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摘要：世界上因为亚硝酸盐残留超标而引发的卫生问题、安全事故时有发生,因此控制亚硝酸盐含量、降低亚硝酸盐的方法成为了食品安全的重中之重。本课题选用短乳杆菌（ Lactobacillus brevis）作为菌种，首先对短乳杆菌的生长特性进行研究，选取稳定期12h的培养液作为诱变用菌种；加入亚硝酸钠6小时后的亚硝酸盐降解量作为判别菌株能力的依据。其次，选择120s为紫外诱变照射时间，经紫外诱变选育后，获得突变株亚硝酸盐降解量为982.76nmol/ml，比出发菌株的降解量提高了29.5%。再次选用40min处理时间进行硫酸二乙酯诱变，获得突变株的降解量为1134.54nmol/ml，经紫外线和硫酸二乙酯诱变后得到的菌株比出发菌株降解能力提高了近45%。最后经过PCR鉴定验证得到的条带与文献中短乳杆菌相符。22481
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毕业论文关键词： 短乳杆菌；亚硝酸盐；亚硝酸还原酶；紫外诱变；硫酸二乙酯诱变
Study on Mutagenesis and Identification of Nitrite Reductase
Abstract: Caused by excessive nitrite, the problems of health and safety accidents occur frequently in the world. Therefore, methods to control and reduce the content of nitrite has become a top priority to food security . We used Lactobacillus brevis in this paper, and we studied the growth characteristics of Lactobacillus brevis. We selected to culture the Lactobacillus brevis 12H for the next experiment, and we used the amount of nitrite degradation after adding sodium nitrite 6 hours as the basis of distinguishing strain’s capacity. Secondly, we chose 120s as the UV irradiation time, The amount of nitrite degradation of mutant strain was 982.76nmol/ml，which increased 29.5% than that of the original strain. Next, we selected 40min for diethyl sulfate mutagenesis, the amount of nitrite degradation of mutant strain was 1134.54nmol/ml. After UV and diethyl sulfate mutagenesis ,the amount of nitrite degradation of mutant strain was almost 45% higher than the starting strain. Finally, the band from PCR identification proved to be consistent with Lactobacillus brevis in the literature.
Keywords: Lactobacillus brevis;nitrite;Nitrite reductase;UV mutagenesis; diethyl sulfate mutagenesis
目 录
1. 引言 1
1.1 亚硝酸盐简介 1
1.2 亚硝酸盐的主要来源 1
1.2.1 来源于蔬菜中的亚硝酸盐 1
1.2.2 来源于水中的亚硝酸盐 2
1.2.3 来源于肉制品中的亚硝酸盐 2
1.3亚硝酸盐国内外研究现状 2
1.3.1 亚硝酸盐的危害 2
1.3.2目前国内外对于亚硝酸盐的降解方法 3
1.4本课题研究的目的和意义 5
1.4.1研究的目的 5
1.4.2研究的意义 5
1.5研究的主要内容 6
2.实验所用菌种、材料和方法 6
2.1实验菌种 6
2.2实验材料 6
2.3培养基 7
2.3.1MRS固体培养基 7
2.3.2种子培养基 7
2.3.3MRS液体培养基——发酵产酶培养基 7
2.4实验设备 7
3.实验方法 8
3.1亚硝酸盐标准曲线 8
3.1.1标准曲线所需试剂 8
3.1.2测定亚硝酸钠标准曲线——盐酸萘乙二胺法[3] 9
3.2菌种特性研究 9
3.2.1革兰氏染色镜检 9
3.2.2菌体生长曲线 9
3.2.3亚硝酸盐降解曲线 9
3.3 紫外诱变 10
3.3.1 悬浮液的制备[22-23] 10