Location: Characterization and Interventions for Foodborne Pathogens
2024 Annual Report
Objectives
Objective 1: Complete the industrial/commercial implementation of the Radio Frequency (RF) technology (with partners) to enhance the safety of shell eggs. Ensure that the technology is optimized, appreciating the complexity of the intervention process, the processing conditions, the equipment necessary, and the sensory and nutritional qualities of the food product.
Objective 2: Utilization of Cold Plasma, a novel non thermal technology to inactivate microbial contamination on various food products, which can include and is not limited to produce, nuts, meats and ready-to-eat foods. Optimize the technology to allow scale-up to commercial treatment levels, appreciating the complexity of the intervention process in terms of the food to be treated, the processing conditions, the equipment necessary, and the sensory and nutritional qualities of the foods types to be treated.
Sub-objective 2.A: Develop and optimize combined treatments of cold plasma with high-intensity pulsed light-based technologies and/or other antimicrobial processes, including chemical sanitizers to enhance microbial food safety and quality.
Approach
Our research will develop and optimize interventions for reducing the microbial load associated with produce, meats, shell eggs, and other food products. The ARS-patented radio frequency pasteurization (RFP) process for shell eggs is 3X faster and produces a higher quality egg than currently available commercial technologies. We will optimize the existing RFP process to improve efficiency and throughput, leading to a commercial-scale prototype. Research will focus on RFP operating parameters such as: power applied; RF pulse frequency and duration; electrode contact profile; cooling water conductivity, temperature, and flow rate. For many other foods, sanitizing technologies currently available fall short of desirable efficacy goals, such as the FDA’s target of a 5 log kill for fresh and fresh-cut produce. Cold plasma is a novel nonthermal sanitizing process which uses ionized air to inactivate pathogens on a variety of foods and food contact surfaces. We will build on our existing cold plasma expertise to develop and optimize applications, focusing on sanitizing protocols suitable for commercially promising commodities such as meats, fruits and vegetables, and low-moisture foods. We will optimize several different types of cold plasma in parallel, as dictated by the most suitable commodity/pathogen application. The treatments will establish commercialization potential for cold plasma as a standalone process and in combination with other antimicrobial treatments, including a primary focus of combining cold plasma with high-intensity light-based treatments. After we evaluate the combined intervention strategies for their effects on product quality and shelf-life, the most effective, practical treatment combinations will be transferred to industry to reduce the risk of foodborne illness. The outcomes of this project will be new validated means/technologies for producers, processors, and distributors to produce safer shell eggs, meats, fruits and vegetables, and other food related commodities.
Progress Report
The 4th generation of the radio frequency (RF) pasteurizer operates at 40 MHz, using a matching network with an energy efficiency of 97 - 99%. The electrodes are embedded in 3D-printed custom-designed paddles, and eggs are placed on top of rollers, allowing a speed of 42 rpm. The temperature is recorded during the processing using fiber optic sensors inserted in the albumen and the yolk. The temperature during processing is controlled by a temperature water - spray system (38.5°C) using nozzles (90°) on top of the eggs. Validate by using used a comprehensive evaluation of the effect of RF energy on the overall quality of the egg contents and the eggshell. Four batches of fresh large white eggs (30 doz. eggs each) were screened for cracks and sorted by weight. Eggs from 58 to 61 g were chosen to process them with RF (55% of the total received eggs). Eggs were warmed up for 45 min (35°C) to simulate ambient conditions in the hatcheries. Quality experiments were conducted at 40 W (5 min) and 16 W (3 min); the total processing time was 8 min. Egg quality was assessed regarding Haugh unit, yolk index, yolk color, albumen absorbance, yolk and albumen pH, and eggshell breaking strength. Results showed that RF does not change the egg quality after processing. Haugh unit, albumen absorbance, eggshell strength, and yolk index remained constant after processing. Minor variations were observed in the yolk color, which can be due to natural variation between each egg. The quality of the RF-treated eggs did not present significant differences (p > 0.05) compared to raw eggs. To study the possible effects on eggshell strength or mineral composition caused by rotation between the paddles, RF-treated samples were compared to raw and thermally treated eggs (57°C, 60 min). The RF system was operated at 40 W, 24.3 °C for 5.5, 8.5, and 10 min, and compared to the non-energized control. The breakage strength was tested by a compression test, and the mineral content (Ca, P, Mg) was quantified using energy-dispersive X-ray spectrometry. Results showed that RF energy and/or rotational movement of eggs does not affect the eggshell breakage strength or mineral content. The microbial validation for egg RF - pasteurization was conducted in large white eggs (~58 g each) inoculated with Salmonella Typhimurium (105 CFU/egg) using the optimum placement of inoculum, the center of the yolk. The eggs were sanitized, inoculated, sealed, and stored overnight at 4°C. Optimal antimicrobial processing conditions for RF - pasteurization are 40 MHz, 35 W, 4.5 min (water drench of 38.5°C, 14 l/min), followed by hot water spray at 56.7°C (20 min). The cooling of eggs was conducted using ice water or forced air cooling (30 min). Salmonella counts were evaluated after processing and the presence of sub-lethal injured cells and cell recovery during storage (7°C). The overall quality of the RF plus heat eggs was evaluated using the above-mentioned parameters. The pasteurization standard (5-log reduction) was achieved for Salmonella in eggs after 24.5 min after RF + heat processing. No sub-lethal injured cells were detected, and no viable cells were recovered after 7 days at 7°C (LOD < 1 log CFU/g). The quality of the RF + heat was preserved after processing, and only the albumen turbidity showed some differences between raw and processed eggs. Research focused on the Cleaning-in-place (CIP) system for the RF pasteurizer continued, and the effect of an enzymatic treatment was evaluated on liquid egg biofilms containing Salmonella cells. The biofilm formation, attachment, and removal were studied in stainless steel, silicone, and nylon coupons, representing the 3 main components of the RF pasteurizer. Cells were allowed to grow and attach at 37°C for 1, 24, and 48 h (static conditions). Biofilm removal and sanitization were conducted with conventional sodium hypochlorite treatment (200 ppm) and peracetic acid (PAA, 200 ppm). Enzymatic treatment ficin (6.25 U/ml) was studied before applying PAA at lower concentrations (90 and 120 ppm). Contact time for all PAA treatments was 1-, 5- and 10-min. Scanning Electron Microscope (SEM) was used to assess the biofilm attachment in the different materials. Results showed a quick growth and biofilm formation of Salmonella in the three working materials after 24 h, reaching more than 7 log. Nylon presented the highest cell attachment after 48 h. SEM images confirmed these findings, showing a thick biofilm with many cells embedded in the egg matrix. Chorine removed the biofilm, and sanitized stainless steel after 1 min (p < 0.05), but all surfaces were Salmonella-free after 5 min; similar results were observed for PAA after 10 min. The enzymatic pre-treatment with ficin opened the egg biofilm matrix and reduced the contact time with the sanitizer, showing the best results for PAA at 120 ppm and 1 min of treatment with no viable cells detected in any of the three materials. The use of a two-step sanitization process, using an enzymatic treatment followed by contact with organic acids, such as PAA, represents a viable option for the removal of LWE biofilms and inactivation of Salmonella cells in stainless steel, silicone, and nylon. Cold plasma is a novel, nonthermal antimicrobial process. By injecting cold plasma directly from the plasma jet electrodes into a liquid, much of the reactive chemical species created by the ionization process are captured, creating plasma activated water (PAW). PAW functions as a sanitizing solution when created exogenously and applied to fresh and fresh-cut produce. Wash tanks are the first stop for produce from the field. With high levels of suspended organic matter, wash tanks represent a known risk factor for cross contamination. To address the potential for PAW as a sanitizing approach for wash tank water, we project scientists injected plasma jet discharges directly into sterile water (H2O), buffered peptone water (BPW), and into simulated wash tank water (SWT). The SWT was prepared by blending 10g of fresh green leaf lettuce in 500ml sterile water, creating a suspension of high particulates and suspended organic matter. Cold plasma was injected into 500ml samples of each liquid with a plasma jet at a distance of 2 cm above the liquid surface. A vortex tube cold air discharge was applied to moderate the temperature of the solution. Cold plasma treatment caused a drop in pH for all liquids, with pH dropping from 6.8-7.0 down to 3.8 (H2O), 4.4 (BPW), and 4.7 (SWT) after 10 minutes of plasma treatment. This indicates that the acidification properties of the plasma jet injection are stronger than the buffering capacity of the test solutions. The nonpathogentic surrogate E. coli K12 was inoculated into H20 and SWT to create a suspension of ~10^6 cfu/ml. Injection of cold plasma as PAW reduced the recoverable E. coli in both solutions. PAW reduced the E. coli in H2O to below detection (a ~6 log (99.999+%) reduction) after 3 minutes in H2O and after 10 minutes in SWT. The suspended organic matter in the SWT thus somewhat reduced the efficacy of the PAW treatment. Although the plasma injection increased the temperature of the solutions, the maximum reached (49.2-52.1C after 10 minutes) is below the level of thermal kill for E. coli K12, confirming this form of PAW as a nonthermal antimicrobial process. Research is ongoing to calibrate and quantify the effect of suspended organic matter on the antimicrobial efficacy of PAW, and to expand the research to address E. coli O157:H7, Salmonella, and other pathogens. In collaborative research, cold plasma was applied to suspensions of switchgrass and other organic matter in an attempt to break down lignin into more accessible organic molecules. This is desirable because lignin typically requires strong acids, high pressures, high temperatures and long times to be broken down. A 30 minute room temperature injection of cold plasma as PAW led to significant degradation of lignin, with shorter chain, less complex oligomeric carbohydrates as reaction products. This “green chemistry” approach has the potential to substantially improve the utility and accessibility of lignic biomass in bioreactors and in the production of biofuels and related products. Although nonthermal, emerging technologies like cold plasma, pulsed light, and others can have some thermal effect, depending on the duration of treatment, which is deleterious to produce quality. Hence it is preferred to combine high intensity short time (HIST) nonthermal methods to minimize thermal effect to protect nutritional quality and yet achieve the targeted higher pathogen reduction. Research is being conducted to evaluate the efficacy of combinations of pulsed light (PL) and cold plasma (CP) with a natural edible chitosan based antimicrobial coating to kill pathogens and native microbiota without affecting the nutritive and sensorial quality. The optimized nonthermal treatments combinations of PL and CP with chitosan-based antimicrobial, demonstrated its full effectiveness, killing > 99.999% of target pathogen like Salmonella enterica on a model produce as tomatoes. Results also revealed no resurgence of growth of the Salmonella pathogen on combined treated tomatoes during storage at 10°C for 21 days. Large scale validation work for commercialization of this concept is needed.
Accomplishments
1. Pasteurization of intact eggs using radio frequency plus mild heat. Raw eggs can be contaminated with Salmonella, an ongoing food safety concern. ARS researchers at Wyndmoor, Pennsylvania, combined the ARS-patented radio frequency (RF) egg pasteurization system (40 MHz and 35W for 4.5 min) with mild heat (56.7C for 20 min), achieving a 5-log (99.999%) reduction of Salmonella cells inoculated inside the egg. The total processing time is 24.5 min, less than half the time required for conventional thermal pasteurization of eggs using hot water (> 60 min). No Salmonella was recovered after treatment, either as sub-lethally injured cells immediately after treatment or as viable cells from any treated eggs stored at 7°C for 7 days. Clean, Salmonella-free eggs will be of interest to egg producers and the public.
2. Eggs retain their quality after radio frequency processing. The ARS-patented radio frequency (RF) egg pasteurization system can kill Salmonella inside an unbroken egg, thereby addressing an important, ongoing food safety concern. ARS researchers at Wyndmoor, Pennsylvania, have substantially enhanced the RF system with 1) electrodes re-designed for better contact, 2) fine control and logging of processing temperature, 3) an improved matching network that yields ultra-high (98+%) energy efficiency, and 4) robust, commercial-grade construction and cleanability. Since egg quality is as important as egg safety, tests quantified the impact of RF process parameters (pulse frequency, power levels, time, and temperature) on egg quality measurements such as Haugh unit, yolk index, yolk color, albumen and yolk pH, albumen turbidity, eggshell strength, and mineral content. The RF process was shown to operate at full antimicrobial processing levels and times with no significant impact on the overall quality of RF-treated eggs. Egg producers and the public have an interest in clean, Salmonella-free eggs with quality and functionality characteristics similar to fresh, untreated eggs.
3. Two-stage process to remove liquid whole egg biofilms and inactivate Salmonella. The ARS-patented radio frequency (RF) egg pasteurization system will incorporate a clean-in-place (CIP) system to remove biofilms and inactivate microbial cells. In a first generation CIP design that will work as part of the RF system, ARS researchers in Wyndmoor, Pennsylvania, used enzymes (ficin) to open the liquid egg biofilm matrix and organic acids (peracetic acid, 120 ppm) to remove the biofilm of liquid whole egg and inactivate Salmonella cells in stainless steel, silicone, and nylon. This synergistic two-step process allows faster sanitization with less peracetic acid than the current commercial standard process (200 ppm). These results will improve the commercial operation of the RF process, making it more robust and cleanable for egg processors.
4. Processing pecans with plasma prevents pathogen problems. Tree nuts can become contaminated with human pathogens. Cold plasma is a novel antimicrobial process with broad sanitizing efficacy. ARS scientists in Wyndmoor, Pennsylvania, in collaboration with ARS scientists in Byron, Georgia, have shown that cold plasma, applied in short treatments of 30 s or less, can inactivate Salmonella by up to 95% with no gross changes to pecan color, texture, and aroma. Cold plasma combined with advanced vortex jet-based cooling is a compact, efficient treatment system, adjustable to allow for uniform, rotational treatment of nuts and seeds under uniform temperatures. Ongoing research is optimizing the time, distance, pulse frequency, and other treatment parameters. This combination system has potential to improve the safety of pecans and other nuts and seeds.
5. Cold plasma enhances organic acid inactivation of Salmonella. Tomatoes and other vegetables can become contaminated with Salmonella and other human pathogens. ARS scientists in Wyndmoor, Pennsylvania, combined an organic acid solution with cold plasma, a novel antimicrobial process, to inactivate Salmonella inoculated on tomato stem scars. As individual stand-alone 180 s treatments, organic acid and cold plasma reduced Salmonella by 99.3% (2.2 log CFU/g) and 98.7% (1.9 log CFU/g). Combined treatments for only 120 s reduced Salmonella by 99.998% (4.9 log CFU/g). These data suggest that a combination treatment will lead to improved microbial safety of tomatoes.
6. Cold plasma inactivation of acid tolerant Listeria on blueberries. Berries and other small fruits can become contaminated with Listeria monocytogenes, a dangerous human pathogen. ARS scientists in Wyndmoor, Pennsylvania, in collaboration with colleagues in Valdivia, Chile showed that acid tolerant L. monocytogenes and L. innocua (a non-pathogenic surrogate) were significantly reduced by cold plasma, a novel antimicrobial process. Treatments of 30s and longer demonstrated significant reductions in all pathogens, with 60 s achieved 48-75% reduction of L. monocytogenes. Yeast and molds were significantly reduced by 30, 45 and 60 s treatments, with an average 99.5% reduction from non-treated samples. Quality parameters showed no significant differences among cold plasma treatments during the 11-day shelf life study. Cold plasma is thus an effective antimicrobial treatment that does not significantly affect fruit quality.
7. Microbial safety of George Washington’s cherries. In April 2024, archeologists working at Mount Vernon, the home of President George Washington, made a remarkable discovery: dozens of glass bottles with well-preserved cherries and other small fruits. This find, unlike any other in the history of 18th century archeology, represents a unique, once-in-a-lifetime opportunity to study food processing and preservation techniques used during the days of our nation’s first President. ARS scientists in Wyndmoor, Pennsylvania, working in collaboration with archeologists and curators at Mount Vernon and with ARS botanists, plant geneticists, and chemists, successfully extracted and analyzed samples from many of the preserved glass bottles. Screened for lactic acid bacteria (to detect fermentation), coliforms (for groundwater contamination), yeasts and molds (to quantify spoilage), and total aerobic counts (for overall quality),ARS scientists established that the microbial quality of the preserved cherries and other fruits was comparable to the standards in place for modern preserved foods. This historical finding establishes the skill and expertise of the enslaved laborers who worked at Mount Vernon from 1755-1775, and sheds light on heretofore unexamined food safety and food processing practices in historical domestic settings.
8. Pulsed light plus novel sanitizers means clean lettuce. The produce industry employs chlorine-based chemical sanitizer washes to reduce contamination. However, safer and more effective methods are needed. ARS researchers in Wyndmoor, Pennsylvania, have developed a method to combat the risk of consumption of postharvest Romaine lettuce. They determined that high intensity short time pulsed light (PL) combined with in-house formulated antimicrobial solution (AW) containing organic acid, Ethylenediaminetetraacetic acid (EDTA) and Nisin yielded substantially enhanced kill, demonstrating a synergistic antimicrobial activity rather than merely additive effects. A very short 10 second treatment with pulsed light treatment followed by 2 minute wash in antimicrobial solution killed >99.999% pathogenic Escherichia coli in Romaine lettuce. Additionally, there were no resurgence of growth of pathogenic Escherichia coli and no deterioration in quality observed, either immediately after treatment or after 7 days of refrigerated storage. This combination treatment of PL and AW holds promise as a valuable tool for producers and processors of fresh and fresh-cut fruits and vegetables.
Review Publications
Bermudez-Aguirre, L.D., Niemira, B.A. 2023. Effect of nisin, EDTA, and abuse temperature on the growth of Salmonella Typhimurium in liquid whole egg during refrigerated storage. Food Research International. 174:113568. https://doi.org/10.1016/j.foodres.2023.113568.
Bermudez-Aguirre, L.D., Niemira, B.A. 2023. Radio frequency treatment of food: A review on pasteurization and disinfestation. Foods. 12(16):3057. https://doi.org/10.3390/foods12163057.
Gyawali, R., Mahapatra, A.K., Bardsley, C.A., Niemira, B.A. 2024. Improving microbial safety and quality of nuts using nonthermal technologies. Trends in Food Science and Technology. 81(1). https://doi.org/10.1016/j.jfp.2023.100201.
Niemira, B.A., Bermudez-Aguirre, L.D., Boyd, G., Sites, J.E. 2023. Cold plasma for food processing. In: Smithers, G.W., editor. Encyclopedia of Food Safety. 2nd edition. Elsevier: Academic Press. p. 591-598. https://doi.org/10.1016/B978-0-12-822521-9.00076-9.
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.
Concha-Meyer, A.A., González-Esparza, A., Cullen, P.J., Veloso, F., Favre, M., Valenzuela, J.C., Toloza, L., Niemira, B.A. 2024. Survival of Listeria strains and shelf life determination of fresh blueberries (Vaccinium corymbosum) treated with cold atmospheric plasma. Foods. 13(6):822. https://doi.org/10.3390/foods13060822.