摘要
通过对北冬虫夏草(Cordyceps militaris Link)菌种液体发酵培养,离心分离获得菌丝体菌液,分别从渗透压稳定剂、裂解酶、裂解时间及菌龄4个方面优化原生质体制备的条件.结果表明:原生质体制备的最优渗透压稳定剂为0.6 mol/L MgSO_4,最优酶解液为1%纤维素酶,最佳菌龄为5 d,最优裂解时间为2 h.L_(20)(5~4)的正交试验表明,优化条件下制备原生质体为1.46×10~7个/m L.紫外诱变实验结果表明:北冬虫夏草半致死率时的照射剂量约为40 s,诱变距离与致死率成反比,在诱变总时间相同的情况下,诱变受诱变次数影响较小,分次处理和一次处理效果基本相同.以多糖含量为指标,筛选诱变菌株,诱变株多糖含量高于普通菌株,其中17~#菌株多糖含量达6.01%,筛选菌株的稳定性实验表明:继代培养3次,筛选的菌株产多糖能力下降较少,表明筛选菌株具有较好的稳定性.
After enlarged culture of Cordyceps militaris Link and liquid fermentation, centrifugal separation was carried out to obtain mycelial liquid. The conditions of protoplast preparation were optimized from four aspects:osmotic pressure stabilizer, lysozyme, lysis time and bacterial age. The results showed that the optimal osmotic pressure stabilizer for protoplast preparation was 0.6 mol/L sulfatase, and the optimal osmotic pressure stabilizer was 0.6 mol/L sulfatase. While the optimum enzyme solution was 1% cellulase, the optimum bacterial age was 5 days, and the optimum cracking time was 2 hours. The orthogonal experiment of L 20 (5 4 ) showed that 1.46×10 7 protoplasts could be prepared under the optimum conditions. The results of ultraviolet mutagenesis experiment showed that the irradiation dose was about 40 seconds, and the mutagenesis distance was inversely proportional to the lethality. When the total time of mutagenesis was the same, the number of mutation was less affected, and the effects of multiple and single treatment were similar. The polysaccharide content was then used as an indicator to screen the mutagenized strains, and we successfully got the mutants that showed higher content of the polysaccharide than that of common strains. The polysaccharide content of 17 # was 6.01%. The stability test of the screened strains showed that the ability of polysaccharide production of screened strains decreased less after three subcultures and the screened strains had better stability.
作者
汪军玲
王钊
WANG Junling;WANG Zhao(College of Life Sciences and Technology,Wuhan University of Bioengineering,Wuhan 430415,China)
出处
《信阳师范学院学报(自然科学版)》
CAS
北大核心
2019年第2期201-206,共6页
Journal of Xinyang Normal University(Natural Science Edition)
基金
国家自然科学基金项目(U1404305)
湖北省教育厅项目(B20104301)
武汉市教育局教研项目(2008J25)
关键词
北冬虫夏草
原生质体
紫外诱变
虫草多糖
Cordyceps militaris Link:protoplast
ultraviolet mutagenesis
Cordyceps Polysaccharide