CN102286096A - Scale purification method for lactalbumin - Google Patents
Scale purification method for lactalbumin Download PDFInfo
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- CN102286096A CN102286096A CN2011101917315A CN201110191731A CN102286096A CN 102286096 A CN102286096 A CN 102286096A CN 2011101917315 A CN2011101917315 A CN 2011101917315A CN 201110191731 A CN201110191731 A CN 201110191731A CN 102286096 A CN102286096 A CN 102286096A
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- whey
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- albumin
- lactoglobulin
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Abstract
The invention relates to a scale purification method for lactalbumin in the technical field of the biological engineering, which comprises the following step of: by using human alpha-serum albumin recombinant milk as the raw mateiral, after carrying out pretreatment and purification of a protein, separating by anion-exchange chromatography to obtain recombinant human alpha-serum albumin, cow alpha-serum albumin and cow beta-lactoglobulin. According to the scale purification method for the lactalbumin, the recombinant human alpha-serum albumin can be rapidly, conveniently, simply and massively purified under the low cost, the production cost is reduced and the production efficiency is improved.
Description
Technical field
What the present invention relates to is a kind of method of technical field of bioengineering, specifically is a kind of scale purification process of whey-protein.
Background technology
Alpha-whey albumin extensively is present in the Mammals Ruzhong, is β-1, the adjusting subunit of 4-galactoside transferase, catalysis lactose synthetic.The amino acid of alpha-whey albumin comprises the necessary amino acid of a large amount of human nutritions in forming, and has important nutritive value.Human alpha-whey albumin polymer or folding varient can inducing apoptosis of tumour cell, are expected to be developed to the clinical medicine of treatment cancer.Alpha-whey albumin is a kind of of whey-protein.Chinese patent 200610076242 has been set up the production method of changeing the recombinant clone heavy livestock of human alpha-lactalbumin gene, for scale production recombination human alpha-whey albumen is laid a good foundation as clinical medicine and functional food.Except that the recombinant human whey-protein, also have the whey-protein of animal oneself expression in the transgenic animal emulsion, this class heterologous protein structure of the same race is very similar, and physico-chemical property is approaching, and scale is separated very difficulty of preparation.
Through the Searches of Patent Literature is found, Chinese patent application number 200810118961.7 " separation purification method of heterogenous homogeneous serum albumin ", adopt hydrophobic chromatography and gel permeation chromatography two-step approach purifying alpha-whey albumin, adopt hydrophobic chromatography to obtain the target protein crude product earlier, refining with gel permeation chromatography again; This purification process step is more, complicated operation, inefficiency.It is that the method for the Ultra Performance Liquid Chromatography post of filler is separated alpha-whey albumin with beta-lactoglobulin that Chinese patent application number 200910161153.3 " separation of whey-protein and measuring method " are adopted with ethylene bridge hydridization (BEH) particle; After the U.S. Patent number 7524932 acidic conditions pre-treatment, adopt anion exchange method and gradient elution purifying alpha-whey albumin; U.S. Patent number 5503864 is the methods that combine isoelectric precipitation and membrane filtration, though easy and simple to handle, selectivity is low, and product purity is low.These methods or operation steps is many, otherwise being difficult to carry out scale amplifies and realizes scale production.Therefore, need set up a kind of simple and effective, cost is low, industrial scale is easy to amplify recombinant human whey-protein purification process.
Summary of the invention
The present invention is directed to the prior art above shortcomings, a kind of scale purification process of whey-protein is provided, can be quick, easy, purifying recombination human alpha-whey albumin low-costly and in high volume, reduce production costs, improved production efficiency.
The present invention is achieved by the following technical solutions, the present invention is a raw material by the cow's milk of recombinating with human alpha-whey albumin, behind the pre-treatment purifying protein, obtain recombination human alpha-whey albumin, ox alpha-whey albumin and bovine beta-lactoglobulin with the anion-exchange chromatography separation.
Described human alpha-whey albumin reorganization cow's milk adopts commercially available or takes from the Li Ning of China Agricultural University professor laboratory and provides.
Described pre-treatment is meant: have under protectant condition newborn sample is heated to 60 ℃-95 ℃ and be incubated 10min-60min after, be cooled to room temperature and remove precipitation, collect the clear liquid that clear liquid is rich in beta-lactoglobulin and alpha-whey albumin.
It is the CaCl of 0.05-0.4M that described protective material adopts concentration
2The aqueous solution.
Described anion-exchange chromatography separation is meant: make ion exchange column with commercially available anionic exchange medium, with the ion exchange column of flowing through of the product after the thermal treatment, concrete steps comprise: at 50-150mM, make beta-lactoglobulin and alpha-whey albumin being adsorbed on the ion exchange column in various degree under the equilibrium conditions of the Tris-HCl of pH 6.4-8.4, the NaCl-50mM of 6.9-13.5ms/cm is led in last electricity consumption, the elutriant one-step elution of the Tris-HCl of pH 7.4 is also collected different elution peaks, three kinds of albumen are eluted respectively, it is white to obtain the ox alpha-whey successively, white and the bovine beta-lactoglobulin of human alpha-whey reaches the purpose of separation and purification human alpha-whey albumin.
The present invention reduced to for two steps with step in the whey-protein production, wherein pre-treatment can realize that alpha-whey albumin and beta-lactoglobulin total content reach more than 99%, and ion exchange treatment step one-step elution is obtained recombination human alpha-whey albumin purity more than 90%; Thereby can be quick, easy, purifying recombination human alpha-whey albumin low-costly and in high volume, reduce production costs, improved production efficiency.
Embodiment
Below embodiments of the invention are elaborated, present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
Get the reorganization milk powder, add deionized water by 1: 8 (w/v), centrifugal degreasing is got whey and is distributed into many parts, transfers CaCl respectively
2Final concentration 0.0,0.05,0.1,0.15,0.2,0.25,0.3,0.4M is heated to temperature then respectively: 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, insulation 10min, 20min, 30min, 40min, 50min, 60min; After reaction finished, in 4 ℃, 9800rpm, centrifugal 10min, supernatant were transferred to new pipe and add NaCO with each pipe
3, the centrifugal CaCl that removes
2, with 200mL pH 7.0, the 10mM sodium phosphate buffer is with after the washing of precipitate three times molten.The analytically cleer and peaceful protein precipitation of 13.5%SDS-PAGE is formed, and supernatant is carried out the mensuration of protein quantification.Total alpha-whey albumin of concentrating sample and beta-lactoglobulin total content can reach more than 99%.Table 1 has shown that whey sample is at different concns CaCl
2Heat the protein concentration result of 95 ℃ of insulation 5min; Table 2 has shown that whey sample is at 0.2M CaCl
2The result of heating differing temps insulation 20min under the condition;
The effect of insulation different time that table 3 has shown 75 ℃.
Use the 5KD regenerated cellulose composite film tentatively concentrated again, remove the part salt ion, add the deionized water desalination, get the whey pretreatment sample.
Table 1
Table 2
| Temperature of reaction | Alpha-whey albumin+beta-lactoglobulin total content (%) |
| 70℃ | 85 |
| 75℃ | 94 |
| 80℃ | 94 |
| 85℃ | 94 |
| 90℃ | 90 |
| 95℃ | 84 |
Table 3
Embodiment 2
The whey pretreatment sample of embodiment 1 preparation is regulated pH to 7.4 with 1M HCl or NaOH solution, last sample is to using 50mM, in the Q Sepharose Fast Flow anion-exchange chromatography post (11x22cm) of pH 7.4Tris-HCl damping fluid pre-equilibration, flow velocity 200ml/ml; The last sample into 50mM that finishes, pH 7.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, NaCl-50mM Tris-HCl (pH 7.4) buffer solution elution into 10.2ms/cm is led in electricity consumption.Collect recombination human alpha-whey albumin component 1.5L, the rate of recovery 76%; It is 94.1% that high performance liquid chromatography detects the white egg purity of recombination human alpha-whey; Collect ox alpha-whey albumin peak 1.2L, the technology rate of recovery 79.2%, it is 90.1% that high performance liquid chromatography detects ox alpha-whey albumin purity; Collect bovine beta-lactoglobulin peak 2L, the technology rate of recovery 85.5%, it is 87.9% that high performance liquid chromatography detects bovine beta-lactoglobulin purity.
Embodiment 3
The whey pretreatment sample of embodiment 1 preparation is regulated pH to 6.4 with 1M HCl or NaOH solution, last sample is to using 50mM, in the Q Sepharose Fast Flow anion-exchange chromatography post (11x22cm) of pH6.4Tris-HCl damping fluid pre-equilibration, flow velocity 200ml/ml; The last sample into 50mM that finishes, pH6.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, NaCl-50mM Tris-HCl (pH6.4) buffer solution elution into 10.2ms/cm is led in electricity consumption.Collect recombination human alpha-whey albumin component 1.6L, the rate of recovery 72%; It is 91.2% that high performance liquid chromatography detects the white egg purity of recombination human alpha-whey; Collect ox alpha-whey albumin peak 1.3L, the rate of recovery 75.3%, it is 87.3% that high performance liquid chromatography detects ox alpha-whey albumin purity; Collect bovine beta-lactoglobulin peak 2.1L, the rate of recovery 90.7%, it is 75.8% that high performance liquid chromatography detects bovine beta-lactoglobulin purity.
Embodiment 4
The whey pretreatment sample of embodiment 1 preparation is regulated pH to 8.4 with 1M HCl or NaOH solution, and last sample arrives and uses 50mM, in the Q Sepharose XL anion-exchange chromatography post (11x22cm) of pH8.4Tris-HCl damping fluid pre-equilibration, and flow velocity 200ml/ml; The last sample into 50mM that finishes, pH8.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, NaCl-50mM Tris-HCl (pH8.4) buffer solution elution into 10.2ms/cm is led in electricity consumption.Collect recombination human alpha-whey albumin component 1.6L, the rate of recovery 73.7%; It is 93.1% that high performance liquid chromatography detects the white egg purity of recombination human alpha-whey; Collect ox alpha-whey albumin peak 1.3L, the rate of recovery 76.3%, it is 86.5% that high performance liquid chromatography detects ox alpha-whey albumin purity; Collect bovine beta-lactoglobulin peak 2.1L, the rate of recovery 95.9%, it is 70.3% that high performance liquid chromatography detects bovine beta-lactoglobulin purity.
Embodiment 5
The whey pretreatment sample of embodiment 1 preparation is regulated pH to 7.4 with 1M HCl or NaOH solution, electricity is led 6.9ms/cm, last sample arrives and uses 50mM, in the DEAE-Sepharose CL-6B anion-exchange chromatography post (11x22cm) of pH7.4Tris-HCl damping fluid pre-equilibration, and flow velocity 200ml/ml; The last sample into 50mM that finishes, pH7.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, NaCl-50mM Tris-HCl (pH7.4) buffer solution elution into 6.9ms/cm is led in electricity consumption.Collect recombination human alpha-whey albumin component 1.0L, the rate of recovery 43.2%; The white egg purity of recombination human alpha-whey is 67.2%; Collect ox alpha-whey albumin peak 0.9L, the rate of recovery 42.7%, purity are 76.5%; Collect bovine beta-lactoglobulin peak 2.0L, the rate of recovery 91.3%, purity are 67.8%.
Embodiment 6
The whey pretreatment sample of embodiment 1 preparation is regulated pH to 7.4 with 1M HCl or NaOH solution, electricity is led 3.4ms/cm, last sample arrives and uses 50mM, in the DEAE-Sepharose CL-4B anion-exchange chromatography post (11x22cm) of pH7.4Tris-HCl damping fluid pre-equilibration, and flow velocity 200ml/ml; The last sample into 50mM that finishes, pH7.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, NaCl-50mM Tris-HCl (pH7.4) buffer solution elution into 13.5ms/cm is led in electricity consumption.Collect recombination human alpha-whey albumin component 1.1L, the rate of recovery 45.2%, purity are 65.1%; Collect ox alpha-whey albumin peak 0.9L, the rate of recovery 35.6%, purity are 69.3%; Collect bovine beta-lactoglobulin peak 2.0L, the rate of recovery 87.7%, purity are 64.3%.
Embodiment 7
The whey pretreatment sample of embodiment 1 preparation is regulated pH to 7.4 with 1M HCl or NaOH solution, electricity is led 3.4ms/cm, last sample arrives and uses 50mM, in the DEAE-Sepharose Fast Flow anion-exchange chromatography post (11x22cm) of pH7.4Tris-HCl damping fluid pre-equilibration, and flow velocity 200ml/ml; The last sample into 50mM that finishes, pH7.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, NaCl-50mM Tris-HCl (pH7.4) buffer solution elution into 10.2ms/cm is led in electricity consumption.Collect recombination human alpha-whey albumin component 1.3L, the rate of recovery 62.1%, purity are 84.5%; Collect ox alpha-whey albumin peak 1.1L, the rate of recovery 60.4%, purity are 84.5%; Collect bovine beta-lactoglobulin peak 2.0L, the rate of recovery 90.8%, purity are 82.5%.
Embodiment 8
The whey pretreatment sample of embodiment 1 preparation is regulated pH to 7.4 with 1M HCl or NaOH solution, electricity is led 3.4ms/cm, last sample arrives and uses 150mM, in the TOYOPEARL DEAE-650 anion-exchange chromatography post (11x22cm) of pH7.4Tris-HCl damping fluid pre-equilibration, and flow velocity 200ml/ml; The last sample into 150mM that finishes, pH7.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, the Tris-HCl (pH7.4) that electricity consumption is led to 10.2ms/cm contains 50mM NaCl buffer solution elution.Collect recombination human alpha-whey albumin component 1.2L, the rate of recovery 58.7%, purity are 82.1%; Collect ox alpha-whey albumin peak 1.2L, the rate of recovery 61.9%, purity are 83.5%; Collect bovine beta-lactoglobulin peak 2.0L, the rate of recovery 94.2%, purity are 83.7%.
Embodiment 9
The whey pretreatment sample 100ml of embodiment 1 preparation regulates pH to 7.4 with 1M HCl or NaOH solution, last sample is to using 50mM, in the TOYOPEARL SuperQ-650 anion-exchange chromatography post (2.5x10cm) of pH 7.4Tris-HCl damping fluid pre-equilibration, flow velocity 200ml/ml; The last sample into 50mM that finishes, pH 7.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, NaCl-50mM Tris-HCl (pH 7.4) buffer solution elution into 10.2ms/cm is led in electricity consumption.Collect the recombination human alpha-whey albumin component, the rate of recovery 86%; It is 96.3% that high performance liquid chromatography detects the white egg purity of recombination human alpha-whey; Collect ox alpha-whey albumin component recovery 85.1%, purity is 92.8%; Collect bovine beta-lactoglobulin component recovery 80.5%, purity is 81.7%.
Embodiment 10
The whey pretreatment sample 100ml of embodiment 1 preparation regulates pH to 7.4 with 1M HCl or NaOH solution, last sample is to using 50mM, in the ToyoScreen Q-600C AR anion-exchange chromatography post (2.5x10cm) of pH 7.4Tris-HCl damping fluid pre-equilibration, flow velocity 200ml/ml; The last sample into 50mM that finishes, pH 7.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, NaCl-50mM Tris-HCl (pH 7.4) buffer solution elution into 10.2ms/cm is led in electricity consumption.Collect the recombination human alpha-whey albumin component, the rate of recovery 78%; Purity is 97.6%; Collect ox alpha-whey albumin component recovery 88.2%, purity is 94.5%; Collect bovine beta-lactoglobulin component recovery 83.6%, purity is 86.4%.
Embodiment 11
The whey pretreatment sample 100ml of embodiment 1 preparation regulates pH to 7.4 with 1M HCl or NaOH solution, last sample is to using 50mM, in the TOYOPEARL GigaCap Q-650 anion-exchange chromatography post (2.5x10cm) of pH 7.4Tris-HCl damping fluid pre-equilibration, flow velocity 200ml/ml; The last sample into 50mM that finishes, pH 7.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, NaCl-50mM Tris-HCl (pH 7.4) buffer solution elution into 10.2ms/cm is led in electricity consumption.Collect the recombination human alpha-whey albumin component, the rate of recovery 88%; Purity is 98.7%; Collect ox alpha-whey albumin component recovery 86.6%, purity is 96.5%; Collect bovine beta-lactoglobulin component recovery 76.8%, purity is 89.6%.
Embodiment 12
The whey pretreatment sample 100ml of embodiment 1 preparation regulates pH to 7.4 with 1M HCl or NaOH solution, last sample is to using 50mM, in the PEARL QAE-550 anion-exchange chromatography post (2.5x10cm) of pH 7.4Tris-HCl damping fluid pre-equilibration, flow velocity 200ml/ml; The last sample into 50mM that finishes, pH 7.4Tris-HCl damping fluid wash post to the 280nm absorption signal to baseline, NaCl-50mM Tris-HCl (pH 7.4) buffer solution elution into 10.2ms/cm is led in electricity consumption.Collect the recombination human alpha-whey albumin component, the rate of recovery 92%; Purity is 96.5%; Collect ox alpha-whey albumin component recovery 90.2%, purity is 94.8%; Collect bovine beta-lactoglobulin component recovery 86.5%, purity is 86.3%.
Claims (5)
1. the scale purification process of a whey-protein, it is characterized in that, by being raw material, behind the pre-treatment purifying protein, obtain recombination human alpha-whey albumin, ox alpha-whey albumin and bovine beta-lactoglobulin with the anion-exchange chromatography separation with human alpha-whey albumin reorganization cow's milk.
2. the scale purification process of whey-protein according to claim 1; it is characterized in that; described pre-treatment is meant: have under protectant condition newborn sample is heated to 60 ℃-95 ℃ and be incubated 10min-60min after; be cooled to room temperature and remove precipitation, collect the clear liquid that clear liquid is rich in beta-lactoglobulin and alpha-whey albumin.
3. the scale purification process of whey-protein according to claim 1 is characterized in that, it is the CaCl of 0.05-0.4M that described protective material adopts concentration
2The aqueous solution.
4. the scale purification process of whey-protein according to claim 1, it is characterized in that, described anion-exchange chromatography separates and is meant: make ion exchange column with commercially available anionic exchange medium, with the ion exchange column of flowing through of the product after the thermal treatment.
5. according to the scale purification process of claim 1 or 4 described whey-proteins according to claim 1, it is characterized in that, the isolating concrete steps of described anion-exchange chromatography comprise: at 50-150mM, make beta-lactoglobulin and alpha-whey albumin being adsorbed on the ion exchange column in various degree under the equilibrium conditions of the Tris-HCl of pH 6.4-8.4, the NaCl-50mM of 6.9-13.5ms/cm is led in last electricity consumption, the elutriant one-step elution of the Tris-HCl of pH 7.4 is also collected different elution peaks, three kinds of albumen are eluted respectively, it is white to obtain the ox alpha-whey successively, white and the bovine beta-lactoglobulin of human alpha-whey reaches the purpose of separation and purification human alpha-whey albumin.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102746395A (en) * | 2012-07-04 | 2012-10-24 | 浙江大学 | Method for separating beta-lactoglobulin from raw milk |
| CN110267968A (en) * | 2017-02-16 | 2019-09-20 | 通用电气医疗集团生物工艺研发股份公司 | The purification process of lactalbumin |
| CN111116706A (en) * | 2018-10-31 | 2020-05-08 | 泰州医药城国科化物生物医药科技有限公司 | Purification preparation method for extracting whey protein in breast milk |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104635A (en) * | 2007-04-30 | 2008-01-16 | 北京济普霖生物技术有限公司 | Method for purifying recombination human alpha-whey albumin from transgene cow milk |
| CN101367864A (en) * | 2008-08-27 | 2009-02-18 | 中国科学院过程工程研究所 | Separation purification process for heterogenous homogeneous serum albumin |
| CN101948530A (en) * | 2010-10-15 | 2011-01-19 | 上海交通大学 | Method of preparing lactalbumin scale purification |
-
2011
- 2011-07-08 CN CN2011101917315A patent/CN102286096A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104635A (en) * | 2007-04-30 | 2008-01-16 | 北京济普霖生物技术有限公司 | Method for purifying recombination human alpha-whey albumin from transgene cow milk |
| CN101367864A (en) * | 2008-08-27 | 2009-02-18 | 中国科学院过程工程研究所 | Separation purification process for heterogenous homogeneous serum albumin |
| CN101948530A (en) * | 2010-10-15 | 2011-01-19 | 上海交通大学 | Method of preparing lactalbumin scale purification |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102746395A (en) * | 2012-07-04 | 2012-10-24 | 浙江大学 | Method for separating beta-lactoglobulin from raw milk |
| CN110267968A (en) * | 2017-02-16 | 2019-09-20 | 通用电气医疗集团生物工艺研发股份公司 | The purification process of lactalbumin |
| CN110267968B (en) * | 2017-02-16 | 2024-01-30 | 思拓凡生物工艺研发有限公司 | Purification method of whey protein |
| CN111116706A (en) * | 2018-10-31 | 2020-05-08 | 泰州医药城国科化物生物医药科技有限公司 | Purification preparation method for extracting whey protein in breast milk |
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