Method for rapidly and efficiently extracting ovotransferrin in egg white

Abstract

The invention discloses a method for rapidly and efficiently extracting ovotransferrin in egg white, which comprises the following steps: (1) pre-treating egg white, namely treating egg white liquid through an ultrasonic composite high-voltage pulse electric field, so that the thick protein is thinned, the viscosity is further reduced, molecules are dispersed, the egg white liquid is prevented from being diluted by adding water in the subsequent process, and the production efficiency is favorably improved; (2) removal of ovomucin and ovalbumin: removing the ovomucin and ovalbumin by an isoelectric precipitation method combined with a PEG precipitation method; (3) preparation of ovotransferrin; (4) separating an ovotransferrin crude product; and (5) purifying the ovotransferrin crude product. According to the method disclosed by the invention, high-yield and high-purity ovotransferrin is rapidly and efficiently prepared, and the method is simple, relatively low in cost and suitable for large-scale industrialization.

Description

Technical Field

[0001] The present invention relates to the field of protein separation and purification, and more particularly to a method for rapidly and efficiently extracting ovotransferrin. Background Art

[0002] Ovotransferrin in egg white is similar to lactoferrin in mammalian breast milk and plays an important role in enhancing immune function, resisting bacterial and viral infections, and maintaining normal growth and development of the body in vivo. Especially its high affinity for iron ions enables it to undertake the important task of transporting iron in vivo and plays a crucial role in iron absorption by mammals. Currently, there are various methods for separating and preparing ovotransferrin, but they all have some disadvantages. In the early stage, mainly salting-out method or organic solvent precipitation method was used, and the obtained ovotransferrin had low purity. Moreover, after being treated with high salt and organic solvents, the natural structure and activity of the protein have been damaged. Currently, mainly liquid chromatography method is used to obtain ovotransferrin, but the current chromatography products have low purity and yield. Generally, a method combining ion exchange chromatography with gel filtration chromatography or affinity chromatography is required. In the multi-step chromatography method, it is often necessary to change the buffer or concentrate the solution between each step of chromatography, and the operation is complex. The extraction process is time-consuming and can reach 3 - 5 days. In addition, the processing capacity of gel filtration chromatography is very small, and the cost of affinity chromatography packing is expensive. These problems seriously restrict the extraction of ovotransferrin.

[0003] Polyethylene glycol (PEG) is a linear macromolecular polymer. Because of its strong hydrophilicity, it can destroy the hydration layer of protein molecules to cause dehydration, and can also entangle protein molecules by steric exclusion and depending on the chain length to cause precipitation. The protein obtained by PEG precipitation method does not require subsequent desalting and is not easily denatured. Also, because of its simple operation, mild conditions, and easy scale-up production, it has attracted increasing attention. Therefore, in this paper, PEG method was used to precipitate ovotransferrin (OVT), and a simple OVT separation process was established.

[0004] CN118638213A discloses a method for extracting highly soluble ovotransferrin from egg white. The egg white is diluted with water, FeCl3 solution is added, the pH of the solution is adjusted with NaOH solution, and high-speed shearing is carried out; the pretreated egg white solution is subjected to ultrasonic and high-voltage alternating electric field treatment; anhydrous ethanol is added, the supernatant is taken by centrifugation and ultrafiltered, and water is added for concentration to obtain a crude ovotransferrin solution; resin is added to the crude ovotransferrin solution, the pH is adjusted with citric acid solution, the resin is filtered off to obtain a filtrate; PEG8000 is added, the pH is adjusted, the precipitate is taken by centrifugation, the precipitate is dissolved in water, and ultrafiltration is carried out; however, the steps are complex and the cost is high, which is not conducive to industrialization.

[0005] CN105949300A A method for protein extraction, separation and purification discloses a method for protein extraction, separation and purification, including a method for ovomucoid separation and purification, a method for chicken egg ovotransferrin separation and purification, and a method for chicken egg immunoglobulin extraction and purification; the present invention adopts a method of first diluting and then homogenizing. The egg white solution is homogenized after being diluted with physiological saline, which not only reduces the viscosity of the egg white solution, but also destroys its complex structure, so that the filtration speed of the egg white solution is faster, the membrane cleaning is easier, the degree of membrane pollution is reduced, and the service life of the membrane is extended. When the operating pressure is 0.15 MPa, it is in a relatively stable flow state, and to a certain extent, the concentration polarization phenomenon of the filtrate is reduced. The present invention is based on the water dilution method, and combined with polyethylene glycol precipitation and DEAE-Toyopearl 650M one-step elution ion exchange chromatography for purification to obtain chicken egg yolk IgY with high recovery rate and high purity.

[0006] CN103160482B A method for co-separating and preparing egg white lysozyme and active protein discloses a method with the following steps: collecting egg white at room temperature, adjusting the pH to about 5.5, stirring evenly and then centrifuging; adding polyethylene glycol with a molecular weight between 200 and 20,000 to the supernatant obtained after centrifugation, stirring evenly and then centrifuging; adding saturated ammonium sulfate solution to the supernatant obtained after centrifugation, mixing evenly and then centrifuging; separately collecting the upper supernatant and the lower supernatant obtained after centrifugation; ultrafiltration or dialysis treatment of the upper supernatant can obtain high-purity egg white lysozyme; ultrafiltration or dialysis treatment of the lower supernatant can obtain other active proteins with relatively high purity mainly composed of ovalbumin and ovotransferrin.

[0007] CN102604915B A method for co-extracting multiple proteins from egg white discloses that proteins are fractionated and precipitated by polyethylene glycol 8000, and then separated by Q Sepharose FF anion exchange chromatography, and ovomucoid, lysozyme, ovotransferrin, ovalbumin and ovoflavoprotein can be obtained at one time.

[0008] In order to synchronously extract multiple proteins by the above methods, the loss of ovotransferrin is too large, and the extraction effect and extraction purity of ovotransferrin are not high. Summary of the Invention

[0009] The purpose of the present invention is to provide a method for rapidly and efficiently preparing highly active ovotransferrin in view of the deficiencies of the prior art. The method includes the following steps:

[0010] (1) Pretreatment of egg white: Treat the egg white solution by ultrasonic combined with high-voltage pulsed electric field to thin the thick protein, further reduce the viscosity, disperse the molecules, avoid adding water to dilute the egg white solution in the subsequent process, and help improve production efficiency;

[0011] (2) Removal of ovomucin and ovalbumin: Remove ovomucin and ovalbumin by isoelectric point precipitation combined with PEG precipitation method. Adjust the pH value of the egg white obtained in step (1) to 3.0 - 5.5, add PEG 4000 - 10000 to 1 - 6%, stir at room temperature, perform centrifugation, and collect the egg white supernatant;

[0012] (3) Preparation of ovotransferrin: Prepare the crude ovotransferrin by isoelectric point precipitation combined with PEG precipitation method. Adjust the pH value of the egg white supernatant obtained in step (2) to 6.0 - 7.5, add PEG 4000 - 10000 to 9 - 18%, stir at room temperature, perform centrifugation, and collect the precipitate;

[0013] (4) Separation of crude ovotransferrin: Separate the impurity proteins in the crude ovotransferrin by PEG precipitation method. Dissolve the precipitate obtained in step (3) with Tris - HCl, add PEG 1000 - 6000 to 5 - 12%, stir at room temperature, then perform centrifugation, and collect the precipitate;

[0014] (5) Purification of crude ovotransferrin: After dissolving the precipitate obtained in step (4) with Tris - HCl, perform separation and purification by Q Sepharose FF anion - exchange chromatography to obtain high - purity ovotransferrin.

[0015] As a preferred embodiment of the present invention, in step (1), the conditions for ultrasonic combined with high - voltage pulsed electric field treatment are: the ultrasonic power is 200 - 300W, the conditions for the high - voltage pulsed electric field are the field strength of 15 - 25kV / cm, the number of pulses is 6 - 12, and the treatment time for ultrasonic combined with high - voltage pulsed electric field is 5 - 10min.

[0016] As a preferred embodiment of the present invention, in steps (2) and (3), use citric acid solution to adjust the pH to 3.0 - 5.5.

[0017] As a preferred embodiment of the present invention, in steps (2), (3), and (4), stir at room temperature for more than 0.2h, for example, 1h, without the need for low temperature and without diluting the egg white, which helps to improve production efficiency.

[0018] As a preferred embodiment of the present invention, in steps (2), (3), and (4), the conditions for centrifugation are centrifuging at a rotational speed of 7500 - 12000rpm for 15 - 45min.

[0019] As a preferred embodiment of the present invention, in steps (4) and (5), dissolve the crude ovotransferrin in Tris - HCl buffer solution with a pH of 7 - 9.

[0020] As a preferred embodiment of the present invention, in step (5), separation is carried out using a Q Sepharose FF chromatographic column, and elution is performed with a Tris-HCl buffer solution having a pH of 7 - 9 and containing 0, 0.06 - 0.12, 0.25 - 0.5, and 2 mol / L sodium chloride. The eluate with 0.06 - 0.12 mol / L sodium chloride is collected and freeze-dried to obtain high-purity ovotransferrin.

[0021] The beneficial technical effects of the present invention are as follows:

[0022] (1) The combined use of ultrasonic composite high-voltage pulsed electric field - isoelectric point precipitation method and composite PEG precipitation method can thin the thick protein in egg white, reduce viscosity and surface tension, and at the same time remove a large amount of ovomucin and ovalbumin, thus avoiding adding water to dilute the egg white during the whole operation process, which helps to improve production efficiency; the ovotransferrin of the present invention has a high yield, purity, iron ion binding and antioxidant activity, which can effectively promote the related research of ovotransferrin and its application in the food field. In particular, the ultrasonic composite high-voltage pulsed electric field can fully unfold proteins, which helps ovotransferrin to bind Fe 3+ ; and reduces the binding ability of proteins to water molecules, which helps to use PEG10000 with high biocompatibility to reduce the solubility of ovotransferrin. The treatment effect of the ultrasonic composite high-voltage pulsed electric field is better than that of ultrasonic and high-voltage alternating electric field treatment, and is more suitable for cooperating with the isoelectric point precipitation method and composite PEG precipitation method to obtain high-purity and high-yield ovotransferrin.

[0023] (2) Using the isoelectric point precipitation method and composite PEG precipitation method to first remove ovomucin and ovalbumin, and then continuing to use the isoelectric point precipitation method and composite PEG precipitation method to prepare a crude ovotransferrin product by impurity removal with the PEG precipitation method. The method is simple and the cost is low, which is very suitable for industrialization and is easy for large-scale industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the electrophoresis pattern of the ovotransferrin prepared in Example 1 of the present invention.

[0025] Figure 2 It is the iron ion binding activity of the ovotransferrin prepared in Example 1 of the present invention.

[0026] Figure 3 It is the DPPH free radical scavenging ability of the ovotransferrin prepared in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0027] For the convenience of those skilled in the art to understand, the present invention will be further described below in conjunction with examples. The content mentioned in the embodiments does not limit the present invention.

[0029] Example 1

[0030] (1) Pretreatment of egg white: The egg white liquid is treated by ultrasonic combined with high - voltage pulsed electric field to thin the thick protein, further reduce the viscosity, and disperse the molecules, avoiding the addition of water to dilute the egg white liquid in the subsequent process, which helps to improve production efficiency. The treatment conditions of ultrasonic combined with high - voltage pulsed electric field are: the ultrasonic power is 250W, the conditions of high - voltage pulsed electric field are field strength 20kV / cm, pulse number 8, and the treatment time of ultrasonic combined with high - voltage pulsed electric field is 8min.

[0031] (2) Removal of ovomucin and ovalbumin: Ovomucin and ovalbumin are removed by isoelectric point precipitation combined with PEG precipitation method. The pH value of the egg white obtained in step (1) is adjusted to 5, 4% of PEG 10000 is added, and after stirring at room temperature for 1h, centrifugation is carried out at 8000rpm for 40min, and the egg white supernatant is collected.

[0032] (3) Preparation of ovotransferrin: Crude ovotransferrin is prepared by isoelectric point precipitation combined with PEG precipitation method. The pH value of the egg white supernatant obtained in step (2) is adjusted to 6.5, 15% of PEG 10000 is added, and after stirring at room temperature for 1h, centrifugation is carried out at 8000rpm for 20min, and the precipitate is collected.

[0033] (4) Separation of crude ovotransferrin: The impurity proteins in the crude ovotransferrin are separated by PEG precipitation method. The precipitate obtained in step (3) is dissolved with Tris - HCl with pH 8.5, 10% of PEG 6000 is added, and after stirring at room temperature for 1h, centrifugation is carried out at 8000rpm for 20min, and the precipitate is collected.

[0034] (5) Purification of crude ovotransferrin: After dissolving the precipitate obtained in step (4) with Tris - HCl with pH 8.5, separation and purification, sterilization, drying, etc. are carried out by Q Sepharose FF anion - exchange chromatography to obtain high - purity ovotransferrin. It is separated by Q Sepharose FF chromatographic column, eluted with Tris - HCl buffer solution with pH 7 - 9 containing 0, 0.08, 0.35 and 2mol / L sodium chloride, the eluate with 0.08mol / L sodium chloride is collected, and freeze - dried to obtain high - purity ovotransferrin.

[0035] Example 2

[0036] Based on Example 1, the difference is (1) Ultrasonic treatment: The ultrasonic power is 250W, and the ultrasonic treatment time is 10min; others are the same as Example 1.

[0037] Example 3

[0038] On the basis of Example 1, the differences are as follows: (1) High-voltage pulse treatment: The conditions of the high-voltage pulsed electric field are an electric field strength of 20 kV / cm, 8 pulses, and the treatment time of the ultrasonic combined high-voltage pulsed electric field is 8 min; other conditions are the same as those in Example 1.

[0039] Example 4

[0040] On the basis of Example 1, the differences are as follows: (2) Removal of impurity proteins by isoelectric point precipitation combined with PEG precipitation: The pH of the egg white is adjusted to 3, 4% of PEG 10000 is added, and after stirring at room temperature for 1 h, centrifugation is carried out at a speed of 8000 rpm for 40 min; other conditions are the same as those in Example 1.

[0041] Example 5

[0042] On the basis of Example 1, the differences are as follows: (2) Removal of impurity proteins by isoelectric point precipitation combined with PEG precipitation: The pH of the egg white is adjusted to 4, 4% of PEG 10000 is added, and after stirring at room temperature for 1 h, centrifugation is carried out at a speed of 8000 rpm for 40 min; other conditions are the same as those in Example 1.

[0043] Example 6

[0044] On the basis of Example 1, the differences are as follows: (2) Removal of impurity proteins by isoelectric point method: The pH of the egg white is adjusted to 3.0, stirred for 1 h, and centrifuged at 8000 rpm for 40 min; other conditions are the same as those in Example 1.

[0045] Example 7

[0046] On the basis of Example 1, the differences are as follows: (2) Removal of impurity proteins by isoelectric point method: The pH of the egg white is adjusted to 4.0, stirred for 1 h, and centrifuged at 8000 rpm for 40 min; other conditions are the same as those in Example 1.

[0047] Example 8

[0048] On the basis of Example 1, the differences are as follows: (2) Removal of impurity proteins by isoelectric point method: The pH of the egg white is adjusted to 5.0, stirred for 1 h, and centrifuged at 8000 rpm for 40 min; other conditions are the same as those in Example 1.

[0049] Example 9

[0050] On the basis of Example 1, the differences are as follows: (2) Removal of impurity proteins by PEG precipitation method: 2% of PEG 10000 is added, stirred for 1 h, and centrifuged at 8000 rpm for 40 min; other conditions are the same as those in Example 1.

[0051] Example 10

[0052] On the basis of Example 1, the difference lies in (2) removing impurity proteins by PEG precipitation method: adding PEG 10000 to 4%, stirring for 1 h, and centrifuging at 8000 rpm for 40 min; other steps are the same as those in Example 1

[0053] Example 11

[0054] On the basis of Example 1, the difference lies in (2) removing impurity proteins by PEG precipitation method: adding PEG 10000 to 6%, stirring for 1 h, and centrifuging at 8000 rpm for 40 min; other steps are the same as those in Example 1

[0055] Example 12

[0056] On the basis of Example 1, the difference lies in (2) removing impurity proteins by PEG precipitation method: adding PEG 6000 to 4%, stirring for 1 h, and centrifuging at 8000 rpm for 40 min; other steps are the same as those in Example 1.

[0057] Example 13

[0058] On the basis of Example 1, the difference lies in (2) removing impurity proteins by PEG precipitation method: adding PEG 8000 to 4%, stirring for 1 h, and centrifuging at 8000 rpm for 40 min; other steps are the same as those in Example 1

[0059] Example 14

[0060] On the basis of Example 1, the difference lies in (3) extracting ovotransferrin by isoelectric point method combined with PEG precipitation method: adjusting the pH of the supernatant to 6.5, adding PEG 10000 to 12%, stirring for 1 h, and centrifuging at 8000 rpm for 20 min; other steps are the same as those in Example 1.

[0061] Example 15

[0062] On the basis of Example 1, the difference lies in (3) extracting ovotransferrin by isoelectric point method combined with PEG precipitation method: adjusting the pH of the supernatant to 6.5, adding PEG 10000 to 18%, stirring for 1 h, and centrifuging at 8000 rpm for 20 min; other steps are the same as those in Example 1.

[0063] Example 16

[0064] On the basis of Example 1, the difference lies in (3) extracting ovotransferrin by isoelectric point method combined with PEG precipitation method: adjusting the pH of the supernatant to 6.5, adding PEG 6000 to 15%, stirring for 1 h, and centrifuging at 8000 rpm for 20 min; other steps are the same as those in Example 1.

[0065] Example 17

[0066] On the basis of Example 1, the difference lies in (3) extracting ovotransferrin by the isoelectric point method combined with the PEG precipitation method: adjusting the pH of the supernatant to 6.5, adding PEG 8000 to 15%, stirring for 1 h, and centrifuging at 8000 rpm for 20 min; other steps are the same as those in Example 1.

[0067] Example 18

[0068] On the basis of Example 1, the difference lies in (4) removing impurity proteins by the isoelectric point method combined with the PEG precipitation method: dissolving the precipitate with Tris-HCl, adding PEG 6000 to 8%, stirring for 1 h, and centrifuging at 8000 rpm for 20 min; other steps are the same as those in Example 1.

[0069] Example 19

[0070] On the basis of Example 1, the difference lies in (4) removing impurity proteins by the isoelectric point method combined with the PEG precipitation method: dissolving the precipitate with Tris-HCl, adding PEG 6000 to 12%, stirring for 1 h, and centrifuging at 8000 rpm for 20 min; other steps are the same as those in Example 1.

[0071] Example 20

[0072] On the basis of Example 1, the difference lies in (4) removing impurity proteins by the isoelectric point method combined with the PEG precipitation method: dissolving the precipitate with Tris-HCl, adding PEG 4000 to 10%, stirring for 1 h, and centrifuging at 8000 rpm for 20 min; other steps are the same as those in Example 1.

[0073] Example 21

[0074] On the basis of Example 1, the difference lies in (4) removing impurity proteins by the isoelectric point method combined with the PEG precipitation method: dissolving the precipitate with Tris-HCl, adding PEG 8000 to 10%, stirring for 1 h, and centrifuging at 8000 rpm for 20 min; other steps are the same as those in Example 1.

[0075] Comparative Example 1

[0076] (1) Removing ovomucin and ovalbumin: Removing ovomucin and ovalbumin by the isoelectric point precipitation method combined with the PEG precipitation method. Dilute the egg white with 1 volume of water and adjust its pH value to 5, add PEG 10000 to 4%, stir at room temperature for 1 h, and then centrifuge at a speed of 8000 rpm for 40 min to collect the egg white supernatant.

[0077] (2) Preparation of ovotransferrin: Preparing the crude ovotransferrin by the isoelectric point precipitation method combined with the PEG precipitation method. Adjust the pH value of the egg white supernatant obtained in step (1) to 6.5, add PEG 10000 to 15%, stir at room temperature for 1 h, and then centrifuge at a speed of 8000 rpm for 20 min to collect the precipitate.

[0078] (3) Isolation of crude ovotransferrin: The impurity proteins in the crude ovotransferrin were separated by PEG precipitation method. The precipitate obtained in step (2) was dissolved with Tris-HCl at pH 8.5. PEG 6000 was added to 10%, and after stirring at room temperature for 1 h, centrifugation was carried out at 8000 rpm for 20 min, and the precipitate was collected.

[0079] (4) Purification of crude ovotransferrin: After dissolving the precipitate obtained in step (3) with Tris-HCl at pH 8.5, separation, purification, sterilization, drying, etc. were carried out by Q Sepharose FF anion exchange chromatography to obtain high-purity ovotransferrin. It was separated with a Q Sepharose FF chromatographic column and eluted with Tris-HCl buffer solution at pH 7-9 containing 0, 0.08, 0.35 and 2 mol / L sodium chloride. The eluate of 0.08 mol / L sodium chloride was collected and freeze-dried to obtain high-purity ovotransferrin.

[0080] Comparative Example 2

[0081] Based on Comparative Example 1, the difference lies in (1) removing impurity proteins by isoelectric point method: the pH of egg white was adjusted to 5.0, stirred for 1 h, and centrifuged at 8000 rpm for 40 min; others were the same as in Example 1.

[0082] Comparative Example 3

[0083] Based on Comparative Example 1, the difference lies in (1) removing impurity proteins by PEG precipitation method: adding PEG 10000 to 4%, stirring for 1 h, and centrifuging at 8000 rpm for 40 min; others were the same as in Example 1.

[0084] Comparative Example 4

[0085] Based on Example 1, ultrasonic and high-voltage alternating electric field treatments were adopted. The conditions of ultrasonic and high-voltage alternating electric field treatments were: ultrasonic power was 250 W, field strength was 20 kV / cm, frequency was 55 Hz, and the electric field treatment time was 8 min.

[0086] Table 1 Purity and yield of ovotransferrin

[0087]

[0088]

[0089] It can be seen from the experimental data that the combined use of ultrasonic combined with high-voltage pulsed electric field-isoelectric point precipitation method and PEG precipitation method first removes ovomucoid and ovalbumin by the isoelectric point precipitation method combined with PEG precipitation method, and then continues to use the isoelectric point precipitation method combined with PEG precipitation method and PEG precipitation method to remove impurities to prepare the crude ovotransferrin. The method is simple, the cost is low, and the purity and yield of ovotransferrin are both high.

[0090] Those skilled in the art of the present technology can understand that unless otherwise defined, all terms (including technical terms and scientific terms) used herein have the same meaning as the general understanding of those of ordinary skill in the art to which the present invention belongs. It should also be understood that terms such as those defined in a general dictionary should be understood to have a meaning consistent with the meaning in the context of the prior art, and will not be interpreted in an idealized or overly formal sense unless specifically defined as here.

[0091] It should be understood that the detailed description of the technical solutions of the present invention with the aid of the preferred embodiments above is illustrative rather than restrictive. Those of ordinary skill in the art can modify the technical solutions recorded in each embodiment on the basis of reading the specification of the present invention, or perform equivalent substitution on some of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of each embodiment of the present invention.

Claims

1. A method for rapidly and efficiently extracting ovotransferrin from egg white, characterized in that: The method comprises the following steps: (1) Pretreatment of egg white: treating the egg white liquid by ultrasonic composite high-voltage pulse electric field; (2) Removing ovomucin and ovalbumin: adjusting the pH value of the egg white obtained in step (1) to 3.0-5.5, adding PEG4000-10000 to 1-6%, stirring, centrifuging, and collecting the egg white supernatant; (3) Preparation of ovotransferrin: adjusting the pH value of the egg white supernatant obtained in step (2) to 6.0-7.5, adding PEG4000-10000 to 9-18%, stirring, centrifuging, and collecting the precipitate; (4) Isolation of crude ovotransferrin: Dissolve the precipitate obtained in step (3) with Tris-HCl, add PEG 1000-6000 to 5-12%, stir, centrifuge and collect the precipitate; (5) Purification of crude ovotransferrin: After dissolving the precipitate obtained in step (4) with Tris-HCl, it is separated and purified by anion exchange chromatography to obtain high-purity ovotransferrin.

2. The method according to claim 1, characterized in that In step (1), the ultrasonic composite high-voltage pulse electric field treatment conditions are: ultrasonic power is 200-300W, high-voltage pulse electric field conditions are field strength 15-25kV / cm, pulse number 6-12, and ultrasonic composite high-voltage pulse electric field treatment time is 5-10min.

3. The method according to claim 1, characterized in that In steps (2) and (3), the pH is adjusted to 3.0-5.5 using citric acid solution.

4. The method according to claim 1, characterized in that In steps (2), (3) and (4), the mixture is stirred at room temperature.

5. The method according to claim 1, characterized in that In steps (2), (3) and (4), the centrifugal treatment is carried out at a rotation speed of 7500-12000 rpm for 15-45 minutes.

6. The method according to claim 1, characterized in that In steps (4) and (5), the crude ovotransferrin is dissolved in a Tris-HCl buffer having a pH of 7-9.

7. The method according to claim 1, characterized in that Step (5), separating with a QSepharose FF column, eluting with a Tris-HCl buffer having a pH of 7-9 and containing 0, 0.06-0.12, 0.25-0.5 and 2 mol / L sodium chloride, collecting the eluate containing 0.06-0.12 mol / L sodium chloride, and freeze-drying to obtain high-purity ovotransferrin.

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