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摘要：植物乳杆菌原生质体制备和恢复的较优条件为，菌龄8 h，酶浓度3 mg/ml，酶解时间为30 min，酶解温度为37℃，此时原生质体形成率为52.5%，再生率为79.7%。将同属于乳酸菌的短乳杆菌和植物乳杆菌分别制成原生质体悬液，融合3 min后涂布长出菌株，经过筛选得到一株突变株R4，其降解NaNO2比出发菌株降解NaNO2能力提高了39.5%。优化培养基后，测得亚硝酸还原能力，比优化前菌降解NaNO2能力量提高了2.8%。将融合菌株进行紫外诱变，获得紫外诱变后突变株，筛选后得到突变株Z2的降解能力比出发菌株的提高了63.2%。将已构建的具有降解亚硝酸盐能力的工程菌Z2，放于含有亚硝酸盐的泡菜汁中进行应用，发现该菌株比出发菌株降解能力更有强。23820
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毕业论文关键词：原生质体；制备；再生；融合；培养基优化；紫外诱变；应用
Study on Breeding Of Nitrite Degradation Strain
Abstract：Optimal conditions for preparation of Lactobacillus plantarum and recovery of protoplast: age of 8 h, 3 mg/ml enzyme concentration, hydrolysis time was 30 min, enzymolysis temperature is 37 ℃, the protoplast formation rate is 52.5%,the regeneration rate is 79.7%. Then, Lactobacillus brevis and Lactobacillus plantarum are made of protoplast fusion of suspension, after 3 min coating long strain, having a strain of mutant R4 after screening, the increased by 39.5% than the NaNO2 degradation of the starting strain. Optimization of culture medium, the content of NaNO2 degradation is increased by 2.8% than that before optimization bacteria. The fusion strain by UV mutation, after screening, degradation of NaNO2 in mutant Z2 is increased by 63.2% than that of starting strain, the degradation activity was the strongest. The constructed has the ability of nitrite degradation engineering bacteria Z2, putting in the pickled cabbage juice containing nitrite in application, showed that mutant Z2 have the more effectively degradation ability than the starting strain
Key Words: Protoplasts; Preparation; Regeneration; Fusion; Medium optimization; UV mutagenesis; Application
目录
1 绪论 1
引言 1
1.1 食品中亚硝酸盐的主要来源 1
1.1.1 蔬菜中亚硝酸盐的来源 1
1.1.2 肉制品中亚硝酸盐的来源 1
1.2 亚硝酸盐的危害 2
1.2.1 亚硝酸盐急性中毒 2
1.2.2 亚硝酸盐致癌作用 2
1.3 减少亚硝酸盐危害的方法 2
1.3.1 生物降解法 2
1.3.2 酶处理法 2
1.4 研究现状及前景 3
1.4.1 研究现状 3
1.4.2 展望 3
1.5 本课题的研究目的和意义 4
1.6 本课题研究的主要内容 4
2 实验材料和方法 4
2.1 实验材料 4
2.1.1 菌种 4
2.1.2 试剂 4
2.1.3 仪器 5
2.1.4 培养基 5
2.2 试验方法 6
2.2.1 形态观察 6
2.2.2 乳酸菌的培养 6
2.2.3 菌体生长曲线的绘制 6
2.2.4 发酵液亚硝酸盐降解曲线的绘制 6
2.2.5 降解亚硝酸盐能力测定 7
2.2.6 原生质体的制备、再生及融合 7
2.2.7 紫外诱变 8
2.2.8 融合子的发酵条件优化 9
2.2.9 诱变后的菌株在降解亚硝酸还原盐中的应用 9
3 实验结果与分析 10
3.1预实验 10
3.1.1 植物乳杆菌的形态观察 10