虎杖内生菌高效转化白藜芦醇苷的研究

发布时间：2018-04-01 09:18

  本文选题：虎杖　切入点：内生菌　出处：《湖南农业大学》2012年硕士论文

【摘要】：白藜芦醇是一种天然活性多酚化合物,具有抑制肿瘤、保护心血管、抗氧化、抗自由基等多种药理作用,被列为抗心血管病、抗癌最有前途的药物之一。虎杖中白藜芦醇往往以白藜芦醇苷形式存在,干燥虎杖根、茎中白藜芦醇含量仅为0.2%左右,而白藜芦醇苷含量可达2%。目前,直接从虎杖中提取白藜芦醇费时、费力、浪费资源,而有机合成白藜芦醇反应产率低,环境危害性大,为克服以上方法不足,本文采用微生物转化法转化虎杖,筛选并鉴定能有效转化其白藜芦醇苷为白藜芦醇的内生菌株J1,经诱变育种后得到优良突变菌株UJ1,优化菌株UJ1摇瓶发酵培养基及培养条件,并进行10L发酵罐验证。得到以下主要结果： 1.从野生虎杖根、茎中初筛得到6株内生真菌,HPLC检测表明6株内生真菌均具有转化白藜芦醇苷能力,转化力最强菌株J1的转化率为25.6%,转化后发酵液中白藜芦醇含量达14.068μg/mL,为未发酵的2.65倍。 2.结合形态学及分子生物学鉴定方法,鉴定内生菌J1为草酸青霉Penicillium oxalicum,GenBank登录号为HQ732137。 3.以内生菌J1为出发菌株,进行紫外线和60Co Y射线诱变育种,得到优良突变菌株UJ1,其转化率较出发菌株提高8.4%,且遗传稳定。 4.单因素及正交试验优化研究确定菌株UJ1发酵培养基最佳配方为：虎杖粉末1.5%,无水乙醇2%,CMC-Na2.5%,NH4N030.2%,Na2HP040.06%,CaCl20.1%:确定最佳培养条件为：温度28℃,初始pH5.5,接种量9%,装液量30mL/250mL,转速220r/min,时间60h。在以上优化条件下,白藜芦醇苷平均转化率达90.1%,相比未优化前提高56.1%。 5.在摇瓶最佳培养基组合及最佳培养条件基础上,进行10L发酵罐验证试验,结果表明10L发酵罐中UJl菌株56h达到最高转化率82.5%。
[Abstract]:Resveratrol is a natural active polyphenol compound with many pharmacological effects, such as inhibiting tumor, protecting cardiovascular, anti-oxidation, anti-free radical and so on. Resveratrol in Polygonum cuspidatum often exists in the form of resveratrol. The content of resveratrol in dried root and stem of Polygonum cuspidatum is only about 0.2%, but the content of resveratrol can reach 2%. The direct extraction of resveratrol from Polygonum cuspidatum is time-consuming, laborious and wasteful, but the organic synthesis of resveratrol has low yield and great environmental harmfulness. In order to overcome the shortage of the above methods, the microbial transformation method was used to transform Polygonum cuspidatum into Polygonum cuspidatum. The endophytic strain J1, which could effectively transform resveratrol into resveratrol, was screened and identified. After mutagenesis and breeding, the excellent mutant strain UJ1 was obtained, and the fermentation medium and culture conditions of strain UJ1 were optimized. The main results are as follows:. 1. Six strains of endophytic fungi were obtained from the root and stem of Polygonum cuspidatum by HPLC. The results showed that all 6 strains of endophytic fungi had the ability to transform resveratrol. The transformation rate of strain J1 was 25.6 and the content of resveratrol in fermentation broth was 14.068 渭 g / mL, 2.65 times as high as that of unfermented strain. 2. The endophytic bacteria J1 was identified as Penicillium oxalicum Penicillium GenBank accession number HQ732137. 3. Endophytic strain J1 was mutagenic by UV and 60Co Y rays, and the transformation rate of UJ1 was 8.4g higher than that of the original strain, and the inheritance was stable. 4. Single factor and orthogonal experiment optimization study showed that the optimum fermentation medium of the strain UJ1 was as follows: the powder of Polygonum cuspidatum 1.5, the absolute ethanol 2CMC-Na2.5NH _ 4N _ (030.2N) and Na2HP040.06 CaCl _ 20.1.The optimum culture conditions were determined as follows: 28 鈩，

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