Preliminary Study On Antibacterial And Antifungal Activity Of Ginkgo Biloba Endophytic Fungi

Endophyte is a sort of particular fungoid that can inhabit in robust flora organization with medicinal value. Due to the unique environment conditions, the studies were relatively few. Generally, new endophyte can be isolated from the plants with long age or geographical characteristics, so as to provide conditions for research and development with bioactive substances. Ginkgo biloba is a rare plant with antibacterial and antioxidant activity. Because of a variety of active ingredients are existed in its tissues, such as ginkgo flavone, lactone, alkaloid, etc. According to the endosymbiont theory, Ginkgo biloba’s tissue is an important source to dig new endophytic fungi which has antibacterial activity.In this study,30 strains endophytic fungi with antibacterial and antimicrobial activity were isolated from healthy tissue of Ginkgo biloba by using tissue isolation. To screen out 10 strains with high and stable antibacterial activity for rescreening in order to supply farther consult to acquire outstanding bacterial strains. The Y09 strain with high antibacterial activity and stable characteristics was screened by the plate hole well diffusion method.The strain Y09 was identified by the combination of morphological and molecular biology methods. The CTAB method was used to extract DNA and the PCR amplified sequences were analyzed. The result showed that Y09 have the higher homology about rDNA sequences with Penicillium oxalicum, it is up to 99%. Initially identified the strain as Penicillium oxalicum, and named Penicillium oxalicum SPE009.The single factor experiment method was used to determine the conditions of SPE009. The liquid medium volume is 30mL/100mL, inoculation quantity of 5%, temperature of 26℃, rotate speed of 160r/min and the initial fermentation pH of 7. Under these conditions, SPE009 has a higher antibacterial activity against Aspergillus niger.At the same time, the impact of nutritional factors was investigated for SPE009 products’ antibacterial activity. Three factors with significant effect on the fermentation products’ antibacterial activity were selected by Plackett-Burman experimental. There are the initial fermentation pH value, glucose content, and culture temperature. Used response surface methodology to optimize the three conditions, obtained the best combination of them:initial pH was 6.97, the glucose content of 19.55g/ L, the culture temperature of 26.31℃. Under these conditions, the theoretical value of the antibacterial circle was 20.10mm.The metabolites of SPE009 strain were isolated and purified by thin layer chromatography and silica gel column chromatography. The sample No.3 has the highest antibacterial activity and its antibacterial circle is 19.9mm. Samples were merged by C-18 reverse phase column and thin layer spotting. We had got sample 3-1 and measured the antibacterial properties, its inhibition zone was 20.0mm. The sample 3-1 was analyzed by HPLC and LC-MS, the sample 3-1 contained C20H22O9. By contrast with the natural product database, the product is the Populus trichocarpa glycosides.