Study On Isolation, Identification And Fermentative Optimization Of Flavoniods-Producing Endobhytites From Ginkgo Biloba L.

Endophytes, including bacteria, fungi and actinomycetes, refer to microbia colonizing in healthy plant tissues without causing symptom or apparent injury to the host.’Endosymbiotic theory’ has shown that endophytes in symbiosis with the host process, can coevolution with plants, and obtain parts of functional gene, thereby generating some capacity metabolites which are similar to the host. In current, research on plant endophytes has become a hot subject, especially the study on endophyte in medicinal plants. These endophytes has become the source and a new way to find new drugs.In this paper, samples of an ancient relict plant Ginkgo biloba L. were collected from the tissue of roots, stems and leaves. We isolated the endophytes, including fungi and bacteria, from the samples in the way of tissue culture. We ensured the microorganisms are endogenous microbial in ginkgo, by combing 75% ethanol with 3% sodium hypochlorite to disinfect the surface of samples. The results showed that the best disinfection time of sodium hypochlorite at Ginkgo roots, stems and leaves, were respectively 5min,3min and lmin. Under this condition, we isolated 664 strains of endophytes from ginkgo, of which 217 strains were fungi, and 447 strains were endophytic bacteria. Endophytic fungi belonged to 5 classes,10 orders,11 families and 13 generas, identified through the morphological identification of fungi. These generas were Fusarium, Corynespora, Alternaria, Fusariumsolani, Stemphylium, Phomopsis, Nectria, Pseudocercospora, Cercospora, Guignardia, Mucor, Diaporthe and Pythium. Endophytic bacteria belonged to 5 classes,8 orders,10 families,18 generas, identified by using molecular biology methods to amplify its 16SrDNA. These generas were Pseudomonas, Chryseobacterium, Brevundimonas, Phyllobacterium, Pantoea, Leclercia, Curtobacterium, Kluyvera, Serratia, Xanthomonas, Psychrobacter, Novosphingobium, Stenotrophomonas, Flavobacterium, Rhodococcus, Brevibacterium, Microbacterium, Bacillus.We tested whether these strains could yield flavonoids, by the way of characteristic color reaction of flavonoid, thin layer chromatography (TLC) and High Performance Liquid Chromatography (HPLC). In addition, we measured the production of flavonoids via UV-spectrophotometry, and identified these strains according to the morphological characteristic and the similarity of nucleotide sequence of internal transcribed spacer (ITS) between rDNAs. The results showed that strain GF111 and GF521 were proved to yield flavonoids. The amounts of total flavonoids in GF111 and GF521 were 14.50±1.30 mg/L and 21.10±1.30 mg/L, respectively. The strain GF111 and GF521 are classified as Mucor circinelloid.es and Fusarium oxysporum.In addition, we used single factor and orthogonal experiments to optimize the medium and culture conditions of strains GF521. The results showed that the optimum carbon source for strains GF521 was maltose, nitrogen source was yeast extract, and inorganic salts was ferrous sulfate. Five factors, incubation temperature (A), medium volume (B), rotation speed (C), inoculum size (D) and initial pH (E) have a very significant impact on (P<0.01) biomass and total flavonoids of strains GF521.