Progress 05/01/24 to 04/30/25

Outputs
Target Audience:Small and very small fresh produce growers in West Virginia who are utilizing the triple wash procedure with antimicrobial solutions for postharvest processing both during and after the project period typically the stakeholder Preston County Workshop. The project also appeals to individuals, organizations, and policymakers interested in reducing food safety hazards of fresh produce at West Virginia. Changes/Problems:The progress of the project is severely affected by the WVU academic transformation process. In FY 2024, the unexpected academic transformation process with multiple faculty and staff left WVU including WVU OSP members. This unexpected situation made our faculty and students were unable to fully focus on research and extension activities. The starting of this project was 8 months delayed and the subaward for two of our Co-PDs Dr. Xiaoli Etienne from WVU to University of Idaho and Dr. Yangchao Luo from WVU to University of Connecticut took 1-year long to finally processed. They just started their shared research and outreach objectives related to proposed agri-economic topics and nanotechnology. Also, this is an integrated research and extension project, and we originally scheduled a plant onset test of triple-wash at Preston County Workshop, which, to date, has not been able to be conducted. Therefore, we have requested for the none-cost request extension of this project for another year. What opportunities for training and professional development has the project provided?We recruited 3PhD students Mr. Corey Coe, Ms. Jesica Temple and Mr. Md Shafiul Islam Rion to conduct the proposed research obejctives. Dr. Corey Coewas sucessfully defended on Spring 2025 and will obtain his PhD degree in summer 2025. How have the results been disseminated to communities of interest?The PD Dr. Shen is recentlyjoining FSMA-Food Safety Prevent Control Alliance-University Extension Specialist representing West Virginia, Oct. 2024. https://www.fspca.net/university-specialist-directory. Also served as WV Food Safety Training Team member leaded by Mr. Andrea O'Dell from WV Department of Agriculture since Fall 2024. He presented part of the triple-wash research at the FSMA Produce Safety Workshops as supplement material. What do you plan to do during the next reporting period to accomplish the goals?The PD Dr. Shen's group will conduct a plant onsite trial of triple-wash with butternut squashes at the stakeholder Preston County Workshop in September 2025. The Co-PD Dr. Luo is developing a nanoparticle to improve the antimicrobial effeicacy of H2O2-peroxyacetic acid (PAA) for triple-wash applied on fresh produce, which will be used for microbial cross-contamination studies during the next grant cycle. We are also planning to completethe outreach survey for the very small produce growers in WV local community. We are expecting to publish 2 peer-review publications and 2-3 conference presentations during the next grant period.

Impacts
What was accomplished under these goals? The PD Dr. Shen's group conducted a research study toevaluate the survival ofSalmonellaTyphimurium,Enterococcus faecium,Listeria monocytogenes, andListeria innocuaon cantaloupes during storage and after triple-wash exposure (water-water-antimicrobial) with peroxyacetic acid (PAA). Bacteria were spot-inoculated onto cantaloupe surfaces prior to triple-washing for 45 s at each washing stepusing0, 27, 54, and96 ppmof PAA. Spread-plating and a modified most-probable-number (MPN) method were utilized for enumeration of cells. Results showed thatD-values (pre-wash)forS. Typhimurium (2.47 h)on cantaloupeswere lower (P< 0.05) thanE. faecium(28.41 h),and the D-values (pre-wash) ofL. monocytogenes(4.50 h) is also lower (P< 0.05) thanL. innocua(7.24 h).Though all tested concentrations of PAA resulted in significant (P< 0.05) reduction of bacteria cells compared to the inoculated unwashed control, the greatest (P< 0.05) reduction ofS.Typhimurium andE. faecium(4.55 vs 3.23 log10CFU/cm2and 3.38 vs 2.55 log10MPN/mL) occurred at96 ppm.This concentration (96 ppm) also achieved 3-4 log reductions ofL. monocytogenesandL. innocuapopulation.Results suggest thatE. faeciumcould be used as surrogate ofS.Typhimurium andL. monocytogenesfor antimicrobial washing validation studies on cantaloupes. The PD Dr. Shen' group also completed a systematic review. "Food Safety Practices Among FSMA-Exempt Small Producers: A Systematic Review of Knowledge, Attitudes, and Behaviors from Cross-Sectional Studies in Three High-Risk Domains--Agricultural Water, Cleaning and Sanitation, and Storage and Transport". The reviewassessed research on FSMA-exempt produce growers' food safety knowledge, attitudes, and behaviors (KAB) across three high-risk domains: agricultural water (AW), cleaning and sanitation (CS), and storage and transport (ST). A structured search of six databases identified five eligible cross-sectional studies. Using a PICO framework, each study was evaluated for small-scale producers (Population), willingness to adopt practices (Intervention), self-reported behavior versus standards (Comparison), and implementation (Outcome). FSMA exemption was inferred when not stated, based on farm size, direct-to-consumer sales, or lack of third-party audits. While awareness of contamination risks and interest in training were high, fewer than 40% of growers reported using tested water sources, sanitizers, or temperature control. The willingness to adopt practices was evident but often limited by cost, infrastructure, and labor. Findings reveal persistent gaps between knowledge and implementation. All studies relied on self-reported data, underscoring the need for low-cost, context-specific interventions and targeted outreach to support voluntary compliance among FSMA-exempt small-scale producers. The Co-PD Dr. Luo's group completed a systematic review about nano and machine learning technology for the objectives related to applying nanotechnology with triple-wash to improve microbial safety of fresh produce.Food quality and safety are critical to global health and economic stability, but traditional assessment methods, such as chemical assays and microbial culturing, are often destructive, time-consuming, and unsuitable for realtime and high-throughput applications. Optical non-destructive techniques, including imaging methods (e.g., red-green-blue (RGB) imaging, hyperspectral imaging (HSI)) and spectral methods (e.g., near-infrared (NIR) spectroscopy), offer real-time, precise, and non-invasive assessments while preserving sample integrity. However, the complex datasets generated by these techniques require advanced machine learning (ML) models for effective analysis. These methods generate complex, multidimensional datasets that align with ML approaches, unlocking advanced capabilities in data interpretation and decision-making. By integrating optical nondestructive techniques with ML models, ranging from classical algorithms like random forests (RF) and support vector machines (SVM) to deep learning architectures such as convolutional neural networks (CNNs), notable progress has been achieved in automating feature extraction, classification, and prediction tasks. This integration enhances the precision, scalability, and applicability of food quality and safety assessments, enabling tasks such as real-time grading, sorting, and microbial detection in diverse food systems. Advanced models like YOLO further expand the potential for real-time object detection in dynamic settings such as smart farms and food processing lines. Despite these advances, challenges remain in addressing the variability of food matrices, real-time processing limitations, and the need to integrate data from multiple optical models. This survey explores the integration of ML with optical non-destructive methods to enhance food quality and safety assessments, highlighting recent advancements and future opportunities. The Co-PD Dr. Etienne's group in University of Idaho completed a Agri-economic surveystudy related toWillingness-To-Pay for Food Safety of applying triple-wash technology.Foodborne illnesses are costly to society and have been associated with local produce. The affordable "3-step wash" cleaning procedure was designed to reduce pathogens on produce. We estimate consumer willingness to pay (WTP) for food safety (i.e. 3-step washed), prepackage, and sales location attributes in locally grown produce (e.g., lettuce). On average, consumers are willing to pay $1.46 more for 3-step washed and $0.30 more for prepackaged lettuce. Additionally, consumers are willing to pay $0.16 more for fresh produce sold in natural stores and farmers markets compared to supermarkets, but $0.22 less for produce sold in other direct-to-consumer locations such as roadside stands. Higher WTP for the food safety attribute is associated with consumers who have greater risk aversion, less knowledge of foodborne illness, and stricter food safety cleaning and handling practices. Consumers highly concerned about foodborne risks also show higher WTP for both food safety and prepackage attributes. These findings can guide local farmers in making decisions about adopting pathogen-reduction cleaning procedures, selecting sales locations, and developing effective marketing strategies.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Md Shafiul Islam Rion, Corey Coe, Joseph Katz, Gary Freshour, Carly Long, Kristen Matak, Annette Freshour, Jacek Jaczynski, Timothy Boltz, Xue Tang, and Cangliang Shen. 2025. Survival of Salmonella Typhimurium, Listeria monocytogenes, and Their Surrogates on Cantaloupes after Triple-Washing with Peroxyacetic Acid. LWT - Food Science and Technology, 224, Article 117821.
• Type: Other Journal Articles Status: Published Year Published: 2025 Citation: Jesica Temple, Jessica Blythe, Christopher Ashwell, Tim Boltz, Cangliang Shen. Food Safety Practices Among Small-Scale Produce Growers Exempt from Federal Regulation: A Systematic Review of Knowledge, Attitudes, and Behaviors. PROSPERO 2025 CRD420251033206. Available from https://www.crd.york.ac.uk/PROSPERO/view/CRD420251033206.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Xinhao Wang, Yihang Feng, Yi Wang, Honglin Zhu, Dongjin Song, Cangliang Shen, Yangchao Luo. 2025. Enhancing optical nondestructive methods for food quality and safety assessments with machine learning techniques: A survey. Journal of Agriculture and Food Research 19, Article 101734.
• Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2025 Citation: Huang, K.-M., X. Etienne, and A.C. SantAnna (2025). How Do Preference and Perception of Risks Affect Willingness-To-Pay for Food Safety? Journal of Agricultural and Applied Economics. https://doi.org/10.1017/aae.2025.14

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:Small and very small fresh produce growers in West Virginia who are utilizing the triple wash procedure with antimicrobial solutions for postharvest processing both during and after the project period typically the stakeholder Preston County Workshop. The project also appeals to individuals, organizations, and policymakers interested in reducing food safety hazards of fresh produce at West Virginia. Changes/Problems:Due to the WVU-OSP delaying the processing of this grant funds, the budget was available to use until Oct 2023, although the start date was set at May 1st, 2023. Also due to the very slow process of WVU-OSP to our Co-PD's institions, our subawardees just got their budget to conduct proposed objectives. Therefore, in this short period of project time, we tried our best to complete the proposed objectives as much as we can. What opportunities for training and professional development has the project provided?We recruited 4PhD students Ms. Rebecca Stearns, Mr. Corey Coe, Ms. Jesica Templeand Mr.Md Shafiul Islam Rion, one MS stduent Peighton Foster to conductthe proposed research obejctives. Dr. Rebecca Stearns was sucessfully defended on Dec 2023, and Ms. Peighton Foster was sucessfully defended her MS dergee in the Spring 2024. How have the results been disseminated to communities of interest?We developed and published 2 video short demonstrations about triple-wash andantimicrobial effeicacy ofH2O2-peroxyacetic acid (PAA) which were adopted by the WV Small Farm Conference Center. What do you plan to do during the next reporting period to accomplish the goals?Since the canteloupe is an emerging subject, we are doing triple-wash on the pathogen control on canteloupes. The Co-PD Dr. Luo is developing a nanoparticle to improve the antimicrobial effeicacy ofH2O2-peroxyacetic acid (PAA) for triple-wash applied on fresh produce, which will be used for microbial cross-contamination studies during the next grant cycle. We are also developing the outreach survey for the very small produce growers in WV local community and will compare the results in California. We are expectingto publish 2 peer-review publications and 2-3 conference presentations during the next grant period.

Impacts
What was accomplished under these goals? We first conducted a research study to 1) evaluate the efficacy of H2O2-peroxyacetic acid (PAA) mixer to inactivate Listeria monocytogenes in 0.1% buffered-peptone-water; 2) evaluate the reduction and mitigation of cross-contamination on peppers. For the study-1, aliquots of 1.0-ml mixer solutions (0.25, 0.50 and 0.84%) were added to the first 3-well of 8-strip-deep-well microplates. Then, the 0.1 ml of 10-fold dilution the Nalidixic-acid-resistant (NaL) L. monocytogenes was added and mixed immediately with a multichannel pipette. After exposure for 0, 5, 15, 30, and up to 120-s, 1 ml of 2×D/E neutralized solution was added to terminate the reaction followed by spread-plating onto tryptic soy agar plus 200 ppm NaL and incubated at 35oC for 48 h. For the study-2, 5 inoculated red peppers were triple-washed with 15 uninoculated green peppers with 0, 0.05, 0.1, and 0.18% H2O2-PAA and analyzed using MPN-method. L. monocytogenes counts were <0.3 log10CFU/ml after exposing to 0.25, 0.50, and 0.84% H2O2-PAA solution for 120, 90 and 60 s, respectively and fit the Linear and Weibull Models. Applying 0.05 to 0.18% of H2O2-PAA reduced L. monocytogenes by 2.03 to 3.32 log10MPN/g and transferred cross-contaminated cell counts by 2.36 to 3.17 log10MPN/g, with no differences (P > 0.05) compared to the water only treatment. Results suggested that applying allowable concentrations (0.08 to 0.15%) of H2O2-PAA during triple-wash does not show promising anti-Listeria activity on peppers. Future studies are needed to determine the minimum concentrations of the H2O2- PAA-mixer required to effectively reduce and mitigate microbial cross-contamination of Listeria monocytogenes on peppers. We alsodeveloped an outreach survey to analyze small and very small produce growers' knowledge of food safety. The questionnaire also assessed producers' familiarity and willingness to implement the triple wash method (TWM). Surveys were conducted at FMs in West Virginia. The survey included demographics, knowledge of microbial cross-contamination on produce, washing strategies, awareness of the TWM, and willingness to attend good agricultural practice/ Food Safety Modernization Act (GAPS/FSMA) and TWM training. Data were analyzed using Chi-square test (P < 0.05) in R-software. A total of 82 vendors participated in the survey. The survey results revealed knowledge gaps about food safety among FM produce vendors. Additionally, 53.7% of FM vendors did not wash their produce due to increased spoilage (52.6%; P < 0.05). Among participants who washed produce, only 28.2% were familiar with the TWM. Most respondents who washed produce (65.3%; P < 0.05) stated they would be interested in attending GAP/FSMA and TWM training. The information obtained from this study will help tailor GAP/FSMA and TWM training provided by regional extension offices. We also published 2 video short demonstrations about triple-wash andantimicrobial effeicacy ofH2O2-peroxyacetic acid (PAA). Our subwardee Co-PD Dr. Luo is developing a nanoparticle to improve the antimicrobial effeicacy ofH2O2-peroxyacetic acid (PAA) for triple-wash applied on fresh produce.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2024 Citation: Rebecca Stearns, Wentao Jiang, Janet Tou, Kristen Matak, Annette Freshour, Timothy Boltz, Jacek Jaczynski, Carly Long, and Cangliang Shen*. 2024. A Preliminary Pilot West Virginia Farmers Market Survey of Very Small Produce Growers' Knowledge of Microbial Contamination and Their Perception of the Triple-Wash Method. Food Protection Trend.
• Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Peighton Foster, Corey Coe, Carly Long, Md Shafiul Islam Rion, Gary Freshour, Annette Freshour, Jacek Jaczynski, Kristen Matak, and Cangliang Shen. 2024.Anti-Listeria Efficacy of a Hydrogen Peroxide-Peroxyacetic acid Mixer in Bacterial Buffered Solution and on Bell Peppers. Journal of Agriculture and Food Research
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Peighton Foster, Rebecca Stearns, Corey Coe, Carly Long, and Cangliang Shen. Anti-Listeria Efficacy of a Peroxyacetic Acid-H2O2 Mixer in Bacterial Buffered Solution and on Peppers. 2023 Annual Meeting of International Association of Food Protection. July 16th to July 19th, Toronto, Canada.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Rebecca Stearns, Carly Long, Corey Coe, Lisa Jones, and Cangliang Shen. Survey of Small Local Produce Growers' Knowledge of Microbial Contamination and Perception of the Triple-Wash Method at Farmers Markets. 2023 Annual Meeting of International Association of Food Protection. July 16th to July 19th, Toronto, Canada.