ENHANCING THE SAFETY AND QUALITY OF FRESH PRODUCE AND LOW-MOISTURE FOODS BY WATERLESS NON-THERMAL TECHNOLOGIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1005440
• Grant No.: 2015-69003-23410
• Project No.: ME0-2014-06144
• Proposal No.: 2014-06144
• Program Code: A4131
• Project Start Date: Apr 15, 2015
• Project End Date: Apr 1, 2020
• Grant Year: 2018

Project Director
Singer, J. T.

Recipient Organization
UNIVERSITY OF MAINE
(N/A)
ORONO,ME 04469

Performing Department
Research & Sponsored Programs

Non Technical Summary
Our food supply could be vulnerable to multiple pathogens such as bacteria, parasites, and viruses. Consumption of produce and low-moisture foods (LMF), such as nuts, cereals and spices, has increased significantly in recent decades. These products are highly susceptible to contamination by pathogens from pre-harvest to post-harvest stages. The challenge for food scientists is to develop new technologies that will improve the safety and extend the shelf life of food products without compromising safety or sensory properties. Most foods are thermally preserved (i.e. cooking) by subjecting them to high temperature or boiling. However, fresh fruits and vegetable are particularly critical because they are commonly eaten without thermal processing. Non-thermal methods allow for the processing of foods below the temperatures used during pasteurization and canning, allowing minimal changes in flavor and quality while removing pathogens. The purpose of this project is to investigate the emerging non-thermal technologies such as decontaminating lights, gaseous treatment, and cold plasma (ionized atmospheric air i.e., lightening). These processes are used on an industrial scale for the manufacturing of electronics and medical instruments. Each non-thermal technology has specific application, and each technology has its merits and limitation. Our approach it to use combinations of technologies to optimize product quality and inactivate pathogens and commercialize the process. With the technology transfer to industry, we expect that processors will spend less money on energy inputs such as energy for heat, water, and chemical passing the savings on the consumer. With the increasing outbreaks associated with fresh produce and LMF, the ability to provide effective microbiological control will be critical to maintaining consumer confidence in the agricultural and food industries, both in the United States and with international trading partners.

Animal Health Component

0%

Research Effort Categories

Basic

25%

Applied

25%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	7299	1100	30%
903	1129	3020	20%
501	1499	1101	20%
723	1212	2020	10%
902	1430	1103	20%

Knowledge Area
902 - Administration of Projects and Programs; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 501 - New and Improved Food Processing Technologies; 903 - Communication, Education, and Information Delivery; 723 - Hazards to Human Health and Safety;

Subject Of Investigation
1499 - Vegetables, general/other; 1212 - Almond; 1129 - Berries and cane fruits, general/other; 1430 - Greens and leafy vegetables; 7299 - Research equipment and methods, general/other;

Field Of Science
2020 - Engineering; 1101 - Virology; 1103 - Other microbiology; 3020 - Education; 1100 - Bacteriology;

Keywords

produce

safety

Goals / Objectives
The goal of this study is to develop an integrated project among academia, government, industry partners, and stakeholders to develop effective waterless, non-thermal processing technologies to provide consumers with safe, nutritious, high-quality produce and low-moisture foods, equip Extension agents with the knowledge and tools that they need to influence a change in consumer perception related to food processing technologies, and facilitate the commercialization of these technologies and dissemination of knowledge through education and outreach.

Project Methods
This research will determine the effects of processing parameters (e.g., concentration, intensity, and time) of light and gaseous treatments, environmental conditions (e.g., temperature and humidity), and product characteristics (e.g., tissue makeup, surface structure, and roughness) on the inactivation and survival of foodborne bacterial, viral, and parasitic pathogens, such as Shiga-toxin producing Escherichia coli, Salmonella spp., Listeria monocytogenes, Toxoplasma gondii, Cryptosporidium parvum, and human norovirus. Food products will include fresh and fresh-cut leafy greens, root and berry produce and LMF, such whole peppercorn and almond. Logarithmic reductions of bacterial pathogens and parasites will be determined using a viable count method and a combination of cell culture and mouse models, respectively. Since human norovirus cannot be grown in cell culture, the inactivation of human norovirus will be determined using a novel published receptor-binding assay and the infectivity will be further verified in a published gnotobiotic pig model. Similarly, work with the protozoan parasite T. gondii will be used to asses Cyclospora, which cannot be grown in cell culture or in animal models. The use of two phenotypically different protozoa (Cryptosporidium and Toxoplasma) will be useful. Mathematical models will be developed to describe inactivation kinetics. The effects of these processing technologies on product quality attributes, such as sensory, color, texture, nutrients, and consumer acceptance, will be evaluated using instruments in conjunction with sensory panels. Technologies that are capable of achieving the desired performance standards for hazard reduction, nutrition, and quality will be optimized for industrial applications. By working with growers, processors, economic researchers, and environmental scientists, the costs, benefits, and environmental impact of implementing the technologies will also be determined.For the education, 2 new courses, "Foodborne Contamination-Impact on Food Safety" course and an "Emerging Nonthermal Technologies" will be developed. Students training in emerging nonthermal technologies, parasitology, microbiology, virology, and food safety will be conducted.Methods for carrying extension component include 1) access consumer perception of food processing technologies, 2) develop educational materials, 3) provide training to Cooperative Extension agents, 4) extension agent training, and 5) eXtension webinar series.The project team has established a plan for evaluating project progress to achieve the research, education, and education objectives and ensure the successful completion of the project. The plan includes a master timeline/milestones that indicates the specific tasks, time, and milestones for each participating institution. A template of performance standard is to be used by the team member to list task, timeline, milestones, and indicators of completion for establishing his/her annual work plan. The effectiveness of the research, education, and extension program will be evaluated.

Progress 04/15/15 to 04/14/20

Outputs
Target Audience:We expect to develop and optimize nonthermal processing technologies to inactivate pathogens on high-risk foods. We plan to target food processors, packagers and retailers that wish to optimize commercial equipment and provide optimal processing parameters for produce and low moisture foods (LMF). Other target audiences include consumer groups; regulators in the US, Canada, EU, and, by extension, regulators globally; and researchers in the field of food safety, with particular emphasis on those developing, evaluating and optimizing nonthermal interventions to inactivate human pathogens. The commercialization of these technologies will significantly improve the safety of the food supply, while benefiting the processors by providing sustainable alternative methods of nonthermal processing. These benefits will be passed on to the consumer by creating products that are safe with high sensory and nutrition quality. Information from this project will be disseminated through websites and surveys and used to develop educational materials for training students, extension agents, consumers, and other educators. Additionally, the results will be published and presented at conferences. Application of the technologies will significantly improve overall food safety and benefit public health and the U.S. economy. Changes/Problems:In this project, we proposed to evaluate our developed interventions against a variety of microorganisms including bacterial, virus, and parasites. Instead of Toxoplasma, Cryptosporidium is our parasite model used to test our interventions, conducted by University of Delaware. What opportunities for training and professional development has the project provided?This CAP project strengthens the collaboration among all the participant institutions. CoPDs, students, post-doc fellows, lab technicians and visiting scholars working on this project present their progress during the weekly lab meetings and quarterly project progress meetings. Members working on this CAP project had opportunities to present their work at the on-site project meeting, grantee meeting, and national and international conferences. These meetings and conferences also provided a great opportunity for graduate students and postdoctoral fellows to improve their presentation skills. In addition to graduate students in UD, VT, OSU, and TTU, we have trained graduate students from Washington State University, University of California at Davis, and University of California at Berkeley. Graduate students, visiting scientists, and postdocs at USDA WRRC have been trained extensively in prototype development, microbiology, molecular biology, virology, and engineering, and have been given the opportunity to present their findings professionally (i.e. IFT 2018 & 2019 and IAFP 2019). MATLAB Fundamentals online training course was also provided to further develop modeling skills. Post-docs and visiting scientists at the USDA ERRC were trained in molecular virology, cell culture techniques, and parasitology. Post-docs and visiting scientist from Ohio State have the opportunity to present their research in the College Research Day, OSU Hayes Graduate Research Forum, and OSU Denman Undergraduate Research Forum. Texas Tech University provided training of one graduate student and two postdocs on either experimental or computational aspects of microbial functional genomics and supported them to present their research in international conferences (e.g., American Society for Microbiology). Graduate and undergraduate students at the University of Delaware are being trained on new methods as part of this work and are supported to present their research findings in the IAFP annual meeting. Virginia Tech has been training one Ph.D. student and one postdoc in conducing the extension outreach activity. How have the results been disseminated to communities of interest?Results have been shown to collaborators on the project and board members during quarterly meetings. Presentations of the results have been made at the grantee meeting and project on-site meeting. Results have been disseminated to target audiences through peer reviewed journal publications and presented in Institute of Food Technology (IFT) Annual Meeting, and International Association for Food Protection (IAFP) meeting, American Society for Virology, International Calicivirus Conference, Ohio Association for Food Protection, and Midwest Food Safety conference, Center for Food Innovation, and Center for Advanced Processing and Packaging Studies (CAPPS). Dissemination has been via direct communication of research progress to stakeholders in industry via telephone and email. We have demonstrated our technologies and laboratory capabilities to several foreign delegations from Taiwan and South Korea. We have also conducted several onsite visits with a RTE, LMF, and produce processors. Visiting their facilities has allowed us to disseminate our development of technologies and research findings, understand their food processing methods, and discuss potential application of our technologies. There have been several opportunities to present research results to FDA and FSIS scientists, which has facilitated technology transfer to these regulatory agencies. Infosheets have begun to be distributed and shared with Extension Agents and consumers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The pilot scale of gaseous antimicrobial interventions had significant accomplishments. The efficacy of gaseous ClO2 generated by dry media and sodium chlorite-hydrogen chloride dosing system in a simulated pilot-scale environment was evaluated at ERRC facility. Both ClO2 generation methods were equally effective. Generally, ClO2(g) caused higher reductions on produce than on LMF. The treatment of 1 mg/L (w/v) ClO2(g) at 70% RH for 3 h reduced 4.9-6.8, 5.1-5.6, and 4.2-6.3 log of STEC, Salmonella, and L. monocytogenes, respectively, on produce. Applying moisture to the surface of almonds increased the reductions to >4.0 log by 1 mg/L ClO2(g) at 95 % RH for 5 h. The treatments did not cause significant color changes on blueberries, tomatoes and peppercorns, however, bleaching of color on baby-cut carrots and almonds were observed. Modeling was conducted at WRRC in preparation of the industrial scale. Validation of the surrogates for the industrial scale up through comprehensive mathematical modeling was accomplished. Using almonds treated in the designed chamber with gaseous ClO2, our model has shown that a high relative humidity and a moisture content of 7% has the most impact on log reduction compared to other environmental factors such temperature and time. Our methods do not require a specific temperature for processing as long as the almonds are able to dry to the original > 5% moisture content. Carrots were treated with ClO2 to model the reduction of pathogens and the amount of gaseous treatment the produce consumes in order to determine the amount of ClO2 precursor necessary to achieve a 4-log reduction within the standard industry treatment time. The inactivation kinetics of L. monocytogenes treated under different water activities (aw) and ClO2 concentrations was investigated at ERRC facility. Results showed that the antimicrobial effect of ClO2 significantly decreased as water activity and ClO2 concentration decreased. These results provide an improvement to the predictive capability of ClO2 models, which ensures the inactivation efficacy for the industrial scale. Our modeling also demonstrated that moisture is more influential on pathogen reduction than temperature. Processors of LMF can benefit from this knowledge by spending fewer resources on temperature control and drying. Further detailed quality evaluation of food models by ClO2 treatment indicated that the treatments did not have any significant effect on appearance, off-odor, firmness, color, or lycopene and vitamin C contents of grape tomatoes during the 21-d storage. Chlorine dioxide was applied to Tulane virus coated blueberries and reduced the virus by 1 to 3.3 logs. Progress was made on ClO2 against human norovirus (HuNoV) conducted at both OSU and WRRC facilities. The results showed that replication of HuNoV in gnotobiotic piglets was dose-dependent. 104 genomic RNA copies of HuNoV was sufficient to cause an infection. The survival of HuNoV after ClO2 treatment using the pig model indicated that severe treatment (2.5 mg/L for 2 h) completely inactivated HuNoV. However, low level of viral RNA was still detectable in feces of piglets received HuNoV with mild treatment (0.4mg/L for 2h). We investigated the mechanism of action and uncovered the molecular genetic underpinnings of bacterial defenses in both WRRC and TTU facilities. We analyzed genetic networks involved in the responses of STEC and L. monocytogenes to the xenobiotic effects of ClO2 and O3 and concluded ClO2 elicits a more potent xenobiotic effects than O3 in both the STEC and Listeria. In STEC, ClO2 triggers responses associated with either defense and injury repair or acclimation, adaptation and selection, depending on the dose and duration of exposure to the xenobiotic agent. A large proportion of genes associated with mechanisms of pathogenesis and stress response were upregulated during the first hour of exposure to 1 ug dose of ClO2, painting a general picture of the genetic responses involved in survival mechanisms under sub-lethal xenobiotic effects. However, continuous exposure to the same dose of ClO2 for 2 and 3 hours, led to a much larger proportion of pathogenesis and defense response genes downregulated. Through our ongoing collaboration with the manufacture of the ClO2 precursors, a successful federal regulation progress has been made through obtaining an exemption status for the EPA for chlorate residues on cantaloupe and tomatoes. Furthermore, the EPA does not anticipate the use ClO2 to contribute any additional residues of potentially toxic on the surface of cantaloupe or tomatoes. Progress made on cold plasma was to evaluate combinations of cold plasma with vortex tube cooling (VTC) vs. conventional air jet cooling (CAJC) for plasma-treated containers and conveyor belt surfaces. In the open container (partial airflow), plasma alone raised temperatures to 117.5 C. VTC-replacement lowered this to 92.8 C. 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. Accomplishments made in photoinactivation conducted in UD facility included a large-scale decontamination system based on a washing process in combination with pulsed light (PL) exposure and H2O2/chlorine (Cl). The combined PL-H2O2 reduced Salmonella in turbid wash water below the detection limit of 2 CFU/mL. Combined PL-Cl is an alternative to the chlorine wash for lettuce decontamination. The water-assisted decontamination systems of pulsed light and ultraviolet had similar decontamination effect on fresh produce, demonstrating that the UV system could replace the pulsed light system to reduce equipment cost. We further evaluated the influence of turbidity (0100 NTU) and chemical oxygen demand (COD; 0-2500 ppm) on UV inactivation of Salmonella in simulated wash water with different produce extracts. In the worst condition tested, Salmonella in simulated wash water could be eliminated by UV (23-28 mW/cm2) in 90 seconds (> 6 log CFU/mL reduction), indicating the potential of UV for fresh produce wash water disinfection. In addition, ultraviolet lamps were installed in different locations (above and/or in the washing tank).Three WUV configurations showed similar Salmonella reduction on blueberries (1.8-2.0 log) and tomatoes (2.4-2.9 log). Pulsed light inactivated C. parvum oocysts by 2-3 log on the contaminated produce. No major visual damage was observed in spinach, mesclun lettuce, or grape tomatoes after treatment. Darkening of the veins of cilantro leaves was observed. We evaluated the impact of cold atmospheric plasma on the viability of Cryptosporidium parvum oocysts on cilantro. Overall, treatments significantly reduced oocyst infectivity compared to the 0 s treatment control (P ≤ 0.02). Log inactivation of oocysts observed on cilantro were 0.8, 1.2, and 2 for the 30 s, 90 s, and 180 s treatment times, respectively. Drying and darkening of cilantro leaves was observed with treatments longer than 30 s. In the outreach activity, we demonstrated our developed interventions to the food industry. Our VT extension team has completed both pilot study of the survey targeting North Carolina Cooperative Extension Agents and completed online survey of Virginia Cooperative Extension Agents' perceptions of various food processing technologies. With input from the whole team members, educational materials including infosheets related to conventional and innovative processing technologies to be distributed to Extension Agents, consumers, and stakeholders are being developed through a peer reviewed process. Infosheets can be found at www.pubs.ext.vt.edu.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Bridges, David F., Bhargavi Rane, and Vivian C.H. Wu. (2018). The effectiveness of closed-circulation gaseous chlorine dioxide or ozone treatment against bacterial pathogens on produce. Food Control 91: 261-267.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Kingsley, D.H., Perez, R., Niemira, B.A., Fan, X. 2018. Evaluation of gaseous chlorine dioxide for the inactivation of Tulane virus on blueberries. International Journal of Food Microbiology. 273:23-32.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gurtler, J., Fan, X., Jin, Z.T., Niemira, B.A. 2019. Effects of antimicrobials on the thermal sensitivity of foodborne pathogens: A Review. Journal of Food Protection.Volume 82, No.4, Page 628-644. https://doi:10.4315/0362-028X.JFP-18-441.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, L., Sokorai, K., Wu, V.C., Fan, X., 2019. Gaseous chlorine dioxide maintained the sensory and nutritional quality of grape tomatoes and reduced populations of Salmonella enterica serovar Typhimurium. Food Control, 96:299-309.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, L., Fan, X., Sokorai K., Sites, J. 2019. Quality deterioration of grape tomato fruit during storage after treatments with gaseous ozone at conditions that significantly reduced populations of Salmonella on stem scar and smooth surface, Food Control,103:9-20.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Erin DiCaprio, Mu Ye, Haiqiang Chen, Jianrong Li. Inactivation of human norovirus and Tulane virus by high pressure processing in simple mediums and strawberry puree. Frontiers in Sustainable Food Systems. 2019 April. https://doi.org/10.3389/fsufs.2019.00026
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Leone CM, Dharmasena M, Tang C, DiCaprio E, Ma Y, Araud E, Bolinger H, Rupprom K, Yeargin T, Li J, Schaffner D, Jiang X, Sharp J, Vinj� J, Fraser A. Prevalence of Human Noroviruses in Commercial Food Establishment Bathrooms. J Food Prot. 2018 May;81(5):719-728.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Elbashir Araud, Miyu Fuzawa, Joanna L. Shisler, Jianrong Li, and Thanh H. Nguyen. Mechanisms of single and triple layered RNA virus inactivation by UV irradiation. Environmental Science & Technology, 2019.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Shu X, Singh M, Karampudi NBR, Bridges DF, Kitazumi A, Wu VCH, De los Reyes BG (2019) Transcriptional networks of Shiga-toxin-producing Escherichia coli O157:H7 in response to the xenobiotic effects of chlorine dioxide on non-host tomato environment: Defenses, supra-optimal effects, and potential adaptation. BMC Genomics
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Huang, R. and Chen, H. 2019. Comparison of water-assisted decontamination systems of pulsed light and ultraviolet for Salmonella inactivation on blueberry, tomato, and lettuce. J. Food Sci. 00: 1-6.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Huang, R. and Chen, H. 2019. Sanitation of tomatoes based on a combined approach of washing process and pulsed light in conjunction with selected disinfectants. Food Res. Int. 116: 778-785.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang, R. and Chen, H. 2018. Evaluation of inactivating Salmonella on iceberg lettuce shreds with washing process in combination with pulsed light, ultrasound and chlorine. Int. J. Food Microbiol. 285: 144-151.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang, R., de Vries, D., and Chen, H. 2018. Strategies to enhance fresh produce decontamination using combined treatments of ultraviolet, washing and disinfectants. Int. J. Food Microbiol.283: 37-44.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Fan, X., Haung, R., Chen, H. 2017. Application of ultraviolet C technology for surface decontamination of fresh produce. Trends in Food Sci. Technol. 70: 9-19.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Cao, X., Huang, R., and Chen. H. 2017. Evaluation of pulsed light treatments on inactivation of Salmonella on blueberries and its impact on shelf-life and quality attributes. Int. J. Food Microbiol. 260:17-26.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Guo, S., Huang, R., and Chen. H. 2017. Application of water-assisted ultraviolet light in combination of chlorine and hydrogen peroxide to inactivate Salmonella on fresh produce. Int. J. Food Microbiol. 257: 101-109.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Tan, J.N., Hwang, C.-A., Huang, L., Wu, V.C.H., and Hsiao, H.-I. A pilot-scale evaluation of using gaseous chlorine dioxide for decontamination of foodborne pathogens on produce and low-moisture foods. 2018 IFT annual meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Tan, J.N., Hwang, C.-A., Huang, L., Wu, V.C.H., and Hsiao, H.-I. In-situ generation of chlorine dioxide for decontamination of sprout seeds. 2018 IAFP annual meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wu, V.C.H. 2018. Gaseous antimicrobial intervention for produce safety. International Symposium on Food and Health (ISFH), Shanghai, China.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Rane, B. D.F. Bridges and V.C.H. Wu. 2018. Reduction of foodborne pathogens on low moisture foods using gaseous chlorine dioxide. International Association for Food Protection (IAFP) Annual Meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Rane, B., A. Lacombe, S. Sablani, D. Bridges, J. Tang, J. Guan, and V.C.H. Wu. 2019. Identifying nonpathogenic Salmonella surrogates for industrial scale treatment of almonds using gaseous chlorine dioxide. International Association for Food Protection (IAFP) Annual Meeting. Louisville, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Guan, J., J. Tang, A. Lacombe, D. F. Bridges, B. Rane, S. Sablani, and V.C.H. Wu. 2019. Identification of nonpathogenic surrogate bacteria applicable for industrial-scale gaseous chlorine dioxide treatment on baby carrots. International Association for Food Protection (IAFP) Annual Meeting. Louisville, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: D. F. Bridges, A. Lacombe, and V.C.H. Wu. 2019. Implementation of fluorescent assays to measure membrane damage to Escherichia coli O157:H7 after exposure to chlorine dioxide. International Association for Food Protection (IAFP) Annual Meeting. Louisville, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Food Science & Technology (FST) 6th Annual Poster Competition Title: Consumer Knowledge, Perceptions, and Purchasing Behaviors Associated with Food Processing Technologies in the United States (poster) Audience: Open to Virginia Tech community, Virginia Tech FST Advisory Board members and other College of Agriculture and Life Sciences faculty served as judges Location: Blacksburg, VA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: International Association for Food Protection 2018 Annual Meeting Title: Consumer Knowledge, Perceptions, and Purchasing Behaviors Associated with Food Processing Technologies in the United States (poster) Audience: Conference attendees Location: Salt Lake City, UT.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Grantees Annual Meeting - Natick Center Title: Outreach Component (poster) Audience: Waterless Nonthermal CAP Grant Stakeholders Location: Natick, MA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Virginia Cooperative Extension Showcasing Scholarship Poster 2nd place in Applied Research category Title: Consumer Knowledge, Perceptions, and Purchasing Intentions Associated with Food Processing Technologies in the United States (poster) Audience: Shared with Virginia Cooperative Extension personnel.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Consumer Food Safety Education Conference Title: Consumer Knowledge, Perceptions, and Purchasing Behaviors Associated with Food Processing Technologies in the United States (oral presentation) Audience: Conference attendees Location: Orlando, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: FST 7th Annual Poster Competition Title: How Virginia Extension Agents Engage with the Public about Food Processing Perceptions (poster) Audience: Open to Virginia Tech community, Virginia Tech FST Advisory Board members and other College of Agriculture and Life Sciences faculty served as judges Location: Blacksburg, VA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: International Association for Food Protection 2019 Annual Meeting Title: How Virginia Extension Agents Engage with the Public about Food Processing Perceptions (poster) Audience: Conference attendees Location: Louisville, KY.
• Type: Other Status: Published Year Published: 2019 Citation: How is Cold Plasma Used to Process Foods Extension publication (peer-reviewed) Posted on Virginia Cooperative Extension website for public viewing. https://www.pubs.ext.vt.edu/FST/FST-314/FST-314.html
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Shu X, Singh M, Karampudi NBR, Kitazumi A, Bridges D, Wu VCH, De los Reyes BG (2019) Transcriptional regulatory networks of Shiga-Toxin Escherichia coli O157:H7 in response to the xenobiotic effects of gaseous chlorine dioxide on non-host tomato environment: Potential adaptation to supra-optimal exposure. Annual Meeting of the American Society for Microbiology, San Francisco, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Singh M, Shu X, Karampudi1 NBR, Kitazumi A, Bridges D, Wu VCH, De los Reyes BG (2019) Analysis of the xenobiotic effects of gaseous ClO2 and O3 on Listeria monocytogenes by transcriptome profiling. Annual Meeting of the American Society for Microbiology, San Francisco, CA.

Progress 04/15/15 to 04/01/20

Outputs
Target Audience:We expect to develop and optimize nonthermal processing technologies to inactivate pathogens on high-risk foods. We plan to target food processors, packagers and retailers that wish to optimize commercial equipment and provide optimal processing parameters for produce and low moisture foods (LMF). Other target audiences include consumer groups; regulators in the US, Canada, EU, and, by extension, regulators globally; and researchers in the field of food safety, with particular emphasis on those developing, evaluating and optimizing nonthermal interventions to inactivate human pathogens. The commercialization of these technologies will significantly improve the safety of the food supply, while benefiting the processors by providing sustainable alternative methods of nonthermal processing. These benefits will be passed on to the consumer by creating products that are safe with high sensory and nutrition quality. Information from this project will be disseminated through websites and surveys and used to develop educational materials for training students, extension agents, consumers, and other educators. Additionally, the results will be published and presented at conferences. Application of the technologies will significantly improve overall food safety and benefit public health and the U.S. economy. Changes/Problems:In this project, we proposed to evaluate our developed interventions against a variety of microorganisms including bacterial, virus, and parasites. Instead of Toxoplasma, Cryptosporidium is our parasite model used to test our interventions, conducted by University of Delaware. What opportunities for training and professional development has the project provided?This CAP project strengthens the collaboration among all the participant institutions. CoPDs, students, post-doc fellows, lab technicians and visiting scholars working on this project present their progress during the weekly lab meetings and quarterly project progress meetings. Members working on this CAP project had opportunities to present their work at the on-site project meeting, grantee meeting, and national and international conferences. These meetings and conferences also provided a great opportunity for graduate students and postdoctoral fellows to improve their presentation skills. In addition to graduate students in UD, VT, OSU, and TTU, we have trained graduate students from Washington State University, University of California at Davis, and University of California at Berkeley. Graduate students, visiting scientists, and postdocs at USDA WRRC have been trained extensively in prototype development, microbiology, molecular biology, virology, and engineering, and have been given the opportunity to present their findings professionally (i.e. IFT 2018 & 2019 and IAFP 2019). MATLAB Fundamentals online training course was also provided to further develop modeling skills. Post-docs and visiting scientists at the USDA ERRC were trained in molecular virology, cell culture techniques, and parasitology. Post-docs and visiting scientist from Ohio State have the opportunity to present their research in the College Research Day, OSU Hayes Graduate Research Forum, and OSU Denman Undergraduate Research Forum. Texas Tech University provided training of one graduate student and two postdocs on either experimental or computational aspects of microbial functional genomics and supported them to present their research in international conferences (e.g., American Society for Microbiology). Graduate and undergraduate students at the University of Delaware are being trained on new methods as part of this work and are supported to present their research findings in the IAFP annual meeting. Virginia Tech has been training one Ph.D. student and one postdoc in conducing the extension outreach activity. How have the results been disseminated to communities of interest?Results have been shown to collaborators on the project and board members during quarterly meetings. Presentations of the results have been made at the grantee meeting and project on-site meeting. Results have been disseminated to target audiences through peer reviewed journal publications and presented in Institute of Food Technology (IFT) Annual Meeting, and International Association for Food Protection (IAFP) meeting, American Society for Virology, International Calicivirus Conference, Ohio Association for Food Protection, and Midwest Food Safety conference, Center for Food Innovation, and Center for Advanced Processing and Packaging Studies (CAPPS). Dissemination has been via direct communication of research progress to stakeholders in industry via telephone and email. We have demonstrated our technologies and laboratory capabilities to several foreign delegations from Taiwan and South Korea. We have also conducted several onsite visits with a RTE, LMF, and produce processors. Visiting their facilities has allowed us to disseminate our development of technologies and research findings, understand their food processing methods, and discuss potential application of our technologies. There have been several opportunities to present research results to FDA and FSIS scientists, which has facilitated technology transfer to these regulatory agencies. Infosheets have begun to be distributed and shared with Extension Agents and consumers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The pilot scale of gaseous antimicrobial interventions had significant accomplishments. The efficacy of gaseous ClO2 generated by dry media and sodium chlorite-hydrogen chloride dosing system in a simulated pilot-scale environment was evaluated at ERRC facility. Both ClO2 generation methods were equally effective. Generally, ClO2(g) caused higher reductions on produce than on LMF. The treatment of 1 mg/L (w/v) ClO2(g) at 70% RH for 3 h reduced 4.9-6.8, 5.1-5.6, and 4.2-6.3 log of STEC, Salmonella, and L. monocytogenes, respectively, on produce. Applying moisture to the surface of almonds increased the reductions to >4.0 log by 1 mg/L ClO2(g) at 95 % RH for 5 h. The treatments did not cause significant color changes on blueberries, tomatoes and peppercorns, however, bleaching of color on baby-cut carrots and almonds were observed. Modeling was conducted at WRRC in preparation of the industrial scale. Validation of the surrogates for the industrial scale up through comprehensive mathematical modeling was accomplished. Using almonds treated in the designed chamber with gaseous ClO2, our model has shown that a high relative humidity and a moisture content of 7% has the most impact on log reduction compared to other environmental factors such temperature and time. Our methods do not require a specific temperature for processing as long as the almonds are able to dry to the original > 5% moisture content. Carrots were treated with ClO2 to model the reduction of pathogens and the amount of gaseous treatment the produce consumes in order to determine the amount of ClO2 precursor necessary to achieve a 4-log reduction within the standard industry treatment time. The inactivation kinetics of L. monocytogenes treated under different water activities (aw) and ClO2 concentrations was investigated at ERRC facility. Results showed that the antimicrobial effect of ClO2 significantly decreased as water activity and ClO2 concentration decreased. These results provide an improvement to the predictive capability of ClO2 models, which ensures the inactivation efficacy for the industrial scale. Our modeling also demonstrated that moisture is more influential on pathogen reduction than temperature. Processors of LMF can benefit from this knowledge by spending fewer resources on temperature control and drying. Further detailed quality evaluation of food models by ClO2 treatment indicated that the treatments did not have any significant effect on appearance, off-odor, firmness, color, or lycopene and vitamin C contents of grape tomatoes during the 21-d storage. Chlorine dioxide was applied to Tulane virus coated blueberries and reduced the virus by 1 to 3.3 logs. Progress was made on ClO2 against human norovirus (HuNoV) conducted at both OSU and WRRC facilities. The results showed that replication of HuNoV in gnotobiotic piglets was dose-dependent. 104 genomic RNA copies of HuNoV was sufficient to cause an infection. The survival of HuNoV after ClO2 treatment using the pig model indicated that severe treatment (2.5 mg/L for 2 h) completely inactivated HuNoV. However, low level of viral RNA was still detectable in feces of piglets received HuNoV with mild treatment (0.4mg/L for 2h). We investigated the mechanism of action and uncovered the molecular genetic underpinnings of bacterial defenses in both WRRC and TTU facilities. We analyzed genetic networks involved in the responses of STEC and L. monocytogenes to the xenobiotic effects of ClO2 and O3 and concluded ClO2 elicits a more potent xenobiotic effects than O3 in both the STEC and Listeria. In STEC, ClO2 triggers responses associated with either defense and injury repair or acclimation, adaptation and selection, depending on the dose and duration of exposure to the xenobiotic agent. A large proportion of genes associated with mechanisms of pathogenesis and stress response were upregulated during the first hour of exposure to 1 ug dose of ClO2, painting a general picture of the genetic responses involved in survival mechanisms under sub-lethal xenobiotic effects. However, continuous exposure to the same dose of ClO2 for 2 and 3 hours, led to a much larger proportion of pathogenesis and defense response genes downregulated. Through our ongoing collaboration with the manufacture of the ClO2 precursors, a successful federal regulation progress has been made through obtaining an exemption status for the EPA for chlorate residues on cantaloupe and tomatoes. Furthermore, the EPA does not anticipate the use ClO2 to contribute any additional residues of potentially toxic on the surface of cantaloupe or tomatoes. Progress made on cold plasma was to evaluate combinations of cold plasma with vortex tube cooling (VTC) vs. conventional air jet cooling (CAJC) for plasma-treated containers and conveyor belt surfaces. In the open container (partial airflow), plasma alone raised temperatures to 117.5 C. VTC-replacement lowered this to 92.8 C. 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. Accomplishments made in photoinactivation conducted in UD facility included a large-scale decontamination system based on a washing process in combination with pulsed light (PL) exposure and H2O2/chlorine (Cl). The combined PL-H2O2 reduced Salmonella in turbid wash water below the detection limit of 2 CFU/mL. Combined PL-Cl is an alternative to the chlorine wash for lettuce decontamination. The water-assisted decontamination systems of pulsed light and ultraviolet had similar decontamination effect on fresh produce, demonstrating that the UV system could replace the pulsed light system to reduce equipment cost. We further evaluated the influence of turbidity (0100 NTU) and chemical oxygen demand (COD; 0-2500 ppm) on UV inactivation of Salmonella in simulated wash water with different produce extracts. In the worst condition tested, Salmonella in simulated wash water could be eliminated by UV (23-28 mW/cm2) in 90 seconds (> 6 log CFU/mL reduction), indicating the potential of UV for fresh produce wash water disinfection. In addition, ultraviolet lamps were installed in different locations (above and/or in the washing tank).Three WUV configurations showed similar Salmonella reduction on blueberries (1.8-2.0 log) and tomatoes (2.4-2.9 log). Pulsed light inactivated C. parvum oocysts by 2-3 log on the contaminated produce. No major visual damage was observed in spinach, mesclun lettuce, or grape tomatoes after treatment. Darkening of the veins of cilantro leaves was observed. We evaluated the impact of cold atmospheric plasma on the viability of Cryptosporidium parvum oocysts on cilantro. Overall, treatments significantly reduced oocyst infectivity compared to the 0 s treatment control (P ≤ 0.02). Log inactivation of oocysts observed on cilantro were 0.8, 1.2, and 2 for the 30 s, 90 s, and 180 s treatment times, respectively. Drying and darkening of cilantro leaves was observed with treatments longer than 30 s. In the outreach activity, we demonstrated our developed interventions to the food industry. Our VT extension team has completed both pilot study of the survey targeting North Carolina Cooperative Extension Agents and completed online survey of Virginia Cooperative Extension Agents' perceptions of various food processing technologies. With input from the whole team members, educational materials including infosheets related to conventional and innovative processing technologies to be distributed to Extension Agents, consumers, and stakeholders are being developed through a peer reviewed process. Infosheets can be found at www.pubs.ext.vt.edu.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Rane, B. D.F. Bridges and V.C.H. Wu. 2018. Reduction of foodborne pathogens on low moisture foods using gaseous chlorine dioxide. International Association for Food Protection (IAFP) Annual Meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Rane, B., A. Lacombe, S. Sablani, D. Bridges, J. Tang, J. Guan, and V.C.H. Wu. 2019. Identifying nonpathogenic Salmonella surrogates for industrial scale treatment of almonds using gaseous chlorine dioxide. International Association for Food Protection (IAFP) Annual Meeting. Louisville, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Guan, J., J. Tang, A. Lacombe, D. F. Bridges, B. Rane, S. Sablani, and V.C.H. Wu. 2019. Identification of nonpathogenic surrogate bacteria applicable for industrial-scale gaseous chlorine dioxide treatment on baby carrots. International Association for Food Protection (IAFP) Annual Meeting. Louisville, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: D. F. Bridges, A. Lacombe, and V.C.H. Wu. 2019. Implementation of fluorescent assays to measure membrane damage to Escherichia coli O157:H7 after exposure to chlorine dioxide. International Association for Food Protection (IAFP) Annual Meeting. Louisville, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Food Science & Technology (FST) 6th Annual Poster Competition Title: Consumer Knowledge, Perceptions, and Purchasing Behaviors Associated with Food Processing Technologies in the United States (poster) Audience: Open to Virginia Tech community, Virginia Tech FST Advisory Board members and other College of Agriculture and Life Sciences faculty served as judges Location: Blacksburg, VA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wu, V.C.H. 2018. Gaseous antimicrobial intervention for produce safety. International Symposium on Food and Health (ISFH), Shanghai, China.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: International Association for Food Protection 2018 Annual Meeting Title: Consumer Knowledge, Perceptions, and Purchasing Behaviors Associated with Food Processing Technologies in the United States (poster) Audience: Conference attendees Location: Salt Lake City, UT.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Grantees Annual Meeting - Natick Center Title: Outreach Component (poster) Audience: Waterless Nonthermal CAP Grant Stakeholders Location: Natick, MA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Virginia Cooperative Extension Showcasing Scholarship Poster 2nd place in Applied Research category Title: Consumer Knowledge, Perceptions, and Purchasing Intentions Associated with Food Processing Technologies in the United States (poster) Audience: Shared with Virginia Cooperative Extension personnel.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Consumer Food Safety Education Conference Title: Consumer Knowledge, Perceptions, and Purchasing Behaviors Associated with Food Processing Technologies in the United States (oral presentation) Audience: Conference attendees Location: Orlando, FL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: FST 7th Annual Poster Competition Title: How Virginia Extension Agents Engage with the Public about Food Processing Perceptions (poster) Audience: Open to Virginia Tech community, Virginia Tech FST Advisory Board members and other College of Agriculture and Life Sciences faculty served as judges Location: Blacksburg, VA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: International Association for Food Protection 2019 Annual Meeting Title: How Virginia Extension Agents Engage with the Public about Food Processing Perceptions (poster) Audience: Conference attendees Location: Louisville, KY.
• Type: Other Status: Published Year Published: 2019 Citation: How is Cold Plasma Used to Process Foods Extension publication (peer-reviewed) Posted on Virginia Cooperative Extension website for public viewing. https://www.pubs.ext.vt.edu/FST/FST-314/FST-314.html
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Shu X, Singh M, Karampudi NBR, Kitazumi A, Bridges D, Wu VCH, De los Reyes BG (2019) Transcriptional regulatory networks of Shiga-Toxin Escherichia coli O157:H7 in response to the xenobiotic effects of gaseous chlorine dioxide on non-host tomato environment: Potential adaptation to supra-optimal exposure. Annual Meeting of the American Society for Microbiology, San Francisco, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Singh M, Shu X, Karampudi1 NBR, Kitazumi A, Bridges D, Wu VCH, De los Reyes BG (2019) Analysis of the xenobiotic effects of gaseous ClO2 and O3 on Listeria monocytogenes by transcriptome profiling. Annual Meeting of the American Society for Microbiology, San Francisco, CA.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Bridges, David F., Bhargavi Rane, and Vivian C.H. Wu. (2018). The effectiveness of closed-circulation gaseous chlorine dioxide or ozone treatment against bacterial pathogens on produce. Food Control 91: 261-267.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Kingsley, D.H., Perez, R., Niemira, B.A., Fan, X. 2018. Evaluation of gaseous chlorine dioxide for the inactivation of Tulane virus on blueberries. International Journal of Food Microbiology. 273:23-32.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, L., Sokorai, K., Wu, V.C., Fan, X., 2019. Gaseous chlorine dioxide maintained the sensory and nutritional quality of grape tomatoes and reduced populations of Salmonella enterica serovar Typhimurium. Food Control, 96:299-309.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, L., Fan, X., Sokorai K., Sites, J. 2019. Quality deterioration of grape tomato fruit during storage after treatments with gaseous ozone at conditions that significantly reduced populations of Salmonella on stem scar and smooth surface, Food Control,103:9-20.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Erin DiCaprio, Mu Ye, Haiqiang Chen, Jianrong Li. Inactivation of human norovirus and Tulane virus by high pressure processing in simple mediums and strawberry puree. Frontiers in Sustainable Food Systems. 2019 April. https://doi.org/10.3389/fsufs.2019.00026
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Leone CM, Dharmasena M, Tang C, DiCaprio E, Ma Y, Araud E, Bolinger H, Rupprom K, Yeargin T, Li J, Schaffner D, Jiang X, Sharp J, Vinj� J, Fraser A. Prevalence of Human Noroviruses in Commercial Food Establishment Bathrooms. J Food Prot. 2018 May;81(5):719-728.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Elbashir Araud, Miyu Fuzawa, Joanna L. Shisler, Jianrong Li, and Thanh H. Nguyen. Mechanisms of single and triple layered RNA virus inactivation by UV irradiation. Environmental Science & Technology, 2019.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Shu X, Singh M, Karampudi NBR, Bridges DF, Kitazumi A, Wu VCH, De los Reyes BG (2019) Transcriptional networks of Shiga-toxin-producing Escherichia coli O157:H7 in response to the xenobiotic effects of chlorine dioxide on non-host tomato environment: Defenses, supra-optimal effects, and potential adaptation. BMC Genomics
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Huang, R. and Chen, H. 2019. Comparison of water-assisted decontamination systems of pulsed light and ultraviolet for Salmonella inactivation on blueberry, tomato, and lettuce. J. Food Sci. 00: 1-6.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Huang, R. and Chen, H. 2019. Sanitation of tomatoes based on a combined approach of washing process and pulsed light in conjunction with selected disinfectants. Food Res. Int. 116: 778-785.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gurtler, J., Fan, X., Jin, Z.T., Niemira, B.A. 2019. Effects of antimicrobials on the thermal sensitivity of foodborne pathogens: A Review. Journal of Food Protection.Volume 82, No.4, Page 628-644. https://doi:10.4315/0362-028X.JFP-18-441.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang, R. and Chen, H. 2018. Evaluation of inactivating Salmonella on iceberg lettuce shreds with washing process in combination with pulsed light, ultrasound and chlorine. Int. J. Food Microbiol. 285: 144-151.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang, R., de Vries, D., and Chen, H. 2018. Strategies to enhance fresh produce decontamination using combined treatments of ultraviolet, washing and disinfectants. Int. J. Food Microbiol.283: 37-44.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Fan, X., Haung, R., Chen, H. 2017. Application of ultraviolet C technology for surface decontamination of fresh produce. Trends in Food Sci. Technol. 70: 9-19.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Cao, X., Huang, R., and Chen. H. 2017. Evaluation of pulsed light treatments on inactivation of Salmonella on blueberries and its impact on shelf-life and quality attributes. Int. J. Food Microbiol. 260:17-26.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Guo, S., Huang, R., and Chen. H. 2017. Application of water-assisted ultraviolet light in combination of chlorine and hydrogen peroxide to inactivate Salmonella on fresh produce. Int. J. Food Microbiol. 257: 101-109.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Tan, J.N., Hwang, C.-A., Huang, L., Wu, V.C.H., and Hsiao, H.-I. A pilot-scale evaluation of using gaseous chlorine dioxide for decontamination of foodborne pathogens on produce and low-moisture foods. 2018 IFT annual meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Tan, J.N., Hwang, C.-A., Huang, L., Wu, V.C.H., and Hsiao, H.-I. In-situ generation of chlorine dioxide for decontamination of sprout seeds. 2018 IAFP annual meeting.

Progress 04/15/18 to 04/14/19

Outputs
Target Audience:We expect to develop and optimize nonthermal processing technologies to inactivate pathogens on high-risk foods. We plan to target food processors, packagers and retailers that wish to optimize commercial equipment and provide optimal processing parameters for produce and low moisture foods (LMF). Other target audiences include consumer groups; regulators in the US, Canada, EU, and, extension, regulators globally; researchers in the field of food safety, with particular emphasis on those developing, evaluating and optimizing nonthermal interventions to inactivate human pathogens. The commercialization of these technologies will significantly improve the safety of the food supply, while benefiting the processors by providing sustainable alternative methods of nonthermal processing. These benefits will be passed on to the consumer by creating products that are safe with high sensory and nutrition quality. Information from this project will be disseminated through websites and surveys and used to develop educational materials for training students, extension agents, consumers, and other educators. Additionally, the results will be published and presented at conferences. Application of the technologies will significantly improve food safety and benefit public health and the U.S. economy. Changes/Problems:In this project, we proposed to evaluate our developed interventions against a variety of microorganisms including bacterial, virus, and parasites. Instead of Toxoplasma, Cryptosporidium is our parasite model used to test our interventions, conducted by University of Delaware. What opportunities for training and professional development has the project provided?This CAP project strengthens the collaboration among all the participant institutions. Co-PDs, students, post-doc fellows, lab technicians and visiting scholars working on this project present their progress on the weekly lab meetings and quarterly project progress meeting. Members working on this CAP project had opportunities to present their work at the on-site project meeting, grantee meeting, and national and international conferences. These meetings and conferences also provided a great opportunity for graduate students and postdoctoral fellows to improve their presentation skills. In addition to graduate students in UD, VT, OSU, and TTU, we have trained graduate students from Washington State University, University of California at Davis, and University of California at Berkeley. Graduate students, visiting scientists, and postdocs at USDA WRRC have been trained extensively in prototype development, microbiology, molecular biology, virology, and engineering, and have been given the opportunity to present their findings professionally (i.e. IFT 2018 & 2019 and IAFP 2019). MATLAB Fundamentals online training course was also provided to further develop modeling skills. Post-docs and visiting scientists at the USDA ERRC were trained in molecular virology, cell culture techniques, and parasitology. Post-docs and visiting scientist from Ohio State have the opportunity to present their research in the College Research Day, OSU Hayes Graduate Research Forum, and OSU Denman Undergraduate Research Forum. Texas Tech University provided training of one graduate student and two postdocs on either experimental or computational aspects of microbial functional genomics and supported them to present their research in international conferences (e.g., American Society for Microbiology). Graduate and undergraduate students at the University of Delaware are being trained on new methods as part of this work and are supported to present their research findings in the IAFP annual meeting. Virginia Tech has been training one Ph.D. student and one postdoc in conducting the extension outreach activity. How have the results been disseminated to communities of interest?Results have been shown to collaborators on the project and board members during quarterly meetings. Presentations of the results have been made at the grantee meeting and project on site meeting. Results have been disseminated to target audiences through peer-reviewed journal publications and presented in Institute of Food Technology (IFT) Annual Meeting, and International Association for Food Protection (IAFP) meeting, American Society for Virology, International Calicivirus Conference, Ohio Association for Food Protection, and Midwest Food Safety conference, Center for Food Innovation, and Center for Advanced Processing and Packaging Studies (CAPPS). Dissemination has been via direct communication of research progress to stakeholders in industry via telephone and email. We have demonstrated our technologies and laboratory capabilities to several foreign delegations from Taiwan and South Korea. We have also conducted several onsite visits with a RTE, LMF, and produce processors. Visiting their facilities has allowed us to disseminate our development of technologies and research findings, understand their food processing methods, and discuss potential application of our technologies. There have been several opportunities to present research results to FDA and FSIS scientists, which has facilitated technology transfer to these regulatory agencies. Infosheets have begun to be distributed and shared with Extension Agents and consumers. What do you plan to do during the next reporting period to accomplish the goals?The analysis of the Listeria-ClO2 and Listeria-O3 transcriptional regulatory networks will be completed. The determination of the survival of human norovirus in food system after ClO2 treatment using the gnotibiotic pig model and porcine gastric mucin (PGM)-conjugated magnetic beads (MBs) assay will be continued. Research involving the study of Cryptosporidium parvum will focus on the effectiveness of chlorine dioxide. Commodities that will be studied include those most at risk for contamination by protozoan parasites, such as leafy greens, herbs, and similar produce models. Cumulative results will be used to focus scale-up efforts on gaseous antimicrobials, photoinactivation, and cold plasma systems. Optimizing processing conditions to achieve 5 log reductions of Salmonella while maximally maintaining the quality of almonds and whole peppercorns will be continued. The applicability of the developed models are being examined. Data will be put into a simulation and predict the outcome of a large-scale treatment. This prediction can be compared to real time data acquired in industrial settings. From there further optimization can be achieved prior to commercialization. Outreach will continue to potential commercial partners to be involved in scale-up and commercialization. Analysis of Cooperative Extension Agent survey data will be completed. Peer-reviewed infosheets for the remaining intervention technologies will be uploaded and made available on Virginia Cooperative.

Impacts
What was accomplished under these goals? This year, the pilot scale of gaseous antimicrobial interventions had significant accomplishments. The efficacy of gaseous ClO2 generated by dry media (sodium chlorite and ferric chloride) and sodium chlorite-hydrogen chloride dosing system in a simulated pilot-scale environment was evaluated at ERRC facility. Both ClO2 generation methods were equally effective. Generally, ClO2(g) caused higher reductions on produce than on LMF. The treatment of 1 mg/L (w/v) ClO2(g) at 70% RH for 3 h reduced 4.9-6.8, 5.1-5.6, and 4.2-6.3 log of STEC, Salmonella, and L. monocytogenes, respectively, on produce with the highest reductions occurred on baby-cut carrots. Applying moisture to the surface of almonds increased the reductions to >4.0 log by 1 mg/L ClO2(g) at 95 % RH for 5 h. The treatments did not cause significant color changes on blueberries, tomatoes and peppercorns, however, bleaching of color on baby-cut carrots and almonds were observed. The findings are used for designing commercial-scale trials. Modeling has been conducted this year at WRRC in preparation of the industrial scale. Validation of the surrogates for the industrial scale up through comprehensive mathematical modeling was accomplished. It improves prediction of industrial-scale treatments and ensures the inactivation efficacy for industrial scale. The inactivation kinetics was modeled by treatment time, concentration, temperature, and humidity in reference to the log reductions of pathogens and their potential surrogates. Using almonds treated in the designed chamber with gaseous ClO2, our model has shown that a high relative humidity and a moisture content of 7% has the most impact on log reduction compared to other environmental factors such as temperature and time. This is important because processors of LMF spend a lot of resources on temperature control. Our methods do not require a specific temperature for processing as long as the almonds are able to dry to the original > 5% moisture content. Carrots were treated with ClO2 to model the reduction of pathogens and the amount of gaseous treatment the produce consumes in order to determine the amount of ClO2 precursor necessary to achieve a 4-log reduction within the standard industry treatment time. Using these equations, we can predict how much starting material is required to achieve our target inactivation with the target parameters. The inactivation kinetics of L. monocytogenes treated under different water activities (aw) and ClO2 concentrations was investigated at ERRC facility. Results showed that the antimicrobial effect of ClO2 significantly decreased as water activity and ClO2 concentration decreased. The nonlinear models, Chick model was suitable to describe the inactivation kinetics of L. monocytogenes. Further detailed quality evaluation of food models by ClO2 treatment indicated that the treatments did not have any significant effect on appearance, off-odor, firmness, color, or lycopene and vitamin C contents of grape tomatoes during the 21-d storage. Chlorine dioxide was applied to Tulane virus coated blueberries and reduced the virus by 1 to 3.3 logs. Progress made on ClO2 against human norovirus (HuNoV) conducted at both OSU and WRRC facilities include determining the minimum dose of HuNoV required for infection in gnotobiotic piglets and the survival of HuNoV after ClO2 treatment. The results obtained from OSU showed that replication of HuNoV in gnotobiotic piglets was dose-dependent. At high doses of inoculation (107 and 106 RNA copies), more virus shedding was found in stool and more viral antigen expression occurred in small intestine. However, 104 genomic RNA copies of HuNoV was sufficient to cause an infection. The survival of HuNoV after ClO2 treatment using the pig model indicated that severe treatment (2.5 mg/L for 2 h) completely inactivated HuNoV. However, low level of viral RNA was still detectable in feces of piglets received HuNoV with mild treatment (0.4mg/L for 2h). In addition to antimicrobial effects of ClO2 we investigated the mechanism of action and uncover the molecular genetic underpinnings of bacterial defenses in both WRRC and TTU facilities. We analyzed genetic networks involved in the responses of STEC and L. monocytogenes to the xenobiotic effects of ClO2 and O3 and conclude ClO2 elicits a more potent xenobiotic effects than O3 in both the STEC and Listeria. A series of RNA-Seq libraries were constructed at TTU to profile the temporal patterns in the transcriptome of bacteria at different doses. In STEC, ClO2 triggers responses associated with either defense and injury repair or acclimation, adaptation and selection, depending on the dose and duration of exposure to the xenobiotic agent. A large proportion of genes associated with mechanisms of pathogenesis and stress response were upregulated during the first hour of exposure to 1 ug dose of ClO2, painting a general picture of the genetic responses involved in survival mechanisms under sub-lethal xenobiotic effects. However, continuous exposure to the same dose of ClO2 for 2 and 3 hours, led to a much larger proportion of pathogenesis and defense response genes downregulated. Through our ongoing collaboration with the manufacture of the ClO2 precursors, a successful federal regulation progress has been made through obtaining an exemption status for the EPA for chlorate residues on cantaloupe and tomatoes. Furthermore, the EPA does not anticipate the use of ClO2 to contribute any additional residues potentially toxic on the surface of cantaloupe or tomatoes. Progress made on cold plasma this year was to evaluate combinations of cold plasma with vortex tube cooling (VTC) vs. conventional air jet cooling (CAJC) for plasma-treated containers and conveyor belt surfaces. In the open container (partial airflow), plasma alone raised temperatures to 117.5 C. VTC-replacement lowered this to 92.8 C. 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. The mechanical simplicity, small footprint, and non-electrical nature of VTC recommend it for industrial scale-up efforts, and to be incorporated in coupled cold plasma applications for in-line systems and remote/field implementations of plasma-based disinfection systems. Accomplishments made in photoinactivation conducted in UD facility included a large-scale decontamination system based on a washing process in combination with pulsed light (PL) exposure and H2O2/chlorine (Cl). The combined PL-H2O2 reduced Salmonella in turbid wash water below the detection limit of 2 CFU/mL and can be used as an environmentally friendly alternative to chlorine washing for tomato decontamination and cleaning. Combined PL-Cl is an alternative to the chlorine wash for lettuce decontamination. The water-assisted decontamination systems of pulsed light and ultraviolet had similar decontamination effect on fresh produce, demonstrating that the UV system could replace the pulsed light system to reduce equipment cost. Pulsed light inactivated C. parvum oocysts by 2-3 log on the contaminated produce. No major visual damage was observed in spinach, mesclun lettuce, or grape tomatoes after treatment. Darkening of the veins of cilantro leaves was observed. In the outreach activity, we demonstrated our developed interventions to the food industry. Our VT extension team has completed both pilot study of the survey targeting North Carolina Cooperative Extension Agents and completed online survey of Virginia Cooperative Extension Agents' perceptions of various food processing technologies. Data are being analyzed. With input from the whole team members, educational materials including infosheets related to conventional and innovative processing technologies to be distributed to Extension Agents, consumers, and stakeholders are being developed through a peer-reviewed process. Infosheets can be found at www.pubs.ext.vt.edu.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Bridges, David F., Bhargavi Rane, and Vivian C.H. Wu. (2018). The effectiveness of closed-circulation gaseous chlorine dioxide or ozone treatment against bacterial pathogens on produce. Food Control 91: 261-267.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Kingsley, D.H., Perez, R., Niemira, B.A., Fan, X. 2018. Evaluation of gaseous chlorine dioxide for the inactivation of Tulane virus on blueberries. International Journal of Food Microbiology. 273:23-32.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Gurtler, J., Fan, X., Jin, Z.T., Niemira, B.A. 2019. Effects of antimicrobials on the thermal sensitivity of foodborne pathogens: A Review. Journal of Food Protection. Volume 82, No.4, Page 628-644. https://doi:10.4315/0362-028X.JFP-18-441.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, L., Sokorai, K., Wu, V.C., Fan, X., 2019. Gaseous chlorine dioxide maintained the sensory and nutritional quality of grape tomatoes and reduced populations of Salmonella enterica serovar Typhimurium. Food Control, 96:299-309.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, L., Fan, X., Sokorai K., Sites, J. 2019. Quality deterioration of grape tomato fruit during storage after treatments with gaseous ozone at conditions that significantly reduced populations of Salmonella on stem scar and smooth surface, Food Control,103:9-20.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Erin DiCaprio, Mu Ye, Haiqiang Chen, Jianrong Li. Inactivation of human norovirus and Tulane virus by high pressure processing in simple mediums and strawberry puree. Frontiers in Sustainable Food Systems. 2019 April. https://doi.org/10.3389/fsufs.2019.00026
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Leone CM, Dharmasena M, Tang C, DiCaprio E, Ma Y, Araud E, Bolinger H, Rupprom K, Yeargin T, Li J, Schaffner D, Jiang X, Sharp J, Vinj� J, Fraser A. Prevalence of Human Noroviruses in Commercial Food Establishment Bathrooms. J Food Prot. 2018 May;81(5):719-728.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Elbashir Araud, Miyu Fuzawa, Joanna L. Shisler, Jianrong Li, and Thanh H. Nguyen. Mechanisms of single and triple layered RNA virus inactivation by UV irradiation. Environmental Science & Technology, 2019.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Shu X, Singh M, Karampudi NBR, Bridges DF, Kitazumi A, Wu VCH, De los Reyes BG (2019) Transcriptional networks of Shiga-toxin-producing Escherichia coli O157:H7 in response to the xenobiotic effects of chlorine dioxide on non-host tomato environment: Defenses, supra-optimal effects, and potential adaptation. BMC Genomics
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Huang, R. and Chen, H. 2019. Comparison of water-assisted decontamination systems of pulsed light and ultraviolet for Salmonella inactivation on blueberry, tomato, and lettuce. J. Food Sci. 00: 1-6.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Huang, R. and Chen, H. 2019. Sanitation of tomatoes based on a combined approach of washing process and pulsed light in conjunction with selected disinfectants. Food Res. Int. 116: 778-785.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang, R. and Chen, H. 2018. Evaluation of inactivating Salmonella on iceberg lettuce shreds with washing process in combination with pulsed light, ultrasound and chlorine. Int. J. Food Microbiol. 285: 144-151.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Huang, R., de Vries, D., and Chen, H. 2018. Strategies to enhance fresh produce decontamination using combined treatments of ultraviolet, washing and disinfectants. Int. J. Food Microbiol.283: 37-44.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Fan, X., Haung, R., Chen, H. 2017. Application of ultraviolet C technology for surface decontamination of fresh produce. Trends in Food Sci. Technol. 70: 9-19. (not included in our previous Annual Reports)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Cao, X., Huang, R., and Chen. H. 2017. Evaluation of pulsed light treatments on inactivation of Salmonella on blueberries and its impact on shelf-life and quality attributes. Int. J. Food Microbiol. 260:17-26. (not included in our previous Annual Reports)
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Guo, S., Huang, R., and Chen. H. 2017. Application of water-assisted ultraviolet light in combination of chlorine and hydrogen peroxide to inactivate Salmonella on fresh produce. Int. J. Food Microbiol. 257: 101-109. (not included in our previous Annual Reports)
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Tan, J.N., Hwang, C.-A., Huang, L., Wu, V.C.H., and Hsiao, H.-I. A pilot-scale evaluation of using gaseous chlorine dioxide for decontamination of foodborne pathogens on produce and low-moisture foods. 2018 IFT annual meeting.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Tan, J.N., Hwang, C.-A., Huang, L., Wu, V.C.H., and Hsiao, H.-I. In-situ generation of chlorine dioxide for decontamination of sprout seeds. 2018 IAFP annual meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wu, V.C.H. 2018. Gaseous antimicrobial intervention for produce safety. International Symposium on Food and Health (ISFH), Shanghai, China.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Rane, B. D.F. Bridges and V.C.H. Wu*. 2018. Reduction of foodborne pathogens on low moisture foods using gaseous chlorine dioxide. International Association for Food Protection (IAFP) Annual Meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Rane, B., A. Lacombe, S. Sablani, D. Bridges, J. Tang, J. Guan, and V.C.H. Wu*. 2019. Identifying nonpathogenic Salmonella surrogates for industrial scale treatment of almonds using gaseous chlorine dioxide. International Association for Food Protection (IAFP) Annual Meeting. Louisville, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Guan, J., J. Tang, A. Lacombe, D. F. Bridges, B. Rane, S. Sablani, and V.C.H. Wu*. 2019. Identification of nonpathogenic surrogate bacteria applicable for industrial-scale gaseous chlorine dioxide treatment on baby carrots. International Association for Food Protection (IAFP) Annual Meeting. Louisville, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: D. F. Bridges, A. Lacombe, and V.C.H. Wu*. 2019. Implementation of fluorescent assays to measure membrane damage to Escherichia coli O157:H7 after exposure to chlorine dioxide. International Association for Food Protection (IAFP) Annual Meeting. Louisville, KY.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Shu X, Singh M, Karampudi NBR, Kitazumi A, Bridges D, Wu VCH, De los Reyes BG (2019) Transcriptional regulatory networks of Shiga-Toxin Escherichia coli O157:H7 in response to the xenobiotic effects of gaseous chlorine dioxide on non-host tomato environment: Potential adaptation to supra-optimal exposure. Annual Meeting of the American Society for Microbiology held in June 2019, San Francisco, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Singh M, Shu X, Karampudi1 NBR, Kitazumi A, Bridges D, Wu VCH, De los Reyes BG (2019) Analysis of the xenobiotic effects of gaseous ClO2 and O3 on Listeria monocytogenes by transcriptome profiling. Annual Meeting of the American Society for Microbiology held in June 2019, San Francisco, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 4/27/18 Food Science & Technology (FST) 6th Annual Poster Competition Title: Consumer Knowledge, Perceptions, and Purchasing Behaviors Associated with Food Processing Technologies in the United States (poster) Audience: Open to Virginia Tech community, Virginia Tech FST Advisory Board members and other College of Agriculture and Life Sciences faculty served as judges Location: Blacksburg, VA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 7/10/18 International Association for Food Protection 2018 Annual Meeting Title: Consumer Knowledge, Perceptions, and Purchasing Behaviors Associated with Food Processing Technologies in the United States (poster) Audience: Conference attendees Location: Salt Lake City, UT.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 8/14/18 Grantees Annual Meeting - Natick Center Title: Outreach Component (poster) Audience: Waterless Nonthermal CAP Grant Stakeholders Location: Natick, MA
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 1/25/19 Virginia Cooperative Extension Showcasing Scholarship Poster 2nd place in Applied Research category Title: Consumer Knowledge, Perceptions, and Purchasing Intentions Associated with Food Processing Technologies in the United States (poster) Audience: Shared with Virginia Cooperative Extension personnel.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 3/7/19 Consumer Food Safety Education Conference Title: Consumer Knowledge, Perceptions, and Purchasing Behaviors Associated with Food Processing Technologies in the United States (oral presentation) Audience: Conference attendees Location: Orlando, FL
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 4/26/19 FST 7th Annual Poster Competition Title: How Virginia Extension Agents Engage with the Public about Food Processing Perceptions (poster) Audience: Open to Virginia Tech community, Virginia Tech FST Advisory Board members and other College of Agriculture and Life Sciences faculty served as judges Location: Blacksburg, VA
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 7/22/19 International Association for Food Protection 2019 Annual Meeting Title: How Virginia Extension Agents Engage with the Public about Food Processing Perceptions (poster) Audience: Conference attendees Location: Louisville, KY.
• Type: Other Status: Published Year Published: 2019 Citation: How is Cold Plasma Used to Process Foods Extension publication (peer-reviewed) Posted on Virginia Cooperative Extension website for public viewing.

Progress 04/15/17 to 04/14/18

Outputs
Target Audience:We expect to develop and optimize nonthermal processing technologies to inactivate pathogens on high-risk foods. We plan to target food processors, packagers and retailers that wish to optimize commercial equipment and provide optimal processing parameters for produce and low moisture foods (LMF). Other target audiences include consumer groups; regulators in the US, Canada, EU, and, extension, regulators globally; researchers in the field of food safety, with particular emphasis on those developing, evaluating and optimizing nonthermal interventions to inactivate human pathogens. The commercialization of these technologies will significantly improve the safety of the food supply, while benefiting the processors by providing sustainable alternative methods of nonthermal processing. These benefits will be passed on to the consumer by creating products that are safe with high sensory and nutrition quality. Information from this project will be disseminated through websites and surveys and used to develop educational materials for training students, extension agents and others. Additionally, the results will be published and presented at the conferences. Application of the technologies will significantly improve the food safety and benefit public health and the U.S. economy. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This CAP project strengthens the collaboration among all the participant institutions. Co-PDs, students, post-doc fellows, lab technicians and visiting scholars working on this project present their progress on the weekly lab meetings and quarterly project progress meeting. Members working on this CAP project had opportunities to present their work at the on-site project meeting, grantee meeting, and national and international conferences. These meetings and conferences also provided a great opportunity for graduate students and postdoctoral fellows to improve their presentation skills. Graduate students at the University of Maine and visiting scientists at USDA WRRC have been trained extensively in prototype development, microbiology, molecular biology, virology, and have been given the opportunity to present their findings professionally (i.e. IFT 2017, Chicago IL). Post-docs and visiting scientists at the USDA ERRC were trained in molecular virology, cell culture techniques, and parasitology. Post-docs and visiting scientist from Ohio State have the opportunity to present their research in the College Research Day, OSU Hayes Graduate Research Forum, and OSU Denman Undergraduate Research Forum. Texas Tech University provided training of one graduate student for: 1) Handling BSL2-type experiments; 2) Delivering ozone and chlorine dioxide treatments to STEC and Listeria monocytogenes on host tomato fruits; 3) Prokaryotic RNA extraction and purification for transcriptomic works; and 3) Basic bioinformatics analytical procedures. Graduate and undergraduate students at the University of Delaware are being trained on new methods as part of this work. Virginia Tech recruited one Ph.D. student in conducing the extension outreach activity. How have the results been disseminated to communities of interest?Results have been shown to collaborators on the project and board members during quarterly meetings. Presentations of the results have been made at the grantee meeting and project on site meeting. Results have been presented and disseminated to 2017 Institute of Food Technology (IFT) Annual Meeting, and International Association for Food Protection (IAFP) meeting, American Society for Virology, International Calicivirus Conference, Ohio Association for Food Protection, and Midwest Food Safety conference. Dissemination has been via direct communication of research progress to stakeholders in industry via telephone and email. Interviews (with Niemira) have been completed with the Washington Post and WIRED Magazine. Articles will feature information arising from NIFA-funded research on cold plasma, high-intensity blue light and other technologies associated with this project. Publications dates for Washington Post are TBD. WIRED Magazine article will appear in the March 2018 issue. The trainees utilize multiple platforms to disseminate the knowledge of this CAP project to scientific community at OSU. These platforms include Work-in-Progress (WIP) meetings in the Institute of Infectious Diseases at OSU, the monthly RNA virus group meeting, the monthly Ohio Virology Association (OVA) meeting at the OSU Medical Library. What do you plan to do during the next reporting period to accomplish the goals?The USDA ARS WRRC and UMaine team has completed phase I work with gaseous ClO2 and O3 treatment of produce and LMF in the closed-chambered treatment system. The team will continue on the phase II scale up study, and conduct surrogate validation in order to prepare for industrial scale up in the phase III. We will use fluorescence-based assays to look at membrane damage to bacteria cells after exposure to gaseous chlorine dioxide or ozone and examine the effects of treatment on the ultrastructural morphology of pathogens. Working with Co-PDs on the project, studies regarding the mechanism behind pathogen susceptibility or resistance to ClO2 and O3 will be conducted. We will assist Co-PDs to optimize ClO2 treatment condition to achieve full inactivation of human NoV. The cold plasma + blue light prototype built in Years 1, 2, and 3 in ERRC is based on a modified version of an AC plasma jet device combined with 405 nm light sources from Ultra High Power LED Lights. In Years 4 and 5, the combined treatments will be scaled up for industrial simulations and subsequent pilot scale development. For optimized retention of product sensory qualities, cooling systems can be incorporated. The temperature development in pilot-scale equipment will guide this decision and implementation. While air jets and directional blowers were adequate for lab-scale studies, engineering controls for temperature will guide the selection and construction of pilot-scale equipment that is capacity-matched to the thermal load of the cold plasma + blue light systems. Effort will go to validate ClO2 treatment in the industrial simulation scale. Similar developmental approach will be used for the scale-up of the ozone treatments. The University of Delaware will continue to study various pulsed light and UV processing parameters on the inactivation of bacteria pathogens proposed foods. The planned activities for next reporting period are to: 1) test the new stirring system utilizing a motor powered mixer, 2) use the large scaled water-assisted pulsed light and UV systems to test large sample size (up to 3 kg). Only fresh-cut lettuce, spinach, carrot, blueberry and tomato will be tested using the water-assisted system, and 3) use dry PL and UV treatments for whole peppercorn and almond since the water-assisted pulsed light and UV treatments might not be applicable for these two items. Work will also be continued to evaluate the waterless technology of UV light and ClO2 on the C. parvum oocyst viability using the ICC-PCR assay. As the project progresses, closer connections are made between the researchers and the use of technologies identified by the team as the most likely to be commercially acceptable. Oocysts will be tested at WRRC for the effectiveness of ClO2. The Ohio State University will continue to estimate the survival of human norovirus by ClO2 treatment, determine the minimum dose of human norovirus required for infection in gnotobiotic piglets, and determine the survival of human norovirus after chloride dioxide treatment using the gnotibiotic pig model. Human NoV GII.4 strain will be inoculated into tomato at the inoculation level of 107 genome copies/g, and will be treated by chloride dioxide. Texas Tech University will examine the global transcriptional changes elicited by food-borne bacteria as affected by abiotic stresses imposed by non-thermal microbial intervention methods and perform additional biochemical and/or cell biological experiments as confirmatory studies to validate the genetic mechanisms of microbial susceptibility or induced resistance to the abiotic stresses imposed by the non-thermal intervention methods. Virginia Tech will conduct the same survey that was conducted with consumers (with slight modifications) with Cooperative Extension Agents since they will ultimately be the educators disseminating research based content related to these food processing technologies. This survey will differ from that of the National Survey conducted with consumers by including a section addressing what Cooperative Extension Agents are currently doing to educate consumers about processing technologies.

Impacts
What was accomplished under these goals? The USDA WRRC and University of Maine team has been focusing on the developing a closed-circulation gaseous treatments of chlorine dioxide (ClO2) and ozone (O3) to control shiga-toxin producing Escherichia coli strains (STEC), Salmonella, and Listeria monocytogenes inoculated on five produce models and two low-moisture food (LMF) models. The 2kg sample scale has been completed. WRRC has also been working with co-PDs at Texas Tech and Ohio State University to provide gaseous treatments for their objectives. Chlorine dioxide was generated using fast-activating sachets containing sodium chlorite and reducing acids and circulated within a treatment chamber containing produce inoculated with pathogens. Equal parts of dry precursor were used to generate 0.03, 0.06, and 0.12 mg ClO2/g of produce for a 2.5hr exposure and 0.04, 0.07, and 0.15 mg ClO2/g of produce for a 5hr exposure time. Overall, ClO2 treatment resulted in maximum bacterial reductions of > 7 log CFU/g observed on carrots and tomatoes and 3.7 CFU/g on blueberries. Ozone was generated through corona-discharge of a dry oxygen feed and either 0.86 or 1.71 µg O3/g produce concentrations were used to treat the produce-mass for 2.5 and 5.0 h. Exposure O3 resulted in observed reductions of 1.2-1.8 log CFU/g and simultaneously resulted in noticeable bleaching carrot and tomato tissue as well. Treatment at 0.27mg of ClO2/g of almonds for 4hr, hurdled with heat at 65oC obtained 4.10 ± 0.27-4.58 ± 0.32 log CFU/g bacterial reduction. These results are valuable to the industrial scale up which will be conducted in year 4 and 5. USDA ERRC has converted the original conveyor belt system into a rotary system with a gyratory/vibrational component that will allow for the continuous (non-batch) treatment of blueberries. Cold plasma was shown to break down Salmonella biofilms, dispersing them. In partial vacuum applications, cold plasma resulted in >1.8 logs reduction of E. coli K12 on blueberries, in combination with water vapor. Mechanism is believed to be via generation of hydrogen peroxide vapor in the cold plasma plume. Research with 405 light and effects of incorporated photoactivator compounds has shown reductions of MNV and TV. Quality evaluation of food models by ClO2 and O3 treatment showed no negative impact. The efficacy of gaseous ClO2 in a simulated pilot-scale environment for was evaluated. Inoculated 90 kg of samples in perforated cardboard boxes were placed in a 0.6 × 1.2 ×1.2 m (W×H×L) chamber and exposed to 1 mg/L (w/v; 360 ppm) ClO2 gas, generated and maintained by a ClO2 generator, 70% relative humidity, and 21°C for 1 (360 ppm-h), 2 (720 ppm-h), and 3 h (1080 ppm-h) or the equivalent ppm-h. Reductions of 2.5-5.7, 3.0-6.7, and 4.0-6.8 log were observed for produce treated for 360, 720, and 1080 ppm-h, respectively, while mostly 1.3-4.0 log reductions were for almonds and peppercorns. The highest reductions occurred in baby carrot (3.0-6.8 log), while the lowest reductions occurred in almonds (1.3-4.0 log). The appearance and color of treated products were not significantly different from the untreated. The University of Delaware has been investigating the effect of dry UV (samples were treated by UV directly), wet UV (samples were dipped in water briefly and then exposed to UV), and water-assisted UV (samples were treated by UV while being immersed in agitated water) on inactivation of Salmonella inoculated on tomatoes and fresh-cut lettuce. The water-assisted UV treatment was the most effective for both produce. Neither chlorine nor hydrogen peroxide significantly enhanced water-assisted UV inactivation. The single water-assisted UV treatment and the combined treatment of water-assisted UV and chlorine (Cl2) were similar or more effective than the chlorine washing treatment. In most of the cases, no Salmonella was detected in the wash water when the single water-assisted UV treatment was used to decontaminate tomatoes. In a few cases when Salmonella was detected in the wash water, the populations were very low, ≤ 2 CFU/mL. Therefore, the single water-assisted UV treatment could potentially be used as an environmentally friendly and non-chemical alternative to Cl2 washing for tomatoes after validation in industrial scale. For lettuce, spinach and baby-cut carrots, the combined treatment of water-assisted UV treatment and Cl2 was needed to maintain a pathogen free environment in the wash water. Research was further initiated on the inactivation of the protozoan parasite Cryptosporidium parvum. Studies have been initiated with cold plasma and pulsed light. Future studies will include ClO2. Pulsed light was used to determine the effect on the viability of C. parvum oocysts on the surface of mesclun lettuce. Oocysts (1.56 x 106oocysts/per 125ul PBS) were applied to glass slides (control) or mesclun lettuce (0.5g) in duplicate and were treated with pulsed light for 0s, 10s, 45s, and 90s. Data suggests pulsed light effects C. parvum oocyst viability without major changes in the physical appearance of mesclun lettuce. The Ohio State University compared the survival of norovirus surrogates, Tulane virus (TV) and murine norovirus (MNV-1) by gaseous ClO2 treatment and is optimizing protocol to estimate the survival of human norovirus by ClO2 treatment. Briefly, 107 PFU of TV or MNV-1 were inoculated onto the surface of 35mm petri dishes, dried and sent to the USDA WRRC for the treatment of ClO2gas at the concentration of 0.4mg/L. After 20 min treatment, a 2-3 log virus reduction was achieved for TV. After 40 min treatment, a 5 log virus reduction was achieved for TV. TV was completely inactivated after 60 min treatment. MNV-1 also exhibited similar level of susceptibility. Thus, ClO2 is capable of inactivating both TV and MNV-1 at a low concentration with a longer exposure time. Since human norovirus cannot be cultivated, anin vitroassay with porcine gastric mucin (PGM)-conjugated magnetic beads (MBs) was optimized to estimate the infectivity of human norovirus. It was found that 99% of human NoV was bound to PGM-MBs at a concentration of 107 genomic RNA copies. Next, 107 genomic RNA copies of human NoV were inoculated onto the surface of 35mm petri dishes, dried and sent to the USDA ARS for the treatment of ClO2gas at various concentrations. After 20 min exposure, a 2 logs reduction in human NoV RNA copies was achieved at the concentration of 0.1 and 0.2 mg/L. A 3 log reduction was observed at a higher concentration (0.4mg/L) of chloride dioxide. Experiments are ongoing to optimize the treatment condition to achieve full inactivation of human NoV. Texas Tech University, which is in charge of genomics experiments started in June 2017. Progress has been made in optimizing the protocol for delivering intervention treatments of O3 and ClO2 to STEC and L. monocytogenes on host tomato for examining mechanisms of bacterial resistance or susceptibility by functional genomics. Protocols for RNA isolation has been optimized and few candidate genes have been preliminary screened by real-time PCR for assessing RNA quality for transcriptomic experiments. Members of the Outreach group at Virginia Tech completed draft version and full implementation of the nationwide survey to the general public, with the objective of assessing consumer knowledge, perceptions, and purchasing behaviors related to food processing technologies. After the elimination of all the ineligible records described above, the remaining number of eligible sample members was 21,384. 505 survey completions were met at the close of the survey. A draft survey for the assessment of Extension agent knowledge, perceptions, and behaviors surround processing technologies has been drafted and will be piloted.

Publications

• Type: Book Chapters Status: Published Year Published: 2017 Citation: Erin DiCaprio, Fangfei Lou, Ashley Predmore, Jianrong Li*, "Strategies for control of food- and water-borne viruses". Viral Foodborne Pathogens. Boca Raton: CRC Press, 2017. ISBN 9781466579507.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Fan, X., Haung, R., Chen, H. 2017. Application of ultraviolet C technology for surface decontamination of fresh produce. Trends in Food Sci. Technol. 70, 9-19.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Cao, X., Huang, R., and Chen. H. 2017. Evaluation of pulsed light treatments on inactivation of Salmonella on blueberries and its impact on shelf-life and quality attributes. Int. J. Food Microbiol. 260:17-26.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Guo, S., Huang, R., and Chen. H. 2017. Application of water-assisted ultraviolet light in combination of chlorine and hydrogen peroxide to inactivate Salmonella on fresh produce. Int. J. Food Microbiol. 257: 101-109.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Lacombe A., B.A. Niemira*, J.B. Gurtler, J. Sites, G. Boyd, D. H. Kingsley, X. Li, H. Chen. 2017. Nonthermal Inactivation of Norovirus Surrogates on Blueberries using Atmospheric Cold Plasma. Food Micro 63:15.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Hertrich S.M, G. Boyd, J. Sites, B.A. Niemira*. 2017. Cold plasma inactivation of Salmonella in pre-packaged, mixed salads is influenced by cross-contamination sequence. J Food Prot 80 (12): 2132-2136.
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Kingsley, D., R. Perez, G. Boyd, J. Sites, B.A. Niemira. 2017. Evaluation of 405 nm Monochromatic Light for Inactivation of Tulane Virus on Blueberry Surfaces. J Appl Micro.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Kingsley, D.H., R.E. P�rez-P�rez, B.A. Niemira, X. Fan. Evaluation of Gaseous Chlorine Dioxide for the Inactivation of Tulane virus on Blueberries. Int J Food Micro
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhihong Yang, Heather Chambers, Erin DiCaprio, Gary Gao, Jianrong Li*. Internalization and Dissemination of Human Norovirus and Tulane virus in fresh produce is plant dependent. Food Microbiology, 2018, 69:25-32.
• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Bridges, D.F., B. Rane, and V.C.H. Wu*. 2018. The effectiveness of closed-circulation gaseous chlorine dioxide or ozone treatment against bacterial pathogens on produce. Food Control.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bridges, D.F., B. Rane, and V.C.H. Wu*. 2017. The effectiveness of a closed-circulation gaseous chlorine dioxide treatment system against bacterial pathogens on produce. Institute of Food Technologist Annual Meeting. Chicago, IL
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Bridges, D.F., B. Rane, and V.C.H. Wu*. 2017. Gaseous ozone fumigation treatment of produce in a closed-circulation treatment chamber to reduce foodborne pathogens. Institute of Food Technologist Annual Meeting. Chicago, IL.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Arnold N., Drape T., Boyer R. National Institute of Food and Agriculture Food Safety Project Directors Meeting: Educating Consumers About Food Processing Technologies Using Cooperative Extension as a Delivery System in Tampa, FL, July 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Arnold N., Drape T., Boyer R. National Institute of Food and Agriculture Food Safety Project Onsite Meeting at the Eastern Regional Research Center: A Survey of Consumer Perceptions of Various Food Processing Technologies: Pilot Survey Results and Next Steps in Wyndmoor, PA, September 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Craighead, S., Shearer, A.E.H., Hertrich, S., Boyd, G., Sites, J., Niemira, B., Kniel, K. 2017. The Use of an Atmospheric Cold Plasma Jet to Inactivate Cryptosporidium parvum Oocysts on Cilantro, International Association for Food Protection Annual Meeting, Tampa FL, July 9-12. P1-21.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Niemira, B.A. 2017. From Chemicals to Cold Plasma: Non-Thermal Food Processing Technologies Research at the USDAs Eastern Regional Research Center. SAAFoST Biennial Congress, Cape Town, South Africa, Sept 3-6, 2017.
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2018 Citation: Rane, B. D.F. Bridges and V.C.H. Wu*. 2018. Reduction of foodborne pathogens on low moisture foods using gaseous chlorine dioxide. International Association for Food Protection (IAFP) Annual Meeting.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Rane, B. 2017. DEVELOPMENT OF GASEOUS ANTIMICROBIAL INTERVENTIONS: CHLORINE DIOXIDE AND OZONE AGAINST FOODBORNE PATHOGENS ON PRODUCE AND LOW-MOISTURE FOODS. University of Maine.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Bridges, D.F. 2017. THE EFFECTIVENESS OF A CLOSED-CIRCULATION GASEOUS CHLORINE DIOXIDE OR OZONE TREATMENT AGAINST SHIGA TOXIN-PRODUCING ESCHERICHIA COLI, SALMONELLA SPECIES, AND LISTERIA MONOCYTOGENES ON PRODUCE. University of Maine.

Progress 04/15/16 to 04/14/17

Outputs
Target Audience:We expect to develop and optimize nonthermal processing technologies to inactivate pathogens on high-risk foods. We plan to target food processors that wish to optimize commercial equipment and provide optimal processing parameters for produce and low moisture foods (LMF). The commercialization of these technologies will significantly improve the safety of the food supply, while benefiting the processors by providing sustainable alternative methods of nonthermal processing. These benefits will be passed on to the consumer by creating products that are safe with high sensory and nutrition quality. Information from this project will be disseminated through websites and surveys and used to develop educational materials for training students, extension agents and others. Additionally, the results will be published and presented at the conferences. Application of the technologies will significantly improve the food safety and benefit public health and the U.S. economy. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students at the University of Maine have been trained extensively in prototype development, experimental design, and have been given the opportunity to present their findings professionally (i.e. IFT 2016, Chicago IL). Post-docs at the USDA ERRC were trained in molecular virology, cell culture techniques, and parasitology. Post-docs and visiting scientist from Ohio State attended Ohio Association for Food Protection, American Society for Virology Conference and American Society for Virology Conference at Virginia Tech on June of 2016. Project has provided postdoctoral training and professional development for Dr. Alison Lacombe (with Niemira & Gurtler) from USDA ERRC, now a researcher at National College of Natural Medicine, Portland OR and Erin DiCaprio from Ohio State has recently become an Assistant Professor in the Department of Food Science and Technology at University of California-Davis. How have the results been disseminated to communities of interest?Results have been shown to collaborators on the project and board members during quarterly meetings. Presentations of the results have been made at 2016 Institute of Food Technology (IFT) Annual Meeting. Abstracts have been submitted to be presented 2017 Institute of Food Technology (IFT) Annual Meeting, American Society for Virology, International Calicivirus Conference, Ohio Association for Food Protection, and Midwest Food Safety conference. In addition, the research results were the subject of an interview with SCIENCE, discussing the NIFA-funded research on how cold plasma can inactivate norovirus on blueberries, with potential applications to a wider array of fresh and fresh-cut fruits and vegetables. (SCIENCE: "Healthier blueberries, thanks to a blast of purple plasma" by Ula Chrobak, Dec. 5, 2016 http://www.sciencemag.org/news/2016/12/healthierblueberries-thanks-blast-purple-plasma). This "general audience" science communication was followed by an industry specific write-up in Food Safety News, which led to numerous opportunities for discussion of the NIFA-funded research with stakeholders in industry and regulatory agencies, and dissemination of the peerreviewed journal articles related to the work. (Food Safety News: "Cold plasma proving to be hottest new food safety treatment" December 7, 2016 http://www.foodsafetynews.com/2016/12/cold-plasma-proving-to-be-hottest-new-foodsafety-treatment/#.WEiJqX36VP3) What do you plan to do during the next reporting period to accomplish the goals?The USDA ARS WRRC and UMaine team has made significant in ClO2 and O3 treatment of carrot, blueberries and tomato. In the next year, this team plan to finish phase I work with gaseous ClO2 and O3 treatment of leafy green and LMF in the closed-chambered treatment system and move to phase II scale up study. Working with a Co-PDs on the project, studies regarding the mechanism behind ClO2 and O3 induced bacterial death and reduction should be underway as well. We will examine the effects of each treatment on the ultrastructural morphology of pathogens. Damage will be graded based on the continuity of the cell membrane/capsid/cell wall, consistency of the inner cellular components, and shape with respect to the control. Relative intactness will be determined by the degree of perforation of the outer structures and its ability to retain the inner constituents. Once cellular damage is confirmed, we will identify the specific cellular sites and quantify the extent of damage using the LIVE/DEAD permeability assay and TEM For next year, USDA ERRC's primary experiments will include the manual manipulation of the rotating bed cold plasma / 405nm blue light system in order to determine the optimal setting for the variable including: the number of berries to be included in each section, the optimal rate that the system will rotate to establish greatest kill while maintaining sensory quality, the optimal cold plasma discharge rate and the distance at which the plasma is applied, and the rate at which the system will rotate to ensure full exposure of berries to the treatment. Once the limits for these variables are established, the design will be further scaled up to an automated continuous system. The manual system is currently being built and preliminary experiments are scheduled to begin next month. A permeable sachet containing an equal amount of sodium chlorite and activating acids will be used to generate ClO2. Ozone is generated from the labels placed inside film bags via dielectric barrier discharge when high voltage is applied on the labels outside. In addition to O3 produced in the bags, other reactive oxidative chemical species such as H2O2 (from water vapor), NO2 and singlet oxygen, may be produced. Ozone concentrations will be measured using an O3 detector tube. Parameters evaluated will include voltage of power supply, treatment time, treatment temperature (low temperature favors O3 production and stability), film thickness, vibration/mixing during or immediately after treatments for uniform O3 exposure. Based on results from microbial inactivation studies, the most effective treatment(s) will be evaluated for effects of these treatments on sensory quality (appearances, odor, color, texture, nutrients) of fresh produce using instrumentation analysis and sensory panel tests. The log reductions of pathogens, parasites, and viruses obtained from produce and LMF treated with light- and gas-treatments of selected factors and parameters will be analyzed using nonlinear or the Weibull models to determine the rate of inactivation, which will then be described by mathematical. The University of Delaware will study various pulsed light and UV processing parameters on the inactivation of bacteria pathogens on high risk foods. For the past year, tomatoes and lettuce are used as model food and Salmonella as a model pathogen in the system development. We will continue verify these results and eventually identify the optimum water-assisted processing conditions for some of the high risked foods proposed in this study, fresh-cut lettuce and spinach (leafy green); carrot (root vegetable); and blueberry and tomato (fruits). The water-assisted pulsed light and UV treatments might not be applicable for whole peppercorn and almond, the other high risk food items that were proposed in this study. For those two items, we will use dry PL and UV treatments. A small-scale water-assisted UV light system is used for the small-scale experiments. The system incorporates a washing unit inside a UV chamber. The washing unit consisted of a glass container with wash water and a stirrer bar sitting on a magnetic stirrer. During treatments, the stir bar is able to agitate the water in the glass container to create turbulent flow so that random rotation and movement of food samples could be achieved. The UV chamber (Reyco UVC Emitter Table Top Test System, Medirian, Idaho, U.S.A.) has four 16-inch long low pressure mercury lamps emitting 254 nm light. Adjusting the distance between the lamps and the surface of the samples could change the UV intensity received by samples. A scaled-up UV light system has been built in our laboratory. The UV system consists of a circulating flume washer inside the UV treatment chamber. The circulating flume washer was fabricated using a stainless steel hotel pan and an inline aquarium pump. The UV chamber has four 90 cm long amalgam UV lamps rated at 265 W power/lamp (Heraeus Noblelight, Buford, GA) on the top of the flume washer. The pulsed light system with a dimension of 47.6 cm (deep) x 63.5 cm (wide) x 66 cm (high) has also been built. Washing setups similar to the UV system, agitation with a stirrer bar and circulating flume washer, are also used for the pulsed light system. After development of the pulsed light and UV systems and identification of optimum processing conditions for inactivation of Salmonella inoculated on the high risked foods, the systems and processing conditions will be tested on other bacterial pathogens proposed in this study which include Listeria monocytogenes, and STEC (E. coli O157:H7, E. coli O145, O111, and O104:H4). Analysis of inactivation on the protozoan parasites will also be initiated. This work will begin using the parameters optimized for reduction and successful inactivation of bacteria and viruses. UDel group will travel to USDA-ARS to use cold plasma along with blue light treatments and will work with Dr. Chen to assess UV light. We will begin evaluation with Cryptosporidium parvum using a combined cell culture (HCT-8 cells) and quantitative RT-PCR detection method. We will then use the aviab protozoa Eimeria tenella as a surrogate for Cyclospora cayetanensis since this C. cayetanensis only infects humans and there is not a good means of collecting and bringing viable oocysts into the laboratory. We will assess sporulation and excystation of the E. tenella oocysts to better understand the biological response of the oocysts to the treatments. This knowledge will expand our understanding concerning the likelihood of effectiveness on other protozoa. The Ohio State University will utilize real-time RT-PCR, RNase treatment, human norovirus receptor binding assay, and gnotobiotic pig (2 litters) model to determine the survival of human norovirus by food processing technologies. This CAP project will identify most promising waterless nonthermal processing technology. Human norovirus clinical isolate will be treated by the most promising waterless nonthermal processing technology. The survival of human norovirus will be first estimated by human norovirus receptor binding assay coupled with real-time RT-PCR, and will be verified in newborn gnotobiotic piglets. Ohio State will also determine the mechanism of human norovirus inactivation. Using these novel tools, we expect that we will obtain precise information about the survival of human norovirus Virginia Tech will focus on survey targeting consumers that will assess their knowledge, understanding and opinions about various food processing technologies including the new technologies evaluated in this grant proposal will be conducted.

Impacts
What was accomplished under these goals? The USDA WRRC and University of Maine team have been focusing on the evaluation of the effectiveness that gaseous treatments of chlorine dioxide (ClO2) and ozone (O3) at reducing shiga-toxin producing Escherichia coli strains (STEC), Salmonella species, and Listeria monocytogenes inoculated on five fresh produce models and two low-moisture food models with large sample sizes. The treatment setup consisted of a five-gallon plastic buckets containing an internal fan (110/120v; All Electronics: CF-120) attached to the lid for gas circulation during treatment. Produce mass consisted of 2 kg aliquoted between two layers of five containers, making ten containers total with 200 g of produce in each. Pure ClO2 gas was generated by mixing sodium chlorite with and acid activator in a 1:1 ratio. For treatment, 7.5, 15, and 30g of each dry precursor was used to generate 0.03, 0.06, and 0.12 mg ClO2/g produce for a 2.5 h exposure and 0.04, 0.07, and 0.15 mg ClO2/g produce for a 5.0 h exposure time. Gaseous O3 was generated from a pure oxygen feed using an LG-7 O3 generator (Del Ozone). Ozonated oxygen was then fed through hosing into the chambers and released at a flow rate of 2 l/min for 2 min, allowing for 4 l of gas to enter the chamber. Produce samples were treated with 200 ppm and 400 ppm O3 and concentration was measured using a 49i O3 analyzer. Chlorine dioxide treatment was much more effective at reducing pathogenic bacteria populations on the carrots than O3 was. E. coli was the most susceptible to the treatment with reductions of >7.0 log CFU/g with treatments of 0.04 and 0.07 mg ClO2/ g carrot for 5.0 h. Salmonella and L. monocytogenes had a maximum reduction of 5.08 ± 0.30 log CFU/g for 2.5 h exposure at 0.12 mg ClO2/g carrot and 5.50 ± 0.24 log CFU/g at 0.15 mg ClO2/g carrot for 5.0 h exposure respectively. Ozone treatment of 400 ppm with a 5.0 h exposure reduced E. coli at a maximum of 1.17 ± 0.11 log CFU/g while causing simultaneous bleaching of some carrots. Salmonella and Listeria at comparative concentrations and exposure times saw maximum reductions of 0.50 ± 0.05 and 0.82 ± 0.07 log CFU/g respectively. Comparatively, gaseous ClO2 was not as effective as reducing bacterial pathogen population on blueberries as it was on the other two models. However, ClO2 treatment was still more effective than O3. Overall, gaseous ClO2 treatment of tomatoes was the most successful treatment to reduce bacterial pathogen concentrations. For all species and gas concentrations, a five hour exposure was enough to reduce the bacterial load down to undetectable levels with reductions ≥7 log CFU/g for each species. Ozone was, comparatively, not nearly as effective with a maximum reduction of 1.60 ± 0.26 log CFU/g seen after a 5.0 h exposure of E. coli to 400 ppm O3. At the same concentration and exposure times, Salmonella and L. monocytogenes has maximum reductions of 1.06 ± 0.27 log CFU/g and 1.14 ± 0.47 log CFU/g respectively. Overall, there was little measured ClO2 residue on the blueberries and tomatoes. Carrots initially had a high level of residue immediately post treatment, but the remaining ClO2 predictably broke down over time and reduced the acceptable levels (< 3 mg/L) after 24 h. There was no measured residual O3. Almonds were treated with gaseous ClO2 using a closed circulatory system arranged with PVC pipes. Reduction of Salmonella found after treatment is up to 1 log CFU/ml, dipping the almonds in water pre-treatment gave a reduction of 2.50 - 3 log CFU/ml. Further heating post-treatment to up to 65oC gave up to 4 log reduction. This technique is being optimized according to industrial needs. Similar work was done using O3, O3 was pumped through the generator into the circulatory system for 10mins, the flowrate of the gas was 2l/min and the reduction obtained after treatment was 1.54 log CFU/ml. The system is being optimized currently according to the suitable requirements. The USDA ERRC has integrated nonthermal technologies for pilot scale studies based on a modified tunnel carrot washing line and/or vibrating convey line to treat produce and low moisture foods. Results from the most recent studies the design for this system has been further adapted to optimize treatment of technology-compatible produce commodities with cold plasma and blue light (405nm) to reduce the number of pathogens present without disrupting organoleptic or sensory properties of the product. The new design has converted the original conveyor belt system into a rotary system with a gyratory/vibrational component that will allow for the continuous (non-batch) treatment of blueberries. The Ohio State has identified human norovirus (NoV) clinical isolates from recent outbreaks of acute gastroenteritis in Ohio and established a gnotobiotic pig model for human norovirus infection assay. The full-length genome of some of viral isolates has been sequenced, and can be found in GeneBank. The titer of human NoV isolates ranges from 7×106 to 7×109 genomic RNA copies/ml. For gnotobiotic pig model for human norovirus infection assay, results showed that mild diarrhea was observed in most 5M and 7I inoculated piglets, but not control piglets. Human NoV infected piglets developed histologic lesion in duodenum, jejunum, and ileum. In addition, a large number of norovirus antigens were detected in jejunum from 7I group, although relatively less antigen positive cells were detectable in duodenum and ileum segments in 7I group. Together, these results demonstrated that human NoV caused mild diarrhea, fecal viral shedding, histologic lesion and antigen expression in small intestines. Control of human norovirus surrogates in fresh foods by gaseous O3 and a proposed mechanism of inactivation was established. Foods were treated with gaseous O3 at 6% wt/wt O3 in oxygen for 0, 10, 20, 30, and 40min, and surviving viruses were quantified by viral plaque assay. Our results showed that gaseous O3 inactivatednorovirusin both liquid media and fresh produce in a dose-dependent manner. The University of Delaware has been investigating two emerging decontamination technique, pulse light (PL) and ultraviolet (UV), in combination with washing. Blueberries, grape tomatoes and iceberg lettuce shreds were tested in this study to represent fresh vegetables and fruits with smooth and rough surfaces. Salmonella spp. were used as targets due to its prevalence in outbreaks and spot and dip inoculation were used to imitate different contamination scenarios. Two levels of pulse light (~ 0.15 and 0.3 J/(cm2*pulse); 3 pulses/s) and ultraviolet ( ~ 13 and 28 mW/cm2) were tested for 1 and 2 min in combination with tap water washing (~ 1 ppm free chlorine). For blueberries with either spot or dip inoculation, PL and UV showed similar inactivation effect of Salmonella. PL and UV could reduce Salmonella on blueberries with spot inoculation by 4.8 - 5.4 log and 5.0 - 5.3 log, respectively. As for blueberries with dip inoculation, PL and UV could achieve 1.9 - 2.2 log and 1.8 - 2.3 log reduction of Salmonella, respectively. For grape tomato with spot inoculation, PL and UV could reduce Salmonella by 4.4 - 5.4 log and 4.8 - 5.3 log, respectively. As for tomatoes with dip inoculation, PL (2.3 -2.5 log) showed better inactivation of Salmonella than UV (1.9 - 2.2 log). For lettuce with spot inoculation, PL and UV could reduce Salmonella by 2.5 - 3.1 log and 1.9 - 2.7 log, respectively. As for lettuce with dip inoculation, PL (2.2 -2.6 log) showed better inactivation of Salmonella than UV (1.9 - 2.4 log). Overall, PL and UV showed similar inactivation of Salmonella on blueberries, tomatoes and lettuce with spot inoculation. For tomatoes and lettuce with dip inoculation, PL showed slightly better inactivation effect than UV. In most treatments, UV showed less Salmonella residuals in washing water than PL.

Publications

• Type: Book Chapters Status: Published Year Published: 2016 Citation: Erin DiCaprio, Fangfei Lou, Ashley Predmore, Jianrong Li*. Strategies for control of food- and water-borne viruses". Foodborne Viral Pathogens. Boca Raton: CRC Press, 2016.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Hwang, C.-A., L. Huang, and V.C.H. Wu. 2016. In-situ generation of chlorine dioxide for surface decontamination of produce. J. Food Prot. In press.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Lacombe A., B.A. Niemira*, J. Sites, G. Boyd, J.B. Gurtler, B. Tyrell, M. Fleck. 2016. Reduction of Enteric Pathogens and Potential Surrogates using Integrated, High Intensity 405nm LED Light on the Surface of Almonds. J Food Prot 79(11):1840-1845
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Lacombe A., B.A. Niemira*, J.B. Gurtler, J. Sites, G. Boyd, D. H. Kingsley, X. Li, H. Chen. 2017. Nonthermal Inactivation of Norovirus Surrogates on Blueberries using Atmospheric Cold Plasma. Food Micro 63:15
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Fangfei Lou, Erin DiCaprio, Xinhui Li, Xianjun Dai, Yuanmei Ma, John Hughes, Haiqiang Chen, David Kingsley, Jianrong Li*. Variable high pressure processing sensitivities for GII human noroviruses. Applied Environmental Microbiology, 2016, 82(19):6037-6045.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ashley Predmore, Gabriel Sanglay G, Jianrong Li, Ken Lee. Control of human norovirus surrogates in fresh foods by gaseous ozone and a proposed mechanism of inactivation. Food Microbiology 2015, 50:118-25. 2
• Type: Journal Articles Status: Published Year Published: 2016 Citation: McHugh, T.H., Niemira, B.A. 2016. Cold plasma technology close-up. Food Technology. 70(3):68-69,72.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Tadepalli, S., R. Anderson, and V.C.H. Wu*. 2016. Synergistic effect of multiple low-dosage chemical sanitizers used at industrial practical treatment times in combination with freezing against foodborne pathogens on blueberries. Institute of Food Technologist Annual Meeting. Saint Louis, MO.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Rane, B., D. Bridges, and V.C.H. Wu*. 2016. Development of an optimal dry inoculation method of for low-moisture foods. Institute of Food Technologist Annual Meeting. Chicago, IL.

Progress 04/15/15 to 04/14/16

Outputs
Target Audience:We expect to develop and optimize nonthermal processing technologies to inactivate pathogens on high-risk foods. We plan to target food processors that wish to optimize commercial equipment and provide optimal processing parameters for produce and low moisture foods (LMF). The commercialization of these technologies will significantly improve the safety of the food supply, while benefiting the processors by providing sustainable alternative methods of nonthermal processing. These benefits will be passed on to the consumer by creating products that are safe with high sensory and nutrition quality. Information from this project will be disseminated through websites and surveys and used to develop educational materials for training students, extension agents and others. Additionally, the results will be published and presented at the conferences. Application of the technologies will significantly improve the food safety and benefit public health and the U.S. economy. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students at the University of Maine have been trained extensively in prototype development, experimental design, and have been given the opportunity to present their findings professionally. Post-docs at the USDA ERRC were trained in molecular virology, cell culture techniques, and parasitology. How have the results been disseminated to communities of interest?Preliminary results have been shown to collaborators on the project and board members during quarterly meetings. An abstract has been submitted to be presented 2016 Institute of Food Technology (IFT) Annual Meeting where preliminary results will be presented. What do you plan to do during the next reporting period to accomplish the goals?In the next year, the USDA ARS WRRC and UMaine team hopes to finish lab-scale work with gaseous chlorine dioxide and ozone treatment of blueberries, cherry tomatoes, and baby carrots in the closed-chambered treatment system. The residue study and the vibratory mixer study will be underway and the pneumatic conveyer should be built as well. Working with a Co-PDs on the project, studies regarding the mechanism behind chlorine dioxide and ozone induced bacterial death and reduction should be underway as well. At ERRC, the prototype technologies will be integrated for scale-up studies, combining CP emitter(s), 405 nm light, pulse electric light strips, and/or gas ports for introduction of O3/ClO2. CP and light-emitting apparatus will be adapted to commercial needs, such as by mounting within a cooling tunnel as be part of a modified tunnel carrot washing line and/or a vibrating convey line for LMF. The design will incorporate two continuous-feed, enclosed chambers with gas injection nozzles. Food samples will continuously exposed to antimicrobial treatments combining CP and photoinactiving lights. The proposed treatment system will include a plastic link vibratory bed conveyor with low profile cleats and a variable conveyor speed to optimize product residence times and intensity of exposure. The conveyor will be encapsulated in a cooling tunnel that will include localized cooling jets. The interior surface of the cooling tunnel will be highly reflective to maximize the effects of the high power LED lights bars. The total output of the LEDs will be varied by energizing different banks of LEDs. The University of Delaware, will develop an effective pulsed light decontamination system in this coming year. Blueberries and spinach will be used as model food in the system development. The system developed will be tested on the high risk foods proposed in this study. Virginia Tech will be hiring a PhD student to develop and implement a research focused survey targeting consumers assessing their knowledge, understanding and opinions about various food processing technologies including the new technologies evaluated in this grant proposal. This will be an important part in dissemination process for the results of our scientific studies by giving the investigators insight into consumer opinions of nonthermal processing.

Impacts
What was accomplished under these goals? The USDA WRRC and University of Maine focused on setting up standard protocols for inoculation, recovery, and enumeration of virus, protozoa, and bacteria utilized during the course of the grant. Preliminary work was done to establish protocols that will sufficiently inoculate the produce models with bacterial pathogens at concentrations of at least 6.5-7 log CFU/ml. Three treatment systems have been optimized for the delivery of gaseous treatment on inoculated produce. The systems, described below, are designed to be a scaled-down versions of industrial realistic setup. Each system is in a different stage of development. Blueberries, carrots, and tomatoes were treated using a scaled-down, closed-chambered gaseous treatment system. for gaseous produce treatment. The treatment system itself consisted of standard-sized (5-gallon) plastic buckets containing an internal fan for circulation and a port to allow for gas ventilation and sampling. Current treatment capacity is at 1.8-2.0 kg (depending on food model) of produce aliquoted between nine clam-shells. The clam-shells are divided into three layers with three shells on each layer. Chlorine dioxide is generated in-chamber using a two-part dry media system (ICA TriNova Inc.; Atlanta, Georgia) consisting of sodium chlorite reacting with iron (III) chloride. Concentration of generated ClO2 was determined using a titration based method provided by the manufacturer. The University of Delaware is currently evaluating the effect of pulsed light (PL) treatment in combination with hydrogen peroxide on the inactivation of Salmonella. Peptone water (0.1%) was inoculated with a 4-strain cocktail of Salmonella to a final population of ~7.8 log. Hydrogen peroxide (30%) was added to the inoculated peptone water to achieve concentrations of 0.5%, 1%, and 2%. The peptone water solutions without hydrogen peroxide and with 0.5%, 1%, and 2% of hydrogen peroxide were treated with low and high intensity of PL for 15 s. Without PL treatment, 0.5% - 2% hydrogen peroxide reduced Salmonella by 0.1-0.3 log. PL alone (without hydrogen peroxide) reduced Salmonella by 5.0 log and 6.6 log for low PL and high PL intensities, respectively. PL treatment in combination with 0.5% - 2% hydrogen peroxide reduced Salmonella by 5.5-5.8 log and 6.8-7.1 log for low PL and high PL intensities, respectively. The results demonstrated that PL treatment alone was very effective in inactivating Salmonella in 0.1% peptone water. Addition of hydrogen peroxide to the peptone water only slightly increased the effectiveness of PL treatment. Regarding the testing of protozoa, mammalian cell lines are growing for the assessment of Cryptosporidium parvum. The team is re-evaluating current protocols involving combination of cell culture infectivity and quantitative PCR using C. parvum a oocysts purchased from Waterborne that were propagated in calves and mice. Genetic similarities between Eimera spp. and Cyclospora cayetanensis are also being explored for inclusion in testing on protozoa when team members identify appropriate parameters. The USDA ERRC has established a prototype design that will incorporate two cold plasma jets, each operated via an independent power supply. This will allow for enhanced simultaneous treatment either before or after exposure to the MLB array, or sequential treatment of samples with the MLB array before, between, or after. The precise arrangement of the different treatments will be optimized to take advantage of synergy, with sublethal injury from one treatment being targeted as a way to enhance kill level from subsequent treatment(s). The inoculated food commodities will be processed via conveyor belt. A prototype conveyor system is in place, with a variable stepper motor to control speed and, therefore, time of exposure. Critical variables of treatment time, time between sequential treatments, distance from cold plasma emitter heads and injection of temperature control gases will all be optimized with the adjustable mechanical positioning armatures for the cold plasma and MLB systems. The Ohio State Univeristy has developed a gnotobiotic pig model to determine the survival of human NoV upon treatment by food processing technologies. The pathogenesis of two human NoV GII.4 strains 7I and 5M was determined in newborn gnotobiotic piglets. Gnotobiotic piglets inoculated with 7 log genomic RNA copies of human NoV GII.4 strain 7I or 5M developed mild to moderate diarrhea at day 3 post-inoculation. A high level of viral RNA shedding was detected in feces at days 2-7 post-inoculation. At day 7 post-inoculation, all piglets were terminated and intestinal tissues were collected for the examination of histopathological changes. It was found that moderate to severe histologic lesions including villous atrophy, necrosis and apoptosis was observed in duodenum and jejunum. In addition, a large number of norovirus antigen-containing cells were detected in duodenum and jejunum tissues using an immunofluorescent assay. Collectively, piglets infected by human NoV developed diarrhea, viral RNA shedding in feces, pathological changes, and human NoV antigen expression in small intestine. Thus, gnotobiotic piglets are susceptible for human NoV infection. Next, Ohio State tested the survival of a human NoV genogroup II, genotype 4 (GII.4) strain in seeded oyster homogenates after high-pressure processing (HPP) using the gnotobiotic pig model. Pressure conditions of 350 MPa at 0°C for 2 min led to a 3.7-log10 reduction in the number of viral RNA copies in oysters, as measured by the porcine gastric mucin-conjugated magnetic bead (PGM-MB) binding assay and real-time RT-PCR, whereas pressure conditions of 350 MPa at 35°C for 2 min achieved only a 1-log10 reduction in the number of RNA copies. Newborn gnotobiotic piglets orally fed oyster homogenate treated at 350 MPa and 0°C for 2 min did not have viral RNA shedding in feces, histologic lesions, or viral replication in the small intestine. In contrast, gnotobiotic piglets fed oysters treated at 350 MPa and 35°C for 2 min had high levels of viral shedding in feces and exhibited significant histologic lesions and viral replication in the small intestine. Collectively, these data demonstrate that human NoV survival estimated by an in vitro PGM-MB virus binding assay is consistent with the infectivity determined by an in vivo gnotobiotic piglet model; and gnotobiotic piglet is a good model to estimate the survival of human NoV survival in vivo.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Fangfei Lou, Mu Ye, Yuanmei Ma, Xinhui Li, Erin DiCaprio, Haiqiang Chen, Steven Krakowka, John Hughes, David Kingsley, and Jianrong Li*. A gnotobiotic pig model for determining human norovirus inactivation by high-pressure processing. Applied Environmental Microbiology 2015, 81(19):6679-6687.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Fangfei Lou, Hudaa Neetoo, Haiqiang Chen, Jianrong Li*. High hydrostatic pressure processing: a promising nonthermal technology to inactivate viruses in high-risk foods. Annual Review of Food Science and Technology, 2015, 6:389-409.
• Type: Conference Papers and Presentations Status: Submitted Year Published: 2016 Citation: Rane, B., Bridges, D. F., Wu, V.C.H. Development of an optimal dry inoculation method of for low-moisture foods" (ID number 14733), for consideration to present at IFT16 in Chicago, IL, July 16-19, 2016