Studies On Endophytic Fungi With Active Production Ability From A Vulnerable Medicinal Plant Eucommia Ulmoides Oliv

Forty-four endophytic fungi strains were isolated from the stems,roots and leaves of a rare medicinal plant Eucommia Ulmoides Oliv.The further researches were as follows.According to the characteristic color reactions of flavonoids and iridoids compounds,three strains,DZ2,DZY5,DZ11,which could produce flavonoids compounds were screened out in the preliminary selection.By UV-visible spectroscopy scanning and stability detection of its quercetin production,DZ11 was selected.Through drawing growth curve of DZ11 strain and yield curve of quercetin,the maximum yields of quercetin which were 0.418mg/L and 0.340mg/L were got by UV spectrophotometry and TLC method,respectively.After HPLC analyzing,it could be found that quercetin exists in both inside and outside of hyphae,but the former had a lower content.Through column chromatography,6 components with different Rf-value were obtained from DZ11 fermentation broth,the fifth ingredient among which had the same UV-visible spectrogram as standard quercetin.After further purification to the fifth part and mass spectrography,the mass over charge ratio(m/z)of the[M+H]⁺ was 303.38,which was the same as the molecular weight of quercetin.Each functional group and chemical bond absorption band of quercetin was found in infrared spectrum.These results proved that one of secondary metabolite produced by DZ11 strain was quercetin.According to the growth rates and growth potential of the colonies,bean sprouts solid medium was chosen.The bean sprouts liquid medium was also suitable for the mycelial growth of the strain DZ11 and the output of quercetin.Based on this liquid medium,by changing different carbon source,nitrogen source,pH-value and culture temperature,the culture conditions were optimized.The optimized conditions of both mycelia growth and quercetin-production were sucrose,urine,pH7.0 and the temperature 28℃.The fermentation conditions of DZ11 were optimized further by the orthogonal experiment of L16(4³).The results showed that the suitable conditions for its mycelial growth were an improved bean sprouts liquid medium with 25g.L^-1as sucrose, 2g.L^-1urine as nitrogen and 120ml/300ml as the liquid medium quantity;the suitable conditions for querctetin production were an improved bean sprouts liquid medium with 30g.L^-1sucrose as carbon source,2.5 g.L^-1urine as nitrogen and 120ml/300ml as the liquid medium quantity.The strain DZ11 was classified mainly according to its microscopic morphological characters.DZ11 should be a strain belonging to the Fungi Imperfecfi, Coelelomyctes,Sphaeropsidales,Sphaeropsidaceae,Chaetophoma Cke.