CN102241767A - Extraction method of bovine serum albumin - Google Patents
Extraction method of bovine serum albumin Download PDFInfo
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- CN102241767A CN102241767A CN2011101684777A CN201110168477A CN102241767A CN 102241767 A CN102241767 A CN 102241767A CN 2011101684777 A CN2011101684777 A CN 2011101684777A CN 201110168477 A CN201110168477 A CN 201110168477A CN 102241767 A CN102241767 A CN 102241767A
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Abstract
The invention relates to an extraction method of bovine serum albumin, aiming to solve the technical problems of complexity, low purity, high production cost and difficulty in large-lot production of the production process of bovine serum albumin. The extraction method comprises the following steps of: adding a proper amount of sodium palmitate to the separated serum, standing, regulating the pH of the solution, heating in water bath for 1-2 hours, filtering, sequentially adding 3MHCl and sodium chloride to the filtrate, standing at 4 DEG C, filtering to obtain precipitate, adding a proper amount of water to the precipitate, regulating the pH for dissolving the precipitate, then regulating the pH of the solution to 8.0-9.0, dialyzing and lyophilizing. The extraction method of bovine serum albumin has low process materials input, short process flow, simplicity of operation, high production efficiency and suitability for mass production, and the obtained product has high purity and good quality and is easy to preserve.
Description
Technical field
The present invention relates to albuminous preparation method, be specifically related to a kind of bovine serum albumin extracting method.
Background technology
Bovine serum albumin (BSA), claim the 5th component again, it is a kind of sphaeroprotein in the bovine serum, comprise 583 amino-acid residues, molecular weight is 66.430 kDa, iso-electric point is 4.7, is the biochemical reagents of all multidisciplinary and doctors of biology, each field widespread use of pharmacy, also can be used as medicines and health protection food, seasonings.
The most frequently used preparation method of bovine serum albumin is to be that raw material is isolated serum with the ox blood, behind ammonium sulfate precipitation, makes with extra care and gets through sad processing; Chinese patent literature CN 1544470C discloses a kind of bovine serum albumin heat ethanol methods extraction process, its method is to adopt 3.8% Sodium Citrate anticoagulation to obtain blood plasma, heating adds Sodium octoate, adds ethanol behind the adjust pH, more after filtration, desalination, concentrate, after the freeze-drying bovine serum albumin.Need to add higher salt of many costs (as Sodium Citrate, Sodium octoate etc.) and organic chemicals (alcohol solvent etc.) among the above-mentioned bovine serum albumin preparation method, preparing albuminous purity is difficult to guarantee, and production technique is complicated, is difficult to produce in enormous quantities.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of purity height, technical process is simple, cost is low bovine serum albumin extracting method.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
A kind of bovine serum albumin extracting method may further comprise the steps:
(1) gets fresh ox blood, get supernatant liquor after the centrifugation, add and the isopyknic aseptic pyrogen-free deionized water of supernatant liquor, press the 0.024M sodium palmitate solution that 1/4～3/4 of supernatant liquor volume adds 60～70 ℃ again, leave standstill 50～100 min behind the mixing;
(2) upwards go on foot in the gained solution and add dilute hydrochloric acid, regulator solution pH to 4.0～6.5,60～70 ℃ following water-bath 1～2h filters to get filtrate;
(3) upwards go on foot adding 3 mol/L HCl in the gained filtrate by 40% of supernatant volume, add sodium-chlor behind the mixing again, make its mass concentration in solution reach 4%, 0～4 ℃ and place 30～60min down;
(4) leaving standstill after-filtration must precipitate, and presses 4～6% of supernatant volume and add aseptic pyrogen-free deionized water in precipitation, and the solution that adds dilute alkali is transferred pH value of solution to 9.0～10.0;
(5) treat that precipitation fully after the dissolving, adds dilute hydrochloric acid again and transfers pH value of solution to 8.0～9.0,10～16h dialyses in aseptic pyrogen-free deionized water;
(6) dialyzate is put into Freeze Drying Equipment, takes out after 45～50 hours, gets protein powder.
In described step (1), the rotating speed of centrifugation is 2800～3200r/m, and the time is 10～20 min.
In described step (2), (5), the concentration of used dilute hydrochloric acid is 1mol/L.
In described step (4), used dilute alkaline soln is a 1mol/L NaOH solution.
The present invention has actively useful effect:
1. purity height, quality are good, are easy to preserve, and only add a small amount of protective material in process of production, do not contain other salt and chemical substance, thereby have guaranteed the purity of finished product, but preserve under the product room temperature.
2. extract albumin in the ordinary method and adopt 3.8% Sodium Citrate anticoagulation to obtain blood plasma, and the present invention adopts serum, avoids adding Sodium Citrate, saves cost on the one hand, reduces participating in of chemical feedstocks in addition, has improved albuminous purity; The albuminous sad method of conventional extraction, sad price is far above Palmiticacid, and Palmiticacid is hexadecanoic acid simultaneously, and the ability of sadder albumin-binding is strong, protects the corresponding also raising of albuminous effect.
3. materials of the present invention are few, and technical process is short, and is simple to operate, are fit to produce in batches, and production efficiency has improved 5 times than cold salting-out process (need consuming time 5 days); The yield height is generally at 1.8～2.0 %; The price of Palmiticacid is far below sad, so production cost reduces greatly, as compare cost low 50% with cold salting-out process.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Used raw material and equipment among the following embodiment if no special instructions, is commercially available.
1 one kinds of bovine serum albumin extracting method of embodiment may further comprise the steps:
Get fresh ox blood, the centrifugal 10min of 3000r/m gets supernatant liquor, and-20 ℃ frozen standby.Get the bovine serum 30ml after the abundant thawing, add deionized water 30ml, fully stir evenly, add 70 ℃ 0.024M sodium palmitate solution 20ml again, place 75min.The pH that records mixing solutions is 8.76, adds 1M HCl 1.6ml, makes pH to 5.16, and water-bath 1.5h(temperature rises to 70 ℃ from 65 ℃).Filter with the common filter paper of individual layer, get filtrate 60ml, add 3M HCl 12ml, NaCl 2.88g, place 30min for 4 ℃, use double medium filtration, precipitation is added water 1.7ml, stir into pasty state, add 1M NaOH 3.5ml, pH to 9.98 adds 1M HCl 0.11ml to pH8.45 again, and 12h dialyses in deionized water.With dialyzate 13.5ml, put into Freeze Drying Equipment, take out after 48 hours, get protein powder.
2 one kinds of bovine serum albumin extracting method of embodiment may further comprise the steps:
Get fresh ox blood, the centrifugal 10min of 3000r/m gets supernatant, and-20 ℃ frozen standby.
Get the bovine serum 30ml after the abundant thawing, add deionized water 30ml, fully stir evenly, add 70 ℃ 0.024M sodium palmitate solution 20ml again, place 75min.The pH that records mixing solutions is 8.78, adds 1M HCl 1.7ml, makes pH to 5.17, and water-bath 1.5h(temperature rises to 70 ℃ from 65 ℃).Filter with individual layer filter paper, get filtrate 60ml, add 3M HCl 12ml, NaCl 2.88g, place 30min for 4 ℃, use double medium filtration, precipitation is added water 1.5ml, stir into pasty state, add 1M NaOH 3.0ml, pH to 10.04 adds 1M HCl 0.20ml to pH8.51 again, and 12h dialyses in deionized water.With dialyzate 12.5ml, put into Freeze Drying Equipment, take out after 48 hours, get protein powder.
The main performance index of prepared bovine serum albumin is as follows in the foregoing description 1,2:
1. outward appearance: the canescence porous solid, do not have the phenomenon of thawing; After redissolving is faint yellow clear liquid, does not have muddy;
2. yield (Tot Prot in lyophilized powder quality/former serum * 100 %):＞26.0%;
3. water content (vacuum decompression desiccating method):＜0.3%;
4. pH value (physiological sodium chloride solution is diluted to 10g/L with the trial-product protein content): 7.3～7.4;
5. water-soluble (in aseptic deionized water, add protein product, ultrasonic 30min, level vibration 24h as there not being precipitation, adds protein sample again in 25 ℃ of thermostat containers, so repeatedly, until not dissolving, gets supernatant, surveys protein content):＞8%;
6. purity (concentration of albuminous concentration/total protein * 100 %):＞95.0%.
Claims (4)
1. bovine serum albumin extracting method may further comprise the steps:
(1) gets fresh ox blood, get supernatant after the centrifugation, add and the isopyknic aseptic pyrogen-free deionized water of this supernatant, add 60～70 ℃ 0.024M sodium palmitate solution again by 1/4～3/4 of described supernatant volume, leave standstill 50～100min behind the mixing;
(2) upwards go on foot in the gained solution and add dilute hydrochloric acid, regulator solution pH to 4.0～6.5,60～70 ℃ following water-bath 1～2h filters to get filtrate;
(3) upwards go on foot adding 3 M HCl in the gained filtrate by 40% of described supernatant volume, add sodium-chlor behind the mixing again, make its mass concentration in solution reach 4%, 0～4 ℃ and place 30～60min down;
(4) leaving standstill after-filtration must precipitate, and adds aseptic pyrogen-free deionized water by 4～6% of described supernatant volume in precipitation, and the solution that adds dilute alkali is transferred pH value of solution to 9.0～10.0;
(5) treat that precipitation fully after the dissolving, adds dilute hydrochloric acid again and transfers pH value of solution to 8.0～9.0,10～16h dialyses in aseptic pyrogen-free deionized water;
(6) dialyzate is put into Freeze Drying Equipment, takes out behind 45～50h, gets protein powder.
2. bovine serum albumin extracting method according to claim 1 is characterized in that, in described step (1), the rotating speed of centrifugation is 2800～3200r/m, and the time is 10～20 min.
3. bovine serum albumin extracting method according to claim 1 is characterized in that, in described step (2), (5), the concentration of used dilute hydrochloric acid is 1mol/L.
4. bovine serum albumin extracting method according to claim 1 is characterized in that, in described step (4), used dilute alkaline soln is a 1mol/L NaOH solution.
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| CN 201110168477 CN102241767B (en) | 2011-06-22 | 2011-06-22 | Extraction method of bovine serum albumin |
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| CN 201110168477 CN102241767B (en) | 2011-06-22 | 2011-06-22 | Extraction method of bovine serum albumin |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103433484A (en) * | 2013-08-22 | 2013-12-11 | 福建医科大学 | Bovine serum albumin-platinum composite nanomaterial mimetic peroxidase |
| CN104209506A (en) * | 2013-08-22 | 2014-12-17 | 福建医科大学 | Platinum nanoparticle-bovine serum albumin core shell structure and preparation method thereof |
| 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 |
| CN107474131A (en) * | 2017-09-26 | 2017-12-15 | 广州中国科学院先进技术研究所 | One kind restructuring bovine serum albumin mature peptide and its preparation method and application |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
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| CN1406247A (en) * | 2000-10-24 | 2003-03-26 | 财团法人化学及血清疗法研究所 | Method of monomerizing human serum albumin polymers |
| CN1544470A (en) * | 2003-11-17 | 2004-11-10 | 孙福森 | Bovine serum albumin hot ethanol extracting process |
| CN101367865A (en) * | 2008-09-26 | 2009-02-18 | 广州倍绣生物技术有限公司 | Production process for high purity porcine blood albumin and uses thereof |
| CN101857635A (en) * | 2010-04-28 | 2010-10-13 | 天津商业大学 | Continuous separation method for three proteins in bovine plasma |
-
2011
- 2011-06-22 CN CN 201110168477 patent/CN102241767B/en not_active Expired - Fee Related
Patent Citations (4)
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|---|---|---|---|---|
| CN1406247A (en) * | 2000-10-24 | 2003-03-26 | 财团法人化学及血清疗法研究所 | Method of monomerizing human serum albumin polymers |
| CN1544470A (en) * | 2003-11-17 | 2004-11-10 | 孙福森 | Bovine serum albumin hot ethanol extracting process |
| CN101367865A (en) * | 2008-09-26 | 2009-02-18 | 广州倍绣生物技术有限公司 | Production process for high purity porcine blood albumin and uses thereof |
| CN101857635A (en) * | 2010-04-28 | 2010-10-13 | 天津商业大学 | Continuous separation method for three proteins in bovine plasma |
Non-Patent Citations (1)
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| 刘永东等: "重组人血清白蛋白生产工艺研究进展", 《微生物学通报》, vol. 30, no. 5, 31 December 2003 (2003-12-31), pages 128 - 132 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103433484A (en) * | 2013-08-22 | 2013-12-11 | 福建医科大学 | Bovine serum albumin-platinum composite nanomaterial mimetic peroxidase |
| CN104209506A (en) * | 2013-08-22 | 2014-12-17 | 福建医科大学 | Platinum nanoparticle-bovine serum albumin core shell structure and preparation method thereof |
| CN103433484B (en) * | 2013-08-22 | 2015-06-17 | 福建医科大学 | Bovine serum albumin-platinum composite nanomaterial mimetic peroxidase, preparation and application thereof |
| CN104209506B (en) * | 2013-08-22 | 2016-03-30 | 福建医科大学 | Nano platinum particle-bovine serum albumin(BSA) nucleocapsid structure and preparation method thereof |
| 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 |
| WO2019062709A1 (en) * | 2017-09-26 | 2019-04-04 | 广州中国科学院先进技术研究所 | Recombinant bovine serum albumin mature peptide, preparation method therefor, and application thereof |
| CN107474131A (en) * | 2017-09-26 | 2017-12-15 | 广州中国科学院先进技术研究所 | One kind restructuring bovine serum albumin mature peptide and its preparation method and application |
| US11634257B2 (en) | 2017-10-09 | 2023-04-25 | Terumo Bct Biotechnologies, Llc | Lyophilization container and method of using same |
| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11609042B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11609043B2 (en) | 2019-03-14 | 2023-03-21 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| US11994343B2 (en) | 2019-03-14 | 2024-05-28 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
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Inventor after: He Meixia Inventor after: Wen Zhanying Inventor after: Zhang Pengjun Inventor after: Ge Shiwen Inventor after: Du Bin Inventor after: Ding Lijing Inventor after: Cui Xiguo Inventor after: He Shilin Inventor after: Li Zhigang Inventor after: Zhu Jianli Inventor after: Li Xiaotian Inventor after: Han Quancheng Inventor after: Zhang Yong Inventor after: Zhang Lirong Inventor after: Li Yang Inventor before: He Meixia Inventor before: Cui Xiguo Inventor before: Ding Lijing Inventor before: He Shilin Inventor before: Hua Haiying Inventor before: Li Zhigang Inventor before: Zhang Lirong Inventor before: Zhang Hongmei Inventor before: Wang Fang Inventor before: Zhang Yong Inventor before: Gao Yuanfa |
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Free format text: CORRECT: INVENTOR; FROM: HE MEIXIA HE SHILIN HUA HAIYING LI ZHIGANG ZHANG LIRONG ZHANG HONGMEI WANGFANG ZHANG YONG GAO YUANFA CUI XIGUO DING LIJING TO: HE MEIXIA HE SHILIN LI ZHIGANG ZHU JIANLI LI XIAOTIAN KAN QUANCHENG ZHANG YONG ZHANG LIRONG LI YANG WEN ZHANYING ZHANG PENGJUN GE SHIWEN DU BIN DING LIJING CUI XIGUO |
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