CN102746392A

CN102746392A - Method for preparing high purity Casein Glycomacropeptide by using ion exchange method

Info

Publication number: CN102746392A
Application number: CN2011101015918A
Authority: CN
Inventors: 王艳萍; 赵运星
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2011-04-22
Filing date: 2011-04-22
Publication date: 2012-10-24

Abstract

The invention relates to a method for separating and purifying Casein Glycomacropeptide, and concretely relates to a method for further purifying and refining Casein Glycomacropeptide by ion exchange resin. According to the invention, the price of the ion exchange resin used for separating Casein Glycomacropeptide is low, the ion exchange resin enables high adsorption quantity on the Casein Glycomacropeptide, and the ion exchange resin enables easy desorption. The ion exchange resin used for separating has the advantages of less washing steps, high purity of the washed Casein Glycomacropeptide. The resin with low price saves the industrial production cost. The ion exchange resin possesses high adsorption quantity and enables easy desorption, the purity of the washed Casein Glycomacropeptide is high, under the condition that the high purity can be ensured, the product yield can be increased, and the method brings benefits for industrial production of Casein Glycomacropeptide.

Description

Ion exchange method prepares highly purified casein PROVON 190

Technical field

The invention belongs to bioengineering field, relate to the separation purification method of casein PROVON 190, especially a kind ofly utilize macropore highly basic benzene property alkene series anion exchange resin separation and purification casein PROVON 190, and utilize spraying drying to prepare finished product.

Technical background

CMP (CaseinGlycomacropeptide; CGMP) be a kind of small peptide that obtains by the peptide bond of the Phe105-Met106 of κ-casein in rennin (or stomach en-) caseinhydrolysate, have multiple biological activity, for example suppress bacterium and the murder by poisoning of virus the host with 64 amino-acid residues; Regulate gi tract; The prevention intestinal tract disease, antithrombotic with function such as bring high blood pressure down, and have higher solvability and thermostability and good emulsifying property and gelation or the like.

Preparation about CGMP is the topic that people pay close attention to always at present, and a lot of methods are attempted being used for preparing and producing CGMP, but most methods can not be applied in the suitability for industrialized production.Tanimoto (1991) makes foreign protein sex change, deposition produce PROVON 190 with the method for high-temperature heat treatment.But this method needs long-time pyroprocessing, and energy consumption is more.Etzel (calendar year 2001), Yuan Yongjun etc. utilize gel chromatography separation and purification CGMP.But the gel chromatography filler costs an arm and a leg, and is not suitable for industry production.Saito T equals to report in 1991 a kind of technological method production CGMP of cold ethanol precipitation, but this method needs a large amount of cold ethanol, and energy consumption is many.Adopt the film of 20kDa and 5kDa that the fresh cheese sweet-whey is held back respectively in the patented technology of U.S. Holst application in 2002; Preparation casein PROVON 190; But also there is weak point in membrane separation technique when preparation CGMP; Cleaning like film need consume lot of energy and water, and the while, more for a long time, the problems such as pollution of its separating effect and film were more outstanding for foreign protein equal size in the target solution.

Summary of the invention

The object of the present invention is to provide a kind of method of suitable industrial separation purifying casein PROVON 190, the ion exchange resin that present method provides is separation and purification casein PROVON 190 preferably, and cost is low, good separating effect.Can be applied in the suitability for industrialized production of casein PROVON 190.

The present invention realizes that the technical scheme of purpose is following:

1, come the separation and purification casein by PROVON 190 by macropore highly basic benzene property alkene series anion exchange resin among the present invention, the ion exchange resin low price reduces industrial production cost.

2, the ion exchange resin of the casein PROVON 190 of separation and purification is high to the adsorptive capacity of casein PROVON 190 among the present invention, resolves and attaches easily.Adsorptive capacity to other foreign proteins etc. is few.Through the purity of 215nm and 280nm absorbancy calculating casein PROVON 190, the purity of using the isolating casein PROVON 190 of this kind ion exchange resin is more than 90%, and the recovery of casein PROVON 190 is more than 80%.The higher casein PROVON 190 purity and the recovery have proved that fully the method for separation and purification casein PROVON 190 of the present invention has purification effect preferably.The price that ion exchange resin is lower saves production cost, and brings benefit for the suitability for industrialized production of casein PROVON 190.

Description of drawings

Below in conjunction with accompanying drawing and embodiment this patent is further specified.

Fig. 1 is casein PROVON 190 wash-out result curve figure in the separation and purification of the present invention;

Fig. 2 is casein PROVON 190 characteristic component sialyl proof diagram among the separation and purification wash-out result of the present invention;

Fig. 3 is separation and purification casein PROVON 190 electrophoresis purity qualification result figure of the present invention;

Wherein Fig. 2 from left to right is respectively: the liquid concentrator of the 27mL～90mL elutriant; The liquid concentrator of the 93mL～138mL elutriant; The liquid concentrator of the 141mL～207mL elutriant; The 210mL～267mL elutriant merges liquid concentrator; The 270mL～327mL elutriant merges liquid concentrator.Negative blank.

Wherein Fig. 3 from left to right is followed successively by for collecting liquid electrophoresis detection figure as a result, maker, and 143mL, 149mL, 155mL, 161mL, 167mL, 173mL, 179mL, 185mL, 191mL, CGMP marks article

Embodiment

Below in conjunction with specific embodiments the present invention is further specified, its specific embodiments is construed as to be merely and illustrates, and is not determinate, can not limit protection scope of the present invention with following illustrating.

The separation purification method of instance 1 casein PROVON 190, step is following:

(1) the configuration casein food grade aqueous solution is adjusted the pH that its pH is an enzymolysis, and enzymolysis obtains enzymolysis solution, and the centrifugal supernatant that obtains of deposition foreign protein concentrates through tubular fibre etc.;

(2) liquid concentrator that in step (1), obtains adsorbs through ion exchange resin D280, and the ion exchange resin of absorption casein PROVON 190 is carried out wash-out with the NaCl solution of different concns, and the elution volume of each concentration NaCl solution is 1BV.

(3) collect the target elutriant, after tubular fibre concentrated desalination, the spray-dried casein PROVON 190 product that obtains was preserved.

The preparation method of instance 2 casein PROVON 190s, step is following:

(2) liquid concentrator that in step (1), obtains adsorbs through ion exchange resin D296, and the ion exchange resin of absorption casein PROVON 190 is carried out wash-out with the NaCl solution of different concns, and the elution volume of each concentration NaCl solution is 1BV.

Explain: 1, be applied in the invention: the source of casein food grade (purchase of Gansu casein food grade head factory) 2, separation spent ion exchange resin are not tailor-make products; Can buy 3 from any ion exchange resin production company, spray-drier, tubular fibres etc. can be bought from any company.

Be the effect of flocculation agent in the separation and purification of casein PROVON 190 among checking the present invention, the spy carries out following proof test:

Proof test 1: the elute effect test of ion exchange resin separation and purification casein PROVON 190

Referring to Zhang Weijie, saccharide complex Biochemical Research technology, the Resorcinol described in the pp.455-456-salt acid system carries out the characteristic component-sialyl of casein PROVON 190 to be identified, characterizes the variation of casein PROVON 190 with the variation of sialic acid content.Concrete test is as follows: 2% Resorcinol solution, and the aqueous solution 0.25ml of 0.1mol/l copper sulfate, concentrated hydrochloric acid 80mL adds water to 100ml and is mixed and puts room temperature, is stored in 4 ℃ behind the 4h, and this working fluid is yellow or green; To the collection liquid of wash-out elution curve according to Fig. 1, be divided into 5 groups, the collection liquid of each group is carried out the ultrafiltration desalination.Solution 2ml behind the desalination adds Resorcinol reagent working fluid 2ml respectively; Test tube adds and is placed on 100 ℃ of boiling water bath 15min, fast cooling; Add acetic acid propyl carbinol ester-propyl carbinol (85: 14) 4ml, vibration is extracted the back at 37 ℃ of following water-bath 15min, gets upper organic phase, measures its absorbancy under the 580nm wavelength; With the contrast of water belongs with yin property, the result sees Fig. 1, Fig. 2.

Fig. 2 detects through Resorcinol-salt acid system, has only the liquid concentrator upper organic phase of the 141mL～207mL elutriant of collection to show and is bluish voilet, and this is the sialyl characteristic color, explains after the IX to contain CGMP in the elutriant.

Proof test 2: the casein PROVON 190 purity after the IX separation and purification in the target elution peak is identified

Getting 10 μ L target solution, is 15% SDS-PAGE electrophoresis through resolving gel concentration, detects the purity of casein PROVON 190 in the target peak, and the result sees Fig. 3.

Referring to Guo Yaojun. protein electrophorese experimental technique (1999 editions): the proteinic purity method of SDS-PAGE electrophoresis detection among the pp92-118., detect casein PROVON 190 purity in the target peak.The result sees Fig. 3.

Test-results shows that the ion exchange resin and the separation purification method that provide among the present invention can play separation and purification effect preferably in the separation and purification of casein PROVON 190.

Claims

1. a casein PROVON 190 separation purification method is characterized in that: in separation and purification CGMP, utilize separation purifying casein PROVON 190s such as ion exchange resin.

2. ion exchange resin according to claim 1 is characterized in that: ion exchange resin is macropore highly basic benzene property alkene series anion exchange resin.

3. separation purification method according to claim 1, its characteristic comprises: utilize salts solution, change pH etc. resolve the casein PROVON 190 attaches wash-out.

4. separation purification method according to claim 3, its characteristic comprises: utilize the gradient elution of different concns salts solution, linear elution etc.Utilize gradient to change the wash-out of pH or the linear pH of change etc.

5. separation purification method that removes the described casein PROVON 190 of claim 1 is characterized in that purification procedures is following:

The target solution that contains CGMP, adjustment pH is 5～9, utilizes the damping fluid counterion exchange resin of identical pH, last appearance, last appearance volume is 1/20～1/4 column volume (BV), last appearance flow velocity is 0.2～4BV/h.Wash-out adopts linear elution or gradient elution.Collection contains the elutriant of target component, crosses the tubular fibre desalination, the spraying drying preparing product.

6. the separation purification method of casein PROVON 190 according to claim 5 is characterized in that: use spraying drying and prepare the casein PROVON 190, spray is done condition and is: 100～250 ℃ of EATs, air blast are 40～80m ³/ h, charging peristaltic pump flow velocity is: 25～125rpm.