SummaryThere is a diverse range of microbiological challenges facing the food, healthcare and clinical sectors. The increasing and pervasive resistance to broad-spectrum antibiotics and health-related concerns with many biocidal agents drives research for novel and complementary antimicrobial approaches. Biofilms display increased mechanical and antimicrobial stability and are the subject of extensive research. Cold plasmas (CP) have rapidly evolved as a technology for microbial decontamination, wound healing and cancer treatment, owing to the chemical and bio-active radicals generated known collectively as reactive oxygen and nitrogen species. This review outlines the basics of CP technology and discusses the interactions with a range of microbiological targets. Advances in mechanistic insights are presented and applications to food and clinical issues are discussed. The possibility of tailoring CP to control specific microbiological challenges is apparent. This review focuses on microbiological issues in relation to food-and healthcareassociated human infections, the role of CP in their elimination and the current status of plasma mechanisms of action.

Atmospheric cold plasma (ACP) represents a potential alternative to traditional methods for non-thermal decontamination of foods. In this study, the antimicrobial efficacy of a novel dielectric barrier discharge ACP device against Escherichia coli, Salmonella enterica Typhimurium and Listeria monocytogenes inoculated on cherry tomatoes and strawberries, was examined. Bacteria were spot inoculated on the produce surface, air dried and sealed inside a rigid polypropylene container. Samples were indirectly exposed (i.e. placed outside plasma discharge) to a high voltage (70kV RMS) air ACP and subsequently stored at room temperature for 24 h. ACP treatment for 10, 60 and 120 s resulted in reduction of Salmonella,

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.

Aims: The main objective of this study was to determine the inactivation efficacy of dielectric barrier discharge atmospheric cold plasma (DBD-ACP) generated inside a sealed package for Escherichia coli ATCC 25922. Methods and Results: A plasma discharge was generated between two circular aluminium electrodes at 40 kV. E. coli suspensions (10 7 CFU ml À1 ) in either maximum recovery diluent (MRD) or phosphate buffered saline (PBS) were treated in a 96-well microtitre plate inside a sealed package. The effects of treatment time, post-treatment storage time, either direct or indirect samples exposure to the plasma discharge and suspension media were studied. Regardless of the media tested, 20 s of direct and 45 s of indirect plasma treatment resulted in complete bacterial inactivation (7 log CFU ml À1 ). At the lower plasma treatment times (10-30 s) investigated, the effects of suspension media and mode of exposure on the inactivation efficacy were evident. The inactivation efficacy was also influenced by the post-treatment storage time.Conclusions: It was demonstrated that the novel DBD-ACP can inactivate high concentrations of E. coli suspended in liquids within sealed packages in seconds.Significance and impact of the Study: A key advantage of this in-package nonthermal novel disinfection approach is the elimination of post-processing contamination.

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.

Recent reports indicate that atmospheric cold plasma (ACP) treatment of seeds can enhance their germination, however, the mechanisms of action are not yet entirely clear. In the present work, we report on the effects of plasma treatment on wheat seed germination and seedling growth. Additionally, changes in the surface chemistry and characteristics of the wheat seeds exposed to plasma were investigated. Treatments of 30–60 s significantly enhanced the germination rate and showed positive effects on seedling growth. ACP resulted in changes of seed surface and chemical characteristics including water uptake and contact angle values. Changes in seed pH and total titratable acidity, as well as nitrites, nitrates, and malondialdehyde concentrations were also recorded.

Microbial biofilms and bacteria internalised in produce tissue may reduce the effectiveness of decontamination methods. In this study, the inactivation efficacy of in-package atmospheric cold plasma (ACP) afterglow was investigated against Salmonella Typhimurium, Listeria monocytogenes and Escherichia coli in the forms of planktonic cultures, biofilms formed on lettuce and associated bacteria internalised in lettuce tissue. Prepared lettuce broth (3%) was inoculated with bacteria resulting in a final concentration of ~7.0 log 10 CFU/ml. For biofilm formation and internalisation, lettuce pieces (5 x 5 cm) were dip-inoculated in bacterial suspension of ~7.0 log 10 CFU/ml for 2 h and further incubated for 0, 24 and 48 h at either 4°C or room temperature (~22°C) in combination with light/dark photoperiod or at 4°C under dark conditions. Inoculated samples were sealed inside a rigid polypropylene container and However, the existence of biofilms and internalised bacteria should be considered for further optimisation of ACP treatment parameters in order to achieve an effective control of the realistic challenges posed by foodborne pathogens.