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bAtmospheric cold plasma (ACP) is a promising nonthermal technology effective against a wide range of pathogenic microorganisms. Reactive oxygen species (ROS) play a crucial inactivation role when air or other oxygen-containing gases are used. With strong oxidative stress, cells can be damaged by lipid peroxidation, enzyme inactivation, and DNA cleavage. Identification of ROS and an understanding of their role are important for advancing ACP applications for a range of complex microbiological issues. In this study, the inactivation efficacy of in-package high-voltage (80 kV [root mean square]) ACP (HVACP) and the role of intracellular ROS were investigated. Two mechanisms of inactivation were observed in which reactive species were found to either react primarily with the cell envelope or damage intracellular components. Escherichia coli was inactivated mainly by cell leakage and low-level DNA damage. Conversely, Staphylococcus aureus was mainly inactivated by intracellular damage, with significantly higher levels of intracellular ROS observed and little envelope damage. However, for both bacteria studied, increasing treatment time had a positive effect on the intracellular ROS levels generated.A tmospheric cold plasma (ACP) refers to nonequilibrated plasma generated at near-ambient temperatures and pressure. ACP is composed of particles, including free electrons, radicals, and positive and negative ions, but it is low in collision frequency of gas discharge compared to that with equilibrated plasma (1, 2). ACP technologies have widely been applied for many surface treatments and environmental processes. Recently, they have been studied for food sterilization and plasma medicine (2-5).ACP provides inactivation effects against a wide range of microbes, mainly by the generation of cell-lethal reactive species (6-8). By discharging in air, groups of reactive species are generated, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), UV radiation, energetic ions, and charged particles (5). However, the inactivation efficacy can be varied by changing the working gases, which results in different types or amounts of reactive species generated (9-11). ROS are often identified as the principal effecting species, with a relatively long half-life and strong antimicrobial effects, which are generated in oxygen-containing gases (12).ROS generated during plasma discharge in air or oxygen-containing mixtures are assemblies of ozone, hydrogen peroxide, and singlet and atomic oxygen, while ozone is considered the most microbicidal species (13). With strong oxidative stress, cells are damaged by lipid peroxidation, enzyme inactivation, and DNA cleavage. The generation of plasma in air or a nitrogen-containing gas mixture can also generate NO x species. However, higher inactivation efficacy has been reported with the combined application of NO and H 2 O 2 on Escherichia coli than that with a treatment with NO or H 2 O 2 alone (14). Reactive nitrogen species are highly toxic and can lead to cell death by increas...

Cold plasma science and technology is increasingly investigated for translation to a plethora of issues in the agriculture and food sectors. The diversity of the mechanisms of action of cold plasma, and the flexibility as a standalone technology or one that can integrate with other technologies, provide a rich resource for driving innovative solutions. The emerging understanding of the longer-term role of cold plasma reactive species and follow-on effects across a range of systems will suggest how cold plasma may be optimally applied to biological systems in the agricultural and food sectors. Here we present the current status, emerging issues, regulatory context, and opportunities of cold plasma with respect to the broad stages of primary and secondary food production.

The exposure of aqueous solutions to atmospheric plasmas results in the generation of relatively long-lived secondary products such as hydrogen peroxide which are biologically active and have demonstrated anti-microbial and cytotoxic activity. The use of plasma-activated solutions in applications such as microbial decontamination or anti-cancer treatments requires not only adequate performance on target cells but also a safe operating window regarding the impact on surrounding tissues. Furthermore the generation of plasma-activated fluids needs to be considered as a by-stander effect of subjecting tissue to plasma discharges. Cytotoxicity and mutagenicity assays using mammalian cell lines were used to elucidate the effects of solutions treated with di-electric barrier discharge atmospheric cold plasma. Plasma-treated PBS inhibited cell growth in a treatment time-dependent manner showing a linear correlation to the solutions' peroxide concentration which remained stable over several weeks. Plasma-treated foetal bovine serum (FBS) acting as a model for complex bio-fluids showed not only cytotoxic effects but also exhibited increased mutagenic potential as determined using the mammalian HPRT assay. Further studies are warranted to determine the nature, causes and effects of the cyto-and genotoxic potential of solutions exposed to plasma discharges to ensure long-term safety of novel plasma applications in medicine and healthcare.Non-thermal plasma, generated by the ionisation of gases, consists of free electrons, radicals, positive and negative charged particles has shown strong effects on living cells. Plasma can cause both cell death and stimulate cell proliferation and has found applications in microbial decontamination, wound healing and cancer treatment 1-4 . The inactivation of bacteria and fungi by cold plasma, and similar cytotoxic effects on eukaryotic cells have been suggested to result predominantly from oxidative damage to the cells' membrane and intra-cellular components including DNA through the action of reactive oxygen species (ROS) 5,6 . A number of recent publications have reported on the biological activity of plasma treated solutions such as plasma-activated water (PAW), plasma-activated PBS (PAPBS) or plasma-activated medium (PAM) which were found to possess anti-microbial and/or cytotoxic activity [7][8][9] . These solutions are of interest as novel anti-microbial agents for decontamination of surfaces, wounds and food products 7,8,10 and may offer similar antimicrobial effects as observed with direct exposure to the plasma discharge. The retention of efficacy in solution would provide the advantages of off-site production, storability and ease of application over direct treatment. Plasma-activated medium has furthermore shown selective cytotoxicity in several cancerous cell lines, including chemotherapy resistant types, making it a novel candidate treatment for anti-cancer therapies 9,11,12 . Atmospheric cold plasmas which may be generated by a range of different plasma devices such as plasma...

Plasma‐activated liquids (PAL) attract increasing interest with demonstrated biological effects. Plasma exposure in air produces stable aqueous reactive species which can serve as chemical diagnostics of PAL systems. Here, we tailor aqueous reactive species inside plasma‐activated water (PAW) through treating water with AC air spark and glow discharges in contact with water. Chemical probing demonstrated species specificity between two types of PAW. Spark discharge PAW contains H2O2 and NO3−, while NO2−and NO3− are generated in glow discharge PAW. Species formation in different PAWs have been discussed in terms of discharge mechanisms and liquid phase chemistry process. Species specificity can provide richer parametric spaces for producing PALs with controlled impact and dosage achievable by combining discharge modes or mixing different PALs.

Contamination of cereal grains as a key global food resource with insects or microorganisms is a persistent concern for the grain industry due to irreversible damage to quality and safety characteristics and economic losses. Atmospheric cold plasma presents an alternative to conventional grain decontamination methods owing to the high antimicrobial potential of reactive species generated during the treatment, but effects against product specific microflora are required to understand how to optimally develop this approach for grains. This work investigated the influence of ACP processing parameters for both cereal grain decontamination and grain quality as important criteria for grain or seed use. A high voltage (HV) (80 kV) dielectric barrier discharge (DBD) closed system was used to assess the potential for control of native microflora and pathogenic bacterial and fungal challenge microorganisms, in tandem with effects on grain functional properties. Response surface modelling of experimental data probed the key factors in relation to microbial control and seed germination promotion. The maximal reductions of barley background microbiota were 2.4 and 2.1 log CFU/g and of wheat - 1.5 and 2.5 log CFU/g for bacteria and fungi, respectively, which required direct treatment for 20 min followed by a 24 h sealed post-treatment retention time. In the case of challenge organisms inoculated on barley grains, the highest resistance was observed for Bacillus atrophaeus endospores, which, regardless of retention time, were maximally reduced by 2.4 log CFU/g after 20 min of direct treatment. The efficacy of the plasma treatment against selected microorganisms decreased in the following order: E. coli > P. verrucosum (spores) > B. atrophaeus (vegetative cells) > B. atrophaeus (endospores). The challenge microorganisms were more susceptible to ACP treatment than naturally present background microbiota. No major effect of short term plasma treatment on the retention of quality parameters was observed. Germination percentage measured after 7 days cultivation was similar for samples treated for up to 5 min, but this was decreased after 20 min of direct treatment. Overall, ACP proved effective for cereal grain decontamination, but it is noted that the diverse native micro-flora may pose greater resistance to the closed, surface decontamination approach than the individual fungal or bacterial challenges, which warrants investigation of grain microbiome responses to ACP.

The main objectives of this work were to investigate the effect of atmospheric cold plasma (ACP) against a range of microbial biofilms commonly implicated in foodborne and healthcare associated human infections and against P. aeruginosa quorum sensing (QS)-regulated virulence factors, such as pyocyanin, elastase (Las B) and biofilm formation capacity post-ACP treatment. The effect of processing factors, namely treatment time and mode of plasma exposure on antimicrobial activity of ACP were also examined. Antibiofilm activity was assessed for E. coli, L. monocytogenes and S. aureus in terms of reduction of culturability and retention of metabolic activity using colony count and XTT assays, respectively. All samples were treated ‘inpack’ using sealed polypropylene containers with a high voltage dielectric barrier discharge ACP generated at 80 kV for 0, 60, 120 and 300 s and a post treatment storage time of 24 h. According to colony counts, ACP treatment for 60 s reduced populations of E. coli to undetectable levels, whereas 300 s was necessary to significantly reduce populations of L. monocytogenes and S. aureus biofilms. The results obtained from XTT assay indicated possible induction of viable but non culturable state of bacteria. With respect to P. aeruginosa QS-related virulence factors, the production of pyocyanin was significantly inhibited after short treatment times, but reduction of elastase was notable only after 300 s and no reduction in actual biofilm formation was achieved post-ACP treatment. Importantly, reduction of virulence factors was associated with reduction of the cytotoxic effects of the bacterial supernatant on CHO-K1 cells, regardless of mode and duration of treatment. The results of this study point to ACP technology as an effective strategy for inactivation of established biofilms and may play an important role in attenuation of virulence of pathogenic bacteria. Further investigation is warranted to propose direct evidence for the inhibition of QS and mechanisms by which this may occur.