甘薯水溶性糖蛋白的提取纯化及降血脂机理的研究
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摘要
甘薯是旋花科甘薯属的一个重要栽培品种,原产于南美洲,为世界第七大农作物。中国甘薯年产量为1.17亿t,占世界甘薯产量90%以上。本论文以甘薯为原料,对甘薯水溶性糖蛋白进行了分离、纯化,并系统的研究了甘薯糖蛋白的理化性质、组成、结构、降血脂作用及降血脂机理。为更好的利用甘薯,开发新型降血脂保健食品提供理论支持。
本文通过水浸提法,采用单因素实验和正交试验确定了甘薯糖蛋白的最佳提取条件;研究了DEAE-纤维素52柱层析对甘薯糖蛋白的纯化条件;采用丙烯葡聚糖凝胶S-300对甘薯糖蛋白进一步纯化。用SDS-聚丙烯酰胺凝胶电泳对甘薯糖蛋白进行纯度鉴定;研究了甘薯糖蛋白的理化性质;对甘薯糖蛋白中氨基酸及单糖组成进行了分析;采用差示扫描量热对甘薯糖蛋白的热力学性质进行研究;采用核磁共振、红外光谱、紫外光谱对甘薯糖蛋白的结构进行分析。通过动物实验,研究甘薯糖蛋白对高脂血症大鼠血脂水平的影响,并探讨其降血脂作用机理及各种加工因素对甘薯糖蛋白降血脂作用的影响。主要研究结论如下:
1.在单因素实验的基础上,利用正交试验确定了甘薯水溶性糖蛋白的最佳提取条件:即室温下,粉碎程度40目,料水比1:15,提取时间1h,提取次数3次时,糖蛋白得率最高,为提取甘薯糖蛋白较理想的工艺条件。
2.用0.00,0.05,0.10,0.50mol/L的碳酸氢钠溶液作为DEAE-52纤维素离子交换柱层析的阶段洗脱剂,效果较好。甘薯糖蛋白纯化采用DEAE-52纤维素柱层析进行第一步纯化,初步分成各级分后,再分别用丙烯葡聚糖凝胶S-300柱层析进行第二步纯化。
3.甘薯水溶性糖蛋白冷冻干燥制品为白色絮状固体。易溶于水,其水溶液为透明淡黄色,不溶于高浓度的乙醇、丙酮等有机溶剂。在浓度为1.02g/mL时,pH=5.6,特征性粘度为2.95mPa.s,比旋光度[α]_D~(25℃)=+46°(C=0.29,H_2O),紫外吸收峰为280nm。经测定计算得知,甘薯糖蛋白样品中蛋白质的含量为71.65%,还原糖的含量为27.41%。
4.甘薯水溶性糖蛋白几乎含有人体所必需的各种氨基酸,其中天冬氨酸、谷氨酸、精氨酸、半胱氨酸、苯丙氨酸、亮氨酸含量较高。甘薯水溶性糖蛋白含有8种必需氨基酸,其含量和比例都接近WHO的推荐值。因此,甘薯水溶性糖蛋白是一种优质的植物蛋白。甘薯糖蛋白样品中的单糖种类是:鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖、半乳糖。
5.甘薯水溶性糖蛋白的热变性温度为45℃,而去糖基后的热变性温度为38.5℃,因此糖蛋白上的寡糖分子对于蛋白质具有较好的稳定作用,具有增强蛋白质抗变性功能。
6.甘薯糖蛋白经NaOH处理后,在240nm处产生明显的吸收,说明分子中存在O-糖肽键。
7.甘薯糖蛋白能够有效降低高脂血症大鼠的血脂水平,改善血脂代谢,降低动脉粥样硬化发生的危险性。主要特征表现为:灌胃甘薯糖蛋白后,高脂血症大鼠的TG、TC、LDL-C显著下降,HDL-C显著上升,动脉硬化指数显著下降,并呈现剂量.效应关系。表明甘薯糖蛋白可明显改善高脂血症大鼠的血脂代谢水平,这对预防动脉硬化的发生具有积极的作用。
8.甘薯糖蛋白降血脂的作用机理是:升高HDL-C和载脂蛋白AⅠ(ApoAⅠ)的水平,激活LCAT,识别HDL受体,促进脂质的转动和排泄;抑制胆固醇合成的关键性限速酶——羟甲基戊二酸单酰辅酶A还原酶(HMG-C_oA还原酶)的活性,从而阻断肝细胞的内源性胆固醇合成;提高卵磷脂胆固醇酰基转移酶(LCAT)的活性,从而促进胆固醇的酯化作用,促进细胞内的游离胆固醇酯化成胆固醇酯,加速胆固醇在体内的分解。
甘薯水溶性糖蛋白使LCAT活性升高的原因,可能是促进肝脏制造LCAT分子;或因升高HDL-C和载脂蛋白AⅠ(ApoAⅠ)的水平,激活LCAT。
甘薯糖蛋白可能是通过与羟甲基戊二酸单酰辅酶A还原酶(HMG-C_oA还原酶)结合,对羟甲基戊二酸单酰辅酶A还原酶(HMG-C_oA还原酶)有较强的竞争性抑制作用,通过对羟甲基戊二酸单酰辅酶A还原酶(HMG-C_oA还原酶)的抑制,使LDL-C受体表达增加,加强血浆中LDL和ApoB的清除,减少胆固醇的生成。
9.甘薯水溶性糖蛋白的降胆固醇活性受温度影响较大,在甘薯糖蛋白的加工中,应尽量避免60℃以上的高温。甘薯水溶性糖蛋白在pH 3-6下处理后,其降血脂功能基本稳定。紫外光处理对甘薯水溶性糖蛋白降血脂功能几乎没有影响。
Sweet potatoe which originated from South Africa belongs to the morning-glory family and ranks as the world's seventh most important food crop. More than 117 million tons are produced in China annually—accounts for 90 percent of worldwide production. Sweet potato was used as raw material in this dissertation, and sweet potato water-soluble glycoprotein (SPG) was isolated and purified. Physical and chemical properties, composition, structure, and mechanism of hypolipidemic were systematic studied in order to make better use of sweet potato in order to develop new hypolipidemic health food.
In this paper, SPG was extracted from sweet potato by water. The single factor and orthogonal experiment were used to determine the best extracting conditions; studied DEAE-cellulose 52 chromatography for the purification of sweet potato glycoprotein conditions; then using Sephacryl S-300 of the sweet potato glycoprotein further purification. By SDS-polyacrylamide gel electrophoresis of sweet potato glycoprotein for purity; studied the sweet potato glycoprotein of physical and chemical properties; the composition of sugar and amino acids of SPG were analyzed. Sweet potato glycoprotein thermodynamic properties was studied by DSC; using nuclear magnetic resonance, infrared spectroscopy, UV and mass spectrometry for the Sweet Potato structural glycoprotein analysis. Through animal experiments, research on sweet potato glycoprotein hyperlipidemia rats' blood lipid levels and explore the mechanism of hypolipidemic and a variety of factors on the processing of hypolipidemic effect of sweet potato. The main conclusions of the study are as follows:
1. The best extraction conditions of sweet potato water-soluble glycoprotein were chosen by the single-factor test: grinding degree is 40 mesh, the ratio of raw material to water is 1:15, extraction time is 1h, extraction times is 3, at room temperature.
2. The 0.00, 0.05, 0.10, 0.50 mol / L solution of sodium bicarbonate as DEAE-52 cellulose ion-exchange chromatography stage eluting agent is better. Sweet Potato glycoprotein purification purified by using DEAE-52 cellulose chromatography for the first step, then purified by Sephacryl S-300 gel chromatography.
3. Sweet potato water-soluble Glycoprotein freeze-dried products is white solid flocculation. Soluble in water and the solution is yellow, insoluble in high concentrations of ethanol, acetone, and other organic solvents. Under the concentration of SPG is 1.02g/mL, pH = 5.6, glutinosity is 2.95mPa.s, degree of opticity is [α]_D~(25℃)=+46o (C=0.29,H_2O), UV absorption is 280nm. Sweet potato glycoprotein samples of the protein content of 71.65 percent, reducing sugar content of 27.41 percent.
4. Sweet potato water-soluble glycoprotein almost contains a variety of amino acids, aspartic acid, glutamic acid, arginine, cysteine, phenylalanine, leucine with a high level. Sweet potato water-soluble glycoprotein with 8 kinds of essential amino acids and the content is close to the WHO recommended which shows sweet potato water-soluble glycoprotein is a high-quality plant protein. The single type of sugar in sweet-potato Glycoprotein sample is rhamnose, arabinose, xylose, mannose, glucose and galactose.
5. Sweet potato water-soluble glycoprotein thermal denaturizing temperature is 45°C, and after the deglycosylated , the thermal denaturizing temperature is 38.5℃, therefore glycoprotein oligosaccharides on the protein molecules has better stability which enhanced anti-denatured function.
6. With the treatment of NaOH , Sweet potato Glycoprotein was significant absorpt at 240nm which shows that glycopeptide linkage existed in SPG.
7. Sweet potato Glycoprotein can effectively reduce hyperlipidemia rats blood lipid levels and improve lipid metabolism and reduce atherosclerosis risk. The main features as follows: oral administration of different doses of the sweet potato glycoprotein, hyperlipidemia rats TG, TC, LDL-C decreased significantly, HDL-C increased significantly, the atherogenic index decreased significantly and shows dose-response relationship. The result shows that the sweet potato glycoprotein can improve hyperlipidemia rats' lipid metabolism, which prevent the occurrence of atherosclerosis.
8. Sweet potato Glycoprotein hypolipidemic mechanism are: increase the level of HDL-C and Apolipoprotein AⅠ(ApoAⅠ), activate LCAT, identify HDL receptor which promote the lipid rotation and excretion; inhibit cholesterol synthesis key rate-limiting enzyme HMG-coenzyme A reductase (HMG-C_oA reductase), which block the liver cells of endogenous synthesis of cholesterol; raise lecithin cholesterol acyltransferase (LCAT) activity which promote Esterification of cholesterol, to increase cells free cholesterol esterification into cholesterol ester, speed up the cholesterol decomposition in the body.
Sweet potato water-soluble glycoprotein increased activity of LCAT which reason may be that promote the liver manufacturing more LCAT molecular; or increase the level of HDL-C and Apolipoprotein AⅠ(ApoAⅠ), activate LCAT.
Sweet potato glycoprotein may be adopted with the HMG coenzyme A reductase (HMG-C_oA reductase), has Strong competitive inhibition on HMG coenzyme A reductase (HMG-C_oA reductase) which increase LDL-C receptor expression, strengthen the cleaning of plasma ApoB and LDL, and reduce the formation of cholesterol.
9. The temperature has a greater impact on sweet potato water-soluble glycoprotein activity. During sweet potato processing should avoid the high temperature above 60°C. Sweet potato water-soluble glycoprotein is steady when pH is 3～6. UV-treated to the sweet water-soluble glycoprotein almost no significant effect on hypolipidemic compared with the former.
本文通过水浸提法,采用单因素实验和正交试验确定了甘薯糖蛋白的最佳提取条件;研究了DEAE-纤维素52柱层析对甘薯糖蛋白的纯化条件;采用丙烯葡聚糖凝胶S-300对甘薯糖蛋白进一步纯化。用SDS-聚丙烯酰胺凝胶电泳对甘薯糖蛋白进行纯度鉴定;研究了甘薯糖蛋白的理化性质;对甘薯糖蛋白中氨基酸及单糖组成进行了分析;采用差示扫描量热对甘薯糖蛋白的热力学性质进行研究;采用核磁共振、红外光谱、紫外光谱对甘薯糖蛋白的结构进行分析。通过动物实验,研究甘薯糖蛋白对高脂血症大鼠血脂水平的影响,并探讨其降血脂作用机理及各种加工因素对甘薯糖蛋白降血脂作用的影响。主要研究结论如下:
1.在单因素实验的基础上,利用正交试验确定了甘薯水溶性糖蛋白的最佳提取条件:即室温下,粉碎程度40目,料水比1:15,提取时间1h,提取次数3次时,糖蛋白得率最高,为提取甘薯糖蛋白较理想的工艺条件。
2.用0.00,0.05,0.10,0.50mol/L的碳酸氢钠溶液作为DEAE-52纤维素离子交换柱层析的阶段洗脱剂,效果较好。甘薯糖蛋白纯化采用DEAE-52纤维素柱层析进行第一步纯化,初步分成各级分后,再分别用丙烯葡聚糖凝胶S-300柱层析进行第二步纯化。
3.甘薯水溶性糖蛋白冷冻干燥制品为白色絮状固体。易溶于水,其水溶液为透明淡黄色,不溶于高浓度的乙醇、丙酮等有机溶剂。在浓度为1.02g/mL时,pH=5.6,特征性粘度为2.95mPa.s,比旋光度[α]_D~(25℃)=+46°(C=0.29,H_2O),紫外吸收峰为280nm。经测定计算得知,甘薯糖蛋白样品中蛋白质的含量为71.65%,还原糖的含量为27.41%。
4.甘薯水溶性糖蛋白几乎含有人体所必需的各种氨基酸,其中天冬氨酸、谷氨酸、精氨酸、半胱氨酸、苯丙氨酸、亮氨酸含量较高。甘薯水溶性糖蛋白含有8种必需氨基酸,其含量和比例都接近WHO的推荐值。因此,甘薯水溶性糖蛋白是一种优质的植物蛋白。甘薯糖蛋白样品中的单糖种类是:鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖、半乳糖。
5.甘薯水溶性糖蛋白的热变性温度为45℃,而去糖基后的热变性温度为38.5℃,因此糖蛋白上的寡糖分子对于蛋白质具有较好的稳定作用,具有增强蛋白质抗变性功能。
6.甘薯糖蛋白经NaOH处理后,在240nm处产生明显的吸收,说明分子中存在O-糖肽键。
7.甘薯糖蛋白能够有效降低高脂血症大鼠的血脂水平,改善血脂代谢,降低动脉粥样硬化发生的危险性。主要特征表现为:灌胃甘薯糖蛋白后,高脂血症大鼠的TG、TC、LDL-C显著下降,HDL-C显著上升,动脉硬化指数显著下降,并呈现剂量.效应关系。表明甘薯糖蛋白可明显改善高脂血症大鼠的血脂代谢水平,这对预防动脉硬化的发生具有积极的作用。
8.甘薯糖蛋白降血脂的作用机理是:升高HDL-C和载脂蛋白AⅠ(ApoAⅠ)的水平,激活LCAT,识别HDL受体,促进脂质的转动和排泄;抑制胆固醇合成的关键性限速酶——羟甲基戊二酸单酰辅酶A还原酶(HMG-C_oA还原酶)的活性,从而阻断肝细胞的内源性胆固醇合成;提高卵磷脂胆固醇酰基转移酶(LCAT)的活性,从而促进胆固醇的酯化作用,促进细胞内的游离胆固醇酯化成胆固醇酯,加速胆固醇在体内的分解。
甘薯水溶性糖蛋白使LCAT活性升高的原因,可能是促进肝脏制造LCAT分子;或因升高HDL-C和载脂蛋白AⅠ(ApoAⅠ)的水平,激活LCAT。
甘薯糖蛋白可能是通过与羟甲基戊二酸单酰辅酶A还原酶(HMG-C_oA还原酶)结合,对羟甲基戊二酸单酰辅酶A还原酶(HMG-C_oA还原酶)有较强的竞争性抑制作用,通过对羟甲基戊二酸单酰辅酶A还原酶(HMG-C_oA还原酶)的抑制,使LDL-C受体表达增加,加强血浆中LDL和ApoB的清除,减少胆固醇的生成。
9.甘薯水溶性糖蛋白的降胆固醇活性受温度影响较大,在甘薯糖蛋白的加工中,应尽量避免60℃以上的高温。甘薯水溶性糖蛋白在pH 3-6下处理后,其降血脂功能基本稳定。紫外光处理对甘薯水溶性糖蛋白降血脂功能几乎没有影响。
Sweet potatoe which originated from South Africa belongs to the morning-glory family and ranks as the world's seventh most important food crop. More than 117 million tons are produced in China annually—accounts for 90 percent of worldwide production. Sweet potato was used as raw material in this dissertation, and sweet potato water-soluble glycoprotein (SPG) was isolated and purified. Physical and chemical properties, composition, structure, and mechanism of hypolipidemic were systematic studied in order to make better use of sweet potato in order to develop new hypolipidemic health food.
In this paper, SPG was extracted from sweet potato by water. The single factor and orthogonal experiment were used to determine the best extracting conditions; studied DEAE-cellulose 52 chromatography for the purification of sweet potato glycoprotein conditions; then using Sephacryl S-300 of the sweet potato glycoprotein further purification. By SDS-polyacrylamide gel electrophoresis of sweet potato glycoprotein for purity; studied the sweet potato glycoprotein of physical and chemical properties; the composition of sugar and amino acids of SPG were analyzed. Sweet potato glycoprotein thermodynamic properties was studied by DSC; using nuclear magnetic resonance, infrared spectroscopy, UV and mass spectrometry for the Sweet Potato structural glycoprotein analysis. Through animal experiments, research on sweet potato glycoprotein hyperlipidemia rats' blood lipid levels and explore the mechanism of hypolipidemic and a variety of factors on the processing of hypolipidemic effect of sweet potato. The main conclusions of the study are as follows:
1. The best extraction conditions of sweet potato water-soluble glycoprotein were chosen by the single-factor test: grinding degree is 40 mesh, the ratio of raw material to water is 1:15, extraction time is 1h, extraction times is 3, at room temperature.
2. The 0.00, 0.05, 0.10, 0.50 mol / L solution of sodium bicarbonate as DEAE-52 cellulose ion-exchange chromatography stage eluting agent is better. Sweet Potato glycoprotein purification purified by using DEAE-52 cellulose chromatography for the first step, then purified by Sephacryl S-300 gel chromatography.
3. Sweet potato water-soluble Glycoprotein freeze-dried products is white solid flocculation. Soluble in water and the solution is yellow, insoluble in high concentrations of ethanol, acetone, and other organic solvents. Under the concentration of SPG is 1.02g/mL, pH = 5.6, glutinosity is 2.95mPa.s, degree of opticity is [α]_D~(25℃)=+46o (C=0.29,H_2O), UV absorption is 280nm. Sweet potato glycoprotein samples of the protein content of 71.65 percent, reducing sugar content of 27.41 percent.
4. Sweet potato water-soluble glycoprotein almost contains a variety of amino acids, aspartic acid, glutamic acid, arginine, cysteine, phenylalanine, leucine with a high level. Sweet potato water-soluble glycoprotein with 8 kinds of essential amino acids and the content is close to the WHO recommended which shows sweet potato water-soluble glycoprotein is a high-quality plant protein. The single type of sugar in sweet-potato Glycoprotein sample is rhamnose, arabinose, xylose, mannose, glucose and galactose.
5. Sweet potato water-soluble glycoprotein thermal denaturizing temperature is 45°C, and after the deglycosylated , the thermal denaturizing temperature is 38.5℃, therefore glycoprotein oligosaccharides on the protein molecules has better stability which enhanced anti-denatured function.
6. With the treatment of NaOH , Sweet potato Glycoprotein was significant absorpt at 240nm which shows that glycopeptide linkage existed in SPG.
7. Sweet potato Glycoprotein can effectively reduce hyperlipidemia rats blood lipid levels and improve lipid metabolism and reduce atherosclerosis risk. The main features as follows: oral administration of different doses of the sweet potato glycoprotein, hyperlipidemia rats TG, TC, LDL-C decreased significantly, HDL-C increased significantly, the atherogenic index decreased significantly and shows dose-response relationship. The result shows that the sweet potato glycoprotein can improve hyperlipidemia rats' lipid metabolism, which prevent the occurrence of atherosclerosis.
8. Sweet potato Glycoprotein hypolipidemic mechanism are: increase the level of HDL-C and Apolipoprotein AⅠ(ApoAⅠ), activate LCAT, identify HDL receptor which promote the lipid rotation and excretion; inhibit cholesterol synthesis key rate-limiting enzyme HMG-coenzyme A reductase (HMG-C_oA reductase), which block the liver cells of endogenous synthesis of cholesterol; raise lecithin cholesterol acyltransferase (LCAT) activity which promote Esterification of cholesterol, to increase cells free cholesterol esterification into cholesterol ester, speed up the cholesterol decomposition in the body.
Sweet potato water-soluble glycoprotein increased activity of LCAT which reason may be that promote the liver manufacturing more LCAT molecular; or increase the level of HDL-C and Apolipoprotein AⅠ(ApoAⅠ), activate LCAT.
Sweet potato glycoprotein may be adopted with the HMG coenzyme A reductase (HMG-C_oA reductase), has Strong competitive inhibition on HMG coenzyme A reductase (HMG-C_oA reductase) which increase LDL-C receptor expression, strengthen the cleaning of plasma ApoB and LDL, and reduce the formation of cholesterol.
9. The temperature has a greater impact on sweet potato water-soluble glycoprotein activity. During sweet potato processing should avoid the high temperature above 60°C. Sweet potato water-soluble glycoprotein is steady when pH is 3～6. UV-treated to the sweet water-soluble glycoprotein almost no significant effect on hypolipidemic compared with the former.
引文
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