丁酸梭菌培养工艺及方法研究

发布时间：2018-06-22 20:23

  本文选题：丁酸梭菌 + 培养工艺　； 参考：《湖北工业大学》2017年硕士论文

【摘要】：丁酸梭菌,即丁酸梭状芽孢杆菌,又名酪酸菌、宫入菌,是存在于人和畜禽肠道的一种厌氧益生菌,可以改善宿主肠道细菌生态系统的平衡,对治疗因各种原因所致的肠道疾病有很好的效果。除此之外,丁酸梭菌对提高猪、鸡、牛、羊等肉质也有一定的效果,因此被作为微生态制剂广泛应用于医药领域和饲料行业当中。丁酸梭菌在使用过程中主要以活菌制剂状态存在,因此本文以提高丁酸梭菌菌体浓度、芽孢生成率为主要目标,利用传统发酵培养方法以及结合数学分析软件优化丁酸梭菌的发酵培养工艺。由于丁酸梭菌属于严格厌氧菌,所以在此过程中还探究了一种利用Fe-C原电池除氧方法来培养丁酸梭菌。1.采用数学分析方法对提高丁酸梭菌生物量的发酵工艺进行了优化。在单因素实验的基础上利用Plackett-Burman实验设计筛选出影响菌体浓度的显著性因素。之后,运用最陡爬坡实验找出中心组合实验(Central Compose Design,CCD)的中心点,最后通过响应面法分析获得最佳发酵培养基组成成份。结果表明:酵母浸粉、FeSO_4和K_2HPO_4为影响菌体量的3个显著性因素。最终确定可溶性淀粉2%、酵母浸粉6%、FeSO_4 1.74%、K_2HPO_4 0.37%、NaCl 0.2%、MgSO_4 0.024%为最佳培养基组分。最佳培养条件为:温度34℃,pH7.0,接种量为4%。优化后的菌体数可达1.01×109个/m L,与优化前(2.3×108个/mL)相比,提高了4.39倍。2.在提高芽孢率过程中发现乳糖为最佳碳源,并以芽孢中2,6-吡啶二羧酸(2,6-Pyridine Dicarboxylic Acid,DPA)的含量为检测指标来测定芽孢的数量。在此基础上,利用Plackett-Burman实验设计筛选出乳糖、MgSO_4为影响芽孢生成率的两个显著因素,用最陡爬坡路径逼近最大DPA浓度区域后,利用响应面中心组合设计对显著因素进行优化,结果表明乳糖、MgSO_4最佳添加量分别为0.3%、0.035%。优化后培养基组分为乳糖0.3%、MgSO_4 0.035%、酵母浸粉1.0%、NaHCO_30.2%、K_2HPO_4 0.3%、KH_2PO_4 0.1%。最佳培养条件为温度43℃、pH7.0,接种量为2%。优化后的DPA含量为15.16μM,与优化前(2.02μM)相比提高了7.5倍,换算成芽孢数后为6.31×107个/mL,芽孢转化率为60%。3.探究了Fe-C原电池系统对丁酸梭菌生长的影响。首先确定了Fe-C原电池系统的最佳组成成份:Fe粉0.5%,C粉0.3%。结果表明:Fe-C原电池系统可以维持丁酸梭菌培养液长时间处于一个较低的的氧化还原电位环境下。与对照相比(无Fe-C系统),丁酸梭菌的最大比生长速率从(h-1)0.23提高到0.43,细胞得率(g cell/g glucose)从0.10提高到0.14,最终生物量提高从0.71g/L提高到了1.31g/L。相反,有机酸(丁酸和乙酸)的比生产率都有一定的下降:丁酸比生产速率(g/g glucose·h)从0.15下降到0.10;乙酸比生产速率(g/g glucose·h)从0.28下降到0.18。
[Abstract]:Clostridium butyrate, also known as Clostridium butyricum, is a kind of anaerobic probiotics that exists in human and animal intestines, which can improve the balance of intestinal bacterial ecosystem. It has a good effect on the treatment of intestinal diseases caused by various causes. In addition, Clostridium butyrate can improve meat quality of pigs, chickens, cattle and sheep, so it is widely used in medicine and feed industry. Clostridium butyrate mainly exists in the form of living bacteria in the process of use. Therefore, the main aim of this paper is to increase the concentration of Clostridium butyrate and the sporulation rate. The fermentation culture technology of Clostridium butyrate was optimized by traditional fermentation method and mathematical analysis software. Because Clostridium butyrate belongs to strict anaerobes, a method of deoxidizing Clostridium butyrate by Fe-C primary battery has been explored in this process to culture Clostridium butyrate. The fermentation process for improving the biomass of Clostridium butyrate was optimized by mathematical analysis. On the basis of single factor experiment, Plackett-Burman experiment was used to screen the significant factors affecting the bacterial concentration. After that, the center point of the Central composition Design CCD was found out by the steepest climbing experiment, and the optimum composition of the fermentation medium was obtained by response surface analysis. The results showed that FeSOs _ 4 and K _ 2HPOs _ 4 were the three significant factors affecting the biomass. Finally, soluble starch 2 was determined, and yeast extract powder 6 / FeSOs 41.74 + K2HPO4 0.37 + NaCl 0.2% MgSO4 0.024% was the best medium component. The optimum culture conditions were as follows: temperature 34 鈩，

本文编号：2054121