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20 strains of endophytic fungi isolated from the roots,stems and leaves of a vulnerable medicinal plant Gentiana macrophylla were studied. By the deposition reaction with Dragendorff’s agent, five strains (QJ2,QJ8,QJ12,QJ16 and QJ18) with alkaloid production ability were screened out. Then the strain QJ18 was further selected out by thin layer chromatography (TLC) because of its gentiopicrin production ability. The procedure of gentiopicrin extraction was optimized. And then the Rf value of concentrated extract from the culture filtrate of QJ18 was compared with that of the standard gentiopicrin by TLC, scanned under UV and visible spectrum. The absorption curve of the extract was basically the same as that of the standard sample. The results of IR, LC-MS and HPLC shown that the sample should contain the ingredient gentiopicrin.Through analyzing the amount of biomass and the yield of gentiopicrin, an optimum liquid medium was selected out; then, by changing different carbon source, nitrogen source, pH and culture temperature, each culture condition was selected, respectively. The optimized conditions of both mycelial growth and gentiopicrin production are sucrose, urea, pH about 7.0 and the temperature about 28°C, respectively.The fermentation conditions of QJ18 were then further optimized by the orthogonal experiment of L16 (4~3). The results showed that sucrose 30g/L, urea 2g/L, pH about 7.0 and the temperature about 28℃, shake rate 80 times per minute were the optimum conditions for its mycelial growth; sucrose 25g/L, urea 2.5g/L, pH about 7.0 and the temperature about 28℃, shake rate 80 times per minute were suitable forgentiopicrin production.The strain QJ18 was classified mainly according to its microscopic morphological characters. QJ18 should be a strain belonging to the Phycomycetes, Mucorales, Mucoraceae, Rhizopus Ehrenberg
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