Isolation And Identification Of Swainsonsine-secreting Fungal And Its Optimization Of Fermentation Condition

Swainsonine belong to one of poisonous indolizidine alkaloid which is the main toxin component in locoweed plants. Feeding locoweeds with swaisonine in a long term may cause animal poisoning, that bring a great damage to prairie stock raising both in domestic and overseas. The recent study show swaisonine have potential bio-activity of anti-tumor, enhance immunity, restrain transforming of cancer cell, it makes swaisonine become the hotspot of new anti-tumor medicinal screening in recent years. But the medicinal use of swaisonine is restricted because of its rare source. In 1985, the researcher of US Braun isolate the pure swaisonine from M. anisopliae, after that, many researchers isolated swaisonine producing endophyte from various locoweed, they also studied the relationship between endophytci fungu and swaisonine in locoweed and recognise it's very intimate. Because of low-costing, high control ability and short producing period, fermentation of swaisonine-producing fungi offer a new way to solve the question of swaisonine source. There is a great quantity locoweed distribute in northwest China, such as Inner Mongolia, Qinghai province, the report about whether locoweed in those area is infected by swaisonine-producing fungu is rarely. To ascertain this problem and offer a theory foundation to swaisonine fermentation, we isolate fungus from O. glabra, A. variabilis, O. kansuensis, O. deflexa which collected from Inner Mongolia and Qinghai province, identify their genus, determine swaisonine yield and optimize the fermentation condition.1. The isolation of locoweed endophyte and its soil fungi.We isolate fungus from locoweed and soil sample by tissue separation and linedraw separation, then pure the strains and foster them in liquid culture. The chemical composition(SW) of hyphae and liquid were detected by thin layer chromatography and gas chromatography, screening the target fungus and determine the yield. As a result, we gain 2 swaisonine-producing strains, one was isolated from O. kansuensis, another was isolated from soil, named EFG-7 and FS-5 respectively, their swaisonine yield was 0.773 mg.g^-1 and 0.11 mg.g^-1. Two strains endophytic fungi EFG-8 and B-1 which were isolated by vice professor Lai Hang-xian(college of resource and environment, Northwest A&F University), their swaisonine yield was 1.578 mg/g and 0.047 mg/g.2. The identification and store of swaisonine-producing fungiIdentification of fungus Used morphology observe and Molecular biology approach. morphology observe include the hyphae shape, spore structure , shape and color.Refer the observe result to Manual of Fungi Identification to identify the genus of strains. Molecular biology methods: strain DNA was extracted by using modified CTAB, fungal universal primers designed by White were as follows ITS1 (5'-TCCGTAGGTGAACCT-3 '), and ITS2 (5'-TCCTCCGCTTATTGATATGC-3'). The amplified sequence was determined by 1% agarose gel electrophoresis。DNA fragment after PCR recovered from agrose gel were inserted into pMD-18 vector and transferred into E. coli DH5α.The positive recombinant clones were identified by PCR using colonies directly as templates, restriction endonuclease digestion analysis and DNA sequencing. Blast with sequences in Genebank, then do sequence alanysis by Mega 4.0 and Clustal W. The strains stored by galycerol and paraffins. The identification result show that FS-5, EFG-7, EFG-8, B-1 was Schizophyllum sp, Fusarium tricinctum, Emericella rugulosa and Aspergillus sp, respectively.3. The fermentation optimize of swaisonine producing fungiThe nutrition factors is optimized by orthogonal experiment, include carbon source, nitrogen source and culture medium, the index is growth speed. But hyphae growth speed can't reflect lateration of swaisonine yield, so another 2 experiment through liquid fermentation process is did to optimizing the nutrition factor and environment factor which may affect swaisonine yield, which include process time, temperature, contain and pH. After fermentation, measure swaisonine yield in liquid by GC, and do statistics analyse with GC data to determine the best condition. The result display the best suitable condition of EFG-8 is 10 % bean pulp medium, 6 % peptone, 4 weeks fermentation, 28℃, 250 mL contain in 500 mL conical flask, pH 6.0. B-1 is 10 % glucose, 4 % KNO₃, 2 weeks fermentation process, 28℃, 250 mL contain, pH 6.0.