Research Project: Intervention Strategies to Mitigate the Food Safety Risks Associated with the Fresh Produce Supply Chain

Location: Environmental Microbial & Food Safety Laboratory

2022 Annual Report

Objectives
Objective 1: Evaluate the microbial safety of alternative fresh produce production systems and the efficacy of preventive practices. Sub-objective 1.a: Investigate sources of pathogen contamination on fresh produce grown by aquaponics (APs). Sub-objective 1.b: Determine the relationship of AP design and operational practices on microbiome characteristics and efficacy of water and probiotic treatments in reducing pathogen risks on produce. Sub-objective 1.c: Develop mitigation strategies for controlling pathogens in microgreen and sprouts. Objective 2: Develop and evaluate novel mitigation strategies to control foodborne pathogens at pre- and post-harvest levels. Sub-objective 2.a. Develop pre-harvest interventions to control pathogens on leafy greens. Sub-objective 2.b. Develop intervention strategies to control pathogens on fresh-cut leafy greens while maintaining quality and shelf life. Sub-objective 2.c. Optimize fresh-cut process to reduce pathogen presence on tomatoes. Objective 3: Characterize pathogen interactions with multi-species biofilms and efficacy of interventions on abiotic surfaces. Sub-objective 3.a. Investigate single and multi-species biofilm formation by foodborne bacterial pathogens in fresh produce processing environments. Sub-objective 3.b. Develop mitigation strategies to control biofilm formation in fresh produce processing environment.

Approach
Sources of pathogen contamination of fresh produce grown using aquaponics production (AP) system will be investigated. The aquaponics system inputs (seeds, transplant media, water, fish, feed) will be analyzed for fecal coliforms, spoilage bacteria, and foodborne pathogens (Shiga-toxigenic E.coli, Salmonella spp., Listera monocytogenes). Fresh produce (basil, cilantro, brassica leafy greens, pepper, pak choi) grown by AP system will be evaluated for spoilange and pathogenic bacteria, and produce yield. The effect of probiotics in controlling surrogate pathogens (avirulent E. coli, L. innocua, Salmonella spp.) on fresh produce seeds/seedlings and APs water will be determined. Nanobubble, UV light, and plasma functionalized water will be evaluated for seed germination. plant growth promotion, and system disinfection. Changes in fish and plant microbiome due to antimicrobial treatments will be evaluated. Sprout seeds (alfalfa, broccoli, clover, mung bean, radish) treated with glucosonilate compounds will be evaluated for rate of seed germination, inhibition of foodborne pathogens, and potential transfer of inoculated pathogens to sprouts. Seeds of microgreens contaminated with pathogens will be grown in different growing media to determine the effect of growth media on pathogen survival, and yield and quality of microgreens. Biocontrols and plant-based nanoemulsions will be investigated as pre-harvest interventions to control pathogen surrogate E. coli O157:H12 at the farm level. The effect of size distribution of particulates generated during commercial fresh-cut vegetables (baby spinach, diced cabbage, shredded lettuce) operation on wash water turbidity and subsequent pathogen cross-contamination will be investigated. Te effect of wash-cut sequence on pathogen cross-contamination on tomatoes will be evaluated and fresh-cut processing of tomatoes will be optimized to minimize pathogen contamination. Multi-species biofilm formation of L. monocytogenes and E. coli O157:H7 with promotor strain Ralstonia insidiosa will be assessed on different equipment surfaces at variable flow rates. Biofilm studies will be simulated in pilot plant to identify potential hotspots of biofilm formation. Novel antimicrobials such as plant-based nanoemulsion, antagonistic bacteria; and new technologies (dry steam, nonflammable ethanol, surface coating) will be investigated for biofilm removal in laboratory and pilot plant studies, respectively.

Progress Report
Progress was made on all objectives and their sub-objectives, which fall under National Program 108, Component 1, Foodborne Contaminants. Activities of this project focus on Problem 1, Population Systems, and Problem 5, Intervention and Control Strategies. Under Objective 1, facilities operating aquaponic and hydroponic leafy green and vegetable production systems were evaluated for water quality, design and operational factor impacts on the microbial (bacterial and protozoan) populations. Pathogens were not detected in roots, shoots, or nutrient solutions. Post-harvest handling and potential contact of consumable harvest with roots and inadvertent droplet contact with the ‘Ag water’ component was identified as a potential food safety risk requiring evaluation. Inoculated challenge experiments revealed a rapid decline in E. coli and Listeria in planktonic water samples, but persistence in system biofilters and on roots. Results from multiple systems reveal growth inhibition. The effect of microgreen production on pathogen persistence was investigated. Salmonella populations increased significantly on microgreens grown on recycled Biostrate pads. Microgreens grown in composted cattle manure-soil had higher yield and lower persistence of E. coli O157:H7. Prevalence of fecal indicator bacteria and pathogens in sediment samples collected from Conococheague creek, Pennsylvania, was determined. Salmonella, E. coli O157:H7, and L. monocytogenes werte recovered from 6%, 0.5% and 70% sediment samples, respectively. Under Objective 2, ARS scientists evaluated Benzyl isothiocyanate (BIT), a naturally occurring compound in Brassica vegetables, to control pathogens on spinach at the farm level. Nano-emulsified BIT was effective in controlling surrogate E. coli on all four spinach cultivars at the farm prior to harvest. The color of BIT-treated spinach leaves was comparable to non-treated leaves 3 days after treatment. ARS scientists in collaboration with scientists from the University of Florida assessed data on dynamic changes in water quality and bacterial populations in wash flumes and on tomatoes during the commercial operation of tomato packinghouses. The turbidity, chemical oxygen demand, and total dissolved solids increased over time in the flumes of all three packinghouses while oxygen-reduction potential and free chlorine concentration fluctuated and varied among packing houses. Additionally, while aerobic bacterial populations varied among packing houses and over time, E. coli was not detected in any water samples. Washing with this new sanitizer formulation containing peracetic acid efficiently reduced Salmonella on diced tomatoes and the transfer to non-inoculated tomato diced after washing. This new sanitizer inhibited proliferation of most dominant spoilage bacteria such as Erwiniaceae, Curtobacterium, Pantoea, Erwinia and Enterobacterales spp. on diced tomatoes during storage. The case distribution patterns of romaine lettuce associated enterohemorrhagic E. coli (EHEC) outbreaks from 2017 to 2020 suggested the “forward processing”, an industry practice of shipping raw commodity from production area in California and Arizona to distant facilities (eg. East Coast) for processing and regional marketing, could be a factor in these outbreaks. ARS scientists continued collaboration with commercial romaine producers/processors to conduct research characterizing the forward processing practices and potential risks on the survival of EHEC and effect on cell physiology and infectivity. ARS scientists collaborated with scientists from Georgia Institute of Technology to evaluate the formation of chlorine disinfection byproducts (DBPs) during commercial washing of fresh-cut produce. Total 33 conventional and emerging DBPs were investigated in water and produce samples; concentrations of four trihalomethanes (THMs) and nine haloacetic acids (HAAs) on the washed products were further used to estimate potential exposure via consumption of fresh-cut produce. L. monocytogenes inactivation was observed on cut carrot surfaces, as well as in water soaked with carrot pieces. In collaboration with scientist at National Institute of Standards and Technology (NIST), ARS scientists identified chemical compounds in carrot which was inhibitory effect against L. monocytogenes. Under Objective 3, Listeria monocytogenes biofilm formation in cantaloupe juice was investigated. L. monocytogenes showed strong aggregation and biofilm formation after growth in cantaloupe juice but not in bacterial growth medium. In collaboration with the FDA, RNA was extracted from L. monocytogenes planktonic cells which revealed significant up/down regulation of 1247 differentially expressed genes. Surface topography, shear stress, and presence of promotor bacteria influenced biofilm formation of L. monocytogenes on equipment surfaces. Ralstonia insidiosa promoted stronger biofilm formation of this pathogen at lower shear stress. Biofilm formation was affected by surface skewness of stainless steel, polycarbonate, Teflon, and rubber materials.

Accomplishments
1. Control of pathogen contamination in tomatoes during washing. Tomatoes have been associated with numerous foodborne illnesses outbreaks due to contamination with food-borne human pathogens. The industry needs scientific information regarding effective preventive control measures against pathogen contamination and spread during tomato wash operations. ARS scientists in Beltsville, Maryland, collaborated with scientists from the University of Florida in evaluating the effects of antimicrobial agents on pathogen survival, inactivation, and cross-contamination. Results indicated that a free chlorine concentration of 25 ppm suffices to prevent pathogen spread via tomato wash water containing a typical organic matter load. These findings will benefit the tomato industry in establishing science- and risk-based food safety regulations and industry standards that can improve food- and worker-safety associated with tomato industry practices.

2. Defining survival and growth of L. monocytogenes on whole and fresh-cut produce. In responding to industry call to extend current knowledge of L. monocytogenes growth on fresh produce, ARS scientists assessed the survival and growth of this pathogen on 20 types whole and fresh-cut produce under simulated commercial storage/retail display conditions. L. monocytogenes populations showed a sustained decrease on all tested whole fruits, including avocado, blueberry, grape, mango, peach, green pepper, and tomato. Significant increase in L. monocytogenes populations was observed on fresh-cut cantaloupe and fresh-cut mango. Exposure to temperature abuse conditions did not change the overall trends of L. monocytogenes survival on the whole fruits but resulted in significant growth on several fresh-cut products. The information is useful for the fresh produce industry in L. monocytogenes risk assessment.

3. Biocontrol of bacterial pathogens on strawberries. Contaminated strawberries have been implicated in outbreaks of foodborne illnesses. Lactic acid bacteria (LAB) were used as a biocontrol to control bacterial pathogens on strawberries during refrigerated storage. Strawberries inoculated with Salmonella or L. monocytogenes were dipped in LAB and stored under refrigeration temperature (4 and 10°C). LAB treatment significantly reduced both pathogens during storage at 7 days. The findings are useful to strawberry packers in controlling pathogens on strawberries using biocontrol.

4. Natural antimicrobials inhibit pathogens on fresh produce. The incidence of foodborne illnesses associated with consumption of fresh produce has continued to increase over the past decade. Natural antimicrobials: fruit extracts and carvacrol (essential oil from oregano) were evaluated to control pathogens on cucumber and fresh produce, respectively. Whole and fresh-cut cucumbers inoculated with Salmonella were washed in extracts of lemon, yuzu, grapes, or citrus fruits. Similarly, spinach and lettuce inoculated with E. coli O157:H7 were washed in nano-emulsified carvacrol (NC) and stored under refrigeration. Lemon extract significantly reduced Salmonella on whole and fresh-cut cucumbers during storage. Likewise, nano-emulsified carvacrol was effective in reducing E. coli O157:H7 on spinach and lettuce (romaine and iceberg) during 14 days of storage at 10°C. The NC treatment did not significantly affect the color of fresh produce leaves. The results benefit processors in controlling pathogens on fresh produce.

5. Water soluble corn zein coating inhibits Listeria on fruits. Pathogen contamination on fresh produce is a major source of foodborne illness outbreaks. In collaboration with Akorn Technology, ARS scientist in Beltsville, Maryland, used edible coating incorporated with natural antimicrobial nisin to control L. monocytogenes on nectarine and apple. L. monocytogenes populations were reduced by 1 log on nectarine with coating. The coating resulted in rapid decline of L. monocytogenes populations on Gala apples during the earlier storage; and it was superior to conventional wax throughout the entire storage period against L. monocytogenes. The findings are useful to fresh produce packers in controlling pathogen contamination, especially on tree fruits.

6. Shift in produce microbiome with pathogen contamination. The indigenous microorganisms on fresh produce could strongly influence the survival and growth of contaminating foodborne pathogens. The dynamics of inoculated L. monocytogenes and native microbiome on fresh-cut cantaloupe and romaine lettuce during storage at refrigerated and abused temperatures. L. monocytogenes rapidly grew on fresh-cut cantaloupe to overtake indigenous bacteria. Microbial diversity of fresh-cut cantaloupe was significantly lower than that of fresh-cut romaine lettuce. Multiple indigenous bacteria, such as Leuconostoc and Weissella spp. on romaine lettuce had potential anti-listeria effect. Better understanding of the microbial dynamics on fresh produce could lead to more efficient controls to foodborne pathogen contaminations.

Review Publications
Aytac, Z., Xu, J., Kumar, S., Eitzer, B., Xu, T., Vaze, N., Ng, K., White, J., Chan-Parkb, M., Luo, Y. 2021. Enzyme- and relative humidity-responsive antimicrobial fibers as active food packaging materials. ACS Applied Materials and Interfaces. 13(42):50298. https://doi.org/10.1021/acsami.1c12319.
Bertoldi, B., Bardsley, C.A., Baker, A.C., Pabst, C.R., Gutierrez, A., De, J., Luo, Y., Schneider, K.R. 2021. Determining bacterial load and water quality of tomato flume tanks in Florida packinghouses. Journal of Food Protection. 84(10):1784–1792. https://doi.org/10.4315/JFP-21-100.
Bertoldi, B., Bardsley, C.A., Pabst, C.R., Baker, C., Gutierrez, A., De, J., Luo, Y., Schneider, K.R. 2021. The influence of organic load and free chlorine on Salmonella cross-contamination of tomatoes in a model flume system. Journal of Food Protection. https://doi.org/10.4315/jfp-21-212.
Byun, S., Chen, C., Yin, H., Patel, J.R. 2022. Antimicrobial effect of natural fruit extracts against Salmonella on whole and fresh-cut Cucumbers. Journal of Food Safety. https://doi.org/10.1111/jfpp.16437.
Chen, C., Yin, H., Teng, Z., Byun, S., Guan, Y., Luo, Y., Upadhyay, A., Patel, J.R. 2021. Nanoemulsified carvacrol as a novel washing treatment reduces Escherichia coli O157:H7 on fresh produce. Journal of Food Protection. 84(12):2163-2173.
Cohen, Y., Mwangi, E., Tish, N., Xu, J., Vaze, N., Falik, E., Luo, Y., Demokritou, P., Rodov, V., Poverenov, E. 2022. Biopolymer-based sanitizers for fresh produce, traditional application vs dry engineered water nanostructures approach. Food Chemistry. 378:132056. https://doi.org/10.1016/j.foodchem.2022.132056.
Gu, G., Kroft, B., Lichtendwald, M., Luo, Y., Millner, P.D., Patel, J.R., Nou, X. 2022. Dynamics of Listeria monocytogenes and microbiome on fresh-cut cantaloupe and romaine lettuce during storage at refrigerated and abusive temperatures. International Journal of Food Microbiology. 364:109531. https://doi.org/10.1016/j.ijfoodmicro.2022.109531.
Gu, T., Meesrisom, A., Luo, Y., Dinh, Q.N., Lin, S., Yang, M., Sharma, A., Tang, R., Zhang, J., Jia, Z., Millner, P.D., Pearlstein, A.J., Zhang, B. 2021. Listeria monocytogenes biofilm formation as affected by stainless steel surface topography and coating composition. Food Control. 130:108275. https://doi.org/10.1016/j.foodcont.2021.108275.
Liu, Z., Sun, J., Teng, Z., Luo, Y., Yu, L., Simko, I., Chen, P. 2021. Identification of marker compounds for predicting browning of fresh-cut lettuce using untargeted UHPLC-HRMS metabolomics. Postharvest Biology and Technology. 180.Article 111626. https://doi.org/10.1016/j.postharvbio.2021.111626.
Ma, P., Zhang, J., Teng, Z., Zhang, Y., Bauchan, G.R., Luo, Y., Liu, D., Wang, Q. 2021. Development of metal-organic framework stabilized high inner phase Pickering emulsions based on computer simulation for curcumin encapsulation. Food Hydrocolloids. 6(40):2655626565. https://doi.org/10.1021/acsomega.1c03932.
Mendes-Oliveira, G., Gu, G., Luo, Y., Zografos, A., Nou, X. 2021. Edible and water solvable corn zein coating impregnated with nisin for Listeria monocytogenes reduction on nectarine and apple. Postharvest Biology and Technology. 185:111811. https://doi.org/10.1016/j.postharvbio.2021.111811.
Peng, H., Luo, Y., Teng, Z., Zhou, B., Bornhorst, E.R., Fonseca, J.M., Simko, I. 2021. Phenotypic characterization and inheritance of enzymatic browning on cut surfaces of stems and leaf ribs of romaine lettuce. Postharvest Biology and Technology. 181. Article 111653. https://doi.org/10.1016/j.postharvbio.2021.111653.
Tan, J., Zhou, B., Luo, Y., Karwe, M. 2021. Numerical simulation and experimental validation of bacterial detachment using a spherical produce model in an industrial-scale flume washer. Food Control. 130:108300. https://doi.org/10.1016/j.foodcont.2021.108300.
Wang, T., Wusigale, Kuttappan, D., Amalaradjou, M., Luo, Y., Luo, Y. 2021. Polydopamine-coated chitosan hydrogel beads for synthesis and immobilization of silver nanoparticles to simultaneously enhance antimicrobial activity and adsorption kinetics. Advanced Composites and Hybrid Materials. 4:696–706. https://doi.org/10.1007/s42114-021-00305-1.
Xue, J., Luo, Y., Li, B., Wang, X., Xiao, Z., Luo, Y. 2022. Antimicrobial effects of thymol-loaded phytoglycogen/zein nanocomplexes against foodborne pathogens on fresh produce. Food Control. 209:1188-1196.
Xue, J., Luo, Y., Balasubramanian, R., Upadhyay, A., Li, Z., Luo, Y. 2021. Development of novel biopolymer-based dendritic nanocomplexes for encapsulation of phenolic bioactive compounds: a proof-of-concept study. Food Hydrocolloids. 120:106987. https://doi.org/10.1016/j.foodhyd.2021.106987.
Zhang, T., Lee, W., Luo, Y., Hunag, C. 2021. Flume and single-pass washing systems for fresh-cut produce processing: Disinfection by-products evaluation. Food Control. 133:108578. https://doi.org/10.1016/j.foodcont.2021.108578.
Yin, H., Chen, C., Colorado-Suarez, S., Patel, J.R. 2022. Biocontrol of Listeria monocytogenes and Salmonella enterica on fresh strawberries with lactic acid bacteria during refrigerated storage. Foodborne Pathogens and Disease. 19(5):324-331. https://doi.org/10.1089/fpd.2021.0091.