CN102241765A - Production method for removing fatty acid from bovine serum albumin - Google Patents
Production method for removing fatty acid from bovine serum albumin Download PDFInfo
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- CN102241765A CN102241765A CN2011101243632A CN201110124363A CN102241765A CN 102241765 A CN102241765 A CN 102241765A CN 2011101243632 A CN2011101243632 A CN 2011101243632A CN 201110124363 A CN201110124363 A CN 201110124363A CN 102241765 A CN102241765 A CN 102241765A
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Abstract
The invention discloses a production method for removing fatty acid from bovine serum albumin, comprising the following steps of: dissolving: weighing bovine serum albumin, adding distilled water 10 times of the bovine serum albumin in weight for dissolving at the temperature of 23-27 DEG C; resolving: regulating the pH to 3-3.5 by HCl with the concentration of 0.2N, cooling the solution; absorption: adding activated carbon 4-5% of the solution in weight, putting the suspension into ice water bath, stirring, filtering, and recovering the filter residue for reuse; concentration: regulating the pH value of the filtrate to 7.0 by NaOH of 0.2N, then concentrating by ultrafiltration with an ultrafiltration device below 20000 in molecular weight; and lyophilization: lyophilizing the concentrated solution according to a lyophilizing curve, and subpackaging into finished products. The method has small material input, low cost, simple process and strong operability and is easy in industrialized production. The finished product has high purity and very low content of fatty acid, and can meet the standards required by cell culture and biological engineering experiments. The method has high recovery rate which can reach 90%.
Description
Technical field the present invention relates to a kind of method of purification of biotechnological formulation, relates to a kind of production method of removing residual lipid acid in the bovine serum albumin leaching process specifically.
The present bovine serum albumin of background technology is widely used in diagnostic reagent industry, scientific research fields such as biotechnology, cell cultures.The method for preparing bovine serum albumin is a lot, but all need add the protective material of Sodium octoate as bovine serum albumin in preparation process, and product exists preparation purity not high, the drawback that the lipid acid residual quantity is too high.Some biomedical researches require to use the bovine serum albumin that does not contain free fatty acids, otherwise the lipid acid of the contained high density of bovine serum albumin will influence cell cultures, differentiation, metabolic reaction, biochemical measurement etc.For example, in 3T3-L1 adipocyte atomization, can not use common bovine serum albumin reagent, otherwise influence differentiation and cellular metabolism state; When the free-fat acid concentration that the histocyte of measure cultivating discharges, also must be with the bovine serum albumin of fatty acids not, otherwise be difficult to accurately measure triglyceride hydrolysis and free fatty acids discharges.
Therefore, the bovine serum albumin that traditional technology is extracted carries out the purity that the acid treatment of degreasing fat can improve bovine serum albumin, enlarges the range of application of bovine serum albumin product.
Summary of the invention the object of the present invention is to provide a kind of production method of removing lipid acid in the bovine serum albumin, its materials is few, cost is low, technology is simple, too high in order to fatty acid content in the bovine serum albumin that solves the traditional technology extraction, can not be applicable to the difficult problem of cell cultures, physiotechnology experiment, improve product quality.
Technical scheme of the present invention is as follows:
Production method of the present invention is made up of following steps:
A, dissolving take by weighing bovine serum albumin and are put in the container, add the dissolved in distilled water of 10 times of its weight, are stirred well to the solution clarification, and solvent temperature is at 23～27 ℃;
B, parsing are the HCl of 0.2N with concentration, regulate PH to 3～3.5, carry out ice-water bath or are cooled to 0～4 ℃ being equipped with in the container dislocation mixture of ice and water of the bovine serum albumin solution after the abundant dissolving, continue simultaneously to stir, and keep 30min;
C, absorption, 4～5% the gac that adds the steps A solution weight, mixing obtains suspension, and the container of dress suspension is placed mixture of ice and water to carry out ice-water bath or is cooled to 0～4 ℃, continue simultaneously to stir, kept 1 hour, D, filtration are filtered suspension with qualitative filter paper, collect filtrate, utilize again after filter residue reclaims;
E, concentrated, with the NaOH adjusting pH value to 7.0 of the rapid filtrate of previous step with 0.2N, the ultra-filtration equipment with 10000～20000 molecular weight carries out ultrafiltration and concentration then;
F, freeze-drying are pressed the freeze-drying curve freeze-drying with concentrated solution, and the concentrated solution after the ultrafiltration soon is refrigerated to-40 ℃ rapidly, continue 50～70min, be raised to-25 ℃, continue 10～20 hours, slowly be warming up to 0 ℃ again, kept 1～4 hour, be warming up to 20 ℃ of storage temperatures, kept 30～60 minutes, be packed as finished product.
The temperature of the mixture of ice and water among the above-mentioned steps C is 0～4 ℃.
The invention has the advantages that: 1, materials are few, cost is low, technology is simple.Present method utilizes a spot of gac whip attachment under 0～4 ℃ of temperature can finish in 1 hour.2, workable, be easy to suitability for industrialized production.3, the finished product purity height that is produced, fatty acid content is atomic, can satisfy the standard of cell cultures, biotechnology requirement of experiment.4, the rate of recovery height of present method, the rate of recovery can reach 90%.5, low, the remarkable in economical benefits of cost.
Table 1 is handled forward and backward quality standard contrast table with the inventive method
With the bovine serum albumin after the method processing of the present invention, its fatty acid content is atomic, and other index also reaches the standard before handling.
Description of drawings Fig. 1 is the freeze-drying curve of step F of the present invention.
Embodiment
Embodiment 1
Processing step is as follows:
A, bovine serum albumin are handled: get 1kg bovine serum albumin crude product and put into container, be dissolved in the distilled water of 1OL (10kg), solvent temperature is stirred well to the solution clarification at 23～25 ℃;
B, parsing are the HCl of 0.2N with concentration, regulate PH to 3.2, and the container that above-mentioned solution is housed is placed mixture of ice and water, stir water-bath 30min, and temperature is at 0～4 ℃;
C, absorption take by weighing gac 500g, slowly add while stirring, and stirring and evenly mixing obtains suspension, place mixture of ice and water to carry out ice-water bath in the container of adorning suspension, continue simultaneously to stir, and keep 1 hour;
D, filtration are filtered suspension with qualitative filter paper, collect filtrate, can reuse after the gac recycling;
E, concentrate, filtrate is regulated pH value to 7.0 with the NaOH of 0.2N, is that 20000 Hollow Fiber Ultrafiltration post carries out ultrafiltration and concentration with filtrate with molecular weight then;
F, freeze-drying are pressed the freeze-drying curve freeze-drying with concentrated solution, and the concentrated solution after the ultrafiltration soon is refrigerated to-40 ℃ rapidly, continue 1 hour, are raised to-25 ℃, continue 12 hours, slowly are warming up to 0 ℃ again, keep 4 hours, are warming up to 20 ℃ of storage temperatures, keep 30 minutes; Behind the recycling of packaging weighing products, be finished product after according to the packing specifications packing with Plastic Bottle.
Can see the freeze-drying curve of step of freeze drying of the present invention by Fig. 1, its concentrated solution temperature is refrigerated to-40 ℃ rapidly, continues 1 hour, is raised to-25 ℃, continues 10 hours, slowly is warming up to 0 ℃ again, keeps 1 hour, is warming up to 20 ℃ of storage temperatures, keeps 60 minutes.
A, bovine serum albumin are handled: get 1kg bovine serum albumin crude product, be dissolved in the distilled water of 10L, temperature is 25 ℃, is stirred well to the solution clarification;
B, parsing, with the HCl of 0.2N, regulator solution PH to 3.0, it is 4 ℃ refrigerator chamber that solution is placed refrigerating temperature, stirs 30min;
C, absorption take by weighing gac 450g, slowly add while stirring, add the back and continue to stir 1 hour;
D, filtration are filtered suspension with qualitative filter paper, collect filtrate, can reuse after the gac recycling;
E, concentrate, filtrate is shifted out refrigerator chamber, at room temperature the NaOH with 0.2N regulates pH value to 7.0, is that 20000 ultrafiltration post carries out ultrafiltration and concentration with filtrate with molecular weight then;
F, freeze-drying are pressed the freeze-drying curve freeze-drying with concentrated solution, and the concentrated solution after the ultrafiltration soon is refrigerated to-40 ℃ rapidly, continue 70min, are raised to-25 ℃, continue 18 hours, slowly are warming up to 0 ℃ again, keep 4 hours, are warming up to 20 ℃ of storage temperatures, keep 30 minutes; Behind the recycling of packaging weighing products, be finished product after according to the packing specifications packing with Plastic Bottle.
Above-mentioned finished product packing is the Plastic Bottle of 100g capacity.
The needle-use activated carbon that the used gac (200 order) of the foregoing description is produced for Shanghai gac Co., Ltd., Factory.
The used hydrochloric acid (analytical pure) of the foregoing description is produced for the Bohai Sea, Huludao City chemical reagent factory.
The used sodium hydroxide of the foregoing description (analytical pure) is produced for sky, Tianjin power chemical reagent company limited.
The used bovine serum albumin crude product of the foregoing description is produced by the peculiar biological high-tech art in Inner Mongol (group) company limited, and its fatty acid content is 0.2-0.3%.
Claims (2)
1. production method of removing lipid acid in the bovine serum albumin is characterized in that it is made up of following steps:
A, dissolving take by weighing bovine serum albumin and are put in the container, add the dissolved in distilled water of 10 times of its weight, are stirred well to the solution clarification, and solvent temperature is at 23～27 ℃;
B, parsing are the HCl of 0.2N with concentration, regulate PH to 3～3.5, place mixture of ice and water to carry out ice-water bath in the container that the bovine serum albumin solution after the abundant dissolving is housed or are cooled to 0～4 ℃, continue simultaneously to stir, and keep 30min;
C, absorption add 4～5% gac of steps A solution weight, and mixing obtains suspension, place mixture of ice and water to carry out ice-water bath in the container of dress suspension or are cooled to 0～4 ℃, continue simultaneously to stir, and keep 1 hour;
D, filtration are filtered suspension with qualitative filter paper, collect filtrate, and filter residue reclaims;
E, concentrated, the filtrate that previous step is rapid is regulated pH value to 7.0 with the NaOH of 0.2N, and the ultra-filtration equipment with 10000～20000 molecular weight carries out ultrafiltration and concentration then;
F, freeze-drying are pressed the freeze-drying curve freeze-drying with concentrated solution, and the concentrated solution after the ultrafiltration soon is refrigerated to-40 rapidly
℃, continue 50～70min, be raised to-25 ℃, continue 10～20 hours, slowly be warming up to 0 ℃ again, kept 1～4 hour, be warming up to 20 ℃ of storage temperatures, kept 30～60 minutes, be packed as finished product.
2. the production method of lipid acid in the removal bovine serum albumin according to claim 1 is characterized in that: the temperature of the mixture of ice and water among its step C is 0～4 ℃.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011101243632A CN102241765A (en) | 2011-05-18 | 2011-05-18 | Production method for removing fatty acid from bovine serum albumin |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011101243632A CN102241765A (en) | 2011-05-18 | 2011-05-18 | Production method for removing fatty acid from bovine serum albumin |
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| CN102241765A true CN102241765A (en) | 2011-11-16 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911147A (en) * | 2015-06-15 | 2015-09-16 | 英普乐孚生物技术(上海)有限公司 | Serum-free medium and preparation method thereof |
| CN109734796A (en) * | 2019-02-01 | 2019-05-10 | 广州蕊特生物科技有限公司 | A kind of technique separating albumin from haemolysis serum |
| CN112759643A (en) * | 2021-03-17 | 2021-05-07 | 华兰生物工程股份有限公司 | Degreasing method of serum albumin |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544470A (en) * | 2003-11-17 | 2004-11-10 | 孙福森 | Bovine serum albumin hot ethanol extracting process |
-
2011
- 2011-05-18 CN CN2011101243632A patent/CN102241765A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1544470A (en) * | 2003-11-17 | 2004-11-10 | 孙福森 | Bovine serum albumin hot ethanol extracting process |
Non-Patent Citations (2)
| Title |
|---|
| RAYMOND F. CHEN: "Removal of Fatty Acids from Serum Albumin by Charcoal Treatment", 《THE JOURNAL OF BIOLOQICAL CHEMSTRY》 * |
| 傅亚珍 等: "脂肪酸对牛血清白蛋白的水合及变性的影响", 《生物化学与生物物理学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911147A (en) * | 2015-06-15 | 2015-09-16 | 英普乐孚生物技术(上海)有限公司 | Serum-free medium and preparation method thereof |
| CN109734796A (en) * | 2019-02-01 | 2019-05-10 | 广州蕊特生物科技有限公司 | A kind of technique separating albumin from haemolysis serum |
| CN109734796B (en) * | 2019-02-01 | 2022-04-15 | 广州蕊特生物科技有限公司 | Process for separating albumin from haemolytic serum |
| CN112759643A (en) * | 2021-03-17 | 2021-05-07 | 华兰生物工程股份有限公司 | Degreasing method of serum albumin |
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Application publication date: 20111116 |