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

Abstract

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.

Keywords: Aspergillus flavus; cold plasma; grains; mycotoxin.

FIG 1

ACP treatment efficacy against A. flavus spores. The effects of GP (a) and PAW (b) on spore culturability (log₁₀ CFU per milliliter) and metabolic activity (percent) are shown. Error bars show the standard deviations. Different letters indicate a significant difference at the 0.05 level between various treatment parameters.

FIG 2

Effect of ACP treatment of A. flavus spores on MDA concentrations (nanomolar) using GP (a) and PAW (b). Different letters indicate a significant difference at the 0.05 level between various treatment parameters.

FIG 3

Effect of ACP treatment of A. flavus spores on concentrations of extracellular DNA (micrograms per milliliter) and protein (milligrams per milliliter) using GP (a) and PAW (b). Different letters indicate a significant difference at the 0.05 level between various treatment parameters.

FIG 4

Effects of acidified water and PAW-20 on A. flavus spores. Effects on spore culturability (log₁₀ CFU per milliliter) and metabolic activity (percent) are shown. Error bars show the standard deviations. Different letters indicate a significant difference at the 0.05 level between various treatment parameters.

FIG 5

Effect of gas plasma treatment on biomass production (optical density at 590 nm [OD₅₉₀]) of A. flavus biofilms. Different letters indicate a significant difference at the 0.05 level between the initial biofilm biomass (after 24 h of incubation) and those of untreated control and ACP-treated samples.

FIG 6

Effect of gas plasma treatment on A. flavus biofilms. The effects on spore culturability (log₁₀ CFU per milliliter) and metabolic activity (percent) are shown. Error bars show the standard deviations. Different letters indicate a significant difference at the 0.05 level between untreated control and ACP-treated samples.

FIG 7

Effect of gas plasma treatment of A. flavus biofilms on MDA concentrations (nanomolar). Different letters indicate a significant difference at the 0.05 level between untreated control and ACP-treated samples.

FIG 8

Effect of gas plasma treatment of A. flavus biofilms on concentrations of extracellular DNA (micrograms per milliliter) and protein (milligrams per milliliter). Different letters indicate a significant difference at the 0.05 level between untreated control and ACP-treated samples.

FIG 9

Structure of A. flavus biofilms before and after ACP treatment. Optical microscope images (magnification, ×400) at initial inoculum concentrations of 6 log₁₀ CFU/ml for control (a) and ACP-treated (b) samples and 7 log₁₀ CFU/ml for control (c) and ACP-treated samples (d) are shown.

FIG 10

Structure of A. flavus biofilms before and after ACP treatment. Images from SEM analysis (magnification, ×2,000) at initial inoculum concentrations of 6 log₁₀ CFU/ml for control (a) and ACP-treated (b) samples and of 7 log₁₀ CFU/ml for control (c) and ACP-treated (d) samples are shown. White arrows indicate spores with visibly deformed structures.

FIG 11

Mechanisms of fungal inactivation by cold plasma.

FIG 12

Experimental setup (a) and schematic diagram (b) of the DBD ACP reactor. Either petri dishes containing sterile deionized water or A. flavus spore suspensions or a 6-well plate containing A. flavus biofilm was treated inside a polypropylene container. For direct exposure, samples were placed within the plasma discharge, and for indirect exposure, they were placed outside the discharge.