Research Project: Integration and Validation of Alternative and Multiple Intervention Technologies to Enhance Microbial Safety, Quality, and Shelf-life of Food

Location: Microbial and Chemical Food Safety

2023 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 Sub-objectives 1, 2 and 3, which falls under National Program 108, Component I, Foodborne Contaminants, National Action Plan Problem Statement 5, Intervention and Control Strategies, and USDA Science and Research Strategy, 2023-2026, Priority 3: Bolstering Nutrition Security & Health. A detailed description of progress is as follows. Advanced oxidation process involves the generation and utilization of hydroxyl radicals-the most reactive oxygen species. Experiments were conducted to produce hydroxyl radicals using Fenton reaction by combination of iron (III), hydrogen peroxide and ultraviolet light (UV) to inactivate Salmonella on tomato fruits. Response surface methodology was used to discover the best combinations. The formation of hydroxyl radicals was measured using two specific methods. Results showed the combinations reduced the population of Salmonella on dip-inoculated tomatoes by more than 99.99%. The formation of hydroxyl radicals is being analyzed in relationship to the reductions of pathogens. Earlier results showed that lauric arginate ethyl ester (LAE) increased curcumin photoinactivation against Listeria. The mechanism of LAE to enhance photoinactivation was then examined by measuring curcumin stability, bacterial morphology, viability and recovery using electron microscopy methods, and Live/Dead cell assays. Results showed that LAE increased the permeability of cells and subsequently promoted the interaction of reactive oxygen species produced by photo-sensitized curcumin with the cell components. Phenolic branched-chain fatty acid (PBC-FA) is bio-based material synthesized from vegetable oil and natural phenolic compounds. The antimicrobial efficacy of PBC-FA emulsion was investigated against Listeria and Escherichia coli (E. coli) in vitro and on apple fruit. In addition, the stability of PBC-FA emulsion was evaluated during 30-day storage at 4 and 20°C. Results showed that the PBC-FA was effective against Listeria innocua with minimum bacteria concentration of 14 part per millions (ppm), but not effective against E. coli O157:H7 at the tested concentrations. The results on the apple study showed that the PBC-FA at levels of 500 ppm or higher had similar or better antimicrobial activity against L. innocua on apple fruit as the 20 ppm chlorine solution, a sanitizer commonly used by food industry. The quality of the apples was not affected by PBC-FA over the 14-day storage period at 20 C. Overall, that PBC-FA may be used as a sanitizer for apple washing without affecting fruit quality. The use of biodegradable and renewable products with antimicrobial property is of interest as sustainable technology can enhance food safety with minimal impact on the environment. The efficacy of cottonseed protein isolates (CSPI) derived from glanded and glandless kernels for inactivation of foodborne pathogenic bacteria, in comparison to conventional sanitizer (generally recognized a safe compound) were assessed in various experiments. Treatments were efficacious for pathogen inactivation in co-inoculation assays resulting in 3.24-4.82 logs/mL reductions of Salmonella Typhimurium and Listeria (L.) monocytogenes populations. Efficacy of CSPI on pathogen-inoculated post-harvest seeds such as alfalfa, soybean and mungbean showed remarkable Salmonella reductions. The CSPI derived from glandless kernels had low gossypol content, indicating limited toxicity. As an abundant byproduct of cotton kernels, utility of cottonseed protein isolates could be expanded because treatment of seeds designated for sprouting appears plausible, due to its alkaline nature and limited effects on seed germination. Application of MXene in antimicrobial food packaging is being evaluated. In this study, three major foodborne pathogens and two non-pathogens were investigated for their survival on stainless steel disk surfaces coated with MXene. Ten microliters of each culture, with a concentration of 7.5 log CFU/ml, were placed on the center of both MXene coated and non-coated stainless disks and allowed to incubate at room temperature for 48 hours. The results demonstrated that disks coated with MXene displayed significant antimicrobial properties and reduced the bacterial populations by 99% to approximately 99.9% compared with non-coated disks. Wash-resistance and antifouling MXene coatings will be further developed and evaluated. Survival and inactivation of mesophilic bacteria and inoculated populations of Salmonella and L. monocytogenes were investigated on Romaine lettuce, tomatoes and apples after treatments with organic acid-based sanitizers. The effectiveness of the organic acid-based sanitizers was compared with chlorine. Results showed that the efficacy depended on the type of produce with populations of L. monocytogenes and Salmonella on fresh produce being reduced by up to 4 logs.

Accomplishments
1. An organic acid-based sanitizer inactivates pathogens and inhibits browning of fresh-cut fruits. Researchers have advocated for lesser use of chlorine, a common sanitizer, due to concerns on the formation of harmful chlorogenic compounds. In addition, browning of fresh-cut fruits continues to be a problem for consumers and fresh produce industry. Therefore, a new single solution that can act as both antimicrobials and antioxidants for killing bacteria and inhibiting browning of fresh-cut fruits is needed. ARS researchers in Wyndmoor, Pennsylvania, developed a novel, organic acid-based formulation that can simultaneously kill bacteria and inhibit browning of fresh-cut fruits. The multi-organic acids solution killed 97% and 77% of attached Salmonella and Listeria monocytogenes on protected sites of tomatoes and apples, respectively, compared to 82% and 29% achieved with chlorinated water. Furthermore, the browning of cut surface of fruits was inhibited for 7 days. This study is important to ARS partners and stakeholders in addressing food safety problems, in reducing foodborne illness outbreak due to bacterial contaminations, and minimizing food wastes resulting from browning or discoloration of foods.

2. Antimicrobial film improves the safety and shelf-life of grape tomatoes. Grape tomatoes have been associated with outbreaks due to the contamination of foodborne pathogens such as Escherichia coli (E. coli) O157:H7, Listeria monocytogenes and Salmonella spp. Chlorine dioxide is known to be an excellent disinfectant. However, chlorine dioxide is a volatile gas that must be produced on-site and requires sophisticated chemical generation equipment. ARS scientists in Wyndmoor, Pennsylvania, developed packaging films with sodium chlorite (NC) and polylactic acid (PLA) polymer, which were placed inside of containers and released gaseous chlorine dioxide to inactivate pathogens and spoilage microflora on packaged tomatoes. The film treatments reduced the populations of E. coli O157:H7, Listeria monocytogenes, and Salmonella by 99% to 99.99%. While non-treated samples showed moldy surfaces after 2 weeks, film-treated tomatoes still had fresh-like appearances after 2 months at 4 C. This study demonstrates that the use of NC-PLA antimicrobial films is a simple and effective way to enhance microbial safety and extend shelf life of grape tomatoes.

3. Undesirable chemical changes occur in almonds after chlorine dioxide treatments. Almonds, which are rich in “good” lipids and other nutrients, have been implicated in several Salmonella outbreaks. Earlier studies conducted by ARS researchers, have demonstrated that gaseous chlorine dioxide is effective in reducing populations of Salmonella on almonds, especially at elevated treatment temperatures. However, whether the treatments lead to undesirable chemical changes in almonds is unclear. ARS scientists in Wyndmoor, Pennsylvania, evaluated effects of gaseous chlorine dioxide fumigation on volatile compounds and lipid oxidation of almonds during post-fumigation storage. Results showed that gaseous chlorine dioxide increased lipid oxidation by up to 4 times compared with non-treated controls, and led to the formation of several chlorine-containing compounds that were not found in non-treated samples. The results may help the tree nut industry in deciding on the application of the technology, in order to improve the safety of almonds.

Review Publications
Yang, W., Duan, X., Sun, H., Fan, X., Wang, H., Wang, W. 2022. Encapsulation of TA in edible nanofibrous mat improves antioxidant efficiency and their modulation of fatty acids profile in flaxseed oil. International Journal of Food Science and Technology. https://doi.org/10.1111/ijfs.16137.
Guo, M., Zhang, X., Jin, Z.T. 2023. Active Food Packaging. In: Smithers, N.E. editor. Reference Module in Food Science. Amsterdam, Netherlands: Elsevier. https://doi.org/10.1016/B978-0-12-822521-9.00078-2.
Ryu, V.N., Chuesiang, P., Lew, H.N., Ashby, R.D., Fan, X. 2022. Sustainable bio-based antimicrobials derived from fatty acids: synthesis, safety, and efficacy. Critical Reviews in Food Science and Nutrition. https://doi.org/10.1080/10408398.2022.2160430.
Ryu, V.N., Chuesiang, P., Corradini, M., McLandsborough, L., Jin, Z.T., Lew, H.N., Fan, X. 2022. Synergistic photoinactivation of Escherichia coli and Listeria innocua by curcumin and lauric arginate ethyl ester micelles. LWT - Food Science and Technology. 173:114317. https://doi.org/10.1016/j.lwt.2022.114317.
Jin, Z.T., Yadav, M.P., Qi, P.X. 2023. Antimicrobial and physiochemical properties of films and coatings prepared from bio-fiber gum and whey protein isolate conjugates. Food Control. 148:109666. https://doi.org/10.1016/j.foodcont.2023.109666.
Jin, Z.T., Aboelhaggag, R.M. 2022. Combined pulsed electric field with antimicrobial packaging for extending shelf life of orange juice. Beverages. https://doi.org/10.3390/beverages8040072.
Wang, W., Smith, D.J., Lew, H.N., Jin, Z.T., Mitchell, A., Fan, X. 2023. Lipid oxidation and volatile compounds of almonds as affected by gaseous chlorine dioxide treatment to reduce salmonella populations. Journal of Agricultural and Food Chemistry. 71(130):5345-5357.
Sui, X., Meng, Z., Dong, T., Fan, X., Wang, Q. 2023. Enzymatic browning and polyphenol oxidase control strategies. Current Opinion in Biotechnology. 81:102921.
Anto, P., Mu, R., Jin, Z.T., Li, D., Pan, Z., Rakshit, S., Cui, S.W., Wu, Y. 2022. Application of yellow mustard mucilage and starch in nanoencapsulation of thymol and carvacrol by emulsion electrospray. Carbohydrate Polymers. 298:120148.
Mukhopadhyay, S., Ukuku, D.O., Jin, Z.T., Olanya, O.M., Fan, X. 2023. Evaluation of pulsed light treatment for inactivation of Salmonella in packaged cherry tomato and impact on background microbiota and quality. Journal of Food Safety. https://doi.org/10.1111/jfs.13035.
Wang, L., Fan, X., Gurtler, J. 2022. Reduction of Salmonella enterica Typhimurium populations and quality of grape tomatoes treated with dry and humidified gaseous ozone. Postharvest Biology and Technology. 193:112061. https://doi.org/10.1016/j.postharvbio.2022.112061.
Fan, X., Gurtler, J., Mattheis, J.P. 2023. Possible sources of Listeria monocytogenes contamination of fresh-cut apples and antimicrobial interventions during antibrowning treatments: A review. Journal of Food Protection. 86:100100. https://doi.org/10.1016/j.jfp.2023.100100.