发酵工程硕士论文.doc

发酵工程硕士论文


谷胱甘肽发酵条件的优化扩大及其分离纯化

英文题目：  The Optimization and Amplification of Fermentation
Conditions of Glutathione and its Separation and Purification

申请学位学科专业：发酵工程

摘   要
谷胱甘肽(GSH)是由谷氨酸、半胱氨酸和甘氨酸组成的三肽化合物，具有清除自由基、解毒、抑制衰老、预防糖尿病和癌症、抑制艾滋病毒、提高人体免疫力等重要生理功能，在医学、食品、化妆品等领域具有广泛的市场前景。现有GSH生产能力远不能满足市场需求，所以采用GSH高产菌株发酵廉价原料转化为GSH，建立适合工业化分离纯化GSH的方法会带来巨大的社会和经济效益。
本文以富含GSH的啤酒酵母Y-14为材料，分别从以下四个方面进行研究：
(1) 利用Plackett-Burman 试验设计、Box-Behnken试验设计法对啤酒酵母Y-14产GSH培养基进行优化，得到的最适培养基配方为: 葡萄糖20g/L，酵母膏10g/L，(NH4)2SO4 8g/L，KH2PO4 3g/L，MgSO4 0.15g/L。对优化结果进行验证试验，GSH产量可达到124.77mg/L。
(2) 利用单因素试验对啤酒酵母Y-14产GSH发酵条件进行研究，结果得到摇瓶最适发酵条件为培养温度30℃、初始pH6.0、摇床转速180rpm、摇瓶装液量30mL/300mL、补料培养基添加时间20h。在此条件下，菌体胞内GSH含量达到31.56mg/g，GSH的产量可达249.2mg/L，较原来提高了102%。
(3) 利用10L自动发酵罐补料培养啤酒酵母Y-14，①采用恒速流加单一葡萄糖培养基，20h时添加前体氨基酸和ATP的方法，测得GSH的产量在52h时，达到最大值243mg/L。②采用恒速流加全营养培养基，36h时添加补料培养基的方法，测得GSH产量在52h达到最大值520.742mg/L。比较发现，第二批发酵罐培养菌体干重、GSH产量较第一批分别提高了122.5%、114.2%。
(4) 采用D001大孔树脂粗分离GSH，得到最佳分离条件为：上样pH3.0，浓度为61.4mg/100mL，流速为0.5mL/min，洗杂质用液为无水乙醇，洗脱液为1.5% HCl + 0.3mol/L NaCl，流速为3mL/min。GSH的平均收得率为69.11%，蛋白质去除率为64.8%。然后通过SephadexG-10脱盐纯化GSH，得到最适条件：Φ1.6cm×25cm玻璃层析柱，上样浓度为116.66mg/100mL，上样体积为20mL。结果GSH的平均收得率为96.21%。最后SephadexG-10分级分离纯化GSH，采用Φ1.6cm×50cm玻璃层析柱，最适上样体积为2mL，GSH的平均收得率为99.26%，蛋白质去除率为56.5%。GSH浓缩倍数平均为9.07倍，纯化倍数为8.67倍。
关键词： 啤酒酵母Y-14，谷胱甘肽，发酵罐，D001大孔树脂，SephadexG-10
 
Abstract
Glutathione (GSH) is a tripeptide formed by glutamic acid, cysteine and glycin, it has important physiological functions such as removing free radical, detoxification,  anti-aging, diabetes and cancer prevention, inhibition of HIV and improve the effectiveness of the human immune system, and has a wide range of market prospects in the fields of medical, food and cosmetics. But the existing production capacity of GSH is far from the market demand.Use microbial fermentation to translate cheap raw materials into high yielded GSH, and establish suitable industrialization separation and purification of GSH will bring enormous social and economic benefits. In this paper, the main research content and results are as follows:
(1) Using Plackett-Burman and Box-Behnken experimental design optimized medium of Saccharomyces cerevisiae Y-14 which producing GSH. The optimum medium was glucose 20g/mL, yeast extract 10g/mL, (NH4)2SO4 8 g/mL, KH2PO4 3 g/mL, MgSO4 0.15 g/mL. The optimum experiment showed that GSH yield reached to 124.77mg/L.
(2) Using single factor test studied the fermentation conditions of yeast Y-14 producing GSH. The optimum shake-flask fermentation conditions was culture temperature 30 ℃, initial pH6.0, shake speed 180rpm, liquid volume 30mL / 300mL, medium batch add time 20h. GSH content reached to 31.56mg/g, GSH yield reached to 249.2mg/L, increased 102% compare to the original.
(3) Using 10L automated fermentor, ① using single constant speed fed glucose medium, added precursors of amino acids and ATP at 20h, and the GSH yield reached to maximum 243mg /L at 52h. ② using constant flow increases total nutrient medium, added fed medium at 36h , GSH yield maximum reached to 520.742mg/L. It showed that dry cell weight and GSH yield increased 122.5%and 114.2% respectively compared to the first fermentor cultivation.
    (4) Using D001 macroporous resin rough to separate GSH, the optimum separation conditions was:sample pH3.0, sample concentration was 61.4mg/100mL, sample flow rate of 0.5mL/min, impurities washing liquid was absolute alcohol, eluate was 1.5% HCl and 0.3 mol/L NaCl, elution flow rate was 3mL/min, the average yield of GSH was 69.11%, protein removal rate was 64.8%. Then using Sephadex G-10 to desalt and purify GSH, the optimum conditions was:Φ1.6cm×25cm chromatography columns, sample concentration was 116.66mg/100mL, sample volume was 20mL. The average yield of GSH was 96.21%. In the end,using Sephadex G-10 to purify GSH, using Φ1.6cm × 50cm chromatography columns, the optimum sample volume was 2mL, the average yield of GSH was 99.26%, protein removal rate was 56.5%. The average of concentration multiple and purification multiple was 9.07 and 8.67 respectively.
Keywords：Saccharomyces cerevisiae Y-14, Glutathione, Fermentor, D001 macroporous resin,  Sephadex G-10
 
目   录
摘   要 I
Abstract II
目   录 III
第1章 引 言 1
1.1 谷胱甘肽概述 1
1.1.1 谷胱甘肽结构和性质 1
1.1.2 谷胱甘肽的生理功能及其应用 3
1.2 发酵法生产谷胱甘肽的国内外研究动态 4
1.3 本研究的意义、目的及内容 9
1.3.1 意义 9
1.3.2 目的 9
1.3.3 研究内容 10
1.4 常用研究指标的说明 10
第2章 啤酒酵母Y-14发酵GSH培养基的优化 11
2.1 材料 11
2.1.1 菌种： 11
2.1.2 培养基： 11
2.1.3 主要试剂 12
2.1.4 主要仪器 12
2.2 方法 12
2.2.1 菌株活化 13
2.2.2 种子摇瓶培养 13
2.2.3 摇瓶发酵培养 13
2.2.4 菌种的保藏 13
2.2.5 DTNB法检测GSH含量 13
2.2.6 试验设计方法 13
2.3 结果与分析 14
2.3.1 显著影响因素的筛选结果 14
2.3.2 Box-Behnken设计进一步优化培养基试验结果 16
2.4 结论 19
第3章 啤酒酵母Y-14产GSH发酵条件的优化 20
3.1 材料 20
3.1.1 菌种： 20
3.1.2 培养基： 20
3.1.3 主要试剂 21
3.1.4 主要仪器 21
3.2 方法 21
3.2.1 啤酒酵母Y-14生长曲线的测定 21
3.2.2 发酵周期的优选试验 21
3.2.3 发酵温度的优选试验 21
3.2.4 发酵初始pH的优选试验 22
3.2.5 发酵摇床转速的优选试验 22
3.2.6 发酵摇瓶装液量的优选试验 22
3.2.7 摇瓶发酵产GSH的进程试验 22
3.3 结果与分析 22
3.3.1 啤酒酵母Y-14生长曲线的绘制 22
3.3.2 发酵周期的优选试验结果 23
3.3.3 发酵温度的优选试验结果 24
3.3.4 发酵初始pH值的优选试验结果 24
3.3.5 发酵摇床转速的优选试验结果 25
3.3.6 发酵摇瓶装液量的优选试验结果 26
3.3.7 摇瓶发酵产GSH进程曲线的绘制 27
3.4 结论 28
第4章10L自动发酵罐扩大发酵的初探 29
4.1 材料 29
4.1.1 菌种： 29
4.1.2 培养基 29
4.1.3 主要试剂 30
4.1.4 主要仪器 30
4.2 方法 30
4.2.1 斜面种子培养 30
4.2.2 一级种子摇瓶培养 31
4.2.3 二级种子摇瓶培养 31
4.2.4发酵罐流加培养试验 31
4.2.5 分析方法 31
4.3 结果与讨论 32
4.3.1 第一批发酵罐补料培养 32
4.3.2 第二批发酵罐补料培养 36
4.4 结论 40
第5章 啤酒酵母Y-14中还原型GSH的分离纯化 41
5.1 材料 41
5.1.1 菌种 41
5.1.2 主要材料 42
5.1.3 主要设备 42
5.2 方法 42
5.2.1 GSH提取液的制备 42
5.2.2 检测方法 42
5.2.3 相关数据的计算公式 42
5.2.4 D001大孔树脂离子交换柱的试验流程 43
5.2.5 D001大孔树脂分离GSH的研究 43
5.2.6 SephadexG-10凝胶柱的试验流程 44
5.2.7 SephadexG-10纯化GSH的研究 45
5.3结果与分析 46
5.3.1 D001大孔树脂分离GSH的研究结果 46
5.3.2 SephadexG-10脱盐纯化GSH的研究结果 50
5.3.3 SephadexG-10分级分离纯化GSH的研究结果 53
5.4 结论 56
第6章 总结与展望 57
6.1 总结 57
6.2 创新点 58
6.3 展望 58
参考文献 59
致  谢 64