CN110066778B - Combined separation method of lysozyme and ovotransferrin in egg white - Google Patents

Combined separation method of lysozyme and ovotransferrin in egg white Download PDF

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CN110066778B
CN110066778B CN201910384796.8A CN201910384796A CN110066778B CN 110066778 B CN110066778 B CN 110066778B CN 201910384796 A CN201910384796 A CN 201910384796A CN 110066778 B CN110066778 B CN 110066778B
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egg white
ovotransferrin
lysozyme
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resin
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CN110066778A (en
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杨严俊
苏宇杰
常翠华
李俊华
顾璐萍
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Abstract

The invention discloses a combined separation method of lysozyme and ovotransferrin in egg white, which belongs to the technical field of protein separation and purification, and comprises the steps of (1) adjusting the pH of egg white liquid to 6.5-9.5, and adsorbing lysozyme and ovotransferrin by adopting metal chelating cationic resin; (2) crystallizing the eluent to obtain lysozyme crystals; (3) and collecting the crystallized supernatant, adsorbing by using anion exchange resin, and performing ultrafiltration and drying treatment on the eluent to obtain the ovotransferrin. The method can simultaneously realize the high-efficiency separation of lysozyme and ovotransferrin in the egg white, the yield reaches over 75 percent, the purity reaches over 95 percent, and the used method has the advantages of simple operation and low cost and is easy for industrialized application.

Description

Combined separation method of lysozyme and ovotransferrin in egg white
Technical Field
The invention relates to a combined separation method of lysozyme and ovotransferrin in egg white, belonging to the technical field of protein separation and purification.
Background
The egg white protein is the most ideal high-quality protein in food and mainly comprises egg white protein, ovotransferrin, ovomucin and lysozyme. The lysozyme and the ovotransferrin are bioactive proteins which are concerned by scientific researchers, and have great application potential.
Lysozyme is an essential antibacterial protein for the growth and development of infants, can kill putrefying enterococcus, can enhance the immune function of immunoglobulin, improves the anti-infection capability of the infants, has the effects of preventing weight loss, preventing digestive organ diseases, increasing weight and the like particularly for premature infants, and is a good auxiliary material of functional foods for the infants.
Ovotransferrin is also a soluble amorphous glycoprotein, whose molecular structural domains are linked by antiparallel beta-strands, which can be unfolded and closed by a hinge mechanism, facilitating the binding with ferric ions. The ovotransferrin deprives a plurality of ferric ions necessary for the growth and the reproduction of bacteria by combining with the ferric ions, thereby achieving the purpose of inhibiting the growth and the reproduction of the bacteria, having broad-spectrum bacteriostasis and stronger inhibiting effect on pseudomonas, escherichia coli and salmonella. The antibacterial property of the ovotransferrin is similar to that of human lactoferrin in quantity and quality, the antiviral property is even better than that of the lactoferrin and serum transferrin, and the ovotransferrin also shows excellent antioxidant and anticancer activity.
However, the existing method for separating lysozyme and ovotransferrin generally has the problems of low separation efficiency and difficult industrialization, so that the development of a method for simply, conveniently and effectively separating lysozyme and ovotransferrin from egg white is urgent in market demand.
Disclosure of Invention
In order to solve the problem, the invention provides a metal chelating cation exchange resin adsorption-crystallization-anion exchange resin adsorption method, which can simultaneously obtain high-purity lysozyme and ovotransferrin, has simple operation method and easy industrialization, can realize effective utilization of resources and promote the increase of the added value of egg product enterprises.
The first purpose of the invention is to provide a method for separating protein in egg white, which comprises the following steps:
(1) adjusting the pH value of the egg white to 6.5-9.5, and then adding cation exchange resin and a ferric iron source for adsorption separation; wherein the molar concentration of iron ions in the ferric iron source relative to the egg white liquid is 0.05-0.4 mmol/L;
(2) eluting the resin to obtain an eluent A; adjusting the pH value of the eluent A, concentrating and crystallizing to obtain lysozyme crystals and a clear liquid B;
(3) and adjusting the pH value of the clear liquid B, purifying and concentrating to obtain the ovotransferrin.
In one embodiment of the present invention, the cation exchange resin in step (1) comprises a weak cation exchange resin of carboxyl type.
In one embodiment of the present invention, the ferric iron source in step (1) comprises soluble ferric salts such as ferric chloride, ferric sulfate, ferric nitrate, etc.
In one embodiment of the present invention, the mass ratio of the resin to the egg white in the step (1) is 1: (2-5).
In one embodiment of the present invention, the mass ratio of the resin to the egg white in the step (1) is preferably 1: 3.
in one embodiment of the present invention, before the resin is eluted in step (2), the resin is washed by a material-liquid ratio of 1: and (2-5) washing with an aqueous NaCl solution, wherein the mass concentration of the aqueous NaCl solution is 0.5% -0.9%.
In one embodiment of the present invention, the elution resin is prepared by mixing a raw material and a solvent in a ratio of 1: and (2-5) eluting with NaCl solution, wherein the mass concentration of the NaCl solution is 4% -7%.
In one embodiment of the present invention, the pH of the eluent a in step (2) is adjusted to 9.5-10.8.
In one embodiment of the invention, the concentration and crystallization in the step (2) are performed by adding NaCl to 3-9% of the mass concentration of the eluent A, concentrating and crystallizing at 5-10 ℃.
In one embodiment of the present invention, the pH of the clear solution B in the step (3) is adjusted to 6 to 8.
In one embodiment of the present invention, the step (3) further comprises: and when the pH value of the clear liquid B is 6-8, mixing the clear liquid B with anion exchange resin according to the material-liquid ratio of 1 (2-5), removing the supernatant, and cleaning, eluting and drying the resin to obtain the ovotransferrin.
In an embodiment of the present invention, the method specifically includes the following steps:
(1) egg liquid pretreatment: stirring the egg white liquid, and adjusting the pH value to 6.5-9.5;
(2) adsorption of metal chelating cation resin: mixing the resin and the pretreated egg white according to a feed-liquid ratio of 1:5, adding 0.05-0.4mM ferric chloride solution, adsorbing for 3-5h, and removing supernatant A after adsorption is completed;
(3) cleaning: adding 0.9% NaCl solution into the resin after adsorption according to the material-liquid ratio of 1:3, shaking and mixing uniformly, removing supernatant, and repeatedly cleaning for 3-5 times;
(4) and (3) elution: adding a NaCl solution with the concentration of 4-7% into the washed resin according to the material-liquid ratio of 1:3, oscillating and eluting for 30min, collecting clear liquid, repeating the operation for 3-5 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent A;
(5) and (3) crystallization: adjusting pH of the eluent A to 9.5-10.8, adding NaCl until the salt concentration is 3% -9%, concentrating the eluent by adopting an ultrafiltration concentration technology until the protein concentration is 2-5%, standing at 10 ℃ to form crystals, and respectively collecting the lysozyme crystals and the supernatant B;
(6) adsorption on anion exchange resin: taking supernatant B, adjusting the pH value to 6-8, mixing with anion exchange resin according to the material-liquid ratio of 1:3, carrying out adsorption reaction for 3-5h, and removing the supernatant;
(7) and (3) elution: washing resin with 0.9% NaCl solution, shaking for 30min, eluting with 4-7% NaCl solution, collecting clear liquid, repeating the operation for 3-5 times, collecting all eluates, mixing to obtain eluates;
(8) ultrafiltration and drying: and (3) carrying out ultrafiltration desalination on the eluent to obtain ultrafiltrate, and drying the ultrafiltrate to obtain the ovotransferrin.
The second purpose of the invention is to apply the method to the purification of lysozyme in egg white.
The third purpose of the invention is to apply the method to the purification of the ovotransferrin in egg white.
A fourth object of the invention is to apply the above method in the field of egg white processing.
Based on the application, the added value of the egg white is increased, and the utilization rate of the egg white is improved.
Has the advantages that:
the method can simultaneously realize the high-efficiency separation of lysozyme and ovotransferrin in the egg white, the yield reaches over 75 percent, the purity reaches over 95 percent, and the used method has the advantages of simple operation and low cost and is easy for industrialized application.
Drawings
FIG. 1 is a flow chart of a method for separating lysozyme and ovotransferrin.
Detailed Description
The method is based on the combination of metal chelating cationic resin and a crystallization method to separate a plurality of proteins in egg white, and the specific flow is shown in figure 1.
Wherein the pH range in the step (1) is 6.5-9.5 to realize the effective adsorption of lysozyme and ovotransferrin, the obtained supernatant A is rich in ovalbumin and ovomucin, and the precipitate B is rich in lysozyme and ovotransferrin.
The resin type selected in the step (1) is metal chelating cation resin, and is carboxyl weak cation exchange resin, wherein the carboxyl can realize the adsorption of the egg-to-iron through metal chelating action, and the lysozyme adsorption is realized through electrostatic interaction. Adding 0.05-0.4mM ferric chloride solution in the resin adsorption process, wherein the adsorption time is 3-5 hours, so as to realize the effective adsorption of lysozyme and ovotransferrin simultaneously.
And (3) eluting for 30min by using a NaCl solution with the concentration of 4-7% in the step (2), wherein the collected eluent A contains lysozyme and ovotransferrin.
The crystallization conditions in the step (2) are pH 9.5-10.8, protein concentration 2-5% and NaCl concentration 3% -9% to ensure effective crystallization and precipitation of lysozyme. The clear solution B contains ovotransferrin and part of uncrystallized lysozyme.
And (3) adsorbing the clear liquid B by using anion exchange resin under the condition of pH 6-8 for 3-5h to ensure the effectiveness of the ovotransferrin, removing the lysozyme which is not crystallized and adsorbed, and eluting the anion exchange resin under the same elution condition as the step (2), wherein the obtained eluent B is the ovotransferrin.
Protein purity calculation method: and quantitatively analyzing the lysozyme and the transferrin obtained by separation by SDS-PAGE, collecting each protein band by a gel imaging system, and calculating the proportion of the target protein in the total protein to obtain the protein purity.
The protein yield calculation method comprises the following steps: separated eggs are tested by adopting Kjeldahl methodCalculating the total content of white, calculating the content m of target protein according to purity, and calculating the total amount m of target protein in processed egg white according to the same method0According to the formula, the protein yield is m/m0100 calculate the yield of the target protein.
Example 1:
(1) egg liquid pretreatment: stirring the egg white liquid, and adjusting the pH value to 8;
(2) adsorption of metal chelating cation resin: mixing carboxyl weak cation exchange resin and the pretreated egg white according to a feed-liquid ratio of 1:5, adding ferric chloride to ensure that the molar concentration of the ferric chloride in the egg white is 0.2mmol/L, adsorbing for 5 hours, and removing a supernatant A after the adsorption is finished;
(3) cleaning: adding 0.9% NaCl solution into the resin after adsorption according to the material-liquid ratio of 1:3, shaking and mixing uniformly, removing supernatant, and repeatedly cleaning for 5 times;
(4) and (3) elution: adding a NaCl solution with the concentration of 5% into the washed resin according to the material-liquid ratio of 1:3, oscillating and eluting for 30min, collecting clear liquid, repeating the operation for 5 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent A;
(5) and (3) crystallization: adjusting the pH value of the eluent A to 10, adding NaCl to the salt concentration of 5%, concentrating the eluent to the protein concentration of 3% by adopting an ultrafiltration concentration technology, standing at 10 ℃ to form crystals, and respectively collecting the obtained lysozyme crystals and a supernatant B;
(6) adsorption on anion exchange resin: taking supernatant B, adjusting the pH value to 6, mixing with anion exchange resin according to the material-liquid ratio of 1:3, carrying out adsorption reaction for 4h, and removing the supernatant;
(7) and (3) elution: after the resin is washed by 0.9% NaCl solution, adopting 5% NaCl solution, shaking and eluting for 30min, collecting clear liquid, repeating the operation for 5 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent B;
(8) ultrafiltration and drying: and (4) carrying out ultrafiltration desalination on the eluent B to obtain ultrafiltrate, and drying the ultrafiltrate to obtain the ovotransferrin.
The purity and yield calculation results of the lysozyme and the ovotransferrin are shown in table 1:
TABLE 1 results of 2 proteins isolated in example 1
Figure BDA0002054477520000041
Example 2:
(1) egg liquid pretreatment: stirring the egg white liquid, and adjusting the pH value to 9.5;
(2) adsorption of metal chelating cation resin: mixing carboxyl weak cation exchange resin and the pretreated egg white according to a feed-liquid ratio of 1:5, adding ferric chloride to ensure that the molar concentration of the ferric chloride in the egg white is 0.1mmol/L, adsorbing for 5 hours, and removing a supernatant A after the adsorption is finished;
(3) cleaning: adding 0.9% NaCl solution into the resin after adsorption according to the material-liquid ratio of 1:3, shaking and mixing uniformly, removing supernatant, and repeatedly cleaning for 4 times;
(4) and (3) elution: adding a NaCl solution with the concentration of 7% into the washed resin according to the material-liquid ratio of 1:3, oscillating and eluting for 30min, collecting clear liquid, repeating the operation for 4 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent A;
(5) and (3) crystallization: adjusting the pH value of the eluent A to 10.5, adding NaCl to the salt concentration of 7%, concentrating the eluent by adopting an ultrafiltration concentration technology to the protein concentration of 4%, standing at 10 ℃ to form crystals, and respectively collecting the lysozyme crystals and the supernatant B;
(6) adsorption on anion exchange resin: taking supernatant B, adjusting the pH value to 7, mixing with anion exchange resin according to the material-liquid ratio of 1:3, carrying out adsorption reaction for 5 hours, and removing the supernatant;
(7) and (3) elution: after the resin is washed by 0.9% NaCl solution, adopting 7% NaCl solution, shaking and eluting for 30min, collecting clear liquid, repeating the operation for 5 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent B;
(8) ultrafiltration and drying: and (4) carrying out ultrafiltration desalination on the eluent B to obtain ultrafiltrate, and drying the ultrafiltrate to obtain the ovotransferrin.
The purity and yield calculation results of the lysozyme and the ovotransferrin are shown in Table 2:
table 2 example 2 results of isolating the 2 proteins obtained
Figure BDA0002054477520000051
Example 3:
(1) egg liquid pretreatment: stirring the egg white liquid, and adjusting the pH value to 6.5;
(2) adsorption of metal chelating cation resin: mixing carboxyl weak cation exchange resin and the pretreated egg white according to a feed-liquid ratio of 1:5, adding ferric chloride to ensure that the molar concentration of the ferric chloride in the egg white is 0.2mmol/L, adsorbing for 5 hours, and removing a supernatant A after the adsorption is finished;
(3) cleaning: adding 0.9% NaCl solution into the resin after adsorption according to the material-liquid ratio of 1:3, shaking and mixing uniformly, removing supernatant, and repeatedly cleaning for 5 times;
(4) and (3) elution: adding a NaCl solution with the concentration of 5% into the washed resin according to the material-liquid ratio of 1:3, oscillating and eluting for 30min, collecting clear liquid, repeating the operation for 5 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent A;
(5) and (3) crystallization: adjusting the pH value of the eluent A to 10, adding NaCl to the salt concentration of 5%, concentrating the eluent to the protein concentration of 3% by adopting an ultrafiltration concentration technology, standing at 10 ℃ to form crystals, and respectively collecting the obtained lysozyme crystals and a supernatant B;
(6) adsorption on anion exchange resin: taking supernatant B, adjusting the pH value to 6, mixing with anion exchange resin according to the material-liquid ratio of 1:3, carrying out adsorption reaction for 4h, and removing the supernatant;
(7) and (3) elution: after the resin is washed by 0.9% NaCl solution, adopting 5% NaCl solution, shaking and eluting for 30min, collecting clear liquid, repeating the operation for 5 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent B;
(8) ultrafiltration and drying: and (4) carrying out ultrafiltration desalination on the eluent B to obtain ultrafiltrate, and drying the ultrafiltrate to obtain the ovotransferrin.
The purity and yield calculation results of the lysozyme and the ovotransferrin are shown in Table 3:
TABLE 3 results of example 3 isolation of 2 proteins obtained
Figure BDA0002054477520000061
Example 4:
(1) egg liquid pretreatment: stirring the egg white liquid, and adjusting the pH value to 8;
(2) adsorption of metal chelating cation resin: mixing carboxyl weak cation exchange resin and the pretreated egg white according to a feed-liquid ratio of 1:5, adding ferric chloride to ensure that the molar concentration of the ferric chloride in the egg white is 0.05mmol/L, adsorbing for 5h, and removing a supernatant A after the adsorption is finished;
(3) cleaning: adding 0.9% NaCl solution into the resin after adsorption according to the material-liquid ratio of 1:3, shaking and mixing uniformly, removing supernatant, and repeatedly cleaning for 5 times;
(4) and (3) elution: adding a NaCl solution with the concentration of 5% into the washed resin according to the material-liquid ratio of 1:3, oscillating and eluting for 30min, collecting clear liquid, repeating the operation for 5 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent A;
(5) and (3) crystallization: adjusting the pH value of the eluent A to 10, adding NaCl to the salt concentration of 5%, concentrating the eluent to the protein concentration of 3% by adopting an ultrafiltration concentration technology, standing at 10 ℃ to form crystals, and respectively collecting the obtained lysozyme crystals and a supernatant B;
(6) adsorption on anion exchange resin: taking supernatant B, adjusting the pH value to 6, mixing with anion exchange resin according to the material-liquid ratio of 1:3, carrying out adsorption reaction for 4h, and removing the supernatant;
(7) and (3) elution: after the resin is washed by 0.9% NaCl solution, adopting 5% NaCl solution, shaking and eluting for 30min, collecting clear liquid, repeating the operation for 5 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent B;
(8) ultrafiltration and drying: and (4) carrying out ultrafiltration desalination on the eluent B to obtain ultrafiltrate, and drying the ultrafiltrate to obtain the ovotransferrin.
The purity and yield calculation results of the lysozyme and the ovotransferrin are shown in Table 4:
table 4 results of 2 proteins isolated in example 4
Figure BDA0002054477520000062
Example 5:
(1) egg liquid pretreatment: stirring the egg white liquid, and adjusting the pH value to 8;
(2) adsorption of metal chelating cation resin: mixing carboxyl weak cation exchange resin and the pretreated egg white according to a feed-liquid ratio of 1:5, adding ferric chloride to ensure that the molar concentration of the ferric chloride in the egg white is 0.4mmol/L, adsorbing for 5h, and removing a supernatant A after the adsorption is finished;
(3) cleaning: adding 0.9% NaCl solution into the resin after adsorption according to the material-liquid ratio of 1:3, shaking and mixing uniformly, removing supernatant, and repeatedly cleaning for 5 times;
(4) and (3) elution: adding a NaCl solution with the concentration of 5% into the washed resin according to the material-liquid ratio of 1:3, oscillating and eluting for 30min, collecting clear liquid, repeating the operation for 5 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent A;
(5) and (3) crystallization: adjusting the pH value of the eluent A to 10, adding NaCl to the salt concentration of 5%, concentrating the eluent to the protein concentration of 3% by adopting an ultrafiltration concentration technology, standing at 10 ℃ to form crystals, and respectively collecting the obtained lysozyme crystals and a supernatant B;
(6) adsorption on anion exchange resin: taking supernatant B, adjusting the pH value to 6, mixing with anion exchange resin according to the material-liquid ratio of 1:3, carrying out adsorption reaction for 4h, and removing the supernatant;
(7) and (3) elution: after the resin is washed by 0.9% NaCl solution, adopting 5% NaCl solution, shaking and eluting for 30min, collecting clear liquid, repeating the operation for 5 times, collecting all the eluates, merging and uniformly mixing to obtain an eluent B;
(8) ultrafiltration and drying: and (4) carrying out ultrafiltration desalination on the eluent B to obtain ultrafiltrate, and drying the ultrafiltrate to obtain the ovotransferrin.
The purity and yield calculation results of the lysozyme and the ovotransferrin obtained are shown in Table 5:
TABLE 5 results of 2 proteins isolated in example 5
Figure BDA0002054477520000071
Comparative example 1:
referring to example 1, the results of separating a plurality of proteins in the processed egg white were shown in Table 6, except that the molar concentration of ferric trichloride in step (2) was changed to 0.02mM and the conditions were not changed.
TABLE 6 results of 3 proteins isolated in comparative example 1
Figure BDA0002054477520000072
Comparative example 2:
referring to example 1, the pH in step (1) was changed to 5 and 10, respectively, and the other conditions were not changed, and a plurality of proteins in the processed egg white were separated, and the results are shown in table 7.
TABLE 7 results of 2 proteins isolated in comparative example 2
Figure BDA0002054477520000073
Figure BDA0002054477520000081

Claims (10)

1. A method for separating lysozyme and ovotransferrin from egg white is characterized by comprising the following steps:
(1) adjusting the pH value of the egg white to 6.5-9.5, and then adding cation exchange resin and a ferric iron source for adsorption separation; wherein the molar concentration of iron ions in the ferric iron source relative to the egg white liquid is 0.05-0.4 mmol/L;
(2) eluting the resin to obtain an eluent A; adjusting the pH value of the eluent A, concentrating and crystallizing to obtain lysozyme crystals and a clear liquid B;
(3) adjusting the pH value of the clear liquid B, purifying and concentrating to obtain ovotransferrin;
adjusting the pH of the eluent A in the step (2) to 9.5-10.8;
and (4) adjusting the pH value of the clear liquid B in the step (3) to 6-8.
2. The method according to claim 1, wherein the mass ratio of the resin to the egg white in the step (1) is 1: (2-5).
3. The method of claim 1, wherein the ferric iron source comprises one or more of ferric chloride, ferric sulfate, and ferric nitrate.
4. The method of claim 2, wherein the ferric iron source comprises one or more of ferric chloride, ferric sulfate, and ferric nitrate.
5. The method according to any one of claims 1 to 4, wherein the eluting resin is prepared by mixing the following components in a ratio of 1: and (2) eluting with NaCl aqueous solution with the mass concentration of 4-7%.
6. The method according to any one of claims 1 to 4, wherein the concentration and crystallization in step (2) are carried out by adding NaCl to an eluate A to a concentration of 3 to 9% by mass, concentrating, and then crystallizing at 5 to 10 ℃.
7. The method according to claim 5, wherein the concentration and crystallization in the step (2) are performed by adding NaCl to 3-9% by mass of the eluate A, concentrating the eluate, and then crystallizing the eluate at 5-10 ℃.
8. Use of the method of any one of claims 1 to 7 in the field of egg white processing.
9. Use of the method of any one of claims 1 to 7 for the purification of lysozyme from egg white.
10. Use of the method of any one of claims 1 to 7 for the purification of ovotransferrin in egg white.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN103160482A (en) * 2013-03-14 2013-06-19 江苏大学 Method for preparing egg white lysozyme and active protein by adopting coseparation
CN108118044A (en) * 2018-02-02 2018-06-05 华南理工大学 A kind of method of separating-purifying egg white lysozyme

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103160482A (en) * 2013-03-14 2013-06-19 江苏大学 Method for preparing egg white lysozyme and active protein by adopting coseparation
CN108118044A (en) * 2018-02-02 2018-06-05 华南理工大学 A kind of method of separating-purifying egg white lysozyme

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Separation of Ovotransferrin from Egg White by;A. Al-Mashdchi and Shuryo Nakai;《Agric. Biol. Chem.》;19871231;第2881-2887页 *
纤维素金属螯合亲和膜用于蛋清中;张亚辉等;《食品与生物技术学报》;20060331;摘要，参见第42页右栏第1段，第46页右栏倒数第1段 *
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