CN114409762B

CN114409762B - Concentration and separation method of alpha-lactalbumin

Info

Publication number: CN114409762B
Application number: CN202210094198.9A
Authority: CN
Inventors: 刘雅文; 陈勃然; 王艺默; 王鹏
Original assignee: Individual
Current assignee: Yongheng Xinghe (Qingdao) Biotechnology Co.,Ltd.
Priority date: 2022-01-26
Filing date: 2022-01-26
Publication date: 2024-03-08
Anticipated expiration: 2042-01-26
Also published as: CN114409762A

Abstract

The invention relates to the technical field of protein purification and preparation, and discloses a concentration and separation method of alpha-lactalbumin, which comprises the following steps: (1) adjusting the pH value in stages: in the first stage, alkali is regulated, and the pH value is regulated to 11.0-13.0; in the second stage, acid is regulated, and the pH value is regulated to be 4.4-5.5; (2) membrane separation or centrifugation; (3) re-dissolving and drying; (4) membrane separation or adsorption: ensuring that the alpha-lactalbumin is adsorbed in the anion exchange resin; (5) eluting for a plurality of times; (6) adsorption, desalination and drying: the alpha-lactalbumin product is obtained. The invention takes the recovery of whey protein in whey water as raw material, and the high purity alpha-lactalbumin is industrially produced on the basis of concentrating the alpha-lactalbumin while concentrating and separating the alpha-lactalbumin, beta-lactoglobulin and immunoglobulin G products through human intervention.

Description

Concentration and separation method of alpha-lactalbumin

Technical Field

The invention relates to the technical field of protein purification and preparation, in particular to a concentration and separation method of alpha-lactalbumin, namely a method for producing concentration and separation of whey protein and preparing the alpha-lactalbumin by taking milk powder such as cow or sheep whey water, whey powder and the like as raw materials.

Background

Whey is a byproduct in the milk cheese processing process, exists in the water phase in the milk cheese protein solidification process, and accounts for 85-95% of the milk volume. Whey is rich in lactose (45-50 g/L), soluble protein (6-8 g/L), lipid (4-5 g/L), mineral salt (80-100 g/L) and the like, and contains 55% of milk nutrition. Whey is considered to be a serious environmental contaminant because it has a Biochemical Oxygen Demand (BOD) of 27 to 60g/L and a Chemical Oxygen Demand (COD) of 50 to 102 g/L. About 9X 10 of annual production whey worldwide ⁷ t, about 50% of which is treated as wastewater discharge, which not only wastes resources but also pollutes the environment. With the increase of the domestic cheese production scale, the whey water discharge becomes a 'old and difficult' problem of cheese enterprises, the Biological Oxygen Demand (BOD) and the Chemical Oxygen Demand (COD) content of the whey water are higher, the pollution is serious after the direct discharge, and the requirements of energy conservation and emission reduction are not met.

The polarity of the alpha protein and the beta protein is similar, and the alpha protein and the beta protein are difficult to separate by directly separating with resin; the protein content ratio of whey is "congenital setting", and the alpha-lactalbumin molecular weight is about 14K, the beta-lactoglobulin molecular weight is about 18K, the separation is difficult by a membrane separation method, and the purification investment by resin alone is large and the industrial production cannot be realized.

Disclosure of Invention

Aiming at the technical problems, the invention provides a concentration and separation method of alpha-lactalbumin, which comprises the following steps:

(1) Adjusting the pH value in stages: the whey water or whey powder dissolving liquid in the cheese production process is utilized to adjust the pH value in stages by utilizing the characteristic that the beta-lactoglobulin has gel; in the first stage, alkali is regulated, and the pH value is regulated to be 11.0-13.0, so that the disulfide bonds of beta-lactoglobulin are changed; in the second stage, acid is regulated, the pH value is regulated to be 4.4-5.5, and the beta-lactoglobulin in the solution generates flocculation precipitation at the isoelectric point of the protein;

(2) Membrane separation or centrifugation: carrying out membrane separation or centrifugal separation on the flocculated and precipitated material in the step (1); the membrane retentate or the centrifugal precipitate is mainly beta-lactoglobulin; the membrane permeation solution or the centrifugal clear solution is mainly mixed solution of alpha-lactalbumin and immunoglobulin G;

(3) Drying after redissolution: regulating the pH value of the membrane concentrate or the centrifugal precipitate in the step (2) to 5.5-7.0, re-dissolving, and directly drying to obtain concentrated beta-lactoglobulin;

(4) Membrane separation or adsorption: performing membrane separation on the mixed solution of the alpha-lactalbumin and the immunoglobulin G obtained in the step (2), intercepting the concentrated solution into a concentrated immunoglobulin G solution, and drying to obtain a concentrated immunoglobulin G product; concentrating the clear solution into concentrated alpha-lactalbumin solution by an ultrafiltration membrane, and drying to obtain a concentrated alpha-lactalbumin product, or injecting the concentrated alpha-lactalbumin solution into a chromatographic column or a stirring tank filled with anion exchange resin for dynamic or static adsorption, and adjusting the pH value of the concentrated alpha-lactalbumin solution to 4.2-7.0 before injection to ensure that target protein can be adsorbed in the anion exchange resin;

(5) And (3) eluting for a plurality of times: eluting the anion exchange resin in the step (4) at least twice by using a salt solution as an eluent; eluting for the first time to obtain target protein alpha-lactalbumin solution; eluting after the second time, collecting the solution after the completion to prepare the next batch for continuous use;

(6) Adsorption, desalting and drying: directly injecting the alpha-lactalbumin solution obtained in the step (5) into a second chromatographic column or a stirring tank which is provided with anion exchange resin, adjusting the pH value of the alpha-lactalbumin solution to 3.2-4.0 before injection, determining the supersaturation of the anion exchange resin, ensuring that impurities can be sufficiently adsorbed in the anion exchange resin, collecting the flowing-through liquid, adjusting the flowing-through liquid to be neutral, carrying out membrane desalination concentration, and drying to obtain the alpha-lactalbumin product.

The alkali used for alkali adjustment in the first stage of the step (1) is food-grade sodium hydroxide.

The acid used for the second-stage acid regulation in the step (1) is food-grade hydrochloric acid, acetic acid or citric acid.

The pore diameter of the membrane used in the step (2) membrane separation is 0.2-1.2 mu m.

And (3) adding dialysis water with the pH value of 4.0-5.5 during membrane separation in the step (2) so as to avoid redissolution of flocculated proteins.

The rotating speed of the centrifugal separation in the step (2) is more than or equal to 1000r/min.

And (3) membrane separation in the step (4) adopts a membrane with a molecular weight cutoff of 50-300K.

The anion exchange resin in the step (4) and the step (6) is any one or more of agarose type ion exchange resin, acrylic acid type ion exchange resin and polystyrene type ion exchange resin; the particle size of the anion exchange resin is 30-300 microns.

The first elution in the step (5) adopts a salt solution with the mass concentration of 0.3-0.8% as an eluent, and the second and subsequent elution adopts a salt solution with the mass concentration of 3.5-5% as an eluent.

The salt solution in the step (5) refers to sodium salt, potassium salt or calcium salt solution.

The invention has the following beneficial technical effects:

1. the invention can concentrate and separate high-purity whey protein by taking the whey protein recovered from the whey water as a raw material, and prepare alpha-lactalbumin, thereby realizing waste recycling.

2. The invention concentrates and separates alpha-lactalbumin, beta-lactoglobulin and immunoglobulin G products by human intervention, and simultaneously industrially produces high-purity alpha-lactalbumin on the basis of concentrating the alpha-lactalbumin.

3. The invention enriches the beta-protein, reduces the adsorption strength of alpha-lactalbumin, and realizes the effective separation between proteins.

4. Low production cost and simple operation.

5. The total protein content of the produced alpha-lactalbumin powder reaches more than 90 percent, and the alpha-lactalbumin content in the total protein reaches more than 90 percent.

Drawings

FIG. 1 is a liquid phase detection profile of concentrated whey (10.038 min alpha-lactalbumin peak; 10.609min beta-lactoglobulin peak);

FIG. 2 is a schematic diagram of the centrifugal effect of the method of the invention;

FIG. 3 is a report of the detection of the commercial production of alpha-lactalbumin and immunoglobulin G powder mix according to the invention;

FIG. 4 is one of the liquid phase detection spectra of the alpha-lactalbumin and immunoglobulin G mixture (10.47 min: alpha-lactalbumin peak; 11.29min: beta-lactoglobulin peak);

FIG. 5 is a second liquid phase detection spectrum of an alpha-lactalbumin and immunoglobulin G mixture (13.42 min: immunoglobulin G peak);

FIG. 6 is one of the membrane permeation supernatant liquid phase detection spectra (10.84 min: alpha-lactalbumin peak; 11.38: min beta-lactoglobulin peak);

FIG. 7 is a second membrane permeation supernatant liquid phase detection spectrum (13.24 min: immunoglobulin G peak);

FIG. 8 is a liquid phase detection spectrum of concentrated immunoglobulin G (13.28 min: immunoglobulin G peak);

FIG. 9 is one of the liquid-phase detection spectra of alpha-lactalbumin (10.90 min: alpha-lactalbumin peak);

FIG. 10 is a second analysis chart of a liquid phase detection of alpha-lactalbumin (10.57 min: alpha-lactalbumin peak);

FIG. 11 is a schematic flow chart of the method of the present invention;

and (3) notes: because no national detection standard exists at present, the high performance liquid detection spectrum is arranged according to the chromatographic gradient of the national standard GB1903.17-2016, and the detection wavelength is 220 nm; immunoglobulin G was determined according to the national standard GB/T5009.194-2003.

Detailed Description

The present invention will be described in detail with reference to examples. The percentage concentrations described below are mass concentrations.

Example 1

Step 1: after milk passes through curd, filtering and collecting whey water, and performing lactose removal and concentration by a 5K ultrafiltration membrane to improve the content of whey protein;

step 2: the concentrated whey protein liquid (a 1) was adjusted to pH 11.22 with 0.25mol/L food grade sodium hydroxide and then to pH 4.86 with 0.5mol/L food grade hydrochloric acid, and the protein in the solution flocculated.

Step 3: the solution was separated by a 0.8 μm ceramic membrane, the pH of the dialysis water was adjusted to 4.76, and proteins that did not flocculate were washed out as much as possible by continuous addition of dialysis water. Obtaining a concentrated solution (b 1) mainly comprising concentrated beta-lactoglobulin solution; the clear solution (c 1) is mainly a mixed solution of alpha-lactalbumin and immunoglobulin G.

Step 4: the clear liquid (c 1) passes through a 100K ultrafiltration membrane and mainly intercepts large molecular proteins such as immunoglobulin G and the like in the solution. Obtaining a concentrated solution (d 1) mainly comprising concentrated immunoglobulin G; the resulting supernatant (e 1) was mainly concentrated alpha-lactalbumin solution.

Step 5: the supernatant (e 1) was pH-adjusted to 4.61 and passed through a column packed with 30 μm polystyrene type weak anion exchange resin at a constant flow rate of 120L/h for an adsorption time of 20min. After the adsorption is finished, 4 column volumes of RO water are used for flushing resin packing in the chromatographic column, and after the flushing is finished, 4 column volumes of 0.5% sodium chloride solution (first eluent) are used for eluting the chromatographic column, so that alpha-lactalbumin solution (f 1) is obtained; and eluting the chromatographic column by adopting 4 column volumes of 3.5% sodium chloride solution (second eluent) to obtain a second elution collection liquid, and preparing the next batch for use after the elution is completed.

Step 6: and (3) adjusting the pH value of the alpha-lactalbumin solution (f 1) subjected to 0.5% salt analysis in the step (5) to 3.72, passing through a second chromatographic column sleeved with 80 mu m agarose type weak anion exchange resin at a constant flow rate of 120L/h, collecting the flow-through liquid, adjusting the pH value to 6.36, desalting and concentrating by using a 5K ultrafiltration membrane device to obtain the alpha-lactalbumin solution (G1), and drying to obtain an alpha-lactalbumin product.

See table 1 for test data for this example.

Table 1 example 1 test data sheet

Example 2

Step 1: WPC80 whey protein powder was dissolved (a 2) at a concentration of 4%, pH adjusted to 11.17 with 0.25mol/L food grade sodium hydroxide, and pH adjusted to 4.92 with 0.25mol/L food grade acetic acid to flocculate the protein in solution.

Step 2: the solution was separated by a 0.8 μm ceramic membrane, the pH of the dialysis water was adjusted to 5.16, and proteins that did not flocculate were washed out as much as possible by continuous addition of dialysis water. Obtaining a concentrated solution (b 2) mainly comprising concentrated beta-lactoglobulin solution; the clear solution (c 2) is mainly a mixed solution of alpha-lactalbumin and immunoglobulin G.

Step 3: and (2) passing the clear solution through a 100K ultrafiltration membrane to mainly intercept large molecular proteins such as immunoglobulin G and the like in the solution. Obtaining a concentrated solution (d 2) mainly comprising concentrated immunoglobulin G; the resulting supernatant (e 2) was mainly concentrated alpha-lactalbumin solution.

Step 4: after pH4.66 was adjusted to the clear liquid (e 2), the mixture was poured into a stirring tank containing 200 μm acrylic weak anion exchange resin at a rotation speed of 40r/min for 20min. After the adsorption is finished, evacuating the residual materials in the resin tank, repeatedly flushing and draining the residual materials by using pure water, and injecting and preparing 0.5% calcium chloride solution (first eluent) to analyze the resin when the detection value of a flushing water refractometer is 0, so as to obtain a first elution collection liquid, namely alpha-lactalbumin liquid (f 2); after completion, 5% sodium chloride solution (second eluent) is injected and prepared for resin analysis, and the next batch is prepared for standby after the second elution collection liquid is obtained.

Step 5: and (3) adjusting the pH value of the alpha-lactalbumin solution (f 2) subjected to 0.5% salt analysis in the step (4) to 3.68, passing through a second chromatographic column sleeved with 80 mu m agarose type weak anion exchange resin at a constant flow rate of 120L/h, collecting the flow-through liquid, adjusting the pH value to 6.36, desalting and concentrating by using a 5K ultrafiltration membrane device to obtain the alpha-lactalbumin solution (G2), and drying to obtain an alpha-lactalbumin product.

See table 2 for test data for this example.

Table 2 example 2 test data sheet

Example 3

Step 1: WPI90 separated whey protein powder was dissolved (a 3) at a concentration of 5%, pH was adjusted to 11.22 with 0.25mol/L food grade sodium hydroxide, pH was adjusted to 4.86 with 0.2mol/L food grade citric acid, and flocculation of the protein in solution occurred.

Step 2: separating the flocculation solution by a centrifugal machine at the rotating speed of 2000r/min for 10min, and obtaining a precipitate (b 3) which is mainly a concentrated beta-lactoglobulin product after the centrifugation is completed; the clear solution (c 3) is mainly a mixed solution of alpha-lactalbumin and immunoglobulin G.

Step 3: and (3) passing the clear solution (c 3) through a 50K ultrafiltration membrane to mainly intercept large molecular proteins such as immunoglobulin G and the like in the solution. Obtaining a concentrated solution (d 3) mainly comprising concentrated immunoglobulin G; the resulting supernatant (e 3) was mainly concentrated alpha-lactalbumin solution.

Step 4: after Ph4.28 was adjusted to the clear liquid (e 3), the mixture was poured into a stirring tank containing 200 μm polystyrene type weak anion exchange resin at a rotation speed of 40r/min for 30min. After the adsorption is finished, evacuating the residual materials in the resin tank, repeatedly flushing and draining the residual materials by using pure water, and injecting and preparing 0.4% potassium chloride solution (first eluent) to analyze the resin when the detection value of a flushing water refractometer is 0, so as to obtain a first elution collection liquid, namely alpha-lactalbumin liquid (f 3); after completion, 4% potassium chloride solution (second eluent) is injected and prepared for resin analysis, and the next batch is prepared for standby after the second elution collection liquid is obtained.

Step 5: and (3) adjusting the pH value of the alpha-lactalbumin solution (f 3) with 0.4% of salt analysis in the step (4) to 3.93, injecting the solution into a second stirring tank sleeved with 200 mu m polystyrene type weak anion exchange resin, stirring and adsorbing for 20min, collecting the flow-through liquid, adjusting the pH value to 6.36, desalting and concentrating the solution by using 5K ultrafiltration membrane equipment to obtain alpha-lactalbumin solution (G3), and drying to obtain an alpha-lactalbumin product.

See table 3 for test data for this example.

Table 3 example 3 test data sheet

Example 4

Step 1: WPC80 whey protein powder was dissolved (a 4) at 5% concentration, pH adjusted to 12.97 with 0.25mol/L food grade sodium hydroxide, and pH adjusted to 4.48 with 0.25mol/L food grade acetic acid to flocculate the protein in solution.

Step 2: the solution was separated by a 1.2 μm ceramic membrane, the pH of the dialysis water was adjusted to 5.48, and proteins that did not flocculate were washed out as much as possible by continuous addition of dialysis water. Obtaining a concentrated solution (b 4) mainly comprising concentrated beta-lactoglobulin solution; the clear solution (c 4) is mainly a mixed solution of alpha-lactalbumin and immunoglobulin G.

Step 3: and (3) passing the clear solution (c 4) through a 100K ultrafiltration membrane to mainly intercept large molecular proteins such as immunoglobulin G and the like in the solution. Obtaining a concentrated solution (d 4) mainly comprising concentrated immunoglobulin G; the resulting supernatant (e 4) was mainly concentrated alpha-lactalbumin solution.

Step 4: after Ph6.93 was adjusted to the clear liquid (e 4), the mixture was poured into a stirring tank containing 300 μm polystyrene type weak anion exchange resin at a rotation speed of 40r/min for 30min. After the adsorption is finished, evacuating the residual materials in the resin tank, repeatedly flushing and draining the residual materials by using pure water, and injecting and preparing 0.8% calcium chloride solution (first eluent) to analyze the resin when the detection value of a flushing water refractometer is 0, so as to obtain a first elution collection liquid, namely alpha-lactalbumin liquid (f 4); after the completion, 5% calcium chloride solution (second eluent) is injected and prepared for resin analysis, and the next batch is prepared for standby after the second elution collection liquid is obtained.

Step 5: and (3) adjusting the pH value of the alpha-lactalbumin solution (f 4) with 0.8% of salt analysis in the step (4) to 3.28, injecting the solution into a second stirring tank which is sleeved with 200 mu m acrylic weak anion exchange resin, stirring and adsorbing for 20min, collecting the flow-through liquid, adjusting the pH value to 6.36, desalting and concentrating the solution by using 5K ultrafiltration membrane equipment to obtain alpha-lactalbumin solution (G4), and drying to obtain an alpha-lactalbumin product.

See table 4 for test data for this example.

Table 4 example 4 test data sheet

Example 5

step 2: the concentrated whey protein liquid (a 5) was adjusted to pH 12.02 with 0.25mol/L food grade sodium hydroxide and then to pH 5.01 with 0.2mol/L food grade citric acid, and the proteins in the solution flocculated.

Step 3: the solution was separated by a 0.2 μm ceramic membrane, the pH of the dialysis water was adjusted to 4.02, and proteins that did not flocculate were washed out as much as possible by continuous addition of dialysis water. Because the pore diameter of the 0.2 mu m ceramic membrane is smaller, the immunoglobulin G cannot permeate, and only part of alpha-lactalbumin permeates, the obtained concentrated solution (b 5) is mainly whey protein concentrated solution; the clear solution (c 5) was obtained as a predominantly alpha-lactalbumin solution.

Step 4: after the pH of the clear liquid (c 5) was adjusted to 4.63, it was poured into a stirring tank containing 200 μm acrylic weak anion exchange resin at a rotation speed of 40r/min for an adsorption time of 30min. After the adsorption is finished, evacuating the residual materials in the resin tank, repeatedly flushing and draining the residual materials by using pure water, and injecting and preparing 0.3% potassium chloride solution (first eluent) to analyze the resin when the detection value of a flushing water refractometer is 0, so as to obtain a first elution collection liquid, namely alpha-lactalbumin liquid (d 5); after the completion, pouring and preparing 3.5% potassium chloride solution (second eluent) to analyze the resin, and preparing the next batch for later use after obtaining second elution collection liquid.

Step 5: and (3) adjusting the pH value of the alpha-lactalbumin solution (d 5) with 0.3% of salt analysis in the step (4) to 3.41, injecting the solution into a second stirring tank which is sleeved with 200 mu m acrylic weak anion exchange resin, stirring and adsorbing for 20min, collecting the flow-through liquid, adjusting the pH value to 6.36, desalting and concentrating the solution by using 5K ultrafiltration membrane equipment to obtain alpha-lactalbumin solution (e 5), and drying to obtain an alpha-lactalbumin product.

See table 5 for test data for this example.

Table 5 example 5 test data sheet

Example 6

Step 1: WPI90 separated whey protein powder was dissolved (a 6) at a concentration of 5%, pH was adjusted to 11.18 with 0.25mol/L food grade sodium hydroxide, pH was adjusted to 5.46 with 0.2mol/L food grade citric acid, and flocculation of the protein in solution occurred.

Step 2: separating the flocculation solution by a centrifugal machine at the rotating speed of 1000r/min for 15min, and obtaining a precipitate (b 6) which is mainly a concentrated beta-lactoglobulin product after the centrifugation is completed; the clear solution (c 6) is mainly a mixed solution of alpha-lactalbumin and immunoglobulin G.

Step 3: the clear liquid (c 6) passes through a microfiltration membrane with the molecular weight of 300K and mainly intercepts large molecular proteins such as immunoglobulin G and the like in the solution. Obtaining a concentrated solution (d 6) mainly comprising concentrated immunoglobulin G; the resulting supernatant (e 6) was mainly concentrated alpha-lactalbumin solution.

Step 4: after pH4.63 was adjusted to the clear liquid (e 6), the mixture was poured into a stirring tank containing 200 μm acrylic weak anion exchange resin at a rotation speed of 40r/min for adsorption time of 30min. After the adsorption is finished, evacuating the residual materials in the resin tank, repeatedly flushing and draining the residual materials by using pure water, and injecting and preparing 0.5% sodium chloride solution (first eluent) to analyze the resin when the detection value of a flushing water refractometer is 0, so as to obtain a first elution collection liquid, namely alpha-lactalbumin liquid (f 6); after completion, 3.5% sodium chloride solution (second eluent) is injected and prepared for resin analysis, and the next batch is prepared for standby after the second elution collection liquid is obtained.

Step 5: and (3) adjusting the pH value of the alpha-lactalbumin solution (f 6) with 0.5% of salt analysis in the step (4) to 3.63, injecting the solution into a second stirring tank sleeved with 200 mu m polystyrene type weak anion exchange resin, stirring and adsorbing for 20min, collecting the flow-through liquid, adjusting the pH value to 6.36, desalting and concentrating the solution by using 5K ultrafiltration membrane equipment to obtain alpha-lactalbumin solution (G6), and drying to obtain an alpha-lactalbumin product.

See table 6 for test data for this example.

Table 6 example 6 test data sheet

Claims

1. A method for concentration and separation of α-lactalbumin, characterized in that it includes the following steps:

(1) Adjust the pH in stages: Use the whey water or whey powder solution in the cheese production process to adjust the pH in stages by utilizing the gel properties of β-lactoglobulin; in the first stage, adjust the alkali and adjust the pH value to 11.0-13.0 to change the disulfide bonds of β-lactoglobulin in the solution; the second stage is acid adjustment, adjusting the pH value to 4.4-5.5 to cause the β-lactoglobulin in the solution to flocculate at the protein isoelectric point. precipitation;

(2) Membrane separation or centrifugal separation: The material after flocculation and precipitation in step (1) is subjected to membrane separation or centrifugal separation to obtain the first membrane retentate or centrifugal precipitate and the first membrane permeate or centrifugal supernatant; the third A membrane retentate or centrifugal sediment includes β-lactoglobulin, and the first membrane permeate or centrifugal supernatant includes a mixture of α-lactalbumin and immunoglobulin G;

(3) Drying after reconstitution: Adjust the pH value of the membrane retentate or centrifugal sediment in step (2) to 5.5-7.0 to obtain a reconstituted solution, and directly dry the reconstituted solution to obtain concentrated β-lactospheres. protein;

(4) Membrane separation or adsorption: The membrane permeate or centrifugal supernatant obtained in step (2) is subjected to membrane separation to obtain the second membrane retentate and the second membrane permeate; the second membrane retentate is concentrated Immunoglobulin G solution, the concentrated immunoglobulin G solution is dried to obtain a concentrated immunoglobulin G product; the second membrane permeate is a concentrated α-lactalbumin solution, and the concentrated α-lactalbumin solution is Concentrate and dry through ultrafiltration membrane to obtain a concentrated α-lactalbumin product, or inject the concentrated α-lactalbumin solution into the first set of chromatography columns or stirring tanks equipped with anion exchange resin for dynamic or static adsorption. Before injection, The pH value of the concentrated α-lactalbumin solution is adjusted to 4.2-7.0 to ensure that the target protein is adsorbed in the anion exchange resin;

(5) Fractional elution: Use salt solution as the eluent to elute the anion exchange resin in step (4) at least twice; obtain the target protein α-lactalbumin solution during the first elution; after the second elution Elution, after completion, collect the solution to prepare the next batch for further use; and

(6) Adsorption, desalting and drying: Directly inject the α-lactalbumin solution obtained in step (5) into the second set of chromatography columns or stirring tanks equipped with anion exchange resin. Before injection, adjust the pH value of the α-lactalbumin solution. Adjust to 3.2-4.0 and confirm that the anion exchange resin is supersaturated to ensure that impurities can be sufficiently adsorbed in the anion exchange resin. Collect the flow-through liquid and adjust it to neutral. Perform membrane desalination and concentration, and dry to obtain the α-lactalbumin product.

2. A method for concentration and separation of α-lactalbumin according to claim 1, characterized in that the alkali used in the first stage of alkali adjustment in step (1) is food grade sodium hydroxide.

3. A method for concentration and separation of α-lactalbumin according to claim 1, characterized in that the acid used in the second stage of acid adjustment in step (1) is food grade hydrochloric acid, acetic acid or citric acid.

4. A method for concentration and separation of α-lactalbumin according to claim 1, characterized in that the membrane pore size used in the membrane separation in step (2) is 0.2-1.2 μm.

5. A method for concentration and separation of α-lactalbumin according to claim 1, characterized in that, during the membrane separation in step (2), dialysis water is added with a pH value of 4.0-5.5 to avoid redissolution of the flocculated protein. .

6. A method for concentration and separation of α-lactalbumin according to claim 1, characterized in that the rotation speed during centrifugal separation in step (2) is ≥1000 r/min.

7. A method for concentration and separation of α-lactalbumin according to claim 1, characterized in that the membrane separation in step (4) adopts a membrane with a molecular weight cutoff of 50-300K.

8. A method for concentrating and separating α-lactalbumin according to claim 1, characterized in that the anion exchange resin of steps (4) and (6) is agarose type ion exchange resin, acrylic type ion exchange resin Any one of exchange resin and polystyrene exchange resin; the particle size of anion exchange resin is 30-300 microns.

9. A method for concentration and separation of α-lactalbumin according to claim 1, characterized in that, in the first elution of step (5), a salt solution with a mass concentration of 0.3%-0.8% is used as the eluent. For dehydration, a salt solution with a mass concentration of 3.5%-5% is used as the eluent for the second and subsequent elutions.

10. A method for concentration and separation of α-lactalbumin according to claim 9, characterized in that the salt solution is a sodium salt, potassium salt or calcium salt solution.