L cysteine

C. difficile VPI10463 (ATCC 43,255), Escherichia coli (ATCC 25,922), Staphylococcus aureus (ATCC 25,923), Enterococcus faecalis (ATCC 29,212), Candida albicans (ATCC 90,028), and THP-1 cells (ATCC TIB-202) were purchased from the American Type Culture Collection (ATCC). The non-toxigenic strain NTCD was isolated clinically and identified as negative for toxin genes, such as tcdA, tcdB, cdtA, and cdtB, previously in our laboratory.^{53 (link)} THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Gibco, USA) and 50 mg/L penicillin/streptomycin (Gibco, USA). C. difficile strains were cultured in brain heart infusion (Oxoid Ltd., USA) broth supplemented with 5 g/L yeast extract (Oxoid Ltd.), 0.1% L-cysteine (Sangon Biotech, China), and 0.1% sodium taurocholate (Sangon Biotech, China) for 48 hours at 37°C anaerobically. The other bacterial strains and C. albicans were cultured in Luria-Bertani (Sangon Biotech, China) and yeast peptone dextrose broth (Sangon Biotech, China), respectively, for 24 hours aerobically. The liquid culture was centrifuged at 1500 g for 5 minutes, and the pellet was washed twice with sterile phosphate-buffered saline (PBS). The inoculum was adjusted to approximately 5 × 10^{6 (link)} Colony Forming Units (CFU)/ml.

Four commercial volatile compounds were used to detect the nematicidal activity of VOCs: N-methylformamide (99%; Aladdin, Shanghai, China), propenamide (97%; Macklin, Nanjing, China), 3-(methylthio)propionic acid (98%; Macklin, Shanghai, China), and phenylmalonic acid (98%; Aladdin, China). Compounds with a similar structure to 3-(methylthio)propionic acid were used to analyze their differences in nematicidal activity: 3-methoxypropionic acid (98%; Macklin, China), L-cysteine (98.5%; Sangon Biotech, Shanghai, China), valeric acid (99%; Macklin, China), and isovaleric acid (99.5%; Aladdin, China). The volatile compounds and structural analogs of 3-(methylthio)propionic acid were diluted with distilled water for subsequent experiments.

L-cysteine (Cys), L-histidine (His), L-leucine (Leu), L-lysine hydrochloride (Lys), L-methionine (Met), L-phenylalanine (Phe), L-proline (Pro), L-tryptophan (Trp), L-tyrosine (Tyr) were purchased from Sangon Biotech Corp. (shanghai, China). Acetonitrile was obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Ultra-pure water was collected from a Milli-Q SP TOC system water purifier (Millipore Corp., Bedford, MA, USA). All amino acid samples were dissolved in ultra-pure water till the concentration reached 1 mmol/L. Then, 50 ml of amino acid solution was transferred to the discharge vessel at room temperature for plasma treatments which were performed at V_P = 4.0 kV within different treatment periods.