Method for synchronously separating ovalbumin, ovotransferrin and lysozyme from egg white

Abstract

The invention discloses a method for synchronously separating ovalbumin, ovotransferrin and lysozyme from egg white, which comprises the following steps: firstly, adsorbing ovalbumin from egg white loading liquid by taking chitosan-polybutyl methacrylate nanocrystal gel with an anion exchange function as a primary separation medium, and carrying out gradient elution to obtain ovalbumin; taking a primary separation loading effluent as a secondary separation loading solution; hydroxyethyl methylacrylate-polyglycidyl methacrylate nanocrystal gel with a cation exchange function is taken as a secondary separation medium to adsorb ovotransferrin and lysozyme from a secondary separation loading solution, gradient elution is performed by gradually increasing salt concentration, and separation of ovotransferrin and lysozyme is realized. The method provided by the invention can realize synchronous separation of ovalbumin, ovotransferrin and lysozyme, has the advantages of high separation efficiency, simplicity and convenience in operation, low energy consumption and the like, and is suitable for industrial large-scale production, and the separation medium can be repeatedly used.

Description

Technical Field

[0001] This invention belongs to the field of bioseparation technology, specifically relating to a method for simultaneously separating ovalbumin, ovotransferrin and lysozyme from egg white. Background Technology

[0002] Ovalbumin is the main protein in egg white, accounting for approximately 54% of the total protein content. It is a glycoprotein with a molecular weight of about 45 kDa and possesses important nutritional and functional properties. Ovalbumin is widely used in the food industry, for example, as an emulsifier, gelling agent, and foaming agent. Furthermore, due to its immunogenicity, ovalbumin is used in vaccine development and biomedical research. In recent years, with the development of biotechnology, ovalbumin isolation and purification techniques have been continuously optimized to meet the demand for high-purity protein in the pharmaceutical and food fields.

[0003] Ovotransferrin is an important functional protein in egg white, accounting for approximately 12% of the total egg white protein. It is an iron-binding protein with a molecular weight of approximately 77 kDa, possessing various biological activities including antibacterial, antioxidant, and immunomodulatory effects. Ovotransferrin can inhibit bacterial growth by chelating iron ions, thus showing potential application value in food preservation and pharmaceuticals. Furthermore, ovotransferrin is being researched for the development of functional foods and nutritional supplements. Efficient separation and purification techniques are crucial for its large-scale application.

[0004] Lysozyme is a natural antimicrobial protein found in egg white, accounting for approximately 3.5% of the total egg white protein. It is an enzyme-like protein with a molecular weight of approximately 14.4 kDa, capable of killing bacteria by hydrolyzing peptidoglycan in the bacterial cell wall. Lysozyme is widely used as a natural preservative in the food industry and is also used in the pharmaceutical field to treat infectious diseases. In recent years, with the increasing demand for natural antimicrobial agents, the isolation and purification technology of lysozyme has become a research hotspot to improve its purity and activity.

[0005] Ovalbumin, ovotransferrin, and lysozyme are three important functional proteins in egg white, each possessing unique nutritional value and bioactivity. With the rapid development of the food, pharmaceutical, and biotechnology fields, the demand for these high-purity proteins is increasing daily. Traditional separation and purification methods typically yield only one protein per process, resulting in low efficiency and high costs. Simultaneous separation, however, allows for the separation of multiple proteins in a single process, simplifying the operation, reducing separation steps and time, and significantly improving raw material utilization and overall separation efficiency. Furthermore, simultaneous separation reduces the amount of reagents such as buffers and eluents, shortens separation time, lowers equipment operating and labor costs, and enhances overall economic benefits. Therefore, developing an efficient, economical, and environmentally friendly separation and purification technology to achieve the efficient simultaneous separation and extraction of ovalbumin, ovotransferrin, and lysozyme has significant industrial application value and scientific research significance. Summary of the Invention

[0006] To address the aforementioned problems in the prior art, the purpose of this invention is to provide a method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white.

[0007] The technical solution adopted in this invention is as follows: A method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white involves first using chitosan-polybutyl methacrylate nanocrystals with anion exchange function as the primary separation medium. Ovalbumin is adsorbed from the pretreated egg white sample solution by anion exchange chromatography, and the nanocrystals are then eluted with a gradient of NaCl solution to obtain ovalbumin. The effluent from the primary separation is collected and used as the secondary separation medium. Ovotransferrin and lysozyme are adsorbed from the secondary separation sample solution by cation exchange chromatography. Based on the difference in electrostatic interaction strength between the two proteins at their ligand binding sites, a gradient elution with progressively increasing salt concentration is used to separate ovotransferrin and lysozyme.

[0008] Furthermore, the preparation method of chitosan-polybutyl methacrylate nanocrystal gel with anion exchange function is as follows: (1) Disperse chitosan and polybutyl methacrylate sodium gel solution in acetic acid aqueous solution to prepare monomer solution, and stir at room temperature for 3-6 h; (2) Add the crosslinking agent glutaraldehyde to the monomer solution in step (1) and place it in a chromatography column. Set the temperature of the cooling system of the chromatography column to -25~-15 ℃ and react continuously at low temperature for 20~30 h to obtain the crystal gel matrix. (3) Under the catalysis of cerium ammonium nitrate, functional monomers with tertiary amino groups are grafted onto the crystal matrix obtained in step (2) through a grafting reaction to obtain chitosan-polybutyl methacrylate nanocrystal gel with anion exchange function. In steps (1) to (2), the total mass of all raw materials is 100%, water is used as the solvent, and the mass fractions of other different components are: chitosan 1.2-2%, polybutyl methacrylate sodium gel 2.4-4%, acetic acid 1.5-3%, and glutaraldehyde 0.08-0.12%, preferably chitosan 1.4-1.6%, polybutyl methacrylate sodium gel 2.6-3%, acetic acid 1.8-2.2%, and glutaraldehyde 0.085-0.09%.

[0009] Further, in step (3), firstly, 5-7 times the volume of the crystal matrix of 0.03-0.06 M cerium ammonium nitrate solution is introduced into the chitosan-polybutyl methacrylate nanocrystalline matrix, and the reaction temperature is controlled at 45-50 ℃ and the reaction time is 35-45 min; then, 5-7 times the volume of the crystal matrix of 0.5-1.5 M dimethylaminoethyl methacrylate monomer solution is introduced to replace the cerium ammonium nitrate solution, and the reaction temperature is controlled at 45-50 ℃ and the reaction time is 1.5-2.5 h; finally, 0.1 M HCl solution is introduced to replace the dimethylaminoethyl methacrylate monomer solution.

[0010] Furthermore, the preparation method of hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystals with cation exchange function is as follows: (1) Disperse hydroxyethyl methacrylate and polyethylene glycol diacrylate in an aqueous solution, and add polyglycidyl methacrylate sodium gel solution to prepare a monomer solution; (2) Add the initiator ammonium persulfate and the accelerator tetramethylethylenediamine to the monomer solution in step (1), and place it in a chromatography column. Set the temperature of the cooling system of the chromatography column to -25~-15 ℃ and react continuously at low temperature for 20~30 h to obtain the crystal gel matrix. (3) Under the catalysis of potassium diperiodate copper(III), the functional monomer with sulfonic acid group is grafted onto the crystal matrix obtained in step (2) through a grafting reaction to obtain hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal gel with cation exchange function. In the preparation of hydroxyethyl methacrylate nanocrystal gel, the total mass of all raw materials in steps (1) to (2) is 100%, water is used as solvent, and the mass fractions of other different components are as follows: hydroxyethyl methacrylate 7.7%, polyethylene glycol diacrylate 2.3%, polyglycidyl methacrylate nanogel 1-4%, ammonium persulfate 0.15%, tetramethylethylenediamine 0.15%, and preferably polyglycidyl methacrylate nanogel 1.8-2.2%.

[0011] Further, in step (3), firstly, 5-7 times the volume of the hydroxyethyl methacrylate-glycidyl methacrylate nanocrystalline matrix is ​​introduced into the matrix, followed by a 0.05-0.07 M potassium diperiodate solution, and the reaction temperature is controlled at 45-50 °C for 35-45 min. Then, 5-7 times the volume of the crystalline matrix is ​​introduced into the matrix, followed by a 0.5-1.5 M 2-acrylamido-2-methylpropanesulfonic acid monomer solution to replace the potassium diperiodate solution, and the reaction temperature is controlled at 45-50 °C for 1.5-2.5 h. Finally, 0.1 M HCl solution is introduced into the matrix to replace the 2-acrylamido-2-methylpropanesulfonic acid monomer solution.

[0012] In the preparation of crystalline gels, adding nanogels can effectively increase the specific surface area of ​​the gel, compensating for the small effective adsorption area of ​​the crystalline gel medium, and improving the adsorption capacity for target analytes by leveraging the adsorption advantages of nanogels. Both polybutyl methacrylate (PBMA) and glycidyl methacrylate (GMA) nanogels increase the specific surface area of ​​the chromatography column and enlarge the adsorption sites to bind more target proteins, but PBMA and GMA have different properties. PBMA contains inert butyl ester groups, relying solely on physical adsorption or hydrophobic interactions. The longer butyl ester side chains of PBMA form a looser network structure during polymerization, which is beneficial for the intercalation of chitosan molecular chains and the formation of a macroporous structure. PBMA forms physical cross-links with chitosan through hydrophobic interactions, avoiding irreversible network solidification caused by covalent bonds, thus preserving the pH-responsive swelling of chitosan and improving the reversible deformability of the nanogel. Polyglycerol methacrylate (PGA) contains epoxy groups, and the cross-linking of these epoxy groups can form a dense network that may clog the pores of chitosan and reduce mass transfer efficiency. Simultaneously, the epoxy groups of PGA react with the -NH2 groups of chitosan to form irreversible covalent bonds, which may lead to the breakage of chitosan molecular chains and a decrease in the biodegradability of the chitosan network.

[0013] The reason for using 2-hydroxyethyl methacrylate crosslinked polyethylene glycol diacrylate as the monomer in the secondary separation process is as follows: Proteins are composed of amino acids and carry positive or negative charges at different pH values. When pH < pI, the protein carries a positive charge and can bind to an anion exchange column; when pH > pI, it carries a negative charge and can bind to a cation exchange column. The pH value selected for the primary separation is greater than the isoelectric point of ovalbumin, and ovalbumin carries a negative charge. Ovalbumin in egg white can be separated using a chromatography column with anion exchange function prepared by grafting dimethylaminoethyl methacrylate onto chitosan. The pH value selected for the secondary separation is less than the isoelectric points of ovotransferrin and lysozyme, and the proteins carry a positive charge. Ovotransferrin and lysozyme can be separated using a chromatography column with cation exchange function prepared by grafting 2-acrylamido-2-methylpropane sulfonic acid onto 2-hydroxyethyl methacrylate. The 2-hydroxyethyl methacrylate monomer used in the secondary separation itself does not carry a charge. After grafting 2-acrylamido-2-methylpropane sulfonic acid, -SO3⁻ becomes the only source of charge, and the negative charge density per unit volume is higher, resulting in better adsorption performance for cations. Therefore, changing the monomer is beneficial to improving the protein adsorption capacity of the chromatography column. A chromatography column prepared by grafting 2-acrylamido-2-methylpropane sulfonic acid onto chitosan as the monomer can also separate ovotransferrin and lysozyme. However, chitosan molecules are rich in amino (-NH2) groups, which make it carry a positive charge (-NH3⁺) under acidic conditions. After grafting 2-acrylamido-2-methylpropane sulfonic acid, -NH3⁺ will neutralize some of the -SO3⁻ charges, reducing the net negative charge amount and decreasing the protein adsorption capacity of the chromatography column.

[0014] Furthermore, the steps of the primary separation are as follows: (1) Preparation of the pretreated egg white sample loading solution: Manually separate the egg white from the egg yolk, then add deionized water to the egg white for dilution, adjust the pH value of the mixture to 6.0 - 7.0 with hydrochloric acid solution, place the adjusted solution in a low-temperature environment of 0 - 4 °C, continuously stir for 8 - 10 h, and then, at 4 °C, centrifuge at 7000 - 9000 rpm / min for 10 min. After centrifugation, collect the supernatant; (2) Dilute the supernatant with acetic acid / sodium acetate buffer as the sample loading solution, and flow it through the chitosan-polybutyl methacrylate nanocrystal gel with anion exchange function, so that ovalbumin is adsorbed by the chitosan-polybutyl methacrylate nanocrystal gel, and at the same time, collect the sample loading flow-through solution as the secondary separation sample loading solution; (3) Rinse the chitosan-polybutyl methacrylate nanocrystal gel after chromatographic separation in step (2) with acetic acid / sodium acetate buffer to remove the residual egg white sample loading solution on the chitosan-polybutyl methacrylate nanocrystal gel and the impurity proteins not adsorbed in the pores; (4) Wash the chitosan-polybutyl methacrylate nanocrystal with eluents of different gradients to elute the adsorbed ovalbumin and restore the adsorption chromatography performance of the chitosan-polybutyl methacrylate nanocrystal.

[0015] Furthermore, in step (2), the sample loading flow rate is 0.5~2 cm / min, and the sample loading volume is 4~10 times the volume of the crystal gel, preferably 5-6 times. In steps (2)~(3), the pH range of the acetic acid / sodium acetate buffer is 3.8~5.8, and the concentration is 5~25mM, preferably pH 5.0-5.2, and the concentration is 5-10mM.

[0016] Further, in step (4), the elution buffer is an acetate / sodium acetate buffer containing 0.03-2 M NaCl. The elution buffer is used for gradient elution according to the NaCl concentration from low to high. Specifically, ovalbumin and lysozyme have different strengths of electrostatic interaction at the ligand binding sites. The electrostatic interaction of ovalbumin is weaker, while that of lysozyme is stronger. The separation of ovalbumin and lysozyme can be achieved by gradually increasing the salt gradient. Some impurities with weak electrostatic interaction are eluted with NaCl at a concentration of 0.03-0.05 M to improve the purity of ovalbumin. Ovalbumin is eluted with NaCl at a concentration of 0.8-1 M. The chromatography column is regenerated with NaCl at a concentration of 1.5-2 M.

[0017] Furthermore, the secondary separation includes the following steps: (1) The secondary separation loading solution is passed through hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal gel, so that ovotransferrin and lysozyme are adsorbed by hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal gel; (2) Rinse the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal after chromatography separation in step (1) with acetic acid / sodium acetate buffer to remove the secondary separation loading solution and unadsorbed impurity proteins remaining on the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal. (3) Wash the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal with eluents of different gradients to wash off the adsorbed ovotransferrin and lysozyme and restore the adsorption chromatography performance of the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal.

[0018] Further, in step (1), the sample loading flow rate is 0.5~2 cm / min, and the sample loading volume is 4~10 times the volume of the crystal gel, preferably 6-8 times; in step (2), the pH range of the acetate / sodium acetate buffer is 3.8~5.8, and the concentration is 5~25 mM, preferably pH 5.0-5.2, and the concentration is 5-10 mM.

[0019] Further, in step (3), the eluent is an acetate / sodium acetate buffer containing 0.1-2 M NaCl. The eluent is used in a gradient elution process from low to high NaCl concentration. Specifically, ovotransferrin with weak electrostatic interactions is eluted with 0.1-0.15 M NaCl; some impurities are eluted with 0.25-0.3 M NaCl to improve lysozyme purity; lysozyme with strong electrostatic interactions is eluted with 0.6-0.8 M NaCl; and the chromatography column is regenerated with 1.5-2 M NaCl. By employing the above techniques, compared with existing technologies, the present invention has the following advantages: (1) This invention employs crystalline gel anion-cation exchange chromatography to simultaneously separate ovalbumin, ovotransferrin, and lysozyme from egg white. The crystalline gel material has a high specific surface area, uniform pore size, and tunable charge characteristics, enabling the efficient separation of multiple proteins by utilizing anion-cation exchange mechanisms simultaneously. The high adsorption capacity and selectivity of the crystalline gel material reduce the loss of target proteins and improve the recovery rate. The uniform pore size and charge characteristics of the crystalline gel material improve the separation resolution and reduce cross-contamination between target proteins. Simultaneous separation reduces separation steps and time, significantly improving the overall separation efficiency.

[0020] (2) The crystal gel anion and cation chromatography technique used in this invention does not require multiple replacements of chromatography media or adjustment of conditions, which simplifies the operation process and reduces the difficulty of operation; the crystal gel material has good mechanical strength and chemical stability, can be reused, and reduces operating costs; the simultaneous separation reduces the amount of reagents such as buffer and elution solution used, shortens the separation time, reduces equipment operation and labor costs, and improves the overall economic benefits.

[0021] (3) The method used in this invention has a wide range of applications. This technology is not only applicable to the separation of ovalbumin, ovotransferrin and lysozyme, but can also be extended to other proteins or multi-component systems with different charge properties. By adjusting conditions such as pH and ionic strength, it can be adapted to the separation requirements of different samples. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0023] The preparation of the poly(glycidyl methacrylate) nanogel solution in this embodiment of the invention includes the following steps: Based on the total mass of all raw materials (100%), 4.93% glycidyl methacrylate and 0.07% ethylene glycol dimethacrylate are added to pure water, followed by 1% sodium dodecyl sulfate. The aqueous solution containing glycidyl methacrylate, ethylene glycol dimethacrylate, and sodium dodecyl sulfate is ultrasonically emulsified at room temperature for 30 minutes until it becomes a white emulsion, with continuous stirring during the ultrasonication process. Then, 0.6% ammonium persulfate and 1.2% tetramethylethylenediamine are added, and the mixture is ultrasonicated for another 5 minutes with continuous stirring. The white emulsion is then transferred to a 70°C water bath for polymerization. After approximately 9-12 minutes, the emulsion changes from white to a translucent light blue. The reaction continues for another 20 minutes, after which the emulsion is removed and cooled to room temperature, thus obtaining the poly(glycidyl methacrylate) nanogel solution.

[0024] The preparation of chitosan-polybutyl methacrylate nanocrystalline gel with anion exchange function in this embodiment of the invention includes the following steps: Based on a total raw material content of 100%, 1.4% chitosan, 2.8% polybutyl methacrylate sodium gel, 2% acetic acid, and 93.713% deionized water were stirred at room temperature for 4 h. 0.087% glutaraldehyde was added and placed in a chromatography column. The cooling system of the chromatography column was set to -20 ℃, and the reaction was carried out at a low temperature for 24 h. The chromatography column was then removed and thawed to obtain the chitosan-polybutyl methacrylate crystal matrix.

[0025] First, a 0.05 M cerium ammonium nitrate solution was used as a catalyst and flowed through a chitosan-polybutyl methacrylate (PPMA) nanocrystalline matrix. The reaction was carried out at 48 °C for 40 min. Then, a 1 M dimethylaminoethyl methacrylate (DMME) monomer solution was flowed through the chitosan-PPMA nanocrystalline matrix, and the reaction was carried out at 48 °C for 2 h to graft the chitosan-PPMA nanocrystalline matrix framework. The volumes of the cerium ammonium nitrate solution and the DMME monomer solution were each 5 times the volume of the chitosan-PPMA nanocrystalline matrix. The final product was a chitosan-PPMA nanocrystalline gel (i.e., anion exchange gel) with a column volume of 5.02 cm³. 3 The adsorption rate of BSA was 30.21 mg / mL.

[0026] The preparation of hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystals with cation exchange function in this invention includes the following steps: Based on a 100% total raw material content, 7.7% hydroxyethyl methacrylate, 2.3% polyethylene glycol diacrylate, 2% polyglycidyl methacrylate sodium gel, and 87.7% deionized water were mixed and pre-cooled at 0 °C for 30 min. 0.15% ammonium persulfate initiator and 0.15% tetramethylethylenediamine accelerator were added to the mixture, and the mixture was placed in a chromatography column. The cooling system of the chromatography column was set to -20 °C, and the reaction was carried out continuously at low temperature for 24 h. The chromatography column was then removed and thawed to obtain the hydroxyethyl methacrylate-polyglycidyl methacrylate crystal gel matrix.

[0027] First, a 0.056 M potassium diperiodide (β-C)-copper(III) solution was used as a catalyst and flowed through a hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal matrix. The reaction was carried out at 48 °C for 40 min. Then, a 1 M 2-acrylamido-2-methylpropanesulfonic acid monomer solution was flowed through the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal matrix and reacted at 48 °C for 2 h to graft the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal matrix framework. The volumes of the potassium diperiodide (β-C)-copper(III) solution and the 2-acrylamido-2-methylpropanesulfonic acid monomer solution were each 5 times the volume of the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal matrix. Finally, a hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal (i.e., a cation exchange nanocrystal) was obtained with a column volume of 5.13 cm³. 3 The adsorption of lysozyme was 4.92 mg / L.

[0028] The method for preparing the supernatant in this embodiment of the invention is as follows: Egg whites and yolks were separated manually. Deionized water was then added to the eggs for dilution. The pH of the mixture was adjusted to 6 with hydrochloric acid solution. The adjusted solution was placed in a low-temperature environment at 0 °C and stirred continuously for 10 h. Then, it was centrifuged at 8000 rpm / min for 10 min at 4 °C. The supernatant was collected after centrifugation.

[0029] Example 1 The supernatant was diluted 1:15 by volume with 5 mM acetate / sodium acetate buffer at pH 5.2 to obtain the loading solution. The loading volume was 30 mL, and the sample was loaded at a flow rate of 1 cm / min to allow adsorption through the anion exchange gel. The flow-through solution was collected as a secondary loading solution. Then, the gel was washed with 5 mM acetate / sodium acetate buffer at pH 5.2 to remove unadsorbed substrate and other impurities. Gradient elution was performed sequentially with elution buffers containing 0.03 M, 1 M, and 2 M NaCl (using 5 mM acetate / sodium acetate buffer at pH 5.2 as the solvent). The elution peak containing ovalbumin was collected to obtain ovalbumin. 0.03 M was used to elute other proteins, 1 M was used to elute ovalbumin, and 2 M was used for column regeneration. SDS-PAGE and Quantity One analysis showed that the obtained ovalbumin was a single band with a relative molecular weight of 45.0 kDa. The purity of ovalbumin reached 98.2%, and the recovery rate was 97.7%.

[0030] 30 mL of the secondary loading solution was loaded at a flow rate of 1 cm / min to allow adsorption through the cation exchange gel. The column was then washed with a 5 mM acetate / sodium acetate buffer (pH 5.2) to remove unadsorbed substrate and other impurities. A gradient elution was then performed using 0.15 M, 0.3 M, 0.8 M, and 2 M NaCl eluents (using a 5 mM acetate / sodium acetate buffer at pH 5.2 as the solvent). 0.15 M was used to elute ovotransferrin, which has weak electrostatic binding; 0.3 M was used to elute some other proteins; 0.8 M was used to elute lysozyme, which has strong electrostatic binding; and 2 M was used for column regeneration. The elution peaks containing ovotransferrin and lysozyme were collected to obtain ovotransferrin and lysozyme. SDS-PAGE and QuantityOne analysis showed that the obtained ovotransferrin was a single band with a relative molecular weight of 76.0-78.0 kDa, and the purity of ovotransferrin reached 96.9%, with a recovery rate of 95.9%. The obtained lysozyme was a single band with a relative molecular weight of 14.3 kDa. The purity of lysozyme reached 93.1%, with a recovery rate of 92.1%.

[0031] Example 2 The supernatant was diluted 1:15 by volume with 20 mM acetate / sodium acetate buffer at pH 3.8 as the loading solution. Ovalbumin in the supernatant was separated by chromatography using the regenerated anion exchange gel from Example 1. The loading volume was 30 mL, and the sample was loaded at a flow rate of 0.5 cm / min to allow adsorption through the anion exchange gel. The flow-through was collected as a secondary loading solution. The gel was then washed with 20 mM acetate / sodium acetate buffer at pH 3.8 to remove unadsorbed substrate and other impurities. A gradient elution was performed sequentially with elution buffers containing 0.05 M, 0.8 M, and 2 M NaCl (using 20 mM acetate / sodium acetate buffer at pH 3.8 as the solvent). The elution peak containing ovalbumin was collected to obtain ovalbumin. 0.05 M was used to elute other proteins, 0.8 M was used to elute ovalbumin, and 2 M was used for column regeneration.

[0032] 30 mL of the secondary loading solution was loaded at a flow rate of 0.5 cm / min and allowed to pass through the regenerated cation exchange gel from Example 1 for adsorption. The column was then washed with a 20 mM acetate / sodium acetate buffer at pH 3.8 to remove unadsorbed substrate and other impurities. A gradient elution was then performed using 0.1 M, 0.25 M, 0.6 M, and 2 M NaCl eluents (using a 20 mM acetate / sodium acetate buffer at pH 3.8 as the solvent). 0.1 M was used to elute ovotransferrin, which has weak electrostatic binding; 0.25 M was used to elute some other proteins; 0.6 M was used to elute lysozyme, which has strong electrostatic binding; and 2 M was used for column regeneration. The elution peaks containing ovotransferrin and lysozyme were collected to obtain ovotransferrin and lysozyme. The final results obtained in Example 2 were as follows: ovalbumin purity reached 97.2% with a recovery rate of 28.4%; oval transferrin purity reached 73.3% with a recovery rate of 34.9%; and lysozyme purity reached 75.3% with a recovery rate of 47.3%.

[0033] Example 3 The supernatant was diluted 1:15 by volume with 5 mM acetate / sodium acetate buffer at pH 5.8 as the loading solution. Ovalbumin in the supernatant was separated by chromatography using the regenerated anion exchange gel from Example 1. The loading volume was 30 mL, and the sample was loaded at a flow rate of 2 cm / min to allow adsorption through the anion exchange gel. The flow-through was collected as a secondary loading solution. The gel was then washed with 5 mM acetate / sodium acetate buffer at pH 5.8 to remove unadsorbed substrate and other impurities. A gradient elution was performed sequentially with elution buffers containing 0.05 M, 0.8 M, and 2 M NaCl (using 5 mM acetate / sodium acetate buffer at pH 5.8 as the solvent). The elution peak containing ovalbumin was collected, yielding ovalbumin. 0.05 M was used to elute other proteins, 0.8 M was used to elute ovalbumin, and 2 M was used for column regeneration.

[0034] 30 mL of the secondary loading solution was loaded at a flow rate of 2 cm / min and allowed to pass through the regenerated cation exchange gel from Example 1 for adsorption. The column was then washed with a 5 mM acetate / sodium acetate buffer at pH 5.8 to remove unadsorbed substrate and other impurities. A gradient elution was then performed using 0.1 M, 0.25 M, 0.6 M, and 2 M NaCl eluents (using a 5 mM acetate / sodium acetate buffer at pH 5.8 as the solvent). 0.1 M was used to elute ovotransferrin, which has weak electrostatic binding; 0.25 M was used to elute some other proteins; 0.6 M was used to elute lysozyme, which has strong electrostatic binding; and 2 M was used for column regeneration. The elution peaks containing ovotransferrin and lysozyme were collected to obtain ovotransferrin and lysozyme. The final results obtained in Example 3 were as follows: ovalbumin purity reached 87.7% with a recovery rate of 98.3%; oval transferrin purity reached 96.9% with a recovery rate of 91.6%; and lysozyme purity reached 85.3% with a recovery rate of 87.3%.

[0035] Example 4 The supernatant was diluted 1:15 by volume with 20 mM acetate / sodium acetate buffer at pH 5.2 as the loading solution. Ovalbumin in the supernatant was separated by chromatography using the regenerated anion exchange gel from Example 1, with a loading volume of 20 mL and a flow rate of 1 cm / min. The sample was allowed to pass through the anion exchange gel for adsorption, and the flow-through was collected as a secondary loading solution. The gel was then washed with 20 mM acetate / sodium acetate buffer at pH 5.2 to remove unadsorbed substrate and other impurities. A gradient elution was performed sequentially with elution buffers containing 0.03 M, 1 M, and 2 M NaCl (using 20 mM acetate / sodium acetate buffer at pH 5.2 as the solvent). The elution peak containing ovalbumin was collected, yielding ovalbumin. 0.03 M was used to elute other proteins, 1 M was used to elute ovalbumin, and 2 M was used for column regeneration.

[0036] 20 mL of the secondary loading solution was loaded at a flow rate of 1 cm / min and allowed to pass through the regenerated cation exchange gel from Example 1 for adsorption. The column was then washed with a 20 mM acetate / sodium acetate buffer at pH 5.2 to remove unadsorbed substrate and other impurities. A gradient elution was then performed using 0.15 M, 0.3 M, 0.8 M, and 2 M NaCl eluents (using a 20 mM acetate / sodium acetate buffer at pH 5.2 as the solvent). 0.15 M was used to elute ovotransferrin, which has weak electrostatic binding; 0.3 M was used to elute some other proteins; 0.8 M was used to elute lysozyme, which has strong electrostatic binding; and 2 M was used for column regeneration. The elution peaks containing ovotransferrin and lysozyme were collected to obtain ovotransferrin and lysozyme. The final separation results in Example 4 showed that ovalbumin purity reached 95.8% with a recovery rate of 92.51%; oval transferrin purity reached 99.8% with a recovery rate of 38.9%; and lysozyme purity reached 26.6% with a recovery rate of 38.3%. The buffer concentration used in Example 4 was 20 mM, which was higher than that in Example 1. The elution peak of lysozyme was broad. When eluting impurities with 0.3 M NaCl elution buffer, the higher concentration of buffer increased the ionic strength of the elution buffer to some extent, resulting in a large amount of lysozyme being eluted during 0.3 M elution. Subsequently, the purity and recovery rate of lysozyme decreased during 0.8 M elution.

[0037] Example 5 The supernatant was diluted 1:15 by volume with 10 mM acetate / sodium acetate buffer at pH 5.2 as the loading solution. Ovalbumin in the supernatant was separated by chromatography using the regenerated anion exchange gel from Example 1. The loading volume was 50 mL, and the sample was loaded at a flow rate of 2 cm / min to allow adsorption through the anion exchange gel. The flow-through was collected as a secondary loading solution. The gel was then washed with 10 mM acetate / sodium acetate buffer at pH 5.2 to remove unadsorbed substrate and other impurities. A gradient elution was performed sequentially with elution buffers containing 0.05 M, 1 M, and 2 M NaCl (using 10 mM acetate / sodium acetate buffer at pH 5.2 as the solvent). The elution peak containing ovalbumin was collected, yielding ovalbumin. 0.05 M was used to elute other proteins, 1 M was used to elute ovalbumin, and 2 M was used for column regeneration.

[0038] 50 mL of the secondary loading solution was loaded at a flow rate of 2 cm / min and allowed to pass through the regenerated cation exchange gel from Example 1 for adsorption. The column was then washed with a 10 mM acetate / sodium acetate buffer at pH 5.2 to remove unadsorbed substrate and other impurities. A gradient elution was then performed using 0.15 M, 0.3 M, 0.8 M, and 2 M NaCl eluents (using a 10 mM acetate / sodium acetate buffer at pH 5.2 as the solvent). 0.15 M was used to elute ovotransferrin, which has weak electrostatic binding; 0.3 M was used to elute some other proteins; 0.8 M was used to elute lysozyme, which has strong electrostatic binding; and 2 M was used for column regeneration. The elution peaks containing ovotransferrin and lysozyme were collected to obtain ovotransferrin and lysozyme. The final separation results in Example 5 showed that ovalbumin purity reached 95.8% with a recovery rate of 63.7%; ovaltransferrin purity reached 46.9% with a recovery rate of 39.3%; and lysozyme purity reached 38.9% with a recovery rate of 77.3%. In this example, the loading volume was 50 mL, an increase compared to 30 mL in Example 1. While the increased loading volume did not change the adsorption performance of the chromatography column, the column reached adsorption saturation, and excess loading solution flowed through. However, the total protein content remained unchanged, leading to a decrease in the recovery rates of ovaltransferrin and lysozyme. Increased loading volume also resulted in competition for binding sites between easily bound contaminating proteins and the target proteins, leading to a decrease in the purity of ovaltransferrin and lysozyme. Example 5 uses a flow rate of 2 cm / min, which is higher than the 1 cm / min in Example 1. At a high flow rate, the mass transfer time between the liquid and the chromatography medium is reduced, and the adsorption of ovotransferrin, which has a weak binding capacity, is reduced. The high sample volume and high flow rate cause the chromatography column to be compressed or blocked, causing some sample liquid to flow out without sufficient contact, resulting in a decrease in the purity and recovery rate of ovotransferrin.

[0039] Example 6 The supernatant was diluted 1:15 by volume with 5 mM acetate / sodium acetate buffer at pH 4.8 as the loading solution. Ovalbumin in the supernatant was separated by chromatography using the regenerated anion exchange gel from Example 1. The loading volume was 30 mL, and the sample was loaded at a flow rate of 2 cm / min to allow adsorption through the anion exchange gel. The flow-through was collected as a secondary loading solution. The gel was then washed with 5 mM acetate / sodium acetate buffer at pH 4.8 to remove unadsorbed substrate and other impurities. A gradient elution was performed sequentially using elution buffers containing 0.05 M, 0.8 M, and 2 M NaCl (using 5 mM acetate / sodium acetate buffer at pH 4.8 as the solvent). The elution peak containing ovalbumin was collected to obtain ovalbumin. 0.05 M was used to elute other proteins, 0.8 M was used to elute ovalbumin, and 2 M was used for column regeneration.

[0040] 30 mL of the secondary loading solution was loaded at a flow rate of 2 cm / min and allowed to pass through the regenerated cation exchange gel from Example 1 for adsorption. The column was then washed with a 5 mM acetate / sodium acetate buffer (pH 4.8) to remove unadsorbed substrate and other impurities. A gradient elution was then performed using 0.15 M, 0.25 M, 0.8 M, and 2 M NaCl eluents (using a 5 mM acetate / sodium acetate buffer at pH 4.8 as the solvent). 0.15 M was used to elute ovotransferrin, which has weak electrostatic binding; 0.25 M was used to elute some other proteins; 0.8 M was used to elute lysozyme, which has strong electrostatic binding; and 2 M was used for column regeneration. The elution peaks containing ovotransferrin and lysozyme were collected to obtain ovotransferrin and lysozyme. The final results obtained in Example 6 were as follows: ovalbumin purity reached 84.3% with a recovery rate of 47.2%; oval transferrin purity reached 97.3% with a recovery rate of 93.4%; and lysozyme purity reached 78.6% with a recovery rate of 88.3%.

[0041] At pH 4.8, ovalbumin has a negative net charge (pH > pI), but some areas on its surface may still carry a localized positive charge. This localized positive charge prevents it from binding to the anion exchange chromatography column, so some ovalbumin remains unadsorbed. With the overall ovalbumin concentration unchanged, the amount adsorbed onto the chromatography column decreases, resulting in a low recovery rate during elution.

[0042] Similarly, in Example 2, the pH value used was 3.8, and the net charge of ovalbumin was positive (pH < pI). However, some areas on its surface may still carry localized negative charges. These localized negative charges cause it to bind to the anion exchange chromatography column, but only a small amount of ovalbumin is adsorbed. The overall ovalbumin concentration remains unchanged, the amount of protein adsorbed by the chromatography column is small, and the recovery rate during elution is low.

Claims

1. A method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white, characterized in that, First, chitosan-polybutyl methacrylate nanocrystals with anion exchange function were used as the primary separation medium. Ovalbumin was adsorbed from the pretreated egg white loading solution by anion exchange chromatography, and the crystals were eluted with NaCl solution in a gradient to obtain ovalbumin. The effluent from the primary separation was collected as the loading solution for the subsequent secondary separation. Then, hydroxyethyl methacrylate-glycidyl methacrylate nanocrystals with cation exchange function were used as the secondary separation medium. Ovalbumin and lysozyme were adsorbed from the secondary separation loading solution by cation exchange chromatography. Based on the difference in electrostatic interaction strength between the two proteins at the ligand binding sites, a gradient elution with gradually increasing salt concentration was used to separate ovalbumin and lysozyme.

2. The method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white as described in claim 1, characterized in that, The preparation method of chitosan-polybutyl methacrylate nanocrystal gel with anion exchange function is as follows: (1) Disperse chitosan and polybutyl methacrylate sodium gel solution in acetic acid aqueous solution to prepare monomer solution, and stir at room temperature for 3-6 h; (2) Add the crosslinking agent glutaraldehyde to the monomer solution in step (1) and place it in a chromatography column. Set the temperature of the cooling system of the chromatography column to -25~-15 ℃ and react continuously at low temperature for 20~30 h to obtain the crystal gel matrix. (3) Under the catalysis of cerium ammonium nitrate, the functional monomer with tertiary amino group is grafted onto the crystal matrix obtained in step (2) through grafting reaction to obtain chitosan-polybutyl methacrylate nanocrystal gel with anion exchange function. In steps (1) to (2), the total mass of all raw materials is 100%, water is used as the solvent, and the mass fractions of other different components are: chitosan 1.2-2%, polybutyl methacrylate sodium gel 2.4-4%, acetic acid 1.5-3%, and glutaraldehyde 0.08-0.12%.

3. The method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white as described in claim 1, characterized in that, The preparation method of hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystals with cation exchange function is as follows: (1) Disperse hydroxyethyl methacrylate and polyethylene glycol diacrylate in an aqueous solution, and add polyglycidyl methacrylate sodium gel solution to prepare a monomer solution; (2) Add the initiator ammonium persulfate and the accelerator tetramethylethylenediamine to the monomer solution in step (1), and place it in a chromatography column. Set the temperature of the cooling system of the chromatography column to -25~-15 ℃ and react continuously at low temperature for 20~30 h to obtain the crystal gel matrix. (3) Under the catalysis of potassium diperiodate copper(III), the functional monomer with sulfonic acid group is grafted onto the crystal matrix obtained in step (2) through a grafting reaction to obtain hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal gel with cation exchange function. In the preparation of hydroxyethyl methacrylate nanocrystal gel, the total mass of all raw materials in steps (1) to (2) is 100%, water is used as solvent, and the mass fractions of other different components are: hydroxyethyl methacrylate 7.7%, polyethylene glycol diacrylate 2.3%, polyglycidyl methacrylate nanogel 1-4%, ammonium persulfate 0.15%, and tetramethylethylenediamine 0.15%.

4. The method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white as described in claim 1, characterized in that, The steps for a single separation are as follows: (1) Preparation of pretreated egg white loading solution: Egg white and yolk were separated manually, and then deionized water was added to the egg white for dilution. The pH of the mixture was adjusted to 6.0~7.0 with hydrochloric acid solution. The adjusted solution was placed in a low temperature environment of 0~4℃ and stirred continuously for 8~10 h. Then, it was centrifuged at 7000~9000 rpm / min for 10 min at 4℃. The supernatant was collected after centrifugation. (2) The supernatant was diluted with acetic acid / sodium acetate buffer and used as the loading solution. It was then flowed through chitosan-polybutyl methacrylate nanocrystals with anion exchange function, so that ovalbumin was adsorbed by chitosan-polybutyl methacrylate nanocrystals. At the same time, the loading effluent was collected as the secondary separation loading solution. (3) Rinse the chitosan-polybutyl methacrylate nanocrystal after chromatography separation in step (2) with acetic acid / sodium acetate buffer to remove the egg white sample solution and unadsorbed impurity proteins remaining on the chitosan-polybutyl methacrylate nanocrystal. (4) Wash the chitosan-polybutyl methacrylate nanocrystal with eluents of different gradients to elute the adsorbed ovalbumin and restore the adsorption chromatography performance of the chitosan-polybutyl methacrylate nanocrystal.

5. The method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white as described in claim 4, characterized in that, In step (2), the sample loading flow rate is 0.5~2 cm / min, and the sample loading volume is 4~10 times the volume of the crystal gel. In steps (2)~(3), the pH range of the acetic acid / sodium acetate buffer is 3.8~5.8, and the concentration is 5~25 mM.

6. The method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white as described in claim 4, characterized in that, Step (4) The elution buffer is an acetate / sodium acetate buffer containing 0.03-2 M NaCl. The elution buffer is used for gradient elution according to the NaCl concentration from low to high. Specifically, some impurities with weak electrostatic interactions are eluted with NaCl concentration of 0.03-0.05 M to improve the purity of ovalbumin; ovalbumin is eluted with NaCl concentration of 0.8-1 M; and the chromatography column is regenerated with NaCl concentration of 1.5-2 M.

7. The method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white as described in claim 4, characterized in that, The secondary separation includes the following steps: (1) The secondary separation loading solution is passed through hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal gel, so that ovotransferrin and lysozyme are adsorbed by hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal gel; (2) Rinse the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal after chromatography separation in step (1) with acetic acid / sodium acetate buffer to remove the secondary separation loading solution and unadsorbed impurity proteins remaining on the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal. (3) Wash the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal with eluents of different gradients to wash off the adsorbed ovotransferrin and lysozyme and restore the adsorption chromatography performance of the hydroxyethyl methacrylate-polyglycidyl methacrylate nanocrystal.

8. The method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white as described in claim 7, characterized in that, In step (1), the sample loading flow rate is 0.5~2 cm / min, and the sample loading volume is 4~10 times the volume of the crystal gel; in step (2), the pH range of the acetate / sodium acetate buffer is 3.8~5.8, and the concentration is 5~25 mM.

9. The method for simultaneously separating ovalbumin, ovotransferrin, and lysozyme from egg white as described in claim 7, characterized in that, Step (3) The elution buffer is an acetate / sodium acetate buffer containing 0.1-2 M NaCl. The elution buffer is used for gradient elution according to the NaCl concentration from low to high. Specifically, ovotransferrin with weak electrostatic interaction is eluted with NaCl concentration of 0.1-0.15 M; some impurities are eluted with NaCl concentration of 0.25-0.3 M to improve the purity of lysozyme; lysozyme with strong electrostatic interaction is eluted with NaCl concentration of 0.6-0.8 M; and the chromatography column is regenerated with NaCl concentration of 1.5-2 M.