本文關(guān)鍵詞：鹽霉素高產(chǎn)菌的高通量篩選及油控制發(fā)酵工藝　出處：《華東理工大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鹽霉素發(fā)酵 高通量篩選 低場核磁共振技術(shù) 油脂定量測定

【摘要】：鹽霉素(Salinomycin)是一種聚醚類抗生素,對家禽類動物有抗球蟲病、促進生長作用,且對腫瘤細(xì)胞具有殺傷作用。同時鹽霉素作為抗癌藥物也受到矚目,需求逐步擴大,未來的市場前景不可限量。鹽霉素的發(fā)酵產(chǎn)業(yè)已經(jīng)接近成熟,優(yōu)質(zhì)高產(chǎn)菌株的獲得和發(fā)酵工藝的優(yōu)化卻是這個產(chǎn)業(yè)不可停止探索的內(nèi)容。本論文從菌種誘變與高通量篩選、發(fā)酵過程油在線控制的技術(shù)等方面進行研究,具體結(jié)果如下:1.通過將等溫等壓等離子(ARTP)和基因組重排(Genome Shuffling)育種技術(shù)與高通量(high through-put screening,HTS)培養(yǎng)篩選技術(shù)相結(jié)合來改良鹽霉素生產(chǎn)菌種。經(jīng)ARTP誘變后篩選到五株高產(chǎn)且穩(wěn)定的菌株,然后作為基因組重排的出發(fā)菌,經(jīng)過三輪原生質(zhì)體融合,篩選到三株鹽霉素高產(chǎn)菌株。對比了 ARTP和Genome Shuffling在鹽霉素高產(chǎn)菌篩選中的應(yīng)用,發(fā)現(xiàn)ARTP技術(shù)操作簡單方便,適合在企業(yè)使用,而Genome Shuffling操作復(fù)雜要求高,更適合在實驗室進行。2.為了對發(fā)酵液中油的檢測,建立了一種方便、快捷、無損的低場核磁共振(LF-NMR)技術(shù)檢測發(fā)酵液中油的方法。通過對培養(yǎng)基中各成分的檢測,確定了油脂的弛豫時間約100ms;單因素實驗表明除了黃豆粉和胚芽粉造成取樣不均對油峰面積有一定的影響,其它成分對油峰面積無明顯影響;含油量不同的梯度實驗結(jié)果表明了 LF-NMR檢測油時有良好的穩(wěn)定性(相對偏差約為4.4%)和精確性(約1.0%);正己烷萃取法測定油濃度與LF-NMR法測定的油濃度進行線性相關(guān)分析,相關(guān)性良好(R2=0.9581)。3.把建立的LF-NMR檢測油濃度的方法應(yīng)用于鹽霉素發(fā)酵過程中的油流加調(diào)控研究。對比了分光光度計法、正己烷萃取法和LF-NMR法對發(fā)酵液中油濃度的檢測。分光光度法在本實驗中基本失效,分辨不出發(fā)酵液中的油梯度;正己烷萃取法得到的油含量不精確,與理論值相差稍大;LF-NMR法測得的油濃度準(zhǔn)確性高且樣品處理簡單�；贚F-NMR方法,比較了恒定速率流加補油策略和批補油策略,發(fā)現(xiàn)在發(fā)酵過程缺油的時候批量補油,促進了鹽霉素生產(chǎn)菌的二次生長,維持了產(chǎn)鹽霉素能力,從而提高了鹽霉素的產(chǎn)量;而恒定速率流加方式補油對提高鹽霉素產(chǎn)量沒有明顯效果。
[Abstract]:Salinomycin is a polyether antibiotic which can resist coccidiosis and promote growth in poultry. At the same time, salinomycin, as a anticancer drug, has been attracting attention, the demand is gradually expanding, the future market prospects are unlimited. The fermentation industry of salinomycin has been nearly mature. The production of high quality and high yield strains and the optimization of fermentation process are the contents of this industry. This paper studies the mutagenesis and high throughput screening of bacteria and the technology of on-line control of oil in fermentation process. The results are as follows: 1. Using isothermal isobaric plasma column (ARTP) and genome rearrangement Genome Shuffling (Genome Shuffling) breeding techniques and high throughput (. High through-put screening. After ARTP mutagenesis, five high-yielding and stable strains were screened, and then they were used as genomic rearrangement bacteria. After three rounds of protoplast fusion, three salinomycin producing strains were screened. The application of ARTP and Genome Shuffling in the screening of salinomycin producing bacteria was compared. It is found that ARTP technology is easy to operate and suitable for use in enterprises, while Genome Shuffling operation is complex and demanding. In order to detect the oil in fermentation broth, a convenient and fast method was established. The method of detecting oil in fermentation broth by non-destructive low field nuclear magnetic resonance (LF-NMR) technique. The relaxation time of oil was determined to be about 100ms by detecting the components of culture medium. Univariate experiments showed that except soybean powder and germ powder caused by uneven sampling on the oil peak area has a certain effect, other components have no significant effect on the oil peak area; The results of gradient experiments with different oil content show that LF-NMR has good stability (relative deviation is about 4.4) and accuracy (about 1.0%) in oil detection. The linear correlation analysis was carried out between the oil concentration determined by n-hexane extraction method and the oil concentration determined by LF-NMR method. The established LF-NMR method for oil concentration detection was applied to the regulation of oil flow in the process of salinomycin fermentation. The spectrophotometer method was compared. Determination of oil concentration in fermentation broth by n-hexane extraction method and LF-NMR method. The oil content obtained by n-hexane extraction method is not accurate and slightly different from the theoretical value. The oil concentration measured by LF-NMR method is accurate and sample processing is simple. Based on the LF-NMR method, the strategies of constant rate flow filling and batch filling are compared. It was found that batch oil supplementation promoted the secondary growth of salinomycin producing bacteria and maintained the ability of producing salinomycin, thus increasing the yield of salinomycin. However, the constant rate flow addition had no obvious effect on increasing the yield of salinomycin.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：TQ920.6

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