Dana Ziuzina

Atmospheric cold plasma inactivation of Escherichia coli in liquid media inside a sealed package.

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.

Cold plasma inactivation of internalised bacteria and biofilms for Salmonella enterica serovar Typhimurium, Listeria monocytogenes and Escherichia coli.

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 log10 CFU/ml. For biofilm formation and internalisation, lettuce pieces (5 × 5 cm) were dip-inoculated in bacterial suspension of ~7.0 log10 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 indirectly exposed (i.e. placed outside plasma discharge) to a high voltage (80 kVRMS) air ACP with subsequent storage for 24 h at 4 °C. ACP treatment for 30s reduced planktonic populations of Salmonella, L. monocytogenes and E. coli suspended in lettuce broth to undetectable levels. Depending on storage conditions, bacterial type and age of biofilm, 300 s of treatment resulted in reduction of biofilm populations on lettuce by a maximum of 5 log10 CFU/sample. Scanning electron and confocal laser microscopy pointed to the incidence of bacterial internalisation and biofilm formation, which influenced the inactivation efficacy of ACP. Measured intracellular reactive oxygen species (ROS) revealed that the presence of organic matter in the bacterial suspension might present a protective effect against the action of ROS on bacterial cells. This study demonstrated that high voltage in-package ACP could be a potential technology to overcome bacterial challenges associated with food produce. 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.

Cold Plasma Inactivation of Bacterial Biofilms and Reduction of Quorum Sensing Regulated Virulence Factors

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.

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.

The Potential of Cold Plasma for Safe and Sustainable Food Production

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.

Characterization of a Novel Cold Atmospheric Air Plasma System for Treatment of Packaged Liquid Food Products

The technology of atmospheric plasma (ionized gas), widely used in material processing, offers one of the most significant breakthroughs in food processing and safety. The technology allows the generation of bactericidal molecules very efficiently with low power requirements. Non-thermal atmospheric plasma has been used to effectively decontaminate surfaces, but has received limited investigation for in-package decontamination. This study demonstrates the potential for in-package plasma treatment of food products in sealed packages. The advantage of in-package cold plasma treatment is that the bactericidal molecules are generated and contained in the package allowing extended exposure to bacteria while reverting back to the original package gas within 24 hr storage. The study treated liquid food surrogates containing bacteria and oxidation sensitive dye (methylene blue) inside sealed packages. The surrogates were placed in 96 well plates and were packaged in air and exposed directly and indirectly to the plasma field in a 2.2 cm high, 23 cm x 31 cm sealed polypropylene container for up to five minutes of treatment. Treatments were carried out using a prototype Dielectric Barrier Discharge (DBD) operating at a voltage of 40 kV, and frequency of 50 Hz. The UV-Vis emission spectra of plasma were collected and analyzed. The spectra showed emission bands for nitrogen and oxygen species including strong emission lines for excited states of the atomic species O, O+, N and N+. The results from the evaluation of methylene blue suggest that direct exposure to the plasma ionization field produces a greater oxidative effect compared to indirect exposure. For five minutes treatment, direct exposure of methylene blue resulted in a 90% reduction in absorbance and indirect exposure of methylene blue resulted in a 75% reduction in absorbance. These reductions may result from conversion of ozone into hydroxyl radicals which reduces the methylene blue from dark blue color to clear. This process appears to be non-reversible. Additionally, bacterial studies examining treatment of E. coli ATCC 25922 suspended in maximum recovery diluent inside of 96 well plates found a 7 log reduction after 50 s treatment and 24 hours storage for both direct and indirect plasma exposure. Ozone concentrations measured immediately after five minutes of ionization were approximately 1600 ppm. The goal of this research is to maximize bacterial reductions and minimize quality (oxidative) loss for liquid food products inside a sealed package.

Improving microbiological safety and quality characteristics of wheat and barley by high voltage atmospheric cold plasma closed processing

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 log10 CFU/g and of wheat - 1.5 and 2.5 log10 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 log10 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.

Chapter 12 – Inactivation of Staphylococcus aureus in Foods by Thermal and Nonthermal Control Strategies

Abstract Despite the technological interventions and efforts from food processors to improve the food quality, the microbiological safety of foods remains a major challenge for food industry globally. A significant portion among the reported foodborne illnesses has been attributed to staphylococcal infection. Staphylococcus aureus is an opportunistic human pathogen, which can cause a variety of infections varying from minor skin infection to severe life-threatening diseases. Its pathogenicity is attributed to toxin-mediated virulence, invasiveness, and antimicrobial resistance. To assure microbiologically safe food that is free from microbial toxins, adequate processing and preservation technologies must be applied. This chapter focuses on the control of S. aureus associated with various food systems and reviews the role of new thermal and nonthermal technologies in the microbiological safety of foods.

Atmospheric Cold Plasma as a Tool for Microbiological Control

Outbreaks of foodborne human illnesses resulting from contaminated raw or minimally processed fruits and vegetables have been widely reported globally. The microbiological challenges associated with fresh produce are diverse and respond differently to minimal processing technologies. Atmospheric cold plasma is a relatively new technology and represents a potential alternative to traditional methods for decontamination of foods. The objective of this work was to determine the influence of extrinsic atmospheric cold plasma (ACP) treatment control parameters and to optimize treatment parameters for decontamination with respect to different forms of key safety challenges pertinent to fresh produce. The optimisation studies demonstrated that inactivation efficacy of treatment, when tested against high populations of E. coli suspended in liquid media, was governed by the processing parameter of mode of exposure, treatment time, post treatment storage time, voltage levels, working gas and media composition. Post treatment storage time emerged as a critical treatment parameter for consistency and efficiency of bacterial inactivation with the system. The effect of media complexity was evident with higher inactivation rates achieved in media with simpler composition. Antimicrobial efficacy of ACP increased when voltage level and gas mixture with higher oxygen content was utilised, nullifying the effect of mode of ACP exposure and media composition. High voltage in-package indirect ACP treatment with 24 h of post treatment storage time, selected as the more favourable treatment approach in terms of produce quality retention, was highly effective for decontamination of cherry tomatoes and strawberries inoculated with Salmonella, E. coli and L. monocytogenes monocultures and against background microflora of produce. However, the produce type and the contaminating pathogen influenced decontaminating effect of ACP with higher inactivation rates achieved for Gramnegative bacteria and bacteria associated with smooth surface of produce. The antimicrobial potential of high voltage either direct or indirect in-package atmospheric air ACP treatment with subsequent 24 h of storage was proven to be effective for inactivation of pathogens in the form of monoculture biofilms commonly implicated in foodborne and healthcare associated human infections, E. coli, L. monocytogenes, S. aureus, P. aeruginosa established during 48 h on abiotic surface. However, the efficiency of ACP treatment was again bacterial type dependant. Although complete inactivation of metabolic activity of Gram-negative bacteria could not be achieved, electron microscopy analyses confirmed the destructive action of ACP treatment. In-package high voltage indirect ACP treatment was effective against Salmonella, L. monocytogenes and E. coli biofilms developed on lettuce. This study also demonstrated that produce storage conditions, such as temperature, light and storage time had interactive effects on bacterial proliferation, internalisation, 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. Significant reductions of P. aeruginosa quorum sensing (QS)-regulated virulence factors, such as pyocyanin and elastase production, were achieved, suggesting that ACP technology could be a potential QS inhibitor and may play an important role in attenuation of virulence of pathogenic bacteria. Despite the varying parameters that influenced plasma bactericidal activity, high voltage in-package atmospheric air ACP decontamination approach showed an efficient reduction of high concentrations of bacteria in liquids, associated with produce and bacteria in their most resistant, biofilm form. These results represent significant technological advances in non-thermal bactericidal treatment with a key advantage of elimination of post-processing contamination of the product, thereby increasing microbiological safety and extension of produce shelf life. Declaration I certify that this thesis which I now submit for examination for the award of Doctor of Philosophy, is entirely my own work and has not been taken from the work of others, save and to the extent that such work has been cited and acknowledged within the text of my work. This thesis was prepared according to the regulations for postgraduate study by research of the Dublin Institute of Technology and has not been submitted in whole or in part for another award in any other third level institution. The work reported on in this thesis conforms to the principles and requirements of the DITs guidelines for ethics in research. DIT has permission to keep, lend or copy this thesis in whole or in part, on condition that any such use of the material of the thesis be duly acknowledged. Signature __________________________________ Date _______________ Acknowledgments I wish to express my gratitude to my supervisors, Dr. Paula Bourke and Dr. PJ Cullen, for providing me opportunity to join their research group in the first instance and for their tremendous amount of valuable guidance and encouragement throughout the work. I would like to extend my sincere thanks to Dr. Sonal Patil, who kindly shared her knowledge and experience with me and provided valuable suggestions and corrections during my research. I would like to thank all academic and technical staff for their assistance and help. I would like to acknowledge the financial support from the European Community ́s Seventh Framework Program (FP7/2207-2013). Finally, many special thanks to my family and friends for their patience, understanding and support over the past years. Abbreviations °C degrees Celsius CFU colony forming units cm centimetres CO2 carbon dioxide g gram h hour H2O2 hydrogen peroxide HCl hydrochloric acid Hz hertz kV kilovolts L litre M molar mg milligram MHz megahertz min minute mJ/cm mega joule per cm square ml millilitre mM millimolar mm millimetre NaCl sodium chloride nm nanometre p-p peak to peak ppm parts per million RMS root mean square rpm revolutions per minute s second V volts v/v volume per volume w/v weight per volume μl microliter μm micrometre