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).补充表S1：预测性代谢物**气相色谱-质谱（GC/MS）分析** 分别收集5 d、12 d和19 d的蛹虫草（*C. militaris*）表面液体发酵培养液各50 mL，于-80℃保存待用。取每份样品15 mL，用35 mL水稀释后，在4℃、16000×g条件下离心10 min，收集沉淀进行冷冻干燥。向样品中加入25 μL吡啶溶解的盐酸甲氧胺溶液（20 mg/mL），涡旋混匀30 s后，于90℃孵育90 min。最后加入75 μL N-甲基-N-三甲基硅基三氟乙酰胺（MSTFA），将混合物于37℃孵育30 min。将混合物在4℃、13000×g条件下离心10 min，取上清液（每份样品设置7个生物学重复）进行气相色谱/质谱（GC/MS）分析：采用安捷伦科技7890A气相色谱系统（Agilent Technologies, Inc., 美国特拉华州威尔明顿市），搭配惰性帽5MS毛细管柱（5%苯基甲基聚硅氧烷：30 m×0.25 mm内径，0.25 μm膜厚；GL-science Co. Ltd., 日本东京）以及JEOL JMS-T100GCv飞行时间质谱仪（JEOL, 日本东京）。 气相色谱以氦气为载气，恒流流速1 mL/min，进样口温度250℃，离子源与传输线温度280℃。程序升温条件如下：40℃保持4 min，以15℃/min速率升温至300℃并保持10 min。进样分流比设置为100:1，电离方式采用电子轰击正离子模式，检测质量范围为m/z 50–600。为初步鉴定化合物，将质谱图与实测精确质量与光谱库（NIST）进行比对，并通过精确质量进行模拟验证。若有对照标准品，则进一步比对质谱图、精确质量与保留时间。 **数据统计** 将GC-MS原始结果转换为.cdf格式，基于默认参数，使用XCMS在线程序（https://xcmsonline.scripps.edu/landing_page.php?pgcontent=mainPage）（Tautenhahn等，2012）进行分析。最终，采用美国国家标准与技术研究院（NIST）的自动处理与鉴定系统（AMDIS）数据库完成GC-MS代谢物注释。