. The treatment helped increase the shelf life of produce by reducing the spoilage microorganism populations on the surface. This process is a feasible method for enhancing the safety and shelf life of tomatoes and cantaloupe.
Impacts
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Publications
- Niemira, B.A., Cooke, P.H. 2010. Escherichia coli O157:H7 biofilm formation and internalization on lettuce and spinach leaf surfaces reduces efficacy of irradiation and sodium hypochlorite washes. Journal of Food Science. 75(5):M270-M277.
- Liao, C., Cooke, P.H., Niemira, B.A. 2010. Localization, growth, and inactivation of Salmonella Saintpaul on jalapeno peppers. Journal of Food Science. 75(6):M377-M382.
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