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Inocutis tamaricis is a medicinal fungus with significant pharmacological activity. In this study, ultra‐high performance liquid chromatography‐high resolution mass spectrometry (UPLC/MS) was used to conduct a comprehensive non‐targeted metabolomics analysis of the metabolites of wild fruiting body (WF), cultured fruiting body (CF), and liquid cultured mycelium (LCM). Orthogonal partial least square discriminant analysis (OPLS‐DA) was used to compare the three culture methods, to describe the regulatory effect of specific culture methods on metabolite distribution and yield of I. tamaricis, and the differential metabolites were enriched and analyzed to identify key metabolic pathways. The results showed significant differences in the types and abundances of active substances produced in different culture methods. There were 35 compounds in common, the WF, CF, and LCM had their unique metabolites, which were 39, 16, and 29 respectively, confirming the significant differences in the metabolites in different cultures, six key metabolic pathways were identified, including unsaturated fatty acid biosynthesis, linoleic acid, and α‐linolenic acid metabolism, etc.
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