Location: Microbial and Chemical Food Safety
2024 Annual Report
Objectives
Objective 1: Utilize novel biological, chemical, and physical technologies to inactivate microbial contamination in and on various food products, which can include and is not limited to produce, nuts, meats and ready-to-eat foods. Directly utilize the hurdle concept to develop processing methods which have a direct application to the need of the industry. Optimize the processes to allow scale-up to commercial treatment levels, appreciating the complexity of the interventions in terms of the food to be treated, the processing conditions, the equipment necessary, and the sensory and nutritional qualities of the food types treated.
Sub-objective 1. Investigate surface characteristics of food and bacteria, bacterial attachment, biofilm formation, and pathogen inactivation mechanisms for potential effective chemical and physical interventions.
Sub-objective 2. Develop and optimize biological, chemical, physical and packaging decontamination interventions that do not affect food quality, making use of pathogen microbial ecology information generated under sub-objective 1
Sub-objective 3. Establish protocols for combination treatments and develop hurdle interventions to achieve additive or synergistic effects on pathogen reduction by combining biological, chemical and physical interventions developed in sub-objective 2, while maintaining or improving the quality and shelf-life of foods.
Sub-objective 4. Conduct scaled-up studies of effective single or hurdle interventions demonstrated in sub-objectives 2 and 3, to facilitate commercialization of the technologies.
Approach
This project will progress in four phases: elucidate pathogen ecology and inactivation mechanisms; evaluate single interventions; apply a combination of interventions; and conduct pilot scale studies (Fig. 1). Initially, we will investigate bacterial attachment, biofilm formation, and bacterial inactivation mechanisms as affected by potential interventions. This knowledge will aid us in choosing and optimizing biological, chemical, packaging and physical control strategies. Selection of intervention technologies will also be based on earlier research conducted by our own group and by others. The optimized interventions will be strategically integrated to achieve additive and synergistic effects on pathogen reduction. The effects of these processing technologies on product quality attributes will be evaluated using instruments to measure quality aspects in conjunction with sensory panels. Both individual and combination intervention technologies capable of achieving the desired performance standards for pathogen reduction, quality and shelf life will be optimized and validated in scaled up studies in our unique BSL-2 pilot plant for large-scale trials where large volumes of foods can be treated. The types of foods evaluated in the project will be those frequently involved in outbreaks of foodborne illnesses or associated with emerging pathogens, fresh produce items that are hard to sanitize due to surface characteristics, and foods that cannot be subjected to traditional wash treatments. To facilitate commercialization of effective interventions, collaborations with the food industry will be established by actively fostering interactions with stakeholders. Stakeholders will be updated via direct interactions, site visits, annual scientific meetings and trade shows regarding research goals and objectives of the project, and inputs will be solicited to identify key problems to be solved so that the technologies will be more relevant and applicable. Although much of the effort will be on hurdle technologies that combine various individual interventions, effective individual treatments along with integrated ones will be tested in pilot scale studies. In addition, even though the project will be conducted in a progressive 4-stage process, the flow or order of research accomplishments will not be strictly chronological as there will be opportunities for technologies to be implemented by the industry at every stage of the project via establishment of research and development agreements and technology transfer.
Progress Report
Progress has been made on Objective 1, which falls under National Program 108, Component I, Foodborne Contaminants, and USDA Science and Research Strategy, 2023-2026, Priority 3: Bolstering Nutrition Security & Health. A detailed description of the progress is as follows.
Combinations of different non-thermal interventions have been evaluated for the inactivation of pathogens on fresh produce. A new organic acid-based sanitizer developed in our laboratory was able to reduce about 99.99% of bacterial pathogens on produce surfaces. A combination of the sanitizer with cold plasma led to 99.999% reduction of attached pathogens. Modification of the sanitizer solution and a combination with other non-thermal technologies such as ultraviolet (UV) light, pulse light etc. will be tested and the mechanism of inactivation will be investigated.
UV-assisted Fenton reaction, i. e. combinations of hydrogen peroxide (H2O2), iron ion and UV light, was tested against Salmonella, Escherichia coli (E. coli) and Listeria on cherry tomatoes. Cherry tomatoes inoculated with cocktails of Salmonella Typhimurium, E. coli O157:H7 and Listeria monocytogenes were washed with solutions of H2O2, ferric ion (Fe3+) and/or exposed to UV-C during washing. Results showed that the reductions of Salmonella populations by the H2O2+Fe3++UV-C treatment were greater than H2O2 or UV-C alone. However, for E. coli and Listeria, the reductions by the combined treatment were not significantly different from individual treatments (H2O2 or UV-C).
Storage temperatures, lauric acid amide pyrrolidine (LAPY, a biosurfactant), Bacillus (B.) subtilis (a competitive exclusion microbe), and water rinse were determined for their effects on the attachment and survival of Salmonella Typhimurium on post-harvest produce. Microbial populations on untreated produce varied within produce types. Types of produce also influenced Salmonella attachment, with greater attachment strength observed on pistachio and soybean than on alfalfa and carrot. When applied on produce, the biosurfactant LAPY, significantly reduced Salmonella populations by 99-99.9% on alfalfa, pistachio and soybean seeds, but not on carrots (< 97% reduction). While the application of B. subtilis on produce and seed did not result in any significant reductions in Salmonella populations, combining B. subtilis and an organic acid-based sanitizer resulted in low to moderate reductions of pathogens.
Cotton seed protein isolate (CSPI) and an organic acid-based sanitizer were investigated for the inactivation of Salmonella Typhimurium and Listeria monocytogenes in-vitro and on post-harvest seeds. In the in-vitro assays at 25°C (room temperature), the organic acid-based sanitizer reduced Salmonella populations by more than 99.999%, while CSPI reduced Salmonella populations by 99.9%. When the sanitizer and CSPI were applied on seeds (alfalfa, almond, soybean and mungbean), Salmonella reductions ranged from 66% to 99.999%. The pH values of CSPI exceeded 12.5, indicating CSPI being highly alkaline, while the sanitizer was highly acidic (pH<1.6). CSPI did not have any adverse effects on seed germination, but the sanitizer had detrimental effects on seed germination. The sanitizer and CSPI demonstrated antimicrobial activity against Salmonella and Listeria monocytogenes in the in-vitro assays and on post-harvest seed.
Activated hydrogen peroxide aerosols produced by passing hydrogen peroxide droplets through a cold plasma arc, were tested against Salmonella Typhimurium and E. coli. To simulate commercial conditions, the activated aerosols were applied as an overhead spray for less than 1 min (5-60 seconds) onto food contact surfaces, lettuce and tomatoes. Results showed their efficacy against Salmonella and E. coli depended on the surface characteristics of fresh produce and materials. On smooth surface, more than 99.999% reductions of bacteria could be inactivated with a few seconds of treatments.
The efficacy of antimicrobial coating and acid washing to inactivate microorganisms on mung bean sprouts was investigated. Populations of E. coli and Listeria inoculated on seeds were significantly lower following coating treatments. Coated samples had lower native bacterial populations, and yeast and mold counts than control samples. There were no significant differences in germination rate or sprout yield among all treatments. These results demonstrate a new approach to reducing microbial contaminants for growing sprouts, without negatively impacting germination rate or yield.
Accomplishments
1. Minimizing formation of undesirable chlorine byproducts in fresh produce wash water. To maximize the antimicrobial efficacy of chlorine, a common sanitizer used by the produce industry, the pH of chlorinated water is often adjusted using citric acid. Earlier ARS results indicated that citric acid reacted with chlorine and generated high amounts of trichloromethane, a probable carcinogen. Alternatives to citric acid are needed to minimize formation of chlorine byproducts. ARS scientists in Wyndmoor, Pennsylvania, evaluated 13 organic and inorganic pH adjusters as potential alternatives to citric acid. Results showed that inorganic acids and many organic acids other than citric and malic acids maintained the levels of effective chlorine in wash water and led to little formation of trichloromethane (less than 5 parts per billion). Therefore, it is recommended to the food industry that inorganic acids and certain organic acids should replace citric acid as pH control agents in chlorinated water to stabilize the chlorine level in wash water, and to minimize the formation of undesirable chlorine byproducts.
2. Combination of antimicrobial treatments and packaging improves microbial safety and shelf-life of fresh root produce. Fresh baby carrots are often used in salad and consumed without cooking, which could lead to illnesses if carrots were contaminated with foodborne pathogens. ARS scientists in Wyndmoor, Pennsylvania, investigated the efficacy of antimicrobial washing, antimicrobial released film, packaging methods (pouch vs. tray), and their combinations in reducing populations of Escherichia coli (E. coli), spoilage bacteria and fungi. Antimicrobial solution washing combined with antimicrobial film showed the most antimicrobial effectiveness for both pouch and tray packaging, and there was no survival of E. coli on carrots after treatments. There were no significant changes in texture and weight loss during storge at 4 degree C for 8 months. The combined methods can enhance the safety and extend the shelf life of baby carrots and other fresh root produce.
3. Bio-based antimicrobials from fatty acids and phenolic compounds. Foodborne disease outbreaks and recalls have been linked with fresh apples due to Listeria monocytogenes contamination occurring during postharvest handling. Sustainable and effective anti-Listeria antimicrobials are needed. ARS scientists at Wyndmoor, Pennsylvania, developed formulations based on ARS-patented antimicrobials to reduce the population of Listeria on apples. Results demonstrated that antimicrobials from phenolic compounds and fatty acids reduced populations of Listeria on apple fruit by up to 99.99% when applied by wash or as a coating. The sanitizing or coating treatment did not negatively impact the quality of apples during 2-week shelf life at ambient temperature. The bio-based antimicrobials provide the apple industry a sustainable approach to enhance microbial safety and maintain quality of fresh apples.
4. Limiting Salmonella attachment on produce and seed. Reducing Salmonella attachment on post-harvest produce and seed is crucial for mitigating pathogen contamination and its survival. ARS scientists in Wyndmoor, Pennsylvania, determined the factors that impact the attachment and survival of Salmonella on fresh produce and seed. Bacillus subtilis, a bacterial competitor, had limited effect on Salmonella attachment, but produce types and biosurfactant influenced the strength of bacterial attachment. Water rinses of fresh produce significantly reduced the attachment of Salmonella cells. Selective application of post-harvest decontamination measures may limit pathogen survival and enhance produce safety.
Review Publications
Ryu, V.N., Uknalis, J., Corradini, M.G., Chuesiang, P., McLandsborough, L., Lew, H.N., Jin, Z.T., Fan, X. 2023. Mechanism of synergistic photoinactivation utilizing curcumin and lauric arginate ethyl ester against Escherichia coli and Listeria innocua. Food Chemistry. 12(23):4195. https://doi.org/10.3390/foods12234195.
Ryu, V.N., Chuesiang, P., Uknalis, J., Lew, H.N., Jin, Z.T., Fan, X. 2024. Bio-based phenolic branched-chain fatty acid emulsion achieved similar reductions of Listeria innocua population on apple fruit as chlorinated water. Food Control. 10(3):e24901. https://doi.org/10.1016/j.heliyon.2024.e24901.
Fan, X., Gurtler, J. 2024. Depletion of free chlorine and generation of trichloromethane in the presence of pH control agents in chlorinated water at pH 6.5. Journal of Food Protection. 87(7):100296. https://doi.org/10.1016/j.jfp.2024.100296.
Ryu, V.N., Uknalis, J., Lew, H.N., Jin, Z.T., Fan, X. 2024. Coating with phenolic branched-chain fatty acid reduces Listeria innocua populations on apple fruit. International Journal of Food Microbiology. 419:110748. https://doi.org/10.1016/j.ijfoodmicro.2024.110748.
Kazem Rostami, M., Ryu, V.N., Wagner, K., Jones, K.C., Mullen, C.A., Wyatt, V.T., Wu, C., Ashby, R.D., Fan, X., Lew, H.N. 2023. Antibacterial agents from waste grease: Arylation of brown grease fatty acids with beechwood creosote and derivatization. ACS Sustainable Chemistry & Engineering. https://doi.org/10.1021/acssuschemeng.3c05767.
Mukhopadhyay, S., Ukuku, D.O., Olanya, O.M., Niemira, B.A., Jin, Z.T., Fan, X. 2023. Combined treatment of pulsed light and nisin-organic acid based antimicrobial wash for inactivation of Escherichia coli O157:H7 in Romaine lettuce, reduction of microbial loads, and retention of quality. Food Microbiology. https://doi.org/10.1016/j.fm.2023.104402.
Olanya, O.M., Yosief, H.O., Ashby, R.D., Niemira, B.A., Sarker, M.I., Ukuku, D.O., Mukhopadhyay, S., Msanne, J.N., Fan, X. 2023. Inactivation of foodborne and other pathogenic bacteria with pyrrolidine based fatty acid amide derivatives. Journal of Food Safety. https://doi.org/10.1111/jfs.13079.
Olanya, O.M., Mukhopadhyay, S., Ukuku, D.O., Niemira, B.A., Uknalis, J. 2024. Attachment of Salmonella Typhimurium and survival on post-harvest produce and seed. Food Science and Technology Research. 2024(3):457-465. https://doi.org/10.3136/fstr.FSTR-D-24-00032.
Inman, C.A., Shevchuk, K., Anayee, M., Hammill, B., Lee, J., Saraf, M., Shuck, C., Armstrong, C.M., He, Y., Jin, Z.T., Shekhirev, M., Capobianco Jr, J.A., Gogotsi1, Y. 2023. High-yield and high-throuhput delamination of multilayer MXene via high-pressure homogenization. Chemical Engineering Journal. 475:146089. https://doi.org/10.1016/j.cej.2023.146089.