A method for preparing bovine serum albumin

Abstract

The present invention relates to the technical field of protein separation and preparation, and specifically discloses a method for preparing bovine serum albumin. The preparation method of the present invention comprises: (1) acidifying bovine whey with citric acid at a pH value of 3.0-3.4, and aging; (2) microfiltration of a whey protein solution, with a permeate as the first feed liquid; (3) diafiltration of the first feed liquid after adjusting the pH value to 6.7-7.0 with water using an ultrafiltration membrane, with a retentate as the second feed liquid, the protein concentration of which is 4%-7%, and the conductivity is less than 250μS / cm; the pore size of the ultrafiltration membrane is 3000-10000Da; (4) separating the second feed liquid by isoelectric focusing-free flow electrophoresis, and using glycine-hydrochloric acid as an amphoteric electrolyte system. The present invention can realize BSA preparation using bovine milk / whey with a wide source of raw materials and low cost, and the obtained BSA has high purity, and high-purity β-lactoglobulin can be obtained at the same time.

Description

Technical Field

[0001] The invention relates to the technical field of protein separation and preparation, in particular to a method for preparing bovine serum albumin. Background Art

[0002] Bovine serum albumin (BSA) is a major protein found in bovine plasma and has a high degree of structural homology with human serum albumin (hSA). BSA consists of approximately 583 amino acid residues, with a molecular weight of approximately 66.43 kDa. It has a heart-shaped three-dimensional structure and contains three homologous domains (I, II, and III), each of which is further divided into two subdomains A and B. This protein plays an important role in biochemical and medical research and is widely used in cell culture, drug delivery, enzyme immobilization, biosensors, and as a food additive.

[0003] BSA plays a key role in maintaining blood osmotic pressure, pH buffering, acting as a carrier for drugs and nutrients, and participating in a variety of metabolic processes. Due to its abundant amino acid residues and specific binding sites, BSA can interact with a variety of compounds, including fatty acids, metal ions, drug molecules, etc. In addition, BSA also has important applications in clinical diagnosis and biopharmaceuticals, such as as a standard protein for immunochemical detection, a culture medium component for vaccine production, and as a nanoparticle matrix in drug delivery systems.

[0004] At present, all the raw materials for the preparation of BSA are bovine blood or bovine serum, which has high production costs and large source restrictions. Raw milk or whey also contains BSA. If BSA can be prepared using them as raw materials, the production cost and the difficulty of raw material sources can be greatly reduced. However, compared with bovine blood or bovine serum, the material environment of raw milk or whey is more complex, which is not conducive to the separation of BSA. There is no report on the preparation of BSA using raw milk or whey as raw materials.

[0005] Currently, there are two main preparation methods for BSA separation - precipitation method and molecular chromatography, but they have obvious problems. The precipitation method destroys the protein structure and leads to reduced BSA activity. The molecular chromatography method has extremely high preparation costs and cannot solve the problem of raw materials being heated during the preparation process. The original structure of the protein is destroyed to varying degrees during the preparation process.

[0006] Specifically, the current preparation method of BSA includes:

[0007] 1. Ammonium sulfate salting-out method: This is a traditional BSA separation method that precipitates the protein by adding ammonium sulfate. However, this method has a cumbersome production process and may cause protein aggregation or denaturation.

[0008] 2. Cold ethanol precipitation method: Using ethanol as a precipitant, the temperature is lowered to promote protein precipitation. Although this method is more common in the production of human serum albumin, there are risks of low purity and possible protein denaturation in BSA preparation.

[0009] 3. PEG precipitation method: Polyethylene glycol (PEG) is a non-ionic polymer that promotes protein precipitation by increasing the viscosity of the solution. However, PEG is difficult to remove and may leave residues in the BSA solution.

[0010] 4. Chromatography: including hydrophobic chromatography, molecular sieve chromatography and ion exchange chromatography, which separate proteins by different physical or chemical properties. Chromatography can effectively improve the purity of BSA, but it is still limited in removing pyrogens or exogenous viruses and other potential contaminants, which is crucial for the safety of BSA in clinical applications.

[0011] In addition, the ammonium sulfate salting-out method and the cold ethanol precipitation method may also cause small molecular substances to remain in BSA, such as sodium chloride, ammonium sulfate, amino acids, etc. These residues may affect the biological activity and application effect of BSA. Although the PEG precipitation method avoids protein aggregation or denaturation, its removal problem in industrial production limits its scope of application, and PEG residues may affect the safety of BSA.

[0012] Therefore, it is necessary to further study the separation and preparation methods of BSA. Summary of the invention

[0013] One of the objects of the present invention is to provide a method for effectively separating high-purity BSA from milk or whey.

[0014] The present invention provides a method for preparing bovine serum albumin, which uses bovine milk or bovine whey as a raw material to prepare bovine serum albumin, and specifically comprises:

[0015] (1) Preparation of whey protein solution:

[0016] When the raw material is cow milk, the raw material is first prepared into cow whey, then acidified with citric acid at a pH value of 3.0-3.4, and then aged to fully precipitate the impurity protein to obtain an aged whey protein solution;

[0017] When the raw material is bovine whey, it is directly acidified with citric acid at a pH value of 3.0-3.4, and then aged to fully precipitate the impurity protein to obtain an aged whey protein solution;

[0018] (2) microfiltration of the whey protein solution, wherein the pore size of the microfiltration membrane is 0.1-0.4 μm, and the permeate is used as the first feed liquid;

[0019] (3) After adjusting the pH value of the first feed liquid to 6.7-7.0, the first feed liquid after the pH value adjustment is diafiltered with water using an ultrafiltration membrane, and the retentate after diafiltration is used as the second feed liquid, wherein the protein concentration of the second feed liquid is 4%-7%, and the conductivity is less than 250 μS / cm; the pore size of the ultrafiltration membrane is 3000-10000 Da;

[0020] (4) Separating the second feed liquid by isoelectric focusing-free flow electrophoresis, and collecting the separated bovine serum albumin; in the isoelectric focusing-free flow electrophoresis, the electrophoresis solution includes the second feed liquid and an amphoteric electrolyte system, and the amphoteric electrolyte system is glycine-hydrochloric acid.

[0021] The present invention has found that BSA is also contained in cow's milk or whey, and the source is simple and easy to obtain, the price is cheap, the cost is low, the output is large, the source is wide, and it is a more stable source of raw materials. If they can be used as raw materials for the preparation of BSA, the production cost can be greatly reduced. However, compared with cow blood or bovine serum, the material environment of raw cow's milk or whey is more complex, and the various components therein will affect the separation of BSA. The use of traditional precipitation method and molecular chromatography method is not conducive to the acquisition of high-purity and high-activity BSA.

[0022] After research, the present invention found that FFE-IEF (Free Flow Electrophoresis Isoelectric Focusing) combines the advantages of isoelectric focusing (IEF) and free flow electrophoresis (FFE), and can achieve high-resolution separation of proteins, peptides or other charged biomolecules. In FFE-IEF technology, the sample is first pre-treated by isoelectric focusing in a gel-free electrophoresis tank, so that the molecules are aggregated in a specific pH region according to their isoelectric point (pI). Subsequently, these molecules flow along the electrophoresis tank under the action of the electric field to achieve further separation by charge and size. The key features of FFE-IEF technology include its gel-free separation environment, which not only avoids nonspecific interactions between the sample and the gel matrix, but also provides milder separation conditions, which is particularly suitable for analysis sensitive to protein conformation and activity. In addition, FFE-IEF has the characteristics of continuous flow, allowing real-time and dynamic analysis of samples, thereby improving the throughput and efficiency of the separation process. The technology is also highly customizable and can be optimized for different sample characteristics by adjusting parameters such as electric field strength, pH gradient, and electrophoresis time. Therefore, FFE-IEF technology is expected to achieve the effect of separating high-purity BSA from more complex milk / whey environments.

[0023] However, further research in the present invention found that although FFE-IEF technology has a good advantage in protein separation, there are many proteins in the milk / whey environment, some of which are similar to BSA in properties. When separated by FFE-IEF technology, the purity of BSA is significantly affected, and the ideal preparation effect cannot be obtained.

[0024] To this end, the present invention performs specific pretreatment settings on the milk / whey raw materials, enriches the target protein substances with higher purity as much as possible before free-flow electrophoresis, removes substances that affect electrophoretic separation, and cooperates with the selection of specific amphoteric electrolytes in FFE-IEF, ultimately achieving an ideal separation effect and obtaining a high-purity BSA product.

[0025] Specifically, the present invention uses citric acid to adjust the pH value of the raw material during pretreatment, removes impurities (impurities) that affect BSA isoelectric focusing-free flow electrophoresis separation by acidification aging combined with microfiltration, and then adjusts the pH value to adjust the target protein concentration by ultrafiltration to remove salts and other impurities in the raw material, further avoiding the impact on subsequent electrophoresis separation. In particular, it is found that if other acids are used during acidification, it is not conducive to the precipitation and removal of impurities, and is not conducive to the temperature control of the isoelectric focusing-free flow electrophoresis system, and will affect the separation efficiency, and ultimately affect the overall separation and purification effect of BSA, and cannot obtain a high-purity product.

[0026] In addition, the present invention also found that the amphoteric electrolyte system commonly used in protein electrophoresis separation, when applied to the preparation method of the present invention, will result in reduced separation efficiency and reduced purity of the separation target. The present invention ultimately explored and found that the use of glycine-hydrochloric acid as an amphoteric electrolyte, in conjunction with a specific pretreatment process, can improve the separation efficiency of BSA, improve the purity of the final separated target protein, and ensure the effective preparation of BSA.

[0027] In the method of the present invention, when the bovine whey is acidified and aged with citric acid, the pH value of the bovine whey is first adjusted to 3.0-3.4, the temperature is adjusted to 50-70°C, and maintained for 40-80 minutes to achieve acidification, and then aged at 30-40°C for 10-60 hours (preferably, 22-26 hours).

[0028] Preferably, when acidifying and aging bovine whey with citric acid, the pH value of the bovine whey is first adjusted to 3.1-3.3, the temperature is adjusted to 63-67°C, and maintained for 50-70 minutes to achieve acidification, and then aged at 35-40°C for 22-26 hours.

[0029] The acidification and aging conditions of the present invention are mild, which can effectively precipitate the impurity proteins and help maintain the activity of the target protein. Preferably, the acidification temperature is higher than the aging temperature to promote rapid denaturation of the impurity proteins during acidification and better aggregation in the subsequent aging stage.

[0030] In the method of the present invention, the temperature during microfiltration is 10-70°C;

[0031] The temperature during the diafiltration is 10-50° C., the pressure is 0.3-0.7 MPa, and the volume ratio of the first liquid to water after the pH value is adjusted is 1:(0.5-3).

[0032] When water is used for diafiltration in the present invention, too much water will affect the treatment efficiency, and too little water will not be conducive to sufficient washing and impurity removal, affecting the subsequent preparation effect. Those skilled in the art can adjust the amount of water and ultrafiltration pressure accordingly according to the diafiltration and impurity removal conditions, the protein content of the retentate and production requirements.

[0033] Preferably, in the method of the present invention, the temperature during microfiltration is 45-55° C.; the pore size of the microfiltration membrane is 0.1-0.2 μm;

[0034] The temperature during the diafiltration is 45-55° C., the volume ratio of the first liquid to water after the pH value is adjusted is 1:(0.8-1.2); and the pore size of the ultrafiltration membrane is 5000-6000 Da.

[0035] In the method of the present invention, in the isoelectric focusing-free flow electrophoresis, the electrophoresis solution includes a glycine-hydrochloric acid solution and the second feed liquid; in the glycine-hydrochloric acid solution, the concentration of glycine is 0.05mol / L-0.3mol / L, and the concentration of hydrochloric acid is 0.05mol / L-0.3mol / L; the volume ratio of the glycine-hydrochloric acid solution to the second feed liquid is (0.3-1):100; the process voltage is 100-600v, the separation temperature is 2-25°C, the circulation flow rate of the electrophoresis solution is 10ml / min-30ml / min, the number of cycles is 1-7 times, the component with a pH value of 5.4 collected is β-lactoglobulin, and the component with a pH value of 4.7-4.8 collected is bovine serum albumin.

[0036] In the glycine-hydrochloric acid solution, the molar ratio of glycine to hydrochloric acid is preferably 1:1, so as to provide a suitable ionic environment (obtaining an amphoteric electrolyte system that provides a wide pH range of 1-14 at the lowest ion concentration).

[0037] According to the liquid obtained by specific pretreatment, the present invention sets the setting parameters of isoelectric focusing-free flow electrophoresis with the goal of effectively separating bovine serum albumin and β-lactoglobulin and maintaining their activity as much as possible. Under the coordination of the above voltage, circulation flow rate and separation cycle number of the present invention, it can be ensured that the separation and purification is completed before the current is reduced to the point where the separation cannot be continued, which is conducive to ensuring production efficiency and product activity. In addition, in the isoelectric focusing-free flow electrophoresis of the present invention, it is preferred to collect bovine serum albumin and β-lactoglobulin within the above pH range. If it is changed to only collect at the conventional bovine serum albumin isoelectric point pH value of 4.7 in the art, the recovery rate will be reduced. If it is changed to collect at the conventional β-lactoglobulin isoelectric point pH value of 5.1-5.3 in the art, the recovery rate will also be reduced.

[0038] In the isoelectric focusing-free flow electrophoresis of the present invention, a stable electrophoresis voltage is provided and maintained by continuously circulating the positive electrode electrolyte and the negative electrode electrolyte. A person skilled in the art can select the positive electrode electrolyte and the negative electrode electrolyte according to common sense. For example, the positive electrode electrolyte includes sulfuric acid, phosphoric acid, and acetic acid, and the negative electrode electrolyte includes sodium hydroxide, ammonia water, and the like.

[0039] The method of the present invention can prepare high-purity and high-activity BSA and at the same time prepare high-purity and high-activity β-lactoglobulin.

[0040] Preferably, in the isoelectric focusing-free flow electrophoresis, the process voltage is 350-450V, the separation temperature is 2-6°C, and the separation cycle is 4-5 times.

[0041] In the method of the present invention, when the raw material is cow's milk, the cow's milk is firstly defatted and pasteurized, and then the pH value is adjusted to 4.2-4.6 with citric acid to separate the cow's whey.

[0042] The present invention preferably uses citric acid to prepare bovine whey, so as to maintain the activity of the protein, and the pH range is set to 4.2-4.6, which is conducive to ensuring the purification efficiency of whey protein.

[0043] In the present invention, defatting, pasteurization and whey separation can be carried out by conventional methods in the art.

[0044] In the method of the present invention, the fat content of the milk after defatting is less than 0.06% (it can be carried out by conventional centrifugation in the art, such as centrifugation at a temperature of 50°C and a centrifugal speed of 5000 rpm); the pasteurization temperature is 70-90°C and the time is 10-60s.

[0045] In the method of the present invention, bovine whey can be separated by centrifugation, the centrifugal temperature is 20-70°C, the rotation speed is 5000-10000rpm, and the time is 20-60min. Preferably, the centrifugal separation temperature is 25°C, the centrifugal time is 30-60min, and the centrifugal speed is 8000-10000rpm. More preferably, the centrifugal temperature is 25°C, the rotation speed is 8000rpm, and the time is 30min.

[0046] The beneficial effects of the present invention are at least:

[0047] The preparation method of the invention can prepare BSA by using cow milk / whey which is a widely available raw material with low cost, and the obtained BSA has high purity, and high-purity β-lactoglobulin can be obtained at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The figure is a schematic flow chart of the method for preparing BSA of the present invention. DETAILED DESCRIPTION

[0049] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0050] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0051] The cheese whey of the specific implementation mode of the present invention is prepared by the conventional method in the art, specifically as follows: thermophilic streptococci are added to skim milk for acidification, then rennet is added to coagulate the milk, the coagulated part is separated to obtain crude cheese whey, and the crude cheese whey is centrifuged to remove impurities to obtain cheese whey.

[0052] Example 1

[0053] This embodiment provides a method for preparing BSA, and the schematic diagram of the process is shown in Figure 1 , as follows:

[0054] 1. Degreasing: Raw milk is degreased by centrifugation at a temperature of 50°C and a centrifugal speed of 5000 rpm, and the fat content is adjusted to 0.05%;

[0055] 2. Pasteurization: pasteurize the skim milk at a temperature of 75°C and a pasteurization time of 20 seconds;

[0056] 3. Preparation of whey liquid: adjust the pH value of skim milk to 4.6 with citric acid, let it stand for 5 minutes, and take the supernatant to obtain whey liquid after centrifugation, wherein the centrifugation temperature is 25°C, the centrifugation time is 30 minutes, and the centrifugation speed is 8000 rpm;

[0057] 4. Acidification of whey liquid: Use citric acid to adjust the pH of the prepared whey liquid to 3.2, heat the solution to 65°C, and keep it for 60 minutes;

[0058] 5. Aging: The acidified whey protein was aged at 40°C for 24 hours;

[0059] 6. Microfiltration separation: The whey protein solution after static aging is separated by a microfiltration membrane, and the permeate is collected and recorded as the first feed liquid, wherein the membrane separation parameters are: separation temperature 50°C, microfiltration membrane pore size 0.1 micron;

[0060] 7. Adjustment of pH of the first feed liquid: Use NaOH to adjust the pH of the whey liquid (first feed liquid) separated by the microfiltration membrane to 6.8;

[0061] 8. Ultrafiltration and impurity removal of the first feed liquid: The first feed liquid after pH adjustment is diafiltrated with water using an ultrafiltration membrane, and then the retentate after diafiltration is collected and recorded as the second feed liquid, wherein the ultrafiltration membrane separation parameters are: separation temperature of 50°C, pressure of 0.5MPa, membrane pore size of 5000Da, and the volume ratio of the amount of washing water added to the first feed liquid after pH adjustment is 1:1;

[0062] 9. Isoelectric focusing-free flow electrophoresis separation of the second feed liquid: The second feed liquid is separated by isoelectric focusing free flow electrophoresis, and the protein content of the second feed liquid is 4.15%, and the conductivity is 223μS / cm. The FFE-IEF process parameters are: using glycine-hydrochloric acid as an amphoteric electrolyte system, preparing a glycine-hydrochloric acid aqueous solution, wherein the concentration of glycine is 0.2mol / L, and the concentration of hydrochloric acid is 0.2mol / L, the glycine-hydrochloric acid aqueous solution is mixed with the second feed liquid at a volume ratio of 0.5:100 to obtain an electrophoresis solution, the circulation flow rate of the electrophoresis solution is 20ml / min, the number of cycles is 4 times, the process voltage is 400v, the separation temperature is 4°C, β-lactoglobulin is collected in the first collection tube, the pH value is 5.4, and bovine serum albumin is collected in the second collection tube, the pH value is 4.7-4.8.

[0063] Example 2

[0064] This embodiment provides a method for preparing BSA, which is as follows:

[0065] 1. Degreasing: Raw milk is degreased by centrifugation at a temperature of 50°C and a centrifugal speed of 5000 rpm, and the fat content is adjusted to 0.05%;

[0066] 2. Pasteurization: pasteurize the skim milk at a temperature of 70°C and a pasteurization time of 60 seconds;

[0067] 3. Preparation of whey liquid: adjust the pH value of skim milk to 4.2 with citric acid, let it stand for 5 minutes, and take the supernatant to obtain whey liquid after centrifugation, wherein the centrifugation temperature is 25°C, the centrifugation time is 60 minutes, and the centrifugation speed is 10000 rpm;

[0068] 4. Acidification of whey liquid: Use citric acid to adjust the pH of the prepared whey liquid to 3.0, heat the solution to 70°C and keep it for 40 minutes;

[0069] 5. Aging: The acidified whey protein was aged at 35°C for 22 hours;

[0070] 6. Microfiltration separation: The whey protein solution after static aging is separated by a microfiltration membrane, and the permeate is collected and recorded as the first feed liquid, wherein the membrane separation parameters are: separation temperature 70°C, microfiltration membrane pore size 0.2 μm;

[0071] 7. Adjustment of pH of the first feed liquid: Use NaOH to adjust the pH of the whey liquid (first feed liquid) separated by the microfiltration membrane to 6.8;

[0072] 8. Ultrafiltration of the first feed liquid to remove impurities: The first feed liquid after pH adjustment is diafiltrated with water using an ultrafiltration membrane, and then the intercepted liquid after diafiltration is collected and recorded as the second feed liquid, wherein the ultrafiltration membrane separation parameters are: separation temperature of 10°C, pressure of 0.7MPa, membrane pore size of 3000Da, and the volume ratio of the amount of washing water added to the first feed liquid after pH adjustment is 1:3;

[0073] 9. Isoelectric focusing-free flow electrophoresis separation of the second feed liquid: The second feed liquid is separated by isoelectric focusing free flow electrophoresis, and the protein content of the second feed liquid is 5.38%, and the conductivity is 205μS / cm. The FFE-IEF process parameters are: using glycine-hydrochloric acid as an amphoteric electrolyte system, preparing a glycine-hydrochloric acid aqueous solution, wherein the concentration of glycine is 0.05mol / L, the concentration of hydrochloric acid is 3mol / L, the glycine-hydrochloric acid aqueous solution is mixed with the second feed liquid at a volume ratio of 1:100 to obtain an electrophoresis solution, the circulation flow rate of the electrophoresis solution is 30ml / min, the number of cycles is 1, the process voltage is 600v, the separation temperature is 25°C, β-lactoglobulin is collected in the first collection tube, the pH value is 5.4, and bovine serum albumin is collected in the second collection tube, the pH value is 4.7-4.8.

[0074] Example 3

[0075] This embodiment provides a method for preparing BSA, which is as follows:

[0076] 1. Degreasing: Raw milk is degreased by centrifugation at a temperature of 50°C and a centrifugal speed of 5000 rpm, and the fat content is adjusted to 0.05%;

[0077] 2. Pasteurization: pasteurize the skim milk at a temperature of 90°C and a pasteurization time of 20 seconds;

[0078] 3. Preparation of whey liquid: adjust the pH value of skim milk to 4.6 with citric acid, let it stand for 5 minutes, and take the supernatant to obtain whey liquid after centrifugation, wherein the centrifugation temperature is 25°C, the centrifugation time is 30 minutes, and the centrifugation speed is 8000 rpm;

[0079] 4. Acidification of whey liquid: Use citric acid to adjust the pH of the prepared whey liquid to 3.4, heat the solution to 50°C and keep it for 80 minutes;

[0080] 5. Aging: The acidified whey protein was aged at 30°C for 26 hours;

[0081] 6. Microfiltration separation: The aged whey protein solution is separated by a microfiltration membrane, and the permeate is collected as the first feed liquid, wherein the membrane separation parameters are: separation temperature 10°C, microfiltration membrane pore size 0.4 μm;

[0082] 7. Adjustment of pH of the first feed liquid: Use NaOH to adjust the pH of the whey liquid (first feed liquid) separated by the microfiltration membrane to 6.8;

[0083] 8. Ultrafiltration and impurity removal of the first feed liquid: The first feed liquid after pH adjustment is diafiltrated with water using an ultrafiltration membrane, and then the intercepted liquid after diafiltration is collected and recorded as the second feed liquid, wherein the ultrafiltration membrane separation parameters are: separation temperature of 45°C, pressure of 0.3MPa, membrane pore size of 10000Da, and the volume ratio of the amount of washing water added to the first feed liquid after pH adjustment is 1:0.5;

[0084] 9. Isoelectric focusing-free flow electrophoresis separation of the second feed liquid: The second feed liquid is separated by isoelectric focusing free flow electrophoresis, and the protein content of the second feed liquid is 6.91%, and the conductivity is 247μS / cm. The FFE-IEF process parameters are: using glycine-hydrochloric acid as an amphoteric electrolyte system, preparing a glycine-hydrochloric acid aqueous solution, wherein the concentration of glycine is 3mol / L, and the concentration of hydrochloric acid is 0.05mol / L, the glycine-hydrochloric acid aqueous solution is mixed with the second feed liquid at a volume ratio of 0.3:100 to obtain an electrophoresis solution, the circulation flow rate of the electrophoresis solution is 10ml / min, the number of cycles is 7 times, the process voltage is 100v, the separation temperature is 2°C, β-lactoglobulin is collected in the first collection tube, the pH value is 5.4, and bovine serum albumin is collected in the second collection tube, the pH value is 4.7-4.8.

[0085] Example 4

[0086] This embodiment provides a method for preparing BSA, which is basically the same as the method in Example 1, except that in step 9, during isoelectric focusing free-flow electrophoresis separation, the circulation flow rate of the electrophoresis solution is changed to 50 ml / min, the process voltage is 450 V, and the number of cycles is 6 times.

[0087] Example 5

[0088] This embodiment provides a method for preparing BSA, which is basically the same as the method in Example 1, except that in step 9, during isoelectric focusing free-flow electrophoresis separation, the pH value for collecting β-lactoglobulin is changed to 5.3, and the pH value for collecting bovine serum albumin is changed to 4.7.

[0089] Example 6

[0090] This embodiment provides a method for preparing BSA, which is basically the same as the method in Example 1, except that the raw material is cheese whey, and steps 4-9 in Example 1 are directly performed using cheese whey.

[0091] Comparative Example 1

[0092] This comparative example provides a method for preparing BSA, which is basically the same as the method in Example 1, except that in step 4, lactic acid is used instead for pH adjustment.

[0093] Comparative Example 2

[0094] This comparative example provides a method for preparing BSA, which is basically the same as the method in Example 1, except that in step 4, citric acid is used to adjust the pH of the prepared whey liquid to 3.6.

[0095] Comparative Example 3

[0096] This comparative example provides a method for preparing BSA, which is basically the same as the method in Example 1, except that in step 9, during the isoelectric focusing free-flow electrophoresis separation, Ampholine (purchased from Cytiva, with a pH applicable range of 2-13) is used as the electrolyte system. Specifically, an aqueous solution of Ampholine is prepared, wherein the concentration of Ampholine is 0.2 mol / L, and the aqueous solution of Ampholine is mixed with the second feed liquid at a volume ratio of 0.5:100 to obtain an electrophoresis solution.

[0097] Comparative Example 4

[0098] This comparative example provides a method for preparing BSA, which is basically the same as the method in Example 1, except that in step 8, by increasing the ultrafiltration pressure and reducing the proportion of filtration water, the protein content of the second feed liquid is finally 8.12% and the conductivity is 240 μS / cm. The remaining steps are the same as in Example 1.

[0099] Experimental example

[0100] This experimental example tests the effects of the methods of the above embodiments and comparative examples, as follows:

[0101] 1. BSA purity and recovery rate

[0102] Test method:

[0103] (1) BSA purity (concentration) was detected by capillary electrophoresis. The detection method was based on the detection method used in the article (Loughney JW, Lancaster C, has, et al. Residual bovine serum albumin (BSA) quantitation in vaccinations using automated Capillary Western technology [J]. Analytical Biochemistry, 2014, 461: 49-56. DOI: 10.1016 / j.ab.2014.05.004.).

[0104] (2) Recovery rate detection method: The BSA concentration in the second feed liquid and the BSA concentration in the first collection tube before electrophoresis were detected according to the above method, and the BSA content in the second feed liquid and the first collection tube were calculated respectively. The BSA recovery rate was calculated as the ratio of the BSA content in the first collection tube to the BSA content in the second feed liquid × 100%. The results are shown in Table 1.

[0105] Table 1

[0106]

[0107] 2. Purity and recovery rate of β-LG

[0108] Test method:

[0109] (1) The purity of β-LG was determined by capillary electrophoresis. The detection method was based on the article (Shengbo Yu, Zhishen Mu, Xuena Dong, Zengli Gao, Guozhi Ji, Yu Liu, Yun Chen. Comparative Analysis of Yak Milk and Holstein Cow Milk in Composition of Protein, Amino Acid and Fatty Acid. Journal of Food and Nutrition Research .vol. 11, No.11, 2023,pp672-677.https: / / pubs.sciepub.com / jfnr / 11 / 11 / 2)

[0110] (2) Recovery rate detection method: The concentration of β-LG in the second feed liquid and the concentration of β-LG in the first collection tube before electrophoresis were detected according to the above method, and the β-LG content in the second feed liquid and the first collection tube were calculated respectively. The β-LG recovery rate was calculated as the ratio of the β-LG content in the second collection tube to the β-LG content in the second feed liquid × 100%. The results are shown in Table 2.

[0111] Table 2

[0112]

[0113] 3. Centrifugal precipitation rate of BSA and β-LG

[0114] Test method:

[0115] In the present invention, the centrifugal sedimentation rate, i.e., the insolubility index, is used as an evaluation parameter for evaluating denatured or inactive BSA and β-LG.

[0116] The detection method is to accurately weigh the mass of the protein solution in the first collection tube and the second collection tube, centrifuge, and the centrifugation parameters are centrifugation temperature 25°C, centrifugation speed 5000rpm, and centrifugation time 30min. The precipitate separated by centrifugation is freeze-dried, and the mass of the precipitate after freeze-drying is accurately weighed. The centrifugal precipitation rate is calculated as the mass of the precipitate / the mass of the protein solution × 100%. The results are shown in Table 3.

[0117] Table 3

[0118]

[0119] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing bovine serum albumin, characterized in that: The preparation of bovine serum albumin using cow milk or cow whey as raw materials specifically includes: (1) Preparation of whey protein solution: When the raw material is cow milk, the raw material is first prepared into cow whey, then acidified with citric acid at a pH value of 3.0-3.4, and then aged to fully precipitate the impurity protein to obtain an aged whey protein solution; When the raw material is bovine whey, it is directly acidified with citric acid at a pH value of 3.0-3.4, and then aged to fully precipitate the impurity protein to obtain an aged whey protein solution; (2) microfiltration of the whey protein solution, wherein the pore size of the microfiltration membrane is 0.1-0.4 μm, and the permeate is used as the first feed liquid; (3) After adjusting the pH value of the first feed liquid to 6.7-7.0, the first feed liquid after the pH value adjustment is diafiltered with water using an ultrafiltration membrane, and the retentate after diafiltration is used as the second feed liquid, wherein the protein concentration of the second feed liquid is 4%-7%, and the conductivity is less than 250 μS / cm; the pore size of the ultrafiltration membrane is 3000-10000Da; (4) Separating the second feed liquid by isoelectric focusing-free flow electrophoresis, and collecting the separated bovine serum albumin; in the isoelectric focusing-free flow electrophoresis, the electrophoresis solution comprises a glycine-hydrochloric acid solution and the second feed liquid; in the glycine-hydrochloric acid solution, the concentration of glycine is 0.05mol / L-0.3mol / L, and the concentration of hydrochloric acid is 0.05mol / L-0.3mol / L; the volume ratio of the glycine-hydrochloric acid solution to the second feed liquid is (0.3-1):100; the process voltage is 100-600v, the separation temperature is 2-25°C, the circulation flow rate of the electrophoresis solution is 10ml / min-30ml / min, the number of cycles is 1-7 times, the component with a pH value of 5.4 collected is β-lactoglobulin, and the component with a pH value of 4.7-4.8 collected is bovine serum albumin.

2. The method for preparing bovine serum albumin according to claim 1, characterized in that: When using citric acid to acidify and age the bovine whey, the pH value of the bovine whey is first adjusted to 3.0-3.4, the temperature is adjusted to 50-70°C, and the acidification is maintained for 40-80 minutes, and then the bovine whey is aged at 30-40°C for 10-60 hours.

3. The method for preparing bovine serum albumin according to claim 2, characterized in that: When the bovine whey is acidified and aged with citric acid, the pH value of the bovine whey is first adjusted to 3.1-3.3, the temperature is adjusted to 63-67°C, and maintained for 50-70 minutes to achieve acidification, and then aged at 35-40°C for 22-26 hours.

4. The method for preparing bovine serum albumin according to claim 1, characterized in that: The temperature during the microfiltration is 10-70°C; The temperature during the diafiltration is 10-50° C., the pressure is 0.3-0.7 MPa, and the volume ratio of the first liquid to water after the pH value is adjusted is 1:(0.5-3).

5. The method for preparing bovine serum albumin according to claim 4, characterized in that: The temperature during microfiltration is 45-55°C; the pore size of the microfiltration membrane is 0.1-0.2 microns; The temperature during the diafiltration is 45-55° C., the volume ratio of the first liquid to water after the pH value is adjusted is 1:(0.8-1.2); and the pore size of the ultrafiltration membrane is 5000-6000 Da.

6. The method for preparing bovine serum albumin according to any one of claims 1 to 5, characterized in that: In the isoelectric focusing-free flow electrophoresis, the process voltage is 350-450V, the separation temperature is 2-6°C, and the separation cycle is 4-5 times.

7. The method for preparing bovine serum albumin according to any one of claims 1 to 5, characterized in that: When the raw material is cow's milk, the cow's milk is first defatted and pasteurized, and then the pH value is adjusted to 4.2-4.6 with citric acid, and then the whey is separated.

8. The method for preparing bovine serum albumin according to claim 7, characterized in that: The fat content of the skimmed milk is less than 0.06%; the pasteurization temperature is 70-90° C. and the time is 10-60 seconds.

9. The method for preparing bovine serum albumin according to claim 7, characterized in that: The bovine whey is separated by centrifugation, wherein the centrifugation temperature is 20-70°C, the rotation speed is 5000-10000 rpm, and the time is 20-60 minutes.

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