Project : USDA ARS

ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Characterization and Interventions for Foodborne Pathogens » Research » Research Project #439577

Research Project: Validation and Commercialization of Innovative Processing Technologies

Location: Characterization and Interventions for Foodborne Pathogens

2025 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
Objective 1: Research continued on the industrial/commercial implementation of the Radio Frequency (RF) technology to enhance the safety of shell eggs. The newest generation of the RF successfully pasteurizes large eggs in only 24.5 minutes, achieving a 5-log reduction of Salmonella Typhimurium inoculated inside the egg while preserving the quality. ARS researchers in Wyndmoor, Pennsylvania, developed fiber-optic temperature sensors (highly accurate and fast responding) which were inserted through the eggshell into the yolk and albumen to validate RF process control. A cost engineering analysis of the integrated RF system was based on the optimized protocol for microbial inactivation of S. Typhimurium. RF optimization and validation was based in part on process logistical input from our stakeholders technical information for projected annual output of RF pasteurized eggs (~14 million). The RF process was divided into three sub-processes to model energy consumption and cost engineering: 1) RF heating (35W, 4.5 min), applied together with 2) water spray (38.5 °C, 4.5 min), and 3) hot water spray (56.7 °C, 20 min). Baseline comparison is conventional thermal processing of hot water spray (56.7 °C, 60 min). ARS researchers in Wyndmoor, Pennsylvania, experimentally determined RF efficiency to be >98%, vs. ~90% for conventional thermal processing. Using validated industrial prices for electricity in Pennsylvania as of March 2025, applied to a model process annual run of 40 million eggs, the final cost of the entire RF process was modeled and found to be considerably more economical, costing ~1/3 as much as conventional thermal. Modeled RF cost is $742,209 per year vs. $1,991,154 per year for conventional thermal treatment. RF adds only $0.05/egg or $0.63/dozen, while conventional thermal adds $0.14/egg or $ 1.70/dozen. These calculations demonstrate the significant cost savings of using RF; eggs can be pasteurized for nearly a third of the conventional pasteurization cost. Furthermore, the total process is shorter (24.5 min) compared to traditional thermal pasteurization (60 min), and the egg quality is better. The research related to the Cleaning-Place (CIP) system for the RF pasteurizer expanded significantly during this fiscal year. A bioengineered hydrolase (CAase) at 0.150 mg/ml, alone and in combination with peracetic acid (PAA), effectively dissolved microbial attachment structures to remove ~2 log cfu of biofilms of Pseudomonas fluorescens and Salmonella in liquid whole egg (LWE) on three food contact surfaces commonly found in the egg industry were studied: stainless steel, silicone and nylon. We developed a rapid method to identify S. Typhimurium cells during the early stages of cell attachment in biofilm formation. By detecting pathogen biofilms in only 1 hour, instead of the 3 hours required by conventional methods, industry will be better able to find the right interventions to sanitize FCS sooner and more effectively. A comprehensive study with eight different serotypes of Salmonella explored the different attachment and biofilm formation capability from Salmonella serotypes from different foodborne outbreaks. This study used a standard microbiological medium to start the biofilm on stainless steel, silicone or nylon, at two temperatures: refrigeration (10°C) and abusive (37°C). Growth at 37°C created a stronger biofilm with higher cell attachment (~ 7 log) compared to refrigerated temperature (~ 5 log). Stainless steel supported the strongest biofilms. Meanwhile, the strongest Salmonella serotypes for biofilm formation were S. Enteritidis, S. Saint Paul and S. Montevideo. The weakest and most distinct serotype was S. Senftenberg. scanning electron microscopy images showed the high production of exopolymeric substances from S. Enteritidis after 24 hours at 37°C. A collaborative genomic analysis showed that a microorganism’s ability to form strong or weak biofilms is related to single nucleotide polymorphisms in the curli (csg) genes. This information can help to develop the right sanitizing interventions, particularly those related to S. Enteritidis and the egg industry. We evaluated essential oils (cinnamaldehyde, garlic, ginger and rosemary oil at 1% (v/v)) as natural antimicrobials for LWE inoculated with S. Typhimurium (103 cfu/ml) and stored at abuse refrigerated temperature (10°C) for 21 days. Cinnamaldehyde and garlic oil inhibited microbial growth with only minor variations of pH and color. Objective 2: Research continued on the use of cold plasma (CP) to improve safety and extend shelf life of dried/low moisture meat products. Spoilage of beef jerky and other dried meat products typically manifests as outgrowth of fungi. CP is a relatively novel food processing intervention suitable for application to low-moisture foods. ARS researchers in Wyndmoor, Pennsylvania, evaluated two types of CP (pre- and post-packaging) as control strategies for fungal spoilage of beef jerky. Samples of commercially prepared beef jerky (42g) were extensively inoculated with a mixed culture of spoilage fungi, including Aspergillus app., Penicillium spp., and Rhizopus spp. Inoculated samples were treated with two types of cold plasma: 1) corona discharge (less aggressive, can be applied after packaging) or 2) plasma jet (more aggressive, applied to product before packaging). For corona discharge treatments, inoculated beef jerky was loaded into clean commercial-grade pouches, sealed, and treated with 36kV, 900 watts corona for 3 minutes, after confirming generation of plasma inside the pouches with no loss of packaging integrity. For plasma jet, inoculated samples were loaded onto a lab-scale conveyor belt, treated running under 4 plasma emitter heads of 40kV, 540 watts each at 9cm gap spacing. Samples treated for 1 minute, flipped, treated again (total treatment time, 2 minutes). Control samples received no plasma. Samples were loaded into clean commercial packaging, pouches sealed. Infrared imaging during treatments confirmed all plasma processes to be nonthermal. Samples were stored at 25°C for up to 16 days. At 5 days of storage, slight mold growth was visible on control samples. This increased rapidly during storage, with complete fungal contamination and extensive growth by day 7. Corona discharge-treated samples remained clean at 5 days but showed slight signs of mold growth by 7 days, with more rapid growth thereafter. In plasma jet treated samples, mold was effectively suppressed, with samples remaining clean until 12 days, when single points of fungal growth were visible. Mold growth expanded on treated samples until complete contamination was observed on Day 14. These results suggest that plasma treatment of beef jerky pieces may be an effective control for fungal contaminants, either as a plasma jet or in combination with corona discharge, applied post-packaging. Future research will optimize CP (singly or in combinations) for sanitizing efficacy, operational parameters, and sensory impact. Based on these promising results, research will continue on the combination of CP with conventional thermal convection dehydration or with advanced vacuum desiccation on meat products as well as on dried fruit and vegetable products. Project staff have conducted equipment calibration and protocol development for vacuum desiccation of apples, banana, ready-to-eat foods in support of this effort. The antimicrobial mode of action of plasma activated water was elucidated for treatment of E. coli O157:H7 inoculated lettuce. Collaborations with ARS researchers at Wyndmoor, Pennsylvania, continue in applying CP for advantageous chemical treatment of bio-oils and suspended organic matter. Sub-objective 2A: Research continued on combined treatments of CP with high-intensity pulsed light-based technologies and/or other antimicrobial processes, including chemical sanitizers to enhance microbial food safety and quality. ARS researchers in Wyndmoor, Pennsylvania, evaluated CP, pulsed light (PL), and their combinations for inactivating E. coli O157:H7 on Romaine lettuce. Lettuce leaves were spot inoculated and then treated with PL (1–60 s), CP (15–60 s) or their optimized treatment combinations. A 30 s treatment with PL (fluence dose of 31.5 J/cm2), was optimum which provided 2.7 log CFU/g reduction of the pathogen, while 45 s treatment of CP was optimum, that delivered 2.1 log CFU/g log reduction. Combinations of PL and CP treatments were investigated for enhanced inactivation. For PL-CP combination, inoculated lettuce was treated with PL for 30 s followed by 45 s of CP exposure. While for CP-PL combination, treatments sequences were 45 s of CP treatment followed by 30 s PL treatment. Both combination treatments, PL-CP and CP-PL, resulted in synergistic inactivation of E. coli cells with > 5 log reductions of the pathogen. These combination treatments significantly (p < 0.05) reduced native microbiota and slowed their growth during storage. Treated lettuce retained fresh-like quality. PL and CP are both non-aqueous, flexible technologies that can enhance microbial safety and preserve the quality of Romaine lettuce. Optimized in-package nonthermal treatment combinations of PL and CP without added chemical antimicrobial demonstrated excellent in-package inactivation capability killing > 99.9% of Salmonella on grape tomatoes and packaged lettuce.

Accomplishments
1. Egg sanitation process validation with advanced temperature monitoring. The ARS-patented radio frequency (RF) pasteurization process can kill 99.999% of Salmonella inside an intact shell egg with minimal impact on egg quality and nutrition. Monitoring the RF process is a key part of ensuring operation at peak efficiency. ARS researchers in Wyndmoor, Pennsylvania developed advanced fiber-optic temperature sensors (highly accurate and fast responding) which were inserted through the eggshell into the yolk and albumen to validate RF process control. These sensors successfully recorded the temperature profiles throughout the egg during the entire RF kill step. Egg producers can use this information to reliably and more precisely implement the RF process to improve egg safety for American consumers.

2. Safer eggs – faster and cheaper with radio frequency processing. The ARS-patented radio frequency (RF) pasteurization process can kill 99.999% of Salmonella inside an intact shell egg with minimal impact on egg quality and nutrition. A key question is the relative cost of the RF process compared with conventional thermal processing of intact shell eggs. ARS researchers in Wyndmoor, Pennsylvania, used industry standard logistical data and consumer egg price information to develop an engineering cost model. For a modeled annual production of 40 million eggs, RF pasteurized eggs had a price premium 1/3 that of conventionally pasteurized eggs: $0.05/egg vs. $0.14/egg. Processing time with RF is shorter (24.5 min) compared to thermal processing (60 min), and the quality of RF treated eggs is higher. Egg producers can use this information to build robust business models for the RF process to improve egg safety for American consumers.

3. Removing biofilms on food contact surfaces with advanced enzymes. Biofilms are firmly attached havens for pathogen and are a major concern for food contact surfaces. ARS researchers in Wyndmoor, Pennsylvania, used a formulated enzyme treatment (CAase), alone and in combination with peracetic acid, to effectively dissolve microbial attachment structures that protect pathogens in biofilms. The ARS enzyme treatment removed 99% of biofilms of Pseudomonas fluorescens and Salmonella in liquid whole egg on three food contact surfaces commonly found in the egg industry: stainless steel, silicone and nylon. These results will provide egg processors with new tools to reduce the risks associated with pathogen biofilms on equipment and processing environments, improving the health and safety of the American consumer.

4. Detecting salmonella biofilms faster to make sanitizers more effective. Once pathogens get firmly established on food contact surfaces and form durable biofilms, they are much harder to remove with conventional sanitizers. ARS researchers in Wyndmoor, Pennsylvania, developed a more rapid method to identify pathogenic Salmonella Typhimurium cells during the early stages of cell attachment in biofilm formation. By detecting pathogen biofilms in only 1 hour, instead of the 3 hours required by conventional methods, industry will be better able to find the right interventions to sanitize food contact surfaces sooner and more effectively. This technological advance will help improve commercial sanitizing protocols and better protect the health and safety of the American consumer.

5. Blocking the behavior of burgeoning biofilms. Once pathogens get firmly established on food contact surfaces and form durable biofilms, they are much harder to remove with conventional sanitizers. Key aspects of this problem are knowing what makes a pathogen a strong biofilm former and what conditions are most problematic for food processing environments. ARS researchers in Wyndmoor, Pennsylvania, conducted a comprehensive study of attachment and biofilm formation in eight serotypes of Salmonella on three food contact surfaces (stainless steel, silicone and nylon), at two temperatures: refrigeration (10°C) and abusive (37°C). The researchers determined that the serotypes ranged in biofilm production, that growth at 37°C was worse than 10°C, and that stainless steel supported stronger biofilms than other surfaces. A detailed genomic analysis showed that variation of a single nucleotide in the curli (csg) gene determined a microorganism’s ability to form strong or weak biofilms. This information will help to develop the food processing industry to the right sanitizing interventions, in the right place, at the right time, against the right pathogens, thereby improving sanitation efficiency and efficacy and protecting the health and safety of the American consumer.

6. Essential oils block pathogen growth in liquid whole egg. Liquid whole egg is a widely used food product which can sometimes be contaminated with Salmonella or other pathogens. To combat this risk, ARS researchers in Wyndmoor, Pennsylvania, used a variety of essential oils as natural antimicrobials to suppress the growth of Salmonella Typhimurium cells during 21 days of refrigerated storage. Among the essential oils tested at a level of 1% (v/v), researchers discovered that cinnamaldehyde and garlic oil inhibited microbial growth with only minor variations of pH and color of the liquid whole egg. These results give new tools to egg producers and food formulation manufacturers that can help stop Salmonella and improve the food safety of a wide range of products for the American consumer.

7. Preserving and protecting packaged protein with precision plasma processing. Beef jerky and other dried meat products can suffer from fungal spoilage, creating health hazards for consumers. ARS researchers in Wyndmoor, Pennsylvania, addressed this problem with cold plasma, a relatively novel food processing intervention suitable for application to low-moisture foods. The researchers inoculated commercially prepared beef jerky with a mixture of spoilage fungi and treated it with two different cold plasma systems: 1) corona discharge (less aggressive, applied to jerky after packaging) or 2) plasma jet (more aggressive, applied to jerky before packaging). Treatment times were 2-3 minutes. Infrared imaging during treatments confirmed all plasma processes to be nonthermal. The samples were stored at 25°C for up to 16 days. The untreated samples began spoiling at 5 days of storage and were completely spoiled by day 7. Corona discharge extended shelf life by 2 days, with no mold visible until after day 7. The plasma jet treatment kept the beef jerky spoilage-free until day 12, after which single points of fungal growth were visible. Plasma treatment of beef jerky pieces may thus be an effective control for fungal contaminants, either as a plasma jet or in combination with corona discharge, applied post-packaging. These results give new tools for producers of low moisture protein foods that can help stop spoilage and reduce risk for the American consumer.

8. Cold plasma and pulsed light – a one-two punch for pathogens. Bagged salads, including Romaine lettuce, continue to be a source of concern for foodborne pathogens. ARS researchers in Wyndmoor, Pennsylvania, combined two advanced food processing technologies to reduce pathogenic E. coli O157:H7 on lettuce by more than 99.999%. Pulsed light is U.S. Food and Drug Administration approved and effective but can take unduly extended treatment times for best antimicrobial efficacy. Cold plasma is quicker and gentler than pulsed light but is not always as potent. Combining the two methods in treatments lasting 15 to 45 seconds resulted in synergistic inactivation of pathogens on lettuce, yielding antimicrobial efficacy far greater than either method alone and comparable to that obtained with industry standard chemical sanitizing treatments. Treated lettuce retained excellent quality. This chemical free, low input combination technology is a promising approach for treating Romaine lettuce, as well as tomatoes, fruits, and other special commodities. These results give new tools for producers and processors of fresh and fresh-cut fruits and vegetables to improve food safety and reduce risk for the American consumer.

U.S. DEPARTMENT OF AGRICULTURE

Characterization and Interventions for Foodborne Pathogens: Wyndmoor, PA