无性型冬虫夏草腺苷高产菌株及指纹图谱的研究
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摘要
冬虫夏草为名贵中药材，味甘性平，有保肺肾、补精髓、化痰止劳咳，以及调节免疫功能、增强人体对多种疾病抵抗力的功效。但是天然虫草受自然条件限制，产量有限，满足不了国内外日益增长的需要，已成为稀世之珍，因而货源奇缺，供不应求，价格昂贵。
目前国内对冬虫夏草的研究，主要以分析天然虫草和虫草菌丝体的腺苷、D-甘露醇和虫草多糖的含量，从自然环境分离冬虫夏草的无性型菌株，以及无性型菌株深层发酵的培养基和培养条件的研究居多。本课题以提高冬虫夏草菌丝体的主要活性化学成分腺苷的含量为目的，对冬虫夏草Cordyceps sinensis(Berk.)Sacc.菌株CS-JS进行了菌种选育和发酵的研究，并初步建立了冬虫夏草发酵菌丝体的毛细管电泳指纹图谱，为冬虫夏草菌丝体产品的研发和产品的鉴别打下了基础。
实验建立了无性型冬虫夏草(Cordyceps sinesis(Berk.)Sacc.)CS-JS菌株菌丝体的两种活性成分D-甘露醇和腺苷的含量测定方法。用80℃水浴40min制备人工虫草菌丝体D-甘露醇样品，比色法测定D-甘露醇，在10μ/ml～50μg／ml内呈良好的线性关系(r=0.9999)，RSD为1.47％，平均回收率为104.08％，该法简便准确，重现性好。用超声处理的方法制备虫草菌丝体腺苷样品，HPLC测定其含量，腺苷保留时间为7.6min，在0.016m/ml-0.24mg/ml浓度范围内，呈良好的线性关系(r=0.9999)，RSD=0.77％。
通过酶解细胞壁的方法，优化了冬虫夏草(Cordyceps sinesis(Berk.)Sacc.)CS-JS菌丝体的原生质体制备和再生条件：6天左右菌龄的菌丝体，1％蜗牛酶和1％纤维素酶的酶组合，在pH6.4的甘露醇稳渗环境，36℃酶解1.5h，可获得原生质体2.04×10~9个／ml。再生培养基为RM3时，再生率为0.091％。
以腺苷的高产为主要筛选目标，D-甘露醇和多糖为辅助筛选指标，选取120秒为诱变时间进行原生质体诱变。应用8-AG筛选AMP脱氨酶缺陷型或低活性的突变株的办法，筛选到腺苷含量提高8.24倍的诱变株CS-JS-16。该筛选模型首次应用于虫草腺苷高产菌株的筛选，可提高筛选效率。
通过对培养条件的研究，确定了冬虫夏草(Cordyceps sinesis(Berk.) Sacc.)CS-JS-16最佳发酵培养基是：蛋白胨0.75％、葡萄糖1.5％、pH6.3、MgSO_40.15％、KH_2PO_40.2％。发酵6天后，最大发酵量是0.1746g/10ml，最大AR含量是1.0826mg／g。CS-JS-16菌丝体急性毒理学实验表明，昆明种小鼠最大耐受剂量为26.4g／Kg。
应用毛细管电泳的方法，比较了冬虫夏草(Cordyceps sinesis(Berk0)Sacc0)同一菌系和不同菌系菌丝体的毛细管电泳图谱，通过分析共有特征峰、重叠率以及特征峰的相对峰面积范围，建立了冬虫夏草无性型菌株的毛细管电泳指纹图谱。冬虫夏草CS-JS菌系共有特征峰7个，特征峰的重叠率大于70％，特征峰的相对峰面积范围小于10％。
Cordyceps sinesis (Berk.) Sacc is a Chinese tranditional medicinal fungus. It has been studied by many countries for its characteristic of medicinal value. It is a famous and costly medicinal fungus, and it was investigated clearly in its biological feature and chemical components. Now Cordyceps sinesis (Berk.) Sacc can be produced by unnatural cultivation, not native collection, which brings many advantages of its output and application. Furthermore, its medicinal was studied better, and its applied extent was increased greatly. Therefore, it has important significance to explore and develope Cordyceps sinensis for national, even international medicine.
The methods of measuration of mannitol and adenosine in the dry mycelia of Cordyceps sinesis (Berk.) Sacc. were optimized.The results showed that the concentration of manntiol in the range of 10 u g/ml to 50 u g/ml was closely linked with the value of absorbance linearly(r=0.9999) by the colorimetry method. The method that the mycelia were extracted at 80X3 for 40 minutes was simple, accuracy and had good reappearance, and the average recovery ratio was 104.08%.The concentration of adenosine in the range of 0.016mg/ml to 0.24mg/ml was closely linked with the peak area (r=0.99991) when the sample was analysised by HPLC.The retent time was 7.6min.
The conditions of preparation and regeneration of Cordyceps sinesis (Berk) Sau) were also studied. When the mycelia were cultured for 6 days and were enzymelysed in mannitol solution for 2.5h at 36℃ by mixture solution of Snailase(1%) and Cellulase(1%) in 6.4 pH, the forming number of protoplst was the highest. Finally,the forming number of protoplast is 2.04 ×109/ml.The highest regeneration ratio of protoplast is 9.1 × 10-2 on RM3 medium.
The protoplast were irradiated by UV for 120 seconds and then were regenerated. The strain CS-JS-16 was gained in these regeneration colonies of Cordyceps sinesis (Berk.) Sacc according to the content of adenosine, mannitol and polysaccharides.The adaptivest culture medium was established through researching the ferment ation conditions: 0.75%peptone, 1.5%glucose, pH6.3, 0.15%MgSO4, 0.2%KH2PO4 The maximum biomass of mycelium and the content of adenosine was respectively 0.1746g/ml and 1.0826mg/g after cultivating for 6 days.The toxicological experiment showed that the maximum tolerance dose in mice was 26.4g/kg.
The fingerprints of Cordyceps sinesis CS-JS strains were established after identifying different strains by making use of the capillary electrophoresis. Finally there were 7 common characteristic peaks, overlap ratio of the characteristic peaks were more than 70%, and the range of relative peak aera was less than 10%.
目前国内对冬虫夏草的研究，主要以分析天然虫草和虫草菌丝体的腺苷、D-甘露醇和虫草多糖的含量，从自然环境分离冬虫夏草的无性型菌株，以及无性型菌株深层发酵的培养基和培养条件的研究居多。本课题以提高冬虫夏草菌丝体的主要活性化学成分腺苷的含量为目的，对冬虫夏草Cordyceps sinensis(Berk.)Sacc.菌株CS-JS进行了菌种选育和发酵的研究，并初步建立了冬虫夏草发酵菌丝体的毛细管电泳指纹图谱，为冬虫夏草菌丝体产品的研发和产品的鉴别打下了基础。
实验建立了无性型冬虫夏草(Cordyceps sinesis(Berk.)Sacc.)CS-JS菌株菌丝体的两种活性成分D-甘露醇和腺苷的含量测定方法。用80℃水浴40min制备人工虫草菌丝体D-甘露醇样品，比色法测定D-甘露醇，在10μ/ml～50μg／ml内呈良好的线性关系(r=0.9999)，RSD为1.47％，平均回收率为104.08％，该法简便准确，重现性好。用超声处理的方法制备虫草菌丝体腺苷样品，HPLC测定其含量，腺苷保留时间为7.6min，在0.016m/ml-0.24mg/ml浓度范围内，呈良好的线性关系(r=0.9999)，RSD=0.77％。
通过酶解细胞壁的方法，优化了冬虫夏草(Cordyceps sinesis(Berk.)Sacc.)CS-JS菌丝体的原生质体制备和再生条件：6天左右菌龄的菌丝体，1％蜗牛酶和1％纤维素酶的酶组合，在pH6.4的甘露醇稳渗环境，36℃酶解1.5h，可获得原生质体2.04×10~9个／ml。再生培养基为RM3时，再生率为0.091％。
以腺苷的高产为主要筛选目标，D-甘露醇和多糖为辅助筛选指标，选取120秒为诱变时间进行原生质体诱变。应用8-AG筛选AMP脱氨酶缺陷型或低活性的突变株的办法，筛选到腺苷含量提高8.24倍的诱变株CS-JS-16。该筛选模型首次应用于虫草腺苷高产菌株的筛选，可提高筛选效率。
通过对培养条件的研究，确定了冬虫夏草(Cordyceps sinesis(Berk.) Sacc.)CS-JS-16最佳发酵培养基是：蛋白胨0.75％、葡萄糖1.5％、pH6.3、MgSO_40.15％、KH_2PO_40.2％。发酵6天后，最大发酵量是0.1746g/10ml，最大AR含量是1.0826mg／g。CS-JS-16菌丝体急性毒理学实验表明，昆明种小鼠最大耐受剂量为26.4g／Kg。
应用毛细管电泳的方法，比较了冬虫夏草(Cordyceps sinesis(Berk0)Sacc0)同一菌系和不同菌系菌丝体的毛细管电泳图谱，通过分析共有特征峰、重叠率以及特征峰的相对峰面积范围，建立了冬虫夏草无性型菌株的毛细管电泳指纹图谱。冬虫夏草CS-JS菌系共有特征峰7个，特征峰的重叠率大于70％，特征峰的相对峰面积范围小于10％。
Cordyceps sinesis (Berk.) Sacc is a Chinese tranditional medicinal fungus. It has been studied by many countries for its characteristic of medicinal value. It is a famous and costly medicinal fungus, and it was investigated clearly in its biological feature and chemical components. Now Cordyceps sinesis (Berk.) Sacc can be produced by unnatural cultivation, not native collection, which brings many advantages of its output and application. Furthermore, its medicinal was studied better, and its applied extent was increased greatly. Therefore, it has important significance to explore and develope Cordyceps sinensis for national, even international medicine.
The methods of measuration of mannitol and adenosine in the dry mycelia of Cordyceps sinesis (Berk.) Sacc. were optimized.The results showed that the concentration of manntiol in the range of 10 u g/ml to 50 u g/ml was closely linked with the value of absorbance linearly(r=0.9999) by the colorimetry method. The method that the mycelia were extracted at 80X3 for 40 minutes was simple, accuracy and had good reappearance, and the average recovery ratio was 104.08%.The concentration of adenosine in the range of 0.016mg/ml to 0.24mg/ml was closely linked with the peak area (r=0.99991) when the sample was analysised by HPLC.The retent time was 7.6min.
The conditions of preparation and regeneration of Cordyceps sinesis (Berk) Sau) were also studied. When the mycelia were cultured for 6 days and were enzymelysed in mannitol solution for 2.5h at 36℃ by mixture solution of Snailase(1%) and Cellulase(1%) in 6.4 pH, the forming number of protoplst was the highest. Finally,the forming number of protoplast is 2.04 ×109/ml.The highest regeneration ratio of protoplast is 9.1 × 10-2 on RM3 medium.
The protoplast were irradiated by UV for 120 seconds and then were regenerated. The strain CS-JS-16 was gained in these regeneration colonies of Cordyceps sinesis (Berk.) Sacc according to the content of adenosine, mannitol and polysaccharides.The adaptivest culture medium was established through researching the ferment ation conditions: 0.75%peptone, 1.5%glucose, pH6.3, 0.15%MgSO4, 0.2%KH2PO4 The maximum biomass of mycelium and the content of adenosine was respectively 0.1746g/ml and 1.0826mg/g after cultivating for 6 days.The toxicological experiment showed that the maximum tolerance dose in mice was 26.4g/kg.
The fingerprints of Cordyceps sinesis CS-JS strains were established after identifying different strains by making use of the capillary electrophoresis. Finally there were 7 common characteristic peaks, overlap ratio of the characteristic peaks were more than 70%, and the range of relative peak aera was less than 10%.
引文
1李志超，杨姗姗．食药用菌生产与消费指南[M]．北京：中国：农业出版社，1997，288～295．
2王伟，陈特灿，李琼英，等．中国虫草研究[J]．食用菌学报，1998，5(4)：17～22．
3小林义雄，清水大典．冬虫夏草菌图谱[M]．大阪：保育社，1983，163～165．
4梁宗琦．虫草的无性型及其确定[．J]．西南农学报，1991，4(4)：1～8．
5赵锦，王宁，陈月琴，等．冬虫夏草无性型的分子鉴别[J]．中山大学学报，1999，38(1)：121～123．
6李增智，黄勃，李春如等．确证冬虫夏草无性型的分子生物学证据[J]．菌物系统，2000，19(1)：60～64．
7孙毓庆，阮婧华，马欣．中药的毛细管电泳指纹图谱研究[J]．色谱，2003，21(4)303～306．
8沈晓云，李兆田，田军．冬虫夏草与虫草菌丝有效成分分析比较[J]．山西大学学报，1998，21(1)：80～85．
9宋金娣，徐荷芬，邵向明等．冬虫夏草菌丝体免疫特性测试[J]．食用菌学报，1999，6(4)：52～54．
10龚晓健，季晖，卢顺高等．人工虫草多糖对小鼠免疫功能的影响[J]．中国药科大学学报，2000，31(1)：53～55．
11夏益平．冬虫夏草口服液治疗慢乙肝36例疗效观察[J]．右江医学，2000，28(2)：91～92．
12许周善，周晓燕．冬虫夏草多糖的研究进展[J]．工业微生物，2000，30(1)：56～57．
13蒲春翔，赵建强．液体培养虫草子座初报[J]．中国食用菌，1992，11(6)：44．
14尚德静，黄孝敏．冬虫夏草深层发酵的研究[J]．中国食用菌，1996，15(6)：48～49．
15梁淑娃，方展瑞，翁照南等．冬虫夏草菌丝体深层发酵技术的研究[J]．广东食品工业科技，2000，16(1)：25～27．
16杨淑全，王澄澈．冬虫夏草液体培养初报[J]．中国食用菌，1999，18(5)：28～29．
17刘丽丽，周剑书，王姝等．虫草菌最适培养基的正交试验研究[J]．天津师大学报，1999，19(2)：40～4．
2王伟，陈特灿，李琼英，等．中国虫草研究[J]．食用菌学报，1998，5(4)：17～22．
3小林义雄，清水大典．冬虫夏草菌图谱[M]．大阪：保育社，1983，163～165．
4梁宗琦．虫草的无性型及其确定[．J]．西南农学报，1991，4(4)：1～8．
5赵锦，王宁，陈月琴，等．冬虫夏草无性型的分子鉴别[J]．中山大学学报，1999，38(1)：121～123．
6李增智，黄勃，李春如等．确证冬虫夏草无性型的分子生物学证据[J]．菌物系统，2000，19(1)：60～64．
7孙毓庆，阮婧华，马欣．中药的毛细管电泳指纹图谱研究[J]．色谱，2003，21(4)303～306．
8沈晓云，李兆田，田军．冬虫夏草与虫草菌丝有效成分分析比较[J]．山西大学学报，1998，21(1)：80～85．
9宋金娣，徐荷芬，邵向明等．冬虫夏草菌丝体免疫特性测试[J]．食用菌学报，1999，6(4)：52～54．
10龚晓健，季晖，卢顺高等．人工虫草多糖对小鼠免疫功能的影响[J]．中国药科大学学报，2000，31(1)：53～55．
11夏益平．冬虫夏草口服液治疗慢乙肝36例疗效观察[J]．右江医学，2000，28(2)：91～92．
12许周善，周晓燕．冬虫夏草多糖的研究进展[J]．工业微生物，2000，30(1)：56～57．
13蒲春翔，赵建强．液体培养虫草子座初报[J]．中国食用菌，1992，11(6)：44．
14尚德静，黄孝敏．冬虫夏草深层发酵的研究[J]．中国食用菌，1996，15(6)：48～49．
15梁淑娃，方展瑞，翁照南等．冬虫夏草菌丝体深层发酵技术的研究[J]．广东食品工业科技，2000，16(1)：25～27．
16杨淑全，王澄澈．冬虫夏草液体培养初报[J]．中国食用菌，1999，18(5)：28～29．
17刘丽丽，周剑书，王姝等．虫草菌最适培养基的正交试验研究[J]．天津师大学报，1999，19(2)：40～4．