本文選題：虎杖　切入點：內(nèi)生菌　出處：《湖南農(nóng)業(yè)大學(xué)》2012年碩士論文

【摘要】：白藜蘆醇是一種天然活性多酚化合物,具有抑制腫瘤、保護心血管、抗氧化、抗自由基等多種藥理作用,被列為抗心血管病、抗癌最有前途的藥物之一�；⒄戎邪邹继J醇往往以白藜蘆醇苷形式存在,干燥虎杖根、莖中白藜蘆醇含量僅為0.2%左右,而白藜蘆醇苷含量可達2%。目前,直接從虎杖中提取白藜蘆醇費時、費力、浪費資源,而有機合成白藜蘆醇反應(yīng)產(chǎn)率低,環(huán)境危害性大,為克服以上方法不足,本文采用微生物轉(zhuǎn)化法轉(zhuǎn)化虎杖,篩選并鑒定能有效轉(zhuǎn)化其白藜蘆醇苷為白藜蘆醇的內(nèi)生菌株J1,經(jīng)誘變育種后得到優(yōu)良突變菌株UJ1,優(yōu)化菌株UJ1搖瓶發(fā)酵培養(yǎng)基及培養(yǎng)條件,并進行10L發(fā)酵罐驗證。得到以下主要結(jié)果： 1.從野生虎杖根、莖中初篩得到6株內(nèi)生真菌,HPLC檢測表明6株內(nèi)生真菌均具有轉(zhuǎn)化白藜蘆醇苷能力,轉(zhuǎn)化力最強菌株J1的轉(zhuǎn)化率為25.6%,轉(zhuǎn)化后發(fā)酵液中白藜蘆醇含量達14.068μg/mL,為未發(fā)酵的2.65倍。 2.結(jié)合形態(tài)學(xué)及分子生物學(xué)鑒定方法,鑒定內(nèi)生菌J1為草酸青霉Penicillium oxalicum,GenBank登錄號為HQ732137。 3.以內(nèi)生菌J1為出發(fā)菌株,進行紫外線和60Co Y射線誘變育種,得到優(yōu)良突變菌株UJ1,其轉(zhuǎn)化率較出發(fā)菌株提高8.4%,且遺傳穩(wěn)定。 4.單因素及正交試驗優(yōu)化研究確定菌株UJ1發(fā)酵培養(yǎng)基最佳配方為：虎杖粉末1.5%,無水乙醇2%,CMC-Na2.5%,NH4N030.2%,Na2HP040.06%,CaCl20.1%:確定最佳培養(yǎng)條件為：溫度28℃,初始pH5.5,接種量9%,裝液量30mL/250mL,轉(zhuǎn)速220r/min,時間60h。在以上優(yōu)化條件下,白藜蘆醇苷平均轉(zhuǎn)化率達90.1%,相比未優(yōu)化前提高56.1%。 5.在搖瓶最佳培養(yǎng)基組合及最佳培養(yǎng)條件基礎(chǔ)上,進行10L發(fā)酵罐驗證試驗,結(jié)果表明10L發(fā)酵罐中UJl菌株56h達到最高轉(zhuǎn)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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