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ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Food Safety and Intervention Technologies Research » Research » Research Project #430150

Research Project: Development of Alternative Intervention Technologies for Fresh or Minimally Processed Foods

Location: Food Safety and Intervention Technologies Research

2021 Annual Report

1. Develop and evaluate novel antagonists (e.g. Bacteriovorax, Bdellovibrio and non-pectolytic Pseudomonas) for biological-based intervention strategies, and identify means of combining pathogen microbial ecology with effective chemical and physical interventions. 2. Develop and optimize chemical decontamination interventions (e.g. novel sanitizer formulations and advanced gas-phase antimicrobial treatments), making use of pathogen microbial ecology information generated under Objective 1. 3. Develop nonthermal technologies (e.g. cold plasma, high-intensity monochromatic light and irradiation) as effective, waterless physical treatments, and establish protocols for combination treatments with interventions developed in the first two objectives.

A holistic approach to the development of new effective intervention technologies will be followed. In an iterative process of technology development, increased knowledge of pathogen ecology on produce surfaces will be used to optimize biological, chemical and physical control strategies; combinatorial use of intervention technologies will be examined for additive and synergistic effects while maintaining product quality. More rapid and successful commercialization will be fostered by determining the equipment and infrastructure required for large-scale implementation, as well as economic costs and benefits expected from the use of the new technologies. A key aspect of this effort to facilitate commercialization will be collaboration and information sharing with industry, including direct contact with potential end-users of technologies. Stakeholders will be updated on research goals and objectives of the project, and input will be sought from them to identify key problems to be solved. By proactively fostering these interactions in conjunction with site visits, annual scientific meetings, industry trade shows and similar venues, there will be opportunities in the early, mid-phase and in late stages of research will allow for the practical needs of industry to be addressed as the research is formulated and conducted.

Progress Report
Objective 1. Pathogen contamination of fresh and fresh-cut produce remains a primary concern for foodborne illness. Studies were conducted to establish post-harvest survival and reduction of Listeria monocytogenes and Escherichia coli O157:H7 on carrot and tomato by hurdle treatments. A cocktail of two pathogens - L monocytogenes stains (008, 2625, and 2634) and E. coli O157:H7 strains (43894, 43895, and 35150) – and a cocktail of non-infectious biocontrol strains of Pseudomonas fluorescens (strains 2-79, 1801 and 1609) were used in this project. All cultures were from the Eastern Regional Research Center (ERRC). Carrot and tomato were inoculated with ~ 6 log inocula of Listeria and E. coli O157:H7 in separate experiments by dipping produce in 20 mL inocula suspension for 5 min (biosafety hood), then dried for 30min. Treatments with single and combinations were: 1) Radiation (R), 2) Sanitizer (S), 3) Biocontrol (B), 4) R+S, 5) S+R, 6) R+B, 7) S+B, 8) R+S+B, 9) S+R+B, and 10) Un-treated, with 2 produce types (carrot and tomato) and at 5 or 20 C storage. Radiation treatment was applied at 0.5 kGy, while sanitizer was at 100% (10mL) and P. fluorescens biocontrol was at ~6 logs. In this study, the main (single) treatments were applied on produce separately, while hurdle treatments were applied sequentially. Treatments were randomized and appropriately replicated. The survival of L. monocytogenes and E. coli O157:H7 differed significantly among treatments and between carrot and tomato. After storage for 7 days (20C), radiation treatment reduced Listeria by 2.9 and 1.8 logs/g (99.8 % and 98.5%) on carrot and tomato respectively. The reduction of Listeria populations at (5C, day 7) was by 2.8 logs (99.8%) and did not differ between carrot and tomato. E. coli O157:H7 reductions on carrot and tomato (20C, day 0) were 3.11 and 2.20 logs (99.93% and 99.4%), respectively. Pathogen reductions after 7 days of storage were 1.82 log (98.5%) on carrot and 1.37 logs (95.8%) on tomato at 5C and did not differ, while at 20C, these were 1.09 and 1.50 logs (94% and 96.9%), on carrot and tomato, respectively. This suggests that low-dose radiation treatment could be useful in hurdle treatments. The competitive exclusion (non-infectious, biocontrol) microbes showed a moderate number of surviving Listeria populations and consequently the lowest number of pathogen reductions. Listeria reductions were <1 log (~90%) after treatment (20C, day 0) on carrot and tomato. After 7 days, reduction of E. coli populations did not vary on carrot and tomato, and storage temperatures. Overall, the biocontrol interventions applied against L. monocytogenes and E. coli O157:H7 resulted in low pathogen reductions, implying that initial biocontrol populations need to be significantly in excess of the pathogen in order to achieve desired effects in the absence of other intervention hurdles. The sanitizer treatment by itself applied against L. monocytogenes and E. coli O157:H7 had the greatest efficacy. Listeria reductions on carrot and tomato ranged from 2.3-4.0 logs (99.5 – 99.99%) across days and storage temperatures. The survival of E. coli O157:H7 populations as a consequent of sanitizer treatment was lower, with reductions of 1.77 logs (98.4%) on carrot and 2.21 logs (99.4%) on tomato (5C, day 7), but these were 3.13 logs (99.95%) on carrot and 2.81 logs (99.8%) on tomato at 20C (day 7). Variation in sanitizer treatment may be expected due to different produce types, temperatures, storage times and sanitizer application methods. Generally, two and three hurdle treatments had greater effects on Listeria survival and pathogen reductions than single treatments. The two hurdle treatments (R+B and S+R) had additive effects on Listeria reductions, while some 3 hurdle combinations (e.g. R+S+B and S+R+B) treatments resulted in synergistic effects on Listeria on carrot and tomato after 7 days of storage. Reductions of Listeria on carrot by hurdle applications were greater than on tomato. The survival and reductions of E. coli O157:H7 on the same produce by single and hurdle treatments were lower than that recorded in the case of Listeria pathogen. Objective 2. Despite new practices and intervention technologies, blueberries and other produce are contaminated with foodborne pathogens, such as Salmonella spp. The aim of this study was to evaluate the efficacy of chlorine dioxide gas (CDG) against Salmonella enterica serovars Newport, Stanley, Muenchen, and Anatum artificially contaminated on whole fresh blueberries. Blueberries were dip inoculated into a 400 ml bath containing either ca. 6 or 9 log10 CFU/ml of a Salmonella serovar cocktail. Samples were dried for either 2 or 24 h before being treated with 1.5 or 3 mg CDG/L air to a final treatment of 3.55 to 6 ppm-h/g blueberry. Salmonella cells were recovered by stomaching, CDG-treated and non-treated control samples, with 0.1% peptone, and enumerated on xylose-lysine-Tergitol-4 agar. CDG treatments achieved up to a 5.63 log (99.999+%) reduction of the cocktail using 5.5 ppm-h/g while the least efficacious treatment, 4 ppm-h/g (1.5 mg/L), was still capable of a 4.45 log (99.997%) reduction. Incubation time significantly (p < 0.001) impacted CDG efficacy against both inoculation concentrations. Additionally, all serovars responded similarly to CDG treatment when tested independently (p > 0.0691). Finally, the availability of a water reservoir during treatments did not have significant impact (p = 0.9818) on CDG efficacy in this study. Our results demonstrated that CDG can be an efficacious treatment option for whole blueberry decontamination. Objective 3. Many studies on the development of new and/or value added processing intervention techniques to improve food safety for U.S. consumers have been reported. However, information on the effect of treatment parameters on microbial inactivation on some of these technologies is limited. In this study, we investigated the effect of integrated treatments of nisin-based antimicrobial and cold plasma treatments in reducing Listeria monocytogenes inoculated on apple surfaces. Apples were inoculated at 5.8±0.24 log CFU/farm apples and 4.6±0.12 log CFU/supermarket apples and they were treated with antimicrobial solution for 30 s, 40s, 3 min (180s) and 1h (3,600s), cold plasma treatments for 30 and 40s, and a combination of antimicrobial and cold plasma treatments. Efficacy of the treatments on microbial reduction were investigated by enumerating surviving colony forming units on selective and non-selective agar plates. Effect of treatments on surface structure of apples and bacterial populations were examined using scanning electron microscopy (SEM). Cold plasma treatments, alone at 30 and 40 s yielded 0.3 log (50%) reduction of L. monocytogenes. Similarly, antimicrobials alone led to slightly higher bacterial reduction at 30 and 40 s. Cold plasma treatment at 40s, followed immediately with antimicrobial treatments at 180s and 3,600s led to 2.5 and 4.6 log (99.7% and 99.997%) inactivation of L. monocytogenes, respectively. SEM observation showed changes on apple surface structures but not on bacterial cell structure. These results suggests that a pre-treatment with cold plasma at 40s reduces sanitizer treatment time from 1 h to 3 min to achieve approximately 3.5 to 4 log (99.97 - 99.99%) inactivation of L. monocytogenes on apples. In another study, the capacity of cold plasma corona discharge to inactivate Salmonella on pre-packaged, tomato-and-lettuce mixed salads was established. Two different inoculation methods were evaluated to address cross-contamination: in separate studies, either cherry tomatoes (55g) or romaine lettuce (10g) were inoculated with a Salmonella cocktail and placed into a commercial PET plastic container and thoroughly mixed together with its non-inoculated counterpart. Mixed salads were allowed to dry in a biosafety cabinet for 1h. Samples were treated with 35 kV cold plasma corona discharge inside plastic containers for 3 min. Samples were stomached and serially diluted in BPW then plated onto APC Petrifilm and incubated for 18 h at 37°C. When lettuce was the inoculated counterpart, log kill of Salmonella was significantly greater on tomatoes (0.75 log, 83%) compared to lettuce (0.34 log, 56%) (p=0.0001). Salmonella was reduced on mixed salad only when lettuce was the inoculated counterpart (0.29 log, 49%) (p=0.002). Cold plasma can kill Salmonella in a pre-packaged mixed salad, with efficacy dependent on the nature of contamination, direction of transfer, and on the surface topography of the contaminated commodity. In studies of a different pathogen, the dielectric barrier discharge atmospheric cold plasma (DACP) treatment was evaluated for the inactivation of Escherichia coli O157:H7, surface morphology, color, carbon dioxide generation, and weight loss of bulk Romaine lettuce in a commercial plastic clamshell container. The lettuce samples were packed in a model bulk packaging configuration (three rows with either 1, 3, 5, or 7 layers) in the container and treated by DACP (42.6 kV, 10 min). DACP treatment reduced the number of E. coli O157:H7 in the leaf samples in the 1-, 3-, and 5-layer configurations by 0.4 to 0.8 log (40% - 84%) lettuce, with no significant correlation to the sample location (P > 0.05). In the largest bulk stacking with 7 layers, a greater degree of reduction (1.1 log, 94%) was observed at the top layer, but shaking the container increased the uniformity of the inhibition. DACP did not significantly change the surface morphology, color, respiration rate, or weight loss of the samples, nor did these properties differ significantly according to their location in the bulk stack. DACP treatment inhibited E. coli O157:H7 on bulk lettuce in clamshell containers in a uniform manner, without affecting the physical and biological properties and thus holds promise as a post-packaging process.

1. Pathogen reduction by a predator. Bdellovibrio is a predator of Gram-negative bacteria such as E. coli and Salmonella, thereby potentially enhancing food safety. ARS researchers at Wyndmoor, Pennsylvania, determined and quantified the predation and subsequent reductions of E. coil O157:H7 and Salmonella in buffer, on carrot and on tomato. Predation ranged from 0.50 to 3.85 logs (69% - 99.98%). They also found that Bdellovibrio does not survive irradiation at a dose of 0.75 kGy, implying that only low-dose radiation could be combined with predator treatment for bacterial inactivation and improved food safety.

Review Publications
Oyeyinka, S.A., Olanya, O.M., Ade-Omowaye, B.I., Niemira, B.A. 2020. Non-thermal processing techniques for innovative food processing. Food Science and Technology Trends and Future Prospects. Berlin, Germany: De Gruyter.
Olanya, O.M., Niemira, B.A., Cassidy, J.M., Boyd, G., Uknalis, J. 2020. Pathogen reduction by predatory bacteria and survival of Bdellovibrio bacteriovorus and Escherichia coli on produce and buffer treated with low-dose gamma radiation. LWT - Food Science and Technology. 130:1-7.