CN102746394A - Method for separating milk-derived alphas-casein by using ion exchange chromatography - Google Patents
Method for separating milk-derived alphas-casein by using ion exchange chromatography Download PDFInfo
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- CN102746394A CN102746394A CN201210001803XA CN201210001803A CN102746394A CN 102746394 A CN102746394 A CN 102746394A CN 201210001803X A CN201210001803X A CN 201210001803XA CN 201210001803 A CN201210001803 A CN 201210001803A CN 102746394 A CN102746394 A CN 102746394A
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Abstract
The present invention discloses a method for separating milk-derived alphas-casein by using ion exchange chromatography. The method of the present invention has advantages of high product purity, simple operation, good repeatability, high resolution, low cost, and the like, and is an important means and method for casein purification and separation. In addition, the alphas-casein separated by using ion exchange chromatography can be used for productions of casein-derived bioactive peptides.
Description
Technical field
The present invention relates to the component stripping technique, be specifically related to a kind of ion-exchange chromatography and separate newborn source α
s-caseic method.
Background technology
Milk-product are abundant because of its milk protein content; Be of high nutritive value; Absorbed by human consumption easily and be acknowledged as the heath food that the person and intelligence growth is had good effect, the composition of Ruzhong most worthy is milk-protein just, mainly comprises casein and whey-protein; Also have the little fat sphaeroprotein, wherein casein content accounts for 80%～82% of milk-protein.By the mixture that multiple proteins is formed, (casein CN) is called casein food grade again to casein, is made up of four kinds of components, comprises α
s-casein, beta-casein and κ-casein, α
s-casein is divided into α again
S1-casein and α
S2-casein accounts for 48% of casein total amount, is the main source of newborn source casein biologically active peptides.
α
s-casein can be used as foodstuff additive, is applied to the production of food, improves flavours in food products; α
s-casein is the phosphor protein complex body, can produce phosphopeptide caseinate (CPP) through hydrolysis, promotes the human calcium to absorb, so α
s-casein can be used as the optimum raw material of phosphopeptide caseinate preparation; α in addition
s-casein hydrolysis can produce antibacterial peptide, immune peptide, opioid peptides, antihypertensive peptide and promote the mineral tissue absorption, promotes immunity, and hormone regulation is antiviral, biologically active peptidess such as reducing blood-fat, and some biologically active peptidess have applied to suitability for industrialized production.α now
s-casein stripping technique is broadly divided into three kinds: chemical precipitation classification, chromatography and calcium cryogenic system partition method.Though chemical precipitation classification and calcium cryogenic system partition method are comparatively commonly used, its isolating conditional request strict (high to the dependence of temperature), and isolated α like calcium cryogenic system partition method
s-casein usually is mixed with caseic other components; In the Application Research of casein biologically active peptides, receive more restriction, (number of patent application: 200510096244.5) name is called a kind of Caseinum componemt separation method and people's such as application and Zhang Liebing patent of invention (number of patent application: 201010144728.3) be called a kind of α like the patent of invention of Lu Xiaomin
s-caseic separation method, isolated α
s-casein purity is up to 97.8%.And chromatography has resolving power high, operate simple and easy, good reproducibility, low cost and other advantages widespread use in the protein purification process.
Ion-exchange chromatography (Ion exchange chromatography; IEC); Also be ion exchange chromatography; Be to utilize Ion Exchange Medium different to various ionic avidity, so as to a kind of chromatographic technique of various ionic in the separating mixture, its principal feature is the effect that relies on the magnetism between the particle that has opposite charges.The stationary phase of ion-exchange chromatography is an ionite; It is by one type of water-fast inertia high molecular polymerization material; Form through certain electric charge group on certain chemical reaction covalent attachment, can be divided into three parts: high molecular polymer matrix, electric charge group and counterion.Electric charge group and high molecular polymer covalent attachment, the group of the charged IX carried out of formation.Counterion is the counter ion that is incorporated on the electric charge group; It can with other ionic group generation reversible permutoid reaction in the solution, be divided into cationite (with the group exchange of positively charged) and anionite (exchanging) again according to the ionic group kind difference of its exchange with electronegative group.Moving phase is the electrolyte solution with certain pH value and certain ionic strength.Protein is in different pH buffer, and can have free amino or carboxyl can exchange absorption with the group of ionite.Carry out wash-out with damping fluid, when the ionic group in the damping fluid was competed with the protein group on being combined in ionite mutually, the little protein molecule of avidity was eluted by desorb earlier, and big back of avidity got off by desorb.
Summary of the invention
The objective of the invention is to provides a kind of α according to the above-mentioned deficiency that exists in the prior art
s-casein ion-exchange chromatography partition method.Present method is a raw material with the caseidin, uses ion exchange chromatography, through the discontinuous gradient wash-out, seeks the concentration that can wash-out goes out the salt that proteic ionic strength contains, with α
sOther components of-casein and casein are separated, and obtain highly purified α
s-casein.
Above-mentioned purpose of the present invention is achieved through following technical scheme:
Casein is an acidic protein; At pH>electronegative during pI, can select anionite to separate, and the electrically charged difference of casein different components; Different with the ionite combination degree, the ionic strength that therefore changes elutriant can make different components separate.The Ion Exchange Medium DEAE-Sepharose CL-6B that the present invention is used, the α that obtains through its separation
s-casein purity can be 100%.
Present method is a raw material with the full element of casein that purchase obtains.Ion-exchange chromatography separates α
sThe separation method of-Caseinum componemt may further comprise the steps:
(1) get the complete plain reference liquid of sample buffer A liquid compound concentration as 4mg/ml that use of standard casein, refrigerator is placed 12h for 4 ℃, and is subsequent use behind the 0.45 μ m membrane filtration.
(2) get chromatographic column and clean after with 20% acetate immersion treatment 24h subsequent usely, get DEAE-Sepharose CL-6B and soak with sample buffer A liquid, the dress post, the about 30cm of height is with sample buffer A liquid balance.
(3) go up appearance; The about 10ml of sample solution; Continue to go out unconjugated albumen, treat that using sample buffer B liquid instead after the complete wash-out of unconjugated albumen goes out carries out the discontinuous gradient wash-out, flow velocity 2mL/min with sample buffer A liquid wash-out; Detecting wavelength is the 280nm ultraviolet-visible, collects the peak that each gradient elution comes out and detects.
The elutriant at each peak of (4) collecting, the SDS-PAGE electrophoresis detection, separation gel is 12%, concentrated glue is 5%.
The protein solution dialysis at each peak that (5) will collect is after 0.45 μ m carries out reverse performance liquid chromatography (RP-HPLC) detection after filtering.Chromatographic column: Jupiter 300 C18 anti-phase albumen posts, 5 μ m, 250mm * 4.6mm i.d; Mobile phase A: the 0.1% TFA aqueous solution; Mobile phase B: 100% acetonitrile solution; Gradient condition: 0 ~ 40min, 32% ~ 49% Mobile phase B; Operational condition: flow velocity is 1.0ml/min; The detection wavelength is 214nm; Sampling volume is 70 μ l.
(6) in the technique scheme sample buffer A liquid for containing 3M urea, 0.1% beta-mercaptoethanol, the pH value is the tris-HCl damping fluid of 8.0 50mM.Sample buffer B liquid is the sample buffer A liquid that contains different concns NaCl.
(7) the dynamic adsorption amount is about 9.2mg/ml in the step (3), and applied sample amount is about 40mg.
The inventive method can be used for the caseic separation in different newborn sources, if raw material is a milk-protein, needs to remove whey-protein through isoelectric point precipitation (pI is about 4.6), obtains casein.
Compared with prior art, the present invention has following beneficial effect:
Present method has simple to operate, and resolving power is high, good reproducibility, and the purity of protein that obtains is high, the recovery is high, and ionite such as can reuse at advantage.In 2h, can obtain purity and be 100% α
s-casein.Separate the α that obtains
s-casein class carries out the preparation and the deep processing of newborn source activity peptide, and the purpose bioactive peptide ratio of acquisition is improved greatly, also can be used for the industry needs through enlarging.The casein that the present invention makes can be used as the raw material of Casein in Milk biologically active peptides research, for the research of function Casein in Milk bioactive peptide lays the foundation.
Description of drawings
Fig. 1 is a treatment scheme synoptic diagram of the present invention;
Fig. 2 is the ion-exchange chromatography figure in embodiment 1 step (1), and wherein I is step (a 3) chromatographic peak, and II is step (a 4) chromatographic peak, and III is step (a 5) chromatographic peak;
Fig. 3 is the electrophoresis detection result of embodiment 1, wherein, and 1: α
s-casein standard substance, 2: beta-casein standard substance, 3: κ-casein standard substance, 4: the albumen that step (3) is collected, 5: the albumen that step (4) is collected, 6: the albumen that step (5) is collected;
Fig. 4 is embodiment 1 α
sThe high-efficient liquid phase chromatogram of-casein standard substance;
Fig. 5 is the high-efficient liquid phase chromatogram of embodiment 1 beta-casein standard substance;
Fig. 6 is the high-efficient liquid phase chromatogram of embodiment 1 κ-casein standard substance;
Fig. 7 is the high-efficient liquid phase chromatogram of embodiment 1 blank solution;
Fig. 8 is the proteic high-efficient liquid phase chromatogram that embodiment 1 step (3) is collected;
Fig. 9 is the proteic high-efficient liquid phase chromatogram that embodiment 1 step (4) is collected;
Figure 10 is the proteic high-efficient liquid phase chromatogram that embodiment 1 step (5) is collected.
Embodiment
Come further to explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
(1) getting the standard casein complete plain is the reference liquid of 4mg/ml with sample buffer A liquid (contain 3M urea, 0.1% beta-mercaptoethanol, the pH value is the tris-HCl damping fluid of 8.0 50mM) compound concentration, and 4 ℃ of placements of refrigerator 12h is subsequent use behind the 0.45 μ m membrane filtration.
(2) getting chromatographic column cleans after with 20% acetate immersion treatment 24h subsequent use; Getting DEAE-Sepharose CL-6B soaks with sample buffer A liquid; The dress post; High about 30cm with going up appearance standard protein sample solution 10ml (applied sample amount is 40mg) after the sample buffer A liquid balance, continues to go out unconjugated albumen with sample buffer A liquid wash-out.
(3) treat to use instead after the complete wash-out of unconjugated albumen goes out the sample buffer A liquid that contains 0.1mol/L concentration NaCl and carry out gradient elution, flow velocity is 2mL/min, and detecting wavelength is the 280nm ultraviolet-visible, collects the peak that this gradient elution comes out and detects.
(4) treat that the complete wash-out of albumen goes out under this gradient after, use the sample buffer A liquid that contains 0.2mol/L concentration NaCl instead according to step (3) and carry out gradient elution, collect the peak that this gradient elution comes out and detect.
(5) use the sample buffer A liquid that contains 0.3mol/L concentration NaCl instead according to step (4) and carry out gradient elution, collect the peak that this gradient elution comes out and detect (particular flow sheet is seen Fig. 1, and the color atlas of chromatography is seen Fig. 2).
(6) albumen of collecting is carried out electrophoresis detection, separation gel is 12%, and concentrated glue is 5% (see figure 3).
The protein solution dialysis at each peak that (7) will collect is after 0.45 μ m carries out reverse performance liquid chromatography detection.Chromatographic column: Jupiter 300 C18 anti-phase albumen posts, 5 μ m, 250mm * 4.6mm i.d; Mobile phase A: the 0.1% TFA aqueous solution; Mobile phase B: 100% acetonitrile solution; Gradient condition: 0 ~ 40min, 32% ~ 49% Mobile phase B; Operational condition: flow velocity is 1.0ml/min; Detecting wavelength is the 214nm ultraviolet-visible; Sampling volume is 70 μ l.The α that its separation obtains
s-casein purity is 100% (see figure 4).
(1) get fresh commercially available buffalo milk, the centrifugal 30min of 4000r/min collects lower floor's skimming milk, and 1mol/L HCl transfers pH to 4.6, the centrifugal 15min of 4000r/min.Deposition is with distilled water wash 2 times, washing with acetone 2-3 time, and the centrifugal 10min of 4000r/min at every turn, it is subsequent use to precipitate in 20 ℃ of refrigerators of natural air drying ， – preservation at last.
(2) the water intaking cow milk casein is the reference liquid of 4mg/ml with sample buffer A liquid (contain 3M urea, 0.1% beta-mercaptoethanol, the pH value is the tris-HCl damping fluid of 8.0 50mM) compound concentration, and 4 ℃ of placements of refrigerator 12h is subsequent use behind the 0.45 μ m membrane filtration.
(3) getting chromatographic column cleans after with 20% acetate immersion treatment 24h subsequent use; Getting DEAE-Sepharose CL-6B soaks with sample buffer A liquid; The dress post, high about 30cm is with going up appearance standard protein sample solution 10ml (applied sample amount is 40mg) after the sample buffer A liquid balance; Continue to go out unconjugated albumen with sample buffer A liquid wash-out
(4) treat to use instead after the complete wash-out of unconjugated albumen goes out the sample buffer A liquid that contains 0.1mol/L concentration NaCl and carry out gradient elution, flow velocity is 2mL/min, and detecting wavelength is the 280nm ultraviolet-visible, collects the peak that this gradient elution comes out and detects.
(5) treat that the complete wash-out of albumen goes out under this gradient after, use the sample buffer A liquid that contains 0.2mol/L concentration NaCl instead according to step (4) and carry out gradient elution, collect the peak that this gradient elution comes out and detect.
(6) use the sample buffer A liquid that contains 0.3mol/L concentration NaCl instead according to step (5) and carry out gradient elution, collect the peak that this gradient elution comes out and detect.
(7) albumen of collecting is carried out electrophoresis detection, separation gel is 12%, and concentrated glue is 5%.
The protein solution dialysis at each peak that (8) will collect is after 0.45 μ m carries out reverse performance liquid chromatography detection.Chromatographic column: Jupiter 300 C18 anti-phase albumen posts, 5 μ m, 250mm * 4.6mm i.d; Mobile phase A: the 0.1% TFA aqueous solution; Mobile phase B: 100% acetonitrile solution; Gradient condition: 0 ~ 40min, 32% ~ 49% Mobile phase B; Operational condition: flow velocity is 1.0ml/min; The detection wavelength is 214nm; Sampling volume is 70 μ l.The α that its separation obtains
s-casein purity is 100%.
Claims (3)
1. an ion-exchange chromatography separates newborn source α
s-caseic method is characterized in that comprising the steps:
(1) be raw material with the full element of standard casein, use the reference liquid of sample buffer A compound concentration as 4mg/ml, refrigerator is placed 12h for 4 ℃, and is subsequent use behind the 0.45 μ m membrane filtration;
(2) get chromatographic column, subsequent use with cleaning behind the 20% acetate immersion 24h; Get DEAE-Sepharose CL-6B again, soak with sample buffer A, the dress post highly is 30cm, uses sample buffer A balance again;
(3) go up appearance; Sample solution is 10ml; Continue to go out unconjugated albumen with sample buffer A wash-out, treat that using sample buffer B instead after the complete wash-out of unconjugated albumen goes out carries out the discontinuous gradient wash-out, sulfuric acid is 2ml/min; Collect the peak that each gradient elution comes out and detect, detecting wavelength is the 280nm ultraviolet-visible;
(4) collect the elutriant at each peak, the SDS-PAGE electrophoresis detection, separation gel is 12%, concentrated glue is 5%;
After the protein solution dialysis at each peak that (5) will collect, filter through 0.45 μ m, carry out reverse performance liquid chromatography then, wherein, moving is the 0.1%TFA aqueous solution mutually, and moving phase is 100% acetonitrile solution; Gradient condition is 32 ~ 49% moving phases, 0 ~ 40min; Operational condition is that flow velocity is 1.0ml/min, and the detection wavelength is 214nm, and sampling volume is 70 μ l;
Wherein, sample buffer A contains the tris-HCl damping fluid that 3M urea, 0.1% beta-mercaptoethanol, pH value are 8.0 50mM; Sample buffer B is the sample buffer A that contains different concns NaCl.
2. separate newborn source α according to the said ion-exchange chromatography of claim 1
s-caseic method is characterized in that the dynamic adsorption amount in the step (3) is 9.2mg/ml, and applied sample amount is 40mg.
3. separate newborn source α according to the said ion-exchange chromatography of claim 1
s-caseic method is characterized in that when raw material is milk-protein, needs to remove the whey-protein in the milk-protein through isoelectric point precipitation (pI is 4.6), obtains casein then, and then carries out subsequent processing steps.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107840883A (en) * | 2017-12-07 | 2018-03-27 | 甘肃农业大学 | The method that 3 kinds of key components of bovine casein separate simultaneously |
| CN112625110A (en) * | 2019-09-24 | 2021-04-09 | 泰州医药城国科化物生物医药科技有限公司 | Purification preparation method of beta-casein in cow milk |
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| WO2007028210A1 (en) * | 2005-09-09 | 2007-03-15 | Murray Goulburn Co-Operative Co Limited | Milk derived composition and use to enhance muscle mass or muscle strength |
| 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 |
| EP2038296A2 (en) * | 2006-05-31 | 2009-03-25 | LFB Biotechnologies | Method for the extraction of one or several proteins present in milk |
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2012
- 2012-01-05 CN CN201210001803XA patent/CN102746394A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007028210A1 (en) * | 2005-09-09 | 2007-03-15 | Murray Goulburn Co-Operative Co Limited | Milk derived composition and use to enhance muscle mass or muscle strength |
| EP2038296A2 (en) * | 2006-05-31 | 2009-03-25 | LFB Biotechnologies | Method for the extraction of one or several proteins present in milk |
| 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 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107840883A (en) * | 2017-12-07 | 2018-03-27 | 甘肃农业大学 | The method that 3 kinds of key components of bovine casein separate simultaneously |
| CN107840883B (en) * | 2017-12-07 | 2022-12-20 | 甘肃农业大学 | Method for simultaneously separating 3 main components of bovine milk casein |
| CN112625110A (en) * | 2019-09-24 | 2021-04-09 | 泰州医药城国科化物生物医药科技有限公司 | Purification preparation method of beta-casein in cow milk |
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Application publication date: 20121024 |