CN114349842B - Concentration and separation method of beta-lactoglobulin - Google Patents
Concentration and separation method of beta-lactoglobulin Download PDFInfo
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- 108010060630 Lactoglobulins Proteins 0.000 title claims abstract description 72
- 238000000926 separation method Methods 0.000 title claims abstract description 19
- 102000000119 Beta-lactoglobulin Human genes 0.000 title 1
- 102000008192 Lactoglobulins Human genes 0.000 claims abstract description 71
- 239000012528 membrane Substances 0.000 claims abstract description 37
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 33
- 102000007544 Whey Proteins Human genes 0.000 claims abstract description 27
- 108010046377 Whey Proteins Proteins 0.000 claims abstract description 27
- 229940027941 immunoglobulin g Drugs 0.000 claims abstract description 23
- 235000013305 food Nutrition 0.000 claims abstract description 21
- 102000004407 Lactalbumin Human genes 0.000 claims abstract description 19
- 108090000942 Lactalbumin Proteins 0.000 claims abstract description 19
- 235000021241 α-lactalbumin Nutrition 0.000 claims abstract description 19
- 238000001035 drying Methods 0.000 claims abstract description 18
- 239000005862 Whey Substances 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 15
- 230000001105 regulatory effect Effects 0.000 claims abstract description 14
- 238000011033 desalting Methods 0.000 claims abstract description 11
- 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 abstract description 7
- 239000003513 alkali Substances 0.000 claims abstract description 7
- 239000003729 cation exchange resin Substances 0.000 claims abstract description 7
- 239000002253 acid Substances 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 92
- 239000007788 liquid Substances 0.000 claims description 28
- 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
- 102000004169 proteins and genes Human genes 0.000 claims description 24
- 108090000623 proteins and genes Proteins 0.000 claims description 24
- 239000003480 eluent Substances 0.000 claims description 18
- 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
- 239000000843 powder Substances 0.000 claims description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- 238000000502 dialysis Methods 0.000 claims description 10
- 239000012266 salt solution Substances 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 9
- 239000002244 precipitate Substances 0.000 claims description 9
- 239000000047 product Substances 0.000 claims description 7
- 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
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- 239000013566 allergen Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 159000000007 calcium salts Chemical class 0.000 claims description 2
- 239000012535 impurity Substances 0.000 claims description 2
- 239000011148 porous material 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
- 239000012466 permeate Substances 0.000 claims 3
- 239000012530 fluid Substances 0.000 claims 2
- 230000003750 conditioning effect Effects 0.000 claims 1
- 235000021119 whey protein Nutrition 0.000 abstract description 11
- 238000002360 preparation method Methods 0.000 abstract description 2
- 238000001742 protein purification Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 abstract description 2
- 238000010828 elution Methods 0.000 description 20
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 16
- 239000011347 resin Substances 0.000 description 13
- 229920005989 resin Polymers 0.000 description 13
- 238000000108 ultra-filtration Methods 0.000 description 13
- 238000001514 detection method Methods 0.000 description 11
- 238000011010 flushing procedure Methods 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 238000001179 sorption measurement Methods 0.000 description 8
- 235000013336 milk Nutrition 0.000 description 6
- 239000008267 milk Substances 0.000 description 6
- 210000004080 milk Anatomy 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 239000012608 weak cation exchange resin Substances 0.000 description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 239000000919 ceramic Substances 0.000 description 4
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- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 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
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
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- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
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- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to the technical field of protein purification and preparation, and discloses a concentration and separation method of beta-lactoglobulin, 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) drying or adsorbing after redissolution: ensuring that the beta-lactoglobulin is adsorbed in the cation exchange resin; (4) eluting for a plurality of times; (5) drying after desalting: obtaining a beta-lactoglobulin product; (6) obtaining a desensitized food. The invention takes the recovery of whey protein in whey water as raw material, and the high-purity beta-lactoglobulin is industrially produced on the basis of concentrating the beta-lactoglobulin while concentrating and separating the alpha-lactalbumin, the beta-lactoglobulin and the immunoglobulin G products through human intervention; desensitized foods are produced by a method of enriching beta-lactoglobulin.
Description
Technical Field
The invention relates to the technical field of protein purification and preparation, in particular to a concentration and separation method of beta-lactoglobulin.
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 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 beta-lactoglobulin, 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 or adsorbing 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; or injecting into chromatographic column or stirring tank with cation exchange resin, and adjusting pH of the complex solution to 4.3-5.2 before injection to ensure that beta-lactoglobulin is adsorbed in cation exchange resin;
(4) And (3) eluting for a plurality of times: eluting the cation exchange resin in the step (3) at least twice by using a salt solution as an eluent; eluting for the first time to remove impurities, and eluting for the second time to obtain target protein beta-lactoglobulin solution;
(5) Desalting and drying: desalting, concentrating and drying the beta-lactoglobulin solution obtained in the step (4) to obtain a beta-lactoglobulin product;
(6) Obtaining desensitized food: concentrating and drying the alpha-lactalbumin and immunoglobulin G mixed solution obtained in the step (2) to obtain the desensitized food for removing the allergen beta-lactoglobulin.
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 0.5-1.2 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.
The first elution in the step (4) adopts salt solution with the mass concentration of 0-1% as eluent, and the second and subsequent elution adopts salt solution with the mass concentration of 1.5% -5% as eluent.
The salt solution refers to sodium salt, potassium salt or calcium salt solution.
The invention has the following beneficial technical effects:
1. the invention can recycle whey protein in whey water as raw material, concentrate and separate high-purity whey protein, and prepare beta-lactoglobulin, thereby realizing waste recycling.
2. The invention concentrates and separates alpha-lactalbumin, beta-lactoglobulin and immunoglobulin G products by human intervention, and simultaneously, the invention industrially produces high-purity beta-lactoglobulin on the basis of concentrating the beta-lactoglobulin.
3. The invention produces 'desensitized food' through the method of enriching beta-lactoglobulin in industrial production and stripping the allergen of the infant beta-lactoglobulin.
4. Low production cost and simple operation.
5. The total protein in the produced beta-lactoglobulin powder reaches more than 90 percent, the content of the beta-lactoglobulin in the total protein reaches more than 92 percent, and the production recovery rate can reach more than 80 percent by calculating through an area method.
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 industrial production of concentrated beta-lactoglobulin products according to the present invention;
FIG. 4 is a liquid phase detection spectrum of concentrated beta-lactoglobulin (10.454 min. Alpha. -lactalbumin peak; 11.307 min. Beta. -lactoglobulin peak);
FIG. 5 is a graph of a liquid phase assay for beta-lactoglobulin;
FIG. 6 is a report of the detection of an industrial production "desensitized food" product according to the invention;
FIG. 7 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.
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, the whey water is collected by filtration, and lactose is removed and concentrated by a 5K ultrafiltration membrane, so that the content of whey protein is improved.
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 0.8um ceramic membrane, the pH of the dialysis water was adjusted to 4.76, and the unflocculated proteins 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 pH of the concentrated solution (b 1) was adjusted to 5.62 to re-dissolve it, and then to 5.02, and the concentrated solution was passed through a column packed with a weak cation exchange resin having a particle size of 200 μm at a constant flow rate of 120L/h for an adsorption time of 20min. Eluting the chromatographic column by adopting 4 column volumes of 0.3% sodium chloride solution (first eluent) to obtain a first elution collection liquid; 4 columns of 3.5% sodium chloride solution (second eluent) are adopted for eluting the chromatographic column, and a second elution collection liquid, namely beta-lactoglobulin solution, is obtained.
Step 6: desalting the second elution collection liquid by using 5K ultrafiltration membrane equipment to obtain beta-lactoglobulin solution (f 1), and drying to obtain beta-lactoglobulin powder.
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 5%, pH adjusted to 11.17 with 0.25mol/L food grade sodium hydroxide, and then 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: the pH of the concentrated solution (b 2) is firstly adjusted to 5.56 to be redissolved, then the pH is adjusted to 4.98, and the mixture is injected into a stirring tank filled with weak cation exchange resin with the particle size of 200 mu m, the rotating speed is 40r/min, and the adsorption time is 30min. After the adsorption is finished, evacuating the residual materials in the resin tank, repeatedly flushing and evacuating by using pure water, and injecting and preparing 0.4% 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; and (3) pouring pure water again, and when the refractometer value is 0, pouring into a prepared 4% sodium chloride solution (second eluent) to analyze the resin, so as to obtain a second elution collection liquid, namely the beta-lactoglobulin solution.
Step 5: desalting the second elution collection liquid by using 5K ultrafiltration membrane equipment to obtain beta-lactoglobulin solution (f 2), and drying to obtain beta-lactoglobulin powder.
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 100K 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: injecting the precipitate (b 3) into pure water for dissolution, adjusting the pH to 5.42 for redissolution, adjusting the pH to 4.87, injecting into a stirring tank filled with weak cation exchange resin with the particle size of 200 μm, and adsorbing for 30min at the rotating speed of 40 r/min. After the adsorption is finished, evacuating the residual materials in the resin tank, repeatedly flushing and evacuating by using pure water, and injecting 1% sodium chloride solution (first eluent) for resolving the resin when the detection value of a flushing water refractometer is 0, so as to obtain a first elution collection liquid; and (3) pouring 3.5 percent of sodium chloride solution (second eluent) prepared by injection when the value of the refractometer is 0 after the solution is drained by pure water for eluting the resin again, so as to obtain a second elution collection liquid, namely the beta-lactoglobulin solution.
Step 5: desalting the second elution collection liquid by using 5K ultrafiltration membrane equipment to obtain beta-lactoglobulin solution (f 3), and drying to obtain beta-lactoglobulin powder.
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: the pH of the concentrated solution (b 4) is firstly adjusted to 5.86 to be redissolved, then the pH is adjusted to 4.37, and the mixture is injected into a stirring tank filled with weak cation exchange resin with the particle size of 200 mu m, the rotating speed is 40r/min, and the adsorption time is 30min. After the adsorption is finished, evacuating the residual materials in the resin tank, repeatedly flushing and evacuating by using pure water, and injecting and preparing 0.1% 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; and (3) pouring pure water again, and when the refractometer value is 0, pouring into 5% sodium chloride solution (second eluent) for resin analysis to obtain second elution collection liquid, namely beta-lactoglobulin solution.
Step 5: desalting the second elution collection liquid by using 5K ultrafiltration membrane equipment to obtain beta-lactoglobulin solution (f 4), and drying to obtain beta-lactoglobulin powder.
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, the whey water is collected by filtration, and lactose is removed and concentrated by a 5K ultrafiltration membrane, so that the content of whey protein is improved.
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 0.5um ceramic membrane, the pH of the dialysis water was adjusted to 4.02, and the unflocculated proteins were washed out as much as possible by continuous addition of dialysis water. Obtaining a concentrated solution (b 5) mainly comprising concentrated beta-lactoglobulin solution; the clear solution (c 5) is mainly a mixed solution of alpha-lactalbumin and immunoglobulin G.
Step 4: and (3) passing the clear solution (c 5) 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 5) mainly comprising concentrated immunoglobulin G; the resulting supernatant (e 5) was mainly concentrated alpha-lactalbumin solution.
Step 5: the pH of the concentrated solution (b 5) was adjusted to 6.02 to re-dissolve the concentrated solution, and then the concentrated solution was subjected to pH5.14 and passed through a column packed with a weak cation exchange resin having a particle size of 200 μm at a constant flow rate of 120L/h for an adsorption time of 20min. Eluting the chromatographic column by adopting 4 column volumes of 0.4% calcium chloride solution (first eluent) to obtain a first elution collection liquid; 4 columns of 1.5% calcium chloride solution (second eluent) are adopted for eluting the chromatographic column, and a second elution collection liquid, namely beta-lactoglobulin solution, is obtained.
Step 6: desalting the second elution collection liquid by using 5K ultrafiltration membrane equipment to obtain beta-lactoglobulin solution (f 5), and drying to obtain beta-lactoglobulin powder.
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: injecting the precipitate (b 6) into pure water for dissolution, adjusting the pH to 6.98 for redissolution, adjusting the pH to 4.87, injecting into a stirring tank filled with weak cation exchange resin with the particle size of 200 mu m, and adsorbing for 30min at the rotating speed of 40 r/min. After the adsorption is finished, evacuating the residual materials in the resin tank, repeatedly flushing and evacuating 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; and (3) pouring pure water again, and when the refractometer value is 0, pouring and preparing a 3% potassium chloride solution (second eluent) to analyze the resin, so as to obtain a second elution collection liquid, namely the beta-lactoglobulin solution.
Step 5: desalting the second elution collection liquid by using 5K ultrafiltration membrane equipment to obtain beta-lactoglobulin solution (f 6), and drying to obtain beta-lactoglobulin powder.
See table 6 for test data for this example.
Table 6 example 6 test data sheet
Claims (8)
1. A method for concentrating and separating beta-lactoglobulin, 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 membrane trapped fluid or centrifugal precipitate and membrane permeate or centrifugal clear liquid; the membrane retentate or the centrifugal precipitate comprises beta-lactoglobulin, and the membrane permeate or the centrifugal clear liquid comprises a mixed solution of alpha-lactalbumin and immunoglobulin G;
(3) Drying or adsorbing after redissolution: regulating the pH value of the membrane trapped fluid or the centrifugal precipitate in the step (2) to 5.5-7.0, and re-dissolving to obtain a re-solution; directly drying the complex solution to obtain concentrated beta-lactoglobulin, or injecting the complex solution into a chromatographic column or a stirring tank filled with cation exchange resin, and adjusting the pH value of the complex solution to 4.3-5.2 before injection to ensure that the beta-lactoglobulin is adsorbed in the cation exchange resin;
(4) And (3) eluting for a plurality of times: eluting the cation exchange resin in the step (3) at least twice by using a salt solution as an eluent; eluting for the first time to remove impurities, and eluting for the second time to obtain target protein beta-lactoglobulin solution;
(5) Desalting and drying: desalting, concentrating and drying the beta-lactoglobulin solution obtained in the step (4) to obtain a beta-lactoglobulin product; and
(6) Obtaining desensitized food: concentrating and drying the membrane permeate or the centrifugated clear liquid obtained in the step (2) to obtain the desensitized food for removing the allergen beta-lactoglobulin.
2. The method of claim 1, wherein the alkali used in the first step of alkali conditioning in step (1) is food grade sodium hydroxide.
3. The method according to claim 1, wherein the acid used in the second-stage acid-regulating step (1) is food-grade hydrochloric acid, acetic acid or citric acid.
4. The method according to claim 1, wherein the membrane used in the separation of step (2) has a pore size of 0.5 to 1.2. Mu.m.
5. The method for concentrating and separating beta-lactoglobulin according to claim 1, wherein the pH value of the dialysis water added in the step (2) is 4.0-5.5 to avoid redissolution of flocculated proteins.
6. The method for concentrating and separating beta-lactoglobulin according to claim 1, wherein the rotational speed of the centrifugal separation in the step (2) is not less than 1000r/min.
7. The method according to claim 1, wherein the first eluting in the step (4) uses a salt solution with a mass concentration of 0-1% as an eluent, and the second and subsequent eluting uses a salt solution with a mass concentration of 1.5% -5% as an eluent.
8. The method for concentrating and separating β -lactoglobulin according to claim 7, wherein said salt solution is a sodium salt, potassium salt or calcium salt solution.
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