Source: UNIVERSITY OF DELAWARE submitted to
INACTIVATION OF ENTERIC FOODBORNE VIRUSES IN HIGH RISK FOODS BY NON-THERMAL PROCESSING TECHNOLOGIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0223954
Grant No.
2011-68003-30005
Project No.
DEL00689
Proposal No.
2014-10160
Multistate No.
(N/A)
Program Code
A4131
Project Start Date
Feb 1, 2011
Project End Date
Jun 30, 2017
Grant Year
2015
Project Director
Chen, H.
Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717
Performing Department
Animal And Food Sciences
Non Technical Summary
Viruses account for more than 67% foodborne illnesses worldwide. Commonly implicated foods include raw or under-cooked shellfish, produce and produce products such as berries, green onions, and salsa. None of the decontamination methods investigated thus far have been shown to effectively control foodborne viruses in high risk foods. Therefore, there is an urgent need to develop novel processing technologies to inactivate viruses. The overall goal of this project is to identify effective processing technologies and to optimize processing parameters to destroy foodborne viruses in high risk foods and disseminate the knowledge through education and outreach. Through this project, we expect to 1) develop effective processing technologies to control foodborne viruses in high risk foods; 2) understand the mechanism of viral inactivation by these processing technologies; 3) develop two courses and disseminate course contents to institutions across the United States; and 4) provide outreach education and training to industry and educators regarding foodborne viruses and food safety impacts of these processing technologies on high risk foods. Through integration of research, education and extension, our study will enhance the safety of high risk foods and reduce foodborne viral illnesses.
Animal Health Component
(N/A)
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5011129110010%
5011129110130%
5010811110010%
5010811110120%
5011451110010%
5011451110120%
Goals / Objectives
This study will focus its efforts on the most significant foodborne enteric viruses, human norovirus, although hepatitis A virus and rotavirus will also be studied. The overall goal is to identify effective non-thermal processing technologies and to optimize processing parameters to destroy these viruses in high risk foods, shellfish (oysters and clams), fresh and frozen berries (strawberries, raspberries and blueberries), berry purees, green onions, and salsa. In addition, the efficacy of these non-thermal processing technologies will be tested on pathogenic bacteria that cause large outbreaks in these high risk foods (Vibrio parahaemolyticus in shellfish and Escherichia coli O157:H7 and Salmonella in other products). The effect of the processing technologies on the quality of high risk foods will be evaluated. The knowledge gained will be disseminated through education curricula and outreach programs.
Project Methods
We will first determine whether an in vitro cultivable enteric calicivirus, the Tulane virus, can be used as a surrogate for human norovirus and develop a culture system to assess the survival of human norovirus. Four non-thermal technologies: 1) high pressure, 2) irradiation, 3) UV, and 4) washing with sanitizers, will be studied on their inactivation of the viruses in high risk foods. Effective technologies for specific foods will be identified and processing parameters for virus inactivation will be established. Mathematical models will be used to predict the inactivation of the viruses. The mechanism of viral inactivation by these four processing technologies will be studied. We will investigate whether viral capsid proteins, viral genome and receptor binding activity are damaged or denatured by the processing technologies. We will also examine the damage of the virion by electron microscope. In addition, the efficacy of these technologies will be tested on the pathogenic bacteria that are related to these high risk foods. The effect of these processing technologies on quality attributes of the high risk foods will be evaluated. Sensory quality, important nutrients in the high risk foods, color, texture, and other important properties will be evaluated. We will develop two courses ("Non-Thermal Food Processing Technologies" and "Foodborne Viruses and Food Safety") and train students in non-thermal processing technologies, food virology, and food safety. Outreach education and training to industry and educators regarding foodborne viruses and food safety impacts of non-thermal technologies on high risk foods will be provided. A variety of approaches to develop and implement the best outreach strategies will include 1) assessment of industry and educators attitudes and knowledge about non-thermal processing and its applications; 2) development of training courses, e.g. webinars and on-site presentations; 3) development of appropriate brochures, pamphlets, or other outreach alternatives as directed by the assessment and 4) evaluation of outreach strategies. The educational resources developed for the target audiences during the program will reflect the results of the research and assessment efforts of the project. Where possible, evaluations will follow Kirkpatrick's four level models for evaluating training.

Progress 02/01/11 to 06/30/17

Outputs
Target Audience:The target audiences are produce and shellfish industries, farmers, food safety and extension educators, food safety manager and regulators, graduate students and undergraduate students in food science and food safety. To reach these target audiences, various approaches have been used. 1. Two videos were developed and posted on the YouTube website in December 2015. The video entitled "Non Thermal or Alternative Processing of Produce and Shellfish: Safe to Eat Without Heat" currently has a total view of 1,744 and the video entitled "Exploring Specific Non Thermal Processes to Enhance the Safety of Produce and Shellfish" a total view of 1,018. Hiperbaric Company, a major manufacturer of high pressure units, adopted the use of the first video for their instructional use. 2. Apamphlet entitled "Non-Thermal or Alternative Food Processing Methods to Enhance Microbial Safety and Quality: Frequently Asked Questions" was developed in November, 2014 and distributed via multiple listserves provided by industry partners, FDA and USDA (food safety extension specialists) and FNSPEC (food and nutrition educators) from Purdue. The brochure is posted on the food safety website of University of California - Davis. 3. A paper entitled "Knowledge and attitudes of produce and seafood processors and food safety educators regarding non-thermal processing" was published in a nationally recognized peer-reviewed journal, J Fd. Sci. Ed., that is for food safety educators and other key demographic groups interested in the information presented. The publication presented the results of the survey and the development, implementation and evaluation of the videos by processors, educators and regulatory personnel. Evaluations clearly showed the applicability of the videos to consumer, educator and food processor audiences. 4. Power Point Presentations (a total of 610 participants). Integrated the final research results related to non-thermal processing of produce and shellfish with general information about non-thermal processing. 5. Seven Seafood HACCP Trainings (165 students) in Oregon between 2012 and 2015 were provided. In addition, co-PD Dr. Jianrong Li has been partnered with Ohio Association for Food Protection, Midwest Food Safety conference, and Ohio Fresh Produce Grower Association for extension and outreach activities. The team also gave extension seminar annually for two major food safety companies (Wendy Inc., Kroger Inc.) to improve fresh produce safety. 6. The results generated from this project have also been presented and disseminated to a number of professional meetings including International Association of Food Protection, Institute of Food Technologists, American Society for Virology, International Conference for Chemical Engineering, International Calicivirus Conference, Midwest Food Safety conference, Ohio Food Protection, Ohio Fresh Produce Grower Association, the Center for Advanced Processing and Packaging Studies (CAPPS), and Kroger food safety meeting. We also organized two symposiums on non-thermal food processing technologies and human norovirus at the 2015 IFT Annual Meeting. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate and undergraduate students have been directly participated in the research activities proposed in this project. The project team has been working very closely, particularly among the University of Delaware (Drs. Chen and Wu), Ohio State University (Dr. Jianrong Li), Delaware State University (Dr. Ozbay) and USDA ARS (Drs. Niemira and Gurtler). Because of this close collaboration, this project provides a unique and ideal opportunity for students to experience a multidisciplinary effort. Training within and between groups has been provided to the professionals, postdocs and graduate students working on this project. The graduate students and postdocs working on this project have become experts in the areas of food safety, non-thermal processing technologies and virology. For example, Dr. Xinhui Li, a former postdoc working with Dr. Chen, Dr. Wenqing Xu, a former Ph.D. student working with Dr. Wu, and Dr. Erin DiCaprio, a former Ph.D. student working with Dr. Li, obtained faculty positions at US universities. Dr. Alison Lacombe, a former postdoc working on this project, obtained a research faculty position at the National College of Natural Medicine. Many graduate students graduated and obtain jobs in the food industry. How have the results been disseminated to communities of interest?To assess the needs, we developed a survey to assess the industry' and educator's knowledge and attitudes about non-thermal processing and its applications. Results demonstrated the need for science-based information regarding non-thermal processing for shellfish and produce to both processors and educators. As delineated in other reports, the on-line needs assessment survey of industry (shellfish/produce) and food safety educators was conducted to assess knowledge/attitudes of non-thermal processing and ascertain willingness to receive information and/or training. The statistical analyses of these surveys showed that all groups surveyed, (produce,/ shellfish (N=106), educators (N=52)) had low knowledge scores which were below proficiency(80%), 48%, 51% and 67% respectively. These findings indicated the need for outreach education to all three target audiences for accurate information regarding the application of non-thermal technologies. While the majority of the respondents were aware of non-thermal processes they were not knowledgeable indicating the need for education and outreach in non-thermal processing to both shellfish/produce industries and food safety educators. The results of this survey, both in information needed and training preferences desired, served as the foundation for the outputs developed by the researchers on this project. The needs assessments showed that workshops were ranked lower by both target audiences as to their training format preferences. While the respondents indicated an interest in receiving training in non-thermal processing, they both preferred PowerPoint, printed materials and web-based approaches. Therefore, based on the survey-based research, we developed a Pamphlet of Frequently Asked Questions, two animated-style videos, PowerPoint presentations, a highly targeted journal paper, and workshops. The results generated from this project have also been presented and disseminated to local food safety organizations such as Center for Food Innovation, Ohio Association for Food Protection, Ohio Fresh Produce Grower Association, and Center for Advanced Processing and Packaging Studies (CAPPS), as well as a number of professional meetings including Institute of Food Technologists, Midwest Food Safety conference, International Association for Food Protection, and International Calicivirus Conference. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1. To determine whether Tulane virus (TV) could be used as a surrogate for human norovirus (HuNoV) and to develop a culture system to assess the survival of HuNoV. Significant Findings 1: We identified and developed a practical assay to selectively quantify only infectious HuNoV in food samples. Without this assay, the decontamination efficacy of a processing method cannot be evaluated. Research on development of control measures for HuNoV had been greatly hindered since there was no cell culture system or assay to determine the number of infectious HuNoV particles in a food sample. Significant Findings 2: We have demonstrated that the use of HuNoV surrogates such as murine norovirus (MNV-1) and TV has its merits in inactivation studies. Using the identified assay for HuNoV and plaque assay for MNV-1 and TV, we demonstrated that MNV-1 and TV behaved similarly to the GII.4 and the GI.1 strains under different HPP treatment conditions. Both the surrogates (MNV-1 and TV) and HuNoV (GI.1 and GII.4) were more sensitive to HPP at lower treatment temperature, higher substrate pH, or in the presence of water. The order of pressure resistance for the virus strains we used in this study was GI.1 > MNV-1 > GII.4 > TV. These findings demonstrate that although surrogates are not perfect, their use in inactivation studies has certain merits. It is often difficult to obtain large quantity of HuNoV for research since it is not culturable. In addition, the results obtained from this study on the pH effect, presence of water and low temperature effect would be very helpful when designing effective HPP processing to inactivate HuNoV, not only for berries and oysters, but also for other foods. Significant Findings 3: Considering the different behavior of HuNoV strains towards HPP treatments (GII.4 was more sensitive to pressure than GI.1), it is important to select relevant strain for target food product when evaluating a technology. To be conservative, selecting the most HPP-resistant strain might be a better alternative. Significant Findings 4: Elucidated that TV recognizes A-type 3 antigen clarifying why TV can bind some, but not all, saliva samples from A-positive individuals. B antigen binding was also confirmed. In addition, we determined that TV is capable of binding to sialic acids, similar to HuNoVs. Surprisingly, TV infectivity in cell culture was also promoted by the Maackia amurensis leukoagglutinin, suggesting that virus-host interactions may involve two host carbohydrate receptors or co-receptors for productive infection. These findings further highlight TV as a valuable surrogate for HuNoVs. Objective 2. To determine the effect of non-thermal processing technologies on inactivation of enteric foodborne viruses and pathogenic bacteria and on food quality Significant Findings 1: We developed a novel approach to enhance high pressure process (HPP) inactivation of HuNoV in shellfish and berries and identified optimum processing conditions. We not only provide a practical HPP technology for the food industry, but also provide regulatory guidelines for the FDA. We demonstrated that in the temperature range of 0 - 35°C the lower the initial sample temperature, the better the HPP inactivation of HuNoV. Since the capital costs of HPP equipment increase exponentially with the operating pressure level and the processing and maintenance costs are also positively correlated to it, it is economically beneficial to use lower pressure levels to achieve the target pathogen reduction. In our study, we came up with a practical idea of mixing ice with water to maintain the pressure chamber temperature at 0°C prior to HPP. We demonstrated that HPP at 350 and 500 MPa at 0°C could achieve more than 4 log reduction of GII.4 and GI.1 HuNoV in oysters. Sensory evaluation showed that pressure-treated oysters received significantly higher quality scores than the untreated control. Commercially, HPP at pressures at 300 MPa or below applied at room temperature is used by the shellfish industry to facilitate oyster shucking, extend shelf life and reduce Vibrio spp. However, these HPP treatment conditions are unlikely to cause substantial GI.1 and GII.4 HuNoV inactivation in oysters as demonstrated in this study. The use of higher pressure levels in combination with refrigeration temperature is needed. In addition, we demonstrated that HPP could be used to inactivate HuNoV in strawberry, blueberry, and raspberry purees without affecting their sensory qualities. Significant Findings 2: We demonstrated that post-HPP frozen storage could substantially enhance pathogenic bacteria inactivation. By utilizing this strategy of post-HPP frozen storage, the HPP capital and operating costs would be considerably reduced and quality of HPP-treated food would improve. Significant Findings 3: We developed a novel water-assisted pulsed light (PL) system to decontaminate fresh produce - a more effective, environmentally friendly, and non-chemical alternative to chlorine washing for fresh produce. Significant Findings 4: We developed a UV appliance that can potentially be use to decontaminate fresh produce at home, restaurants, cafeterias and commercial kitchens. To decontaminate fresh produce before consumption, a user will place the fresh produce and water into a tray inside the UV appliance, close the door and then hit the "Start" button. The UV lamps placed on the top of the tray will be turned on and a mechanical device inside the tray will agitate water. The system will be automatically turned off after the desired decontamination time is reached. Significant Findings 5: We demonstrated that cold plasma was an effective intervention against HuNoV and was the basis for tech transfer to FSIS, FDA and other regulatory agencies for rule-making regarding cold plasma. Significant Findings 6: We demonstrated variations in pressure resistance among V. parahaemolyticus strains and identified a high pressure processing treatment of 250 MPa for 5 min at 5°C can deliver >6.0 log reductions of clinical strains of V. parahaemolyticus in oyster homogenates. In addition, we revealed that inactivation of V. parahaemolyticus cells by HPP is due to cell damage and changes of cellular protein profiles. Significant Findings 7: We demonstrated that the combination of a surfactant with a commonly used sanitizer enhanced the efficiency in removing viruses from fresh produce by approximately 100 times. Implementation of this novel sanitization strategy would be a feasible approach for efficient reduction of the virus load in fresh produce. Significant Findings 8: Our results showed that gaseous ozone was able to inactivate HuNoV on fresh produce, demonstrating that it is an effective means to improve fresh produce safety. Objective 3. To determine the mechanism of viral inactivation by the non-thermal processing technologies. We have elucidate the mechanisms of viral inactivation by non-thermal processing technologies. Mechanism of viral inactivation by HPP includes: disrupt the integrity of viral capsid and damage the receptor binding activity of viral capsid. Mechanism of viral inactivation by gamma irradiation and E-beam include: physically degradation of viral capsid protein and genetic materials. Mechanism of viral inactivation by ozone includes: damage the viral capsid and degradation of viral genomic RNA. Mechanism of viral inactivation by surfactants includes damage viral capsid and alter the shape of virus particles.

Publications

  • 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.


Progress 02/01/16 to 01/31/17

Outputs
Target Audience:The target audiences are produce and shellfish industries, farmers, food safety and extension educators, food safety manager and regulators, graduate students and undergraduate students in food science and food safety. 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. The PD, co-PDs, and members working on this CAP project had an opportunity to present their work at conferences such as those organized by Institute of Food Technologists, International Association for Food Protection, and International Calicivirus Conference. These conferences have provided great opportunities for graduate students and postdoctoral fellows to improve their presentation skills. Below are some examples of training between groups and professional development for Year 6 of this project. The postdoctoral research associate at ARS had opportunity to teach students at Drexel University regarding her USDA virology research and disseminate information to graduate and undergraduate students as well as faculty. The postdoctoral associate was also able to mentor two undergraduate students in laboratory research on the grant-funded project. Dr. Wu's graduate student learned techniques related to tissue culture and virus propagation from Dr. Chen's laboratory. Dr. Chen's graduate student learned from Dr. Wu's research group the methodologies related with fruit quality analyses such as tests for total anthocyanin content. An undergraduate summer education scholar received trainings from postdoctoral researcher Mingming Guo on HPLC determination of anthocyanin compounds in raspberries in 2016. A new M.S. student joined Dr. Wu's research group in September 2016 and received trainings from a postdoctoral researcher on HPLC determination of anthocyanin compounds and use of pulsed light processing. How have the results been disseminated to communities of interest?Research results have been published in various peer-reviewed science journals and data has been disseminated through presentations at numerous conferences and meetings. The results have also been disseminated to local food safety organizations such as Center for Food Innovation, Ohio Association for Food Protection, Ohio Fresh Produce Grower Association, and Center for Advanced Processing and Packaging Studies (CAPPS). What do you plan to do during the next reporting period to accomplish the goals? To continue to evaluate the scaled up pulsed light and UV systems Verification of low-temperature high pressure processing to inactivate V. parahaemolyticus in whole oysters for largescale application.

Impacts
What was accomplished under these goals? Objective 1. To determine whether Tulane virus (TV) can be used as a surrogate for human norovirus (HuNoV) and to develop a culture system to assess the survival of HuNoV. Elucidated that TV recognizes A-type 3 antigen clarifying why TV can bind some, but not all, saliva samples from A-positive individuals. B antigen binding was also confirmed. Impacts future studies using TV as a model for HuNoVs with confirmation of its binding profile. In addition, determined that TV is capable of binding sialic acids, similar to HuNoVs. Surprisingly, TV infectivity in cell culture was also promoted by the Maackia amurensis leukoagglutinin, suggesting that virus-host interactions may involve two host carbohydrate receptors or co-receptors for productive infection. These findings further highlight TV as a valuable surrogate for HuNoVs. Objective 2. To determine the effect of the non-thermal processing technologies on inactivation of enteric foodborne viruses. Sensitivities of genogroup II HuNoV to high-pressure-processing Genogroup II (GII) contains the majority of HuNoVs, with GII genotype 4 (GII.4) being the most prevalent. Recently, GII.1 and GII.6 have emerged and caused many outbreaks worldwide. However, the survival of these GII HuNoVs is poorly understood because they are uncultivable in vitro. Using a novel receptor-binding assay conjugated with real-time RT-PCR, we found that GII HuNoVs had variable susceptibilities to high-pressure processing (HPP), which is one of the most promising food-processing technologies. The resistance of HuNoV strains to HPP ranked as follows: GII.1 > GII.6 > GII.4. This study highlights the ability of HPP to inactivate HuNoV and the need to optimize processing conditions based on HuNoV strain variability and sample matrix. High pressure processing on improving calm quality and safety Using high pressure processing at 350MPa and 21°C can inactivate human norovirus surrogates and microbial spoilage while maintaining major sensory parameters for clam samples. Inactivation of human norovirus (HuNoV) and Tulane virus (TV) by ionizing radiation Electron beam (E-beam) and gamma radiation were evaluated for efficacy against a HuNoV GII.4 strain and TV. Virus survival following ionizing radiation treatments was determined using direct quantitative reverse transcriptase PCR (RT-qPCR), the porcine gastric mucin magnetic bead (PGM-MB) binding assay followed by RT-qPCR, and plaque assay. Both HuNoV and TV were more susceptible to gamma irradiation than E-beam, requiring 22.4kGy to achieve complete inactivation. In whole strawberries, no HuNoV or TV RNA was detected following 28.7kGy of E-beam treatment using the PGM-MB binding assay. Overall, HuNoV and TV are highly resistant to ionizing radiation. In addition, the PGM-MB binding assay is an improved method to detect viral infectivity compared to direct RT-qPCR. Objective 3. To determine the efficacy of the non-thermal processing technologies on inactivating pathogenic bacteria Pulsed light (PL) inactivation of pathogens on strawberries In purpose of strawberry shelf-life extension, PL treatments of low intensity 3J/cm^2 total dose (L3), medium intensity 3J/cm^2 total dose (M3) and medium intensity 5J/cm^2 total dose (M5) were applied to strawberry samples before cold storage. All three PL treatments delayed visible fungal development and overall spoilage, better preserved strawberry surface glossiness and retarded anthocyanin accumulation during storage. Among the three treatments, M3 showed better shelf-life extension effects, although L3 and M5 achieved better decontamination effects and slightly enhanced surface redness right after treatment. No noticeable difference in firmness, pH, titratable acidity and total soluble solid was significantly induced. However, although not obviously reflected in final shelf-life, a tendency of accelerated quality degradation was discerned in the samples treated with higher total dose PL, suggesting cautious use of high total dose treatments in future applications. Pulsed light (PL) inactivation of pathogens on blueberries Our study investigated the effect of PL on the shelf life of blueberries and inactivation of Salmonella inoculated on blueberries. Dry PL (6 J/cm2) and water-assisted PL (samples were agitated in water during PL treatment; 9 J/cm2) were applied to blueberries with subsequent storages at room temperature (3 days) or 5 °C (7 days). Neither dry nor water-assisted PL treatments improved the shelf life of blueberries even though direct inactivation of natural yeasts and molds were achieved. Surface lightness was instantly reduced after both dry and water-assisted PL treatments. Reduced firmness was detected in water-assisted PL treated samples after 7 days of storage at 5 °C, while weight loss increased for dry PL treated samples with both storage conditions. Delayed anthocyanins accumulation and reduced total antioxidant activity were induced by both PL treatments at the end of storage at room temperature, while slight enhancement in total phenolics content was achieved by water-assisted PL treatment. For inactivation of Salmonella, dry PL treatment achieved 0.9 and 0.7 log reduction of Salmonella for spot and dip inoculation, respectively. Water-assisted PL treatment reduced Salmonella by 4.4 log for both inoculation methods and no Salmonella was detected after 7 days of storage at 5 °C. In all, the selected PL treatments, especially the water-assisted PL treatment, may help reducing total yeasts and molds count as well as improving safety and shelf life of blueberries. PL treatment parameters and storage conditions should be further investigated before possible application of PL in blueberry industries. Pulsed light (PL) on anthocyanins of raspberries during storage Compared with untreated control, raspberry extracts treated with PL with fluence of 5.0 J/cm2 or PL with fluence of 14.3 J/cm2 had better protection of one anthocyanin compound, cyanidin-3-glucoside during 10 day of refrigerated storage. Compared with untreated control fruits, raspberry fruits treated with PL with fluence of 5.0 J/cm2 or PL with fluence of 14.3 J/cm2 had retained more of one anthocyanin compound, cyanidin-3-glucoside, during 10 day of refrigerated storage. PL processing with fluence of 14.3 J/cm2 or 5.0 J/cm2 had no impacts on the weight loss of raspberry fruits stored in clamshell packaging during 10 day of refrigerated storage. Objective 4. Outreach component National Needs Assessment Survey of Shellfish and Fruit/Vegetable Producers and Educators was completed and documented the need for science-based information regarding non-thermal processing for shellfish and produce to both processors and educators. As delineated in other reports, the on-line needs assessment survey of industry (shellfish/produce) and food safety educators was conducted to assess knowledge/attitudes of non-thermal processing and ascertain willingness to receive information and/or training. The statistical analyses of these surveys showed that all groups surveyed, (produce/shellfish (N=106), educators (N=52)) had low knowledge scores which were below proficiency (80%), 48%, 51% and 67% respectively. These findings indicated the need for outreach education to all three target audiences for accurate information regarding the application of non-thermal technologies. While the majority of the respondents were aware of non-thermal processes they were not knowledgeable indicating the need for education and outreach in non-thermal processing to both shellfish/produce industries and food safety educators. The results of this survey, both in information needed and training preferences desired, served as the foundation for the outputs developed by the researchers on this project.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Li, X., Huang, R., and Chen. 2017. Evaluation of assays to quantify infectious human norovirus for heat and high pressure inactivation studies using Tulane virus. Food and Environmental Virology. doi: 10.1007/s12560-017-9288-2.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Sido, R.F., Huang, R., Liu, C., and Chen, H. 2017. High hydrostatic pressure inactivation of murine norovirus and human noroviruses on green onions and in salsa. Int. J. Food Microbiol. 242: 1-6.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Lacombe, A., Niemira, B.A., Gurtler, J.B., Sites, J., Boyd, G., Kingsley, D., Li, X., and Chen, H. 2017. Nonthermal Inactivation of Norovirus Surrogates on Blueberries using Atmospheric Cold Plasma. Food Microbiol. 63: 1-5.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Huang, Y., Cao, X., and Chen. H. 2017. Pulsed light inactivation of murine norovirus, Tulane virus, Escherichia coli O157:H7 and Salmonella in suspension and on berry surfaces. Food Microbiol. 61: 1-4.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Lou, F., DiCaprio, E., Li, X., Dai, X, Ma, Y., Hughes, J., Chen, H., Kingsley, D., Li, J. 2016. Variable high pressure processing sensitivities for GII human noroviruses. Appl. Environ. Microbiol. 82: 6037-45.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Pivarnik, LF, Richard, NL, Gable, RK and Worobo, RW. 2016. Knowledge and attitudes of produce and seafood processors and food safety educators regarding non-thermal processing. J Fd. Sci. Ed. 15:120-128. doi:10.1111/1541-4329.12093
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Xu, W., Chen, H., and Wu. C. 2016. Salmonella and Escherichia coli O157:H7 inactivation, color and bioactive compounds enhancement on raspberries during frozen storage after decontamination using new formula sanitizer washing or pulsed light. J. Food Prot. 79: 1107-1114.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: DiCaprio, E. and Li, J. 2016. Inactivation of human norovirus and Tulane virus in simple media and fresh whole strawberries by ionizing radiation. International Journal of Food Microbiology. 232:43-51.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: DiCaprio, E. Lou, F., and Li, J. 2016. Strategies for control of food- and water-borne viruses. Viral Foodborne Pathogens. Boca Raton: CRC Press, 2016.


Progress 02/01/15 to 01/31/16

Outputs
Target Audience:The target audiences are produce and shellfish industries, and food safety and extension educators. Two videos on non-thermal processing technologies have been developed and posted on YouTube, https://www.youtube.com/watch?v=0n0Rom8nBFw, https://www.youtube.com/watch?v=KJd_LJR2ypk. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Within each PD's group, senior people have always helped to train junior people. Our project team has been working very closely. Training between groups has been provided to the professionals, postdocs and graduate students working on this project. Below are some examples of training between groups and professional development for Year 5 of this project. Dr. Wu's graduate student learned techniques related to tissue culture and virus propagation from Dr. Chen's laboratory. Dr. Su's graduate student learned to operate high pressure processing for inactivating Vibrio parahaemolyticus cells. Graduate students were provided funding to go to IFT and IAFP Annual Meetings. Dr. Li's post-doc (Erin DiCaprio) gave several extension seminars for local companies in Ohio including Ohio Association for Food Protection, Wendy Inc., Kroger Inc. Dr. Li's post-doc (Erin DiCaprio) went to Kent State University to learn E-beam technology. Dr. Li's post-doc (Erin DiCaprio) went to OSU Irradiation Facility to learn gamma irradiation technology. Dr. Li's undergraduate student (Douglas Culbertson) went to OSU Irradiation Facility to learn gamma irradiation technology. Dr. Li's undergraduate student (Anastasia Purgianto) learned ozone technologies. How have the results been disseminated to communities of interest?New Mexico State University was successfully hired to collaborate on the two videos each 4+ minutes in length - this included script development, animation development and review by core research team of the project. The two videos are "Non-Thermal or Alternative Processing of Produce and Shellfish: Safe to Eat without Heat" and "Exploring Specific Non-thermal Processes to Enhance the Safety of Produce and Shellfish". Both videos are posted on YouTube. Evaluation template and protocol has been developed. Requests for evaluation implemented through a variety of audiences: e.g. Food safety Educators, Nutrition Educators, Produce Industry, Shellfish Industry, and Regulatory agencies. Evaluation has just begun during this reporting period. Two Seafood HACCP Training Courses were taught in Portland of Oregon in March and September 2015. A total of 65 students from seafood industries and state regulatory agencies completed the courses and received certificates from the Association of Food and Drug Officials. The results generated from this project have been presented and disseminated to a number of professional meetings including International Association of Food Protection, Institute of Food Technologists, Ohio Association for Food Protection, Wendy Inc., Kroger Inc., Midwest Food Safety conference, and Ohio Fresh Produce Grower Association, and Center for Advanced Processing and Packaging Studies (CAPPS). What do you plan to do during the next reporting period to accomplish the goals? To evaluate the scaled up pulsed light and UV systems for processing larger quantity of berries. To evaluate the decontamination effectiveness of combined technologies, UV/pulsed light with sanitizers such as chlorine and hydrogen peroxide. To determine pulsed light treatment on the shelf life or berries. The effect of UV on inactivation of hepatitis A virus on blueberries will be studied. The effect of high pressure processing on the inactivation of in green onions and salsa will be determined. Verification of low-temperature high pressure processing to inactivate V. parahaemolyticus in whole oysters for large-scale application. The effect of high pressure processing on the inactivation of viruses in clams will be determined. The impacts of effective HPP on the sensory and quality of clams during the storage will be evaluated.

Impacts
What was accomplished under these goals? Objective 1. To determine whether Tulane virus (TV) can be used as a surrogate for human norovirus (HuNoV) and to develop a culture system to assess the survival of HuNoV. TV as a surrogate for HuNoV TV appears to be a viable surrogate for HuNoV. It is sensitive to increased temperature and shows dose-dependent decreases in infectivity with exposure to free chlorine and UV-C. When exposed to extreme pH levels, TV appears more sensitive to acidic pH. Unlike murine norovirus (MNV), TV is more resistant to ethanol, similar to HuNoV, indicating that it should be used along with MNV as HuNoV surrogates. In addition, TV also recognizes sialic acids (SAs). Maackia amurensis leukoagglutinin (MAL), a lectin that recognizes the alpha-2,3 linked SAs, bound LLC-MK2 cells, as well as TV, by which MAL promoted TV infectivity in cell culture. Since HuNoVs also recognize sialic acids (SAs) in addition to HBGAs, our findings further highlight TV as a valuable surrogate for HuNoVs, particularly in studying virus-host interactions. Objective 2. To determine the effect of the non-thermal processing technologies on inactivation of enteric foodborne viruses. Effect of high pressure processing (HPP) on HuNoV inactivation and sensory characteristics of berries and berry purees Porcine gastric mucin (PGM)-conjugated magnetic beads (PGM-MBs) and RT-qPCR were utilized for infectious HuNoV discrimination and quantification. HuNoV GI.1 or GII.4 strains were inoculated into three types of berries and their purees and HPP treated. For the purees, the HPP condition needed to achieve > 2.9 log reduction of HuNoV GI.1 strain and > 4.0 log reduction of HuNoV GII.4 strain was found to be ≥ 550 MPa for 2 min at 0 °C. HuNoV GI.1 strain was more resistant to HPP treatment than HuNoV GII.4 strain. Color, pH and viscosity of blueberries and three berry purees showed no or slight changes after HPP treatment. Sensory evaluation demonstrated that HPP treatment of 550 MPa for 2 min at 0 °C did not significantly reduced the sensory qualities of three berry purees. The results demonstrated that the HPP treatment of 550 MPa for 2 min at 0 °C could be a potential nonthermal intervention for HuNoV in berry purees without adversely affecting their sensory qualities and physical properties. Pulsed light (PL) inactivation of pathogens on berries The PL inactivation of two HuNoV surrogates, MNV-1 and TV, and two common bacterial pathogens, E. coli O157:H7 and Salmonella, were evaluated. MNV-1 and TV were significantly (P < 0.05) more resistant to PL treatment than Salmonella and E. coli O157:H7 in PBS suspension. Lower inactivation of each microorganism was achieved on berry surfaces than in PBS suspension. This study shows that PL can induce rapid inactivation of MNV-1, TV, Salmonella and E. coli O157:H7 in clear suspension with viruses more resistant to PL treatment than bacteria. The efficacy of PL treatment is substantially influenced by food surface structure. Water-assisted PL inactivation of pathogens on berries The effects of combinations of water-assisted PL (WPL) treatment with 1% hydrogen peroxide (H2O2) or 100 ppm sodium dodecyl sulfate (SDS) were evaluated. Strawberries and raspberries inoculated with E. coli O157:H7 were treated by WPL for 5-60 s. Significantly higher reductions of E. coli O157:H7 were obtained using 60-s WPL treatment than washing with 10 ppm chlorine. The combination of WPL treatment with 1% H2O2 for 60 s showed significantly higher efficacy by reducing E. coli O157:H7 on strawberries and raspberries by 3.3- and 5.3-log units, respectively. Similarly, Salmonella on strawberries and raspberries was inactivated by 2.8- and 4.9-log units after 60-s WPL-H2O2 treatments. For decontamination of MNV-1, a 60-s WPL treatment reduced the viral titers on strawberries and raspberries by 1.8- and 3.6-log units, respectively. WPL-H2O2 treatment was the most effective treatment in our study for decontamination of bacterial pathogens on berries. Ozone inactivation of HuNoV surrogates on strawberries This study determined the effect of gaseous ozone for the inactivation of MNV-1 and TV from both liquid media and strawberries. Foods were treated with gaseous ozone at 6% wt/wt ozone for 0, 10, 20, 30, and 40min. Ozone inactivatednorovirusin both liquid media and fresh produce in a dose-dependent manner. Viruses are generally more resistant to sanitation treatments than bacteria, thus gaseous ozone is an effective means to improve fresh produce safety. Ionizing radiation inactivation of HuNoV and TV on strawberries Electron beam (E-beam) and gamma radiation were evaluated for efficacy against a HuNoV GII.4 strain and TV. Virus survival following ionizing radiation treatments was determined using direct quantitative reverse transcriptase PCR (RT-qPCR), the PGM-MB binding assay followed by RT-qPCR, and plaque assay. Both HuNoV and TV were more susceptible to gamma irradiation than E-beam. In whole strawberries, no human NoV or TV RNA was detected following 28.7 kGy of E-beam treatment using the PGM-MB binding assay. Overall, HuNoV and TV are highly resistant to ionizing radiation and therefore the technology may not be suitable to eliminate viruses in fresh produce at the currently approved levels. The PGM-MB binding assay is an improved method to detect viral infectivity compared to direct RT-qPCR. HPP inactivation of rotaviruses (RV) This study determined the baro-sensitivity of seven RV strains from four serotypes (G1: Wa, Ku, and K8, G2: S2, G3: SA-11 and YO, and G4:ST3). Ku, K8, S2, SA-11, YO, and ST3 showed enhanced inactivation at 4 °C compared to 20 °C. In contrast, Wa strain was not significantly impacted by the initial treatment temperature. The resistance of RV strains to HPP at 4 °C can be ranked as Wa>Ku=K8>S2>YO>ST3 and in terms of serotype G1>G2>G3>G4. HPP at 400 MPa for 2 min was sufficient to eliminate the Wa strain, the most pressure resistant RV, from oyster tissues. HPP disrupted virion structure, but did not degrade viral protein or RNA. Objective 3. To determine the efficacy of the non-thermal processing technologies on inactivating pathogenic bacteria PL decontamination of raspberries during frozen storage E. coli O157:H7 and Salmonella survivors in PL treated raspberries were significantly lower than the untreated ones and the treated raspberries maintained lower survival pathogen population during three months storage at -20 ?. At the end of 3 months of frozen storage, PL treated raspberries had significantly higher total phenolic content (TPC) and total anthocyanin content (TAC) compared with control samples. PL decreased the total bacterial count (TBC) and total yeast and mold counts (TYMC) on raspberries and maintained the low counts at the end of 3 month storage. At the end of 15 months of frozen storage, 15 s dry PL treated raspberries still had significantly higher TAC. Comparing with control (no treatment), 15 s PL significantly increased total anthocyanin content (TAC) of fresh raspberries during 10 d storage at 4 °C without negatively affecting the color and texture. HPP inactivation mechanism of Vibrio parahaemolyticus Inactivation of V. parahaemolyticus cells by HPP was found to be associated with cell membrane damage and changes of cellular protein profiles. Scanning electron microscopy images showed cell damage of V. parahaemolyticus after pressure treatments, especially at 300 MPa for 5 min. SDS-PAGE analysis of cellular proteins extracted from V. parahaemolyticus cells after pressure treatments revealed apparent changes of cellular protein profiles with protein fragments of 150 - 250 kDa being affected most. In addition, total protein and sulfhydryl contents in cellular protein extracts of V. parahaemolyticus cells decreased as the pressure treatment increased, which confirmed membrane damage of the bacterial cells after pressure treatments.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Lingham, T., Ye, M., Chen, H., Chintapenta, L.K., Handy, E., Zhao, J., Wu, C., Ozbay, G. 2016. Effects of high hydrostatic pressure on the physical, microbial, and chemical attributes of oysters (Crassostrea virginica). J. Food Sci. In press.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Huang, R., Ye, M., Li, X., Ji, L., Karwe, M. and Chen H. 2016. Evaluation of high hydrostatic pressure inactivation of human norovirus on strawberries, blueberries, raspberries and in their purees. Int. J. Food Microbiol. Accepted for publication.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Araud, E., DiCaprio, E., Yang, Z., Li, X., Lou, F., Hughes, J., Chen, H., Li. J., 2015. High-pressure inactivation of rotaviruses: the role of treatment temperature and strain diversity in virus inactivation. Appl. Environ. Microbiol. 81: 6669-6678.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Lou, F., Ye, M., Ma, Y., Li, X., DiCaprio, E., Chen, H., Krakowka, S., Hughes, J., Kingsley, D., Li, J. 2015. A gnotobiotic pig model to determine human norovirus inactivation by high pressure processing. Appl. Environ. Microbiol. 81: 6679-6687.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Huang, Y., Sido, R., and Chen, H. 2015. Application of water-assisted pulsed light treatment to decontaminate raspberries and blueberries from Salmonella. Int. J. Food Microbiol. 208:43-50.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Liu, C., Li, X., and Chen, H. Application of water-assisted ultraviolet light processing on the inactivation of murine norovirus on blueberries. Int. J. Food Microbiol. 214:18-23.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Liu, C., Huang, Y., and Chen, H. 2015. Inactivation of Escherichia coli O157:H7 and Salmonella enterica on blueberries in water using ultraviolet light. J. Food Sci. 80: M1532-1537.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ye, M., Lingham, T., Huang, Y., Ozbay, G., Ji, L., Karwe, M., and Chen, H. 2015. Effects of high-hydrostatic pressure on inactivation of human norovirus and physical and sensory characteristics of oysters. J. Food Sci. 80: M1330-1335.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Huang, Y. and Chen, H. 2015. Inactivation of Escherichia coli O157:H7, Salmonella and human norovirus surrogate on artificially contaminated strawberries and raspberries by water-assisted pulsed light treatment. Food Res. Int. 72: 1-7.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Tan, M., C. Wei, P. Huang, Q. Fan, C. Quigley, M. Xia, H. Fang, X. Zhang, W. Zhong, J. S. Klassen, and X. Jiang 2015. Tulane virus recognizes sialic acids as cellular receptors. Scientific reports. 5:11784.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Phuvasate S, Su Y-C. 2015. Alteration of structure and cellular protein profiles of Vibrio parahaemolyticus cells by high pressure treatment. Food Control 50:831-837.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: DiCaprio, E., Purgianto, A., Ma, Y., Hughes, J., Dai, X., Li, J. 2015. Attachment and localization of human norovirus and animal caliciviruses in fresh produce. Int. J. Food Microbiol. 211:101-108.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Predmore, A., Sanglay G., Li, J., Lee, K. 2015. Control of human norovirus surrogates in fresh foods by gaseous ozone and a proposed mechanism of inactivation. Food Microbiol. 50:118-25.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: DiCaprio, E., Phantkankum, N., Culbertson, D., Ma, Y., Hughes, J.H., Kingsley, D., Uribe, R.M., Li. J., 2016. Inactivation of human norovirus and Tulane virus in simple media and fresh whole strawberries by ionizing radiation. Int. J. Food Microbiol. In press.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Xu, W., Wu, C. 2016. The Impact of Pulsed Light on Decontamination, Quality, and Bacterial Attachment of Fresh Raspberries. Food Microbiol. In press.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Huang, Y., Ye, M., and Chen, H. Pulsed Light Inactivation of Murine Norovirus, Tulane Virus, Escherichia Coli O157:H7, and Salmonella in Suspension and on Berry Surfaces. Presented at the 2015 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Huang, R., Li, X., and Chen, H. High Pressure Inactivation of Human Norovirus GI.1 and GII.4 Strains on Strawberry, Blueberry, Raspberry, and in Purees. Presented at the 2015 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Liu, C., Li, X., and Chen, H. Application of Water-Assisted Ultraviolet Light Processing on the Inactivation of Murine Norovirus on Blueberries. Presented at the 2015 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Sido, R., Guo, X., and Chen, H. High Inactivation of Salmonella on Blueberries by a Novel Ultraviolet Light and Washing Process. Presented at the 2015 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ye, M. and Chen, H. Comparison of Porcine Gastric Mucin Binding and Infectivity Assays for Tulane Virus Treated With Pulsed Light, UV, and Sanitizers. Presented at the 2015 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Xu, W., Chen, H, and Wu, C. Application of Pulsed Light (PL) and Sanitizer Washing to Decontaminate Escherichia Coli O157:H7 and Salmonella and Preserve Quality of Frozen Raspberries. Presented at the 2015 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Xu, W., Chen, H., Wu, C. Attachment of E. coli O157:H7 on Produce and Damage of Bacterial Cell Envelope by Dry Pulsed Light (PL), Wet PL and PL-Surfactant Combinations. IAFP Program and Abstract Book Annual Meeting. Portland, OR, USA. July, 2015.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Worobo & Pivarnik, Non-thermal processing educational videos for the seafood and produce industries. December 10, 2015. Juice HACCP Training. Ithaca, NY.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Worobo & Pivarnik, Non-thermal processing educational videos for the seafood and produce industries. January 19, 2016. Global Food Safety Conference. Bangalore, India.


Progress 02/01/14 to 01/31/15

Outputs
Target Audience: The target audiences are produce and shellfish industries, and food safety and extension educators. A Frequently Asked Questions brochure was developed. It includes general information about non-thermal processes and a summary chart of research findings on produce and shellfish regarding pertinent pathogens (bacterial and viral) of interest in the grant. The brochure has been distributed to multiple audiences - food safety contacts, industry personnel, educators, regulators - through a variety of listserves. Changes/Problems: The needs assessments (educators and produce/shellfish processors) completed showed that workshops were ranked low by both target audiences as to their training format preferences. Based on the survey-based research, the outreach project directors decided to take another approach in an effort to maximize effectiveness, and that would fulfill the training preferences of both industry and educators. The major outputs will be two short videos: 1) general information about non-thermal processing technology and 2) focus on processes that the research has shown to be both effective and available and be dependent upon the research-based portion of this project. The scope of the outreach program and its ultimate goals has not changed - only the output needed to accomplish those goals. What opportunities for training and professional development has the project provided? Within each PD's group, senior people have always helped to train junior people. Our project team has been working very closely. Training between groups has been provided to the professionals, postdocs and graduate students working on this project. Below are some examples of training between groups and professional development for Year 4 of this project. Dr. Wu's graduate student learned techniques related to tissue culture and virus propagation from Dr. Chen's laboratory. The postdoc of Drs. Gurtler and Niemira learned techniques related to tissue culture and virus propagation from Dr. Chen's laboratory. In addition, training was conducted in molecular virology and real time PCR. The postdoc of Drs. Gurtler and Niemira was trained on novel nonthermal technologies for the inactivation of foodborne pathogens Dr. Su's graduate student learned to operate high pressure processing under refrigeration temperatures. Dr. Li's Ph.D. student (Erin DiCaprio) went to Kent State University to learn E-beam technology. Graduate students were provided funding to go to IFT and IAFP Annual Meetings. How have the results been disseminated to communities of interest? A Frequently Asked Questions brochure was developed and distributed to multiple audiences - food safety contacts, industry personnel, educators, regulators - through a variety of listserves. It is also posted on the URI website, http://web.uri.edu/foodsafety/files/2014-FAQ-Non-thermal-processing.pdf. Two Seafood HACCP Training Courses were taught in Portland of Oregon in July and November 2014. More than 40 students from seafood industries and state regulatory agencies completed the courses and received certificates from the Association of Food and Drug Officials. The results generated from this project have been presented and disseminated to a number of professional meetings including International Association of Food Protection, Institute of Food Technologists, American Society for Virology, International Conference for Chemical Engineering, Midwest Food Safety conference, Ohio Food Protection, Ohio Fresh Produce Grower Association, the Center for Advanced Processing and Packaging Studies (CAPPS), and Kroger food safety meeting. What do you plan to do during the next reporting period to accomplish the goals? Plan to study norovirus replication in human enteroids and porcine intestinal epithelial cells in attempt to establish a cell culture system for human norovirus (HuNoV). We have found that the porcine gastric mucin-conjugated magnetic beads binding assay is probably able to correctly quantify infectious HuNoV at < 2 log reduction levels for high pressure processing. This assay will be tested for heat, chlorine, and ethanol treatments. The effect of high pressure processing on the inactivation of viruses in clams will be determined. The impacts of effective HPP on the sensory and quality of clams during the storage will be evaluated. Conduct a comparison between kinetic inactivation rate of Tulane virus, murine norovirus, and HuNoV during cold plasma treatment. Investigate effects of pressure processes on changes of cell wall structure and cellular protein profiles of clinical strains of V. parahaemolyticus. The effect of high pressure processing parameters developed in this study on the sensory and quality of berries will be determined. The effects of pulsed light and UV on inactivation of hepatitis A virus on berries will be studied. To evaluate the scaled up pulsed light and UV systems for processing larger quantity of berries. To determine pulsed light treatment on the shelf life or berries. The effect of high pressure processing on the inactivation of HuNoV in green onions and salsa will be determined.

Impacts
What was accomplished under these goals? Objective 1. To determine whether Tulane virus (TV) can be used as a surrogate for human norovirus (HuNoV) and to develop a culture system to assess the survival of HuNoV. TV as a surrogate for HuNoV It appears that organic material in stool protects TV virions from heat induced damage at 72°C for longer periods of time, but only protects TV RNA from degradation by RNase after ethanol and free chlorine exposure. Study of norovirus replication in immune-deficient mice in an attempt to establish a mouse infection model NOD scid gamma (NSG) and NOD-scid IL2Rgnull-3/GM/SF (NSG-SGM3 or NSGS) mice were transplanted with human cord blood cells containing hematopoietic stem cells and followed by HuNoV challenge to study its replication in vivo. Viral RNA was not detected in stool at any time points. Viral antigen was not detected in any organs post infection by immunofluorescence staining. Among ip-infected mice, no viral RNA was detected in peripheral blood, organs or stools. These negative results indicated that the mouse model did not work. Evaluation of the porcine gastric mucin binding assay We compared high pressure processing (HPP) inactivation of murine norovirus (MNV-1) and TV using the porcine gastric mucin binding followed by PCR assay (referred to as PGM-MB/PCR assay) and plaque assay. The two pressure inactivation curves obtained using the plaque and PGM-MB/PCR assays were almost identical at ≤ ~2-log reduction levels. HPP treatments could achieve maximums of ~3 and 3.5 log reductions of GI.1 and GII.4 assessed by the PGM-MB/PCR assay, respectively. Based on these results, it can be reasonably concluded that the PGM-MB/PCR assay would be very likely able to estimate HPP inactivation of HuNoV at ≤ 2 log-reduction levels. It would also likely to conservatively quantify HPP inactivation of GI.1 strain at 2-3 log-reduction levels and GII.4 strain at 2-3.5 log-reduction levels. Verification of the pressure inactivation of HuNoV using a Gn pig model. We determined the survival of a human NoV GII.4 strain in seeded oyster homogenate after HPP using the PGM-MB/PCR assay and a gnotobiotic pig model. Pressure condition of 350 MPa at 0°C for 2 min led to a 3.7 log reduction in viral RNA copies in oysters measured by the PGM-MB/PCR assay whereas 350 MPa at 35°C for 2 min only achieved 1 log reduction in RNA copies. Newborn gnotobiotic piglets orally fed with oyster homogenate inoculated with the GII.4 strain and treated at 350 MPa at 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 pigs fed oysters treated by 350 MPa at 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 demonstrated that (i) HuNoV survival estimated by the in vitro PGM-MB/PCR assay was consistent with the infectivity determined by the in vivo gnotobiotic pig model; and (ii) HPP is capable of inactivating the GII.4 strain at commercially acceptable pressure levels. Objective 2. To determine the effect of the non-thermal processing technologies on inactivation of enteric foodborne viruses. Effects of HPP on HuNoV inactivation and sensory characteristics of oysters HuNoV was inoculated into oysters and treated at 300-600 MPa at 25 and 0°C for 2 min. After HHP, viral particles were extracted and quantified by the PGM-MB/PCR assay. Lower initial temperature (0°C) significantly enhanced HPP inactivation of HuNoV compared to ambient temperature (25°C). HPP at 350 and 500 MPa at 0°C could achieve more than 4 log reduction of GII.4 and GI.1 HuNoV in oysters, respectively. HHP treatments did not significantly change color or texture of oyster tissue. A 1-5 scale hedonic sensory evaluation showed that pressure-treated oysters received significantly higher quality scores than the untreated control. Inactivation of pathogens on berries by UV A novel set-up using water-assisted UV processing was developed and evaluated for its decontamination efficacy against MNV-1 inoculated on fresh blueberries. Results showed that water-assisted UV treatment generally showed higher efficacies than dry UV treatment. Water-assisted UV showed similar inactivating efficacy as a 10-ppm chlorine wash. Presence of 2% blueberry juice in wash water decreased the decontamination efficacy of water-assisted UV and chlorine washing treatments. Findings of this study suggest that water-assisted UV treatment could be used as an alternative to chlorine washing for blueberries and potentially for other fresh produce. Gamma and E-beam irradiation on inactivation of viruses Ionizing radiation was evaluated for efficacy against a HuNoV GII.4 strain, TV, hepatitis A virus (HAV) and rotavirus (RV). In whole strawberries, TV was completely inactivated by E-beam at 16.3 kGy. No human NoV or TV RNA was detected in strawberry following 28.7 kGy of E-beam treatment using the PGM-MB/PCR assay. A dose of 28 kGy was required for complete inactivation of both HAV and RV. Overall, all tested viruses were found to be highly resistant to ionizing radiation. Objective 3. To determine the efficacy of the non-thermal processing technologies on inactivating pathogenic bacteria Pressure inactivation of Vibrio parahaemolyticus in oyster Pressure inactivation of V. parahaemolyticus in oyster homogenates was greatly enhanced by lowering the processing temperature to 5°C or lower. A treatment of oyster homogenates at 250 MPa for 5 min at 5 or 1.5°C decreased the populations of five clinical strains of V. parahaemolyticus by 6.1 or > 6.5 log CFU/g, respectively. Pulsed light (PL) decontamination of raspberries Dry PL effectively inactivated Salmonella and Escherichia coli O157:H7 on raspberries. PL treated produce also maintained lower pathogen survival population during storage at 4? compared with the untreated control. We developed and evaluated a small scaled-up water-assisted PL (WPL) system, in which berries were washed in a flume washer while being treated by PL. The combination of WPL and 1% H2O2 (WPL-H2O2) was the most effective treatment in clear water. When high organic load and SiO2, as a soil simulator, were added in wash water, the free chlorine level in chlorinated water decreased significantly; however, no significant difference was observed for the decontamination efficacy of 1-min WPL-H2O2 treatment. Even in the presence of high organic load and water turbidity, no viable bacterial cells were recovered from the wash water, which showed that WPL-H2O2 could effectively prevent the risk of cross-contamination during treatment. Objective 4. To determine the mechanism of viral inactivation by the non-thermal processing technologies We first determined if E-beam treatment can damage virus particles using HuNoV virus-like particles (VLPs) as a model. Following 11.8 kGy of treatment, fewer intact VLP particles were observed and the VLPs appeared aggregated as shown by Transmission electron microscopy (TEM). Using SDS-PAGE analysis, the VLP protein detected decreased with E-beam treatment, however VP1 and cVP1 were still observed on the gel following 28.3 kGy of treatment. The PGM-MB binding assay showed that E-beam damaged the ability of VLPs bind to PGM-MB. Next, HuNoV GII.4 and TV were treated by E-beam and irradiation (14.7 kGy and 19.5 kGy). TEM revealed that increased irradiation doses negatively affected the structure of both viruses. Irradiation also degraded viral proteins and genomic RNA. This demonstrated that the inactivation mechanism includes damages to viral receptor binding activity, viral capsid, and viral genome RNA. Similarly, purified HAV and RV were also treated by lethal dose of E-beam and gamma irradiation. It was found that irradiation damaged virus particle, genome RNA and receptor binding activity in cell culture.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, X., Chen, H. 2015. Evaluation of the porcine gastric mucin binding assay for high pressure inactivation studies using murine norovirus and Tulane virus. Appl. Environ. Microbiol. 81: 515-521.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Xu, W., Chen, H., and Wu. C. 2015. Application of pulsed light (pl)-surfactant combination on inactivation of salmonella and apparent quality of green onions. LWT - Food Science and Technology. 61: 596-601.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Lacombe, A., Niemira, B.A., Gurtler, J.B., Fan, X., Sites, J., Boyd, G., and Chen, H. 2015. Atmospheric cold plasma inactivation of aerobic microorganisms on blueberries and effects on quality attributes. Food Microbiology. 46: 479-484.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Li, X. and Chen, H. Evaluation of porcine gastric mucin binding assay for quantification of infectious virus particles using murine norovirus and Tulane virus. Presented at the 2014 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Ye, M. and Chen, H. Inactivation of human norovirus in contaminated oysters and clams by high-hydrostatic pressure. Presented at the 2014 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lacombe, A. , Niemira, B.A., Gurtler, J.B., Sites, J., Boyd, G., Chen, H, and Fan, X. Effects of Nonthermal Atmospheric Cold Plasma on Blueberry Native Microbiota and Sensory Attributes. Presented at the 2014 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Lacombe, A. , Niemira, B.A., Gurtler, J.B., Sites, J., Boyd, G., Chen, H, and Li, X. Nonthermal Inactivation of the Norovirus Surrogate Tulane Virus on Blueberries using Atmospheric Cold Plasma. Presented at the 2014 IFT Annual Meeting.
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Yaoxin Huang, 2015. Inactivation of bacterial and viral pathogens in berry products by high pressure and pulsed light. Ph.D. dissertation.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Phuvasate S, Su Y-C. 2015. Efficacy of low-temperature high hydrostatic pressure processing in inactivating Vibrio parahaemolyticus in culture suspension and oyster homogenate. Int. J. Food Microbiol. 196:11-15.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhang D, Huang P, Zou L, Lowary TL, Tan M, Jiang X. 2015. Tulane virus recognizes the A type 3 and B histo-blood group antigens. J Virol 89:14191427.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Tan M, Jiang X. 2014. Histo-blood group antigens: a common niche for norovirus and rotavirus. Expert Rev Mol Med 2014;16:e5
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Erin DiCaprio, Fangfei Lou, Ashley Predmore, Jianrong Li. 2015. Strategies for control of food- and water-borne viruses". Foodborne Viral Pathogens. In press.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ashley Predmore, Gabriel Sanglay, Erin DiCaprio, Jianrong Li, Robert Uribe, Ken Lee. Electron beam inactivation of Tulane virus on fresh produce, and mechanism of inactivation of human norovirus surrogates by electron beam irradiation. International Journal of Food Microbiology. 2015 Apr 2;198:28-36. doi: 10.1016/j.ijfoodmicro.2014.12.024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Xu, W., Chen, H., Wu, C. Application of Pulsed Light (PL) to Decontaminate Escherichia coli O157:H7 and Salmonella and Preserve Quality on Raw Raspberries. IAFP Program and Abstract Book Annual Meeting. Indianapolis, IN, USA. August, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Xu, W., Chen, H., Wu, C. Application of Pulsed Light (PL)-Surfactant Combination on Inactivation of Salmonella on Green Onions. IFT Program and Abstract Book Annual Meeting. New Orleans, LA, USA. June, 2014.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Pivarnik, LF, Richard, NL, Gable, R and Worobo, RW. 2014. Attitude and knowledge assessment of shellfish and produce industries, and outreach educators regarding non-thermal processing and its applications. Abstract. IFT, New Orleans, LA, July.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Huang, R., Li, X., Huang, Y., and Chen, H. Strategies to enhance high pressure inactivation of murine norovirus in strawberry puree and on strawberries. Presented at the 2014 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Liu, C., Li, X., Ye, M., and Chen, H. Inactivation of murine norovirus (MNV) on blueberries using ultraviolet light irradiation with and without the presence of water. Presented at the 2014 IFT Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Sido, R. and Chen, H. High Pressure Processing of Green Onions and Salsa to Reduce or Eliminate Murine Norovirus. Presented at the 2014 IFT Annual Meeting.
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Chuhan Liu, 2015. Inactivation of Escherichia coli O157:H7, Salmonella enterica and murine norovirus on blueberries using a novel water- assisted ultraviolet light process. MS thesis.
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Runze Huang, 2015. High pressure inactivation of murine norovirus and human noroviruses on strawberries, blueberries or raspberries or in purees. MS thesis.
  • Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Robert Sido, 2015. Inactivation of murine norovirus on green onions and in salsa by high pressure processing, and inactivation of Salmonella on blueberries by a novel ultraviolet light and washing process. MS thesis.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Phuvasate S., Su Y-C. 2014. Efficacy of low-temperature high hydrostatic pressure processing in inactivating Vibrio parahaemolyticus in pure culture and oyster homogenates. Institute of Food Technologists Annual Meeting. June 21-24, New Orleans, LA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Phuvasate S., Su Y-C. 2014. Response of Vibrio parahaemolyticus strains to high hydrostatic pressure processing at low temperatures. The 65th Pacific Fisheries Technologists Annual Meeting, February 23-26, Monterey, CA.
  • Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Xu, W. (Ph.D. dissertation) FOODBORNE PATHOGENIC DECONTAMINATION AND QUALITY ENHANCEMENT OF FRESH PRODUCE USING PULSED LIGHT (PL) AND SANITIZER WASHING
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Huang, Y. and Chen, H. Decontamination of Escherichia coli O157:H7 on Fresh Strawberries and Raspberries Using a Novel Water-assisted Pulsed Light System. Presented at the 2014 IFT Annual Meeting.


Progress 02/01/13 to 01/31/14

Outputs
Target Audience: The target audiences are produce and shellfish industries, and food safety and extension educators. We propose to deliver these education programs through formal classroom instruction, workshops, industry meetings, and information brochures highlighting important concepts and information based on the needs assessment and research findings. Changes/Problems: The human organoid culture system has encountered difficulty of inconsistent results of viral replication and slow growing of organoids for scale-up the cultures to meet the needs for assessment of norovirus inactivation in food. Our current researches are on the development of the mouse model for norovirus using immune-deficient mouse lines. What opportunities for training and professional development has the project provided? Within each PD’s group, senior people have always helped to train junior people. Our project team has been working very closely. Training between groups has been provided to the professionals, postdocs and graduate students working on this project. Below are some examples of training between groups and professional development for Year 3 of this project. One of Dr. Ozbay’s technicians received training in Dr. Chen’s lab on working with oysters and microbial analysis. The postdoc of Drs. Gurtler and Niemira learned techniques related to tissue culture and virus propagation from Dr. Chen’s laboratory. Dr. Chen’s group and Dr. Li’s group have been communicating and teaching each other on some virological and molecular techniques. Dr. Wu’s graduate student learned how to use the pulsed light equipment in Dr. Chen’s laboratory. Postdocs and graduate students were provided funding to go to IFT and/or IAFP Annual Meetings. Dr. Li’s postdoc and student attended the 5th International Conference on Calicivirus held in Beijing, China to present findings from this project. How have the results been disseminated to communities of interest? In year 2, a needs assessment was performed for the shellfish and produce industries, as well as food safety educators and professionals. The data from these surveys have been analyzed to identify training deficiencies and allow for integration of key research results for non-thermal processing into the training and information strategies. The research findings have already been used to guide produce industry companies for safety strategies to enhance the safety of their produce products. These were through consultation and produce workshops. Two Seafood HACCP Training Courses were taught in Salem and Portland of Oregon in April and August 2013. More than 30 persons from the seafood industries and state regulatory agents completed the courses and received certificates from the Association of Food and Drug Officials. The results generated from this project have been presented and disseminated to a number of professional meetings including International Association of Food Protection, Institute of Food Technologists, American Society for Virology, International Conference for Chemical Engineering, Midwest Food Safety conference, Ohio Food Protection, and Ohio Fresh Produce Grower Association. What do you plan to do during the next reporting period to accomplish the goals? Research Continue working on the characterization of the Tulane virus (TV) as a useful surrogate for human norovirus (HuNoV). Plan to study norovirus and TV replication in immune-deficient mice in an attempt to establish a mouse infection model. Using the porcine gastric mucin-conjugated magnetic beads binding assay, we have found the high pressure processing parameters to effectively inactivate HuNoV GI.1 and GII.4 in oysters. We will test these processing conditions using the Gnotobiotic pig model. The effect of high pressure processing parameters developed in this study on the sensory and quality of oysters will be determined. The effects of pulsed light and UV on inactivation of viruses on berries will be studied. We plan to scale up the pulsed light system and UV system to process larger quantity of berries. The effect of high pressure processing on the inactivation of viruses in berries, green onions and salsa will be determined. The effect of washing with sanitizers on inactivation of viruses on fresh berries and green onions will be determined. The effect of cold plasma (CP) treatment on inactivation of viruses on fresh berries will be determined, while considering CP’s effect on compression firmness, visual appearance, and total anthocyanins. Modify CP treatments to be utilized in conjunction with force air cooling processing for fresh berries. Scale up CP treatment process and collaborate with local berry processors to determine the efficacy of CP in an industrial setting. The effects of pressure processes on changes of cell protein profiles of several clinical strains of V. parahaemolyticus will be determined. In addition, we will determine the ability of V. parahaemolyticus cells to attach to surface after various HHP treatments. The effect of treatment temperature on high pressure inactivation of V. parahaemolyticus in oysters will be determined. To determine the effect of irradiation on virus inactivation. To determine the mechanism of viral inactivation by the non-thermal processing technologies. Outreach The data from the needs assessment survey will be used in conjunction with the research findings from the collaborators to develop training and outreach materials as originally proposed in the grant proposal. We will provide 1) outreach education and training to industry and educators regarding foodborne viruses and food safety impacts of non-thermal technologies on high risk foods and 2) communication of results to stakeholders, regulatory agencies, and the public. Year 4 will include the development of training courses, appropriate brochures, pamphlets, or other outreach alternatives as directed by the assessment and evaluation of outreach strategies. The proposed outreach will be reviewed by the grant collaborators for content and accuracy of information. The findings from the on-line needs assessment will be prepared/submitted for publication in a peer-reviewed journal.

Impacts
What was accomplished under these goals? Research Activities: Objective 1. To determine whether Tulane virus (TV) can be used as a surrogate for human norovirus (HuNoV) and to develop a culture system to assess the survival of HuNoV. TV as a surrogate for HuNoV Temperatures of 4 - 72°C were tested and overall higher temperature and longer exposure time resulted in higher inactivation of TV and murine norovirus (MNV). Extreme pH (2, 3, 9, and 10) reduced viral titers. MNV was highly sensitive to ethanol, although TV was quite resistant. Both TV and MNV were able to survive 292 ppm free chlorine and showed dose-dependent decreases in infectivity when exposed to UV. UV had no effect on the viral capsid and no significant differences were observed with chlorine, while ethanol impacted capsid integrity. Objective 2. To determine the effect of the non-thermal processing technologies on inactivation of enteric foodborne viruses. High hydrostatic pressure (HHP) inactivation of HuNoV GI.1 and GII.4 Porcine gastric mucin (PGM)-conjugated magnetic beads (PGM-MBs) were used to collect and quantify potentially infectious HuNoV strains GI.1 and GII.4 treated by HHP. Both GI.1 and GII.4 strains showed increasing pressure sensitivity as judged by loss of PGM binding with decreasing temperature over a range of 1 to 35°C. Both GI.1 and GII.4 were more resistant to pressure at pH 4 than at neutral pH. GI.1 on dry blueberries was very resistant to pressure while immersion of blueberries in water during pressure treatments substantially enhanced the inactivation. Inactivation of MNV in strawberry puree and strawberry by HHP MNV was more sensitive to HHP at lower temperatures. HHP treatments at 350 MPa for 2 min at 0°C achieved > 3.5 log reduction of MNV in both strawberry and strawberry puree. For strawberry, water presence significantly improved the MNV inactivation effect, increasing by ~2 log reduction than without water presence. Inactivation of HuNoV and its surrogates by gamma irridaition The sensitivity of HuNoV GII.4, MNV, and TV was tested by gamma irradiation. A dose of 4 kGy, a FDA-approved dose for treating fresh produce, reduced MNV and TV by < 1.5 log and HuNoV GII.4 by < 2 log genomic RNA copies. Thus, gamma irradiation has limited ability to inactivate HuNoV in foods. Inactivation of HuNoV surrogates by ozone Gaseous ozone inactivated MNV and TV in both liquid media and fresh produce in a dose-dependent manner. Longer treatment time led to more viral reduction. In liquid media, both MNV and TV were undetectable after 40 min of treatment. In produce, there was also significant reduction in both viral titers. Inactivation of TV by cold plasma (CP) Blueberries inoculated with TV were treated with atmospheric CP. CP for 60s reduced TV by 2 log, but increased berry surface temperature to > 56°C. In contrast, berries treated with the combination of CP and normal air remained under 50 °C for up to 120s. This combination reduced TV by1.6 log after 45s and reduced background microflora. Treatments longer than 60s resulted in significant reductions in firmness and a reduction in total anthocyanins and color after 120s. Objective 3. To determine the efficacy of the non-thermal processing technologies on inactivating pathogenic bacteria Pulsed light (PL) decontamination of blueberries and green onions Blueberries inoculated with E. coli O157:H7 or Salmonella were either treated with PL directly (dry PL treatment) or immersed in agitated water during the PL treatment (wet PL treatment). The dry PL treatment adversely affected the appearance of the blueberries. The visual appearance of blueberries remained unchanged after the wet PL treatment. The wet PL treatment was more effective than chlorine washing on inactivating both pathogens. Green onions were cut into two segments, stems and leaves. Stems were more difficult to be decontaminated. Spot and dip inoculation method were compared and dipped inoculated green onions were found to be more difficult to be decontaminated. PL combined with surfactant (SDS) was found to be more effective than single treatment of PL. Inactivation of pathogens on berries by UV The UV inactivation kinetics of produce associated bacterial pathogens has been determined in a water system to serve as a reference for the bacterial pathogens on berries and leafy greens. These results will establish the UV exposure levels for different commodities and pathogens for commercial application. Survival of Vibrio parahaemolyticus cells under pressured conditions HHP inactivation of 5 environmental isolates and 5 clinical strains of V. parahaemolyticus in 2% NaCl solution revealed variations in pressure resistance among strains of V. parahaemolyticus. Studies of temperature effects on HHP discovered that the efficacy of HHP in inactivating V. parahaemolyticus cells can be enhanced by lowering operating temperature from 15 to 5°C. Removal of Salmonella Typhimurium from blueberries by combinations of SDS with organic acids or hydrogen peroxide Maximum reductions of Salmonella after washing with 0.5 mg/ml acetic acid plus 5000ppm SDS and 200ppm hydrogen peroxide containing 5000ppm SDS were 4.0 and 4.2 log CFU/g, respectively. Addition of SDS enhanced Salmonella inactivation of the acetic acid or hydrogen peroxide solutions. These treatments showed similar Salmonella reductions with 200ppm chlorine (P>0.05). None of these washings decreased the total phenolic, anthocyanins content and apparent quality. Objective 4. To determine the mechanism of viral inactivation by the non-thermal processing technologies. Mechanism of viral inactivation by high pressure. After pressure treatment, the virion structure changed from a discrete small round shape to a large amount of undefined protein debris as visualized by electron microscopy. The primary mechanism of norovirus inactivation is the disruption of the virion capsid structure. However, the primary and secondary structures of viral proteins remained intact, although the quaternary and tertiary structures of viral capsid proteins were completely distorted. HHP also impaired the receptor binding capacity of the HuNoV capsid. Mechanism of HuNoV inactivation by HPP using virus-like particles (VLPs) as models VLPs are structurally similar to native virions. The mechanism of viral inactivation involved in disruption of capsid structure and receptor binding ability of VLPs. The VLPs were more sensitive to HHP at neutral pH (6.5) than at acidic pH (2.5-4.5). Additionally, salts can provide baroprotective effects on capsids during HHP treatment. Education Activities: “Food Virology and Food Safety” modules were developed by Dr. Jianrong Li, and have been incorporated into a number of courses. Outreach Activities: The on-line survey of industry (shellfish/produce) and food safety educators was conducted to assess knowledge/atttitudes of non-thermal processing and ascertain willingness to receive information and/or training. The statistical analyses of these surveys showed that all groups, produce,/shellfish (N=113), educators (N=52) had knowledge scores which were below proficiency(80%), 48%, 51% and 67% respectively., Question reliability was >0.9These findings suggest a strong need for education and training for all three groups. There were no differences in attitudes and knowledge between the two industry groups. Both groups indicated a desire for information and/or training – with information dissemination preferred way to obtain knowledge regarding non-thermal processing. For both industry and educators, the preferred formats for information were PowerPoint, printed materials, and internet/web-based materials. Educators identified barriers as equipment cost, lack of information, and lack of product validation.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: DiCaprio, E., Hughes, J., Li, J. 2013. Epidemiology, prevention, and control of human norovirus. Infectious Disease Clinics in North America. 27(3):651-74.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Lou, F., Li, X., Huang, P., Niemira, B., Gurtler, J.B., Jiang, X., Chen, H., Li, J. 2013. Development of approaches to estimate the survival of noncultivable human norovirus by high pressure processing. American Society for Virology, College Station, The Penn State University.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Lou, F., Huang, P., Neetoo, H., Gurtler, J.B., Niemira, B.A. Chen, H., Jiang, X., Li, J. 2012. High pressure inactivation of human norovirus virus-like particles: evidence that human norovirus may be highly pressure resistant. International Association of Food Protection Annual Meeting. Providence, RI.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Lou, F., Huang, P., Neetoo, H., Gurtler, J.B., Niemira, B.A. Chen, H., Jiang, X., Li, J. 2012. High pressure processing of human norovirus virus-like particles: evidence that the capsid of human norovirus is highly pressure resistant. Institute of Food Technologists Annual Meeting. Las Vegas, NV.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Chen, H., Li, J. 2012. Inactivation of enteric foodborne viruses in high risk foods by non-thermal processing technologies. Institute of Food Technologists Annual Meeting. Las Vegas, NV.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Li, J. 2012. High pressure inactivation of human norovirus in foods and environment. International Conference for Chemical Engineering. Ixatapa: Mexico.
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Yue Duan. MS thesis, 2013. Pathogenesis of Tulane virus (a surrogate for human noerovirus) in gnotobiotic pigs. The Ohio State University, Columbus, Ohio.
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Jia Wei Yeap. MS thesis, 2013. Development of norovirus indicators for application in retail food settings. The Ohio State University, Columbus, Ohio.
  • Type: Theses/Dissertations Status: Published Year Published: 2012 Citation: Erin DiCaprio. MS thesis, 2012. Attachment, internalization, and dissenmination of human norovirus in fresh produce. The Ohio State University, Columbus, Ohio.
  • Type: Theses/Dissertations Status: Published Year Published: 2011 Citation: Ashley Predmore. MS thesis, 2011. Enhanced removal of a human norovirus surrogate from fresh vegetables and fruits by a combination of surfactants and sanitizers. The Ohio State University, Columbus, Ohio.
  • Type: Theses/Dissertations Status: Published Year Published: 2011 Citation: Fangfei Lou. MS thesis, 2011. Inactivation of selected nonenveloped and enveloped viruses by high pressure processing. The Ohio State University, Columbus, Ohio.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: Su Y-C, Liu C. 2014. Shellfish Handling and Primary Processing. In: I.S. Boziaris Ed. Seafood Processing: Technology, Quality and Safety (1st ed.), John Wiley & Sons, Ltd., England.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Phuvasate S., Su Y-C. 2013. Vibrio parahaemolyticus persistence in oysters during depuration and survival under pressurized conditions. IFT Meeting. July 13-16, Chicago, IL.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Li, Y., Wu, C. 2013. Enhanced removal of Salmonella Typhimurium from blueberries by combinations of sodium dodecyl sulfate with organic acids or hydrogen peroxide. Food Research International 54 (2),15531559.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Xu, W., Wu, C. Different Efficiency of Ozonated Water Washing to Inactivate Salmonella enterica Typhimurium on Green Onions, Grape Tomatoes, and Green Leaf Lettuces. Journal of Food Science (in press).
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Li, Y. (M.S. thesis) Enhanced removal of Salmonella Typhimurium and E. Coli O157:H7 from blueberries and strawberries by solution containing sodium dodecyl sulfate and organic acids or hydrogen peroxide.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Tan, M., Jiang, X. 2014. Histo-blood group antigens: a common niche for norovirus and rotavirus. Experts in Molecular Medicine, In Press.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Huang, Y. and Chen H. 2014. A novel water-assisted pulsed light processing for decontamination of blueberries. Food Microbiol. 40: 1-8.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Lou, F., Li, X., Huang, P., Gurtler, J., Niemira, B., Jiang, X., Chen, H., Kingsley, D., Li, J. Estimation of human norovirus survival during high pressure processing. 5th International Conference on Calicivirus. Beijing, China. Oct. 2013.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Ma, Y., Krakowka, S., Li. J. A gnotobiotic model for human norovirus infectivity. 5th International Conference on Calicivirus. Beijing, China. Oct. 2013.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Li, X., Chen, H., and Kingsley, D.H. 2013. The influence of temperature, pH, and water immersion on the high hydrostatic pressure inactivation of GI.1 and GII.4 human noroviruses. Int. J. Food Microbiol. 167: 138-143.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Ye, M., Huang, Y., Gurtler, J.B., Niemira, B.A., Sites, J.E. and Chen, H. 2013. Effects of pre- or post-processing storage conditions on high-hydrostatic pressure inactivation of Vibrio parahaemolyticus and V. vulnificus in oysters. Int. J. Food Microbiol. 163: 146-152.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Li, X.; Ye, M., Neetoo, H.; Golovan, S., and Chen, H. 2013. Pressure inactivation of Tulane virus, a candidate surrogate for human norovirus and its potential application in food industry. Int. J. Food Microbiol. 162: 37-42.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Huang, Y., Ye, M. and Chen, H. 2013. Inactivation of Escherichia coli O157:H7 and Salmonella spp. in strawberry puree by high hydrostatic pressure with/without subsequent frozen storage. Int. J. Food Microbiol. 160: 337-343.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Xu, W., Chen, H., Huang, Y. and Wu. C. 2013. Decontamination of Escherichia coli O157:H7 on green onions using pulsed light (PL) and PL-surfactant-sanitizer combinations. Int. J. Food Microbiol. 166: 102-108.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Lou, F., Huang, P., Neetoo, H., Gurtler, J.B., Niemira, B.A., Chen, H., Jiang, X. and Li, J. 2012. High-pressure inactivation of human norovirus virus-like particles provides evidence that the capsid of human norovirus is highly pressure resistant. Appl. Environ. Microbiol. 78: 5320-5327.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Ye, M., Huang, Y., and Chen, H. 2012. Inactivation of Vibrio parahaemolyticus and Vibrio vulnificus in oysters by high-hydrostatic pressure and mild heat. Food Micro. 32: 179-184.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Li, D., Baert, L., Zhong, D., Xia, M., Zhong, W., Van Coillie, E., and Jiang, X. 2012. The Effect of grape seed extract on human noroviruses GII.4 and murine norovirus-1 in viral suspensions, on stainless steel discs, and in lettuce wash water. Appl. Environ. Microbiol. 78: 7572-7578.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Tian, P., Yang, D., Quigley, C., Chou, M. and Jiang, X.. 2013. Inactivation of Tulane virus, a novel surrogate for human norovirus. J. Food Prot. 76: 712-718.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Araud, E., Lou, F., Li, X., Chen, H., and Li, J. 2012. High pressure processing of rotaviruses: the roles of strain diversity and treatment temperature in virus inactivation. IAFP Annual Meeting, Providence, RI.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Lou, F., Huang, P., Neetoo, H., Gurtler, J., Niemira, B., Chen, H., Jiang, X., and Li, J. 2012. High pressure processing of human norovirus virus-like particles: evidence that human norovirus may be highly pressure resistant. IAFP Annual Meeting, Providence, RI.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Phuvasate S., Su Y-C. 2012. Impacts of water salinity and type and size of oysters on depuration for reducing Vibrio parahaemolyticus in raw Pacific oyster (Crassostrea gigas). IFT Annual Meeting, Las Vegas, NV.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Phuvasate S., Su Y-C. 2013. Persistence of Vibrio parahaemolyticus in depurated oysters and survival and injury of cells in pressurized pure culture. The 64th PFT Annual Meeting, Nuevo Vallarta, Mexico.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Xu, W., Wu, C. 2012. Influence of time, temperature and pH on efficiency of bubbling ozone treatment to inactivate salmonella enterica typhimurium on green onions, grape tomatoes, and green leaf lettuces. American Chemical Society (ACS) Annual Meeting, Philadelphia, PA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Huang, Y. and Chen, H. 2013. Inactivation of Escherichia coli O157:H7 and Salmonella on fresh blueberries using pulsed light technology. IAFP Annual Meeting, Charlotte, NC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Ye, M., Li, X., Kingsley, D.H., Chen, H. 2013. Inactivation of human noroviruses in oysters by high-hydrostatic pressure. IFT Annual Meeting, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Li, X., Kingsley, D.H., Chen, H. 2013. Temperature and pH influence high hydrostatic pressure inactivation of human noroviruses. IFT Annual Meeting, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Liu, C., Huang, Y. and Chen, H. 2013. Inactivation of Escherichia coli O157:H7 on blueberries using ultraviolet light with/without the presence of water. IFT Annual Meeting, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Huang, Y., Ye, M., and Chen, H. 2013. Effects of Pulsed Light on the inactivation of Escherichia coli O157:H7 on Fresh Strawberries, Raspberries and Blueberry. IFT Annual Meeting, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Lou, F., Li, X., Huang, P., Gurtler, J., Niemira, B., Jiang, X., Chen, H., Jiang, X., and Li, J. 2013. High pressure inactivation of human norovirus virus-like particles: factors that affect the efficacy. IFT Annual Meeting, Chicago, IL.
  • Type: Theses/Dissertations Status: Published Year Published: 2013 Citation: Ye, M. 2013. Inactivation of Vibrio parahaemolyticus, Vibrio vulnificus and noroviruses in oysters by high-hydrostatic pressure, mild heat and cold storage. Ph.D. Dissertation. University of Delaware, Newark, DE.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Xu, W., Li, Y., Chen, H., Wu, C. 2013. Decontamination of Escherichia coli O157:H7 on Green Onions Using Pulsed Light (PL) and PL-Surfactant-Sanitizer Combinations. IFT Annual Meeting, Chicago, IL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Xu, W., Wu, C. 2013. Inactivation of Salmonella on Cantaloupe and Green onion Using Pulsed Light (PL) and PL-SDS Combination. IAFP Annual Meeting, Charlotte, NC.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Phuvasate S, Su Y-C. 2013. Impact of water salinity and types of oysters on depuration for reducing Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas). Food Control 32: 569-573.
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2013 Citation: Su Y-C, Liu C. 2013. Shellfish Handling and Primary Processing. In: I.S. Boziaris Ed. Seafood Processing: Technology, Quality and Safety, John Wiley & Sons, Ltd., England (In Press).
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Yu, G., D. Zhang, F. Guo, M. Tan, X. Jiang, and W. Jiang 2013. Cryo-EM structure of a novel calicivirus, Tulane virus PloS one. 8:e59817.
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: DiCaprio, E., Lou, F., Li, J. 2014. Strategies for control of foodborne viruses. Foodborne viral pathogens. Editor: Peter White. CRC Press.


Progress 02/01/12 to 01/31/13

Outputs
OUTPUTS: Tulane virus (TV) as a surrogate for human noroviruses (HuNoV): TV was sensitive to heat (63C for 5 min or at 56C for 30 min), UV (60 mJ/cm2) and chlorine (300 ppm). It was stable at pH 3-8 and resistant to ≤ 40% of ethanol, but could be inactivated by 50-70% ethanol. Viral inactivation mechanism: a) High hydrostatic pressure (HHP)- VLPs were more resistant to HHP in their ability to bind type A than type B and O HBGAs. The 23-nm VLPs appeared to be much more stable than the 38-nm VLPs; b) E-beam irradiation- E-beam affected the structure of murine norovirus (MNV-1) and vesicular stomatitis virus. It degraded viral genomic RNA and viral proteins. Inactivation of viruses: a) Pressure inactivation of HuNoV- Pig gastric mucin-conjugated magnetic beads (PGM-MB) were used to bind and collect infectious HuNoV followed by qRT-PCR to quantify it. Using this approach, GI.1 HuNoV was found to be more resistant to HHP than the GII.4 strain. Treatment at 600 MPa achieved a > 4.1 log reduction of GI.1 NoV, which agrees with the results from a recent human volunteer study. HHP inactivation of the two HuNoV strains increased as sample temperature decreased from 35 to 1C. Both strains were more resistant to pressure under acidic conditions; b) E-beam irradiation of MNV-1- Less than 1 log virus reduction was achieved at 2, 4, and 6 kGy for buffer, cabbage, and strawberries, respectively. Inactivation of pathogenic bacteria: a) HHP inactivation of vibrios in oysters- Cold storage at -18, 4 and 10C, prior to HHP, decreased V. parahaemolyticus or V. vulnificus by 1.5 - 3.0 log MPN/g, but did not increase their sensitivity to subsequent HHP treatments. V. parahaemolyticus populations in HHP-treated oysters gradually decreased during post-HHP ice or frozen storage; b) Pulsed light (PL) inactivation of E. coli O157:H7 on blueberries- Inoculated berries were treated by PL alone (dry PL) or immersed in agitated water during the PL treatment (wet PL). The wet PL treatment reduced E. coli O157:H7 on blueberries by > 5 log CFU/g. Both dry and wet PL treatments were more effective than washing with 10 ppm chlorinated water; c) PL inactivation of E. coli O157:H7 on green onions- For dip-inoculated green onions, 60s wet PL treatment was comparable with 100 ppm chlorine washing. PL combined with surfactant (SDS) was more effective than PL alone; d) Sanitizer wash for blueberries- Wash time and storage temperature influenced the reduction of Salmonella Typhimurium. Survey: The survey to assess the industry knowledge and attitudes regarding non-thermal processing has been completed. The database included 113 participants. Both shellfish and produce respondents had very low knowledge scores, 51% and 48%, respectively; indicating the need for education and outreach efforts. While 100% of the produce processors had established food safety training programs, only 50% of the shellfish processors identified themselves as having one. The majority were interested in receiving training and information on non-thermal processing. The survey to assess food safety educator knowledge and attitudes regarding non-thermal processing was recently completed. PARTICIPANTS: 1. PI: Haiqiang Chen (University of Delaware) 2. Co-PIs: Brendan Niemira (USDA, ARS, Eastern Regional Research Center), Changqing Wu (University of Delaware), Gulnihal Ozbay (Delaware State University), Xi Jason Jiang (Cincinnati Children's Hospital Medical Center), Jianrong Li (The Ohio State University), Joshua Gurtler (USDA, ARS, Eastern Regional Research Center), Ken Lee (The Ohio State University), Lori Pivarnik (University of Rhode Island), Randy Worobo (Cornell University), and Yi-Cheng Su (Oregon State University) 3. Research Support Specialist: John J. Churey (Cornell University) 4. Post-doctoral fellows: Xinhui Li (University of Delaware), Christina Quigley (Cincinnati Children's Hospital Medical Center), and Alison Lacombe (USDA ARS) 5. Graduate Students: a) University of Delaware: Mu Ye, Yaoxin Huang, Robert Sido, Chuhan Liu, Wenqing Xu, and Yingying Li; b) Oregon State University: Sureerat Phuvasate; c) The Ohio State University: Erin Dicaprio, Fangfei Lou and Yue Duan; d) Cornell University: Carmen Wickware, Giselle Guron, and Claire Zoellner 6. Undergraduate Student: Robert Sido (summer student, University of Delaware), Melissa Ehrich (University of Delaware), Marissa Chou (summer student, Cincinnati Children's Hospital Medical Center) TARGET AUDIENCES: Two courses have been developed and are currently taught by co-PI, Dr. Jianrong Li. Course 1 - Food Safety and Public Health: Food safety is an important component of public health. Course 2 - Advanced Food Microbiology II - Food Virology and Immunology. PROJECT MODIFICATIONS: None.

Impacts
TV as a surrogate for HuNoV: Although TV behaved similarly to various HHP conditions as MNV-1and the two stains of HuNoV (GI.1 and GII.4), it might not be an optimal surrogate for HuNoV, as it is slightly more sensitive to pressure than the two HuNoV strains. The likely order of pressure resistance is: GI.1>MNV-1>GII.4>TV. Therefore, MNV-1 may be considered the better surrogate for GII.4. Viral inactivation mechanism: a) HuNoV VLPs- Using VLPs as a model to understand the stability of HuNoV, we demonstrated that the HuNoV capsid is highly resistant to HPP; b) E-beam- The mechanism of E-beam inactivation of viruses is similar to that of gamma irradiation. Inactivation of viruses: a) HHP- HHP could be applied as a potential intervention for inactivating HuNoV in raw oysters. To effectively inactivate HuNoV GI.1, the pressure may need to exceed > 500 MPa, which is much higher that pressure levels currently used by the oyster industry (NoV, wMPa). The impact of such high pressures on sensorial properties of oysters remains to be determined. Low treatment temperature could be used to reduce the pressure level needed for effective inactivation of HuNoV; b) Binding assay- Binding to PGM-MB, followed by RT-PCR detection, can be explored as a practical means of evaluating the infectious and non-infectious states of NoV. The assumption is that non-infectious HuNoV was not able to bind to PGM; c) E-beam- In foods, MNV-1 is difficult to inactivate by E-beam irradiation due to the protective effect of the food matrix and small size and the highly-stable viral capsid. The application of E-beam to inactivate HuNoV in foods is probably unlikely. Inactivation of pathogenic bacteria: a) HHP inactivation of vibrios in oysters- Effective combinations of pressure treatment and cold storage were identified. HPP at (1) 300 MPa for 2 min at 21C, followed by 5-day ice storage or 7-day frozen storage, or (2) HPP at 250 MPa for 2 min at 21C, followed by 10-day ice or 7-day frozen storage, completely inactivated V. parahaemolyticus in whole-shell oysters (> 7 log reductions). The combination of HHP at a relatively low pressure (e.g., 250 MPa), followed by short-term frozen storage (7 days), could potentially be applied by the shellfish industry as a post-harvest process to eliminate V. parahaemolyticus in oysters; b) PL treatment- The wet PL treatment could be a promising alternative to traditional chlorine washing for blueberries intended for frozen storage and green onions, to avoid the use of chemicals. Since berries destined for the fresh market are usually not washed, the dry PL treatment can potentially be used to reduce the level of pathogens on fresh berries. In addition, SDS could be used to enhance PL inactivation of E. coli O157:H7 on green onions; c) Sanitizer wash for blueberries- Effective washing solutions and treatment times were identified. The combination of 200 ppm H2O2 and 0.5% SDS had greater efficacy in inactivating S. Typhimurium after a 5 min wash (4.0 log CFU/g reduction) than a 1 min wash (3.2 log CFU/g reduction). Treated blueberries stored at lower temperatures significantly enhanced visual appearance and decreased mold growth.

Publications

  • Peer-reviewed Journal Papers: Huang, Y., Ye, M. and Chen H. 2013. Inactivation of Escherichia coli O157:H7 and Salmonella spp. in strawberry puree by high hydrostatic pressure with/without subsequent frozen storage. Int. J. Food Microbiol. 160: 337-343.
  • Li, D., Baert, L., Zhong, D., Xia, M., Zhong, W., Van Coillie, E., and Jiang, X. 2012. The Effect of grape seed extract on human noroviruses GII.4 and murine norovirus-1 in viral suspensions, on stainless steel discs, and in lettuce wash water. Appl. Environ. Microbiol. 78: 7572-7578.
  • Li, X., Ye, M., Neetoo, H., Golovan, S., and Chen, H. 2013. Pressure inactivation of Tulane virus, a candidate surrogate for human norovirus and its potential application in food industry. Int. J. Food Microbiol. 162: 37-42.
  • Lou, F., Huang, P., Neetoo, H., Gurtler, J.B., Niemira, B.A., Chen, H., Jiang, X. and Li, J. 2012. High-pressure inactivation of human norovirus virus-like particles provides evidence that the capsid of human norovirus is highly pressure resistant. Appl. Environ. Microbiol. 78: 5320-5327.
  • Tian, P., Yang, D., Quigley, C., Chou, M. and Jiang, X.. 2013. Inactivation of Tulane virus, a novel surrogate for human norovirus. J. Food Prot. In press.
  • Ye, M., Huang, Y., Gurtler, J.B., Niemira, B.A., Sites, J.E. and Chen, H. 2013. Effects of pre- or post-processing storage conditions on high-hydrostatic pressure inactivation of Vibrio parahaemolyticus and V. vulnificus in oysters. Int. J. Food Microbiol. In press.
  • Meeting Abstracts: Araud, E., Lou, F., Li, X., Chen, H., and Li, J. 2012. High pressure processing of rotaviruses: the roles of strain diversity and treatment temperature in virus inactivation. IAFP Annual Meeting, Providence, RI.
  • Lou, F., Huang, P., Neetoo, H., Gurtler, J., Niemira, B., Chen, H., Jiang, X., and Li, J. 2012. High pressure processing of human norovirus virus-like particles: evidence that human norovirus may be highly pressure resistant. IAFP Annual Meeting, Providence, RI.
  • Phuvasate S., Su Y-C. 2012. Impacts of water salinity and type and size of oysters on depuration for reducing Vibrio parahaemolyticus in raw Pacific oyster (Crassostrea gigas). IFT Annual Meeting, Las Vegas, NV. Phuvasate S., Su Y-C. 2013. Persistence of Vibrio parahaemolyticus in depurated oysters and survival and injury of cells in pressurized pure culture. The 64th PFT Annual Meeting, Nuevo Vallarta, Mexico. Xu, W., Wu, C. Chen, H. 2012. Effect of sanitizers and ozone combinations against Salmonella enteric Typhimurium on green onions. IAFP Annual Meeting, Providence, RI.
  • Xu, W., Wu, C. 2012. Influence of time, temperature and pH on efficiency of bubbling ozone treatment to inactivate salmonella enterica typhimurium on green onions, grape tomatoes, and green leaf lettuces. American Chemical Society (ACS) Annual Meeting, Philadelphia, PA.


Progress 02/01/11 to 01/31/12

Outputs
OUTPUTS: Tulane virus (TV) as a surrogate for human noroviruses (HuNoV): Heat treatment may disrupt up to 20% of intact viral capsids. Low pH did not disrupt viral capsid integrity. TV was also found to be more sensitive to high pressure (HHP) at neutral pH than in an acidic environment (pH 4), at a lower treatment temperature (4C) than at higher temperatures (21 and 35C), and on wetted blueberries (surrounded by water during HHP) than on dry blueberries. Viral inactivation mechanism: a) HHP: Disruption of viral capsid structure, viral envelope, and receptor binding activity; b) Gamma irradiation: Disruption of virion structure and degradation of viral proteins and genomic RNA. Inactivation of viruses: a) Murine norovirus (MNV): A > 5 log PFU/ml reduction was achieved in aqueous medium and fresh produce when MNV was pressurized at 400 MPa for 2 min at 4C; b) Human rotavirus: A > 3.9 log virus reduction under 350 MPa at 4C for 2 min; c) Human norovirus virus-like particle (VLPs): Pressurization at 500-600 MPa for 60 min was not sufficient to disrupt the structure and function of the VLPs. The times required for complete disruption of VLPs at 700, 800, and 900 MPa were 45, 15, and 2 min, respectively; d) Pressure, pH, temperature, and food matrix affected the virus inactivation by HHP; e) gamma irradiation is not effective against viruses. For MNV, only a 2.0 log virus reduction was achieved at the dose of 5.6 kGy. Inactivation of pathogenic bacteria: a) Pressure inactivation of Vibrio parahaemolyticus in oysters. HHP at 200 to 300 MPa for 2 min resulted in the inactivation of between 1.2 and > 7 log MPN/g of V. parahaemolyticus. Heat treatment at 40 and 45C was not effective at inactivating V. parahaemolyticus, but could enhanced inactivation when applied after HHP treatment; b) Pressure inactivation of E. coli O157:H7 and Salmonella in berry purees. Both pathogens were able to survive in berry puree for 12 weeks under frozen storage. HHP treatment of purees at 450 MPa for 2 min and 21C completely eliminated both pathogens. In addition, frozen storage after HHP enhanced inactivation of the pathogens; c) Pressure inactivation of Salmonella in salsa. Storage time (0, 12 or 24 h) of salsa prior to HHP as well as pH (3.8-5.3) and type of acidulant (i.e., vinegar and lemon juice) all influenced the extent of Salmonella inactivation by HHP; d) Sanitizer wash for blueberries and green onions: A combination of acetic acid and SDS solution had similar Salmonella inactivation as 200 ppm chlorinated water, while having minimal effects on sensory quality, total anthocyanins and total phenolic contents of blueberries. Chlorinated (200 ppm) or ozonated (6.25 ppm) water did not eliminate Salmonella on green onions, but a combination of thymol and acetic acid reduced it to non-detectable level. The needs assessment survey for produce and shellfish processors was drafted and experts reviewed it for content validity. Target launch of the survey is by the end of July. In an effort to maximize industry participation, members of the advisory group, representing both shellfish and produce industries, have agreed to send the survey link to members with their endorsement. PARTICIPANTS: 1. PI: Haiqiang Chen (University of Delaware) 2. Co-PIs: Brendan Niemira (USDA Eastern Regional Research Center), Changqing Wu (University of Delaware), Gulnihal Ozbay (Delaware State University), Xi Jason Jiang (Cincinnati Children's Hospital Medical Center), Jianrong Li (The Ohio State University), Joshua Gurtler (USDA Eastern Regional Research Center), Ken Lee (The Ohio State University), Lori Pivarnik (University of Rhode Island), Randy Worobo (Cornell University), and Yi-Cheng Su (Oregon State University) 3. Post-doctoral fellows: Xinhui Li (University of Delaware), Christina Quigley (Cincinnati Children's Hospital Medical Center) 4. Graduate Students: a) University of Delaware: Hudaa Neetoo, Mu Ye, Yaoxin Huang, Wenqing Xu, Yingying Li, and Chan Ho Jang; b) Oregon State University: Sureerat Phuvasate; c) The Ohio State University: Fangfei Lou and Duan Yue 5. Undergraduate Student: Melissa Ehrich (University of Delaware) TARGET AUDIENCES: Two courses have been developed by co-PI, Dr. Jianrong Li. Course 1 - Food Safety and Public Health: Food safety is an important component of public health. This course is based on presenting the basic principles and practices related to food safety. We will be presenting information regarding foodborne illness transmission, impact on public health, and how illness prevention. The course will have four major components: 1) foodborne pathogens; 2) foodborne chemical and physical hazards; 3) foodborne biological toxins and allergens; and 4) management activities required to ensure food safety and public health. The course will include a discussion of food safety management practices such as Hazard Analysis Critical Control Points (HACCP), public health policies, risk assessment, sanitation, and pathogen and allergen control in foods. Course 2 - Advanced Food Microbiology II - Food Virology and Immunology: This is an advanced food microbiology course covering various cutting-edge topics in food virology and immunology. The course will have four major components: 1) food virology, including basic concepts in virology, biological characteristics of foodborne viruses, mechanisms of viral replication, gene expression and pathogenesis, and prevention and control of foodborne viruses; 2) food immunology, including function and components of the host immune system, host responses to foodborne viral pathogens, foods as a regulator in tumor immunity and immunodeficiency diseases; 3) food-related immunopathology, including allergies, tolerances, and hypersensitivities; and 4) vaccines against foodborne pathogens. The course will also discuss the experimental methods and technologies in food virology and immunology. PROJECT MODIFICATIONS: A proposal to include cold plasma, a non-thermal processing technology, in this project was approved by NIFA. Four research objectives were proposed. 1. To determine the effect of cold plasma on inactivation of human NVs, TV, HAV and RV in high risk foods, 2. To determine the effect of cold plasma on inactivating pathogenic bacteria related to high risk foods, 3. To determine the mechanism of viral inactivation by cold plasma technology, and 4. To determine the effect of cold plasma processing on the quality attributes of high risk foods.

Impacts
TV as a surrogate for HuNoV: Heat treatment appears to disrupt TV viral capsids and decrease its infectivity, low pH exposure decreases infectivity without impacting viral RNA level. Therefore, current RT-PCR assessment of HuNoV RNA with and without RNase treatment may not be an effective assessment of viral infectivity. When subjected to HHP under varying conditions, TV behaved similarly to MNV, a surrogate commonly used for HuNoV. This study provides important information for the use of TV as a surrogate for HuNoV and establishes a foundation for designing effective HHP for inactivating HuNoV in high risk foods. Viral inactivation mechanism: These studies provided mechanistic insight of viral inactivation by food processing technologies. A better understanding of the mechanism of viral inactivation will facilitate the development of effective food processing technologies to eliminate viruses without significantly affecting food quality. Inactivation of viruses: HuNoV surrogates and human rotavirus can be effectively inactivated by HHP, demonstrating that HHP is a novel intervention method to eliminate viruses from high risk foods. Although the dose of gamma irradiation required to eliminate virus contaminants in fresh produce exceeds the limit set by FDA, this technology might have application in sterilization of medical equipment. Inactivation of pathogenic bacteria: a) Pressure inactivation of V. parahaemolyticus in oysters. Effective combinations of pressure treatment and mild heat were identified. Combinations such as HHP at 250 MPa for 2 min, followed by heat treatment at 45C for 15 min and HHP at 200 MPa for 2 min, followed by heat treatment at 50C for 5 min reduced V. parahaemolyticus to non-detectable levels as assessed by the MPN method (< 3 MPN/g). These results may help the shellfish industry establish parameters for processing oysters; b) Pressure inactivation of E. coli O157:H7 and Salmonella in berry purees. The survival of both pathogens during frozen storage demonstrates that they could pose potential health risks for consumers, considering their low infectious dose. Effective combinations of HHP and frozen storage were identified. Combinations, such as HHP of 250 MPa for 2 min at 21C followed by 8 days frozen storage, were sufficient to eliminate these pathogens at both high and low inoculation levels. Therefore, HHP may be a viable alternative to traditional thermal processing for processing of berry purees, while preserving flavors destroyed during thermal processing; c) Pressure inactivation of Salmonella in salsa. These results may be used as a guideline to design product formulations (e.g., pH range and type of acidulants) as well as pressure processing parameters. In addition, the influence of salsa storage time prior to HHP needs to be considered when designing pressure treatment protocols; d) Sanitizer wash for blueberries and green onions: The study evaluated various combinations of GRAS agents to improve Salmonella inactivation. The data could be used as guidelines to develop novel sanitizer wash for blueberries and green onions.

Publications

  • Feng, K., Divers, E., Ma, Y. and Li, J. 2011. Inactivation of human norovirus surrogate, human norovirus virus-like particle, and vesicular stomatitis virus by gamma irradiation. Appl. Environ. Microbiol. 77: 3507-3517.
  • Huang, Y., Ye, M. and Chen. H. 2012. Effect of high hydrostatic pressure on the inactivation of Escherichia coli O157:H7 and Salmonella spp. in strawberry puree. Institute of Food Technologists Annual Meeting. Las Vegas, NV.
  • Li, D., Baert, L., Xia, M., Zhong, W., Coillie , E.V., Jiang, X., and Uyttendaele, M. 2012. Evaluation of methods measuring the capsid integrity and/or functions of noroviruses by heat inactivation. J. Virological Methods, 181-1-5.
  • Li, J., Predmore, A., Divers, E. and Lou, F. 2012. New interventions against human norovirus: progress, opportunities, and challenges. Annual Rev. Food Sci. and Technol. 3: 331-352.
  • Li, X., Neetoo. H. and Chen. H. 2012. Pressure inactivation of Tulane virus, a potential surrogate for human norovirus. Institute of Food Technologists Annual Meeting. Las Vegas, NV.
  • Li, Y., Xu, W., Wu, C. and Ehrich M. 2012. Decontamination of Salmonella enterica on blueberries by washing with organic acid, ozone, and mild heat. Institute of Food Technologists Annual Meeting. Las Vegas, NV.
  • Lou, F., Neetoo, H., Li, J., Chen, H. and Li, J. 2011. Lack of correlation between virus barosensitivity and the presence of a viral envelope during inactivation of human rotavirus, vesicular stomatitis virus, and avian metapneumovirus by high-pressure processing. Appl. Environ. Microbiol.77: 8538-8547.
  • Ma, L. and Su, Y.-C. 2011. Effects of growth temperature and salt concentration on outer membrane proteins profiles of Vibrio parahaemolyticus. Institute of Food Technologists Annual Meeting. New Orleans, LA.
  • Neetoo, H. and Chen, H. 2012. High pressure inactivation of Salmonella on Jalapeno and Serrano peppers destined for direct consumption or as ingredients in Mexican salsa and guacamole. Int. J. Food Microbiol. 156: 197-203.
  • Predmore, A. and Li, J. 2011. Enhanced sanitization of a human norovirus surrogate in fresh vegetables and fruits by a combination of surfactants and sanitizers. Appl. Environ. Microbiol.77: 4829-4838.
  • Xu, W., Wu, C. and Chen, H. 2012. Effect of sanitizers and ozone combinations against Salmonella enteric Typhimurium on green onions. IAFP Annual Meeting. Providence, RI.
  • Ye, M., Huang, Y.. and Chen. H. 2012. Effect of cold storage before and/or after high-hydrostatic pressure on inactivation of Vibrio parahaemolyticus and Vibrio vulnificus in oysters. Institute of Food Technologists Annual Meeting. Las Vegas, NV.