## Results and Discussion
### Characteristics of the Plasma-Activated Water Produced by the Soft Plasma Jet
Since \(H_{2}\)O\({}_{2}\) and \(HNO_{3}\) were stable and long-lived reactive species and were confirmed to be present in the PAW (Figures 1-3), they were considered to be key agents for fungal spore inactivation. To provide evidence that they can work against black pigment-protected  
\[O_{2}+e_{aq}\to O_{2}^{-}\,, \tag{2}\]3 (**a**) \(H_{2}\)O\({}_{2}\) and (**b**) \(NO_{x}\) concentrations of the plasma-activated water (PAW) as a function of plasma activation time. The PAW was prepared by activating de-ionized (DI) water with the soft plasma jet. spores, we investigated the effect of \(H_{2}O_{2}\) and \(HNO_{3}\) solutions on the spores. For this investigation, _A. brasiliensis_ spores were treated with either \(H_{2}O_{2}\) or \(HNO_{3}\) solutions with 0.001%, 0.01%, 0.1%, 0.3%, and 1.0% concentrations. As shown in Figure 4b, both \(H_{2}O_{2}\) and \(HNO_{3}\) treatments reduced spore viability in a concentration-dependent manner. These results demonstrate that \(H_{2}O_{2}\) and \(HNO_{3}\) are key players in fungal spore inactivation. Therefore, we could deduce that plasma-induced \(H_{2}O_{2}\) and \(HNO_{3}\) could lead to irreversible intracellular damage in _A. brasiliensis_ spores. These results agreed with those on the effect of PAW treatment on _Aspergillus flavus_ spores [23]. However, [PERSON] et al. treated the _A. flavus_ spores using PAW generated by an electric discharge between plane-parallel electrodes and investigated the effect of PAW treatment on _A. flavus_ spores with a plasma activation time of 5 min and a PAW treatment time of 24 h. Their plasma device and treatment parameters are different from those of our study, which used a plasma activation time of 3 min and a contact time of 30 min for treating _A. brasiliensis_ spores. Meanwhile, we treated _A. brasiliensis_ spores using PAW generated by an electric shock-free, nonthermal atmospheric pressure air plasma jet. The coaxial electrodes plasma jet can generate high density radicals in a relatively large volume by activating DI water with the plasma jet, but plane-parallel electrodes plasma can activate water between electrodes. In addition, plane-parallel electrodes plasma requires relatively high voltage as compared with plasma jet with coaxial electrodes. When considering plasma treatment effects, it seems that our system is more efficient. However, _A. flavus_ is a different species. Thus, there is the possibility that this different inactivation efficiency could also be due to the different biological properties of the two species. To clarify the discrepancy, a comparative study with the same plasma device will be necessary. Generally, nonenzymatic antioxidants such as carotenoids, polysaccharides, chitosan, polyphenol, polyolsols, mannitol, glucose, and vitamins in fungi have oxygen radical scavenging effects [49; 50; 51; 52]. It was well known that glucose and mannose exist in the cell wall of _A. brasiliensis_ spores [53]. The filamentous fungus _A. niger_ produces several different polyols, including glycerol, erythritol, and D-mannitol [54]. In _A. niger_ spores, D-mannitol is the predominant carbon-containing compound and makes up 10-15% of the dry weight [54]. Physiological functions of mannitol include serving as a reserve carbon source and as an antioxidant to store reducing power. Molecular genetic analysis of \(mpdA\), the gene encoding the first enzyme in the mannitol biosynthesis pathway, revealed that spores of the _A. niger_ mutant strain with a deficiency in \(mpdA\) were extremely sensitive to a variety of stress conditions, including oxidative stress [55]. Furthermore, the \(mpdA\) mutant study indicated that there is no evidence for the role of mannitol as a reserve carbon source in conidia, but mannitol is essential for resistance to a variety of stress conditions. Since certain strains of _A. niger_ were reclassified into _A. brasiliensis_, we need to confirm whether mannitol is present in _A. brasiliensis_ spores [24]. Darkly pigmented _A.