Studies Of Extracellular Polysaccharide Produced From The Filtrate Of Fermention Of Cordyceps

In this thesis,we developed the new method to obtain the exopolysaccharide from a strain G106-1 identified as Acremonium terricola fermentation filtrate by altering filtrate pretreatment and extraction techniques based on the previous report. Furthermore,the exopolysaccharide separation,purity,components,physical and chemical properties,primary structure and biological activities were studied.The assay of B and T lymphocytes proliferation and the assay of life-span of male fruit flies both suggested that the products (EPS) from 50% ethanol fractionation contain active components,which could activate B and T lymphocytes proliferation and prolong life-span of fruit flies at 31.5%.The suitable procedure for extraction exopolysaccharides from G106-1 was determined as follows:the filtrate was concentrated to 1/3 volume at 65 and then precipitated at a final 30% ethanol at 4 overnight. The supernatant after centrifuge was adjusted to the final concentration of 50% ethanol and precipitated at 4 overnight further. Then the pellets were harvested by centrifuge,washed several times with 95% ethanol and 100% ethanol,and dried by vacuum refrigerator.The raw exopolysacchride precipitates (EPS) were further separated into two fractions (EPS I and EPS II) by DEAE-DE52 (Ciy chromatography. The yield rates of EPS I and EPS II were 12.1% and 28.7%,which contained 86.9% and 95.5% total polysaccharides,respectively. EPS I was further purified by Sephadex-150 gel chromatography to obtain the homogenenous component (EPS I a),which was proved by polyacylamide gel electrophoresis,cellulose acetatefilm electrophoresis and Sepharose-6B gel chromatograph. Its yield rate and content of polysaccharides were 63.5% and 96.5%.The average molecular weight of EPS I a was determined by the gel chromatography to be 416KD. The component of EPS I a was determined to be composed of D-Man,D-Glu,D-Gal,D-Ara,D-Xyl in molar ratio of 4.46:0.24:2.38:0.90:0.19 by gas chromatography. EPS I a contains glucuronic acid,sulphate and protein,which were 27.4%,0.92% and 2.23% respectively.The infrared spectrum indicated that glucosidal linkage in EPS I a was pyranose. The assay of dilute alkali suggested that the polysaccharide chain is linked to peptide chain through N-glucosidal bond. Petriodate oxidation and Smith degradation studies showed that EPS I a was composed of 1-3,1-6 and 1-2 linked glucosyl bond.The immunity activity of AA mouse assay showed that the rude extract (EPS),EPS I and EPS II significantly enhanced the proliferation of spleen lymphocytes and increased interleukin-2 (IL-2) level in vitro (PO.01). Moreover,EPS I notably decreased level of interleukin-1 (IL-1) in AA mouse at the concentration of Immol/L,lOmmol/L and 10"2mmol/L,while EPS II did at the concentration of Immol/L,and EPS at Immol/L andlOmmol/L. These results suggested that the exopolysacchrides from G106-1 fermentation filtrate had immunomodulatory activities in vitro.