http://www.paper.edu.cn Study on Endophytic fungi producing Orange 1 Pigment isolated from Ginkgo Biloba L Jia-Jia Liu1, Shu-Juan Chen1, Han-Xiang Gong2 1Department of Pharmacy Engineering, college of chemistry and chemical engineering, central south university , Changsha (410083) 2 Suzhou Changshu bureau of Quality and Technique Supervision, Changshu (215500) E-mail: [email protected] Abstract More than seventy endophytic fungi were isolated by aseptic techniques from the phloem of the root of Ginkgo biloba L.. They were cultured in the potato dextrose (PD) liquid medium and one endophytic fungus (Gh01) was proved can produce orange pigment. The orange pigment was identified as quercetin glycoside by chemical reaction and HPLC. This is the first report on quercetin glycoside produced by endophytic fungus. The effects of the carbon and nitrogen sources, metal ions, initial pH and incubation temperature on pigment production of the endophytic fungus were evaluated. The optimal temperature and initial pH for pigment production in PD liquid medium were 28℃ and 7.0, respectively. Through the orthogonal trial, 20 g/L Glucose and 5 g/L peptone were the most suitable carbon and nitrogen source, 1 g/L chloridize zinc could increase the yield of pigment. Under the optimal conditions established, the maximum yield of the pigment was 27.515 g/L after 120 hours’ successive culture. Keywords: Ginkgo biloba L; Endophytic fungi; Pigment; Quercetin glycoside; Optimization 1. Introduction Plant endophytic fungi are eukaryotic organisms that live inside plant tissues and plant behaves as hosts[1]. The association is symbiotic and both organisms profit from the relationship. That is, the plant is thought to provide nutrient to the microorganisms, while the microorganisms may produce factors that protect the host plant from attack by animals, insects or other microorganisms[2]. Endophytes are presumably ubiquitous in plants, which populations depend on host species and location, and they have been recognized as a valuable source of novel bioactive metabolites[3]. Utilizing microorganisms (especially the fungi) to produce some chemicals and bioactive production by fermentation is promising because it could be further benefited by optimizing the fermentation techniques or meliorating the seed of microorganisms. Ginkgo is an ancient dioecious plant. At present, only Ginkgo biloba L. still exists as a living fossil plant. Botanists have studied it from different aspects in the world due to its unique characteristics[4]. Its fruits and seeds have been used for the treatment of asthma, cough and enuresis[5]. Since 1990s, the standardized extracts of Ginkgo. biloba L. leaves has becoming one of the most popular supplements for memory enhancement. The flavonoids genistein, biochanin A, luteolin, quercetin, and kaempferol are plant natural products with potentially useful pharmacological and nutraceutical activities. These natural products usually exist in plants as glycosides[6]. Quercetin glycoside is derived by enzymatic transglycosylation[7], and it is very important in medicines[8,9] and food industry[10]. The leaves of Chrysanthemum coronarium are shown to be rich in quercetin and its glycosides[11]. A series of Quercetin glycosides are isolated from the translucent bracts of Rheum nobile as UV-absorbing substances [12]. Moreover, quercetin-type glycosides are isolated from the stems and leaves of Delphinium hybridum cv. “Belladonna Casablanca” (Ranunculaceae)[13]. Recently, a new lactoyl glycoside quercetin which is isolated from Melia azedarach leaves are reported[14]. However, there have been no reports on quercetin glycoside from endophytic fungi. 1 Corresponding Author.Tel.:+86-731-8836834; Fax: +86-731-8836834. - 1 - http://www.paper.edu.cn In this study, an endophytic fungus which could produce orange pigment(Quercetin glycoside) is isolated, and its biological characters and culture conditions are discussed. The results will be useful for Gh01’s further using. 2. Materials and Methods 2.1. Plant material The roots of Ginkgo biloba L. trees (ø=50-80 cm) were collected in the Central South University, Hunan province, central China. 2.2. Isolation and identification of endophytic fungi The outer bark of each sample was excised by a sterilized sharp blade after washed with running tap water, sterilized with 75% (v/v) ethanol for 2 min and 2.5% sodium hypochlorous for 10 min, rinsed in steriled water for several times and cut into pieces (about 1cm×1cm). Then, each sample was incubated at 27℃ in the melted potato dextrose agar (PDA) medium supplemented with 200 µg/mL ampicillin and 200 µg/mL streptomycin to inhibit the bacterial growth until the mycelium or colony originating from the newly formed surface of the segments appeared. Several days latter, fungi were observed growing from the inner bark fragments in the plates. Individual hyphal tips of the various fungi were removed from the agar plates, placed on new PDA medium, and incubated at 27℃ for at least 10 d. Each fungal culture was checked for purity and transferred to another agar plate by the hyphal tips method which was described by Stroel G et al[15]. Fungal identification methods were based on the morphology of the fungal culture, the mechanism of spore production, and the characteristics of the spores. All experiments and observations were repeated at least twice. The purified endophytic fungi were numbered and stored in distilled water at 4℃ as agar plugs and 15%(V/V) glycerol at -80℃ as spores and mycelium. 2.3. Fermentation and treatment of the fermentation broth The inoculum was prepared by introducing the periphery of 7-day-old endophyte in Petri dish into 500 mL flasks, which contained 250 mL of potato dextrose liquid medium. After 4 days of incubation at 27℃ on rotary shaker at 120 rpm., 10 mL of incubated liquid (about 106 spores) as the seed was transferred into 250 ml flask which contained 150 mL potato dextrose liquid medium. After 7 days of growth at 27℃ , crude fermentation broth was filtrated thoroughly, distilled in decompressor at 40℃ , added absolute alcohol (v:v=1:1) and centrifuged at 3500 rpm for 10 min. The filtrate was collected and stored. 2.4. Screening of the aim strain During the fermentation, an endophytic fungus whose label was Gh01 producing pigment was screened out. The color of pigment was in red state at first, then turned to orange. This strain was isolated from the phloem of the root of Ginkgo biloba L., and it was the trial material in following researches. 2.5. Identification of the pigment Flasks(500 mL) containing 200 mL PD liquid medium were autoclaved twice at 121℃ for 20 min, then 10 mL of the seed liquid(about 106 spores) was added into per flask. After 7 days of growth at 27℃, crude fermentation broth was treated accordingly. Evaporation of the solvent in vacuo gave a brown residue (51.5 g), which was subjected to chromatography over silica gel - 2 - http://www.paper.edu.cn column (500 g, 200-300 mesh) eluting with a chloroform-ethanol-water (13:6:1, 6 L) to collect the yellow and filemot fraction B (10.1 g). B was further separated over silica gel (100 g, 200-300 mesh) eluting with a methanol-water mixture (6:1, 2 L) to yield compound G-01 (948 mg). The identification of the pigment was carried out by chemical reaction and HPLC. 2.6. Optimizing of the culture medium In order to optimize the cultural conditions, the initial pH in the PDA medium was adjusted to (the desired value ) by addition of either 1 M HCl or 1 M NaOH. The flasks were incubated at various temperatures (20-32℃). 20 g/L Sucrose, maltose, lactose, glucose, rhamnose were studied as carbon sources. 5 g/L Beef extract, peptone, tryptone, ammonium chloride, ammonium nitrate, and yeast extract powder were used in this study to select the best nitrogen source. Metal ions (bluestone、zinc、bitter salt、chloridize calcium) were added into the culture medium to judge whether the existent of metal ions could influence the yield of pigment. During time factor study, the flasks were incubated at design temperature on a rotary shaker at 120 rpm for 7 days. After the appearance of pigment, the UV absorbability of the pigment was mensurated every 12 h. All experiments were performed at least in duplicate to ensure reproducibility. 3. Results and Discussions 3.1. The identification of Gh01 More than seventy endophytic fungi were isolated, and one endophytic fungus (Gh01) was screened out for producing orange pigment. The colony of the Gh01 was white in the initial period, and then turned to gray-green and became the shape of felt. Few protruding fetlocks were in its center while white flosses were on its edge. Few yellow liquid was secreted out on the surface of the colony. At the back of the substrate, bright red pigment could be seen in the earlier period, and its color became yellow-brown latter. The mycelium of hypha developed, with dissepiment, more branching, polynuclear, furthermore, taking on the fancy lotus root burl. The peduncle of conidiophore was about 13-25 µm in length, with diaphragm. Its top did not expand, having broom-like branchs, belonging to the structure of two rounds and asymmetric. The shape of the spores was rotundity or ellipse, bunchiness, lubricity, and the size was 2.0-2.5 µm. The pigment was produced extracellularly (Fig 1). According to those characters, Gh01 would belong to penillicium[16]. Fig. 1: Conidiophore and spore of Gh01 - 3 - http://www.paper.edu.cn 3.2. The identification of the pigment UV measurement The absorbance wavelength of G-01(MeOH) was measured on the UVCRT756 UV spectrophotometer. The range of the scan wavelength was 190-500 nm. The result was shown in Fig 2. The peak absorption was at 252 nm、265 nm、351 nm, those was the characteristic absorbability wavelengths of the flavonol.