【作者】 项小燕；

【导师】 张小平； 郑维发；

【作者基本信息】 安徽师范大学 ， 植物学， 2006， 硕士

【摘要】 桦褐孔菌(Inonotus obliquus (Fr.) Pilat),是珍稀的大型药用真菌,在俄罗斯已有一百多年的应用历史,能治疗多种疾病。目前美国、韩国及日本正加大对这种菌的研究,而我国尚处于初级研究阶段。自美国的Humfeld H等对蘑菇的深层发酵进行了研究,并提出用发酵法来培养蘑菇的菌丝体后,研究者们纷纷用这种方法进行多种菌类的发酵。而国内外对桦褐孔菌的深层发酵研究甚少,国内目前仅有一篇生物量方面的报道。对于其化学成分分析、调控及药理学方面的研究尚属空白。本文通过对野外采集的桦褐孔菌菌核体进行组织分离,得到PDA菌种,然后对该菌进行深层发酵培养。为了得到大量的菌丝体和多糖,应用单因子试验和正交试验相结合分别得到菌丝体和多糖的优化培养基,并用DNS法测定发酵液中还原糖浓度;石油醚提取菌丝体中弱极性物质各成分后用GC-MS对其分析和鉴定。在此试验基础上,为了提高菌丝体中甾醇类化合物含量,首先优化了甾醇类化合物合成培养基,并通过人工干预该类化合物合成途径关键酶,对甾醇类化合物进行上调因子调控研究,以达到进一步提高甾醇类物质含量的目的,结果均用GC-MS分析。同时,对发酵胞内多糖进行了药理学研究,结果用试剂盒检测。得出结论如下:1.在所选的几种碳、氮源中,葡萄糖是桦褐孔菌菌丝体生长、胞内粗多糖和甾醇类化合物合成的最佳碳源,麦芽糖是胞外粗多糖合成的最佳碳源;蛋白胨和酵母膏的混合物是最佳氮源;起始pH 5.5最适合菌丝体生长,起始pH 6.5最有利于胞内、外粗多糖和甾醇类化合物的合成。2. 3%的葡萄糖最适合菌丝体生长和胞外粗多糖的合成,0.4%的蛋白胨和0.2%的酵母膏最适合菌丝体的生长,0.6%的蛋白胨和0.3%的酵母膏最有利于胞外粗多糖的合成。3.菌丝体最佳培养基配方组成为:2.5%葡萄糖,0.4%蛋白胨,0.2%酵母膏,起始pH为5.5;胞外粗多糖最佳培养基配方为:3.5%葡萄糖,0.6%蛋白胨,0.2 %酵母膏,起始pH为6.5。4.在菌丝体最佳发酵培养基中,还原糖的利用率达99.9%,pH由5.48更多还原

【Abstract】 Inonotus obliquus (Fr.) Pilat has been known for over one hundred years as a medicinal fungus in folk medicine, it can treat many difficult diseases. At present, this fungus is being explored extensively in American, Korea and Japan, but is just investigated in China. Since American Humfeld H and other workers studied submerged culture of mushroom and put forward to culture mycelia of mushroom by this method, fungi have been cultured in succession. But the researches about I. obliquus are poor in this aspect, and there is only one report about the mycelia dry weight (MDW). Until now it has not been studied Chemically, manipulately, pharmacologically and so on.In this paper, the purity mycelia used in fermentation were separated from sclerotia of I. obliquus which were collected from fields. To get more MDW and polysaccharides, both the one-factor-at-a-time and the orthogonal matrix methods were applied to obtain optimum media for them, the residual sugar and exo-polysaccharides (EPS) in the media were detected by DNS method, the non-polar constituents from the submerged cultures were extracted with petroleum and analyzed by GC-MS. The optimum medium for steroids accumulation was studied, moreover, steroids were manipulated by adding to stimulated factors of the key enzyme in the synthesized routes, all products were analyzed by GC-MS. The polysaccharides activities in different media were analyzed by pharmacological experiments. The results were summarized as follows:1. In choosed carbon sources and nitrogen sources, glucose was optimum carbon source for the MDW, endo-polysaccharides (IPS) and steroids accumulation; maltose was optimum carbon source for the EPS production; optimum nitrogen source was the mixture of peptone and yeast extracts; the initial pH 5.5 was optimal for the MDW, but 6.5 was optimal for the IPS, EPS and steroids production.更多还原