卵黄多肽的分离纯化及降血脂活性研究
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摘要
我国是农业大国，畜产品资源非常丰富，禽蛋在畜产品总量中占有相当大的比重。禽蛋作为人们日常生活中餐桌的必备佳肴，不仅富含蛋白质、脂类，也是维生素和矿物质的良好来源，特别是其中的蛋白质人体吸收利用率达99.7％，且必需氨基酸十分丰富。传统的禽蛋主要用于烹饪、生产蛋白粉和蛋黄粉等，许多活性物质没有得到充分利用。为合理开发利用禽蛋资源，本研究以卵黄蛋白为试材，系统研究水解多肽的制备工艺、分离纯化方法及其降血脂活性。研究结果如下:
通过单因素和响应面试验优选出提取卵黄蛋白质的最佳提取工艺条件为：脱脂蛋黄粉浓度为30%，乙醇溶液的浓度为45%，pH值为4.4，氯化钠浓度为0.02mol/L。此条件下卵黄蛋白质的提取率达72.20%，相对于使用其他方法提高近20%。
根据响应面实验确定了复合风味蛋白酶和碱性蛋白酶分别水解卵黄蛋白质的最佳条件。通过单因素和响应面试验确定使用以上两种酶分步水解卵黄蛋白质的最佳酶解工艺条件为：底物浓度为10%，碱性蛋白酶与复合风味蛋白酶的比是0.8：0.4，pH6.5，温度为55℃，两种酶水解时间均为2h。此条件下卵黄蛋白质的水解度达13.233%，多肽得率为45.573mg/ml。
运用超滤技术对卵黄蛋白质水解液进行初步分离，得到分子量小于10000的多肽液，冷冻干燥后备用。用Sephadex G-25凝胶层析柱对卵黄蛋白质多肽进行初步分离，最佳条件为上样浓度30mg/ml，上样量2.6ml，流速1.3ml/min，以含有NaCl0.25mol/L的重蒸水为洗脱液时可以得到4个级分，它们的分子量分布为5810D的EYP-1、1330D的EYP-2、897D的EYP-3和557D的EYP-4。
通常情况下，分子量小于5kD的多肽生物活性更高，故实验将EYP-2、EYP-3和EYP-4三个级分进行体外降血脂实验，利用高效液相色谱对以上三个级分结合的胆酸盐量进行测定，结果显示557D的EYP-4的胆酸盐结合能力最强，即体外降血脂效果最佳。
对EYP-4使用高效液相色谱（HPLC）进行纯化，优化纯化条件，得到较高的HPLC纯化条件为：C18色谱柱（5μm，4.6mm×150mm i.d）；流动相为正丙醇与水，采用梯度洗脱（条件见表4-5）；柱温35℃；上样量15uL；上样浓度60mg/ml；流速0.8mL/min；采用紫外检测器；采用以上条件，可以得到三个极性不同的组分EYP-4-1、EYP-4-2和EYP-4-3。
将EYP-4利用高效逆流色谱（HSCCC）进行单体制备，最佳条件为：上样浓度为40mg/ml，流动相为聚乙二醇、磷酸盐的水溶液（配制方法为每1000ml流动相中含有聚乙二醇150g，磷酸盐170g），流动相中磷酸氢二钾和磷酸二氢钾的比例为3:1。EYP-4经HSCCC制备分离得到的三个组分，利用纳升电喷雾-四极杆-飞行时间串联质谱(Nano-ESI-Ms/Ms)技术进行质谱分析，得到三个组分的氨基酸排列顺序分别为Lys-Glu-Pro-Ile、His-Ile-Pro-Leu和Lys-Glu-Tyr。
将EYP-4进行小鼠的体内降血脂研究，将8组小鼠建立高血脂模型后，灌胃EYP-4多肽液四周，每周对小鼠进行称重。四周后对小鼠进行眼球采血，利用试剂盒检测血清中的胆固醇、甘油三酯、低密度脂蛋白、高密度脂蛋白和肝脂中的胆固醇、甘油三酯和总脂肪量，并计算动脉硬化指数。结果发现多肽液可以明显降低血清中胆固醇和甘油三酯的含量（p＜0.05）；降低低密度脂蛋白效果较明显（p＜0.05）；明显升高血清中高密度脂蛋白的浓度（p＜0.05）。具体表现为实验组小鼠的体重增长是空白对照组小鼠体重增长的76.79-90.44%；小鼠血清胆固醇分别是空白对照组的49.68-79.90%，是给药对照组的57.34-92.21%；血清甘油三酯分别是空白对照组的51.20-87.08%、是给药对照组的75.35-128%；血清低密度脂蛋白分别是空白对照组的81.66-177%、是给药对照组的80.23-174%；血清高密度脂蛋白分别是空白对照组的116-140%、是给药对照组的99.40-119%；动脉硬化指数分别是空白对照组的0.31-55.62%、是给药对照组的0.54-96.74%；肝脏中胆固醇分别是空白对照组的29.83-92.89%、是给药对照组的53.89-167%；肝脏中甘油三酯分别是空白对照组的43.10-87.76%、是给药对照组的47.51-96.73%。
China is an agricultural country with rich resources of animal by-product. The yield of eggs ofthe poultry have a considerably proportion in the total output of the animal by-product. Theeggs of the poultry are the general cate on the dining-table in daily life. Egg is not only full ofnutrition such as protein and lipids but also the good resource of vitamin and mineralcomposition. Especially, the ratio of digestion of protein in egg is99.7%and the essentialamino-acid is rich enough. The traditional utilization for egg is to cook, product the power ofyolk and white. Thus, many active ingredients are lost and can not be taken fulladvantagement. Thus the common yolk protein is used as the experimental material in orderto make the resources exploited reasonable. The study emphases on the process of thehydrolysis of the yolk protein, the methods of the isolation and purification and the activity ofreducing the blooding fat of the peptides. The results of the research as follows:
Single factor test and response surface methodology are employed to optimize theextraction condition for the protein in egg yolk. The best conditions of extracting proteinthose have been obtained in the experiment were egg yolk powder concentration30%, ethanolconcentration45%, pH4.4, NaCl concentration0.02mol/L. Under these conditions, theextraction rate of yolk protein is72.20%, rising20%compared to the other methods.
2. The optimal hydrolytic conditions of Alcalase and Flavourzyme those the enzymeswhich have been chosen are assured according to the response surface methodology. Singlefactor test and response surface methodology are employed to optimize the hydrolysispretreatment conditions with two kinds of enzymes step by step. The best conditions for thehydrolysis are substrate concentration10%, the ratio of the amount of enzyme Alcalase andFlavourzyme0.8:0.4, pH6.5, the temperature55℃, hydrolysis time of Alcalase andFlavourzyme2h respectively. Under these conditions the yolk protein is hydrolyzed and thedegree of hydrolysis is up to13.233%and the peptide yield is up to45.573mg/ml.
3. The ultrafiltration is used to separate preliminarily the egg yolk hydrolysate. Thehydrolysate that the molecular weigh less than10kD was obtained and it was powered underthe condition of freeze drying for the next step. Using Sephadex G-25gel chromatographycolumn to purify preliminarily the yolk peptides and the results show that the peptides can bepurified well when the purification sample yield3.5%of the gel volumn, sample thickness30mg/ml, the flow velocity1.3ml/min and the eluent distilled water with NaCl of0.25mol/L,so four parts of peptides were obtained:5810D EYP-1,1330D EYP-2,894D EYP-3and557D EYP-4according to the standard curve that is made by the standard protein.
4. The three peptides1330D EYP-2,894D EYP-3and557D EYP-4were employed theexperiment of reducing the blooding lipid in vitro. The cholate that was not combined by thepeptide was detected on the HPLC. The experiment results showed that the557D EYP-4hasthe best effect to reduce the blooding lipid according to whether the peptide can combine the more cholate.
5. The peptides of EYP-4is purified on the HPLC and optimized the purificationcondition. The better purification conditions are C18(5μm，4.6mm×150mm i.d), themobile phase methanol and ultrapure water, gradient elute(showed in Tab.4-3), columntemperature35℃, sample yield15uL, sample thickness60mg/ml and the flow velocity0.8ml/min，UV detector are get from the experimental results. And three ingredients A, B，andC are obtained on the above condition.
6. HSCCC was used to prepare the monomer with the peptides of EYP-4that has beenget from the preliminary purification. The best preparation conditions are sample thickness40mg/ml, mobile phase the solution of PEG and PBS (There are150g of PEG and170g ogPBS in1000ml solution), the ration of K2HPO4and KH2PO43:1.
7. The three monomers that have been obtained on HPLC are applied the massspectrometry by use of Nano-ESI-Ms/Ms. The order of amino acid of the three monomersare Lys-Glu-Pro-Ile, His-Ile-Pro-Leu and Lys-Glu-Tyr.
8. The peptides of EYP-4were used to the small mice to apply the study of reducing theblooding lipid. The blood of the mice that was took from the eyeball and the cholesterol,triglycerides, low density lipoprotein, high-density lipoprotein and AI in the serum and thecholesterol, triglycerides and the total fat in the liver were detected by use of the kits. Theexperimental results showed that the cholesterol and triglycerides in the serum of the mice ofpouring the peptides into the stomach decreased very obviously, the high-density lipoproteinincreased very obviously, but the low density lipoprotein decreased obviously. They embodedas the weigh of the experimental groups is the76.99-90.44%of blank controller. Thecholesterol in the serum is49.68-79.90%of the blank controller,57.34-92.21%of the drugcontroller respectively. The triglyceride in the serum is51.20-87.08%of the blank controller,75.35-128%of the drug controller respectively. The low density lipoprotein in the serum is81.66-177%of the blank controller,80.23-174%of the drug controller respectively. Thehigh-density lipoprotein in the serum is116-140%of the blank controller,94.40-119%of thedrug controller respectively. The AI is0.31-55.62%of the blank controller,0.54-96.74%ofthe drug controller respectively. The cholesterol in the liver is29.83-92.89%of the blankcontroller,53.89-167%of the drug controller respectively. The triglyceride in the liver is43.10-87.76%of the blank controller,47.51-96.73%of the drug controller respectively.
通过单因素和响应面试验优选出提取卵黄蛋白质的最佳提取工艺条件为：脱脂蛋黄粉浓度为30%，乙醇溶液的浓度为45%，pH值为4.4，氯化钠浓度为0.02mol/L。此条件下卵黄蛋白质的提取率达72.20%，相对于使用其他方法提高近20%。
根据响应面实验确定了复合风味蛋白酶和碱性蛋白酶分别水解卵黄蛋白质的最佳条件。通过单因素和响应面试验确定使用以上两种酶分步水解卵黄蛋白质的最佳酶解工艺条件为：底物浓度为10%，碱性蛋白酶与复合风味蛋白酶的比是0.8：0.4，pH6.5，温度为55℃，两种酶水解时间均为2h。此条件下卵黄蛋白质的水解度达13.233%，多肽得率为45.573mg/ml。
运用超滤技术对卵黄蛋白质水解液进行初步分离，得到分子量小于10000的多肽液，冷冻干燥后备用。用Sephadex G-25凝胶层析柱对卵黄蛋白质多肽进行初步分离，最佳条件为上样浓度30mg/ml，上样量2.6ml，流速1.3ml/min，以含有NaCl0.25mol/L的重蒸水为洗脱液时可以得到4个级分，它们的分子量分布为5810D的EYP-1、1330D的EYP-2、897D的EYP-3和557D的EYP-4。
通常情况下，分子量小于5kD的多肽生物活性更高，故实验将EYP-2、EYP-3和EYP-4三个级分进行体外降血脂实验，利用高效液相色谱对以上三个级分结合的胆酸盐量进行测定，结果显示557D的EYP-4的胆酸盐结合能力最强，即体外降血脂效果最佳。
对EYP-4使用高效液相色谱（HPLC）进行纯化，优化纯化条件，得到较高的HPLC纯化条件为：C18色谱柱（5μm，4.6mm×150mm i.d）；流动相为正丙醇与水，采用梯度洗脱（条件见表4-5）；柱温35℃；上样量15uL；上样浓度60mg/ml；流速0.8mL/min；采用紫外检测器；采用以上条件，可以得到三个极性不同的组分EYP-4-1、EYP-4-2和EYP-4-3。
将EYP-4利用高效逆流色谱（HSCCC）进行单体制备，最佳条件为：上样浓度为40mg/ml，流动相为聚乙二醇、磷酸盐的水溶液（配制方法为每1000ml流动相中含有聚乙二醇150g，磷酸盐170g），流动相中磷酸氢二钾和磷酸二氢钾的比例为3:1。EYP-4经HSCCC制备分离得到的三个组分，利用纳升电喷雾-四极杆-飞行时间串联质谱(Nano-ESI-Ms/Ms)技术进行质谱分析，得到三个组分的氨基酸排列顺序分别为Lys-Glu-Pro-Ile、His-Ile-Pro-Leu和Lys-Glu-Tyr。
将EYP-4进行小鼠的体内降血脂研究，将8组小鼠建立高血脂模型后，灌胃EYP-4多肽液四周，每周对小鼠进行称重。四周后对小鼠进行眼球采血，利用试剂盒检测血清中的胆固醇、甘油三酯、低密度脂蛋白、高密度脂蛋白和肝脂中的胆固醇、甘油三酯和总脂肪量，并计算动脉硬化指数。结果发现多肽液可以明显降低血清中胆固醇和甘油三酯的含量（p＜0.05）；降低低密度脂蛋白效果较明显（p＜0.05）；明显升高血清中高密度脂蛋白的浓度（p＜0.05）。具体表现为实验组小鼠的体重增长是空白对照组小鼠体重增长的76.79-90.44%；小鼠血清胆固醇分别是空白对照组的49.68-79.90%，是给药对照组的57.34-92.21%；血清甘油三酯分别是空白对照组的51.20-87.08%、是给药对照组的75.35-128%；血清低密度脂蛋白分别是空白对照组的81.66-177%、是给药对照组的80.23-174%；血清高密度脂蛋白分别是空白对照组的116-140%、是给药对照组的99.40-119%；动脉硬化指数分别是空白对照组的0.31-55.62%、是给药对照组的0.54-96.74%；肝脏中胆固醇分别是空白对照组的29.83-92.89%、是给药对照组的53.89-167%；肝脏中甘油三酯分别是空白对照组的43.10-87.76%、是给药对照组的47.51-96.73%。
China is an agricultural country with rich resources of animal by-product. The yield of eggs ofthe poultry have a considerably proportion in the total output of the animal by-product. Theeggs of the poultry are the general cate on the dining-table in daily life. Egg is not only full ofnutrition such as protein and lipids but also the good resource of vitamin and mineralcomposition. Especially, the ratio of digestion of protein in egg is99.7%and the essentialamino-acid is rich enough. The traditional utilization for egg is to cook, product the power ofyolk and white. Thus, many active ingredients are lost and can not be taken fulladvantagement. Thus the common yolk protein is used as the experimental material in orderto make the resources exploited reasonable. The study emphases on the process of thehydrolysis of the yolk protein, the methods of the isolation and purification and the activity ofreducing the blooding fat of the peptides. The results of the research as follows:
Single factor test and response surface methodology are employed to optimize theextraction condition for the protein in egg yolk. The best conditions of extracting proteinthose have been obtained in the experiment were egg yolk powder concentration30%, ethanolconcentration45%, pH4.4, NaCl concentration0.02mol/L. Under these conditions, theextraction rate of yolk protein is72.20%, rising20%compared to the other methods.
2. The optimal hydrolytic conditions of Alcalase and Flavourzyme those the enzymeswhich have been chosen are assured according to the response surface methodology. Singlefactor test and response surface methodology are employed to optimize the hydrolysispretreatment conditions with two kinds of enzymes step by step. The best conditions for thehydrolysis are substrate concentration10%, the ratio of the amount of enzyme Alcalase andFlavourzyme0.8:0.4, pH6.5, the temperature55℃, hydrolysis time of Alcalase andFlavourzyme2h respectively. Under these conditions the yolk protein is hydrolyzed and thedegree of hydrolysis is up to13.233%and the peptide yield is up to45.573mg/ml.
3. The ultrafiltration is used to separate preliminarily the egg yolk hydrolysate. Thehydrolysate that the molecular weigh less than10kD was obtained and it was powered underthe condition of freeze drying for the next step. Using Sephadex G-25gel chromatographycolumn to purify preliminarily the yolk peptides and the results show that the peptides can bepurified well when the purification sample yield3.5%of the gel volumn, sample thickness30mg/ml, the flow velocity1.3ml/min and the eluent distilled water with NaCl of0.25mol/L,so four parts of peptides were obtained:5810D EYP-1,1330D EYP-2,894D EYP-3and557D EYP-4according to the standard curve that is made by the standard protein.
4. The three peptides1330D EYP-2,894D EYP-3and557D EYP-4were employed theexperiment of reducing the blooding lipid in vitro. The cholate that was not combined by thepeptide was detected on the HPLC. The experiment results showed that the557D EYP-4hasthe best effect to reduce the blooding lipid according to whether the peptide can combine the more cholate.
5. The peptides of EYP-4is purified on the HPLC and optimized the purificationcondition. The better purification conditions are C18(5μm，4.6mm×150mm i.d), themobile phase methanol and ultrapure water, gradient elute(showed in Tab.4-3), columntemperature35℃, sample yield15uL, sample thickness60mg/ml and the flow velocity0.8ml/min，UV detector are get from the experimental results. And three ingredients A, B，andC are obtained on the above condition.
6. HSCCC was used to prepare the monomer with the peptides of EYP-4that has beenget from the preliminary purification. The best preparation conditions are sample thickness40mg/ml, mobile phase the solution of PEG and PBS (There are150g of PEG and170g ogPBS in1000ml solution), the ration of K2HPO4and KH2PO43:1.
7. The three monomers that have been obtained on HPLC are applied the massspectrometry by use of Nano-ESI-Ms/Ms. The order of amino acid of the three monomersare Lys-Glu-Pro-Ile, His-Ile-Pro-Leu and Lys-Glu-Tyr.
8. The peptides of EYP-4were used to the small mice to apply the study of reducing theblooding lipid. The blood of the mice that was took from the eyeball and the cholesterol,triglycerides, low density lipoprotein, high-density lipoprotein and AI in the serum and thecholesterol, triglycerides and the total fat in the liver were detected by use of the kits. Theexperimental results showed that the cholesterol and triglycerides in the serum of the mice ofpouring the peptides into the stomach decreased very obviously, the high-density lipoproteinincreased very obviously, but the low density lipoprotein decreased obviously. They embodedas the weigh of the experimental groups is the76.99-90.44%of blank controller. Thecholesterol in the serum is49.68-79.90%of the blank controller,57.34-92.21%of the drugcontroller respectively. The triglyceride in the serum is51.20-87.08%of the blank controller,75.35-128%of the drug controller respectively. The low density lipoprotein in the serum is81.66-177%of the blank controller,80.23-174%of the drug controller respectively. Thehigh-density lipoprotein in the serum is116-140%of the blank controller,94.40-119%of thedrug controller respectively. The AI is0.31-55.62%of the blank controller,0.54-96.74%ofthe drug controller respectively. The cholesterol in the liver is29.83-92.89%of the blankcontroller,53.89-167%of the drug controller respectively. The triglyceride in the liver is43.10-87.76%of the blank controller,47.51-96.73%of the drug controller respectively.
引文
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66. Kim SY, J e J Y, Kim SK. Purification and characterization of antioxidant peptide from hoki (Johni usbelengeri i) frame protein by gastrointestinal digestion[J]. Journal of Nutritional Biochemistry,2007(18):31-38
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