A method for extracting high solubility ovotransferrin from chicken egg white

Abstract

The present application relates to the technical field of biological medicine manufacturing, and particularly relates to a method for extracting high-solubility ovotransferrin from egg white, wherein the egg white is diluted with water, FeCl3 solution is added, NaOH solution is used to adjust the pH of the solution, and high-speed shearing is performed; the pretreated egg white solution is subjected to ultrasonic and high-voltage alternating electric field treatment; anhydrous ethanol is added, the supernatant is obtained by centrifugation, and ultrafiltration is performed; water is added for concentration, and a crude ovotransferrin solution is obtained; resin is added to the crude ovotransferrin solution, citric acid solution is used to adjust the pH, and the resin is removed by filtration, so that a filtrate is obtained; PEG8000 is added, the pH is adjusted, and the precipitate is obtained by centrifugation; the precipitate is dissolved in water, and ultrafiltration is performed; water is continuously added for concentration, so that concentrated ovotransferrin is obtained. The extracted ovotransferrin has high solubility and iron binding capacity, can bind with iron elements again to prepare ovotransferrin iron preparation for improving iron deficiency anemia, the whole preparation process is green and environmentally friendly, has low cost, and can be mass-produced in an industrialized manner.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine manufacturing, and particularly relates to a method for extracting high-solubility ovotransferrin from egg white. BACKGROUND

[0002] Iron deficiency anemia (IDA) is a global public health problem, which has attracted high attention from governments around the world. Iron fortification and supplementation is an effective means to treat IDA, but most iron supplements have side effects such as causing intestinal oxidative stress and inflammation, so developing iron supplements with less side effects and higher safety has become a big problem to be solved. Ovotransferrin (ovotransferrin) is also known as conalbumin, which is an iron-binding glycoprotein from egg white. As a member of the transferrin family, ovotransferrin has a strong iron-binding capacity and has the potential to be an ideal candidate for a natural, safe and non-toxic iron supplement.

[0003] Ovotransferrin can be separated from egg white by water phase and ethanol fractionation program, ammonium sulfate fractionation precipitation. However, these technical methods have defects. Firstly, the complex of ovotransferrin, ovalbumin, lysozyme and other proteins is formed, and ovotransferrin is not fully exposed in the system, thereby hindering the separation process of ovotransferrin and reducing the yield and purity of ovotransferrin. Therefore, researchers try to use physical fields such as ultrasonic waves to destroy the protein complex, but due to the special composition of egg white system, the ovomucin is highly glycosylated, which can provide a stable skeleton for the protein complex, making the structure of the protein complex stable and difficult to be dissociated. Secondly, the extraction process involves high-concentration ethanol and extremely acidic environment, and the extracted ovotransferrin is denatured to varying degrees, affecting the solubility and iron-binding capacity of ovotransferrin. In order to overcome these shortcomings, researchers have developed ion exchange chromatography and fixed metal affinity chromatography methods on a laboratory scale, but these technologies are difficult to apply on a pilot scale due to low production efficiency and high cost. Therefore, developing a method for simply, quickly and economically extracting ovotransferrin from egg white is conducive to the development of the activity of ovotransferrin and the development of related research and industry. SUMMARY

[0004] In view of the problems existing in the prior art, the present application provides a method for extracting high-solubility ovotransferrin from egg white, which mainly utilizes the cavitation effect, molecular polarization effect and molecular reciprocating motion of ultrasonic complex high-voltage alternating electric field to destroy the protein complex, fully expose the ovotransferrin, and promote the structure stretching of the ovotransferrin, which is helpful for the ovotransferrin to bind Fe 3+, and then ethanol is used to remove proteins such as ovalbumin, ovomucin and lysozyme in egg white, and then ethanol is removed by ultrafiltration and the protein solution is concentrated. Then, the pH of the system is adjusted to be acidic by using citric acid, so that the ovotransferrin releases Fe 3+ , and AG1-X2 resin is added to remove free Fe 3+ . In addition, the cavitation effect, molecular polarization effect and molecular reciprocating motion generated by ultrasonic complex high-voltage alternating electric field promote the stretching of protein structure, the exposure of internal hydrophobic groups, and the reduction of the water molecule binding capacity, which helps to reduce the solubility of ovotransferrin by using high biocompatibility PEG8000, thereby obtaining ovotransferrin. The ovotransferrin obtained by the extraction method has high solubility and iron binding capacity, and can be used to prepare ovotransferrin iron preparation for improving IDA by recombining iron elements. The whole preparation process is green, low in cost and can be mass-produced in industry.

[0005] The technical scheme adopted by the present application to solve the above technical problems is:

[0006] A method for extracting high-solubility ovotransferrin from egg white, the preparation method comprising the following steps:

[0007] (1) Pretreatment of egg white: dilute egg white with water and stir uniformly;

[0008] (2) Add FeCl3 solution to the diluted egg white solution, adjust the pH of the solution with NaOH solution, and disperse FeCl3 in the solution by high-speed shearing to obtain a pretreated egg white solution;

[0009] (3) Ultrasonic complex high-voltage alternating electric field treatment: the pretreated egg white solution in step (2) is treated by ultrasonic and high-voltage alternating electric field;

[0010] (4) Alcohol extraction: add anhydrous ethanol to the egg white solution treated in step (3) to a final ethanol concentration of 40% to 45%, centrifuge to obtain supernatant, and filter to remove suspended impurities;

[0011] (5) First ultrafiltration: the supernatant is subjected to two times of ultrafiltration, and water is added for concentration to remove ethanol, Fe 3+ not chelated with ovotransferrin, and part of the impure proteins with a molecular weight less than that of ovotransferrin, to obtain a crude ovotransferrin solution;

[0012] (6) Ovotransferrin Fe 3+ removal: add resin to the crude ovotransferrin solution, adjust the pH with citric acid solution to promote the release of Fe 3+ from ovotransferrin, and fully stir until the free Fe 3+ is completely adsorbed, then filter to remove the resin to obtain a filtrate;

[0013] (7) Separation of ovotransferrin: add PEG8000 to the filtrate in step (6) to a final concentration of 10% to 20%, adjust the pH with NaOH solution, and centrifuge to obtain the precipitate;

[0014] (8) Secondary ultrafiltration: dissolve the precipitate in step (7) with water, perform two times of ultrafiltration, and add water to concentrate, thereby removing PEG8000 and obtaining concentrated ovotransferrin.

[0015] (9) Vacuum freeze-drying: freeze-dry the concentrated ovotransferrin solution to obtain the ovotransferrin product.

[0016] As a preferred scheme of the present application, in step (1), the mass ratio of egg white to water is 1: (0.8-1.2), the stirring speed is 500-600 r / min, and the stirring time is 10-15 min.

[0017] As a preferred scheme of the present application, in step (2), the concentration of the FeCl3 solution is 0.4-0.6 mol / L, the addition amount of the FeCl3 solution is 0.8-1.2 mL / L of the egg white solution, the pH of the solution is adjusted to 8-9 with NaOH solution, the concentration of the NaOH solution is 0.3-0.8 mol / L, and the high-speed shearing condition is 11000-12000 r / min for 2.5-3.5 min; preferably, the pH of the solution is adjusted to 8.5 with NaOH solution.

[0018] As a preferred scheme of the present application, in step (3), the ultrasonic compound high-voltage alternating electric field treatment condition is: ultrasonic power is 200-300 W, high-voltage alternating electric field condition is field strength 15-25 kV / cm, frequency is 50-60 Hz, and ultrasonic compound high-voltage alternating electric field treatment time is 5-10 min.

[0019] As a preferred scheme of the present application, in step (4), the filter cloth for filtration is 70-80 mesh nylon filter cloth.

[0020] As a preferred scheme of the present application, in step (5), the ultrafiltration is performed by diluting the supernatant in step (4) with water, using an ultrafiltration membrane with a molecular weight cut-off of 40-60 kDa, and adding water to completely backflow the residual proteins between the collection pipeline and the ultrafiltration membrane after the ultrafiltration; preferably, the ultrafiltration membrane has a molecular weight cut-off of 50 kDa or 60 kDa.

[0021] As a preferred scheme of the present application, in step (6), the pH is adjusted to 2.5-2.7 with citric acid solution.

[0022] As a preferred scheme of the present application, in step (6), the resin is added in an amount of 0.8-1.5 g / 100 mL egg white solution, the resin is a strong base anion exchange resin, the concentration of the citric acid solution is 0.4-0.6 mol / L, the stirring condition is 500-600 r / min for 1.5-2.5 h, and the filter cloth with a mesh size of 70-90 is used for filtration; preferably, the resin type is AG1-X2 resin.

[0023] As a preferred scheme of the present application, in step (7), the pH is adjusted to 4.5-6 using a NaOH solution, the solubility of the NaOH solution is 0.5-0.6 mol / L, and the centrifugation condition is 9000 r / min for 10 min; preferably, the pH is adjusted to 5.5 using the NaOH solution.

[0024] As a preferred scheme of the present application, in step (8), after the precipitate in step (7) is redissolved with water, the ultrafiltration is performed using an ultrafiltration membrane with a molecular weight of 45-60 kDa, and after the ultrafiltration is completed, water is added to completely backflow the residual protein between the collection pipeline and the ultrafiltration membrane.

[0025] As a preferred scheme of the present application, in step (9), the freeze-drying condition is a temperature of -50 to -40 ℃, a vacuum degree of 0.1-0.2 Pa, and a time of 32-38 h.

[0026] As a preferred scheme of the present application, the method further comprises the following steps:

[0027] (10) Recovery of ethanol: the liquid with a molecular weight less than that of the ultrafiltration membrane in step (4) is subjected to suspension evaporation using a rotary evaporator, and the parameter of the rotary evaporator is set as a pressure of 1 MPa and a temperature of 40-45 ℃;

[0028] (11) AG1-X2 resin regeneration: 1.0 mol / L NaOH solution washing.

[0029] (12) Recovery of PEG8000: the liquid with a molecular weight less than that of the ultrafiltration membrane in step (7) is placed into a freeze-drying box and then into a freezer at -20 ℃ to be pre-frozen, and then placed into a freeze dryer to be freeze-dried, and the freeze-drying condition is a temperature of -50 ℃, a vacuum degree of 0.1 Pa, and a time of 36 h.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] 1. The present application overcomes the incomplete unfolding of protein complex and ovotransferrin structure to cause Fe 3+The low chelation efficiency problem is enhanced by the environmental tolerance of ovotransferrin, and the subsequent removal of ovalbumin, ovomucin and lysozyme and other protein impurities, thereby improving the yield and purity of ovotransferrin.

[0032] 2、The present application uses the cavitation effect, molecular polarization effect and molecular reciprocating motion generated by ultrasonic composite high-voltage alternating electric field to promote the protein structure stretching, expose the internal hydrophobic group, and reduce the protein water molecule binding capacity, and then uses PEG8000 with good biocompatibility to extract ovotransferrin, thereby avoiding the adverse effects of strong acid, strong base, high-concentration ethanol or salt ions on the structure of ovotransferrin, effectively improving the solubility of ovotransferrin, and being beneficial to the development of the activity of ovotransferrin, and promoting the development of related research and industry.

[0033] 3、The anhydrous ethanol, AG1-X2 resin and PEG8000 used in the present application can be recycled and reused through rotary evaporation, regeneration and vacuum freeze drying, respectively, and are green and environmentally friendly.

[0034] 4、The present application has simple process, and can mass-produce ovotransferrin with high purity, high yield and good solubility, and has short production cycle, and the extraction process can be completed in 1-2 days. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Effect of different treatment methods on particle size distribution of egg white;

[0036] Figure 2 Effect of different treatment methods on endogenous fluorescence spectrum of egg white;

[0037] Figure 3 Effect of different treatment methods on surface hydrophobicity of egg white;

[0038] Figure 4 Standard curve of iron;

[0039] Figure 5 Effect of different treatment methods on free Fe 3+ concentration in egg white diluent;

[0040] Figure 6 SDS-PAGE electrophoretogram of impurity protein removed by different concentrations of ethanol;

[0041] Figure 7 Residual Fe 3+ in solution after deferrization at different pH values;

[0042] Figure 8 SDS-PAGE electrophoretogram of examples 1-5;

[0043] Figure 9SDS-PAGE electrophoretogram of Example 1, Comparative Examples 1-3;

[0044] Figure 10 Solubility of ovotransferrin;

[0045] Figure 11 Iron content of ovotransferrin;

[0046] Figure 12 Iron binding capacity of ovotransferrin;

[0047] Figure 13 FTIR spectrum of ovotransferrin;

[0048] Figure 14 Second derivative of FTIR spectrum of ovotransferrin;

[0049] Figure 15 Oxidative stress of mouse colon tissue;

[0050] Figure 16 Expression of inflammatory factors of mouse colon tissue. DETAILED DESCRIPTION

[0051] In order to better understand the present application, the following further illustrates the content of the present application in combination with examples and drawings, but the present application is to enable those skilled in the art to fully understand the technical solutions and beneficial effects of the present application, the following further illustrates in combination with specific examples, the examples are only simple examples of the present application, and do not represent or limit the protection scope of the present application, the protection scope of the present application is subject to the claims.

[0052] The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not marked with the manufacturer, which are all conventional products that can be purchased on the market.

[0053] Example 1

[0054] 1) Pretreatment of chicken egg white: fresh chicken egg was washed clean, and egg white was separated out, diluted with 1 volume of water, and stirred uniformly by using an electric mixer;

[0055] 2) Chelation by adding FeCl3: 1.0 mL / L of FeCl3 solution (0.5 mol / L) was added, NaOH solution was used to adjust the pH of the solution to 8.5, and a high-speed shearing disperser was used to treat at 12000 r / min for 3 min to make FeCl3 fully dispersed in the solution;

[0056] 3) Ultrasonic composite high-voltage alternating electric field treatment: the ultrasonic power was 250 W, the high-voltage alternating electric field conditions were field strength 20 kV / cm and frequency 55 Hz, and the ultrasonic composite high-voltage alternating electric field treatment time was 8 min.

[0057] 4) Add ethanol: add anhydrous ethanol slowly to the final concentration of 45%, then centrifuge at 9000 r / min for 10 min to take the supernatant, and filter with 80 mesh nylon filter cloth to remove suspended impurities;

[0058] 5) Primary ultrafiltration: the supernatant in 4) is subjected to two times of ultrafiltration using a 50 kDa ultrafiltration membrane, and water is added for concentration to remove ethanol, excess Fe 3+ and partial impurities with molecular weight less than that of ovotransferrin;

[0059] 6) Ovotransferrin removes Fe 3+ : take the solution with molecular weight greater than the ultrafiltration membrane in 5), dilute with water, add appropriate AG1-X2 resin to the solution, adjust pH to 2.5 with citric acid solution, and filter to remove AG1-X2 resin after sufficient stirring until free Fe 3+ is completely adsorbed;

[0060] 7) Isolation of ovotransferrin: add PEG8000 to the solution to a final concentration of 15%, adjust pH to 5.5 with NaOH solution, and centrifuge to take the precipitate;

[0061] 8) Secondary ultrafiltration: dissolve the precipitate in 7) with 10 volumes of water, and perform two times of ultrafiltration with water added for concentration to remove PEG8000 and partial impurities with molecular weight less than that of ovotransferrin, thereby obtaining ovotransferrin.

[0062] 9) Vacuum freeze-drying: the concentrated ovotransferrin solution is loaded into a freeze-drying box and placed in a -20 ℃ refrigerator for pre-freezing, and then placed in a freeze dryer for freeze-drying, with the freeze-drying conditions being temperature -50 ℃, vacuum degree 0.1 Pa, and time 36 h.

[0063] Example 2

[0064] Based on Example 1, the difference is that 3) ultrasonic treatment: the ultrasonic power is 250 W, and the ultrasonic treatment time is 8 min.

[0065] Example 3

[0066] Based on Example 1, the difference is that 3) high-voltage alternating electric field treatment: the high-voltage alternating electric field conditions are field strength 20 kV / cm and frequency 55 Hz, and the high-voltage alternating electric field treatment time is 8 min; the others are the same as Example 1.

[0067] Example 4

[0068] Based on the embodiment 1, the difference is that 3) high-voltage pulse electric field treatment: the condition of high-voltage pulse electric field is field strength 20 kV / cm, pulse number 8, and ultrasonic composite high-voltage pulse electric field treatment time is 8 min; the others are the same as embodiment 1.

[0069] Example 5

[0070] Based on the embodiment 1, the difference is that not through step (3) ultrasonic composite high-voltage alternating electric field treatment, the others are the same as embodiment 1.

[0071] Example 6

[0072] Based on the embodiment 1, the difference is that 4) adding ethanol: after adding anhydrous ethanol slowly to the solution until the final concentration of ethanol is 40%, centrifuging to take supernatant, the others are the same as embodiment 1.

[0073] Example 7

[0074] Based on the embodiment 1, the difference is that 4) adding ethanol: after adding anhydrous ethanol slowly to the solution until the final concentration of ethanol is 50%, centrifuging to take supernatant, the others are the same as embodiment 1.

[0075] Example 8

[0076] Based on the embodiment 1, the difference is that 4) adding ethanol: after adding anhydrous ethanol slowly to the solution until the final concentration of ethanol is 55%, centrifuging to take supernatant, the others are the same as embodiment 1.

[0077] Example 9

[0078] Based on the embodiment 1, the difference is that 4) adding ethanol: after adding anhydrous ethanol slowly to the solution until the final concentration of ethanol is 60%, centrifuging to take supernatant, the others are the same as embodiment 1.

[0079] Example 10

[0080] Based on the embodiment 1, the difference is that 6) removing Fe of ovotransferrin 3+ : using citric acid solution to adjust pH=2.0, the others are the same as embodiment 1.

[0081] Example 11

[0082] Based on the embodiment 1, the difference is that 6) removing Fe of ovotransferrin 3+ : using citric acid solution to adjust pH=3.0, the others are the same as embodiment 1.

[0083] Example 12

[0084] Based on the embodiment 1, the difference is that 6) removing Fe of ovotransferrin 3+: pH = 3.5 adjusted with citric acid solution, otherwise as in example 1.

[0085] Example 13

[0086] Based on example 1, difference in that 6) Ovotransferrin is removed from Fe 3+ : pH = 4.0 adjusted with citric acid solution, otherwise as in example 1.

[0087] Example 14

[0088] Based on example 1, difference in that 7) Ovotransferrin is separated: PEG 4000 is added to the solution to a final concentration of 15%, pH = 5.5 is adjusted with NaOH solution, the precipitate is taken by centrifugation, otherwise as in example 1.

[0089] Example 15

[0090] Based on example 1, difference in that 7) Ovotransferrin is separated: PEG 6000 is added to the solution to a final concentration of 15%, pH = 5.5 is adjusted with NaOH solution, the precipitate is taken by centrifugation, otherwise as in example 1.

[0091] Example 16

[0092] Based on example 1, difference in that 7) Ovotransferrin is separated: PEG 10000 is added to the solution to a final concentration of 15%, pH = 5.5 is adjusted with NaOH solution, the precipitate is taken by centrifugation, otherwise as in example 1.

[0093] Example 17

[0094] Based on example 1, difference in that 7) Ovotransferrin is separated: PEG 8000 is added to the solution to a final concentration of 9%, the precipitate is taken by centrifugation, otherwise as in example 1.

[0095] Example 18

[0096] Based on example 1, difference in that 7) Ovotransferrin is separated: PEG 8000 is added to the solution to a final concentration of 12%, the precipitate is taken by centrifugation, otherwise as in example 1.

[0097] Example 19

[0098] Based on example 1, difference in that 7) Ovotransferrin is separated: PEG 8000 is added to the solution to a final concentration of 18%, the precipitate is taken by centrifugation, otherwise as in example 1.

[0099] Example 20

[0100] Based on example 1, the difference is that 7) isolation of ovotransferrin: add PEG8000 to the solution to a final concentration of 21%, centrifuge to take the precipitate, and the rest is the same as example 1.

[0101] Example 21

[0102] Based on example 1, the difference is that 7) isolation of ovotransferrin: adjust the pH to 4.5 using an HCl solution, centrifuge to take the precipitate, and the rest is the same as example 1.

[0103] Example 22

[0104] Based on example 1, the difference is that 7) isolation of ovotransferrin: adjust the pH to 5.0 using an HCl solution, centrifuge to take the precipitate, and the rest is the same as example 1.

[0105] Example 23

[0106] Based on example 1, the difference is that 7) isolation of ovotransferrin: adjust the pH to 6.0 using an HCl solution, centrifuge to take the precipitate, and the rest is the same as example 1.

[0107] Example 24

[0108] Based on example 1, the difference is that 7) isolation of ovotransferrin: adjust the pH to 6.5 using an HCl solution, centrifuge to take the precipitate, and the rest is the same as example 1.

[0109] Comparative example 1

[0110] 1) Pretreatment of chicken egg white: fresh chicken egg whites were washed and separated, diluted with 1 volume of water, and stirred uniformly using an electric mixer;

[0111] 2) Chelation with FeCl3: 1.0 mL / L of FeCl3 solution (0.5 mol / L) was added, the pH of the solution was adjusted to 8.5 using an NaOH solution, and the FeCl3 was fully dispersed in the solution using a high-speed shear disperser at 12000 r / min for 3 min;

[0112] 3) First addition of ethanol: anhydrous ethanol was slowly added to a final concentration of 43%, and the supernatant was taken by centrifugation at 9000 r / min for 10 min, and the suspended impurities were removed by filtration using 80-mesh nylon filter cloth;

[0113] 4) Second addition of ethanol: anhydrous ethanol was added to the filtrate in 3) to a concentration of 61%, and the precipitate was taken by centrifugation at 9000 r / min for 10 min;

[0114] 5) Ultrafiltration to remove salt: after the precipitate in 4) is washed thoroughly, 10 volumes of water is added to re-dissolve, 50 kDa ultrafiltration membrane is used to perform ultrafiltration, water is added constantly to concentrate, to remove salt ions and partial impurities with molecular weight less than ovotransferrin, thus obtaining ovotransferrin.

[0115] 6) Vacuum freeze-drying: the concentrated ovotransferrin solution is loaded into a freeze-drying box and placed in a -20 ℃ refrigerator to pre-freeze, then placed in a freeze dryer to freeze-dry, the freeze-drying condition is temperature -50 ℃, vacuum degree 0.1 Pa, time 36 h.

[0116] Comparative Example 2

[0117] 1) Pretreatment of egg white: fresh egg white is washed clean, separated, diluted with 1 volume of 0.025 M NaCl solution, stirred uniformly with an electric mixer, pH is adjusted to 6.0 with 1.0 mol / L HCl solution, kept at 4 ℃ for 12 h, then centrifuged at 9000 r / min at 4 ℃ for 10 min, and the supernatant is taken;

[0118] 2) First precipitation: 5.0% ammonium sulfate and 2.5% citric acid are added, kept at 4 ℃ for 12 h, then centrifuged at 9000 r / min at 4 ℃ for 10 min, and the precipitate is taken;

[0119] 3) Second precipitation: the precipitate in 2) is dissolved with 10 volumes of water, 2.0% ammonium sulfate and 1.5% citric acid are added, kept at 4 ℃ for 12 h, then centrifuged at 9000 rpm at 4 ℃ for 10 min, and the precipitate is taken;

[0120] 4) Ultrafiltration to remove salt: after the precipitate in 3) is washed thoroughly, 10 volumes of water is added to re-dissolve, 50 kDa ultrafiltration membrane is used to perform ultrafiltration, water is added constantly to concentrate, to remove salt ions and partial impurities with molecular weight less than ovotransferrin, thus obtaining ovotransferrin.

[0121] 5) Vacuum freeze-drying: the concentrated ovotransferrin solution is loaded into a freeze-drying box and placed in a -20 ℃ refrigerator to pre-freeze, then placed in a freeze dryer to freeze-dry, the freeze-drying condition is temperature -50 ℃, vacuum degree 0.1 Pa, time 36 h.

[0122] Comparative Example 3

[0123] 1) Pretreatment of egg white: fresh egg white is washed clean, separated, diluted with 1 volume of water, stirred uniformly with an electric mixer;

[0124] 2) Chelation with FeCl3: 1.0 mL / L of FeCl3 solution (0.5 mol / L) was added, the pH of the solution was adjusted to 8.5 using NaOH solution, and the FeCl3 was fully dispersed in the solution using a high-speed shearing disperser at 12000 r / min for 3 min;

[0125] 3) Addition of ethanol: After adding anhydrous ethanol slowly to a final concentration of 45%, the supernatant was obtained by centrifugation at 9000 r / min for 10 min, and the suspended impurities were removed by filtration using a 80-mesh nylon filter cloth;

[0126] 4) Primary ultrafiltration: the supernatant in 3) was subjected to two times of ultrafiltration using a 50 kDa ultrafiltration membrane, and water was added for concentration to remove ethanol, excess Fe 3+ and part of the impurities with a molecular weight less than that of ovotransferrin;

[0127] 5) Precipitation of ovotransferrin: the solution with a molecular weight greater than the molecular weight of the ultrafiltration membrane in 4) was diluted with water, 2.5% ammonium sulfate and 2.5% citric acid were added, and the mixture was kept at 4 ℃ for 12 h, then centrifuged at 9000 r / min for 10 min at 4 ℃, and the precipitate was collected;

[0128] 6) Ultrafiltration to remove salt: after the precipitate in 5) was washed thoroughly, it was redissolved in 10 volumes of water, and subjected to ultrafiltration using a 50 kDa ultrafiltration membrane, and water was added for concentration to remove salt ions and part of the impurities with a molecular weight less than that of ovotransferrin, thereby obtaining ovotransferrin.

[0129] 6) Vacuum freeze-drying: the concentrated ovotransferrin solution was placed in a freeze-drying box and put into a -20 ℃ refrigerator for pre-freezing, and then put into a freeze-drying machine for freeze-drying, with a temperature of -50 ℃, a vacuum degree of 0.1 Pa, and a time of 36 h.

[0130] The following is a description of the determination methods and results of the above examples, Comparative Examples 1 to 3:

[0131] 1. Particle size distribution determination

[0132] The sample was diluted to a protein mass concentration of 1 mg / mL or less, and centrifuged at 5000 r / min for 15 min in a table centrifuge. The particle size distribution of egg white protein was determined by a laser particle size analyzer. The refractive index of the protein was set to 1.46, and the absorption parameter was set to 1.33.

[0133] 2. Zeta potential determination

[0134] The egg white was diluted to 0.1 mg / mL with 0.02 mol / L pH=7.0 phosphate buffer, and the Zeta potential of the egg white was determined using a Zeta potential instrument. All measurements were performed at 25 °C, and repeated 3 times.

[0135] 3. Free Fe in egg white 3+ Determination

[0136] Take 10 mL of diluted egg white, use 10 kDa ultrafiltration tube for ultrafiltration, set the centrifugal speed to 9000 r / min, centrifugal time is 30 min, and the lower clear liquid is used for subsequent iron content determination.

[0137] 4. Iron content determination

[0138] Use ammonium sulfate iron to prepare iron standard solution, and the iron concentration is 0 mg / L, 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, and 10 mg / L, respectively, to draw a standard curve. Accurately weigh 0.5 g of solid sample into a digestion tube, add 10 mL of nitric acid and 0.5 mL of perchloric acid, and digest on a digestion furnace (120 °C / 0.5-1 h, rise to 180 °C / 2-4 h, rise to 200-220 °C). If the digestion solution is brown, add more nitric acid and digest until white smoke appears. The digestion solution is colorless and transparent or slightly yellow. Remove the digestion tube, cool it, and transfer the digestion solution to a 100 mL volumetric flask. Wash with a small amount of water for 2-3 times, combine the washing liquid in the volumetric flask, and dilute to the mark with water. Mix well and use as needed. At the same time, do a blank test (GB5009.90-2016).

[0139] 5. Endogenous fluorescence determination

[0140] Take an appropriate amount of egg white sample, dilute the sample to a protein concentration of 0.1 mg / mL with 0.02 mol / L pH=7.0 phosphate buffer, set the excitation wavelength of the fluorescence spectrophotometer to 280 nm, the scanning range to 300-470 nm, and the slit width to 5 nm, and obtain the endogenous fluorescence spectrum of the egg white.

[0141] 6. Surface hydrophobicity determination

[0142] Adjust the protein concentration to 0.125, 0.250, 0.500, and 1.00 mg / mL, respectively, then mix well with 8 mmol / L 8-aniline-1-naphthalene sulfonic acid (pH=7.4) at a volume ratio of 150:1, incubate in the dark for 5 min, set the emission wavelength of the fluorescence spectrophotometer to 470 nm and the excitation wavelength to 390 nm, and immediately determine the sample after the reaction. The fluorescence intensity results and the slope formed by the sample are used to represent the surface hydrophobicity.

[0143] 7. SDS-PAGE electrophoresis and purity determination

[0144] The sample was dissolved into 0.3 mg / mL protein solution using double distilled water. Then, 80 μL sample solution was taken and 20 μL loading buffer was added. After incubation in boiling water for 5 min, 10 μL per lane was loaded onto an acrylamide gel with 5% concentration of stacking gel and 10% concentration of separating gel. Pre-stained protein molecular weight marker was used as reference of molecular weight. After electrophoresis, the gel was stained with Coomassie blue rapid staining solution at room temperature for 30 min.

[0145] 8. Purity and yield determination of ovotransferrin

[0146] The sample was prepared into solution and its concentration was determined by BCA method to ensure its concentration was 1.0 mg / mL. Purity determination was performed using high performance liquid system equipped with reversed phase (RP) chromatographic column Grace Vydac C4 (214TP, 5 μm, 250 x 4.6 mm). 0.1% trifluoroacetic acid and acetonitrile were used as mobile phase for linear gradient elution, i.e. acetonitrile concentration was increased from 5% to 80% in 15 min, decreased to 5% in 1 min, and then kept constant for 4 min. During the process, photodiode array detector was used for detection at 280 nm, and area normalization method was used to calculate the purity of ovotransferrin. On the basis of the purity obtained, the yield calculation formula was as follows

[0147]

[0148] Wherein, 10% was the theoretical content of protein in egg white; 12% was the theoretical content of ovotransferrin in egg white protein.

[0149] 9. Solubility determination

[0150] Accurately weighed 50 mg ovotransferrin was dissolved in 10 mL double distilled water, after centrifugation at 10000 r / min for 20 min at 4 °C, the supernatant was taken, and the soluble protein concentration was determined using BCA kit, and the solubility calculation formula was as follows.

[0151] ;

[0152] 10. Iron binding capacity determination

[0153] The 5 mg / mL ovotransferrin solution was prepared using Tris-HCl buffer solution with pH = 9.0. 5 mL of the solution was taken, and an appropriate amount of freshly prepared 0.6 mmol / L FeCl3 solution was added to the molar ratio of iron to ovotransferrin of 2.0. The reaction was carried out at 25 °C for 20 min. The free Fe 3+ was collected using a 20 kDa ultrafiltration tube, and the amount of free Fe 3+ was determined. The calculation formula of the iron binding capacity of ovotransferrin is as follows.

[0154] ;

[0155] 11. FTIR determination

[0156] The ovotransferrin and potassium bromide were ground into fine powder with a mass ratio of 1:100 under an infrared lamp. The sample was scanned at 400-4000 cm -1 using a Fourier infrared spectrometer at a data acquisition rate of 4 cm −1 , with potassium bromide as the background. The amide I region was unwound using Omnic and Peak fit software, and the composition and proportion of the secondary structure of the protein were calculated.

[0157] 12. Preparation of ovotransferrin iron preparation (OVT-Fe)

[0158] The purified ovotransferrin was dissolved in a Tris-HCl solution with pH 8.5-9.0, 0.05 mol / L NaHCO3, and 0.02 mol / L, to prepare a 5 mg / mL OVT solution. Then, 0.5 mM FeCl3 solution was added, and the reaction was carried out for 1 h. After centrifugation to remove the precipitate, the sample was dialyzed in deionized water for 24 h and vacuum freeze-dried for standby use.

[0159] 13. Animal experiment process

[0160] 3-week-old ICR male mice were adaptively fed in a standard feeding environment for 7 days, and then fed with low-iron feed for 60 days. The blood samples of the mice were analyzed using a full-automatic blood analyzer, and the content of hemoglobin was less than 100 g / L, which was considered as the successful establishment of an iron deficiency anemia (IDA) model. Subsequently, the mice were intragastrically administered with ovotransferrin iron preparation for 30 days, and the blood biochemical indicators were detected to clarify the application value of ovotransferrin as an iron element carrier.

[0161] Effect of different treatment methods on the particle size of egg white:

[0162] The proteins in the natural egg white system form large particle complexes, which reduces the protein extraction efficiency. Ultrasonic treatment can effectively destroy the protein complexes, but due to the strong viscosity of ovomucin in the egg white system, the ovomucin can provide a stable skeleton for the protein complexes, so that the ultrasonic treatment is not enough to completely destroy the structure. Therefore, on the basis of ultrasonic treatment, the application adopts high-voltage alternating electric field to fully destroy the protein complexes in the system by using molecular polarization effect and reciprocating motion, so as to reduce the particle size of the egg white. The effects of different treatment methods on the particle size distribution of the egg white are shown in Table 1. Figure 1 As shown in Table 1, the particle size distribution of the untreated egg white is in a three-peak distribution, and after the ultrasonic treatment (Example 2), high-voltage alternating electric field treatment (Example 3), high-voltage pulse electric field treatment (Example 4) or ultrasonic combined high-voltage alternating electric field treatment (Example 1), the particle size distribution of the egg white shifts to the small particle size direction, and the effect of the high-voltage alternating electric field is better than that of the high-voltage pulse electric field. The volume-weighted average particle size (d 4,3 ) is calculated by weighting the particle size distribution data, and the results are shown in Table 1. The d 4,3 of the untreated egg white is 629.89 nm, and the d 4,3 of the egg white treated by ultrasonic, high-voltage alternating electric field, high-voltage pulse electric field and ultrasonic combined high-voltage alternating electric field is 464.54 nm, 245.49 nm, 343.34 nm and 118.89 nm respectively. It is shown that the protein complexes in the egg white system in Example 1 are disintegrated under the triple action of cavitation effect, molecular polarization effect and molecular reciprocating motion, so as to form smaller protein particles.

[0163] Table 1 Effects of different treatment methods on the particle size and zeta potential of egg white

[0164]

[0165] Effects of different treatment methods on the Zeta potential of egg white:

[0166] By destroying the protein complexes in the egg white system by ultrasonic combined high-voltage alternating electric field, the contact probability of ovotransferrin and Fe 3+ is improved, and the ovotransferrin chelates Fe 3+ . The test results of the Zeta potential of the egg white protein are shown in Table 1. Under the action of molecular polarization effect, the Zeta potential of the protein is also significantly reduced. The Zeta potential of the untreated egg white is-14.46 mV, and the Zeta potential of the egg white treated by ultrasonic, high-voltage alternating electric field, high-voltage alternating electric field and ultrasonic combined high-voltage alternating electric field is reduced to-19.53 mV, -27.67 mV, -27.23 mV and-36.06 mV respectively, which is helpful for the chelation of ovotransferrin and Fe 3+ .

[0167] Effect of different treatment methods on egg white structure:

[0168] When the excitation wavelength is 280 nm, the aromatic amino acids contained in egg white protein, such as phenylalanine, tyrosine and tryptophan residues, produce fluorescence, i.e. endogenous fluorescence. From Figure 2 It can be seen that the ultrasonic combined high-voltage alternating electric field treatment increases the endogenous fluorescence intensity of egg white, because the cavitation effect and molecular polarization effect act on egg white protein, making the protein structure loose, the egg white protein molecules stretched, and the aromatic amino acids, especially the tryptophan residues, exposed, so that the fluorescence intensity increases. At the same time of the exposure of the aromatic amino acids, the hydrophobic groups inside the protein are also exposed with the unfolding of the protein structure, and the results of the determination of the surface hydrophobicity are shown in Figure 3 . The increase of the surface hydrophobicity effectively reduces the ability of the protein molecules to compete with the system water molecules, which helps the subsequent impurity removal and the use of PEG8000 to separate ovotransferrin.

[0169] Effect of different treatment methods on the chelation of Fe 3+ by ovotransferrin:

[0170] The concentration of free Fe 3+ in the egg white system is used to characterize the chelation of Fe 3+ by ovotransferrin, and the lower the concentration of free Fe 3+ , the more Fe 3+ is chelated by ovotransferrin, which helps to improve the yield of ovotransferrin. The concentration of free Fe 3+ in the egg white system is determined by using the flame atomic absorption spectrometry in the national standard GB5009.90-2016, and the results are shown in Figure 5 . The concentration of free Fe 3+ in the untreated egg white system is 35.87 mg / L, and after the ultrasonic treatment (Example 2), high-voltage alternating electric field treatment (Example 3), high-voltage pulse electric field treatment (Example 4) or ultrasonic combined high-voltage alternating electric field treatment (Example 1), the concentrations of free Fe 3+ in the egg white system are 27.98 mg / L, 29.34 mg / L and 22.31 mg / L respectively, indicating that the ultrasonic combined high-voltage alternating electric field treatment helps the combination of Fe 3+ by ovotransferrin, which is also consistent with the previous conclusion.

[0171] Comparison of the impurity removal effects of different ethanol addition amounts:

[0172] After the combination of Fe 3+ by ovotransferrin, the environmental resistance of ovotransferrin is increased, which can resist the denaturation effect caused by a certain concentration of ethanol. In the present application, different concentrations of ethanol are used to remove other proteins in the egg white system except ovotransferrin, and the precipitate after the addition of ethanol is taken for SDS-PAGE electrophoresis analysis, and the results are shown inFigure 6 No obvious ovotransferrin band was observed when the ethanol concentration was 40% (Example 6) and 45% (Example 1). In addition, a band appeared at 66 kDa when the ethanol concentration was 45%, indicating that the impurity removal effect of 45% ethanol was better than that of 40%. When the ethanol concentration was higher than 45%, different degrees of precipitation of ovotransferrin occurred. In summary, 45% ethanol was selected as the optimal concentration.

[0173] Effect of different pH on ovotransferrin deferrization:

[0174] The filtrate of step 6) in Examples 1, 10-13 was used to determine the Fe 3+ content, and the results are shown in Figure 7 Table 2. Between pH = 2.5-4.0 (Examples 1, 11-13), the residual amount of Fe 3+ was negatively correlated with pH. When pH < 2.5, the residual amount of Fe 3+ in the solution no longer decreased, indicating that pH = 2.5 (Example 1) was the optimal pH for ovotransferrin deferrization.

[0175] Effect of different molecular weight PEG and its addition amount, system pH on yield and purity:

[0176] To determine the effect of different molecular weight PEG and its addition amount on yield and purity, the extraction pH was fixed at 5.5, and the results are shown in Table 2. First, set the addition amount to 15%, and select PEG4000, PEG6000, PEG8000 and PEG10000 to extract ovotransferrin, and then use HPLC to determine its purity and yield. The results show that PEG8000 has the best extraction effect. Subsequently, PEG8000 was used to explore the effect of its addition amount on the purity and yield of ovotransferrin, and the results show that when the addition amount is 15%, the extraction effect is the best. Selecting 15% PEG8000 to extract ovotransferrin, it was found that different pH had no obvious effect on the purity of ovotransferrin, but had a significant effect on its yield. When pH = 5.5, the yield of ovotransferrin was the highest. The data show that when pH = 5.5 and 15% PEG8000 is added (Example 1), the extraction effect is the best, with a yield of 95.79% and a purity of 95.23%.

[0177] Yield and purity of ovotransferrin in Examples 1-5:

[0178] To further clarify the positive role of ultrasonic complex high-voltage alternating electric field in the present application, SDS-PAGE electrophoresis analysis was performed on the products of Examples 1-5, and the results are shown in Figure 8As shown. The molecular weight of ovotransferrin is approximately 77.6 kDa. The band in Example 1 is mainly concentrated in this area, and no other obvious bands were found. However, in Examples 2-5, some small molecule protein bands were still present, indicating that ultrasonic combined with high-voltage alternating electric field treatment helps to improve the purity of ovotransferrin.

[0179] The yield was calculated based on a 12% content of ovotransferrin in egg white protein, and its purity was determined by HPLC. The results are shown in Table 2. The cavitation effect, molecular polarization effect, and molecular reciprocating motion generated by the ultrasonic combined high-voltage alternating electric field increased the yield of ovotransferrin and Fe... 3+ The chelation efficiency was improved, thereby increasing the yield of ovotransferrin. The yield of Example 1 was 95.79%, and the purity was 95.23%, both significantly higher than other examples. The results of SDS-PAGE electrophoresis were further verified. In summary, Example 1 showed a significant improvement in both purity and yield; therefore, the products of Example 1 and Comparative Examples 1-3 were selected for further characterization to explore the differences in their basic properties.

[0180] Table 2. Purity and yield of ovotransferrin extracted in the examples and comparative examples.

[0181]

[0182] Example 1, the yield and purity of ovotransferrin extracted from Comparative Examples 1-3:

[0183] The SDS-PAGE electrophoresis results of Examples 1 and 3 are as follows: Figure 9 As shown in Table 2, the yield and purity of the product are as follows. Ultrasonic treatment combined with a high-voltage alternating electric field significantly reduced the content of proteins smaller than 77.6 kDa in the product, thus improving the yield and purity. Calculations showed that the yield of Example 1 was increased by 14.05%, 16.25%, and 12.07% compared to Comparative Examples 1-3, respectively; the purity was increased by 29.90%, 10.81%, and 7.88% compared to Comparative Examples 1-3, respectively.

[0184] Example 1, Comparative Examples 1-3: Solubility, iron content, and iron-binding capacity of ovotransferrin extracted from the samples.

[0185] The solubility, iron content, and iron-binding capacity of the ovotransferrin extracted in Examples 1 and 1-3 are as follows: Figures 10-12As shown. The solubility of Example 1 reached 97.08%, which was increased by 70.14%, 36.14%, and 18.93% compared to Comparative Examples 1-3, respectively. The iron content of Example 1 was 0.12 mg / g protein, while that of Comparative Examples 1-3 was 1.35 mg / g protein, 0.04 mg / g protein, and 0.18 mg / g protein, respectively. Furthermore, the iron-binding capacity of Example 1 at pH=9.0 was 90.72%, which was stronger than that of Comparative Examples 1-3, indicating that the ovotransferrin obtained in Example 1 had a stronger iron-loading capacity.

[0186] Example 1, Comparative Examples 1-3: FTIR spectra and secondary structures of ovotransferrin extracted from the animal:

[0187] Example 1, FTIR spectra of ovotransferrin extracted from Comparative Examples 1-3 are as follows: Figure 13 As shown. Ovotransferrin extracted in Example 1 was subjected to [a process / concentration] at 1100 cm⁻¹. -1 The significantly enhanced absorption caused by CO stretching vibrations at the region indicates a structural difference between the ovotransferrin extracted in Example 1 and those extracted in Comparative Examples 1-3. Unwinding of the amide I region using Omnic and Peakfit software revealed that the amide I band (1600-1700 cm⁻¹)... -1 There are significant differences, and their second derivatives are as follows: Figure 14 As shown in the table. Further calculations were performed on the composition and proportion of the protein's secondary structure, and the results are shown in Table 3. The α-helix proportion of ovotransferrin extracted in Example 1 was 34.41%, which was higher than that extracted in Comparative Examples 1-3. According to literature reports, the proportion of α-helices in iron-binding proteins such as myoglobin and hemoglobin is significantly higher than in other secondary structures, which may be one of the reasons why the ovotransferrin extracted in Example 1 has a stronger iron-binding capacity.

[0188] The above analysis shows that egg white proteins form protein complexes, hindering component separation and purification. For this unique material system, egg white is treated with a combination of ultrasound and a high-voltage alternating electric field. This overcomes the problem of difficult protein separation caused by the inability of ultrasound or high-voltage pulsed electric field treatment to completely destroy protein complexes. The cavitation effect, molecular polarization effect, and molecular reciprocating motion generated by the ultrasound combined with the high-voltage alternating electric field disrupt the protein complexes, fully exposing ovotransferrin and promoting its structural extension, which facilitates the binding of Fe by ovotransferrin. 3+This process enhances the protein's environmental tolerance, facilitating the subsequent removal of proteins such as ovalbumin, ovomucoid, and lysozyme from egg white using high-concentration ethanol. Furthermore, the exposure of hydrophobic groups within the proteins during processing reduces their water-binding capacity, allowing for the specific use of highly biocompatible PEG8000 to reduce the solubility of ovotransferrin, thus yielding the final ovotransferrin. The ovotransferrin extracted using this invention exhibits high solubility and iron-binding capacity, and is environmentally friendly, low-cost, and suitable for large-scale industrial production.

[0189] Table 3. Secondary structures of ovotransferrin extracted from Examples 1-3 and Comparative Examples 1-3

[0190]

[0191] The iron-supplementing effect of ovotransferrin:

[0192] The above data indicate that the ovotransferrin obtained in Example 1 has good solubility and iron-binding capacity, and has the potential to act as an iron carrier. Therefore, its effect as an iron supplement was verified by constructing iron deficiency anemia (IDA) mice, and the results are shown in Table 4. OVT-Fe effectively improved the decrease in hemoglobin, blood cell count, serum iron, and other indicators caused by IDA, and its effect was similar to that of FeSO4, indicating that OVT-Fe has a good iron supplementation effect. In order to further clarify the difference between OVT-Fe and FeSO4 in iron supplementation, the oxidative stress and inflammatory factor expression in the duodenum of mice were measured, and the results are shown in Table 4. Figures 15-16 As shown, IDA induced a decrease in the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in the duodenum of mice. Oral administration of OVT-Fe significantly increased the activities of SOD and GSH-PX, with effects 42.28% and 83.54% higher than those of oral administration of FeSO4, respectively. Furthermore, oral administration of OVT-Fe also reduced the expression of TNF-α and IL-1β, with effects 22.30% and 30.21% higher than those of oral administration of FeSO4, respectively. In conclusion, OVT-Fe, while supplementing iron, reduces intestinal oxidative stress and the expression of inflammatory factors, contributing to balancing intestinal homeostasis. It is an excellent iron supplement and can be used to improve IDA symptoms.

[0193] Table 4 Blood biochemical indicators of mice

[0194]

[0195] The above embodiments are merely preferred technical solutions of the present application, and should not be regarded as a limitation on the present application. The embodiments in the present application and the features in the embodiments can be combined with each other in any manner without conflict. The protection scope of the present application should be subject to the technical solutions recited in the claims, and include equivalent replacement solutions of the technical features recited in the claims. That is, equivalent replacement improvements within this range are also within the protection scope of the present application.

Claims

1. A method for extracting high solubility ovotransferrin from chicken egg white, characterized by, The method comprises the following steps: (1) Pretreatment of egg white: egg white is diluted with water and stirred uniformly; (2) FeCl3 solution is added to the diluted egg white solution, NaOH solution is used to adjust the pH of the solution, and high-speed shearing is used to make FeCl3 fully dispersed in the solution to obtain pretreated egg white solution; (3) Ultrasonic and high-voltage alternating electric field treatment: the pretreated egg white solution in step (2) is treated by ultrasonic and high-voltage alternating electric field, and the ultrasonic and high-voltage alternating electric field treatment conditions are as follows: ultrasonic power is 200-300 W, high-voltage alternating electric field conditions are field strength of 15-25 kV / cm and frequency of 50-60 Hz, and ultrasonic and high-voltage alternating electric field treatment time is 5-10 min; (4) Alcohol extraction: after the egg white solution treated in step (3) is added with anhydrous ethanol to a final ethanol concentration of 40%-45%, the supernatant is obtained by centrifugation, and suspended impurities are removed by filtration; (5) First ultrafiltration: the supernatant is subjected to ultrafiltration, water is added for concentration, and ethanol, Fe 3 + and part of the impurities with molecular weight less than that of ovotransferrin are removed to obtain a crude ovotransferrin solution; (6) Removal of Fe from Ovotransferrin 3+ : Adding resin to the crude ovotransferrin solution, adjusting pH with citric acid solution to promote the release of Fe from ovotransferrin 3+ , and filtering to remove the resin after the free Fe 3+ is completely adsorbed to obtain the filtrate; (7) Isolation of ovotransferrin: PEG8000 is added to the filtrate in step (6) to a final PEG8000 concentration of 10%-20%, NaOH solution is used to adjust the pH, and the precipitate is obtained by centrifugation; (8) Secondary ultrafiltration: the precipitate in step (7) is dissolved with water, and ultrafiltration is performed, and water is continuously added for concentration to remove PEG8000, so as to obtain concentrated ovotransferrin; (9) Vacuum freeze-drying: the concentrated ovotransferrin solution is freeze-dried to obtain ovotransferrin product.

2. A method of extracting high soluble ovotransferrin from chicken egg white according to claim 1, characterized in that: In step (1), the mass ratio of egg white to water is 1:(0.8-1.2), the stirring speed is 500-600 r / min, and the stirring time is 10-15 min.

3. A method of extracting high soluble ovotransferrin from chicken egg white according to claim 1, characterized in that: In step (2), the concentration of FeCl3 solution is 0.4-0.6 mol / L, the addition amount of FeCl3 solution is 0.8-1.2 mL / L egg white solution, NaOH solution is used to adjust the pH of the solution to 8-9, the concentration of NaOH solution is 0.3-0.8 mol / L, and the high-speed shearing conditions are 11000-12000 r / min for 2.5-3.5 min.

4. A method of extracting high solubility ovotransferrin from chicken egg white according to claim 3, characterized in that: In step (2), NaOH solution is used to adjust the pH of the solution to 8.

5.

5. The method of claim 1, wherein the method is characterized by: In step (4), the filter cloth used for filtration is 70-80 mesh nylon filter cloth.

6. The method of claim 1, wherein the method is characterized by: In step (5), after the supernatant in step (4) is diluted with water, ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cut-off of 40-60 kDa, and after the ultrafiltration is completed, water is added to completely backflow the residual protein between the collection pipeline and the ultrafiltration membrane, and the ultrafiltration is performed twice; the ultrafiltration membrane has a molecular weight cut-off of 50 kDa or 60 kDa.

7. The method of claim 1, wherein the method is characterized by: In step (6), the pH of the citric acid solution is adjusted to 2.5-2.7; the addition amount of the resin is 0.8-1.5 g / 100 mL egg white solution, the resin is a strong base anion exchange resin, the concentration of the citric acid solution is 0.4-0.6 mol / L, the stirring conditions are 500-600 r / min for 1.5-2.5 h, and the filter cloth used for filtration has a mesh size of 70-90; the type of the resin is AG1-X2 resin.

8. The method of claim 1, wherein the method is characterized by: The NaOH solution used in step (7) is used to adjust pH=4.5-6, the solubility of the NaOH solution is 0.5-0.6 mol / L, and the centrifugal condition is 9000 r / min for 10 min.

9. A method of extracting high solubility ovotransferrin from chicken egg white according to claim 8, characterized in that: The NaOH solution used in step (7) is used to adjust pH=5.

5.

10. The method of claim 1, wherein the method is characterized by: In step (8), the ultrafiltration is performed on the re-dissolved precipitate in step (7) using an ultrafiltration membrane with a molecular weight cut-off of 40-60 kDa, and after the ultrafiltration is completed, water is added to completely backflow the residual protein between the collection pipeline and the ultrafiltration membrane, and the number of ultrafiltration is twice.

11. The method of claim 1, wherein the method is characterized by: In step (9), the freeze-drying condition is a temperature of-50~-40 ℃, a vacuum degree of 0.1-0.2 Pa, and a time of 32-38 h.

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