| 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. |
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