压力对谷胱甘肽生物合成的影响及其分离提取工艺研究
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摘要
温和压力(0.1-5.0MPa)条件下,酵母菌受到刺激后发生应激性反应。谷胱甘肽(GSH)是很多生物体受环境胁迫产生的应激性代谢产物之一。GSH作为一种重要的药物,在临床上的用途极广,GSH的抗氧化性又能使得它在食品加工中倍受青睐。本文研究的主要内容为压力条件下面包酵母CICC1447积累GSH最佳条件的确定；加压、常压下酵母生物合成GSH代谢流的变化；温和压力对细胞膜通透性的影响；GSH分离提取工艺研究。主要结果如下：
1.面包酵母CICC1447生物合成GSH加压条件的确定
以高纯空气为加压介质,通过单因素实验对面包酵母CICC1447生物合成GSH加压条件进行优化,确定了最佳条件：加压时间6h、压力0.5MPa、升降压速率0.05-0.10MPa、加压前培养20h为最优加压条件,GSH含量较常压提高了17.21%。
2.面包酵母CICC1447生物合成GSH代谢通量分析
利用代谢通量分析手段研究比较了加压、常压条件下的面包酵母CICC1447生物合成GSH代谢流变化,并通过对代谢通量分布情况的分析,有效地解释了加压提高GSH合成能力的原因。
3.温和压力对面包酵母CICC1447、CICC1339细胞膜通透性的影响
以CO2和N2为加压介质,考察了温和压力(0.1-1.0MPa)及加压时间对面包酵母CICC1447、CICC1339细胞膜通透性的影响。结果表明,在压力条件下面包酵母CICC1447细胞悬浮液的电导率、OD260和OD280显著增加,虽然上升后有些波动,但仍高于常压对照组；压力作用下面包酵母CICC1339细胞悬浮液电导率、OD260和OD280随着压力的增加显著升高。以N2为加压介质,通过对加压(1.0MPa)、常压下PI与DNA结合产生的荧光强度的测定,更有效的说明了温和压力可以显著增强细胞膜的通透性,促进物质的传递。
4.GSH分离提取工艺研究
(1)利用沸水浴抽提法、微波辅助提取法、超声波破碎法和高压均质破碎法对胞内GSH进行抽提,其中高压均质破碎法效果最好,在最佳操作条件下GSH抽提量为3.975mg/g,抽提率达到99.87%。
(2)利用超滤技术去除GSH提取液中的蛋白质,考察了操作压力、温度、提取液pH及料液浓度对超滤的影响。确定了适宜的操作条件：压力为0.04MPa,pH为3,温度为20-25℃,料液不稀释。在此操作条件下GSH损失率相对较小,为25.4%,蛋白质截留率达到46.6%。通过稀释过滤方法有效的提高了GSH回收率,通过稀释1次,使得GSH回收率提高到89.6%。并且通过超滤和稀释过滤方法有效地减少了GSH提取液中细胞色素的含量。
(3)利用离子交换装置对GSH进行分离操作,确定了最佳的上柱pH为3,上柱流速为4mL/min,共上柱1.00L,GSH吸附率达到86.19%,蛋白质吸附率为49.73%,以盐酸为洗脱剂,洗脱浓度为lmol/L,洗脱流速为4mL/min,GSH解吸率为85.23%,共洗脱了464.52mg,蛋白质洗脱率为66.85%,共洗脱了1.936g。GSH浓缩倍数为2.09,峰点浓缩倍数为3.41,纯化倍数为3.48。通过吸附上柱及洗脱操作色素含量明显减少。
(4)GSH提取液经逐步分离提取所得白色粉末状GSH复合产品,其中GSH含量为15.4%。
Under mild pressure(0.1-5.0MPa), the stress response occurred after being stimulated for yeast cells. GSH is one of important stress metabolic products of plenty of microorganisms during they were intimidated by environment. It is widely used in clinic due to its pharmaceutical performance; also it is popular in food industry because of its antioxidant activity. In this paper, the main contents include establishing the optimum conditions of Saccharmyces cerevisiae CICC1447 accumulating GSH in the high pressure circumstance; the variation of metabolic flux of GSH synthesis under mild and normal pressure; effect of mild pressure on membrane permeability; process of separation and extraction of GSH. The results are as follows:
1. Established optimum conditions of Saccharmyces cerevisiae CICC1447 accumulating GSH under mild pressure.
With high purity air as pressure media, the optimum conditions of Saccharmyces cerevisiae CICC1447 accumulating GSH by single-factor-experiment have been established. The optimized conditions were as follows:holding time 6 h, pressure value 0.5 MPa, pressing and depressing rates 0.05-0.10 MPa/min, culture time before pressuring 20 h. The content of GSH was increased by 17.21% than normal pressure.
2. Metabolic flux analysis of synthesis of GSH of Saccharomyces cerevisiae CICC1447
Studied and compared the changes of metabolic flux of synthesis of GSH under mild and normal pressure through metabolic flux analysis. Explained convincingly why mild pressure can improve the content of GSH effectively through the analysis of metabolic flux distribution.
3. Influence of mild pressure on membrane permeability of Saccharomyces cerevisiae CICC1447、CICC1339
With CO2 and N2 as pressure media, the influence of mild pressure(0.1-5.0MPa) and holding time on membrane permeability of Saccharomyces cerevisiae CICC1447、CICC1339 have been determined. The results show that:For Saccharomyces cerevisiae CICC1447, the conductivity of the cell suspension, OD280 and OD260 of the supernatant fluctuated with the pressure increase; whereas, in general, increased with pressure rising and high than the control. For Saccharomyces cerevisiae CICC1339, the membrane permeability increased significantly. With N2 as the pressure media,1.0 MPa of mild pressure remarkably improved the permeability of Saccharomyces cerevisiae cells through comparing the fluorescence intensities produced by combination of PI and DNA under mild pressure and normal pressure.
4. Process of separation and extraction of GSH
(1) The boil water bath extraction, microwave assistant extraction, ultrasonic disruption and high pressure homogenization were used for extracting GSH from intracellular. The best method was high pressure homogenization whose GSH extraction amount was 3.975mg/g and the extraction rate was 99.87% under the best condition.
(2) The optimal operation parameters for removing protein from GSH extracted solution by ultrafiltration have been determined. The best conditions were:transmembrane pressure 0.04MPa, pH=3, temperature 20-25℃, the feed solution non diluted. Eventually, the loss rate of GSH was 25.4%, and the retention rate of protein was up to 46.6%. By diluting one fold, the recovery of GSH was increased to 89.6%, and the content of pigment decreased significantly.
(3) The best condition of cation change process were:adsorption pH=3, adsorption flow rate 4mL/min, adsorption amount 1.00 L, adsorption rate of GSH 86.19%, adsorption rate of protein 49.73%, using HCl as eluent, elution concentration lmol/L, elution flow rate 4mL/min, elution rate of GSH 85.23%, elution amount 464.52 mg, elution rate of protein 66.85%, elution amount 1.936 g, concentration multiple of GSH 2.09, peak concentration multiple of GSH 3.41, purification multiple 3.48. The content of pigment decreased significantly by cation exchange process which OD decreased significantly.
(4) Based on these processes, an end-white-product containing 15.4%(w/w) GSH have been obtained.
1.面包酵母CICC1447生物合成GSH加压条件的确定
以高纯空气为加压介质,通过单因素实验对面包酵母CICC1447生物合成GSH加压条件进行优化,确定了最佳条件：加压时间6h、压力0.5MPa、升降压速率0.05-0.10MPa、加压前培养20h为最优加压条件,GSH含量较常压提高了17.21%。
2.面包酵母CICC1447生物合成GSH代谢通量分析
利用代谢通量分析手段研究比较了加压、常压条件下的面包酵母CICC1447生物合成GSH代谢流变化,并通过对代谢通量分布情况的分析,有效地解释了加压提高GSH合成能力的原因。
3.温和压力对面包酵母CICC1447、CICC1339细胞膜通透性的影响
以CO2和N2为加压介质,考察了温和压力(0.1-1.0MPa)及加压时间对面包酵母CICC1447、CICC1339细胞膜通透性的影响。结果表明,在压力条件下面包酵母CICC1447细胞悬浮液的电导率、OD260和OD280显著增加,虽然上升后有些波动,但仍高于常压对照组；压力作用下面包酵母CICC1339细胞悬浮液电导率、OD260和OD280随着压力的增加显著升高。以N2为加压介质,通过对加压(1.0MPa)、常压下PI与DNA结合产生的荧光强度的测定,更有效的说明了温和压力可以显著增强细胞膜的通透性,促进物质的传递。
4.GSH分离提取工艺研究
(1)利用沸水浴抽提法、微波辅助提取法、超声波破碎法和高压均质破碎法对胞内GSH进行抽提,其中高压均质破碎法效果最好,在最佳操作条件下GSH抽提量为3.975mg/g,抽提率达到99.87%。
(2)利用超滤技术去除GSH提取液中的蛋白质,考察了操作压力、温度、提取液pH及料液浓度对超滤的影响。确定了适宜的操作条件：压力为0.04MPa,pH为3,温度为20-25℃,料液不稀释。在此操作条件下GSH损失率相对较小,为25.4%,蛋白质截留率达到46.6%。通过稀释过滤方法有效的提高了GSH回收率,通过稀释1次,使得GSH回收率提高到89.6%。并且通过超滤和稀释过滤方法有效地减少了GSH提取液中细胞色素的含量。
(3)利用离子交换装置对GSH进行分离操作,确定了最佳的上柱pH为3,上柱流速为4mL/min,共上柱1.00L,GSH吸附率达到86.19%,蛋白质吸附率为49.73%,以盐酸为洗脱剂,洗脱浓度为lmol/L,洗脱流速为4mL/min,GSH解吸率为85.23%,共洗脱了464.52mg,蛋白质洗脱率为66.85%,共洗脱了1.936g。GSH浓缩倍数为2.09,峰点浓缩倍数为3.41,纯化倍数为3.48。通过吸附上柱及洗脱操作色素含量明显减少。
(4)GSH提取液经逐步分离提取所得白色粉末状GSH复合产品,其中GSH含量为15.4%。
Under mild pressure(0.1-5.0MPa), the stress response occurred after being stimulated for yeast cells. GSH is one of important stress metabolic products of plenty of microorganisms during they were intimidated by environment. It is widely used in clinic due to its pharmaceutical performance; also it is popular in food industry because of its antioxidant activity. In this paper, the main contents include establishing the optimum conditions of Saccharmyces cerevisiae CICC1447 accumulating GSH in the high pressure circumstance; the variation of metabolic flux of GSH synthesis under mild and normal pressure; effect of mild pressure on membrane permeability; process of separation and extraction of GSH. The results are as follows:
1. Established optimum conditions of Saccharmyces cerevisiae CICC1447 accumulating GSH under mild pressure.
With high purity air as pressure media, the optimum conditions of Saccharmyces cerevisiae CICC1447 accumulating GSH by single-factor-experiment have been established. The optimized conditions were as follows:holding time 6 h, pressure value 0.5 MPa, pressing and depressing rates 0.05-0.10 MPa/min, culture time before pressuring 20 h. The content of GSH was increased by 17.21% than normal pressure.
2. Metabolic flux analysis of synthesis of GSH of Saccharomyces cerevisiae CICC1447
Studied and compared the changes of metabolic flux of synthesis of GSH under mild and normal pressure through metabolic flux analysis. Explained convincingly why mild pressure can improve the content of GSH effectively through the analysis of metabolic flux distribution.
3. Influence of mild pressure on membrane permeability of Saccharomyces cerevisiae CICC1447、CICC1339
With CO2 and N2 as pressure media, the influence of mild pressure(0.1-5.0MPa) and holding time on membrane permeability of Saccharomyces cerevisiae CICC1447、CICC1339 have been determined. The results show that:For Saccharomyces cerevisiae CICC1447, the conductivity of the cell suspension, OD280 and OD260 of the supernatant fluctuated with the pressure increase; whereas, in general, increased with pressure rising and high than the control. For Saccharomyces cerevisiae CICC1339, the membrane permeability increased significantly. With N2 as the pressure media,1.0 MPa of mild pressure remarkably improved the permeability of Saccharomyces cerevisiae cells through comparing the fluorescence intensities produced by combination of PI and DNA under mild pressure and normal pressure.
4. Process of separation and extraction of GSH
(1) The boil water bath extraction, microwave assistant extraction, ultrasonic disruption and high pressure homogenization were used for extracting GSH from intracellular. The best method was high pressure homogenization whose GSH extraction amount was 3.975mg/g and the extraction rate was 99.87% under the best condition.
(2) The optimal operation parameters for removing protein from GSH extracted solution by ultrafiltration have been determined. The best conditions were:transmembrane pressure 0.04MPa, pH=3, temperature 20-25℃, the feed solution non diluted. Eventually, the loss rate of GSH was 25.4%, and the retention rate of protein was up to 46.6%. By diluting one fold, the recovery of GSH was increased to 89.6%, and the content of pigment decreased significantly.
(3) The best condition of cation change process were:adsorption pH=3, adsorption flow rate 4mL/min, adsorption amount 1.00 L, adsorption rate of GSH 86.19%, adsorption rate of protein 49.73%, using HCl as eluent, elution concentration lmol/L, elution flow rate 4mL/min, elution rate of GSH 85.23%, elution amount 464.52 mg, elution rate of protein 66.85%, elution amount 1.936 g, concentration multiple of GSH 2.09, peak concentration multiple of GSH 3.41, purification multiple 3.48. The content of pigment decreased significantly by cation exchange process which OD decreased significantly.
(4) Based on these processes, an end-white-product containing 15.4%(w/w) GSH have been obtained.
引文
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