In this study, atmospheric pressure air dielectric barrier discharge plasma was used to inactivate Bacillus subtilis (B. subtilis) spores by varying the concentration of plasma-activated hydrogen peroxide ( \({\text{H}}_{2}{{\text{O}}}_{2}\) ). The results showed that the inactivation effect significantly increased as the \({\text{H}}_{2}{{\text{O}}}_{2}\) solution concentration rose from 0 to 30%. The inactivation effect on B. subtilis spores was almost the same for 15% and 30% \({\text{H}}_{2}{{\text{O}}}_{2}\) , and it shows a 6 Logs decrease after 15 s of treatment. When the gas temperature of the chamber was fixed at 85 °C, the \({\text{CFU/cm}}^{2}\) decrease of B. subtilis spores at 15% and 30% \({\text{H}}_{2}{{\text{O}}}_{2}\) is 1.4 and 1.7 Logs, respectively. Compared to the 0% \({\text{H}}_{2}{{\text{O}}}_{2}\) condition, the concentration of reactive species produced by the plasma at 30% \({\text{H}}_{2}{{\text{O}}}_{2}\) increased 57% for 2-hydroxyterephthalic acid, 8% for nitrate and sevenfold for \({\text{H}}_{2}{{\text{O}}}_{2}\) while nitrite production was decreased 24%. To elucidate the inactivation process of B. subtilis spores treated with \({\text{H}}_{2}{{\text{O}}}_{2}\) added to humidified air plasma, we analyzed and compared the chemical stability of 11 representative amino acids. Our LC–MS measurements showed that amino acids can be hydroxylated, nitrated and oxidized by the reactive species in the plasma. The addition of \({\text{H}}_{2}{{\text{O}}}_{2}\) to the plasma promotes the oxidation of Glu, Lys and Arg, leading to an increase the relative abundance of by-products. Our analyses revealed that the addition of \({\text{H}}_{2}{{\text{O}}}_{2}\) to humidified air plasma significantly inactivated B. subtilis spores, which was in close correlation with the chemical stability of amino acids.
