CN101402672A - Method for extracting water-soluble protein component of yolk - Google Patents
Method for extracting water-soluble protein component of yolk Download PDFInfo
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- CN101402672A CN101402672A CNA2008101596952A CN200810159695A CN101402672A CN 101402672 A CN101402672 A CN 101402672A CN A2008101596952 A CNA2008101596952 A CN A2008101596952A CN 200810159695 A CN200810159695 A CN 200810159695A CN 101402672 A CN101402672 A CN 101402672A
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Abstract
The invention provides a method for extracting water soluble protein of yolk, pertaining to the technical field of bioseparation engineering. The method is realized mainly by the application of ultrafiltration separation technology, and four types of water soluble protein of yolk with high purity, which include immunoglobulin of yolk, vitellin, phosvitin and unknown protein with the molecular weight of 50kDa, and can be extracted from the yolk through five simple steps of dilution, freeze thawing, centrifugation, filtration and ultrafiltration. During the extracting process, any organic solvent is not used, thus effectively avoiding the protein denaturation caused by the organic solvent and precluding hidden danger caused by solvent residues on product security.
Description
Technical field
The invention belongs to bioseparation engineering and technical field, specifically is the method for using ultra-filtration technique four kinds of water-soluble protein components of separation and Extraction from Ovum Gallus domesticus Flavus, and product purity all reaches more than 85%, and portioned product purity is higher than 95%.
Background technology
Ovum Gallus domesticus Flavus contains 119 kinds of protein, accounts for 17.8% of total mass.Wherein, the most of combination with fat forms lipoprotein, and water miscible protein is less relatively, common kind lecithality immunoglobulin (Ig), vitellin(Vt), vitellin, phosvitin etc.
Yolk immunoglobulin is meant the main immunoglobulin that exists in the Ovum Gallus domesticus Flavus, is hen 7～10 days after accepting immunity, i.e. yolk ripening stage, optionally transferred in the yolk by the immunoglobulin IgG in the hen blood and form.So Yolk immunoglobulin is equivalent to mammiferous IgG, mammiferous IgG then is a most important antibody in the primary immune response.In anti-infective, play the effect of main force, can promote the phagolysis of mononuclear macrophage, the bacteriotoxic toxicity that neutralizes, and can combine the ability that makes virus lose host cells infected with virus antigen.
At present, obtain antibody by the hen immunity and obtained extensive concern, be considered to one of optimal path that obtains antibody.Because compare with mammiferous IgG, Yolk immunoglobulin has the following advantages: 1. differ bigger owing to plant system's distance, can not intersect serological reaction between the Yolk immunoglobulin of bird and the mammalian immune sphaeroprotein, this helps improving specificity and the susceptibility that immunodiagnosis detects; 2. Yolk immunoglobulin stable in properties.Between pH3-12, below 75 ℃, activity is unaffected substantially; 3. need not blood sampling, only need to collect egg, and cheap and easy to get.Up to the present, obtained to comprise the multiple antibody of SARS virus by the method for hen immunity.
Vitellin and phosvitin are topmost two kinds of phosphorproteins in the Ovum Gallus domesticus Flavus, also are the important nutrient of egg.There is stronger affinity interaction between them, normal in conjunction with forming the cryptomere particle.The molecule of vitellin is spherical in shape, and protein by 80% and 20% fat constitute; Phosvitin accounts for 2% of yolk dry weight, and the amino acid of intramolecularly 50% is Serine, and 90% hexamethyl phosphoric acidization.
At present, the main method of isolated protein has from yolk: salting-out process, polyethylene glycol precipitation, chloroform extraction method, medium chain fatty acid method, surfactant method etc.Yet all there is certain limitation in these technology, and wherein, salting-out process and polyethylene glycol precipitation cause proteinic inactivation and sex change easily; The processing cycle of chloroform extraction method, medium chain fatty acid method, surfactant method is longer, has residue problem.
Membrane separation process is often referred to the film that utilizes natural or synthetic, is impellent with outside energy or chemical potential difference, to two components or multi-component mixture separate, the method for purification and enrichment.Membrane separation technique is a high efficient separation technology that grows up the 1950's, compares with traditional isolation technique, but has separation efficiency height, the low operate continuously of energy consumption, is easy to amplify, advantage such as pollution-free.
Ultra-filtration membrane is meant membrane pore size between 1～100nm, has the composite membrane of unsymmetrical structure.Because the size of its aperture size and biomacromolecule is comparatively approaching, so can be used for the isolation and purification of biomacromolecule such as protein etc.Ultra-filtration and separation is a molecular level, can hold back the macromole solute in the solution, and the small molecules solute is seen through; Sometimes also can make the protein molecule of pre-separation and film surface have electric charge of different nature, by the electrostatic interaction between the protein molecule, between protein and the film surface, reach isolating purpose again by microenvironment in the regulator solution.At present, in the biological processing field, ultra-filtration technique is usually used in protein purification and concentrates, in industrial existing more application, but because kinds of protein complexity in the natural system, and have more isozyme, it is few to use ultrafiltration process directly to separate the example that obtains highly purified functional protein, and the research of this respect still is in laboratory scale.
Summary of the invention
The purpose of this invention is to provide a kind of environmental friendliness, simple to operate, four kinds of yolk water soluble protein ultrafiltration extracting method being easy to amplify, and all product purities can reach all more than 85%, portioned product purity is higher than 95%.
The technological line that the present invention takes is to use ultrafiltration process directly to separate, and realizes as follows:
1. eggshell, egg white and the membrane of yolk of egg are removed, got egg yolk liquid;
2. use deionized water that yolk liquid is diluted to 6～15 times, the best is 7～10 times, fully stir 20～30 minutes after, in-20 ℃ freezing 8～12 hours, thaw again;
Above-mentioned partially thawed liquid is centrifugal under 4 ℃ 3., discard precipitation, get supernatant liquor;
4. utilizing molecular weight cut-off is that the commercially available ultra-filtration membrane of 30～100kDa carries out ultra-filtration and separation to the supernatant liquor that above-mentioned steps obtains, separation condition: 4 ℃, pH4.0～10.0, the best is pH6.0～8.0, obtains to dam liquid A and filtrate A ', and filtrate A ' is phosvitin solution;
5. utilizing molecular weight cut-off is that the commercially available ultra-filtration membrane of 70～150kDa carries out ultra-filtration and separation to the trapped fluid A that step 4 obtains, separation condition: 4 ℃, pH4.0～10.0, the best is pH6.0～8.0, acquisition dam liquid B and filtrate B ', trapped fluid B is highly purified Yolk immunoglobulin solution, and filtrate B ' is a kind of yolk water soluble protein solution of the unknown, and this proteinic molecular weight is 50kDa, and iso-electric point (pI) is 8.0;
6. the partially thawed liquid that step 2 is obtained was placed 1～2 hour under 4 ℃ of conditions, got the supernatant liquid part, utilized the filter paper filtering of 0.22 μ m, got mother liquor;
7. utilize molecular weight cut-off the mother liquor that step 6 obtains to be carried out ultra-filtration and separation, separation condition for the commercially available ultra-filtration membrane of 300kDa: 4 ℃, pH6.0～7.0, gained trapped fluid C is a vitellin solution;
8. resulting filtrate A ', filtrate B ', trapped fluid B and trapped fluid C are distinguished freeze-drying 10～12 hours, promptly obtain four kinds of yolk water soluble protein dry powder.
The present invention directly uses the ultra-filtration and separation technology and extracted four kinds of water-soluble protein components from Ovum Gallus domesticus Flavus, is the agnoprotein of 50kDa comprising Yolk immunoglobulin, vitellin, phosvitin and a kind of molecular weight.Because ultra-filtration membrane is meant membrane pore size at 1～100nm, has the composite membrane of unsymmetrical structure.The size of its aperture size and biomacromolecule is comparatively approaching, so can be used for the isolation and purification of biomacromolecule such as protein etc.Ultra-filtration and separation is a molecular level, can hold back the macromole solute in the solution, and the small molecules solute is seen through, sometimes also can be by microenvironment in the regulator solution, make the protein molecule and the film surface of pre-separation have electric charge of different nature, by the electrostatic interaction between the protein molecule, between protein and the film surface, reach isolating purpose again.
Embodiment
Embodiment 1, extracts the method for water-soluble protein component of yolk from the Calusena lansium egg, step:
1. get eight fresh Calusena lansium eggs, clean eggshell, dry with paper again, behind removal eggshell and the egg white, gently membrane of yolk is punctured, collect egg yolk liquid, about 100mL with syringe needle with tap water;
2. use deionized water that the yolk liquid that above-mentioned steps obtains is diluted to 700mL, put into stirrer, fully stir 20 minutes after, place-20 ℃ freezing 8 hours, thaw again;
3. get above-mentioned thawing solution 400mL, under 4 ℃, with 3000rpm centrifugal, discard precipitation, get supernatant liquor, about 350mL;
4. utilize molecular weight cut-off for the commercially available polysulfone membrane of 30kDa the supernatant liquor that step 3 obtains to be carried out ultra-filtration and separation, separation condition is: 4 ℃, pH6.0, and filtrate is a phosvitin solution, purity 87.3%;
5. utilize molecular weight cut-off for the commercially available regenerated cellulose film of 70kDa the trapped fluid that step 4 obtains to be carried out the ultra-filtration and separation second time, separation condition is: 4 ℃, pH6.0, the trapped fluid of acquisition are Yolk immunoglobulin solution, purity 95.1%; Filtrate is that molecular weight is the agnoprotein solution of 50kDa, purity 96%;
6. the thawing solution 300mL that gets step 2 and obtained placed 1 hour under 4 ℃ of conditions, got the supernatant liquid part, utilized the filter paper filtering of 0.22 μ m, got mother liquor;
7. utilize molecular weight cut-off for the commercially available ultra-filtration membrane of 300kDa the mother liquor that step 6 obtains to be carried out ultra-filtration and separation, separation condition: 4 ℃, pH6.0, the trapped fluid of acquisition are vitellin solution, purity 98.4%;
8. four kinds of protein solns that will obtain place-60 ℃ of cold-trap freeze-drying 10 hours, can obtain the about 730mg of agnoprotein dry powder of the about 406mg of phosvitin dry powder, the about 610mg of Yolk immunoglobulin dry powder, the about 724mg of vitellin dry powder, 50kDa.
Embodiment 2, extract the method for water-soluble protein component of yolk from white skin egg, step:
1. get six fresh white skin eggs, clean eggshell, dry with paper again, behind removal eggshell and the egg white, gently membrane of yolk is punctured, collect egg yolk liquid, about 76mL with syringe needle with tap water;
2. use deionized water that the yolk liquid that above-mentioned steps obtains is diluted to 760mL, put into stirrer, fully stir 30 minutes after, place-20 ℃ freezing 12 hours, thaw again;
3. get above-mentioned thawing solution 420mL, under 4 ℃, with 6000rpm centrifugal, discard precipitation, get supernatant liquor, about 360mL;
4. utilize molecular weight cut-off for the commercially available polysulfone membrane of 100kDa the supernatant liquor that step 3 obtains to be carried out ultra-filtration and separation, separation condition is: 4 ℃, pH8.0, and filtrate is a phosvitin solution, purity 85.2%;
5. utilize molecular weight cut-off for the commercially available polysulfone membrane of 150kDa the trapped fluid that step 4 obtains to be carried out the ultra-filtration and separation second time, separation condition is: 4 ℃, pH8.0, the trapped fluid of acquisition are Yolk immunoglobulin solution, purity 91.8%; Filtrate is that molecular weight is the agnoprotein solution of 50kDa, purity 95.4%;
6. the thawing solution 340mL that gets step 2 and obtained placed 2 hours under 4 ℃ of conditions, got the supernatant liquid part, utilized the filter paper filtering of 0.22 μ m, got mother liquor;
7. utilize molecular weight cut-off for the commercially available ultra-filtration membrane of 300kDa the mother liquor that step 6 obtains to be carried out ultra-filtration and separation, separation condition: 4 ℃, pH7.0, the trapped fluid of acquisition are vitellin(Vt) solution, purity 98.1%;
8. four kinds of protein solns that will obtain place-60 ℃ of cold-trap freeze-drying 12 hours, obtain the about 597mg of agnoprotein dry powder of the about 302mg of phosvitin dry powder, the about 483mg of Yolk immunoglobulin dry powder, the about 658mg of vitellin(Vt) dry powder, 50kDa.
Embodiment 3, extract the method for water-soluble protein component of yolk from egg, step:
1. get ten fresh eggs, clean eggshell, dry with paper again, behind removal eggshell and the egg white, gently membrane of yolk is punctured, collect egg yolk liquid, about 122mL with syringe needle with tap water;
2. use the phosphate buffer soln of pH5.0,200mM that the yolk liquid that above-mentioned steps obtains is diluted to 1000mL, put into stirrer, fully stir 25 minutes after, place-20 ℃ freezing 10 hours, thaw again;
3. get above-mentioned thawing solution 600mL, under 4 ℃, with 5000rpm centrifugal, discard precipitation, get supernatant liquor, about 515mL;
4. utilize molecular weight cut-off for the commercially available polysulfone membrane of 100kDa the supernatant liquor that step 3 obtains to be carried out ultra-filtration and separation, separation condition is: 4 ℃, pH7.0, and filtrate is a phosvitin solution, purity 86.7%;
5. utilize molecular weight cut-off for the commercially available regenerated cellulose film of 100kDa the trapped fluid that step 4 obtains to be carried out the ultra-filtration and separation second time, separation condition is: 4 ℃, pH7.0, the trapped fluid of acquisition are Yolk immunoglobulin solution, purity 93.6%; Filtrate is that molecular weight is the agnoprotein solution of 50kDa, purity 92.8%;
6. the thawing solution 400mL that gets step 2 and obtained placed 1.5 hours under 4 ℃ of conditions, got the supernatant liquid part, utilized the filter paper filtering of 0.22 μ m, got mother liquor;
7. utilize molecular weight cut-off for the commercially available ultra-filtration membrane of 300kDa the mother liquor that step 6 obtains to be carried out ultra-filtration and separation, separation condition: 4 ℃, pH6.0, the trapped fluid of acquisition are vitellin(Vt) solution, purity 98.3%;
8. four kinds of protein solns that will obtain place-60 ℃ of cold-trap freeze-drying 11 hours, obtain the about 972mg of agnoprotein dry powder of the about 558mg of phosvitin dry powder, the about 840mg of Yolk immunoglobulin dry powder, the about 880mg of vitellin(Vt) dry powder, 50kDa.
Technology of the present invention only relates to dilution, freeze thawing, centrifugal, filtration and five easy steps of ultrafiltration, present relatively industry, laboratory extraction process, have simple to operate, separate to concentrate carry out synchronously, loss of activity is little, the rate of recovery is high characteristics, and not with an organic solvent, can effectively avoid the protein denaturation that organic solvent causes in the traditional extraction process, stop the product safety hidden danger that organic solvent residual causes; Energy consumption is low, environmental pollution is little, separation efficiency is high, product purity is adjustable, controlling factor is single, is convenient to industry and amplifies.
Claims (3)
1. the extracting method of a water-soluble protein component of yolk is characterized in that carrying out as follows:
1. eggshell, egg white and the membrane of yolk of egg are removed, got egg yolk liquid;
2. use deionized water that yolk liquid is diluted to 6～15 times, fully stir 20～30 minutes after, in-20 ℃ freezing 8～12 hours, thaw again;
3. above-mentioned partially thawed liquid is centrifugal under 4 ℃, discard precipitation, get supernatant liquor;
4. utilizing molecular weight cut-off is that the commercially available ultra-filtration membrane of 30～100kDa carries out ultra-filtration and separation, separation condition to the supernatant liquor that above-mentioned steps obtains: 4 ℃, pH4.0～10.0, obtain to dam liquid A and filtrate A ', and filtrate A ' is phosvitin solution;
5. utilizing molecular weight cut-off is that the commercially available ultra-filtration membrane of 70～150kDa carries out ultra-filtration and separation to the trapped fluid A that 4. step obtains, separation condition: 4 ℃, pH4.0～10.0, acquisition dam liquid B and filtrate B ', trapped fluid B is highly purified Yolk immunoglobulin solution, and filtrate B ' is a kind of yolk water-soluble protein solution of the unknown, this proteinic molecular weight is 50kDa, and iso-electric point (pI) is 8.0;
6. the partially thawed liquid that 2. step is obtained was placed 1～2 hour under 4 ℃ of conditions, got the supernatant liquid part, utilized the filter paper filtering of 0.22 μ m, got mother liquor;
7. utilize molecular weight cut-off the mother liquor that 6. step obtains to be carried out ultra-filtration and separation, separation condition for the commercially available ultra-filtration membrane of 300kDa: 4 ℃, pH6.0～7.0, gained trapped fluid C is a vitellin solution;
8. resulting filtrate A ', filtrate B ', trapped fluid B and trapped fluid C are distinguished freeze-drying 10～12 hours, promptly obtain four kinds of yolk water soluble protein dry powder.
2. according to the extracting method of the described water-soluble protein component of yolk of claim 1, it is characterized in that using deionized water that yolk liquid is diluted to 7～10 times.
3. according to the extracting method of the described water-soluble protein component of yolk of claim 1, it is characterized in that step 4. with step 5. in the condition of said ultra-filtration and separation be 6.0～8.0,4 ℃ of pH.
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| CNA2008101596952A CN101402672A (en) | 2008-11-06 | 2008-11-06 | Method for extracting water-soluble protein component of yolk |
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| CNA2008101596952A CN101402672A (en) | 2008-11-06 | 2008-11-06 | Method for extracting water-soluble protein component of yolk |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053799A (en) * | 2016-08-03 | 2016-10-26 | 上海市水产研究所 | Gold-labeled test paper for rapid determination of crab vitellin content and application of colloidal-gold test paper |
| CN112931752A (en) * | 2021-02-24 | 2021-06-11 | 朱鹏辉 | Diabetes mellitus non-drug treatment reverse protein |
| CN113527542A (en) * | 2021-08-04 | 2021-10-22 | 广西大学 | Method for efficiently separating bagasse high-yield high-purity high-molecular-weight hemicellulose by freeze thawing assisted with p-toluenesulfonic acid |
| CN117164702A (en) * | 2023-09-13 | 2023-12-05 | 江苏润洁生物科技有限公司 | Preparation method and application of yolk globulin for clinical HPV infection |
-
2008
- 2008-11-06 CN CNA2008101596952A patent/CN101402672A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053799A (en) * | 2016-08-03 | 2016-10-26 | 上海市水产研究所 | Gold-labeled test paper for rapid determination of crab vitellin content and application of colloidal-gold test paper |
| CN112931752A (en) * | 2021-02-24 | 2021-06-11 | 朱鹏辉 | Diabetes mellitus non-drug treatment reverse protein |
| CN113527542A (en) * | 2021-08-04 | 2021-10-22 | 广西大学 | Method for efficiently separating bagasse high-yield high-purity high-molecular-weight hemicellulose by freeze thawing assisted with p-toluenesulfonic acid |
| CN117164702A (en) * | 2023-09-13 | 2023-12-05 | 江苏润洁生物科技有限公司 | Preparation method and application of yolk globulin for clinical HPV infection |
| CN117164702B (en) * | 2023-09-13 | 2024-05-10 | 江苏润洁生物科技有限公司 | Preparation method and application of yolk globulin for clinical HPV infection |
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Open date: 20090408 |