CN100387620C - Bovine serum albumin hot ethanol extracting process - Google Patents
Bovine serum albumin hot ethanol extracting process Download PDFInfo
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- CN100387620C CN100387620C CNB2003101166025A CN200310116602A CN100387620C CN 100387620 C CN100387620 C CN 100387620C CN B2003101166025 A CNB2003101166025 A CN B2003101166025A CN 200310116602 A CN200310116602 A CN 200310116602A CN 100387620 C CN100387620 C CN 100387620C
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Abstract
The present invention discloses an extracting process for using a hot ethanol method to extract bovine albumin. The process is composed of the following steps: separating plasma; adding trisodium citrate dihydrate in bovine blood for centrifugation and extraction; placing the collected plasma into an interlayer reaction tank; heating the interlayer reaction tank; adding sodium octanoate into the tank; slowly mixing the mixture; adding hydrochloric acid at the temperature of 55 to 65 DEG C. to regulate the pH value to 4.0 to 6.5; adding ethyl alcohol; filtering, desalting, concentrating and freezing the mixture to obtain finished products. The process has the advantages of few materials, short process flow, five times of the production efficiency of a cold salting out method for five days, easy control of each step, easy operation, suitable mass production, high yield of 1.8 to 2.0% in general, low cost which is 50% lower than the cold salting out method, and good quality. Only a small quantity of anticaking agent and a small quantity of protecting agent are added in the production process without other salts and chemical substances, and the main extracting agent is ethyl alcohol. Thereby, the pure quality of the finished products is ensured.
Description
Technical field
The present invention relates to a kind of albuminous method of extracting from bovine serum, specifically is a kind of bovine serum albumin heat ethanol methods extraction process.
Background technology
Bovine serum albumin is used for the coating of diagnostic reagent, its preparation method is more, but majority is still at the experimental stage, be applicable to the following method that has of pilot scale and formal production: (1) salting-out process, divide cold salting-out process and hot salting-out process, the main technique flow process of cold salting-out process be fresh ox blood is centrifugal, add pure water, add ammonium sulfate, filtration, filtrate dialysis desalting, add that polyoxyethylene glycol, dialyzate concentrate, freeze-drying is a finished product; Present method materials are many, and adding ammonium sulfate needs 40～50% of ox blood amount, add polyoxyethylene glycol and be 2～4 times of ox blood amount, add 20～40 times of pure water need ox blood amount of the usefulness of dialyse; Time-consuming, cold salting-out process dialysis needs 48 hours, and dehydration needs 24 hours, and whole process flow needs 5 working dayss; Yield is low, is generally 0.8～1.0%; More than 3 make the production efficiency of cold salting-out process low, cost is higher.(2) organic solvent precipitation method, the most frequently used with the E.J.CohnShi method, but present method needs 0 ℃～5 ℃ low temperature to carry out, and wastes time and energy, and one way needs 7, and yield is not high, and about 1%, its cost is also higher.
Summary of the invention
The object of the present invention is to provide a kind of bovine serum albumin heat ethanol methods extraction process, its materials is few, saves time the yield height; Can reduce cost by a relatively large margin with this technology.
Technical scheme of the present invention is as follows:
Technology of the present invention has following steps:
A, separated plasma are got the whizzer that fresh ox blood is put into the band refrigerating function, and 10% the concentration that adds blood volume is 3.8% Sodium Citrate, and centrifugal, temperature is below 2 ℃, and separated plasma, blood cell are collected blood plasma, and blood cell is used in addition;
B, extraction, blood plasma is put into the interlayer retort, interlayer internal heating water, the limit heating edge adds the Sodium octoate of bovine serum volumetrical 0.2-0.3%, slowly stir, temperature is at 55 ℃～65 ℃, and pH value is transferred in the dissolving back, and adding concentration is 1 mole hydrochloric acid, PH is transferred to 4.0～6.5,5.5% the concentration that adds the serum total amount then is 95% ethanol, filters, and filter residue is abandoned it;
C, desalination and concentrated with the ultra-fine filter wash-out salt below 20000 molecular weight and other small molecular weight impurity, concentrate filtrate simultaneously;
D, freeze-drying are pressed the freeze-drying curve freeze-drying with concentrated solution, are about to concentrated solution and are cooled to-40 ℃ rapidly, continue about 1 hour, are raised to-25 ℃, continue 10-20 hour, slowly are warmed up to 0 ℃ again, are raised to 20 ℃ of storage temperatures again in lasting 1-4 hour., be finished product.
Temperature among the above-mentioned step B is 55 ℃～60 ℃.
PH among the step B of above-mentioned extraction is 4.5-6.0.
The used Sodium Citrate of this technology is an antithrombotics, and Sodium octoate is albuminous protective material, makes it not decompose more than 55 ℃ in temperature.
The invention has the advantages that: 1, materials are few, and the used all colder salting-out process of Sodium Citrate, Sodium octoate, hydrochloric acid, ethanol of present method is few, can save input.2, technical process is short, all extracts flow process and only needs 24 hours, needs 5 days to enhance productivity 5 times than cold salting-out process.3, each step of this technology all is easy to grasp, and processing ease is fit to produce in enormous quantities.4, yield height is generally at 1.8-2.0%.5, cost is low, and is lower by 50% than cold salting-out process cost, remarkable in economical benefits.6, quality is good, only adds a small amount of antithrombotics and protective material Sodium Citrate, Sodium octoate in process of production, does not contain other salt and chemical substance, and its main extraction agent is an ethanol, thereby has guaranteed the pure of finished product quality, and its quality examination the results are shown in Table 1.
Finished product albumin of the present invention is through check, and quality meets industry quality standard fully.
Detect: the purity electrophoresis technique determining, the clarity of solution is measured with spectrophotometer 403nm.
Albuminous quality inspection result of table 1 ox blood of the present invention and quality standard synopsis
| Interventions Requested | Albumin of the present invention | Quality standard |
| Outward appearance | White or off-white color crystalline powder | White or off-white color crystalline powder |
| Dissolution experiment | Dissolving in 2 minutes, solution is clear and bright | Dissolving in 2 minutes, solution is clear and bright |
| Purity | Albumin content 〉=95 | Albumin content 〉=90 |
| 1% albumin solution 403nm absorption value | ≤0.1 | ≤0.35 |
| Freeze-drying moisture | Residual water-content≤5.0% | Residual water-content≤5.0% |
| Function test | Qualified | Bag is by profile |
Can see that by table 1 its albuminous content is higher than quality standard, 1% albumin solution 403nm absorption value is lower than standard, and its impurity is few, the clarity height, and other index conformance with standard illustrate that the ox blood albumin quality of this technology extraction is good.
Description of drawings
Fig. 1 is the prior art processes schema;
Fig. 2 is a process flow sheet of the present invention;
Fig. 3 is freeze-drying curve figure of the present invention.
Embodiment
Embodiment 1:
Technology of the present invention has following steps:
A, separated plasma, get the fresh ox blood of the natural cows of herding, through the quarantine of strictness, guaranteeing does not have the disease of infection, ox blood is put into the whizzer of band refrigerating function, 10% 3.8% the Sodium Citrate that adds blood volume, 4000 rev/mins centrifugal, and temperature is below 2 ℃, separated plasma, blood cell, collect blood plasma, blood cell is used in addition;
B, extraction, blood plasma is put into the interlayer retort, add hot water in the interlayer, the limit heating edge adds the Sodium octoate of bovine serum volumetrical 0.2-0.3%, slowly stirs, temperature is at 55 ℃～60 ℃, pH value is transferred in the dissolving back, and adding concentration is 1 mole hydrochloric acid, and PH is transferred to 6,5.5% the concentration that adds the serum total amount then is 95% ethanol, and be 25-40 minute heat-up time; Filter with canvas then, filter residue is abandoned it;
C, desalination and concentrated with the ultra-fine filter wash-out salt below 20000 molecular weight and other small molecular weight impurity, concentrate filtrate simultaneously;
D, freeze-drying are undertaken by freeze-drying curve, are about to concentrated solution and are cooled to-40 ℃ rapidly, continue about 1 hour, are raised to-25 ℃, continue 10-20 hour, slowly are warmed up to 0 ℃ again, are warmed up to storage temperature again in lasting 1-4 hour.Be finished product.
It is bottled or plastics are packed to be packaged as sealing.The albuminous storage temperature of this ox blood is smaller or equal to 20 ℃.The used Sodium Citrate of this technology is antithrombotics, and is commercially available, and Sodium octoate is albuminous protective material, and the Beijing Chemical Plant produces, and hydrochloric acid and ethanol are commercially available.
Claims (3)
1. bovine serum albumin heat ethanol methods extraction process, it is characterized in that: it is made up of following steps:
A, separated plasma are got the whizzer that fresh ox blood is put into the band refrigerating function, and 10% the concentration that adds blood volume is 3.8% Sodium Citrate, and centrifugal, temperature is below 2 ℃, and separated plasma, blood cell are collected blood plasma, and blood cell is used in addition;
B, extraction, blood plasma is put into the interlayer retort, interlayer internal heating water, the limit heating edge adds the Sodium octoate of bovine serum volumetrical 0.2-0.3%, slowly stir, temperature is at 55 ℃～65 ℃, and pH value is transferred in the dissolving back, and adding concentration is 1 mole hydrochloric acid, PH is transferred to 4.0～6.5,5.5% the concentration that adds the serum total amount then is 95% ethanol, filters, and filter residue is abandoned it;
C, desalination and concentrated with the ultra-fine filter wash-out salt below 20000 molecular weight and other small molecular weight impurity, concentrate filtrate simultaneously;
D, freeze-drying are pressed the freeze-drying curve freeze-drying with concentrated solution, are about to concentrated solution and are cooled to-40 ℃ rapidly, continue about 1 hour, are raised to-25 ℃, continue 10-20 hour, slowly are warmed up to 0 ℃ again, are raised to 20 ℃ of storage temperatures in lasting 1-4 hour again, are finished product.
2. bovine serum albumin heat ethanol methods extraction process according to claim 1 is characterized in that: the temperature among its step B is 55 ℃～60 ℃.
3. bovine serum albumin heat ethanol methods extraction process according to claim 1 is characterized in that: the PH among its step B is 4.5-6.0.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2003101166025A CN100387620C (en) | 2003-11-17 | 2003-11-17 | Bovine serum albumin hot ethanol extracting process |
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| CNB2003101166025A CN100387620C (en) | 2003-11-17 | 2003-11-17 | Bovine serum albumin hot ethanol extracting process |
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| CN1544470A CN1544470A (en) | 2004-11-10 |
| CN100387620C true CN100387620C (en) | 2008-05-14 |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101367865B (en) * | 2008-09-26 | 2012-01-04 | 广州倍绣生物技术有限公司 | Production process for high purity porcine blood albumin and uses thereof |
| CN102241765A (en) * | 2011-05-18 | 2011-11-16 | 内蒙古奇特生物高科技术（集团）有限公司 | Production method for removing fatty acid from bovine serum albumin |
| CN102241767B (en) * | 2011-06-22 | 2013-07-10 | 河南省医药科学研究院 | Extraction method of bovine serum albumin |
| CN104231074A (en) * | 2014-09-09 | 2014-12-24 | 青岛康大食品有限公司 | Purification and preparation method of albumin in cony blood |
| CN105085666A (en) * | 2015-09-15 | 2015-11-25 | 江苏锦宇环境工程有限公司 | Preparation method of bovine serum albumin |
| CN105384791B (en) * | 2015-12-09 | 2016-07-06 | 青海北极牦牛生物科技有限公司 | A kind of albuminous production technology of high-purity Serum of Yaks |
| CN112521488A (en) * | 2020-12-26 | 2021-03-19 | 郑州伊美诺生物技术有限公司 | Preparation method of bovine serum albumin |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0826182A (en) * | 1994-07-15 | 1996-01-30 | Hitachi Zosen Corp | Ladder and manufacture thereof |
| JPH08116985A (en) * | 1994-08-31 | 1996-05-14 | Green Cross Corp:The | Purification of human serum albumin produced by gene manipulation |
| CN1137528A (en) * | 1995-10-31 | 1996-12-11 | 江西省博达生物工程研究所 | Production process for modified low temp. ethanolic human serum albumin |
| US6489443B2 (en) * | 1998-07-10 | 2002-12-03 | Aventis Behring Gmbh | Process for the preparation of a protein solution |
-
2003
- 2003-11-17 CN CNB2003101166025A patent/CN100387620C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0826182A (en) * | 1994-07-15 | 1996-01-30 | Hitachi Zosen Corp | Ladder and manufacture thereof |
| JPH08116985A (en) * | 1994-08-31 | 1996-05-14 | Green Cross Corp:The | Purification of human serum albumin produced by gene manipulation |
| CN1137528A (en) * | 1995-10-31 | 1996-12-11 | 江西省博达生物工程研究所 | Production process for modified low temp. ethanolic human serum albumin |
| US6489443B2 (en) * | 1998-07-10 | 2002-12-03 | Aventis Behring Gmbh | Process for the preparation of a protein solution |
Non-Patent Citations (2)
| Title |
|---|
| 热变性与拄层析法给合生产人血蛋白工艺的研究. 罗亮等.药物生物技术，第5卷第1期. 1998 |
| 热变性与拄层析法给合生产人血蛋白工艺的研究. 罗亮等.药物生物技术，第5卷第1期. 1998 * |
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