蛋清源抗凝血肽分离纯化及活性鉴定
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摘要
心脑血管疾病已成为我国发病率致死率最高的疾病，其中大部分疾病与血栓相关。而临床上使用的肝素、香豆素、链激酶、尿激酶等物质易引发出血等副作用。近些年来从水蛭、蜱虫、钩口线虫、蛇毒和蜂毒中提取的抗凝溶栓活性物质具有良好的抗凝血效果，但毒副作用大。因此开发安全、来源广泛的抗凝溶栓类保健品具有较为广阔的研发前景。
生物活性肽近些年来因其安全性高、易吸收、功效好，被广泛应用于功能性因子的开发，例如抗氧化肽、降血压肽、抗疲劳肽、增强免疫力肽和增强记忆力肽等。我国是禽蛋大国，禽蛋多为直接食用，深加工产品较少。由于蛋黄的需求量较大，导致剩余蛋清的浪费。而蛋清中有许多功能活性因子，将其通过酶解可得到具有生物活性功能的肽。有文献表明，蛋清中具有抗凝物质，本文致力于从来源广泛并安全的蛋清中分离纯化具有抗凝活性的物质。
本文对比了Alcalase、中性蛋白酶、风味蛋白酶和胰蛋白酶的水解活性，筛选出最优的水解酶。对比了普通酶解、加热预处理酶解、微波辅助酶解和超声波辅助酶解四种酶解前处理方式对蛋清蛋白质酶解水解度的影响，选择最优的酶解方式。考察了酶解时间、加酶量、底物质量分数、酶解pH和酶解温度等五个单因素，并选取合适的范围以蛋清蛋白质水解度和蛋清源抗凝血肽的抗凝血活性为指标构建多元线性回归方程，确定最优的酶解工艺。运用Sephadex G-50和半制备高效液相色谱对蛋清蛋白质酶解物进行纯化，选取出抗凝血活性高的组分。研究所得抗凝血活性高的组分的pH、温度、金属离子和有机溶剂的稳定性并利用紫外光谱和傅里叶红外光谱研究所得组分可能包含的基团以及二级结构。确定蛋清源抗凝血肽的抗凝血活性IC50值及抗凝的途径。
结果表明采用Alcalase、加热预处理后酶解蛋清蛋白质的水解效果好。根据单因素实验选择2h作为酶解时间。综合考虑蛋清蛋白质水解度及蛋清源抗凝血肽的活性，通过单因素实验选取加酶量为3%、4%、5%，底物质量之比选取1%、3%、5%，pH选取7、8、9，温度选取30℃、40℃、50℃进行实验优化设计。分别以蛋清蛋白质水解度和蛋清源抗凝血肽的抗凝血活性作为指标建立多元线性回归模型。最优的实验方案为加酶量5%，底物质量之比1%，pH8.0，温度50℃时，抗凝血活性为68.92%。以蛋清蛋白质水解度为指标的模型，底物质量之比为显著性水平，以蛋清源抗凝血肽为指标检测，pH为显著性水平。方程的拟合度较好，无多重共线性。将蛋清蛋白质酶解物通过Sephadex G-50纯化后组分4抗凝血活性最高，将组分4经半制备高效液相色谱纯化后组分Ⅶ抗凝血活性最好。酶解物的抗凝血活性为69%、经Sephadex G-50纯化后为85%，经半制备高效液相色谱纯化后为71%，可能是由于有机溶剂对蛋清源抗凝血肽的活性造成了影响。将纯化后的样品通过紫外光谱研究酶解后的样品出现了蓝移，通过红外光谱分析蛋清源抗凝血肽的各构象占总构象的比例。酶解和Sephadex G-50纯化可使α螺旋增加，但半制备高效液相色谱却使样品β折叠的比例有所回升，推测构象的改变可影响其活性的强弱。稳定性实验表明pH为6，温度为30℃时蛋清源抗凝血肽的活性较为稳定。钠盐对蛋清源抗凝血肽的稳定性无显著性影响，钾盐和硫酸铜以及有机溶剂均对其有着不同程度的影响。蛋清源抗凝血肽的IC50值为28.04mg/mL，主要通过内源性途径实现抗凝血的效果。
从结果可以看出酶解蛋白质可获得优良的生物活性肽，以分子量作为分离指标的交联葡聚糖凝胶柱色谱进行纯化适用于蛋清源抗凝血肽的纯化，但是以极性为分离指标的半制备高效液相色谱法虽然能够提高纯度，但是由于有机溶剂对生物活性肽结构的破坏，导致活性的损失。故如何在不影响活性的情况下更好地运用半制备高效液相色谱进行分离需要进一步的研究。通过上述方法分离得到的蛋清源抗凝血肽室温下，pH中性的环境较为稳定，与其他肽类似，重金属盐和有机溶剂对其影响较大。蛋清源抗凝血肽可以通过影响内源性凝血途径达到抗凝的效果，虽然该肽与临床用药相比IC50值较大，体外半衰期较短，但是由于其来源广泛，安全性高，作为针对预防血栓类疾病的保健品具有较为广阔的研发市场及前景。
Cerebrovascular disease has become a disease which has the highest incidenceand lethality, and most of the disease is related to thrombus. However, the clinicalmedicine such as hamocura, tonka bean campor, streptokinase, urokinase are easy tolead to adverse effect such as bleed. In recent years, active compounds with theantithrombotic extracted from leech, tick innsect, ancylostome, snake venom and beevenom have good anticoagulated blood effect, but the adverse effect is also great.Therefore, it is significant to develop antithrombotic effect with high safety and broadsource.
Biologically active peptide such as antioxidant peptide, antihypertensive peptide,antifatigue peptide, strengthening immunity peptide and strengthening rememberancepeptide have been widely used in the development of functional factors for theadvantages of high safety, easy absorption, good function. China is a country withpoultry eggs, most of the poultry eggs are directly used and the deep processingproducts are less. Furthermore, the big demand for the egg yolk lead to the waste ofegg white. However, there are many functional actors in the egg white, and thebiologically active peptides can be obtained by enzymolysis. Literatures show thatthere are antithrombotic compounds in the egg white, and the purpose of this paper isto extract, separate and purify antithrombotic compounds from egg white which havethe advantage of high safety and broad source.
The hydrolytic activity of Alcalase, dispase, flavor protease and trypsinase werecompared, and the optimal hydrolase were selected. The effect of pre-procssingmethod of ordinary enzymolysis, heating pre-processing enzymolysis,microwave-aided enzymolysis and ultrasound aided enzymolysis on degree ofhydrolysis of egg white protein were compared and the best method was optimized.Five single factors like enzymatic hydrolysis time, enzyme and substrate ratio,substrate concentration, pH and hydrolysis temperature were examined, based on the appropriate scope, a multiple linear regression equation with protein degree ofhydrolysis of egg white and anticoagulant activity of egg white source thrombininhibitory peptide as indexes was built to determine the optimal hydrolysis process.Sephadex g-50and semi-preparative HPLC were used to purify egg white proteinhydrolysates and select anticoagulant components.The pH, temperature, metal ionsand stability of organic solubitily were studied, and ultraviolet spectroscopy andFourier transform infrared spectroscopy were used to determine the possiblefunctional group and secondary structure. The anticoagulant activity IC50value andthe anticougulant pathway were determined.
The results indicated that Alcalase has the best enzymolysis effect, and theheating was selected as the pre-processing method for its simplicityin during theexperiment instead of microwave aided enzymolysis which has the best effect as theresults show. The degree of hydrolysis increases with the time of enzymatichydrolysis, but the rate slowed down after2h, so2h was chosen as enzymatichydrolysis time. Take both the the degree of hydrolysis of the egg white protein andthe activity of egg white source thrombin inhibitory peptide into consideration, basedon single factor experiment, the following conditions were set to conduct theExperimental optimization experiment design: enzyme and substrate ratio:3%、4%、5%, substrate concentration:1%、3%、5%,pH:7.0、8.0、9.0，hydrolysis temperature:30℃、40℃、50℃.
Multiple linear regression model was built with the protein degree of hydrolysisof egg white and anticoagulant activity of egg white source thrombin inhibitorypeptide as indexes was built respectively. The optimal experimental program was asfollows: enzyme and substrate ratio:5%, substrate concentration:1%,pH:8.0，hydrolysis temperature:50℃. anticoagulant activity:68.92%. The model with degreeof hydrolysis of egg white protein as an indicator showed significant level onsubstrate concentration level of significance, and the one with egg white source ofthrombin inhibition peptide as indicators detected pH as the significant level.
The equation fitting is good, and no multiple collinearities. component4fromegg white protein hydrolysates puried by Sephadex G-50has the highest anticoagulant activity, and the component VII after the purification of Semi-preparative HPLCpossessed the best anticoagulant activity. The anticoagulant activity of hydrolysateswas69%, after purification by Sephadex G-50is85%, and after Semi-preparativeHPLC was71%, the possible reseason is that organic solvents and the rigid structureof C18caused an impact on the activity of egg white source of thrombin inhibitorypeptide. The study of purified samples by ultraviolet spectroscopy showed redshift,and the infrared spectroscopy indicated that the egg white powder acrylamide I bandsecondary structure were mainly constituded by β conformation and random coil,accounting for85.74%.
In samples after the enzymolysis, α-helix, βcorner, and antiparallel β-sheetincreased, while forward parallel β-folded and random coil decreased. In samples afterSephadex G-50purification, all the four conformation decreased except for theincrease of α-helix. In the samples after purification by semi-preparative HPLC,βcorner and antiparallel β-sheet had gone up, and the rest of conformation werereduced.
Stability experiments showed that activity of egg white source thrombininhibitory peptides was more stable under the condition of pH6.0and the temperatureis30℃. Sodium salt has no significant effect on the stability of egg white sourcethrombin inhibitory peptides, while potassium and copper sulfate and organic solventshave varying degrees of impact.The IC50value of egg white source of thrombininhibition inhibitory peptides is28.04mg/mL and play anticoagulant effect mainlythrough the endogenous pathway.
It can be seen from the results that high quality bioactive peptides can beobtained by enzymolysis of protein, and cross-linked dextran gel columnchromatography with molecular weight as separate indicators was suitable for thepurification of egg white source thrombin inhibitory peptides, while semi-preparativeHPLC with polarity as the separation indicators was not suitable, although it canimprove the purity, organic solvents can also cause the destruction the structure of thebioactive peptides, resulting in loss of activity.
Therefore, how to use semi-preparative high performance to improve the purity without affecting the activity needs further investigation. Egg white source thrombininhibitory peptides obtained by the above method was stable under the roomtemperature and pH-neutral environment., and easily affected by heavy metal saltsand organic solvents compared with other peptides. Egg white source thrombininhibitory peptides can reach the anticoaglant function by affecting the extrinsiccoagulation pathway. Compared with clinical medicine, the IC50of egg white sourcethrombin inhibitory peptides is bigger,and the demiperiod is shorter, but due to theadvantage of broad source and high safety, it has a wide market and prospect asfunctional food aiming prevention of thrombotic diseases.
生物活性肽近些年来因其安全性高、易吸收、功效好，被广泛应用于功能性因子的开发，例如抗氧化肽、降血压肽、抗疲劳肽、增强免疫力肽和增强记忆力肽等。我国是禽蛋大国，禽蛋多为直接食用，深加工产品较少。由于蛋黄的需求量较大，导致剩余蛋清的浪费。而蛋清中有许多功能活性因子，将其通过酶解可得到具有生物活性功能的肽。有文献表明，蛋清中具有抗凝物质，本文致力于从来源广泛并安全的蛋清中分离纯化具有抗凝活性的物质。
本文对比了Alcalase、中性蛋白酶、风味蛋白酶和胰蛋白酶的水解活性，筛选出最优的水解酶。对比了普通酶解、加热预处理酶解、微波辅助酶解和超声波辅助酶解四种酶解前处理方式对蛋清蛋白质酶解水解度的影响，选择最优的酶解方式。考察了酶解时间、加酶量、底物质量分数、酶解pH和酶解温度等五个单因素，并选取合适的范围以蛋清蛋白质水解度和蛋清源抗凝血肽的抗凝血活性为指标构建多元线性回归方程，确定最优的酶解工艺。运用Sephadex G-50和半制备高效液相色谱对蛋清蛋白质酶解物进行纯化，选取出抗凝血活性高的组分。研究所得抗凝血活性高的组分的pH、温度、金属离子和有机溶剂的稳定性并利用紫外光谱和傅里叶红外光谱研究所得组分可能包含的基团以及二级结构。确定蛋清源抗凝血肽的抗凝血活性IC50值及抗凝的途径。
结果表明采用Alcalase、加热预处理后酶解蛋清蛋白质的水解效果好。根据单因素实验选择2h作为酶解时间。综合考虑蛋清蛋白质水解度及蛋清源抗凝血肽的活性，通过单因素实验选取加酶量为3%、4%、5%，底物质量之比选取1%、3%、5%，pH选取7、8、9，温度选取30℃、40℃、50℃进行实验优化设计。分别以蛋清蛋白质水解度和蛋清源抗凝血肽的抗凝血活性作为指标建立多元线性回归模型。最优的实验方案为加酶量5%，底物质量之比1%，pH8.0，温度50℃时，抗凝血活性为68.92%。以蛋清蛋白质水解度为指标的模型，底物质量之比为显著性水平，以蛋清源抗凝血肽为指标检测，pH为显著性水平。方程的拟合度较好，无多重共线性。将蛋清蛋白质酶解物通过Sephadex G-50纯化后组分4抗凝血活性最高，将组分4经半制备高效液相色谱纯化后组分Ⅶ抗凝血活性最好。酶解物的抗凝血活性为69%、经Sephadex G-50纯化后为85%，经半制备高效液相色谱纯化后为71%，可能是由于有机溶剂对蛋清源抗凝血肽的活性造成了影响。将纯化后的样品通过紫外光谱研究酶解后的样品出现了蓝移，通过红外光谱分析蛋清源抗凝血肽的各构象占总构象的比例。酶解和Sephadex G-50纯化可使α螺旋增加，但半制备高效液相色谱却使样品β折叠的比例有所回升，推测构象的改变可影响其活性的强弱。稳定性实验表明pH为6，温度为30℃时蛋清源抗凝血肽的活性较为稳定。钠盐对蛋清源抗凝血肽的稳定性无显著性影响，钾盐和硫酸铜以及有机溶剂均对其有着不同程度的影响。蛋清源抗凝血肽的IC50值为28.04mg/mL，主要通过内源性途径实现抗凝血的效果。
从结果可以看出酶解蛋白质可获得优良的生物活性肽，以分子量作为分离指标的交联葡聚糖凝胶柱色谱进行纯化适用于蛋清源抗凝血肽的纯化，但是以极性为分离指标的半制备高效液相色谱法虽然能够提高纯度，但是由于有机溶剂对生物活性肽结构的破坏，导致活性的损失。故如何在不影响活性的情况下更好地运用半制备高效液相色谱进行分离需要进一步的研究。通过上述方法分离得到的蛋清源抗凝血肽室温下，pH中性的环境较为稳定，与其他肽类似，重金属盐和有机溶剂对其影响较大。蛋清源抗凝血肽可以通过影响内源性凝血途径达到抗凝的效果，虽然该肽与临床用药相比IC50值较大，体外半衰期较短，但是由于其来源广泛，安全性高，作为针对预防血栓类疾病的保健品具有较为广阔的研发市场及前景。
Cerebrovascular disease has become a disease which has the highest incidenceand lethality, and most of the disease is related to thrombus. However, the clinicalmedicine such as hamocura, tonka bean campor, streptokinase, urokinase are easy tolead to adverse effect such as bleed. In recent years, active compounds with theantithrombotic extracted from leech, tick innsect, ancylostome, snake venom and beevenom have good anticoagulated blood effect, but the adverse effect is also great.Therefore, it is significant to develop antithrombotic effect with high safety and broadsource.
Biologically active peptide such as antioxidant peptide, antihypertensive peptide,antifatigue peptide, strengthening immunity peptide and strengthening rememberancepeptide have been widely used in the development of functional factors for theadvantages of high safety, easy absorption, good function. China is a country withpoultry eggs, most of the poultry eggs are directly used and the deep processingproducts are less. Furthermore, the big demand for the egg yolk lead to the waste ofegg white. However, there are many functional actors in the egg white, and thebiologically active peptides can be obtained by enzymolysis. Literatures show thatthere are antithrombotic compounds in the egg white, and the purpose of this paper isto extract, separate and purify antithrombotic compounds from egg white which havethe advantage of high safety and broad source.
The hydrolytic activity of Alcalase, dispase, flavor protease and trypsinase werecompared, and the optimal hydrolase were selected. The effect of pre-procssingmethod of ordinary enzymolysis, heating pre-processing enzymolysis,microwave-aided enzymolysis and ultrasound aided enzymolysis on degree ofhydrolysis of egg white protein were compared and the best method was optimized.Five single factors like enzymatic hydrolysis time, enzyme and substrate ratio,substrate concentration, pH and hydrolysis temperature were examined, based on the appropriate scope, a multiple linear regression equation with protein degree ofhydrolysis of egg white and anticoagulant activity of egg white source thrombininhibitory peptide as indexes was built to determine the optimal hydrolysis process.Sephadex g-50and semi-preparative HPLC were used to purify egg white proteinhydrolysates and select anticoagulant components.The pH, temperature, metal ionsand stability of organic solubitily were studied, and ultraviolet spectroscopy andFourier transform infrared spectroscopy were used to determine the possiblefunctional group and secondary structure. The anticoagulant activity IC50value andthe anticougulant pathway were determined.
The results indicated that Alcalase has the best enzymolysis effect, and theheating was selected as the pre-processing method for its simplicityin during theexperiment instead of microwave aided enzymolysis which has the best effect as theresults show. The degree of hydrolysis increases with the time of enzymatichydrolysis, but the rate slowed down after2h, so2h was chosen as enzymatichydrolysis time. Take both the the degree of hydrolysis of the egg white protein andthe activity of egg white source thrombin inhibitory peptide into consideration, basedon single factor experiment, the following conditions were set to conduct theExperimental optimization experiment design: enzyme and substrate ratio:3%、4%、5%, substrate concentration:1%、3%、5%,pH:7.0、8.0、9.0，hydrolysis temperature:30℃、40℃、50℃.
Multiple linear regression model was built with the protein degree of hydrolysisof egg white and anticoagulant activity of egg white source thrombin inhibitorypeptide as indexes was built respectively. The optimal experimental program was asfollows: enzyme and substrate ratio:5%, substrate concentration:1%,pH:8.0，hydrolysis temperature:50℃. anticoagulant activity:68.92%. The model with degreeof hydrolysis of egg white protein as an indicator showed significant level onsubstrate concentration level of significance, and the one with egg white source ofthrombin inhibition peptide as indicators detected pH as the significant level.
The equation fitting is good, and no multiple collinearities. component4fromegg white protein hydrolysates puried by Sephadex G-50has the highest anticoagulant activity, and the component VII after the purification of Semi-preparative HPLCpossessed the best anticoagulant activity. The anticoagulant activity of hydrolysateswas69%, after purification by Sephadex G-50is85%, and after Semi-preparativeHPLC was71%, the possible reseason is that organic solvents and the rigid structureof C18caused an impact on the activity of egg white source of thrombin inhibitorypeptide. The study of purified samples by ultraviolet spectroscopy showed redshift,and the infrared spectroscopy indicated that the egg white powder acrylamide I bandsecondary structure were mainly constituded by β conformation and random coil,accounting for85.74%.
In samples after the enzymolysis, α-helix, βcorner, and antiparallel β-sheetincreased, while forward parallel β-folded and random coil decreased. In samples afterSephadex G-50purification, all the four conformation decreased except for theincrease of α-helix. In the samples after purification by semi-preparative HPLC,βcorner and antiparallel β-sheet had gone up, and the rest of conformation werereduced.
Stability experiments showed that activity of egg white source thrombininhibitory peptides was more stable under the condition of pH6.0and the temperatureis30℃. Sodium salt has no significant effect on the stability of egg white sourcethrombin inhibitory peptides, while potassium and copper sulfate and organic solventshave varying degrees of impact.The IC50value of egg white source of thrombininhibition inhibitory peptides is28.04mg/mL and play anticoagulant effect mainlythrough the endogenous pathway.
It can be seen from the results that high quality bioactive peptides can beobtained by enzymolysis of protein, and cross-linked dextran gel columnchromatography with molecular weight as separate indicators was suitable for thepurification of egg white source thrombin inhibitory peptides, while semi-preparativeHPLC with polarity as the separation indicators was not suitable, although it canimprove the purity, organic solvents can also cause the destruction the structure of thebioactive peptides, resulting in loss of activity.
Therefore, how to use semi-preparative high performance to improve the purity without affecting the activity needs further investigation. Egg white source thrombininhibitory peptides obtained by the above method was stable under the roomtemperature and pH-neutral environment., and easily affected by heavy metal saltsand organic solvents compared with other peptides. Egg white source thrombininhibitory peptides can reach the anticoaglant function by affecting the extrinsiccoagulation pathway. Compared with clinical medicine, the IC50of egg white sourcethrombin inhibitory peptides is bigger,and the demiperiod is shorter, but due to theadvantage of broad source and high safety, it has a wide market and prospect asfunctional food aiming prevention of thrombotic diseases.
引文
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