盐霉素高产菌的高通量筛选及油控制发酵工艺

发布时间：2018-01-02 21:16

本文关键词：盐霉素高产菌的高通量筛选及油控制发酵工艺　出处：《华东理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：盐霉素(Salinomycin)是一种聚醚类抗生素,对家禽类动物有抗球虫病、促进生长作用,且对肿瘤细胞具有杀伤作用。同时盐霉素作为抗癌药物也受到瞩目,需求逐步扩大,未来的市场前景不可限量。盐霉素的发酵产业已经接近成熟,优质高产菌株的获得和发酵工艺的优化却是这个产业不可停止探索的内容。本论文从菌种诱变与高通量筛选、发酵过程油在线控制的技术等方面进行研究,具体结果如下:1.通过将等温等压等离子(ARTP)和基因组重排(Genome Shuffling)育种技术与高通量(high through-put screening,HTS)培养筛选技术相结合来改良盐霉素生产菌种。经ARTP诱变后筛选到五株高产且稳定的菌株,然后作为基因组重排的出发菌,经过三轮原生质体融合,筛选到三株盐霉素高产菌株。对比了 ARTP和Genome Shuffling在盐霉素高产菌筛选中的应用,发现ARTP技术操作简单方便,适合在企业使用,而Genome Shuffling操作复杂要求高,更适合在实验室进行。2.为了对发酵液中油的检测,建立了一种方便、快捷、无损的低场核磁共振(LF-NMR)技术检测发酵液中油的方法。通过对培养基中各成分的检测,确定了油脂的弛豫时间约100ms;单因素实验表明除了黄豆粉和胚芽粉造成取样不均对油峰面积有一定的影响,其它成分对油峰面积无明显影响;含油量不同的梯度实验结果表明了 LF-NMR检测油时有良好的稳定性(相对偏差约为4.4%)和精确性(约1.0%);正己烷萃取法测定油浓度与LF-NMR法测定的油浓度进行线性相关分析,相关性良好(R2=0.9581)。3.把建立的LF-NMR检测油浓度的方法应用于盐霉素发酵过程中的油流加调控研究。对比了分光光度计法、正己烷萃取法和LF-NMR法对发酵液中油浓度的检测。分光光度法在本实验中基本失效,分辨不出发酵液中的油梯度;正己烷萃取法得到的油含量不精确,与理论值相差稍大;LF-NMR法测得的油浓度准确性高且样品处理简单。基于LF-NMR方法,比较了恒定速率流加补油策略和批补油策略,发现在发酵过程缺油的时候批量补油,促进了盐霉素生产菌的二次生长,维持了产盐霉素能力,从而提高了盐霉素的产量;而恒定速率流加方式补油对提高盐霉素产量没有明显效果。
[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.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ920.6

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