Fermentation Conditions Optimization And Structure Analysis Of Extracellular Polysaccharide Secreted By Endophytic Fungi From Gynostemma Pentaphyllum

Endophytic fungi of medicinal plant are a kind of potential but not exploited microbial resources. Previous experiments confirmed that extracellular polysaccharide(EPS) of endophytic fungi JY25 from the medicinal plant Gynostemma pentaphyllum has anti-cancer,anti-oxidation, hypoglycemic effect, immunity enhancement and other bioactive effects.Studying the conditions of the higher EPS yeild, polysaccharide construction and structure-function relationship have great important significance to its development and utilize.First of all, the single factor analysis and the orthogonal analysis of incubation time,inoculum dose, carbon sources and nitrogen sources were made to optimize the fermentation conditions. The optimized results of single factor showed that the culture cycle was 8 d, the inoculums dose was 7 %, the carbon source was glucose and the nitrogen source was yeast powder. The orthogonal analysis indicated that 8 d culture cycle, 5 %inoculum dose, 40 g/L glucose concentration and 5 g/L yeast powder concentration were the best fermentation conditions, which the yield of extracellular polysaccharide was increased32.2 % than the previous yield to 271.40 mg/L.Afterwards, two kinds of refined polysaccharides(they are named JY25P-O、JY25P-P respectively come from the broth of optimal conditions and the broth of peptone as nitrogen source)were obtained by a series of steps, which were concentration, ethanol precipitation,deproteinization, depigmentation, column chromatography, dialysis, freeze drying. The structure of two kinds of refined polysaccharides were analyzed by Uv scanning, weight analysis, infrared spectra, high performance liquid. Results confirmed that JY25P-O is a glycoprotein of relative molecular weight of 199526.23 Da, and is mainly composed of five kinds of monosaccharide: glucose, arabinose, mannose, galactose and rhamnose(the ratio of63.29:8.24:11.32:13.01:4.14). The JY25P-O structure has β-anomeric carbon configuration,adsorbed water and crystal water. JY25P-P also is a glycoprotein of relative molecular weight of 151356.13 Da, and is mainly composed of four kinds of monosaccharide: glucose,arabinose, galactose and rhamnose(the ratio of 2.64:8.56:13.17:5.63), The JY25P-P structure has β-anomeric carbon configuration, adsorbed water and crystal water.Employing the phenanthroline-Fe2+ and DPPH method to explore the anti-oxidation properties of these two kinds of polysaccharide, the results confirm that the clearance rate of?DPPH of 10 mg/mL JY25P-O polysaccharide reached 39.4 %, ?OH clearance rate reached99.8%, meanwhile, the clearance rate of ?DPPH 10 mg/mL JY25P-P polysaccharide reached37.2 %, ?OH clearance rate reached 99.1 %, which proved excellent anti-oxidation properties of JY25 extracellular polysaccharide.In conclusion, we gained two refined polysaccharides of JY25P-O and JY25P-P by optimizing the fermentation conditions of JY25 endophytic fungi from Gynostemma Pentaphyllum secreted extracellular polysaccharide. Analysising the structure of these two polysaccharides, we found they have very similar structure and higher anti-oxidant properties. These researches can provide basic data for the future research and application.