Added insult to injury? The response of meat-associated pathogens to proposed antimicrobial interventions.

Modern requirements for 'green label' meat products have led to the design of novel antimicrobial innovations which prioritise quality, safety and longevity. Plasma-functionalised water (PFW), ultraviolet light and natural antimicrobial compositions have been investigated and optimised for control of foodborne pathogens like Campylobacter jejuni and Salmonella enterica serovar Typhimurium. However, given the adaptive mechanisms present in bacteria under external stresses, it is imperative to understand the effect that sublethal treatment may have on the bacterial transcriptome. In this study, Salmonella Typhimurium and C. jejuni were treated with sublethal doses of ultraviolet light, a citrus juice/essential oil marinade, and 'spark' or 'glow' cold plasma generation system-produced PFW. Immediately after treatment, cells were lysed and RNA was extracted and purified. mRNA was converted to cDNA by reverse transcription-PCR and sequenced by an Illumina MiSeq® system. Sequences were filtered and analysed using the Tuxedo workflow. Sublethal treatment of Campylobacter jejuni and Salmonella Typhimurium led to increased immediate cellular and metabolic activity, as well as diversification in protein and metabolic functioning. There was further expression of pathogenesis and virulence-associated traits associated with spark PFW and marinade treatment of Salmonella Typhimurium. However, similar concerns were not raised with glow PFW or UV-treated samples. This study provides science-based evidence of the efficacy of multi-hurdle antimicrobial system using green-label marinades and PFW or UV to inactivate pathogens without upregulating virulence traits in surviving cells. This study will inform policymakers and food industry stakeholders and reinforces the need to incorporate in-line novel technologies to ensure consumer safety. KEY POINTS: • Salmonella and C. jejuni showed increased cell activity in immediate response to stress. • Virulence genes showed increased expression when treated with natural antimicrobials and sPFW. • Reduced immediate transcriptomic response to gPFW and UV treatment indicates lower risk.

Combined Effect of Plasma-Activated Water and Topotecan in Glioblastoma Cells.

The increase in cancer diagnoses and cancer deaths, severe side effects of existing treatments and resistance to traditional treatments have generated a need for new anticancer treatments. Glioblastoma multiforme (GBM) is the most common, malignant and aggressive brain cancer. Despite many innovations regarding GBM treatment, the final outcome is still very poor, making it necessary to develop new therapeutic approaches. Cold atmospheric plasma (CAP) as well as plasma-activated liquids (PAL) are being studied as new possible approaches against cancer. The anticancer activity of PAL such as "plasma-activated water" (PAW) is dependent on the reactive chemical compounds present in the solution. Possible combinatory effects with conventional therapies, such as chemotherapeutics, may expand the potential of PAL for cancer treatment. We aim to explore the therapeutic properties of a combination of PAW and topotecan (TPT), an antineoplastic agent with major cytotoxic effects during the S phase of the cell cycle, on a GBM cancer cell line (U-251mg). Combined treatments with PAW and TPT showed a reduction in the metabolic activity and cell mass, an increase in apoptotic cell death and a reduction in the long-term survival. Single applications of PAW+TPT treatments showed a cytotoxic effect in the short term and an antiproliferative effect in the long term, warranting future exploration of combining PAW with chemotherapeutic agents as new therapeutic approaches.

The efficiency of UV light-emitting diodes (UV-LED) in decontaminating Campylobacter and Salmonella and natural microbiota in chicken breast, compared to a UV pilot-plant scale device.

This study investigated the combined effect of Ultraviolet (UV) light-emitting diode (LED) technology treatment with refrigerated storage of chicken breast meat over 7 days on Campylobacter jejuni, Salmonella enterica serovar Typhimurium, total viable counts (TVC) and total Enterobacteriaceae counts (TEC). An optimised UV-LED treatment at 280 nm for 6 min decreased inoculated S. Typhimurium and C. jejuni populations by 0.6-0.64 log CFU/g, and TVC and TEC population by 1-1.2 log CFU/g in chicken samples. During a 7-day storage at 4 °C, a 0.73 log reduction in C. jejuni was achieved compared with non-treated samples. Moreover, the UV-LED effectiveness to reduce TVC and TEC during refrigerated storage was compared with a conventional UV lamp and a similar efficiency was observed. The impact of UV-LED and UV lamp devices on the microbial community composition of chicken meat during storage was further examined using 16 S rRNA gene amplicon sequencing. Although similar bacterial reductions were observed for both technologies, the microbial communities were impacted differently. Treatment with the UV conventional lamp increased the proportion of Brochothrix spp. In meat samples, whilst Photobacterium spp. Levels were reduced.

Product development and X-Ray microtomography of a traditional white pan bread from plasma functionalized flour.

Cold plasma (CP) technology has emerged as a novel non-thermal technology with the potential to improve food quality or impart functionality to ingredients. Our previous studies on wheat flour demonstrated how the structure and functionality of wheat flour might be modified using CP to provide an alternative to chemical additives (Chaple et al., 2020). However, understanding of the further effects of plasma functionalized ingredients in existing or new product formulation is limited. This study investigated the effects of CP treatment of wheat flour on traditional white pan bread development. The bread was formulated using plasma functionalized flour (PFF), and critical product characteristic responses were analyzed. Plasma treatment of flour positively affected the bread's expansion ratio, crust color, and water activity. Farinograph analysis suggests improvement in water absorption capacity, dough development time, and dough stability. X-Ray Microtomography (XRMT) analysis was conducted to understand how plasma functionalising the flour impacted the microstructure of bread. The 3D scans suggested no macro-change in the bread matrix compared to control; however, the porosity decreased in line with the increasing plasma treatment duration of the flour. The texture profile analysis showed an improvement in the gluten network developed in the dough developed from PFF. Sensory analysis results showed overall acceptance for bread formulated with PFF compared with a commercial sample. Overall, CP treatment of the flour improved the functionality in relation to dough and bread preparation and can thus provide an alternative to chemical additives in bread making. The CP processes may be modulated to deliver tailored effects for bread product development.

Emerging green cell disruption techniques to obtain valuable compounds from macro and microalgae: a review.

In the modern era, macro-microalgae attract a strong interest across scientific disciplines, owing to the wide application of these cost-effective valuable bioresources in food, fuel, nutraceuticals, and pharmaceuticals etc. The practice of eco-friendly extraction techniques has led scientists to create alternative processes to the conventional methods, to enhance the extraction of the key valuable compounds from macro-microalgae. This review narrates the possible use of novel cell disruption techniques, including use of ionic liquid, deep eutectic solvent, surfactant, switchable solvents, high voltage electrical discharge, explosive decompression, compressional-puffing, plasma, and ozonation, which can enable the recovery of value added substances from macro-microalgae, complying with the principles of green chemistry and sustainability. The above-mentioned innovative techniques are reviewed with respect to their working principles, benefits, and possible applications for macro-microalgae bioactive compound recovery and biofuel. The benefits of these techniques compared to conventional extraction methods include shorter extraction time, improved yield, and reduced cost. Furthermore, various combinations of these innovative technologies are used for the extraction of thermolabile bioactive compounds. The challenges and prospects of the innovative extraction processes for the forthcoming improvement of environmentally and cost-effective macro-microalgal biorefineries are also explained in this review.

Advances in 3D culture systems for therapeutic discovery and development in brain cancer.

This review focuses on recent advances in 3D culture systems that promise more accurate therapeutic models of the glioblastoma multiforme (GBM) tumor microenvironment (TME), such as the unique anatomical, cellular, and molecular features evident in human GBM. The key components of a GBM TME are outlined, including microbiomes, vasculature, extracellular matrix (ECM), infiltrating parenchymal and peripheral immune cells and molecules, and chemical gradients. 3D culture systems are evaluated against 2D culture systems and in vivo animal models. The main 3D culture techniques available are compared, with an emphasis on identifying key gaps in knowledge for the development of suitable platforms to accurately model the intricate components of the GBM TME.

Efficacy of plasma activated saline in a co-culture infection control model.

Plasma activated liquids have demonstrated antimicrobial effects and receive increasing attention due to the potential to strengthen the armoury of novel approaches against antibiotic resistant bacteria. However, the antibacterial activity and cytotoxic effects of these solutions need to be understood and balanced before exposure to humans. In this study, the antibacterial effects of plasma activated saline (PAS) were tested against Gram negative and positive bacteria, and HaCaT keratinocytes were used for cytotoxicity studies. For the first time, a co-culture model between these bacteria and eukaryotic cells under the influence of PAS has been described. Exposure of saline to plasma resulted in high concentrations of nitrate, hydrogen peroxide and a reduction of pH. PAS caused high antibacterial effects in the co-culture model, accompanied by high cytotoxic effects to the monolayer of mammalian cells. We present evidence and provide a deeper understanding for the hypothesis that upon treatment with PAS, chemical species generated in the liquid mediate high antimicrobial effects in the co-culture setup as well as mitochondrial depolarization and glutathione depletion in HaCaT cells and cell lysis due to acidic pH. In conclusion, PAS retains strong antibacterial effects in a co-culture model, which may have unintended negative biological effects on mammalian cells.

Cell death induced in glioblastoma cells by Plasma-Activated-Liquids (PAL) is primarily mediated by membrane lipid peroxidation and not ROS influx.

Since first identified in 1879, plasma, the fourth state of matter, has been developed and utilised in many fields. Nonthermal atmospheric plasma, also known as cold plasma, can be applied to liquids, where plasma reactive species such as reactive Oxygen and Nitrogen species and their effects can be retained and mediated through plasma-activated liquids (PAL). In the medical field, PAL is considered promising for wound treatment, sterilisation and cancer therapy due to its rich and relatively long-lived reactive species components. This study sought to identify any potential antagonistic effect between antioxidative intracellularly accumulated platinum nanoparticles (PtNPs) and PAL. We found that PAL can significantly reduce the viability of glioblastoma U-251MG cells. This did not involve measurable ROS influx but instead lead to lipid damage on the plasma membrane of cells exposed to PAL. Although the intracellular antioxidative PtNPs showed no protective effect against PAL, this study contributes to further understanding of principle cell killing routes of PAL and discovery of potential PAL-related therapy and methods to inhibit side effects.

Combination of Natural Compounds With Novel Non-thermal Technologies for Poultry Products: A Review.

Ensuring safe, fresh, and healthy food across the shelf life of a commodity is an ongoing challenge, with the driver to minimize chemical additives and their residues in the food processing chain. High-value fresh protein products such as poultry meat are very susceptible to spoilage due to oxidation and bacterial contamination. The combination of non-thermal processing interventions with nature-based alternatives is emerging as a useful tool for potential adoption for safe poultry meat products. Natural compounds are produced by living organisms that are extracted from nature and can be used as antioxidant, antimicrobial, and bioactive agents and are often employed for other existing purposes in food systems. Non-thermal technology interventions such as high-pressure processing, pulsed electric field, ultrasound, irradiation, and cold plasma technology are gaining increasing importance due to the advantages of retaining low temperatures, nutrition profiles, and short treatment times. The non-thermal unit process can act as an initial obstacle promoting the reduction of microflora, while natural compounds can provide an active obstacle either in addition to processing or during storage time to maintain quality and inhibit and control growth of residual contaminants. This review presents the application of natural compounds along with emerging non-thermal technologies to address risks in fresh poultry meat.

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.

The effect of atmospheric cold plasma treatment on the antigenic properties of bovine milk casein and whey proteins.

Casein, ß-lactoglobulin and α-lactalbumin are major milk protein allergens. In the present study, the structural modifications and antigenic response of these bovine milk allergens as induced by non-thermal treatment by atmospheric cold plasma were investigated. Spark discharge (SD) and glow discharge (GD), as previously characterized cold plasma systems, were used for protein treatments. Casein, ß-lactoglobulin and α-lactalbumin were analyzed before and after plasma treatment using SDS-PAGE, FTIR, UPLC-MS/MS and ELISA. SDS-PAGE results revealed a reduction in the casein and α-lactalbumin intensity bands after SD or GD treatments; however, the ß-lactoglobulin intensity band remained unchanged. FTIR studies revealed alterations in protein secondary structure induced by plasma, particularly contents of ß-sheet and ß-turn. The UPLC-MS/MS results showed that the amino acid compositions decreased after plasma treatments. ELISA of casein and α-lactalbumin showed a decrease in antigenicity post plasma treatment, whereas ELISA of ß-lactoglobulin showed an increase in antigenicity. The study indicates that atmospheric cold plasma can be tailored to mitigate the risk of bovine milk allergens in the dairy processing and ingredients sectors.

Effects of cold plasma on wheat grain microbiome and antimicrobial efficacy against challenge pathogens and their resistance.

The safety and quality of cereal grain supplies are adversely impacted by microbiological contamination, with novel interventions required to maximise whole grains safety and stability. The microbiological contaminants of wheat grains and the efficacy of Atmospheric Cold Plasma (ACP) for potential to control these risks were investigated. The evaluations were performed using a contained reactor dielectric barrier discharge (DBD) system; samples were treated for 0-20 min using direct and indirect plasma exposure. Amplicon-based metagenomic analysis using bacterial 16S rRNA gene and fungal 18S rRNA gene with internal transcribed spacer (ITS) region was performed to characterize the change in microbial community composition in response to ACP treatment. The antimicrobial efficacy of ACP against a range of bacterial and fungal contaminants of wheat, was assessed to include individual isolates from grains as challenge pathogens. ACP influenced wheat microbiome composition, with a higher microbial diversity as well as abundance found on the untreated control grain samples. Culture and genomic approaches revealed different trends for mycoflora detection and control. A challenge study demonstrated that using direct mode of plasma exposure with 20 min of treatment significantly reduced the concentration of all pathogens. Overall, reduction levels for B. atrophaeus vegetative cells were higher than for all fungal species tested, whereas B. atrophaeus spores were the most resistant to ACP among all microorganisms tested. Of note, repeating sub-lethal plasma treatment did not induce resistance to ACP in either B. atrophaeus or A. flavus spores. ACP process control could be tailored to address diverse microbiological risks for grain stability and safety.

Direct Plasma Deposition of Collagen on 96-Well Polystyrene Plates for Cell Culture.

A cold atmospheric plasma unit was used to deposit a biologic, in this case collagen, onto a surface. A collagen coating was applied to 96-well polystyrene plates at a range of powers to determine the effects of the plasma power on the coating structure and viability. Plasma characterization was carried out using voltage, current, and power measurements. Coating characterization was completed using gravimetric measurement, cell growth, water contact angle, as well as spectroscopic analysis and compared to commercial collagen-coated plates. Cell culture studies were also undertaken. The plasma coating matched the performance of the commercial plate but dramatically reduced production time and cost. This method could allow for automated inline production of collagen-coated plates for cell culture applications.

Assessing the Biological Safety of Atmospheric Cold Plasma Treated Wheat Using Cell and Insect Models.

Atmospheric cold plasma (ACP) is under investigation for an extensive range of biocontrol applications in food biosystems. However, the development of a novel intervention technology requires a thorough evaluation of the potential for negative effects and the implications for the human and animal food chains' safety. The evaluations were performed using a contained, high-voltage, dielectric barrier discharge plasma system. The cytotoxicity of two types of food models-a liquid model (wheat model medium (WMM)) vs. a solid model (wheat grain extract (WGE)) was compared in vitro using the mammalian cell line CHO-K1. The residual toxicity of ACP treatment of grains for food purposes was assessed using the invertebrate model Tribolium castaneum, by feeding the beetles with flour produced from ACP-treated wheat grains. The cytotoxic effects and changes in the chemistry of the ACP-treated samples were more pronounced in samples treated in a liquid form as opposed to actual wheat grains. The feeding trial using T. castaneum demonstrated no negative impacts on the survivability or weight profiles of insects. Investigations into the interactions of plasma-generated species with secondary metabolites in the food matrices are necessary to ensure the safety of plasma for food applications.

Sex-Specific Differences in Short-Term and Long-Term Outcomes in Acute Stroke Patients from Qatar.

BACKGROUND: Sex differences may determine presentation, utility of treatment, rehabilitation, and occurrences of major adverse cardiovascular events (MACEs) in acute stroke (AS). OBJECTIVE: The purpose of the study was to evaluate the short-term prognosis and long-term outcomes in MACEs in Qatari nationals admitted with AS. METHODS: All AS patients admitted between January 2014 and February 2019 were included. We evaluated the preadmission modified Rankin scale (mRS) score, etiology and severity of symptoms, complications, and functional recovery at discharge and 90 days. MACEs were recorded for 5 years. RESULTS: There were 891 admissions for AS (mean age 64.0 ± 14.2 years) (male, n = 519 [mean age ± SD 62.9 ± 14.1 years]; female, n = 372 [mean age ± SD 65.6 ± 14.2 years] p = 0.005). There were no differences in the preadmission mRS and severity of symptoms as measured on National Institute of Stroke Scale. At discharge, the outcome was better (mRS 0-2) in men (57.8 vs. 46.0%), p = 0.0001. This difference persisted at the 90-day follow-up (mRS 0-2, male 69.4% vs. female 53.2%, p = 0.0001). At the 90-day follow-up, more women died (total deaths 70; women 38 [10.2%] versus men 32 [6.2%], p = 0.03). MACEs occurred in 25.6% (133/519) males and 30.9% (115/372) females over the 5-year follow-up period (odds ratio 0.77, 95% confidence interval 0.57-1.0, p = 0.83). CONCLUSIONS: Female patients have a poor short-term outcome following an AS when corrected for age and comorbidities. While our study cannot explain the reasons for the discrepancies, higher poststroke depression and social isolation in women may be important contributory factors, and requires further studies are required to confirm these findings.

Corneal confocal microscopy identifies greater corneal nerve damage in patients with a recurrent compared to first ischemic stroke.

OBJECTIVES: Corneal nerve damage may be a surrogate marker for the risk of ischemic stroke. This study was undertaken to determine if there is greater corneal nerve damage in patients with recurrent ischemic stroke. METHODS: Corneal confocal microscopy (CCM) was used to quantify corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL) and corneal nerve fiber tortuosity (CNFT) in 31 patients with recurrent ischemic stroke, 165 patients with a first acute ischemic stroke and 23 healthy control subjects. RESULTS: Triglycerides (P = 0.004, P = 0.017), systolic BP (P = 0.000, P = 0.000), diastolic BP (P = 0.000, P = 0.000) and HbA1c (P = 0.000, P = 0.000) were significantly higher in patients with first and recurrent stroke compared to controls. There was no difference in age, BMI, HbA1c, total cholesterol, triglycerides, LDL, HDL, systolic and diastolic BP between patients with a first and recurrent ischemic stroke. However, CNFD was significantly lower (24.98±7.31 vs 29.07±7.58 vs 37.91±7.13, P<0.05) and CNFT was significantly higher (0.085±0.042 vs 0.064±0.037 vs 0.039±0.022, P<0.05) in patients with recurrent stroke compared to first stroke and healthy controls. CNBD (42.21±24.65 vs 50.46±27.68 vs 87.24±45.85, P<0.001) and CNFL (15.66±5.70, P<0.001 vs 17.38±5.06, P = 0.003) were equally reduced in patients with first and recurrent stroke compared to controls (22.72±5.14). CONCLUSIONS: Corneal confocal microscopy identified greater corneal nerve fibre loss in patients with recurrent stroke compared to patients with first stroke, despite comparable risk factors. Longitudinal studies are required to determine the prognostic utility of corneal nerve fiber loss in identifying patients at risk of recurrent ischemic stroke.

Cold Atmospheric Plasma Stimulates Clathrin-Dependent Endocytosis to Repair Oxidised Membrane and Enhance Uptake of Nanomaterial in Glioblastoma Multiforme Cells.

Cold atmospheric plasma (CAP) enhances uptake and accumulation of nanoparticles and promotes synergistic cytotoxicity against cancer cells. However, the mechanisms are not well understood. In this study, we investigate the enhanced uptake of theranostic nanomaterials by CAP. Numerical modelling of the uptake of gold nanoparticle into U373MG Glioblastoma multiforme (GBM) cells predicts that CAP may introduce a new uptake route. We demonstrate that cell membrane repair pathways play the main role in this stimulated new uptake route, following non-toxic doses of dielectric barrier discharge CAP. CAP treatment induces cellular membrane damage, mainly via lipid peroxidation as a result of reactive oxygen species (ROS) generation. Membranes rich in peroxidised lipids are then trafficked into cells via membrane repairing endocytosis. We confirm that the enhanced uptake of nanomaterials is clathrin-dependent using chemical inhibitors and silencing of gene expression. Therefore, CAP-stimulated membrane repair increases endocytosis and accelerates the uptake of gold nanoparticles into U373MG cells after CAP treatment. We demonstrate the utility of CAP to model membrane oxidative damage in cells and characterise a previously unreported mechanism of membrane repair to trigger nanomaterial uptake. This knowledge will underpin the development of new delivery strategies for theranostic nanoparticles into cancer cells.

Prevalence, Characteristics and Risk Factors for Embolic Stroke of Undetermined Source in West and South Asia and North African Population Residing in Qatar.

BACKGROUND AND PURPOSE: Embolic stroke of undetermined source (ESUS) is an important contributor to stroke worldwide. Little is known about ESUS in developing parts of the world such as South Asia, West Asia and North Africa despite the high stroke burden in these areas. The purpose of the study was to characterize the prevalence, demographic, risk factor and clinical aspects of ESUS in patients from South Asia, West Asia and North Africa residing in Qatar. METHODS: Data were retrospectively collected on 3103 stoke patients. Risk factors and clinical features of the ESUS group were compared to all other strokes using Chi-square or student's t-tests. Logistic regression was used to identify factors associated with ESUS. ESUS patients were compared based on ethnicity using Chi-square or one-way ANOVA. RESULTS: 634 patients (30·9%, 95% CI (28·9%-32·9%) met the ESUS criteria. Mean age was 56·3 years ± 13·7 and South Asian ESUS patients were younger than West Asians or North Africans (67·1 ± 13·5 versus 52·1 ± 10·8 versus 53·5 ± 14·2, P = .001). Smoking, diastolic function, prior antiplatelets and wall motion abnormalities were more common in ESUS. Logistic regression showed that South Asian ethnicity (OR 1·50, CI 1·14-1·97, P = ·003), diastolic dysfunction (OR 1·47, CI 1·23-1·75, P = ·005), global (OR 1·79, CI 1·41-2·26, P = ·001) and focal (OR 5·48, CI 3·79-7·92, P = ·001) wall motion abnormalities, predicted ESUS. CONCLUSIONS: ESUS is a major cause of stroke in patients from West Asia, South Asia and North Africa residing in Qatar. The clinical profile and risk factors for ESUS vary based on ethnicity. In South Asians, ESUS occurs at a younger age and is most likely cardiogenic in origin.

Inactivation Efficacies and Mechanisms of Gas Plasma and Plasma-Activated Water against Aspergillus flavus Spores and Biofilms: a Comparative Study.

Atmospheric cold plasma (ACP) treatment is an emerging food technology for product safety and quality retention, shelf-life extension, and sustainable processing. The activated chemical species of ACP can act rapidly against microorganisms without leaving chemical residues on food surfaces. The main objectives of this study were to investigate the efficiency and mechanisms of inactivation of fungal spores and biofilms by ACP and to understand the effects of the gas-mediated and liquid-mediated modes of application against important fungal contaminants. Aspergillus flavus was selected as the model microorganism. A. flavus spores were exposed to either gas plasma (GP) or plasma-activated water (PAW), whereas gas plasma alone was used to treat A. flavus biofilms. This study demonstrated that both GP and PAW treatments independently resulted in significant decreases of A. flavus metabolic activity and spore counts, with maximal reductions of 2.2 and 0.6 log10 units for GP and PAW, respectively. The characterization of the reactive oxygen and nitrogen species in PAW and spore suspensions indicated that the concentration of secondary reactive species was an important factor influencing the antimicrobial activity of the treatment. The biofilm study showed that GP had detrimental effects on biofilm structure; however, the initial inoculum concentration prior to biofilm formation can be a crucial factor influencing the fungicidal effects of ACP.IMPORTANCE The production of mycotoxin-free food remains a challenge in both human and animal food chains. A. flavus, a mycotoxin-producing contaminant of economically important crops, was selected as the model microorganism to investigate the efficiency and mechanisms of ACP technology against fungal contaminants of food. Our study directly compares the antifungal properties of gas plasma (GP) and plasma-activated water (PAW) against fungi as well as reporting the effects of ACP treatment on biofilms produced by A. flavus.