This paper presents a detailed study of the potential physical mechanisms of the microbial inactivation by cold atmospheric plasmas. With the Bacillus subtilis spores as a model microorganism and an atmospheric-plasma plume in helium flow, optical emission spectroscopy and inactivation kinetics are used to demonstrate the dominating role played by the reactive oxygen species (e.g., atomic oxygen and OH) as well as the minor contributions of the UV photons, heat, charged particles, and electric fields. To differentiate the concentrations of the reactive oxygen species, an atmospheric helium-oxygen plasma is also used for the spore inactivation. Results with the helium and the helium-oxygen plasmas are contrasted to highlight how the production of the spore-killing oxygen species may be enhanced

The ability of atmospheric pressure glow discharges (APGD) to inactivate microorganisms has been demonstrated in a number of previous studies. However, most of this work has been performed using microorganisms that do not form biofilms and with the microorganisms supported on abiotic surfaces that discourage cell growth. When microorganisms attach to the surface of a food, they can extract nutrients from the food and proliferate at the surface. Often this growth takes the form of biofilms which comprise three-dimensional (3-D) networks of polysaccharides that attach microorganisms to surfaces and serve to protect them from external stresses; fresh foods, such as salad crops, frequently harbor biofilms. We believe that the use of APGD offers a potential for inactivating microorganisms on the surface of fresh foods that cannot be readily treated by other methods without inducing unacceptable changes to these foods. As a first step toward a full evaluation of the viability of the APGD technology for food safety control, we consider in this paper two key issues, namely: 1) whether atmospheric glow discharges can inactivate biofilm-forming microorganisms and 2) whether plasma treatment causes significant discoloration to food surfaces. Using the biofilm-forming bacterium Pantoea agglomerans and bell peppers (Capsicum annuum) as a typical example of plant tissue, we show that atmospheric He-O/sub 2/ plasmas can be effective inactivation agents without causing unacceptable levels of discoloration to the peppers, and that furthermore they are superior to the use of low-pressure ultraviolet sources.

Biological sterilization represents one of the most exciting applications of atmospheric pressure glow discharges (APGD). Despite the fact that surgical instruments are contaminated by both microorganisms and proteinaceous matters, sterilization effects of APGD have so far been studied almost exclusively for microbial inactivation. This work presents the results of a detailed investigation of the capability of a helium-oxygen APGD to inactivate proteins deposited on stainless-steel surfaces. Using a laser-induced fluorescence technique for surface protein measurement, a maximum protein reduction of 4.5 logs is achieved by varying the amount of the oxygen admixture into the background helium gas. This corresponds to a minimum surface protein of $0.36\text{femtomole}/{\text{mm}}^2$ . It is found that plasma reduction of surface-borne protein is through protein destruction and degradation, and that its typically biphasic reduction kinetics is influenced largely by the thickness profile of the surface protein. Also presented is a complementary study of possible APGD protein inactivation mechanisms. By interplaying the protein inactivation kinetics with optical emission spectroscopy, it is shown that the main protein-destructing agents are excited atomic oxygen (via the 777 and 844 nm emission channels) and excited nitride oxide (via the 226, 236, and 246 nm emission channels). It is also demonstrated that the most effective protein reduction is achieved possibly through a synergistic effect between atomic oxygen and nitride oxide. This study is a useful step toward a full confirmation of the efficacy of APGD as a sterilization technology for surgical instruments contaminated by prion proteins.

Mechanisms of plasma-induced microbial inactivation have commonly been studied with physicochemical techniques. In this letter, Escherichia coli K-12 and its $\Delta \mathit{rec}A$ , $\Delta \mathit{rpo}S$ , and $\Delta \mathit{sox}S$ mutants are employed to discriminate effects of UV photons, OH radicals, and reactive oxygen species produced in atmospheric discharges. This microbiological approach exploits the fact that these E. coli mutants are defective in their resistance against various external stresses. By interplaying bacterial inactivation kinetics with optical emission spectroscopy, oxygen atoms are identified as a major contributor in plasma inactivation with minor contributions from UV photons, OH radicals, singlet oxygen metastables, and nitric oxide.

It is well established that atmospheric pressure glow discharges are capable of bacterial inactivation. Much less known is their ability to destruct infectious proteins, even though surgical instruments are often contaminated by both bacteria and proteinaceous matters. In this letter, the authors present a study of protein destruction using a low-temperature atmospheric dielectric-barrier discharge jet. Clear evidences of protein removal are presented with data of several complimentary experiments using scanning electron microscopy, electron dispersive x-ray analysis, electrophoresis, laser-induced fluorescence microscopy, and protein reduction kinetics. Considerable degradation is observed of protein fragments that remain on their substrate surface after plasma treatment.

Current inactivation studies of Bacillus subtilis spores using atmospheric-pressure glow discharges (APGD) do not consider two important factors, namely microbial loading at the surface of a substrate and sporulation temperature. Yet these are known to affect significantly microbial resistance to heat and hydrogen peroxide. This letter investigates effects of microbial loading and sporulation temperature on spore resistance to APGD. It is shown that microbial loading can lead to a stacking structure as a protective shield against APGD treatment and that high sporulation temperature increases spore resistance by altering core water content and cross-linked muramic acid content of B. subtilis spores.