In order to acquire more accurate and complete intracellular metabolite information,and therefore to more realistically reflect the microbial intracellular metabolism,the systematic optimization of metabolomic sample preparation process was carried out from 3 aspects of quencher,extraction and solvent agent based on the GC-MS platform. The leakage of intracellular metabolites and cell injury with different quenchers was compared,and the results showed that 60% methanol/0.9% NaCl had the least leakage. The effects of extraction and solvent agent on relative abundance of metabolite distribution were investigated,and 60% methanol presented the significant extraction effect, and pyridine had the dissolving effect.The optimal sample pretreatment method for metabolomic analysis of Aureobasidium pullulans was determined as follows:pre-cooled 60% methanol/0.9% NaCl as quencher,ground in liquid nitrogen and repeated freezing and thawing for cell disruption,pre-cooled 60% methanol for the extraction of metabolites,and then the extracts were dissolved by pyridine and derivatized for 90 min under MSTFA at 40 ℃ . By using this optimized protocol,more than 300 kinds of metabolic components were identified by GC-MS,including a large number of amino acids,organic acids and sugars. Thus our established method can be used to effectively analyze the A. pullulansmetabolites.%为了得到更加准确和完整的胞内代谢物信息，从而更加真实的反映微生物胞内代谢情况，基于GC-MS平台，从淬灭剂、提取剂和溶解剂3个方面对代谢组学样品制备过程进行了系统性的优化。对比了不同淬灭剂对胞内代谢物的渗出及细胞损伤情况，结果表明60%甲醇/0.9%NaCl效果最佳。研究了不同提取剂、溶解剂的提取和溶解效果，结果显示60%甲醇提取效果显著，吡啶溶解效果突出。最终确定了出芽短梗霉菌代谢组样品最适前处理方法：预冷的60%甲醇/0.9%NaCl淬灭细胞，液氮研磨和反复冻融破碎细胞，预冷的60%甲醇提取，最后采用吡啶溶解提取物并采用MSTFA40℃衍生90min。采用该方法可以检测出300多种代谢组分，包括大量氨基酸、有机酸和糖类物质。本方法可以有效的应用于出芽短梗霉菌代谢组学样品的研究。
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