微生物法制备还原型谷胱甘肽的工艺研究

发布时间：2018-04-15 04:07

  本文选题：还原型谷胱甘肽 + 发酵　； 参考：《大连工业大学》2011年硕士论文

【摘要】：还原型谷胱甘肽(reduced glutathione, GSH)是由L-谷氨酸、L-半胱氨酸和甘氨酸缩合而成的一种含有巯基的生物活性三肽化合物,是人体细胞内的主要代谢物质,具有清除自由基、解毒、促进肝酶活性等生理功能。在食品工业中,GSH亦可起到增强肉类风味、预防色素沉着及防止水果褐变等作用。日本等一些发达国家很早就开始研究GSH且基本垄断了中国市场,我国主要是以微生物发酵法生产GSH,亟需对其工艺进行优化改进以提高GSH的产率。本文从中科院沈阳应用生态研究所菌种保藏室的酵母菌库中筛选得到一株产GSH能力较高的酿酒酵母Y,其GSH产量达9.60 mg/g。改进发酵培养基配方以促进菌体生长和提高GSH产量,采用添加L-半胱氨酸和补加葡萄糖的策略,使得菌体浓度提高12.4倍,胞内GSH产量达12.94 mg/g干菌体。选用热水抽提法和乙醇提取法提取胞内GSH,通过单因素实验和正交实验对pH值(A)、提取温度(B)、提取时间(0、料液比(D)4个因素进行优化,并对两种方法的提取效率和提取条件进行比较,确定最佳提取方法为热水抽提法,提取条件：料水比1：3,提取液pH 2.0,90℃水浴中提取10min, GSH的提取量可达14.27 mg/g。为了提高GSH纯度,采用离子交换和葡聚糖凝胶层析技术分离纯化提取液中的GSH。通过静态实验优化分离条件：D0017阳离子交换树脂,提取液上柱pH 4.5,用pH 7.0磷酸氢二钠-柠檬酸盐缓冲液+03 mol/L NaCl进行洗脱,将最优条件应用于上柱分离实验中,柱高15 cm,洗脱流速2.0 mL/min, GS H回收率为91.46%。将GSH浓度大于250μmol/L的部分进行冷冻干燥,浓缩后上Sephadex G-10凝胶层析柱,再次纯化,最佳纯化条件为：柱高45 cm,上柱量为柱体积10%,去离子水洗脱,流速为10 mL/min,GSH回收率为86.18%,杂蛋白去除率为87.6%。由实验结果可知,热水抽提法提取GSH耗时短、无污染、提取彻底,用离子交换法和葡聚糖凝胶层析分离纯化GSH效果好、操作简便、能较好的去除杂蛋白,有利于工业化生产。
[Abstract]:Reduced glutathione glutathione (GSH) is a bioactive tripeptide compound containing sulfhydryl group, which is formed by condensation of L-glutamic acid L-cysteine and glycine. It is the main metabolite in human cells and has the ability to scavenge free radicals and detoxify.Promote liver enzyme activity and other physiological functions.GSH can also enhance meat flavor, prevent pigmentation and prevent fruit browning in food industry.Some developed countries, such as Japan, have been studying GSH for a long time and have basically monopolized the Chinese market. In our country, the production of GSH is mainly by microbial fermentation, so it is necessary to optimize and improve the process to increase the yield of GSH.A Saccharomyces cerevisiae Y. with high GSH production capacity was obtained from the yeast bank of Shenyang Institute of Applied Ecology, Shenyang Institute of Applied Ecology, Chinese Academy of Sciences. Its GSH yield was 9.60 mg / g.The fermentation medium formulation was improved to promote the growth of bacteria and increase the GSH yield. By adding L- cysteine and glucose, the cell concentration was increased by 12.4 times, and the intracellular GSH production reached 12.94 mg/g dry bacteria.The intracellular GSH was extracted by hot water extraction and ethanol extraction. The extraction efficiency and extraction conditions of the two methods were compared by single factor experiment and orthogonal experiment. The four factors of pH value, extraction temperature, extraction time and ratio of material to liquid were optimized by single factor experiment and orthogonal experiment.The optimum extraction method is hot water extraction. The extraction conditions are as follows: the ratio of material to water is 1: 3, the pH value of the extract is 2. 0 ~ 90 鈩，

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