| Angelica sinensis,a perennial herb which is widely distributed in China.It s a valuable traditional Chinese medicine.Researches shown that Angelica sinensis has various biological activities such as blood-activating,antioxidant,anti-inflammatory,anti-tumor and so on.Its clinical application value is very high.Endophytic fungi is widely distributed in plants.Secondary metabolites,produced by endophytic fungi in many medicinal plants,have high biological activity.Extracellular polysaccharide is a kind of polysaccharide produced by microbial cells and secreted into culture medium.Studying the basic characteristics of exopolysaccharide(EPS)will provide certain value for the application of Angelica sinensis in medicine industry.In this paper,endophytic fungi were isolated from Angelica sinensis.The medium components of EPS produced by endophytic fungi of Angelica sinensis were optimized by orthogonal experiment.The fermentation conditions of EPS produced by endophytic fungi of Angelica sinensis were optimized by response surface methodology.The EPS produced by endophytic fungi of Angelica sinensis were isolated and purified,and its antioxidant activity was also studied.The main results are as follows:1.Endophytic fungus F1 was isolated from the roots of Angelica sinensis,it was identified as Alternaria tenuissima by analyzing the colony morphology,microscopic characteristics and phylogenetic tree structure.2.Combining single factor experiment and orthogonal experiment to optmize the medium components of EPS produced by the endophytic fungi of Alternaria tenuissima F1 with the yield of exopolysaccharide as the index.The results showed that the effects of various factors on EPS production by endophytic fungi of Alternaria tenuissima F1 were mannose,yeast extract,magnesium ion and VC.The optimum medium formulation were:8%mannose,2%yeast extract,0.04%magnesium ion and 0.005%VC.Under these conditions,the average content of EPS was 2.5657mg/mL,which was close to the predicted value,indicating that the optimized medium formula was effective and feasible.3.Fermentation conditions of EPS production by endophytic fungus Alternaria tenuissima F1 were optimized by single factor test combined with response surface test.The results showed that the order of the factors affecting the content of EPS produced by endophytic fungus Alternaria tenuissima F1 of Angelica sinensis was:fermentation temperature,pH,inoculation quantity,fermentation time.The optimum fermentation conditions were as follows:fermentation temperature 30?,fermentation time 5 days,initial pH=7,inoculation volume 3%.Under the optimum conditions,the yield of EPS was 3.32 mg/mL,which was close to the predicted value,indicating that the optimum process was reasonable and feasible.4.The EPS produced by endophytic fungus Alternaria tenuissima F1 were isolated and purified,and the basic characteristics of the EPS were determined.EPS was purified by DEAE-52 cellulose column chromatography and Sephadex G-100 gel column chromatography.The purified EPS was non reducing sugar,yellow powder,soluble in water at room temperature,insoluble in ethanol,and without starch.The average molecular weight(Mw)and number average molecular weight(Mn)of EPS were 3.246 ×104 g/mol and 2.012 ×104 g/mol,measured by high performance liquid chromatography(HPSEC-MALLS).The purified EPS was determined by gas chromatography-mass spectrometry(GC-MS)to be composed by five different monosaccharides,namely D-galacturonic acid,rhamnose,D-mannose,glucose and D-galactose.Infrared spectroscopy(FT-IR)showed that the EPS was a glycoprotein with pyranose analysis structure.Scanning electron microscopy(SEM)showed that the surface of the EPS was rough and showed a porous network structure.TGA/DSC analysis showed that the EPS had good thermal stability,?H=51.92 J/g.5.The results of antioxidant activity test showed that the EPS had strong hydroxyl radical scavenging activity,superoxide anion scavenging activity and reducing power.It's showed the EPS had strong antioxidant activity. |
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