Atmospheric Cold Plasma Interactions With Microbiological Risks In Fresh Food Processing

Atmospheric cold plasma (ACP) is a novel emerging non-thermal technology that has attracted attention as a decontamination tool in several industrial, food and healthcare sectors. This study investigated the anti-microbial efficacy of ACP against microbiological risks associated with fresh foods. Treatment was performed using in-package ‘dry’ ACP technology and plasma functionalised liquid to decontaminate microorganisms, exploring the responses to real and challenging microbiological risks pertinent to both fresh foods themselves as well as the effluents generated from food processing industry. A range of critical control process parameters were investigated with respect to key pathogenic and spoilage microorganisms commonly implicated in the food environment. The inactivation efficacy of ACP against all applied bacterial strains was depended on applied voltage, treatment time and post treatment storage time (PTST). Greater inactivation was obtained at 80 kV with 24 h of PTST providing greater interaction between the bacteria and the reactive species. Bacterial biofilms were significantly susceptible to ACP. Viable and metabolic active cells in mono and dual biofilms were inactivated within short treatment time. Different inactivation rate was observed, depending on physiological state of the bacteria (planktonic or biofilms, mono or mixed culture). An extended time was required to reduce the challenge mixed culture biofilm formed on lettuce at environmental stress conditions. The study demonstrated that produce storage conditions, such as temperature and storage time had interactive effects on bacterial proliferation, stress response and susceptibility to the ACP treatment, highlighting the importance of preventive measures as key factors for the assurance of microbiological safety of fresh produce. Further, to ascertain the effect of stress conditions on ACP’s bacterial inactivation efficacy, L. monocytogenes and its knockout mutants associated with stress were treated with sub-lethal stress conditions. The gene expression of stress associated genes were significantly increased after 1 min treatment, while long treatment time reduced the gene expression and some cases down-regulated prfA and gadD3 gene expression. By comparing the response of mutants under ACP exposure to key processing parameters, the experimental results presented here provide a baseline for understanding the bacterial genetic response and resistance to plasma stress and offers promising insights for optimizing ACP applications. The impact of the ACP technology on model food surface and wash-water generated from fresh produce processing was also investigated. The ACP treatment reduced microbial load showing similar efficacy as chlorine, providing further advantage of continuously treating the lettuce wash water. Micro-bubbling along with agitation assisted bacterial detachment and distribution of reactive species, thus increasing bacterial inactivation efficacy from fresh produce and wash water. Liquid media complexity was explored as a factor in cold plasma decontamination efficacy for microbiologically safe effluents from food processing. The high nutritive components in the model effluents exerted a protective effect during treatment, showing higher inactivation in phosphate buffer solution (PBS) than in nutrient rich wastewater effluents. ACP was effective to inactivate principle indicator bacteria (mono and mixed culture planktonic bacteria and spores) from model dairy and meat wastewaters. This study also investigated the eco-toxicological impact of cold plasma treatment of the model wastewater using a range of aquatic bioassays. Differing sensitivities were observed to ACP treated effluents across the different test bio-assays; with greater sensitivity retained to plasma treated meat effluent than dairy effluent. The toxic effects were dependent on concentration and treatment time of the ACP treated effluents. ACP shows potential as an efficient decontamination approach against bacteria in their most resistant, biofilm or spore form associated with complex and nutritious food products during food production to wastewaters generated by the food industries

Controlling Brochothrix Thermosphacta as a Spoilage Risk Using in Package Atmospheric Cold Plasma

Brochothrix thermosphacta is a predominant spoilage microorganism in meat and its control in processing environments is important to maintain meat product quality. Atmospheric cold plasma is of interest for control of pathogenic and spoilage microorganisms in foods. This study ascertained the potential of dielectric barrier discharge atmospheric cold plasma (DBD-ACP) for control of B. thermosphacta in response to key parameters such as treatment time, voltage level, interactions with media composition and post treatment storage conditions. Challenge populations were evaluated as suspensions in PBS, as biofilms in meat model medium and surface attached on raw lamb chops under MAP. ACP treatment (80kV) for 30s inactivated B. thermosphacta populations below detection in PBS, while 5 min treatment achieved a 2 Log cycle reduction using a complex meat model medium and attached cells, suggesting useful control for meat processing facilities. A ‘worst case scenario challenge’ of high population density on a nutritious medium in a biofilm matrix was evaluated using a surface inoculated lamb chop and the antimicrobial efficacy of plasma was reduced but still apparent over the 10 day storage period. However, there is scope to further enhance microbial control leading to meat storage life extension through adjusting the modality of treatmen

Inactivation Efficacy of Atmospheric Air Plasma and Airborne Acoustic Ultrasound Against Bacterial Bioflms

Bioflms are complex microbial communities that present serious contamination risks to our environment and health. In this study, atmospheric air plasma and airborne acoustic ultrasound technology were applied to inactivate Escherichia coli and Listeria innocua bioflms. Both technologies were efcient in controlling, or completely inactivating, the target bacterial bioflms. Viability and metabolic assays, along with microscopy analysis, revealed that atmospheric air plasma and airborne acoustic ultrasound damaged both the bacterial bioflm cells and its structural integrity. Scanning electron microscopy images highlighted the disruption of the bioflms and pore formation in bacterial cells exposed to both the plasma and acoustic treatments. Elevated reactive oxygen and nitrogen species in bacterial cells treated with atmospheric air plasma, demonstrated their primary role in the observed bacterial inactivation process. Our fndings provide potential antimicrobial strategies to combat bacterial bioflms in the food and healthcare sectors

Assessment of the disinfection capacity and eco-toxicological impact of atmospheric cold plasma for treatment of food industry effluents

Generation of wastewater is one of the main environmental sustainability issues across food sector industries. The constituents of food process effluents are often complex and require high energy and processing for regulatory compliance. Wastewater streams are the subject of microbiological and chemical criteria, and can have a significant eco-toxicological impact on the aquatic life. Thus, innovative treatment approaches are required to mitigate environmental impact in an energy efficient manner. Here, dielectric barrier discharge atmospheric cold plasma (ACP) was evaluated for control of key microbial indicators encountered in food industry effluent. This study also investigated the eco-toxicological impact of cold plasma treatment of the effluents using a range of aquatic bioassays. Continuous ACP treatment was applied to synthetic dairy and meat effluents. Microbial inactivation showed treatment time dependence with significant reduction in microbial populations within 120 s, and to undetectable levels after 300 s. Post treatment retention time emerged as critical control parameter which promoted ACP bacterial inactivation efficiency. Moreover, ACP treatment for 20 min achieved significant reduction (≥2 Log10) in Bacillus megaterium endospores in wastewater effluent. Acute aquatic toxicity was assessed using two fish cell lines (PLHC-1 and RTG-2) and a crustacean model (Daphnia magna). Untreated effluents were toxic to the aquatic models, however, plasma treatment limited the toxic effects. Differing sensitivities were observed to ACP treated effluents across the different test bio-assays in the following order: PLHC-1 \u3e RTG-2 ≥ D. magna; with greater sensitivity retained to plasma treated meat effluent than dairy effluent. The toxic effects were dependent on concentration and treatment time of the ACP treated effluent; with 30% cytotoxicity in D. magna and fish cells observed after 24 h of exposure to ACP treated effluent for concentrations up to 5%. The findings suggest the need to employ wider variety of aquatic organisms for better understanding and complete toxicity evaluation of long-term effects. The study demonstrates the potential to tailor ACP system parameters to control pertinent microbial targets (mono/poly-microbial, vegetative or spore form) found in complex and nutritious wastewater effluents whilst maintaining a safe eco-toxicity profile for aquatic species

High voltage atmospheric cold air plasma control of bacterial biofilms on fresh produce

Atmospheric cold plasma (ACP) offers great potential for decontamination of food borne pathogens. This study examined the antimicrobial efficacy of ACP against a range of pathogens of concern to fresh produce comparing planktonic cultures, monoculture biofilms (Escherichia coli, Salmonella enterica, Listeria monocytogenes, Pseudomonas fluorescens) and mixed culture biofilms (Listeria monocytogenes and Pseudomonas fluorescens). Biotic and abiotic surfaces commonly occurring in the fresh food industry were investigated. Microorganisms showed varying susceptibility to ACP treatment depending on target and process factors. Bacterial biofilm populations treated with high voltage (80 kV) ACP were reduced significantly (p \u3c 0.05) in both mono- and mixed species biofilms after 60 s of treatment and yielded non-detectable levels after extending treatment time to 120 s. However, an extended time was required to reduce the challenge mixed culture biofilm of L. monocytogenes and P. fluorescens inoculated on lettuce, which was dependent on biofilm formation conditions and substrate. Contained treatment for 120 s reduced L. monocytogenes and P. fluorescens inoculated as mixed cultures on lettuce (p \u3c 0.05) by 2.2 and 4.2 Log10 CFU/ml respectively. When biofilms were grown at 4 °C on lettuce, there was an increased resistance to ACP treatment by comparison with biofilm grown at temperature abuse conditions of 15 °C. Similarly, L. monocytogenes and P. fluorescens exposed to cold stress (4 °C) for 1 h demonstrated increased tolerance to ACP treatment compared to non-stressed cells. These finding demonstrates that bacterial form, mono versus mixed challenges as well as environmental stress conditions play an important role in ACP inactivation efficacy

The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using \u3ci\u3e Listeria monocytogenes\u3c/i\u3e Knockout Mutants

Listeria monocytogenes is an opportunistic intracellular pathogen commonly associated with serious 16 infections and multiple food-borne outbreaks. In this study, we investigated the influence of 17 atmospheric cold plasma (80 kV, 50 Hz) on L. monocytogenes (EGD-e) and its knockout mutants of 18 sigB, rsbR, prfA, gadD and lmo0799 genes at different treatment time intervals. Further, to ascertain if 19 sub-lethal environmental stress conditions could influence L. monocytogenes survival and growth 20 responses, atmospheric cold plasma (ACP) resistance was evaluated for the cultures exposed to cold 21 (4°C) or acid (pH 4) stress for 1 h. The results demonstrate that both wild-type and knockout mutants 22 were similarly affected after 1 min exposure to ACP (p \u3e 0.05), with a difference in response noted 23 only after 3 min of treatment. While all L. monocytogenes strains exposed to acid/cold stress were 24 hypersensitive to ACP treatment and were significantly reduced or inactivated within 1 min of 25 treatment (p \u3c 0.05). The results indicate sigB and prfA are important for general stress resistance and 26 biofilm respectively, loss of these two genes significantly reduced bacterial resistance to ACP 27 treatment. In addition, exposure to sub-lethal 1min ACP increased the gene expression of stress 28 associated genes. SigB showed the highest gene expression, increasing by 15.60 fold, followed by 29 gadD2 (7.19) and lmo0799 (8.6) after 1 min exposure. Overall, an increase in gene expression was 30 seen in all stress associated genes analyzed both at 1 min treatment; while long treatment time reduced 31 the gene expression and some cases down-regulated prfA and gadD3 gene expression. By comparing 32 the response of mutants under ACP exposure to key processing parameters, the experimental results 33 presented here provide a baseline for understanding the bacterial genetic response and resistance to 34 cold plasma stress and offers promising insights for optimizing ACP applications

Controlling Microbial Safety Challenges of Meat Using High Voltage Atmospheric Cold Plasma

Atmospheric cold plasma (ACP) is a non-thermal technology, effective against a wide range of pathogenic microorganisms. Inactivation efficacy results from plasma generated reactive species. These may interact with any organic components in a test matrix including the target microorganism, thus food components may exert a protective effect against the antimicrobial mode of action. The effect of an in-package high voltage ACP process applied in conjunction with common meat processing MAP gas compositions as well as bacteria type and meat model media composition have been investigated to determine the applicability of this technology for decontamination of safety challenges associated with meat products. E. coli, L. monocytogenes and S. aureus in PBS were undetectable after 60 s of treatment at 80 kVRMS in air, while ACP treatment of the contaminated meat model required post treatment refrigeration to retain antimicrobial effect. The nutritive components in the meat model exerted a protective effect during treatment, where 300 s ACP exposure yielded a maximum reduction of 1.5 log using a high oxygen atmosphere, whilst using air and high nitrogen atmospheres yielded lower antimicrobial efficacy. Furthermore, an ROS assay was performed to understand the protective effects observed using the meat model. This revealed that nutritive components inhibited penetration of ROS into bacterial cells. This knowledge can assist the optimization of meat decontamination using ACP technology where interactions with all components of the food matrix require evaluation

Antimicrobial effects of airborne acoustic ultrasound and plasma activated water from cold and thermal plasma systems on biofilms

peer-reviewedBacterial bioflms are difcult to inactivate due to their high antimicrobial resistance. Therefore, new approaches are required for more efective bacterial bioflm inactivation. Airborne acoustic ultrasound improves bactericidal or bacteriostatic activity which is safe and environmentally friendly. While, plasma activated water (PAW) is attracting increasing attention due to its strong antimicrobial properties. This study determined efcacy of combined airborne acoustic ultrasound and plasma activated water from both cold and thermal plasma systems in inactivating Escherichia coli K12 bioflms. The application of airborne acoustic ultrasound (15 min) alone was signifcantly more efective in reducing E. coli counts in 48 and 72 h bioflms compared to 30 min treatment with PAW. The efect of airborne acoustic ultrasound was more pronounced when used in combination with PAW. Airborne acoustic ultrasound treatment for 15 min of the E. coli bioflm followed by treatment with PAW signifcantly reduced the bacterial count by 2.2—2.62 Log10 CFU/mL when compared to control bioflm treated with distilled water. This study demonstrates that the synergistic efects of airborne acoustic ultrasound and PAW for enhanced antimicrobial efects. These technologies have the potential to prevent and control bioflm formation in food and bio-medical applications.Science Foundation Irelan

The Effect of Atmospheric Cold Plasma on Bacterial Stress Responses and Virulence Using Listeria monocytogenes Knockout Mutants

Listeria monocytogenes is an opportunistic intracellular pathogen commonly associated with serious infections and multiple food-borne outbreaks. In this study, we investigated the influence of atmospheric cold plasma (80 kV, 50 Hz) on L. monocytogenes (EGD-e) and its knockout mutants of sigB, rsbR, prfA, gadD, and lmo0799 genes at different treatment time intervals. Further, to ascertain if sub-lethal environmental stress conditions could influence L. monocytogenes survival and growth responses, atmospheric cold plasma (ACP) resistance was evaluated for the cultures exposed to cold (4°C) or acid (pH 4) stress for 1 h. The results demonstrate that both wild-type and knockout mutants were similarly affected after 1 min exposure to ACP (p > 0.05), with a difference in response noted only after 3 min of treatment. While all L. monocytogenes strains exposed to acid/cold stress were hypersensitive to ACP treatment and were significantly reduced or inactivated within 1 min of treatment (p < 0.05). The results indicate sigB and prfA are important for general stress resistance and biofilm, respectively, loss of these two genes significantly reduced bacterial resistance to ACP treatment. In addition, exposure to sub-lethal 1min ACP increased the gene expression of stress associated genes. SigB showed the highest gene expression, increasing by 15.60 fold, followed by gadD2 (7.19) and lmo0799 (8.6) after 1 min exposure. Overall, an increase in gene expression was seen in all stress associated genes analyzed both at 1 min treatment; while long treatment time reduced the gene expression and some cases down-regulated prfA and gadD3 gene expression. By comparing the response of mutants under ACP exposure to key processing parameters, the experimental results presented here provide a baseline for understanding the bacterial genetic response and resistance to cold plasma stress and offers promising insights for optimizing ACP applications.Department of Agriculture, Food and the MarineIrish Research CouncilFood Institutional Research Measure (FIRM