1a. Objectives (from AD-416):
This project will focus on the integration of effective intervention technologies and treatments to enhance microbial safety of fresh fruits and vegetables with a holistic approach addressing major elements (safety, quality, and shelf-life), necessary for the implementation of technologies. The ultimate goal is to reduce the risk of foodborne illnesses associated with consumption of fresh produce, while maintaining acceptable food quality and shelf-life. Specific objectives of the research program are: Objective 1 - Develop and optimize single intervention technologies to reduce pathogen populations, maintain quality, and extend shelf-life of foods. Sub-objective 1.1. Develop and optimize aerosolizing technology and pulsed light to reduce pathogen populations, maintain sensorial and nutritional quality, and extend shelf-life of fresh produce. Sub-objective 1.2. Develop new antimicrobial packaging/coating-based technologies by incorporating natural, modified and novel materials to inactivate foodborne pathogens on fresh produce. Objective 2 - Determine the synergistic/additive effectiveness of combining non-thermal processing, antimicrobial packaging and effective chemical interventions utilizing information generated from the first objective.
1b. Approach (from AD-416):
An integrated approach to enhance microbial safety while maintaining product quality and extending shelf life of fresh produce will be adopted by combining aerosolizing antimicrobials, pulsed light antimicrobial packaging and other interventions. The types of fresh and fresh-cut produce evaluated in the project will be those frequently involved in outbreaks of foodborne illnesses, those that are hard to sanitize due to surface characteristics, and those that cannot be washed. During the first part of the proposed project plan, we will develop and optimize new aerosolization systems, pulsed light technology, novel antimicrobial coating with incorporation of nature and bio-based substances, and antimicrobial packaging materials with controlled-release mechanisms triggered with either acids or pulsed light. The optimized/developed interventions and antimicrobial packaging will then be combined with each other, and with other effective antimicrobial treatments to study the synergistic or additive effects on pathogen inactivation while maintaining quality and shelf-life of fresh produce. When selecting combinations, technologies with different pathogen-inactivation mechanisms or synergistic interactions will be desirable. We will utilize the advanced oxidation, photochemical, and photothermal, and triggered-release mechanisms and other hurdle technologies to increase the efficacy of combined antimicrobial treatments. By combining effective intervention technologies and treatments, synergistic effects with a targeted 5-log reduction of common pathogens may be achieved. Pathogens to be included in the proposed project plan are Salmonella spp., E. coli O157:H7, L. monocytogenes and other emerging pathogens (such as non-O157 STECs). We will use a representative cocktail of 3-5 strains from each genera of bacteria that are associated with outbreaks of relevant fresh produce. Scientifically well-established inoculation, recovery, and enumeration procedures will be used. Appropriate controls will be included in each experiment, and experiments will be replicated independently at least three times. Inoculation of fresh produce will be achieved either by surface ‘spot inoculation’ where specific locations on the produce surface will be inoculated or by a ‘dip inoculation’ technique where the whole produce item will be submerged in the experimental inocula. The inoculated fresh produce will be drained and air dried in a laminar flow hood before being subjected to various treatments. After treatment with various chemicals and physical interventions, the total number of viable and injured bacteria will be determined using amended media. The effects of the individual and combined treatments on the physiochemical and sensorial quality and shelf-life will be evaluated during storage. Shelf-life will be determined based on the deterioration in product quality and increasing populations of microorganisms that render the product unacceptable to consumers.
3. Progress Report:
Progress was made on Objectives 1 and 2, which fall under National Program 108, Component I, Foodborne Contaminants, and National Action Plan Problem Statement 5, Intervention and Control Strategies. Experiments were conducted to develop antimicrobial films that release chlorine dioxide gas, integrate sanitizer washes with pulsed light, and combine aerosolized hydrogen peroxide with gaseous ozone in order to maximize the effectiveness in inactivating various human pathogens and spoilage microorganisms while maintaining sensory quality of fresh produce. Detailed progress to achieve the overall objectives is listed below. Under Objective 1, significant progress was made by continuous development and evaluation of antimicrobial films that release gaseous chlorine dioxide into the headspace of packaging containers. The concentration of sodium chlorite (a chlorine dioxide precursor) in the films and the methods to make the films (press, coating, etc.) were evaluated for their antimicrobial efficacy against foodborne pathogens (Listeria, Salmonella, and E. coli) and spoilage microorganisms in growth media, broccoli, strawberries and tomatoes. Results show that these antimicrobial films placed inside of food containers effectively killed or inhibited the growth of pathogens and spoilage microorganisms. Further studies will be conducted to evaluate the impact of these treatments on the quality of treated foods. Under Objective 2, experiments were conducted to develop and validate a method using pulsed light followed by sanitizer washing for greater than 3 log reductions of common food borne pathogens in fresh leafy green and tomato fruits without significant quality deterioration. Among various sanitizers tested, hydrogen peroxide + EDTA + nisin, and organic acid + EDTA + nisin exhibited synergistic effects with pulsed light, inactivating more than 99.999% of Salmonella and E. coli O157:H7 on tomato and spinach leaves, respectively. In addition, the treatments also reduced initial native microbial populations on these produces and slowed their growth during storage. Furthermore, color and firmness of the produce were not significantly affected by the combination treatment method. Overall, results demonstrate that the integrated pulsed light-sanitizer technology can be used to enhance microbial safety of produce. Also under Objective 2, significant progress was made by an advanced oxidation process which combined aerosolized hydrogen peroxide and gaseous ozone to inactivate Salmonella Typhimurium on tomato fruits. Tomatoes inoculated with a cocktail of Salmonella Typhimurium strains on stem scar and smooth surface were treated simultaneously with combinations of gaseous ozone and hydrogen peroxide aerosolized from various concentrations of hydrogen peroxide solutions. Results showed that combinations of aerosolized hydrogen peroxide with 1,600 ppm ozone frequently reduced the populations of Salmonella by 99.999% on the smooth surface of the fruit. On the stem scar area, combinations of 1,600 ppm ozone and aerosolized hydrogen peroxide achieved 99.99% of Salmonella. Overall, our results suggest that the advanced oxidation process with combinations of hydrogen peroxide and gaseous ozone may be used to inactivate Salmonella on tomato fruits. Studies are underway to evaluate quality of fruits treated with the process. In addition, significant progress has been made on a NIFA-funded project involving applying gaseous antimicrobials to inactivate Salmonella on tomato fruit. Gaseous chlorine dioxide and ozone were evaluated for its effectiveness on reducing populations of Salmonella and native microorganisms on grape tomatoes, and impacts on sensory and nutritional quality. Results showed that chlorine dioxide at headspace concentration of 4.3 mg per liter reduced Salmonella populations by more than 99.99%. The treatments did not have any significant effect on appearance, off-odor, firmness, color, lycopene, or vitamin C contents of grape tomatoes during the 21 days storage at 10°C. While dry ozone was capable of reducing Salmonella populations by 99%, the treatment significantly reduced firmness and decreased lycopene and vitamin C contents of the fruit. Overall, our results showed gaseous chlorine dioxide preserved the sensory or nutritional quality of tomatoes while dry ozone did not. Studies are underway to investigate the effects of humidified ozone on Salmonella populations and quality of the fruit. Furthermore, pulsed electric field technology was used to inactivate foodborne pathogens and spoilage microorganisms in special formulated heath juices. Preliminary results show that 5 log reduction of E. coli O157:H7 can be achieved by adjusting pulsed electric field treatment parameters. More studies will be conducted to evaluate the quality and shelf life of treated foods.
1. Novel antibrowning and antimicrobial formulation for cut apples. There have been a number of recalls of cut apples due to contamination with Listeria monocytogenes. Therefore, there is an urgent need to develop antimicrobial formulations to minimize the risk of Listeria contamination while maintaining the freshness of cut apples. ARS scientists at Wyndmoor, Pennsylvania systemically evaluated the combinations of organic acids and various antioxidants for their anti-listerial and anti-browning properties. Results showed that formulations comprised of citric acid (a fruit acid), ascorbic acid (vitamin C), and N-acetyl-L-cysteine (an amino acid) were able to reduce populations of L. monocytogenes by more than 99.999% and at the same time, inhibited the surface browning of cut apples for at least 21 days. The developed formulations, if adopted by the fresh produce industry, may reduce the risk of Listeria contamination and maintain shelf life of cut apples.
2. Antimicrobial films used for in-package pasteurization. Foodborne pathogens and spoilage fungi may reside in fresh produce after packaging. ARS scientists at Wyndmoor, Pennsylvania developed antimicrobial films that released allyl isothiocyanate vapor (a natural flavor compound from mustard) into headspace of the container and inhibited E. coli and fungi growth in fresh tomatoes stored at 4 and 10 °C for 21 days. The treatment reduced the populations of bacteria and molds by 99 - 99.9% on tomatoes, and treated fruit had less change in quality and nutritional values during storage than the non-treated samples. The developed antimicrobial film has the potential to enhance the safety and extend the shelf-life of perishable fresh produce.
3. Integrated interventions of processing and coating improve microbial food safety. Currently, the produce industry employs chlorine to avoid cross contamination despite its limited ability to eliminate pathogens and to form potentially carcinogenic chlorine by-products in wash water. Hence, there is a need to develop new chlorine-free decontamination technologies. ARS scientists at Wyndmoor, Pennsylvania developed a safe and effective method combining organic acid wash with chitosan-allyl isothiocyanate (natural compounds with broad antimicrobial properties) antimicrobial coating. This integrated technology inactivated more than 99.999% of Salmonella on tomatoes. The treatment was also effective in controlling native microbial loads during storage. Furthermore, the firmness and color of tomatoes were not affected by the treatments. This new integrated method potentially can be used as a replacement for current chlorine-based sanitizers.
4. Enhancing tomato safety with organic acids. Consumption of raw tomatoes has been implicated in multiple Salmonella outbreaks in the U.S. ARS researchers have previously shown that washing round tomatoes with combinations of organic acids reduced populations of Salmonella by 99.999%. However, the impact of the acid washes on fruit quality and factors that influence their efficacy have not been studied. ARS scientists at Wyndmoor, Pennsylvania evaluated the changes in fruit quality after acid washes and compared two types of common tomatoes in response to the treatments. Results demonstrated that the treatments were more effective in reducing populations of Salmonella on large round tomatoes than on small grape tomatoes, and the water rinse after acid treatments negated residual acidic odor caused by the acid wash treatments. The information will help the produce industry in adopting more effective treatments to enhance the microbial safety of tomatoes while maintaining fruit quality.Mukhopadhyay, S., Ukuku, D.O., Juneja, V.K., Nayak, B., Olanya, O.M. 2018. Microbial control and food Preservation: Theory and practice: Principles of food preservation. Book Chapter. https://doi.org/10.1016/j.cofs.2018.01.013.
Guo, M., Yadav, M.P., Jin, Z.T. 2017. Antimicrobial edible coatings and films from micro-emulsions and their food applications. Food Control. 263:9-16. https://doi.org/10.1016/j.ijfoodmicro.2017.10.002.
Mukhopadhyay, S., Sokorai, K.J., Ukuku, D.O., Jin, Z.T., Fan, X., Olanya, O.M., Juneja, V.K. 2018. Inactivation of Salmonella in tomato stem scars by organic acid wash and chitosan-allyl isothiocyanate coating. International Journal of Food Microbiology. 266:234-240.
Mukhopadhyay, S., Ukuku, D.O. 2018. The role of emerging technologies to ensure the microbial safety of fresh produce, milk and eggs. Current Opinion in Food Science. https://doi.org/10.1016/j.cofs.2018.01.013.