Abstract: We established a method for preparing metabolome sample of Streptomyces avermitilis, including cell separation, washing, quenching, disruption and extraction conditions based on GC-MS analysis system, and applied it in the analysis of the metabolome of industrial strain 9-39 and wild type strain. The optimal conditions for sample preparation were as follows: separating cells by centrifugation, washing 3 times, quenching 5 minutes with liquid nitrogen, cell disruption with bead mill（3 cycles and 48 seconds per each cycle）, and extraction of metabolites（chloroform:ethanol:water = 2:2:1）. By using this optimized protocol, 59 differential compounds were identified and quantified, including amino acids, organic acids, fatty acids and saccharides. Our established method can be used to effectively analyze the Streptomyces avermitilis metabolites.

Key words: Streptomyces avermitilis, metabolomics, cell disruption, extraction, GC-MS