Inactivation efficacy of atmospheric air plasma and airborne acoustic ultrasound against bacterial biofilms.

Biofilms 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 biofilms. Both technologies were efficient in controlling, or completely inactivating, the target bacterial biofilms. Viability and metabolic assays, along with microscopy analysis, revealed that atmospheric air plasma and airborne acoustic ultrasound damaged both the bacterial biofilm cells and its structural integrity. Scanning electron microscopy images highlighted the disruption of the biofilms 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 findings provide potential antimicrobial strategies to combat bacterial biofilms in the food and healthcare sectors.

Biomolecules as Model Indicators of In Vitro and In Vivo Cold Plasma Safety.

The potential applications for cold plasma in medicine are extensive, from microbial inactivation and induction of apoptosis in cancer cells to stimulating wound healing and enhancing the blood coagulation cascade. The safe bio-medical application of cold plasma and subsequent effect on complex biological pathways requires precision and a distinct understanding of how physiological redox chemistry is manipulated. Chemical modification of biomolecules such as carbohydrates, proteins, and lipids treated with cold plasma have been characterized, however, the context of how alterations of these molecules affect cell behavior or in vivo functionality has not been determined. Thus, this study examines the cytotoxic and mutagenic effects of plasma-treated molecules in vitro using CHO-K1 cells and in vivo in Galleria mellonella larvae. Specifically, albumin, glucose, cholesterol, and arachidonic acid were chosen as representative biomolecules, with established involvement in diverse bioprocesses including; cellular respiration, intracellular transport, cell signaling or membrane structure. Long- and short-term effects depended strongly on the molecule type and the treatment milieu indicating the impact of chemical and physical modifications on downstream biological pathways. Importantly, absence of short-term toxicity did not always correlate with absence of longer-term effects, indicating the need to comprehensively assess ongoing effects for diverse biological applications.

Impact of cold plasma processing on major peanut allergens.

Cold plasma is emerging as a novel food processing technology, with demonstrated efficacies for microbial inactivation and residual chemical dissipation of food products. Given the technology's multimodal action it has the potential to reduce allergens in foods, however data on the efficacy and mechanisms of action are sparse. This study investigates the efficacy of cold plasma on major peanut allergens (Ara h 1 and Ara h 2). For this purpose, dry, whole peanut (WP) and defatted peanut flour (DPF) were subjected to an atmospheric air discharge using a pin to plate cold plasma reactor for different treatment durations. With increases in plasma exposure, SDS-PAGE analysis revealed reduced protein solubility of the major peanut allergens. Alterations in allergenicity and structure of Ara h 1 and Ara h 2 were examined using ELISA and circular dichroism (CD) spectroscopy. Competitive ELISA with proteins purified from plasma treated WP or DPF revealed reduced antigenicity for both Ara h 1 and Ara h 2. The highest reduction in antigenicity was 65% for Ara h 1 and 66% Ara h 2 when purified from DPF. Results from CD spectroscopy analysis of purified proteins strongly suggests the reduction in antigenicity is due to modifications in the secondary structure of the allergens induced by plasma reactive species. Cold plasma is effective at reducing peanut protein solubility and causes changes in allergen structure leading to reduced antigenicity.

Perspectives from CO+RE: How COVID-19 changed our food systems and food security paradigms.

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Efficacy of cold plasma functionalised water for improving microbiological safety of fresh produce and wash water recycling.

Atmospheric cold plasma (ACP) is an effective method for microbiological decontamination. This study evaluated an alternative water-based decontamination approach for inactivation of bacterial population from fresh produce and in the wash water generated from fresh produce washing. The study characterised ACP inactivation of attached Listeria innocua and Pseudomonas fluorescens inoculated on lettuce in comparison to chlorine treatment. P. fluorescens was sensitive to ACP treatment and was reduced below detection limit within 3 min of treatment. L. innocua population was reduced by ∼2.4 Log10 CFU/g after 5 min of treatment; showing similar inactivation efficacy to chlorine treatment. The microbial load in wash water was continuously decreased and was below detection limits after 10 min of ACP treatment. Micro-bubbling along with agitation assisted the bacterial detachment and distribution of reactive species, thus increasing bacterial inactivation efficacy from fresh produce and wash water. A shift in pH of plasma functionalised water was observed along with high concentration of nitrate and ozone with a relative amount of nitrites which increased with plasma exposure time. Further, L. innocua treated at different independent pH conditions showed minimal or no effect of pH on ACP bacterial inactivation efficacy. Aqueous ACP treatment poses a promising alternative for decontamination of fresh produce and the associated wash-waters which could be applied in the food industry to replace continuous chlorine dosing of process waters.

Improving enzymatic hydrolysis of brewer spent grain with nonthermal plasma.

In this study, a new pre-treatment method based on novel non-thermal plasma technology was developed to improve the enzymatic hydrolysis of brewer's spent grain (BSG) and subsequent bioethanol production. A submerged dielectric barrier discharge plasma reactor system was applied for this purpose. Pre-treatments were performed by taking into account variables including; voltages (22 kV, 25 kV and 28 kV), solvent (acid, alkali and water) and time (5, 10, 15 min). The resulting treated biomass was subjected to enzymatic hydrolysis. A 2.14-fold increase in yield of the reducing sugar was achieved post hydrolysis when the biomass was treated in water for 10 min at a voltage setting of 28 kV (162.90 mg/g of BSG) compared to control (75.94 mg/g of BSG). This research suggests that subjecting lignocellulose to plasma discharges can enhance the efficiency of enzymatic hydrolysis. A high ethanol titre was also obtained upon fermentation of the hydrolysate (25.062 g/l).

Degradation kinetics of cold plasma-treated antibiotics and their antimicrobial activity.

Antibiotics, such as ofloxacin (OFX) and ciprofloxacin (CFX), are often detected in considerable concentrations in both wastewater effluents and surface water. This poses a risk to non-target organisms and to human health. The aim of this work was to study atmospheric cold plasma (ACP) degradation of antibiotics in water and meat effluent and to explore any residual antimicrobial activity of samples submitted to the plasma process. The results revealed that ACP successfully degraded the studied antibiotics and that the reaction mechanism is principally related to attack by hydroxyl radicals and ozone. According to the disk diffusion assay, the activity of both antibiotics was considerably reduced by the plasma treatment. However, a microdilution method demonstrated that CFX exhibited higher antimicrobial activity after ACP treatment than the corresponding control revealing a potentially new platform for future research to improve the efficiency of conventional antibiotic treatments. Importantly, short-term exposures to sub-lethal concentrations of the antibiotic equally reduced bacterial susceptibility to both ACP treated and untreated CFX. As a remediation process, ACP removal of antibiotics in complex wastewater effluents is possible. However, it is recommended that plasma encompass degradant structure activity relationships to ensure that biological activity is eliminated against non-target organisms and that life cycle safety of antibiotic compounds is achieved.

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 < 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 < 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.

Diagnostics of plasma reactive species and induced chemistry of plasma treated foods.

Cold plasma is a promising technique that has been tested as a process technology for a range of food commodities, mainly to destroy microorganisms, but also aimed at toxin degradation, enzyme inactivation, residual pesticide degradation and functionalization of food properties. Plasma has already been employed by industry for food packaging material sterilization and surface modification. As most of the current literature on cold plasma in the field of food science is focused on microbial inactivation efficacy, the information about its chemical influences on food is sparse. To better understand the chemical interactions of with plasma, this review focuses on plasma chemistry diagnostics techniques available to characterize the plasma reactive species generated. Equally important is the detection of induced chemistry in the food and here we present approaches to analyze likely reactions with key food bio-molecules. Such analysis will support mechanistic insights involved in these complex chemical reactions (i.e., DNA, lipid and protein) along with potential physical modifications of the food structure. For successful adoption of plasma as a food processing aid it is critical to elucidate these interactions as they have an important role in demonstrating the technology's safety as a food processing technique along with understanding any effect on food nutrients.

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.

Combating Staphylococcus aureus and its methicillin resistance gene (mecA) with cold plasma.

The increase in antibiotic resistance has become a global challenge to public health. In this study, an atmospheric cold plasma (ACP) system was applied for combating methicillin-resistant Staphylococcus aureus (MRSA) and its methicillin resistance gene (mecA) during food wastewater treatment. The plate count and flow cytometry methods were employed to estimate the damage in MRSA induced by plasma treatment. A quantitative real-time PCR (qPCR) method was used to assess the plasma-induced degradation of the mecA genes. The inactivation of MRSA and degradation of extracellular (e-) and intracellular (i-)mecA genes were investigated in phosphate buffered solution as a function of plasma exposure. A relatively low plasma influence of 0.12 kJ/cm2 accounted for 5-log MRSA and 1.4-log e-mecA genes reduction, while only around 0.19-log degradation for i-mecA genes. As the plasma intensity was accumulated to 0.35 kJ/cm2, the reduction of e- and i-mecA genes was increased to 2.6 and 0.8 logs, respectively. The degradation of i-mecA genes was much slower than that of e-mecA genes due to the protective effects of the outer envelopes or intracellular components against plasma. The matrix effect of wastewater effluents shielded both antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) from plasma disinfection, which led to a lower degradation efficacy. Our results could support the development and optimization of plasma-based wastewater treatment.

Chemical Modifications of Lipids and Proteins by Nonthermal Food Processing Technologies.

A range of nonthermal techniques have demonstrated process efficacy in ensuring product safety, extension of shelf life, and in general a retention of key quality attributes. However, various physical, chemical and biochemical effects of nonthermal techniques on macro and micro nutrients are evident, leading to both desirable and undesirable changes in food products. The objective of this review is to outline the effects of nonthermal techniques on food chemistry and the associated degradation mechanisms with the treatment of foods. Oxidation is one of the key mechanisms responsible for undesirable effects induced by nonthermal techniques. Degradation of key macromolecules largely depends on the processing conditions employed. Various extrinsic and intrinsic control parameters of high-pressure processing, pulsed electric field, ultrasound processing, and cold atmospheric plasma on chemistry of processed food are outlined. Proposed mechanisms and associated degradation of macromolecules, i.e., proteins, lipids, and bioactive molecules resulting in food quality changes are also discussed.

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 > 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.

Recent Advances in the Application of Cold Plasma Technology in Foods.

The past decade has seen a surge in the scientific literature investigating the potential food-related applications of plasma. A multidisciplinary scientific effort has started to demonstrate process efficacy for a range of plasma applications, including antimicrobial, pesticidal, food functionalization, and waste treatment. Insights into the interactions of plasma species with food and the mechanisms of action are also emerging. This review examines the current status of cold plasma technology within the food sector with a particular emphasis on emerging applications. Opportunities and current challenges that need to be addressed for successful adoption of the approach by industry are detailed.

Hydrogen Peroxide and Beyond-the Potential of High-voltage Plasma-activated Liquids Against Cancerous Cells.

BACKGROUND: The use of plasma-activated liquids such as PBS, medium or simply plasma-activated water (PAW) has been receiving increasing attention for applications in cancer treatments. Amongst the reactive species contained in these solutions, hydrogen peroxide appears to play a pivotal role in causing cytotoxic effects. H2O2 concentrations can be correlated with reduced cell viability and growth and used as an indicator of the potential efficacy of a plasma-activated liquid. OBJECTIVE: To investigate the cytotoxic mediators generated in water specific to high-voltage DBD-ACP. METHOD: Using a high-voltage dielectric barrier atmospheric cold plasma (DBD-ACP) system, we examined PAW-mediated cytotoxic effects on different mammalian cell lines employing a set-up where short-lived reactive species can be discounted and activated liquids with long-term stability are generated. RESULTS: The PAW potency could be modulated using voltage level, treatment time and post-treatment storage time and target-related characteristics such as surface to volume ratio. All of these parameters effected cell viability in a hydrogen peroxide concentration correlated manner. The susceptibility of two cancer cell lines to PAW was similar to that observed for two non-cancer cell lines and the toxicity of plasma-activated water exceeded that of the corresponding hydrogen peroxide concentrations. CONCLUSION: In cytotoxic plasma activated water an essential role for H2O2 has been demonstrated multi-fold, yet further contributing factors are apparent and remain to be identified.

Non-invasive 3D and 360° optical imaging of micro-particles.

Scanning electron microscopy and X-ray microtomography are useful methods for high resolution shape imaging. Visible microscopy is also common, however, developing a low-cost and customizable system for surface and shape investigation of optically active particles is challenging. In this work, we demonstrate an assembly offering good light sensitivity, flexibility of illumination and contrasts from varying angles. The design was applied, together with recent programs for focus-stacking, to analyze crystals of taurine, L-glutamic acid, acetylsalicylic acid, and copper sulfate, along with digital 3D-360° modelling of phosphorescent [Ru(bpy)3]Cl2 and strontium aluminate particles. We further tested the approach for real time monitoring of size, shape and texture analysis of fat filled milk particles and acid whey powders. The findings show proof of concept for detailed feature imaging of particles directly from the process environment.

Ferric chloride assisted plasma pretreatment of lignocellulose.

In this study, a novel pretreatment for spent coffee waste (SCW) has been proposed which combines two techniques viz. atmospheric air plasma and FeCl3 to create a superior pretreatment that involves Fenton chemistry. The pretreatment was optimised employing Taguchi Design of Experiments, and five parameters were taken into consideration viz. biomass loading, FeCl3 concentration, H2SO4 concentration, plasma discharge voltage and treatment time. The composition analysis of the pretreated SCW revealed substantial amounts of lignin removal, with a maximum for process conditions of 70kV for 2min in an acidic environment containing 1% H2SO4. FTIR, XRD and DSC were performed to characterise the samples. The pretreated SCW after enzymatic hydrolysis yielded 0.496g of reducing sugar/g of SCW. The hydrolysate was subjected to fermentation by S. cerevisiae and led to the production of 18.642g/l of ethanol with a fermentation efficiency of 74%, which was a two fold increase in yield compared to the control.

Controlling Brochothrix thermosphacta as a spoilage risk using in-package atmospheric cold plasma.

Brochothrix thermosphacta is the 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, taking microbial and food environment factors into consideration, and investigated the shelf-life of lamb chop after in-package plasma treatment in modified atmosphere. Community profiling was used to assess the treatment effects on the lamb microflora. ACP treatment (80 kV) for 30s inactivated B. thermosphacta populations below detection levels in PBS, while 5 min treatment achieved a 2 Log cycle reduction using a complex meat model medium and attached cells. The antimicrobial efficacy of plasma was reduced but still apparent on lamb chop surface-inoculated with high concentrations of B. thermosphacta. Lamb chop treated under modified atmosphere exhibited reduced microbial growth over the product shelf-life and community profiling showed no evident changes to the microbial populations after the treatment. The overall results indicated potential of ACP to enhance microbial control leading to meat storage life extension through adjusting the modality of treatment.

Challenges in Model Development for Meat Composition Using Multipoint NIR Spectroscopy from At-Line to In-Line Monitoring.

This study evaluates the efficiency of multipoint near-infrared spectroscopy (NIRS) to predict the fat and moisture content of minced beef samples both in at-line and on-line modes. Additionally, it aims at identifying the obstacles that can be encountered in the path of performing in-line monitoring. Near-infrared (NIR) reflectance spectra of minced beef samples were collected using an NIR spectrophotometer, employing a Fabry-Perot interferometer. Partial least squares regression (PLSR) models based on reference values from proximate analysis yielded calibration coefficients of determination (Rc2) of 0.96 for both fat and moisture. For an independent batch of samples, fat was estimated with a prediction coefficient of determination (Rp2) of 0.87 and 0.82 for the samples in at-line and on-line modes, respectively. All the models were found to have good prediction accuracy; however, a higher bias was observed for predictions under on-line mode. Overall results from this study illustrate that multipoint NIR systems combined with multivariate analysis has potential as a process analytical technology (PAT) tool for monitoring process parameters such as fat and moisture in the meat industry, providing real-time spectral and spatial information.

Characterisation of cold plasma treated beef and dairy lipids using spectroscopic and chromatographic methods.

The efficacy of cold plasma for inactivation of food-borne pathogens in foods is established. However, insights on cold plasma-food interactions in terms of quality effects, particularly for oils and fats, are sparse. This study evaluated plasma-induced lipid oxidation of model matrices, namely dairy and meat fats. Product characterisation was performed using FTIR, 1H NMR and chromatographic techniques. The oxidation of lipids by cold plasma followed the Criegee mechanism and typical oxidation products identified included ozonides, aldehydes (hexanal, pentenal, nonanal and nonenal) and carboxylic acids (9-oxononanoic acid, octanoic acid, nonanoic acid), along with hydroperoxides (9- and 13-hydroperoxy-octadecadienoylglycerol species). However, these oxidation products were only identified following extended treatment times of 30min and were also a function of applied voltage level. Understanding cold plasma interactions with food lipids and the critical parameters governing lipid oxidation is required prior to the industrial adoption of this technology for food products with high fat contents.