| Flavonoids in Ginkgo biloba L. have wide bioactivities, including antioxidant, anti-tumor, anti-inflammatory. G. biloba extracts (GBE) can be obtained using water or water alcohol solvent extraction method. Although the enzyme-aid extraction has been reported, the use of pure cellulase limited its use for mass production. We try to use the cellulase fermentation broth from endophytic fungi in G. biloba to assist water alcohol extraction of flavonoids. Through the separation of endophyte, screening high-yielding cellulase producing strains and optimizing the conditions for fermentation, we used the fermentation broth in the extraction of flavonoids. Moreover, the fungal strain has been identified by ITS sequence and the enzyme has been purified. Our research provided a new method for the extraction of traditional chinese medicine. The method of endophytic fungal cellulase applicated in the extraction of total flavonoid from G. biloba is not yet reported.In present study, 71 strains of endophytic fungi were isolated from G. biloba, and then a high-yielding cellulase strain Y2 was screened by the Congo red staining. After screening and optimizing the conditions of enzyme production and the culture medium for cellulase production, we found the optimum medium: 20% potato, 2% glucose, 0.3% KH2PO4, 0.15% MgSO4, 10 mg/L vitamin B1, 0.5% Yeast extract, and the best conditions: temperature 28℃, speed of 150 rpm, liquid volume 200 mL/500 mL. The cellulase activity reached 4.47 U/mL after 6 d under this condition.The morphological observation and molecular identification for the fungal strain Y2 were performed. The fungal could be basically confirmed as Penicillium fungi under microcopy observation. The extraction of DNA and molecular identificationis were also performed, total DNA was isolated with CTAB method, and the extracted DNA accorded with the quality standard. The screened PCR amplification conditions as follows: initial denaturation for 5 min at 94℃→94℃40 s→52℃1.0 min→72℃1.0 min, after 30 cycles ,72℃8 min. 5 mL product was separated by 1.5% agarose gels in 100 V, a single, clear band was found in the ultraviolet light. The PCR amplified fragment was sequenced, and got a 564-bp fragment, which has 100% homology with the GenBank nucleotide sequence of Penicillium oxalicum.The CMCase components was purified from the cellulase fermentation broth of P. oxalicum by sub-ammonium sulfate precipitation, dialysis, DEAE-Sephadex A-25 anion-exchange chromatography, Sephadex G-75 molecular sieve gel filtration chromatography. The properties of this protein component were investigated. The final purification results were that total protein decreased by 89.87%, the total activity fell by 30.65 %. The enzyme activity increased to 6.67 times and reached 466.59 U/mg, the recovery of cellulase was 10.13%. The molecular weight of the purified enzyme was about 34.2 kD according to the standard curve. The properties of purified enzyme showed that the optimum temperature 50℃, pH 5.0. Fe3+, Fe2+, Al3+ ions enhanced activity, whereas Zn2+, Cu2+, Hg2+caused obvious inhibition. The stability study showed that the best preservation temperature pH value was less than 50℃, 5.0, respectively.We established a new HPLC method to determinate the total G. biloba flavonoids (quercetin, kaempfe, isorhamnetin). The chromatography conditions selected were: column, Agilent C18 column(4.6 mm×250 mm, 5μm); mobile phase of methanol: 0.4% phosphoric acid = 59:41; flow rate 1.0 mL/min; column temperature was 30℃; injection capacity of 20μL; detection wavelength of 360 nm.We also studied the inhibitory of tannin on cellulase enzyme activity and screened an effective absorbent collagen fiber to remove it. The conditions of the extraction process were optimized by means of single factor experiments and orthogonal design. The optimum conditions was selected as follows: after incubation with 15:1 (mL/g) of crude enzyme (pH5.0, 50℃) to dry flower bud for 3 h, the extraction rate of total flavonoids was 1.33%, an increase of 18.75% than that in regular extraction process.We screened a high-yielding cellulase of endophyte P. oxalic from the G. biloba. The conditions of enzyme production were optimized. The extract rate of G. biloba flavonoids were then successfully improved by using cellulase fermentation broth of this endophyte. Our study provided a new idea for the extraction of traditional Chinese medicinal herbs, and also provided the possibility for large-scale industrialization for Chinese herbal extract with enzymatic aid.
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