【作者】 江艳华；

【导师】 江晓路；

【作者基本信息】 中国海洋大学 ， 食品科学， 2005， 硕士

【摘要】 本文从液体深层发酵产多糖条件的筛选、胞内及胞外多糖的制备与分析和多糖的生物学活性三方面对Isaria farinosa B05菌株进行了研究。 （1） 液体深层发酵产多糖条件中,由单因素实验和正交实验确定了Isaria farinosa B05菌株发酵最佳的培养基组成为:蔗糖3.5%（w/v）、蛋白胨0.5%、酵母膏0.2%、K₂HPO₄ 0.1%、MgSO₄ 0.05%;由单因素实验确定了该菌株最佳的发酵条件为:初始pH7.0、温度25℃、装液量75mL/250mL、接种量5%（v/v）、培养时间5d;在最佳的培养基和培养条件下,该菌株最大的生物量达2.12g/100mL,产水溶性胞外多糖可高达2.14g/L。 （2） 确定了胞外多糖的最佳提取工艺为发酵液在70℃下浓缩至原来的1/7,然后调pH7.0,加入3倍乙醇沉淀多糖;胞内多糖的最佳提取工艺为干菌体加30倍的水,100℃浸提150min,浸提2次。根据确定的工艺从Isaria farinosa B05菌株液体发酵获得的菌丝体及发酵液中提取得到水溶性胞内多糖（IPS）和胞外多糖（EPS）。 IPS和EPS经Q-Sepharose FF阴离子交换柱层析和Sephacryl S-300HR凝胶柱层析分离得到主峰IPS1和EPS1。经Sephacryl S-300HR凝胶柱层析证明纯化后IPS1和EPS1均一性较好。IPS1分子量为42kDa;糖含量为90.3%,含甘露糖、半乳糖、葡萄糖,摩尔比为8.0:4.8:1.0;糖醛酸含量为8.00%,有氨基糖存在;蛋白含量7.15%,含有11种常见氨基酸。EPS1分子量为208kDa;糖含量为93.4%,含甘露糖、半乳糖和少量葡萄糖,摩尔比为21.6:4.7:1.0;糖醛酸含量为8.06%,有氨基糖存在,蛋白含量4.38%,含有13种常见氨基酸。通过稀碱法确定IPS1和EPS1的糖肽键为N-糖苷键,红外光谱显示,二者为β-吡喃糖。 （3） 研究了多糖的生物学活性。IPS1与EPS1对S180实体瘤有明显的抑制作用,IPS1的剂量为200mg/kg时,抑瘤率最大,为61.63%;EPS1的剂量400mg/kg时最大,为63.10%。IPS1和EPS1可以明显促进荷瘤小鼠抗氧化酶的活性,在抗肿瘤活性最高的剂量组中,SOD和CAT酶活普遍较高。进一步研究了虫草棒束孢更多还原

【Abstract】 The selection of the optimal fermentation medium and conditions for polysaccharides production from Isaria farinosa B05, the extraction methods, characterization, components and bioactivities of polysaccharides are studied.（1） The medium components and fermentation conditions are optimized according to the one-at-a-time method, while the concentration of medium components is determined by orthogonal matrix method. The results show that the optimal fermentation medium is as follows: sucrose 3.5% （w/v）, peptone 0.5%, yeast extract 0.2%, K₂HPO₄ 0.1% and MgSO₄ 0.05%. The suitable fermentation conditions are as follows: initial pH 7.0, temperature 25℃, medium volume 75mL/250mL, inoculum volume 5% （v/v）, time 5d. In such optimal nutrition and environmental conditions, the maximal mycelial yield is 2.12g/100mL after 4 day’s fermentation, while maximal water-soluble exo-polysaccharides production reaches 2.14g/L after 5 day’s fermentation.（2） The suitable procedure for extraction exo-polysaccharides is determined as follows: the filtrate is concentrated to 1/7 volume at 70℃, adjusted to pH 7.0, and then precipitated by 3 volume of 95% ethanol. The suitable procedure for extraction intra-polysaccharides is determined as follows: the dried mycelium is added 30 multiple of water, and extracted 150min twice at 100℃. In such suitable procedure for extraction, the water-soluble intra-polysaccharides （IPS） and exo-polysaccharides （EPS） are obtained from the mycelium and filtrate.The active peaks of IPS1 and EPS1 are obtained when IPS and EPS are purified by anion-exchange chromatography on Q-Sepharose FF and gel chromatography on Sephacryl S-300HR, which was proved by Sephacryl S-300HR chromatography. The average molecular weight of IPS1 is determined to be 42kDa. IPS1 is composed ofmannose, galactose and glucose in molar ratio of 8.0: 4.8: 1.0, 90.3% carbohydrate, 8.00% uronic acid, hexosamine, 7.15% protein and 11 kinds of amino acid. The average molecular weight of EPS 1 is determined to be 208kDa. EPS1 is composed of mannose, galactose and glucose in molar ratio of 21.6: 4.7: 1.0, 93.4% carbohydrate, 8.06% uronic acid, hexosamine, 4.38% protein and 13 kinds of amino acid. The assay of dilute alkali suggests that the polysaccharide chain of IPS 1 and EPS1 is N-glucosidal bond. The infrared spectrum indicates that glucosidal linkage in IPS1 and EPS1 is (3-pyranose.（3） The bioactivities of polysaccharides are studied. Both IPS1 and EPS1 exhibit antitumor activities against SI80 solid-type tumor. The antitumor activity of IPS1 reaches maximum of 61.63% at a dose of 200mg/kg, while the one of EPS1 reaches maximum of 63.10% at a dose of 400mg/kg. IPS1 and EPS1 can improve the activities of antioxidase in tumor-bearing mice. Apparently the higher activities of SOD and CAT are, the higher the inhibition effects are obtained in the antitumor assay. The in vitro antioxidant activities are further investigated. The results show that WSIPS and WSEPS have effective antioxidant activities in various systems. WSIPS exhibits strong hydroxyl radical, superoxide radical and hydrogen peroxide scavenging activities; more over, it can chelate Fe2+, has great reductive ability and anti-lipid peroxidation ability. Although WSEPS can not scavenge hydroxyl radical as well as chelate Fe2+, it exactly shows similar effects with WSIPS in other antioxidation systems.The studies of this paper provide a theoretic foundation for further industrialization and clinic application.更多还原