抗氧化剂对Glarea lozoyensis发酵生产纽莫康B0的影响毕业论文
2022-01-28 22:08:16
论文总字数：17610字
摘 要
纽莫康定B0是由丝状真菌Glarea lozoyensis产生的脂肽类化合物，是合成卡泊芬净的中间体。由于过度使用多烯（两性霉素B），唑类药物和氟胞嘧啶引起的真菌感染治疗失败率较高以及临床实践中耐药性问题日益增加，卡泊芬净一经批准用于人类治疗就很流行。纽莫康定B0是通过抑制细胞壁的合成而起到抗真菌感染的效果，由于人类和其他哺乳动物不具有细胞壁，所以此药物不会对人体产生影响，具有副作用小、耐药性小的特点。
在生物氧化反应中，氧是一种必需物质，既有缺点又有优点。一方面，氧是氧化磷酸 化反应的重要中间物；另一方面，氧会随着一系列的化学反应产生对机体有害的氧自由基。活性氧是作为机体内氧化还原反应和各种酶促反应和非酶促反应的副产物出现的。在机体内，低浓度的ROS可以促进细胞增生，高浓度的ROS可导细胞内脂质、蛋白质、DNA受损，以至细胞凋亡或损伤死亡。机体内的抗氧化体统可以保证ROS水平维持动态平衡。
抗氧化剂的添加可以降低过氧化水平，保护细胞免于受损。在发酵过程中添加维生素C可以直接清除部分ROS，还可以激活被ROS抑制的抗氧化酶，以提高总抗氧化能力。
本文以G. lozoyensis菌株为材料。首先考察了添加维生素C、没食子酸、亚麻籽油三种抗氧化剂对G. lozoyensis生产纽莫康定B0的影响。结果表明：维生素C、没食子酸、亚麻籽油都促进了纽莫康定B0的生产。其中当亚麻籽油添加量为0.50 mL/L时，纽莫康定B0产量最高，产量到达最大值396.30 mg/L，相比对照组提高了35.60 %。工业生产中可以添加适量的亚麻籽油以提高纽莫康定B0的产量。但是由于亚麻籽油中含有多种不饱和酸，难以确定发挥作用的关键成分，并且在之前的实验中已发现脂质可以提高纽莫康定B0的发酵产量，因此，接下来的实验中主要研究维生素C对纽莫康定B0发酵产量的影响。维生素C添加量为1.0 g/L时，纽莫康定B0产量最高，为366.58 mg/L，相比对照组提高了25.43 %。结果分析表明，低浓度的维生素C可以发挥抗氧化作用，而高浓度的维生素C则表现出促氧化作用。接下来研究了发酵的不同阶段添加维生素C对纽莫康定B0产量的影响。结果表明：在发酵中段添加维生素C对纽莫康定BO产量的促进作用最明显。
通过对G.lozoyensis菌株发酵过程的研究，找出了最佳抗氧化剂的类型、添加浓度以及添加时间，以求提高纽莫康定B0 的产量。接下来，还要继续研究不同pH、不同温度发酵过程中纽莫康定B0的产量、碳消耗量、各种生物量以及pH的变化。以求找出最佳抗氧化剂的类型及其添加时间、添加量、添加方式，为纽莫康定B0的生产提供更好的发酵环境。
关键词：纽莫康定B0 抗氧化剂 维生素C
ABSTRACT
Pneumocandin B0 is a cell-associated lipopeptide produced by the filamentous fungus Glarea lozoyensis, which is used as an intermediate in the synthesis of caspofungin acetate. Due to the high failure rates during management of fungal infections and increasing resistance problems in clinical practice caused by the overuse of polyenes (amphotericin B), azoles, and flucytosine, caspofungin became popular as soon as it was approved for human therapy. Pneumocandin B0 can inhibit the the synthesis of the cell walls to treat fungal infections.Since humans and other mammals do not have a cell wall, this drug does not affect the human body, and has the characteristics of small side effects and low drug resistance.
In the biological oxidation reaction, oxygen is an essential substance that has both disadvantages and advantages. On the one hand, oxygen is an important intermediate for the oxidative phosphorylation. On the other hand, oxygen will produce active oxygen radicals which are harmful to the body during a series of chemical reactions. Active oxygen appears as a by-product of redox reactions and various enzymatic and non-enzymatic reactions in the body. In the body, low concentration of ROS can promote cell proliferation, and high concentration of ROS can lead to the damage of lipids, proteins and DNA, even lead to the death of cells.The body's antioxidant system ensures that the ROS level maintains its homeostasis.
The addition of antioxidants can reduce the level of peroxidation and protect cells from damage. Adding vitamin C during the fermentation process can directly remove some of the ROS, and can also activate antioxidase that are inhibited by ROS to increase total antioxidant capacity.
In this paper , we use G.lozoyensis strain as material. Firstly, the effects of adding vitamin C, gallic acid and flaxseed oil on the yield of Pneumocandin B0 were investigated. The results showed that all of them promoted the yield of pneumocandin B0. When the amount of flaxseed oil was 0.50 mL/L, the yield of pneumocandin B0 was the highest and the yield reached a maximum of 396.30 mg/L, an increase of 35.60 % compared to the control group.Appropriate amount of linseed oil can be added to industrial production to increase the yield of pneumocandin B0.However, because linseed oil contains a variety of unsaturated acids, it is difficult to determine which of them play the key role , and in previous experiments it has been found that lipids can increase the yield of pneumocandin B0, so the main research in the following experiments is the effect of vitamin C on the yield of pneumocandin B0.When vitamin C was added at 1.0 g/L, the yield of pneumocandin B0 was the highest at 366.58 mg/L, which was 25.43% higher than that of the control group.The results show that low concentration of vitamin C can exert antioxygenation effects, while high concentration of vitamin C appears to prooxidation effects. Because the composition of flaxseed oil is difficult to analyze, the effect of adding vitamin C at different stages of fermentation on the yield of pneumocandin B0 was studied. The results showed that the addition of vitamin C in the middle stage of the fermentation had the most obvious effect on the growth of the pneumocandin B0.
By studying the fermentation process of G.lozoyensis strain, We have found the type, concentration and time of the best antioxidants to increase the yield of pneumocandin B0. Next, we need continue to study the changes in yield, carbon consumption, various biomass, and pH of Pneumocandin B0 during fermentation at different pH and temperature. This is to find the best type of antioxidant and its addition time, amount and method , to provide a better fermentation environment for pneumocandin B0.
Keywords: Pneumocandin B0 antioxidants Vitamin C
目 录
目 录 1
第一章 文献综述 3
1.1 纽莫康定简介 3
1.1.1抗生素 3
1.1.2 棘白菌素类抗生素 3
1.1.3 纽莫康定B0发酵生产的局限性 4
1.2 活性氧（ROS） 4
1.3 活性氧在生物发酵过程中的应用 6
1.4 抗氧化剂 6
1.5 研究意义 6
第二章 实验材料 8
2.1 菌种 8
2.2 主要实验试剂 8
2.3 主要实验仪器 9
2.3 培养基 10
第三章 实验方法 11
3.1 培养方式 11
3.2 检测方法 11
3.2.1 纽莫康定B0的检测方法 11
3.2.2 生物量检测 11
3.2.3 糖耗检测 11
3.2.4 活性氧检测（ROS） 12
3.2.5 总抗氧化能力检测（T-AOC） 12
3.2.6 超氧化物歧化酶检测（SOD） 12
3.2.7 过氧化物酶检测（POD） 13
3.2.8 过氧化氢酶检测（CAT） 13
第四章 实验结果与讨论 14
4.1 不同抗氧化剂对纽莫康定B0发酵产量的影响 14
4.2 发酵不同阶段添加抗氧化剂对纽莫康定B0产量的影响 16
4.3 考察发酵中期维生素C添加对纽莫康定B0产量影响的原因 17
第五章 结论与展望 19
5.1 结论 19
5.2 展望 19
参考文献 20
致谢 22
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