CN114409762A - Concentration and separation method of alpha-lactalbumin - Google Patents
Concentration and separation method of alpha-lactalbumin Download PDFInfo
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- CN114409762A CN114409762A CN202210094198.9A CN202210094198A CN114409762A CN 114409762 A CN114409762 A CN 114409762A CN 202210094198 A CN202210094198 A CN 202210094198A CN 114409762 A CN114409762 A CN 114409762A
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- 102000004407 Lactalbumin Human genes 0.000 title claims abstract description 96
- 108090000942 Lactalbumin Proteins 0.000 title claims abstract description 96
- 235000021241 α-lactalbumin Nutrition 0.000 title claims abstract description 93
- 238000000926 separation method Methods 0.000 title claims abstract description 27
- 239000012528 membrane Substances 0.000 claims abstract description 51
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 32
- 102000007544 Whey Proteins Human genes 0.000 claims abstract description 31
- 108010046377 Whey Proteins Proteins 0.000 claims abstract description 31
- 229940027941 immunoglobulin g Drugs 0.000 claims abstract description 29
- 102000008192 Lactoglobulins Human genes 0.000 claims abstract description 21
- 108010060630 Lactoglobulins Proteins 0.000 claims abstract description 21
- 238000001179 sorption measurement Methods 0.000 claims abstract description 21
- 239000005862 Whey Substances 0.000 claims abstract description 19
- 238000001035 drying Methods 0.000 claims abstract description 18
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000011033 desalting Methods 0.000 claims abstract description 9
- 239000003513 alkali Substances 0.000 claims abstract description 5
- 239000002253 acid Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 111
- 239000007788 liquid Substances 0.000 claims description 33
- 102000004169 proteins and genes Human genes 0.000 claims description 26
- 108090000623 proteins and genes Proteins 0.000 claims description 26
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- 235000018102 proteins Nutrition 0.000 claims description 24
- 239000003480 eluent Substances 0.000 claims description 21
- 238000010828 elution Methods 0.000 claims description 17
- 239000000047 product Substances 0.000 claims description 17
- 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
- 235000013305 food Nutrition 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
- 239000012266 salt solution Substances 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 9
- 239000011259 mixed solution Substances 0.000 claims description 8
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 7
- 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
- 239000000463 material Substances 0.000 claims description 7
- 229920002223 polystyrene Polymers 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
- 239000003456 ion exchange resin Substances 0.000 claims description 6
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 239000002244 precipitate Substances 0.000 claims description 6
- 229920000936 Agarose Polymers 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 239000012466 permeate Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 159000000007 calcium salts Chemical class 0.000 claims description 2
- 238000010612 desalination reaction Methods 0.000 claims description 2
- 238000000502 dialysis Methods 0.000 claims description 2
- 238000005189 flocculation Methods 0.000 claims description 2
- 230000016615 flocculation Effects 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 239000012535 impurity Substances 0.000 claims description 2
- 230000007935 neutral effect Effects 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
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 159000000000 sodium salts Chemical class 0.000 claims description 2
- 230000003068 static effect Effects 0.000 claims description 2
- 235000021119 whey protein Nutrition 0.000 abstract description 12
- 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
- 238000001514 detection method Methods 0.000 description 18
- 239000011347 resin Substances 0.000 description 18
- 229920005989 resin Polymers 0.000 description 18
- 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
- 238000005507 spraying Methods 0.000 description 10
- 238000001228 spectrum Methods 0.000 description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 8
- 235000013336 milk Nutrition 0.000 description 8
- 239000008267 milk Substances 0.000 description 8
- 210000004080 milk Anatomy 0.000 description 8
- 239000000919 ceramic Substances 0.000 description 7
- 239000007791 liquid phase Substances 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 239000012141 concentrate Substances 0.000 description 6
- 230000000717 retained effect Effects 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
- 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
- 239000008101 lactose Substances 0.000 description 3
- 230000006269 (delayed) early viral mRNA transcription Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
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- 238000009776 industrial production Methods 0.000 description 2
- 239000012460 protein solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 235000020247 cow milk Nutrition 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000004134 energy conservation Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 230000006303 immediate early viral mRNA transcription Effects 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
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- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 235000020254 sheep milk Nutrition 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
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- 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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- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
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- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
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- 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 method for concentrating and separating alpha-lactalbumin, which comprises the following steps: (1) adjusting the pH value by stages: adjusting alkali and pH value to 11.0-13.0 in the first stage; adjusting acid in the second stage, and adjusting the pH value to 4.4-5.5; (2) membrane separation or centrifugal separation; (3) drying after redissolution; (4) membrane separation or adsorption: ensuring that the alpha-lactalbumin is adsorbed in the anion exchange resin; (5) eluting for several times; (6) adsorption, desalting and drying: to obtain the alpha-lactalbumin product. The invention recovers the whey protein in the whey water as the raw material, and the high-purity alpha-lactalbumin is industrially produced on the basis of the concentration of the alpha-lactalbumin while the alpha-lactalbumin, the beta-lactoglobulin and the immunoglobulin G are concentrated and separated by human intervention.
Description
Technical Field
The invention relates to the technical field of protein purification and preparation, in particular to a method for concentrating and separating alpha-lactalbumin, namely a method for producing concentrated and separated lactalbumin and preparing alpha-lactalbumin by taking milk powder such as cow or sheep milk clear water, whey powder and the like as raw materials.
Background
Whey is an accessory in milk cheese processingThe product, which is present in the water phase during the coagulation of the cow cheese protein, accounts for 85-95% of the milk volume. The 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. The Biochemical Oxygen Demand (BOD) of the treated whey is 27-60 g/L, and the Chemical Oxygen Demand (COD) is 50-102 g/L, so the whey is considered to be a serious environmental pollutant. About 9X 10 of the annual production of whey in the world7t, about 50% of them are discharged as waste water, which not only wastes resources but also pollutes the environment. With the increase of the production scale of cheese in China, the discharge of whey water becomes a problem of 'big difficulty' of cheese enterprises, the Biological Oxygen Demand (BOD) and Chemical Oxygen Demand (COD) in whey water are high, the pollution is serious after the whey water is directly discharged, and the requirements of energy conservation and emission reduction are not met.
The alpha and beta proteins have similar polarities and are difficult to separate by directly using resin for separation; the content ratio of each protein in whey is set up "innate", because the molecular weight of alpha-lactalbumin is about 14K, the molecular weight of beta-lactoglobulin is about 18K, the separation is difficult by a membrane separation method, the investment is large when resin is used for purification alone, and the industrial production cannot be realized.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for concentrating and separating alpha-lactalbumin, which comprises the following steps:
(1) adjusting the pH value by stages: adjusting the pH value of whey water or whey powder solution in the cheese production process by stages by utilizing the characteristic that beta-lactoglobulin has gel; adjusting alkali and pH value to 11.0-13.0 in the first stage to change disulfide bond of beta-lactoglobulin; adjusting the pH value to 4.4-5.5 to make the beta-lactoglobulin in the solution generate flocculation precipitation at the protein isoelectric point;
(2) membrane separation or centrifugation: performing membrane separation or centrifugal separation on the flocculated and precipitated material in the step (1); the membrane trapped fluid or centrifugal precipitate is mainly beta-lactoglobulin; the membrane permeate or the centrifugal clear liquid is mainly the mixed liquid of alpha-lactalbumin and immunoglobulin G;
(3) drying after redissolution: regulating the pH value of the membrane concentrated solution or the centrifugal precipitate in the step (2) to 5.5-7.0 for redissolution, 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 concentrated solution to be concentrated immunoglobulin G solution, and drying to obtain a concentrated immunoglobulin G product; the clear solution is concentrated alpha-lactalbumin solution, concentrated by an ultrafiltration membrane and dried to obtain a concentrated alpha-lactalbumin product, or the concentrated alpha-lactalbumin solution is injected into a chromatographic column or a stirring tank filled 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 that the target protein can be adsorbed in the anion exchange resin;
(5) and (3) fractional elution: 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 a target protein alpha-lactalbumin solution; eluting for the second time, and collecting the solution after the elution is finished to prepare the next batch for continuous use;
(6) adsorption, desalting and drying: and (3) directly injecting the alpha-lactalbumin solution obtained in the step (5) into a second chromatographic column or a stirring tank which is sleeved with anion exchange resin, adjusting the pH value of the alpha-lactalbumin solution to 3.2-4.0 before injection, determining supersaturation of the anion exchange resin, ensuring that impurities can be sufficiently adsorbed in the anion exchange resin, collecting flow-through liquid, adjusting the flow-through liquid to be neutral, performing membrane desalination and concentration, and drying to obtain an alpha-lactalbumin product.
The alkali used in the first stage of the step (1) is food-grade sodium hydroxide.
The acid used in the second stage of the step (1) is food grade hydrochloric acid, acetic acid or citric acid.
The pore diameter of the membrane used for membrane separation in the step (2) is 0.2-1.2 μm.
And (3) adding dialysis water with the pH value of 4.0-5.5 during membrane separation in the step (2) to avoid redissolving the flocculated protein.
The rotating speed in the centrifugal separation in the step (2) is more than or equal to 1000 r/min.
And (4) membrane separation in the step (4) adopts a membrane with the molecular weight cutoff of 50-300K.
The anion exchange resin in the steps (4) and (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 later elution adopts a salt solution with the mass concentration of 3.5-5% as an eluent.
The salt solution in the step (5) is sodium salt, potassium salt or calcium salt solution.
The invention mainly 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 the alpha-lactalbumin, thereby changing waste into valuable.
2. The invention relates to a method for industrially producing high-purity alpha-lactalbumin on the basis of concentrating alpha-lactalbumin while concentrating and separating alpha-lactalbumin, beta-lactoglobulin and immunoglobulin G products by human intervention.
3. The invention enriches beta-protein, reduces the adsorption strength of alpha-lactalbumin and realizes 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 content of the alpha-lactalbumin in the total protein reaches more than 90 percent.
Drawings
FIG. 1 is a liquid phase detection spectrum of concentrated whey (10.038min alpha-lactalbumin peak; 10.609min beta-lactoglobulin peak);
FIG. 2 is a schematic view of the centrifugal effect of the method of the present invention;
FIG. 3 is a product detection report of the industrial production of alpha-lactalbumin and immunoglobulin G mixed powder of the present invention;
FIG. 4 shows one of the liquid phase detection spectra of the mixture of alpha-lactalbumin and immunoglobulin G (10.47min: alpha-lactalbumin peak; 11.29min: beta-lactoglobulin peak);
FIG. 5 shows the second detection spectrum (13.42min: IgG peak) of the mixed solution of alpha-lactalbumin and IgG;
FIG. 6 is one of the liquid phase detection spectra of the membrane-permeated clear liquid (10.84min: alpha-lactalbumin peak; 11.38 min: beta-lactoglobulin peak);
FIG. 7 shows the second detection spectrum (13.24min: IgG peak) of the membrane-permeated clear liquid phase;
FIG. 8 is a diagram showing a liquid phase detection spectrum of concentrated IgG (13.28min: IgG peak);
FIG. 9 shows one of the liquid phase detection spectra of alpha-lactalbumin (10.90min: alpha-lactalbumin peak);
FIG. 10 shows the second detection spectrum of alpha-lactalbumin liquid phase (10.57min: alpha-lactalbumin peak);
FIG. 11 is a schematic flow chart of the method of the present invention;
and (4) supplementary notes: because no national detection standard exists at present, the high performance liquid detection spectrum is set according to the national standard GB1903.17-2016 chromatographic gradient, and the detection wavelength is 220nm for detection; immunoglobulin G was determined according to the method of national Standard GB/T5009.194-2003.
Detailed Description
The present invention will be described in detail with reference to examples. In the following, the percentage concentrations are mass concentrations.
Example 1
Step 1: after milk is curdled, milk clear water is collected by filtration, and the whey is concentrated by 5K ultrafiltration membrane lactose removal, so that the content of whey protein is increased;
step 2: the concentrated whey protein solution (a1) was adjusted to pH 11.22 with 0.25mol/L food grade sodium hydroxide and then adjusted to pH 4.86 with 0.5mol/L food grade hydrochloric acid to flocculate the protein in the solution.
And 3, step 3: the solution was separated by passing through a 0.8 μm ceramic membrane, the pH of the dialyzed water was adjusted to 4.76, and the nonflocculated protein was washed out as much as possible by continuously adding the dialyzed water. The obtained concentrated solution (b1) is mainly concentrated beta-lactoglobulin solution; the clear solution (c1) is obtained as a mixed solution of mainly alpha-lactalbumin and immunoglobulin G.
And 4, step 4: the clear solution (c1) passes through a 100K ultrafiltration membrane, and macromolecular proteins such as immunoglobulin G in the solution are mainly retained. The resulting concentrate (d1) is mainly a concentrated immunoglobulin G solution; the resulting clear solution (e 1) was mainly a concentrated alpha-lactalbumin solution.
And 5, step 5: the clear solution (e 1) was adjusted to pH4.61 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 20 min. After adsorption, washing the resin filler in the chromatographic column by using 4 column volumes of RO water, and after washing, eluting the chromatographic column by using 4 column volumes of 0.5 percent sodium chloride solution (first eluent) to obtain an alpha-lactalbumin solution (f 1); and eluting the chromatographic column by adopting 4 sodium chloride solutions (second eluent) with the volume of 3.5 percent of the column volume to obtain second elution collecting liquid, and preparing for next batch after the elution is finished.
And 6, step 6: adjusting pH of the alpha-lactalbumin solution (f 1) with 0.5% salt resolution in the step 5 to 3.72, passing through a second chromatographic column sheathed with 80 μm agarose type weak anion exchange resin at a constant flow rate of 120L/h, collecting flow-through liquid, adjusting pH to 6.36, desalting and concentrating with 5K ultrafiltration membrane equipment to obtain alpha-lactalbumin solution (G1), and drying to obtain alpha-lactalbumin product.
For the test data, see table 1.
Table 1 example 1 test data table
Example 2
Step 1: WPC80 whey protein powder was dissolved (a2) at a concentration of 4%, and the pH was adjusted to 11.17 with 0.25mol/L food grade sodium hydroxide and to 4.92 with 0.25mol/L food grade acetic acid to flocculate the protein in the solution.
Step 2: the solution was separated by means of a 0.8 μm ceramic membrane, the pH of the dialyzed water was adjusted to 5.16, and the nonflocculated protein was washed out as much as possible by the continuous addition of the dialyzed water. The obtained concentrated solution (b2) is mainly concentrated beta-lactoglobulin solution; the clear solution (c2) is obtained as a mixed solution of mainly alpha-lactalbumin and immunoglobulin G.
And 3, step 3: the clear solution (c2) passes through a 100K ultrafiltration membrane, and macromolecular proteins such as immunoglobulin G in the solution are mainly retained. The resulting concentrate (d2) is mainly a concentrated immunoglobulin G solution; the resulting clear solution (e 2) was mainly a concentrated alpha-lactalbumin solution.
And 4, step 4: ph4.66 of the clear solution (e 2) is adjusted, and then the clear solution is injected into a stirring tank filled with 200 mu m of acrylic acid type weak anion exchange resin, the rotating speed is 40r/min, and the adsorption time is 20 min. After adsorption is finished, emptying the residual materials in the resin tank, repeatedly spraying by using pure water, emptying, and injecting and preparing 0.5% calcium chloride solution (first eluent) to analyze the resin when the detection value of a spraying water refractometer is 0 to obtain first elution collecting liquid, namely alpha-lactalbumin liquid (f 2); and injecting and preparing a 5% sodium chloride solution (second eluent) to analyze the resin after the completion, and preparing the next batch for later use after the second eluent is obtained.
And 5, step 5: adjusting pH of the alpha-lactalbumin solution (f 2) with 0.5% salt resolution in the step 4 to 3.68, passing through a second chromatographic column sheathed with 80 μm agarose type weak anion exchange resin at a constant flow rate of 120L/h, collecting flow-through liquid, adjusting pH to 6.36, desalting and concentrating with 5K ultrafiltration membrane equipment to obtain alpha-lactalbumin solution (G2), and drying to obtain alpha-lactalbumin product.
For the test data, see table 2.
Table 2 example 2 test data table
Example 3
Step 1: WPI90 isolated whey protein powder was solubilized (a3) at 5% concentration, adjusted to pH 11.22 with 0.25mol/L food grade sodium hydroxide and adjusted to pH 4.86 with 0.2mol/L food grade citric acid to flocculate the protein in solution.
Step 2: separating the flocculated solution by a centrifugal machine at the rotating speed of 2000r/min for 10min to obtain a precipitate (b3) which is mainly a concentrated beta-lactoglobulin product after the centrifugation is finished; the clear solution (c3) is obtained as a mixed solution of mainly alpha-lactalbumin and immunoglobulin G.
And 3, step 3: the clear solution (c3) passes through a 50K ultrafiltration membrane, and macromolecular proteins such as immunoglobulin G in the solution are mainly retained. The resulting concentrate (d3) is mainly a concentrated immunoglobulin G solution; the resulting clear solution (e 3) was mainly a concentrated alpha-lactalbumin solution.
And 4, step 4: ph4.28 of the clear liquid (e 3) is adjusted, and then the clear liquid is injected into a stirring tank filled with 200 mu m polystyrene type weak anion exchange resin, the rotating speed is 40r/min, and the adsorption time is 30 min. After adsorption is finished, emptying the residual materials in the resin tank, repeatedly spraying by using pure water, emptying, and injecting and preparing 0.4% potassium chloride solution (first eluent) to analyze the resin when the detection value of a spraying water refractometer is 0 to obtain first elution collecting liquid, namely alpha-lactalbumin liquid (f 3); and injecting and preparing 4% potassium chloride solution (second eluent) to analyze the resin after the completion, and preparing the next batch for later use after the second eluent is obtained.
And 5, step 5: adjusting the pH value of the alpha-lactalbumin solution (f 3) with 0.4% salt analysis in the step 4 to 3.93, injecting the solution into a second stirring tank which is sleeved with 200 mu m polystyrene type weak anion exchange resin, stirring and adsorbing for 20min, collecting flow-through liquid, adjusting the pH value to 6.36, desalting and concentrating by using a 5K ultrafiltration membrane device to obtain alpha-lactalbumin solution (G3), and drying to obtain alpha-lactalbumin product.
For the test data, see table 3.
Table 3 example 3 test data table
Example 4
Step 1: WPC80 whey protein powder was dissolved (a4) at 5% concentration, and after adjusting pH to 12.97 with 0.25mol/L food grade sodium hydroxide, the pH was adjusted to 4.48 with 0.25mol/L food grade acetic acid, and protein in the solution flocculated.
Step 2: the solution was separated by passing through a 1.2 μm ceramic membrane, the pH of the dialyzed water was adjusted to 5.48, and the nonflocculated protein was washed out as much as possible by continuously adding the dialyzed water. The obtained concentrated solution (b4) is mainly concentrated beta-lactoglobulin solution; the clear solution (c4) is obtained as a mixed solution of mainly alpha-lactalbumin and immunoglobulin G.
And 3, step 3: the clear solution (c4) passes through a 100K ultrafiltration membrane, and macromolecular proteins such as immunoglobulin G in the solution are mainly retained. The resulting concentrate (d4) is mainly a concentrated immunoglobulin G solution; the resulting clear solution (e 4) was mainly a concentrated alpha-lactalbumin solution.
And 4, step 4: ph6.93 of the clear liquid (e 4) is adjusted, and then the clear liquid is injected into a stirring tank filled with polystyrene type weak anion exchange resin with the particle size of 300 mu m, the rotating speed is 40r/min, and the adsorption time is 30 min. After adsorption is finished, emptying the residual materials in the resin tank, repeatedly spraying by using pure water, emptying, and injecting and preparing 0.8% calcium chloride solution (first eluent) to analyze the resin when the detection value of a spraying water refractometer is 0 to obtain first elution collecting liquid, namely alpha-lactalbumin liquid (f 4); and injecting and preparing a 5% calcium chloride solution (second eluent) to analyze the resin after the completion, and preparing the next batch for later use after the second eluent is obtained.
And 5, step 5: adjusting the pH value of the alpha-lactalbumin solution (f 4) with 0.8% 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 acid type weak anion exchange resin, stirring and adsorbing for 20min, collecting flow-through liquid, adjusting the pH value to 6.36, desalting and concentrating by using a 5K ultrafiltration membrane device to obtain an alpha-lactalbumin solution (G4), and drying to obtain an alpha-lactalbumin product.
For the test data, see table 4.
Table 4 example 4 test data table
Example 5
Step 1: after milk is curdled, milk clear water is collected by filtration, and the whey is concentrated by 5K ultrafiltration membrane lactose removal, so that the content of whey protein is increased;
step 2: the concentrated whey protein solution (a5) was adjusted to pH 12.02 with 0.25mol/L food grade sodium hydroxide and then adjusted to pH 5.01 with 0.2mol/L food grade citric acid to flocculate the protein in the solution.
And 3, step 3: the solution was separated by means of a 0.2 μm ceramic membrane, the pH of the dialyzed water was adjusted to 4.02, and the nonflocculated protein was washed out as far as possible by the continuous addition of the dialyzed water. The pore diameter of the 0.2 mu m ceramic membrane is small, immunoglobulin G cannot permeate through the ceramic membrane, and only part of alpha-lactalbumin permeates through the ceramic membrane, so that the obtained concentrated solution (b5) is mainly whey protein concentrated solution; the resulting clear solution (c 5) was mainly an alpha-lactalbumin solution.
And 4, step 4: the pH of the clear solution (c 5) was adjusted to 4.63, and the solution was poured into a stirring tank containing 200 μm of an acrylic acid type weak anion exchange resin at a rotation speed of 40r/min for an adsorption time of 30 min. After adsorption is finished, emptying the residual materials in the resin tank, repeatedly spraying by using pure water, emptying, and injecting and preparing 0.3% potassium chloride solution (first eluent) to analyze the resin when the detection value of a spraying water refractometer is 0 to obtain first elution collecting liquid, namely alpha-lactalbumin liquid (d 5); after the completion, 3.5% potassium chloride solution (second eluent) is injected and prepared to analyze the resin, and second elution collecting liquid is obtained to prepare the next batch for standby.
And 5, step 5: adjusting the pH value of the alpha-lactalbumin solution (d 5) with 0.3% 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 acid type weak anion exchange resin, stirring and adsorbing for 20min, collecting flow-through liquid, adjusting the pH value to 6.36, desalting and concentrating by using a 5K ultrafiltration membrane device to obtain an alpha-lactalbumin solution (e5), and drying to obtain an alpha-lactalbumin product.
For this example, see table 5 for test data.
Table 5 example 5 test data table
Example 6
Step 1: WPI90 isolated whey protein powder was solubilized (a6) at 5% concentration, adjusted to pH 11.18 with 0.25mol/L food grade sodium hydroxide and adjusted to pH 5.46 with 0.2mol/L food grade citric acid to flocculate the protein in solution.
Step 2: separating the flocculated solution by a centrifugal machine at the rotation speed of 1000r/min for 15min to obtain a precipitate (b6) which is mainly a concentrated beta-lactoglobulin product after the centrifugation is finished; the clear solution (c6) is obtained as a mixed solution of mainly alpha-lactalbumin and immunoglobulin G.
And 3, step 3: the clear solution (c6) passes through a microfiltration membrane with the molecular weight of 300K, and macromolecular proteins such as immunoglobulin G in the solution are mainly retained. The resulting concentrate (d6) is mainly a concentrated immunoglobulin G solution; the resulting clear solution (e 6) was mainly a concentrated alpha-lactalbumin solution.
And 4, step 4: ph4.63 of the clear solution (e 6) is adjusted, and then the clear solution is injected into a stirring tank filled with 200 mu m of acrylic acid type weak anion exchange resin, the rotating speed is 40r/min, and the adsorption time is 30 min. After adsorption is finished, emptying the residual materials in the resin tank, repeatedly spraying by using pure water, emptying, and injecting and preparing 0.5% sodium chloride solution (first eluent) to analyze the resin when the detection value of a spraying water refractometer is 0 to obtain first elution collecting liquid, namely alpha-lactalbumin liquid (f 6); after the reaction is finished, 3.5% sodium chloride solution (second eluent) is injected and prepared to analyze the resin, and second elution collecting liquid is obtained to prepare the next batch for standby.
And 5, step 5: adjusting pH of the alpha-lactalbumin solution (f 6) with 0.5% salt analysis in the step 4 to 3.63, injecting the solution into a second stirring tank with 200 μm polystyrene type weak anion exchange resin, stirring and adsorbing for 20min, collecting flow-through liquid, adjusting pH to 6.36, desalting and concentrating by using a 5K ultrafiltration membrane device to obtain alpha-lactalbumin solution (G6), and drying to obtain alpha-lactalbumin product.
For this example, see table 6 for test data.
Table 6 example 6 test data table
Claims (10)
1. A method for concentrating and separating alpha-lactalbumin is characterized by comprising the following steps:
(1) adjusting the pH value by stages: adjusting the pH value of whey water or whey powder solution in the cheese production process by stages by utilizing the characteristic that beta-lactoglobulin has gel; adjusting alkali and pH value to 11.0-13.0 in the first stage to change disulfide bond of beta-lactoglobulin; adjusting the pH value to 4.4-5.5 to make the beta-lactoglobulin in the solution generate flocculation precipitation at the protein isoelectric point;
(2) membrane separation or centrifugation: performing membrane separation or centrifugal separation on the flocculated and precipitated material in the step (1); the membrane trapped fluid or centrifugal precipitate is mainly beta-lactoglobulin; the membrane permeate or the centrifugal clear liquid is mainly the mixed liquid of alpha-lactalbumin and immunoglobulin G;
(3) drying after redissolution: regulating the pH value of the membrane concentrated solution or the centrifugal precipitate in the step (2) to 5.5-7.0 for redissolution, 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 concentrated solution to be concentrated immunoglobulin G solution, and drying to obtain a concentrated immunoglobulin G product; the clear solution is concentrated alpha-lactalbumin solution, concentrated by an ultrafiltration membrane and dried to obtain a concentrated alpha-lactalbumin product, or the concentrated alpha-lactalbumin solution is injected into a chromatographic column or a stirring tank filled 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 that the target protein can be adsorbed in the anion exchange resin;
(5) and (3) fractional elution: 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 a target protein alpha-lactalbumin solution; eluting for the second time, and collecting the solution after the elution is finished to prepare the next batch for continuous use;
(6) adsorption, desalting and drying: and (3) directly injecting the alpha-lactalbumin solution obtained in the step (5) into a second chromatographic column or a stirring tank which is sleeved with anion exchange resin, adjusting the pH value of the alpha-lactalbumin solution to 3.2-4.0 before injection, determining supersaturation of the anion exchange resin, ensuring that impurities can be sufficiently adsorbed in the anion exchange resin, collecting flow-through liquid, adjusting the flow-through liquid to be neutral, performing membrane desalination and concentration, and drying to obtain an alpha-lactalbumin product.
2. The method for concentrating and separating alpha-lactalbumin as claimed in claim 1, wherein the alkali used in the first stage of the step (1) is food-grade sodium hydroxide.
3. The method for concentrating and separating alpha-lactalbumin as claimed in claim 1, wherein the acid used in the second stage of the acid adjustment in step (1) is food grade hydrochloric acid, acetic acid or citric acid.
4. The method for concentrating and separating alpha-lactalbumin as claimed in claim 1, wherein the membrane used in the membrane separation in step (2) has a pore size of 0.2-1.2 μm.
5. The method for concentrating and separating alpha-lactalbumin as claimed in 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 the redissolution of the flocculated protein.
6. The method for concentrating and separating alpha-lactalbumin according to claim 1, wherein the rotation speed of the centrifugal separation in the step (2) is more than or equal to 1000 r/min.
7. The method for concentrating and separating alpha-lactalbumin as claimed in claim 1, wherein the membrane separation in step (4) adopts a membrane with molecular weight cut-off of 50-300K.
8. The method for concentrating and separating alpha-lactalbumin as claimed in claim 1, wherein the anion exchange resin in steps (4) and (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.
9. The method for concentrating and separating alpha-lactalbumin as claimed in claim 1, wherein the first elution in step (5) uses a salt solution with a mass concentration of 0.3% -0.8% as an eluent, and the second later elution uses a salt solution with a mass concentration of 3.5% -5% as an eluent.
10. The method for concentrating and separating alpha-lactalbumin as claimed in claim 1, wherein the salt solution in step (5) is sodium salt, potassium salt or calcium salt solution.
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| CN118271424A (en) * | 2024-06-03 | 2024-07-02 | 中国农业科学院农产品加工研究所 | Separation preparation method of fresh milk-grade high-purity alpha-lactalbumin |
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| WO1992003468A1 (en) * | 1990-08-24 | 1992-03-05 | Baylor College Of Medicine | Method for isolating alpha-lactalbumin from whey |
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| CN110964095A (en) * | 2019-12-26 | 2020-04-07 | 吉林大学 | Method for continuously separating and preparing functional lactoprotein in raw fresh milk |
| CN110973345A (en) * | 2019-12-26 | 2020-04-10 | 吉林大学 | Method for continuously separating and preparing functional lactoprotein in colostrum |
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| CN110964095A (en) * | 2019-12-26 | 2020-04-07 | 吉林大学 | Method for continuously separating and preparing functional lactoprotein in raw fresh milk |
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| CN118271424A (en) * | 2024-06-03 | 2024-07-02 | 中国农业科学院农产品加工研究所 | Separation preparation method of fresh milk-grade high-purity alpha-lactalbumin |
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