亚硝酸盐还原酶高产菌选育及发酵研究

摘要：亚硝酸盐在自然界中广泛存在，是潜在的致癌物质。因此，亚硝酸盐在食品中过量存在问题已引起世界各国科学界极大关注。植物乳杆菌是一种益生菌，可产亚硝酸盐还原酶，利用生物酶解法降解亚硝酸盐是一种安全有效的新方法。本论文利用实验室保存的植物乳杆菌菌种，研究其生长特性，并利用紫外线诱变、硫酸二乙酯诱变获得的高产菌株通过单因素与正交优化实验来优化发酵培养基配方。主要研究成果如下：通过对紫外诱变突变株的筛选，得到亚硝酸盐还原酶活力较高的突变株D8，其酶活力为125.0U，比出发菌株的酶活力提高了22.9%。通过对硫酸二乙酯诱变突变株的筛选，得到亚硝酸盐还原酶活力较高的突变株DS2，其酶活力为160.0U，比出发菌株的酶活力提高了28.0%。通过正交优化的实验结果得到3个因素最优参数值分别为葡萄糖22.0 g/L；牛肉膏 11.0 g/L；硫酸镁0.5 g/L。最后验证实验得到的酶活力为228.3U，比优化前高出13.8%。8820
关键词：植物乳杆菌；亚硝酸盐还原酶；诱变；正交
Nitrite reductase high yield breeding and fermentation research
Abstract：Nitrite is a potential carcinogen which can be widely found in nature. Therefore, the problem of nitrite excessive in food has caused great concern for the scientific community. L.plantarum is the human probiotic that can produce nitrite reductase. Enzymatic degradation of nitrite is a safe and effective new way .In this paper, we used a strains which saved by the laboratory to study the growth characteristics, and use of UV and diethyl sulfate mutagenesis to obtain high yield strain. Mutation breeding high-yield strains by single-factor orthogonal optimization experiment to optimize the fermentation medium. The study is as follows: by UV mutagenesis mutant screening, the high nitrite reductase producing mutant D8 was obtained and its nitrite reductase activity was 125.0U, which was increased by 22.9% as compared with the staring stain. By diethyl sulfate mutagenesis mutant screening, the high nitrite reductase producing mutant DS2 was obtained and its nitrite reductase activity was 160.0U, which was increased by 28.0% as compared with the mutant D8. The response surface optimization of the experimental results of three factors optimal parameter values were glucose 22.0 g / L; beef extract, 11.0 g / L; magnesium sulfate 0.5 g / L. Finally the enzyme activity of the verification experiment was 228.3U, 13.8% higher than before.
Key word: Lactobacillus plantarum; nitrite reductase; mutagenesis; orthogonal
目录
1 绪论   1
1.1 亚硝酸盐的简介   1
1.1.1 亚硝酸盐在食物中的来源   1
1.1.2 亚硝酸盐的危害   2
1.2 微生物诱变育种的简介   3
1.2.1 紫外线——物理诱变   3
1.2.2 硫酸二乙酯——化学诱变   4
1.2.3 微生物诱变育种的应用   4
1.3 国内外研究现状   4
1.3.1 亚硝酸盐的降解与去除的研究进展   4
1.3.2 亚硝酸盐还原酶研究进展   6
1.4 本课题的研究目的和意义   7
1.5 研究内容   7
2 材料与方法   8
2.1材料   8
2.1.1 实验菌种   8
2.1.2 实验试剂   8
2.1.3 培养基   9
2.1.4 实验设备   10
2.2 方法   10
2.2.1 菌体生长曲线的绘制   10
2.2.2 亚硝酸盐降解曲线的绘制   10
2.2.3 亚硝酸盐测定标准曲线的建立   11
2.2.4 亚硝酸盐含量测定   11
2.2.5 酶活力的测定及计算方法   12 亚硝酸盐还原酶高产菌选育及发酵研究:http://www.youerw.com/shengwu/lunwen_7296.html