CN114409762B - Concentration and separation method of alpha-lactalbumin - Google Patents
Concentration and separation method of alpha-lactalbumin Download PDFInfo
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- CN114409762B CN114409762B CN202210094198.9A CN202210094198A CN114409762B CN 114409762 B CN114409762 B CN 114409762B CN 202210094198 A CN202210094198 A CN 202210094198A CN 114409762 B CN114409762 B CN 114409762B
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- 102000004407 Lactalbumin Human genes 0.000 title claims abstract description 94
- 108090000942 Lactalbumin Proteins 0.000 title claims abstract description 94
- 235000021241 α-lactalbumin Nutrition 0.000 title claims abstract description 92
- 238000000926 separation method Methods 0.000 title claims abstract description 29
- 239000012528 membrane Substances 0.000 claims abstract description 58
- 229940027941 immunoglobulin g Drugs 0.000 claims abstract description 34
- 102000007544 Whey Proteins Human genes 0.000 claims abstract description 31
- 108010046377 Whey Proteins Proteins 0.000 claims abstract description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- 102000008192 Lactoglobulins Human genes 0.000 claims abstract description 21
- 108010060630 Lactoglobulins Proteins 0.000 claims abstract description 21
- 239000005862 Whey Substances 0.000 claims abstract description 18
- 238000001035 drying Methods 0.000 claims abstract description 18
- 238000001179 sorption measurement Methods 0.000 claims abstract description 18
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 17
- 230000001105 regulatory effect Effects 0.000 claims abstract description 14
- 239000002253 acid Substances 0.000 claims abstract description 7
- 239000003513 alkali Substances 0.000 claims abstract description 7
- 238000005119 centrifugation Methods 0.000 claims abstract description 5
- 238000010612 desalination reaction Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 102
- 239000007788 liquid Substances 0.000 claims description 41
- 102000004169 proteins and genes Human genes 0.000 claims description 25
- 108090000623 proteins and genes Proteins 0.000 claims description 25
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- 235000018102 proteins Nutrition 0.000 claims description 23
- 239000003480 eluent Substances 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 17
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 15
- 239000000047 product Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- 238000000108 ultra-filtration Methods 0.000 claims description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 12
- 238000000502 dialysis Methods 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 239000012266 salt solution Substances 0.000 claims description 10
- 238000011033 desalting Methods 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 7
- 239000004793 Polystyrene Substances 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 239000012466 permeate Substances 0.000 claims description 7
- 229920002223 polystyrene Polymers 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 7
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 235000013351 cheese Nutrition 0.000 claims description 6
- 238000005189 flocculation Methods 0.000 claims description 6
- 230000016615 flocculation Effects 0.000 claims description 6
- 239000003456 ion exchange resin Substances 0.000 claims description 6
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 6
- 229920000936 Agarose Polymers 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 239000011148 porous material Substances 0.000 claims description 3
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 2
- 159000000007 calcium salts Chemical class 0.000 claims description 2
- 239000012535 impurity Substances 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 239000012465 retentate Substances 0.000 claims description 2
- 159000000000 sodium salts Chemical class 0.000 claims description 2
- 230000003068 static effect Effects 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims 4
- 235000021119 whey protein Nutrition 0.000 abstract description 13
- 239000002994 raw material Substances 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 2
- 238000001742 protein purification Methods 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 abstract 1
- 239000011347 resin Substances 0.000 description 18
- 229920005989 resin Polymers 0.000 description 18
- 238000001514 detection method Methods 0.000 description 17
- 238000010828 elution Methods 0.000 description 14
- 238000011010 flushing procedure Methods 0.000 description 12
- 235000013305 food Nutrition 0.000 description 12
- 239000012610 weak anion exchange resin Substances 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 8
- 239000007791 liquid phase Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 235000013336 milk Nutrition 0.000 description 7
- 239000008267 milk Substances 0.000 description 7
- 210000004080 milk Anatomy 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 238000001228 spectrum Methods 0.000 description 7
- 239000000919 ceramic Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 239000001103 potassium chloride Substances 0.000 description 4
- 235000011164 potassium chloride Nutrition 0.000 description 4
- 230000006269 (delayed) early viral mRNA transcription Effects 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 239000008101 lactose Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000006303 immediate early viral mRNA transcription Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004134 energy conservation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
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
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 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 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 (10)
1. A method for concentrating and separating alpha-lactalbumin, which is characterized by comprising the following steps:
(1) Adjusting the pH value in stages: 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 beta-lactoglobulin has gel; in the first stage, alkali is regulated, the pH value is regulated to be 11.0-13.0, and the disulfide bonds of beta-lactoglobulin in the solution 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) to obtain a first membrane trapped fluid or centrifugal precipitate and a first membrane permeate or centrifugal clear liquid; the first membrane retentate or the centrifugal precipitate comprises beta-lactoglobulin, and the first membrane permeate or the centrifugal clear liquid comprises a mixed solution of alpha-lactalbumin and immunoglobulin G;
(3) Drying after redissolution: regulating the pH value of the membrane trapped fluid or the centrifugal precipitate in the step (2) to 5.5-7.0, re-dissolving to obtain a re-solution, and directly drying the re-solution to obtain concentrated beta-lactoglobulin;
(4) Membrane separation or adsorption: performing membrane separation on the membrane permeate or the centrifugal clear liquid obtained in the step (2) to obtain second membrane trapped fluid and second membrane permeate; the second membrane trapped fluid is a concentrated immunoglobulin G solution, and the concentrated immunoglobulin G solution is dried to obtain a concentrated immunoglobulin G product; the second membrane permeate is a concentrated alpha-lactalbumin solution, the concentrated alpha-lactalbumin solution is concentrated and dried by an ultrafiltration membrane to obtain a concentrated alpha-lactalbumin product, or the concentrated alpha-lactalbumin solution is injected into a first chromatographic column or a stirring tank which is provided with anion exchange resin for dynamic or static adsorption, and the pH value of the concentrated alpha-lactalbumin solution is adjusted to 4.2-7.0 before injection so as to ensure that target protein is 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; and
(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.
2. The method for concentrating and separating alpha-lactalbumin as claimed in claim 1, wherein the alkali used for the first-stage alkali adjustment in the step (1) is food-grade sodium hydroxide.
3. The method for concentrating and separating alpha-lactalbumin according to claim 1, wherein the acid used in the second-stage acid adjustment in the step (1) is food-grade hydrochloric acid, acetic acid or citric acid.
4. The method for concentrating and separating alpha-lactalbumin according to claim 1, wherein the membrane used in the membrane separation in the step (2) has a pore size of 0.2-1.2 μm.
5. The method for concentrating and separating alpha-lactalbumin according to claim 1, wherein the pH value of the dialysis water added in the membrane separation in the step (2) is 4.0-5.5 so as to avoid redissolution of flocculated protein.
6. The method for concentrating and separating alpha-lactalbumin as claimed in claim 1, wherein the rotational speed of the centrifugal separation in the step (2) is more than or equal to 1000r/min.
7. The method for concentrating and separating alpha-lactalbumin according to claim 1, wherein the membrane separation in the step (4) adopts a membrane with a molecular weight cut-off of 50-300K.
8. The method for concentrating and separating α -lactalbumin according to claim 1, wherein the anion exchange resin in step (4) and step (6) is any one 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.
9. The method according to claim 1, wherein the first eluting in the step (5) uses a salt solution with a mass concentration of 0.3% -0.8% as an eluent, and the second and subsequent eluting uses a salt solution with a mass concentration of 3.5% -5% as an eluent.
10. The method for concentrating and separating alpha-lactalbumin according to claim 9, wherein the salt solution is a sodium salt, potassium salt or calcium salt solution.
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