摘要:探究酵母摇瓶发酵过程中辅因子NADH/NAD+的主要影响因子。通过单因素实验探讨各因素对辅酶提取得率的影响。在此基础上,采Plackett-Burman 设计对影响酵母细胞内辅因子NADH/NAD+提取得率的因素进行筛选,PB 试验设计与统计学分析表明,超声时间、提取液的加入量和震荡次数是影响酵母细胞内辅因子NADH/NAD+提取得率的4个关键因素。通过拟合得到响应曲面函数,获得了最佳的提取条件为:超声功率:300瓦、超声时间:20min、提取液的加入量:3ml、震荡次数:10次,在此条件下酵母细胞内辅因子NADH/NAD+的提取得率最高,这表明从实验结果可以看出单因素法、Plackett-Burman试验设计结合响应面分析法,可以很好地优化酵母细胞内辅因子NADH/NAD+的提取工艺。4924
关键词:PB设计;响应面优化;NADH/NAD+;超声波;高效液相色谱
Detection of cofactor NADH / NAD + during the process of biotransformation of  producing 2 – Phenylethanol
   Abstract :The research aimed to explore main influencing factors of extraction NADH / NAD + by yeast in shaking flask fermentation. Single factor test of all kinds of factors for the extraction rate of NADH / NAD + produced by yeast. On this basis, Plackett- Burman (PB) design were applied to screen and optimize influential factors for extraction process of total flavonoids . The ultrasonic time, the amount of added extracts, number of shocks, as three key factors, were found to significantly influence the yields of extraction NADH / NAD + by yeast via PB design and the following statistic analysis, With the hybrid design, response surfaces were optimized and response surfaces function was obtained. Three statistically significant parameters were ultrasonic power:300W、ultrasonic time :20 min、the amount of added extracts:3mL、numbers of shocks:10 times. Under the optimal levels of the extraction conditions the content of NADH / NAD + was the highest. It can be seen from the experimental results show that Single factor test、Plackett-Burman (PB) design with the response surface methodology can optimize the extraction process of cofactor NADH / NAD + in yeast cells .

key words:Plackett–Burman design; response surface methodology; Ultrasonic NADH / NAD + ; HPLC
                     
目录

1前言    1
1.1国内外研究进展    1
1.2酵母细胞内辅因子NAD+、NADH代谢机制    2
1.3辅因子对发酵过程的影响    3
2 材料与方法    4
2.1材料与仪器设备    4
2.1.1仪器设备    4
2.1.2培养基及实验试剂    4
2.2方法    5
2.2.1实验方法    5
2.2.2分析方法    6
3实验条件优化    6
3.1单因素实验    6
3.1.1.细胞破碎法    6
3.1.2提取液配比    8
3.1.3 超声功率    8
3.2 Plackett-Burman实验设计筛选关键因素    9
3.3相应面实验设计    9
3.4 NAD+/NADH检测条件优化    10
3.4.1 高效液相色谱法    10
3.4.2 酶循环法    12
3.4.3 试剂盒法    13
4结果与分析    14
4.1单因素结果分析    14
4.1.1.细胞破碎法    14
4.1.2 提取液配比    15
4.2 PB设计结果分析    17
4.3响应面结果分析    19
5.实验结果分析    22
文献引用    23
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