【作者】 于春秀；

【导师】 樊美珍； 李增智； 唐欣昀；

【作者基本信息】 安徽农业大学 ， 微生物学， 2002， 硕士

【摘要】 本文在地生枝顶孢霉(Acremonium terricola(Miller et al．)Gams)G106-1菌株发酵液胞外多糖的初步研究的基础上，改变虫草发酵液的预处理以及胞外多糖的提取方法，并以多糖的生物活性为检测指标，重新确定了G106-1虫草菌的胞外多糖提取方法，并对多糖组分的分离纯化及纯度鉴定、多糖的理化性质及组成分析、一级结构及生物活性进行了深入的研究。 T、B淋巴细胞的增殖反应试验及果蝇寿命试验结果表明：乙醇分级沉淀所得各部分多糖，调节乙醇终浓度为50％提取出的粗多糖的活性部分，经检测可诱导B和T淋巴细胞增殖，具有免疫增强作用。果蝇寿命试验表明，50％醇沉部分可延长果蝇寿命，其延长率为31.5％。综合活性检测结果，确定50％醇沉部分为进一步研究的目标产物。 不同pH下温度对胞外多糖的影响试验及不同浓缩倍数对胞外多糖的影响试验显示：在中性条件下，虫草胞外多糖的得率和糖含量基本趋于一致；调整pH后的发酵液浓缩至1／3体积时，其胞外多糖的得率达最大值。综合试验结果，确定胞外多糖提取的最佳工艺路线：首先将离心去除菌丝体的发酵液调至中性，4℃放置1h，离心去除沉淀，65℃下浓缩至原体积的1／3，冷却后加入95％乙醇溶液至终浓度30％，4℃静置24h，离心收集上清液。上清液中再加入95％乙醇溶液至终浓度为50％，4℃静置24h后，离心收集沉淀部分。沉淀经95％乙醇和无水乙醇反复洗涤后，低温真空干燥，得多糖粗品(EPS)。 多糖粗品(EPS)经DEAE-DE52色谱柱(Cl~-)分离，得到两个组分(EPSⅠ、EPSⅡ)，得率分别为：12.1％、28.7％，其总糖含量分别为86.9％、95.5％。高效液相色谱分析显示，EPSⅠ含有1个强峰组分和1个弱峰组分，而EPSⅡ中尚含有多个组分，本研究选定EPSⅠ作为深入研究对象。 EPSⅠ经Sephadex-150色谱柱纯化得到强峰组分EPSⅠa，其多糖含量为96.5％，回收率为63.5％。聚丙烯酰胺凝胶电泳、醋酸纤维素薄膜电泳、 Se…se6B柱层析结果表明：纯化后的EPS为均一组分。 凝胶过滤法测得EPS分子量为416KD。气相色谱法分析单糖组成， 结果表明：EPS中含有D－甘露糖、D－葡萄糖、D－半乳糖、D平拉伯糖、 D－木糖，各单糖摩尔比为4．46：0．24：2．38：0．90：0二9。糖醛酸含量分析、硫酸基 及蛋白质含量测定结果表明：EPS中糖醛酸含量为27．4％；硫酸基含量 0．92％；蛋白质含量2．23％。氨基酸组成分析显示：EPS中含有 17种常见 氨基酸，其中天冬氨酸和谷氨酸含量最高，分别为2026％和32．28％。 在EPS的一级结构研究中，红外光谱测得其为p型毗哺糖，P一消 去反应证明EPS中的糖肽键是N一糖苦键，高碘酸氧化、Smith降解结果 表明EPS中含有l—3糖苦键，l—6糖苦键及l—2糖苦键 胞外多糖体外对佐剂性关节炎大鼠免疫功能的影响试验结果表明，多糖 粗品（EPS）以及分离得到的EPS、EP引对AA大鼠低下的增殖反应、过 低的白介素上（IL．2）均具有明显的促进作用0＜0刀1）。对AA大鼠过高的 白介素4 门Ll）产生的试验结果显示，EPS在浓度为 11lllllOMi、10－‘ mmoffe、10－2mmoffe时均具有明显影响，而EPS 11在浓度为 Inunol几、多糖 粗品（EPS）在浓度为Illlllloll、10”‘1lllllol几时具有明显影响。综合结果表 明胞外多糖在体外具有免疫活性。更多还原

【Abstract】 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.更多还原