Metabolic comparison of aerial and submerged mycelia formed in the liquid surface culture of Cordyceps militaris

Supplemeatary Table S1: Predictive metabolites<b>GC/MS analysis</b> Fifty milliliters of <i>C. militaris </i>liquid surface culture broth at 5, 12, and 19 d was separately collected and stored at -80ºCbefore further analysis. Each sample of 15 mL was diluted in 35 mL of water, centrifuged at 16,000 × g for 10 min at 4°C, and the pellets were collected for freeze-drying. Twenty-five microliters of methoxyamine hydrochloride in pyridine (20 mg/mL) was added, and the mixture was vortexed for 30 s and incubated at 90°C for 90 min. Finally, 75 μL of N-methyl-N-trimethyl-silyltrifluoroacetamide (MSTFA)<i></i>was added, and the mixture was incubated at 37°C for 30 min. The mixture was centrifuged at 13,000 × g for 10 min at 4°C, and the supernatant (n=7 for each sample) was subjected to gas chromatography/mass spectrometry (GC/MS)(Agilent Technologies 7890A GC system, Agilent Technologies, Inc., Wilmington, DE, USA) equipped with an inertcap 5MS capillary column (5% phenylmethylsiloxane: 30 m × 0.25 mm internal diameter, 0.25 μm film thickness; GL-science Co. Ltd., Tokyo, Japan)and JEOL JMS-T100GCv Time-of-flight Mass Spectrometer (JEOL, Tokyo, Japan). The GC was operated at a constant flow of helium (1 mL/min), an injector temperature of 250°C, and an ion source and transfer line temperature of 280°C. The oven temperature program was as follows: 40°C for 4 min, increased at 15°C/min to 300°C, and held for 10 min. The samples were injected with a split ratio of 100:1. The ionization was conducted in the EI positive mode. The detection mass range was m/z 50–600. To tentatively identify a compound, the mass spectra and measured exact mass were compared against a spectral library (NIST) and the exact mass simulated. The spectra, exact mass, and retention times were compared with authentic standards when they were available. <b>Data statistics</b> The GC-MS raw results were converted to .<i>cdf</i>format and analyzed using an XCMS online program (https://xcmsonline.scripps.edu/landing_page.php?pgcontent=mainPage) (Tautenhahn et al., 2012) based on default parameters. Finally, the metabolite annotation of GC-MS was performed using the automatic processing and identification system (AMDIS) databases of the National Institute of Standards and Technology (NIST).