【作者】 傅岚；

【导师】 陈作红； 张志光；

【作者基本信息】 湖南师范大学 ， 植物学， 2004， 硕士

【摘要】 本文以产于湖南浏阳市的古尼虫草[Cordyceps gunnii (Berk.)Berk.]及深层发酵的古尼虫草菌丝体为实验材料，利用菌种分离技术，液体深层发酵、PCR、柱层析、纸层析、紫外、红外光谱技术和化学反应等手段，对古尼虫草菌丝体液体发酵培养条件及部分化学成分进行了研究，主要包括以下内容：古尼虫草液体种子培养基及培养条件的筛选，发酵罐深层发酵生长曲线的测定，古尼虫草子座和古尼拟青霉(Paecilomyces gunnii)ITS序列的分析，菌丝体多糖的分离纯化及结构测定，古尼虫草的碱溶性多糖、甘露醇的提取方法及含量研究，获得了如下结果： 1．优化筛选出了古尼虫草菌丝体最适液体种子培养基和最佳培养条件，培养基配方为：土豆汁20％、蛋白胨1％、葡萄糖2％、硫酸镁1‰、磷酸二氢钾0.5％0、磷酸氢二钾1‰；用三因素四水平的正交试验，获得其最佳培养条件：26℃，转速150 r／min，pH 6.5，时间以5d为宜。 2．测定了古尼虫草菌丝体发酵罐深层发酵的生长曲线，确定发酵周期以66h为宜，并根据其生长规律，将其分为四个典型生长时期：生长延迟期、对数生长期、稳定生长期及衰老期。 3．运用PCR技术研究了古尼虫草子座和菌丝体的完整的ITS区(ITS1+5.8S+ITS2)，两者的PCR扩增产物电泳图谱及整个碱基序列完全一致，ITS1为207bp，5.8S为157 bp，ITS2为202 bp，可证明本文分离得到的古尼虫草菌丝体，确为古尼虫草的无性型古尼拟青霉；比较了古尼虫草子座、菌丝与冬虫夏草的rrs序列，并认为它们具有较近的亲缘关系。 4.古尼虫草菌丝体经热水浸提、醇析、脱蛋白，得到粗多糖，再经D王AE一52纤维素柱层析纯化得到纯多糖CG-1，经紫外光谱，柱层析等证明其为单一成分，纸层析表明其单糖组分为葡萄糖，用高碘酸反应证明其糖基是以(1。6)位糖若键型缩合，红外光谱结果显示CG-1具多糖特征吸收峰，为Q一毗喃型葡聚糖。 5.用四因素三水平的正交试验确定了以35℃，加5倍量的0 75mol几的NaOH溶液浸泡提取古尼虫草菌丝体sh，为碱溶性多糖的最适提取方法，从每克菌丝体中提取的多糖最高可含41 .92mg。 6古尼虫草发酵菌丝体含有丰富的甘露醇，在每克干菌粉中含量为60mg，每克古尼虫草子座中含量为61mg，每克僵虫体含量达105mg。更多还原

【Abstract】 The liquid fermentation and partical chemical constituents of Cordyceps gunnii fruitbodies collected from Liuyang Hunan and its cultured mycelium were studied by means of tissue isolation, liquid fermentation, PCR, column chromatography, TLC and spectrum technology, which including riddling of culture condition of C. gunnii, determining the growth survey of the spawn, analyzing the ITS sequence of C.gunnii and Paecilomyces gunnii, separating, purification and structural analyzing of polysaccharide mycelia of C. gunnii, extracting of alkali soluble polysaccharide and mannitol and their content. The result showed as below:1. The optimum liquid fermentation medium of Cordycep gunnii was potato juice 20%, peptone 1%, glucose 2%, magnesium sulfate 0.1%, potassium dihydrogen phosphate 0.05%, dipotassium hydrogen phosphate 0.1%. The optimum culture conditions of C. gunnii was investigated by the orthogonal experiment in three factors and four levels, temperature was 25 ~26 C, rotation rate 150 r/min, pH 6.5 and culture period 5 days.2. Four typical growth periods in the spawn of liquid fermentation were studied: the period of growth hesitation, the rapid growth period, the growth steady period and the old period. We got a best fermentation period of 66 hours.3. The internal transcribed spacer (including ITS1, ITS2 and 5.8S rDNA gene) of nuclear ribosomal DNA from the stroma and mycelium of C. gunnii were amplified by means of PCR technology. The PCR products electrophoretic patterns and nucleotide sequence were complete concordant, the sequences of ITS1 was 207 bp in size, 5.8S was 157 bp and ITS2 was 202 bp. The result indicated that the asexual stage of C. gunnii was the isolated mycelium, namely Paecilomyces gunnii. The sequences of comparison ITS of stroma and mycelium of C. gunnii and Cordyceps sinensis, showed that they had a close predestined relationship.4. Crude polysaccharide was obtained from the cultured mycelium of C. gunnii by extraction with hot water and precipitated by alcohol, The proteins in it was removed by Sevage method, individual constituent CG-1 was isolated and purified by DEAE-52 chromatography. Its homogeneity was verified by UV scan and chromatography, TLC shows that CG-1 was composed by glucose only. The reaction of periodic acid suggested that the sugar residue be interlinked by 1- 6 bonds. The result of IR spectrum shows that there was typical characteristic absorption peaks of polysaccharides and a a - pyranoglucan in CG-1.5. The optimal extractive technology of alkali soluble polysaccharide from mycelium was determined by orthogonal experiment in four factors and three levels, the temperature was 35 C, marinated in 5 multiple volume 0.75 mol/L NaOH solution for 5 hours. A maximum extraction of 41.92 mg / g has been obtained.6. There was plenty of mannitol in the cultured mycelium of C. gunnii,the sum of mannitol was 60 mg / g, the stroma was 61 mg /g, the sclerotium was 105mg/g.更多还原