Method for extracting chicken egg yolk phosvitin

Abstract

The application provides an extraction method of egg yolk phosvitin, which extracts egg yolk phosvitin through a pH-sensitive composite magnetic nanogel to realize magnetic separation, wherein the pH-sensitive composite magnetic nanogel is prepared by taking a magnetic porous gel as an inner core, modifying the surface of the magnetic porous gel with polydopamine and horseradish, forming a shell layer, and connecting iron ions on the shell layer to obtain the pH-sensitive composite magnetic nanogel. In the application, the polydopamine combined with the surface of the magnetic porous gel can be quickly combined with the horseradish, the shell layer is beneficial to the combination of the iron ions, and therefore, the egg yolk phosvitin can be quickly separated under the action of an external magnetic field. Finally, the external magnetic field is removed and placed in an alkaline environment, and the egg yolk phosvitin can be safely and efficiently separated.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of food processing and extraction technology, and particularly relates to an extraction method of egg yolk phosvitin. BACKGROUND

[0002] There are many bioactive components in egg yolk, such as yolk globulin, lipoprotein, egg yolk oil, lecithin, sialic acid, etc., and there is a protein with the highest phosphorylation degree in nature, i.e. phosvitin, which is a complex formed by lipoprotein and yolk phospholipid protein, contains 11.9% of nitrogen, 9.7% of phosphorus, accounts for 80% of the total phosphorus content of egg yolk, and contains 6.5% of carbohydrates. Phosvitin accounts for about 4% of the dry matter of egg yolk, and it is mainly composed of two proteins: alpha-phosvitin and beta-phosvitin, as shown by polyacrylamide gel electrophoresis. Phosvitin is composed of 216 amino acid residues, of which 124 are serine residues, accounting for 56% of the total amino acid content, and more than 90% of the serine residues are phosphorylated. The preparation method of phosvitin is mostly limited to laboratory scale, and almost all uses fresh eggs as raw materials, takes too long time, uses non-food grade solvents and other auxiliary materials during degreasing, and has low recovery rate of phosvitin. Therefore, the current preparation process can only meet the small batch requirements of laboratory research, and cannot meet the production capacity requirements of commercial application.

[0003] In the prior art, the conventional extraction steps of egg yolk phosvitin include removing water-soluble proteins by adding water, removing lipid substances by adding organic solvents, extracting phosvitin by adding NaCl solution, and purifying by heating. The extraction process is complicated and the yield of phosvitin is low. The extraction methods include (NH4)2SO4 precipitation method, NaCl solution leaching method, etc. A large amount of ethyl acetate or non-food additive auxiliary material ammonium sulfate is used in the extraction process, resulting in the prepared phosvitin having use limitations. At present, there are reports on the separation and extraction of phosphopeptides using magnetic microspheres, but there are few reports on the separation of egg yolk phosvitin using magnetic microspheres. Therefore, it is very important to seek a safe and efficient separation method. SUMMARY

[0004] Technical problems to be solved: In view of the above technical problems, the purpose of the present application is to provide an extraction method of egg yolk phosvitin, wherein a magnetic porous gel is taken as the inner core, the surface of the magnetic porous gel is modified by polydopamine and allyl isothiocyanate to form a shell layer, and iron ions are connected to the shell layer to prepare a pH-sensitive composite magnetic nanogel. The polydopamine combined with the surface of the magnetic porous gel can be quickly combined with allyl isothiocyanate, the shell layer is conducive to the combination of iron ions, and egg yolk phosvitin has strong affinity for metal cations, so it is beneficial to magnetically separate egg yolk phosvitin under the action of an external magnetic field. Finally, the external magnetic field is removed and placed in an alkaline environment, which can more efficiently separate egg yolk phosvitin.

[0005] Technical scheme: An extraction method of egg yolk phosvitin, specifically comprising the following steps:

[0006] Step 1. Take egg yolk liquid, add 2 times the mass of distilled water, magnetically stir at 4℃ for 1h, then centrifuge at 15000xg for 15-20min, remove the supernatant, and collect the precipitate to obtain egg yolk particles;

[0007] Step 2. The egg yolk particles are suspended in a 100g / L NaCl solution with a mass to volume ratio of 1:10, stirred overnight at 4℃, heated to 85℃ and kept for 20min, then dialyzed with distilled water for 24h, centrifuged at 15000xg for 15min, the supernatant is collected, the pH is adjusted to 8.0, stirred at 4℃ for 1h and centrifuged again to collect the upper clear liquid, which is freeze-dried to obtain an egg yolk pretreatment sample;

[0008] Step 3. Add acetic acid-sodium acetate buffer solution to the egg yolk pretreatment sample to prepare a sample solution with a pH of 4-4.5 and a concentration of 10mg / mL;

[0009] Step 4. Take the sample solution and add the pH-sensitive composite magnetic nanogel, vortex to mix, and adsorb for 80-100min in a constant temperature shaker at 30℃ to obtain an extraction liquid. The egg yolk phosvitin and the pH-sensitive composite magnetic nanogel are separated under an external magnetic field. The complex is placed in an alkaline solution and the external magnetic field is removed, so that the pH-sensitive composite magnetic nanogel is separated, and the egg yolk phosvitin is obtained.

[0010] Further, the mass to volume ratio of the pH-sensitive composite magnetic nanogel to the sample solution in step 4 is (4-5):1. Further, the preparation steps of the pH-sensitive composite magnetic nanogel are as follows:

[0011] S1. The curdlan was dissolved in 0.1 mol / L sodium hydroxide solution to obtain a concentration of 2-3% curdlan solution, and then Fe3O4 powder was added, stirred and infiltrated, allowed to stand for 1-5h, filtered, washed, and freeze-dried to obtain a magnetic porous gel;

[0012] S2. The magnetic porous gel was mixed with a polydopamine solution having a concentration of 1 mg / mL, ultrasonicated for 20-30 min, stirred at room temperature for 20-24 h, filtered, washed, and dried to obtain a polydopamine-coated magnetic porous gel;

[0013] S3 horseradish was dissolved in an ethanol solution to obtain a horseradish solution, after adding the polydopamine-coated magnetic porous gel, stirring and infiltrating, filtering, washing, and drying to obtain a magnetic porous gel - polydopamine - horseradish nanogel;

[0014] S4. Disperse the magnetic porous gel-polydopamine-horseradish extract nanogel in a 5 mg / mL FeCl3 solution with a pH of 5, stir and soak, filter, wash, and dry to obtain a pH-sensitive composite magnetic nanogel.

[0015] Furthermore, in step S1, the mass volume ratio of Fe3O4 powder to curdlan solution is 1:(2-20).

[0016] Furthermore, in step S2, the mass volume ratio of the magnetic porous gel to the polydopamine solution is 1:(1-2).

[0017] Furthermore, the concentration of the horseradish solution in step S3 is 8-10%; and the mass volume ratio of the polydopamine-coated magnetic porous gel to the horseradish solution is 1:(3-5).

[0018] Beneficial effects:

[0019] 1. The egg yolk phosvitin of the present invention contains a large amount of phosphorylated serine, which makes the molecule carry a negative charge and has a strong affinity for metal cations, and is easy to react with Ca 2+ 、Fe 3+ Mg 2+ 、Mn 2+ The plasma is combined and is very stable after combination, so the Fe 3+ The strong binding force with egg phosvitin allows for safe and efficient magnetic separation of phosvitin from egg yolk;

[0020] 2. Horseradish in the present invention has the ability to form complexes with metal ions. The polydopamine bound to the surface of the composite magnetic nanogel can react with horseradish quickly and efficiently, making Fe 3+Can be more stable combination in the surface of composite magnetic nanogel; in addition, the iron element in the isorhodine is combined with the Fe 3+ , which is beneficial to the combination of egg yolk phosvitin to a greater extent, and improves the extraction rate;

[0021] 3. The bare magnetic nanogel is easy to agglomerate due to interaction, which affects the dispersibility and stability of the nanogel in solution, thereby seriously affecting the application of the magnetic nanogel, and after the surface modification of the composite magnetic nanogel by polydopamine, the solubility, stability and biocompatibility of the composite magnetic nanogel in water are improved, and the isorhodine is beneficial to be connected to the outer layer; 4. In the application, the magnetic porous gel material is prepared from Fe3O4 and dextran, has a high specific surface area, rich active groups and good magnetic responsiveness, thereby being beneficial to the magnetic separation and extraction of egg yolk phosvitin, avoiding the use of a large amount of organic solvent, and being more simple, safe and efficient in the extraction step;

[0022] 5. The composite magnetic nanogel in the application has good adsorption capacity in an acidic environment, therefore, in the desorption process, the complex of egg yolk phosvitin and the pH-sensitive composite magnetic nanogel is placed in an alkaline environment, the binding force between the egg yolk phosvitin and the Fe 3+ is weakened, and at the same time, the external magnetic field is removed, so that the maximum desorption is realized, so as to collect the egg yolk phosvitin. DETAILED DESCRIPTION

[0023] The application will be further described below in combination with examples, and the following examples are an explanation of the application, and the application is not limited to the following examples:

[0024] Example 1

[0025] The preparation steps of the pH-sensitive composite magnetic nanogel are as follows:

[0026] S1. The dextran is dissolved in a 0.1 mol / L sodium hydroxide solution, to obtain a dextran solution with a concentration of 2%, then Fe3O4 powder is added, the mass / volume ratio is 1:2, stirring is performed for immersion, and then standing for 3h, filtration, washing, and freeze-drying are performed, to obtain a magnetic porous gel; S2. The magnetic porous gel is mixed with a polydopamine solution with a concentration of 1 mg / mL, the mass / volume ratio of the magnetic porous gel and the polydopamine solution is 1:1, ultrasonic treatment is performed for 30 min, stirring reaction is performed at room temperature for 24h, and then filtration, washing, and drying are performed, to obtain the polydopamine-coated magnetic porous gel;

[0027] S3. Dissolve allicin in ethanol solution to obtain an allicin solution with a concentration of 8%, then add the polydopamine-coated magnetic porous gel, and stir to infiltrate, and filter, wash, and dry to obtain the magnetic porous gel-polydopamine-allicin nanogel;

[0028] S4. Disperse the magnetic porous gel-polydopamine-allicin nanogel in a 5 mg / mL FeCl3 solution with a pH of 5, stir to infiltrate, and filter, wash, and dry to obtain the pH-sensitive composite magnetic nanogel.

[0029] Example 2

[0030] The pH-sensitive composite magnetic nanogel is prepared by the following steps:

[0031] S1. Dissolve curdlan in a 0.1 mol / L sodium hydroxide solution to obtain a curdlan solution with a concentration of 2%, then add Fe3O4 powder, and stir to infiltrate, and stand for 3 h, filter, wash, and freeze-dry to obtain the magnetic porous gel; S2. Mix the magnetic porous gel with a 1 mg / mL polydopamine solution, and ultrasonically treat for 30 min, and stir to react at room temperature for 24 h, and filter, wash, and dry to obtain the polydopamine-coated magnetic porous gel;

[0032] S3. Dissolve allicin in ethanol solution to obtain an allicin solution with a concentration of 8%, then add the polydopamine-coated magnetic porous gel, and stir to infiltrate, and filter, wash, and dry to obtain the magnetic porous gel-polydopamine-allicin nanogel;

[0033] S4. Disperse the magnetic porous gel-polydopamine-allicin nanogel in a 5 mg / mL FeCl3 solution with a pH of 5, stir to infiltrate, and filter, wash, and dry to obtain the pH-sensitive composite magnetic nanogel.

[0034] Example 3

[0035] The pH-sensitive composite magnetic nanogel is prepared by the following steps:

[0036] S1. Dissolve the gellan gum in 0.1 mol / L sodium hydroxide solution to obtain a gellan gum solution with a concentration of 3%, then add Fe3O4 powder with a mass-volume ratio of 1:10, stir and soak, stand for 3 h, filter, wash, freeze-dry, and obtain a magnetic porous gel; S2. Mix the magnetic porous gel with a polydopamine solution with a concentration of 1 mg / mL, with a mass-volume ratio of the magnetic porous gel to the polydopamine solution being 1:2, ultrasonic for 30 min, stir and react at room temperature for 24 h, filter, wash, and dry to obtain a polydopamine-coated magnetic porous gel;

[0037] S3. Dissolve the isothorugensol in an ethanol solution to obtain an isothorugensol solution with a concentration of 8%, then add the polydopamine-coated magnetic porous gel, with a mass-volume ratio of the polydopamine-coated magnetic porous gel to the isothorugensol solution being 1:5, stir and soak, filter, wash, and dry to obtain a magnetic porous carbon-polydopamine-isothorugensol nanogel;

[0038] S4. Disperse the magnetic porous gel-polydopamine-isothorugensol nanogel in a 5 mg / mL FeCl3 solution with a pH of 5, stir and soak, filter, wash, and dry to obtain a pH-sensitive composite magnetic nanogel.

[0039] Example 4

[0040] The pH-sensitive composite magnetic nanogel is prepared by the following steps:

[0041] S1. Dissolve the gellan gum in 0.1 mol / L sodium hydroxide solution to obtain a gellan gum solution with a concentration of 3%, then add Fe3O4 powder with a mass-volume ratio of 1:15, stir and soak, stand for 3 h, filter, wash, and freeze-dry to obtain a magnetic porous gel; S2. Mix the magnetic porous gel with a polydopamine solution with a concentration of 1 mg / mL, with a mass-volume ratio of the magnetic porous gel to the polydopamine solution being 1:2, ultrasonic for 30 min, stir and react at room temperature for 24 h, filter, wash, and dry to obtain a polydopamine-coated magnetic porous gel;

[0042] S3. Dissolve the isothorugensol in an ethanol solution to obtain an isothorugensol solution with a concentration of 8%, then add the polydopamine-coated magnetic porous gel, with a mass-volume ratio of the polydopamine-coated magnetic porous gel to the isothorugensol solution being 1:5, stir and soak, filter, wash, and dry to obtain a magnetic porous carbon-polydopamine-isothorugensol nanogel;

[0043] S4. Disperse the magnetic porous gel-polydopamine-isothorugensol nanogel in a 5 mg / mL FeCl3 solution with a pH of 5, stir and soak, filter, wash, and dry to obtain a pH-sensitive composite magnetic nanogel.

[0044] Example 5

[0045] The preparation steps of the pH-sensitive composite magnetic nanogel are as follows:

[0046] S1. Dissolve the dextran in a 0.1 mol / L sodium hydroxide solution to obtain a dextran solution with a concentration of 3%, then add Fe3O4 powder with a mass-volume ratio of 1:20, stir and soak, stand for 3 h, filter, wash, and freeze-dry to obtain a magnetic porous gel; S2. Mix the magnetic porous gel with a polydopamine solution with a concentration of 1 mg / mL, and the mass-volume ratio of the magnetic porous gel to the polydopamine solution is 1:2, ultrasonic for 30 min, stir and react at room temperature for 24 h, filter, wash, and dry to obtain a polydopamine-coated magnetic porous gel;

[0047] S3. Dissolve the allyl isothiocyanate in an ethanol solution to obtain an allyl isothiocyanate solution with a concentration of 10%, then add the polydopamine-coated magnetic porous gel, and the mass-volume ratio of the polydopamine-coated magnetic porous gel to the allyl isothiocyanate solution is 1:5, stir and soak, filter, wash, and dry to obtain a magnetic porous gel-polydopamine-allyl isothiocyanate nanogel;

[0048] S4. Disperse the magnetic porous gel-polydopamine-allyl isothiocyanate nanogel in a 5 mg / mL FeCl3 solution with a pH of 5, stir and soak, filter, wash, and dry to obtain the pH-sensitive composite magnetic nanogel.

[0049] Comparative Example 1

[0050] The difference between this comparative example and Example 2 is that no polydopamine is added, and the specific steps are as follows:

[0051] The preparation steps of the pH-sensitive composite magnetic nanogel are as follows:

[0052] S1. Dissolve the dextran in a 0.1 mol / L sodium hydroxide solution to obtain a dextran solution with a concentration of 2%, then add Fe3O4 powder with a mass-volume ratio of 1:5, stir and soak, stand for 3 h, filter, wash, and freeze-dry to obtain a magnetic porous gel; S2. Dissolve the allyl isothiocyanate in an ethanol solution to obtain an allyl isothiocyanate solution with a concentration of 8%, then add the magnetic porous gel, and the mass-volume ratio of the magnetic porous gel to the allyl isothiocyanate solution is 1:4, stir and soak, filter, wash, and dry to obtain a magnetic porous gel-allyl isothiocyanate nanogel;

[0053] S3. Disperse the magnetic porous gel-allyl isothiocyanate nanogel in a 5 mg / mL FeCl3 solution with a pH of 5, stir and soak, filter, wash, and dry to obtain the pH-sensitive composite magnetic nanogel.

[0054] Comparative Example 2

[0055] The comparative example is different from example 2 in that no allyl isocyanate is added, and is as follows:

[0056] The preparation steps of the pH-sensitive composite magnetic nanogel are as follows:

[0057] S1. Dissolve curdlan in a 0.1 mol / L sodium hydroxide solution to obtain a curdlan solution with a concentration of 2%, then add Fe3O4 powder at a mass-volume ratio of 1:5, stir and soak, stand for 3 h, filter, wash, and freeze-dry to obtain a magnetic porous gel;

[0058] S3. Disperse the polydopamine-coated magnetic porous gel in a 5 mg / mL FeCl3 solution with a pH of 5, stir and soak, filter, wash, and dry to obtain a pH-sensitive composite magnetic nanogel.

[0059] (1) Particle size determination:

[0060] Disperse the composite magnetic nanogels prepared in examples 1-5 and comparative examples 1-2 into a certain solvent (water or DMSO), ultrasonicate for 20 min, and test the particle size distribution of the nanogels at 25°C.

[0061] Table 1 Particle size of the composite magnetic nanogels prepared in examples 1-5 and comparative examples 1-2

[0062] Particle diameter (nm) Particle diameter (nm) Example 1 182 Example 5 189 Example 2 170 Comparative Example 1 165 Example 3 200 Comparative Example 2 163 Example 4 185

[0063] As shown in Table 1, the particle sizes of the composite magnetic nanogels prepared in examples 1-5 are 170-200 nm, the particle size of the composite magnetic nanogel prepared in comparative example 1 is 165 nm, and the particle size of the composite magnetic nanogel prepared in comparative example 2 is 163 nm. It can be seen that the particle size increases after the magnetic porous gel is modified and coated layer by layer.

[0064] (2) Fe 3+ adsorption capacity

[0065] Fe 3+ in the FeCl3 solution is denoted as C0, the Fe 3+ concentration in the solution at adsorption equilibrium is denoted as C1, and the Fe 3+ adsorption capacity is calculated according to the following formula:

[0066] Q = (C0-C1)V / m

[0067] In the formula, Q is the Fe 3+Adsorption capacity (mg / g); V is the volume of the solution; m is the mass of the magnetic porous carbon-polydopamine-allitridin nanogel.

[0068] Fe of the composite magnetic nanogel prepared in Examples 1-5 and Comparative Examples 1-2 in Table 2 3+ Adsorption capacity

[0069] Fe 3+ Adsorption capacity (mg / g) Fe 3+ Adsorption capacity (mg / g) Example 1 28.79 Example 5 30.27 Example 2 35.61 Comparative Example 1 29.15 Example 3 31.28 Comparative Example 2 18.42 Example 4 33.46

[0070] As can be seen from Table 2, the Fe of the composite magnetic nanogel prepared in Examples 1-5 is 3+ The adsorption capacity is 28.79-35.61 mg / g, and the Fe of Comparative Example 1 without adding polydopamine has 3+ The adsorption capacity is not greatly affected, and the Fe of Comparative Example 2 without adding allitridin has 3+ The adsorption capacity is decreased to 18.42 mg / g, and it can be seen that allitridin has the ability to form a complex with metal ions, and the polydopamine on the surface of the composite magnetic nanogel can quickly and efficiently react with allitridin, so that the Fe 3+ can be more stably combined on the surface of the composite magnetic nanogel.

[0071] Therefore, the composite magnetic nanogel prepared in Example 2 is selected for application in the subsequent extraction of egg yolk phosvitin.

[0072] Example 6

[0073] An egg yolk phosvitin extraction method, the specific steps are as follows:

[0074] Step 1. Take egg yolk liquid, add 2 times the mass of distilled water, magnetically stir at 4°C for 1 h, then centrifuge at 15000 x g for 20 min, remove the supernatant, and collect the precipitate to obtain egg yolk particles;

[0075] Step 2. The egg yolk particles are suspended in a 100 g / L NaCl solution with a mass to volume ratio of 1:10, stirred at 4°C overnight, heated to 85°C and kept for 20 min, then dialyzed with double distilled water for 24 h, centrifuged at 15000 x g for 15 min, the supernatant was collected, the pH was adjusted to 8.0, stirred at 4°C for 1 h and centrifuged again to collect the upper clear liquid, which was freeze-dried to obtain an egg yolk pretreatment sample;

[0076] Step 3. Add acetic acid-sodium acetate buffer solution to the egg yolk pretreatment sample to prepare a sample solution with a pH of 4-4.5 and a concentration of 10 mg / mL;

[0077] Step 4. Take the sample solution and add the pH-sensitive composite magnetic nanogel at a mass-volume ratio of 4:1, mix by vortex oscillation, adsorb for 80 min in a constant-temperature shaker at 30°C, obtain the extract, separate the complex of egg yolk phosvitin and the pH-sensitive composite magnetic nanogel under an external magnetic field, place the complex in an alkaline solution, and remove the external magnetic field, so that the pH-sensitive composite magnetic nanogel is separated, and the egg yolk phosvitin is obtained.

[0078] Example 7

[0079] A method for extracting egg yolk phosvitin, the specific steps being as follows:

[0080] Step 1. Take egg yolk liquid, add 2 times the mass of distilled water, magnetically stir at 4°C for 1 h, centrifuge at 15000 x g for 20 min, remove the supernatant, and collect the precipitate to obtain yolk particles;

[0081] Step 2. Suspend the yolk particles in a 100 g / L NaCl solution at a mass-volume ratio of 1:10, stir overnight at 4°C, heat to 85°C and maintain for 20 min, then dialyze with distilled water for 24 h, centrifuge at 15000 x g for 15 min, collect the supernatant, adjust the pH to 8.0, stir at 4°C for 1 h, and centrifuge again to collect the upper clear liquid, which is freeze-dried to obtain an egg yolk pretreatment sample;

[0082] Step 3. Add acetic acid-sodium acetate buffer solution to the egg yolk pretreatment sample to prepare a sample solution with a pH of 4-4.5 and a concentration of 10 mg / mL;

[0083] Step 4. Take the sample solution and add the pH-sensitive composite magnetic nanogel at a mass-volume ratio of 4:1, mix by vortex oscillation, adsorb for 80 min in a constant-temperature shaker at 30°C, obtain the extract, separate the complex of egg yolk phosvitin and the pH-sensitive composite magnetic nanogel under an external magnetic field, place the complex in an alkaline solution, and remove the external magnetic field, so that the pH-sensitive composite magnetic nanogel is separated, and the egg yolk phosvitin is obtained.

[0084] Example 8

[0085] A method for extracting egg yolk phosvitin, the specific steps being as follows:

[0086] Step 1. Take egg yolk liquid, add 2 times the mass of distilled water, magnetically stir at 4°C for 1 h, centrifuge at 15000 x g for 20 min, remove the supernatant, and collect the precipitate to obtain yolk particles;

[0087] Step 2. The egg yolk particles were suspended in 100 g / L NaCl solution at a mass-volume ratio of 1:10, stirred overnight at 4°C, heated to 85°C and maintained for 20 min, then dialyzed with double distilled water for 24 h, frozen at 15000 x g for 15 min, the supernatant was collected, the pH was adjusted to 8.0, stirred at 4°C for 1 h and centrifuged again to collect the supernatant, which was freeze-dried to obtain the egg yolk pretreatment sample;

[0088] Step 3. The egg yolk pretreatment sample was added with acetic acid-sodium acetate buffer solution to prepare a sample solution with a pH of 4-4.5 and a concentration of 10 mg / mL;

[0089] Step 4. The sample solution was taken and added with pH-sensitive composite magnetic nanogel at a mass-volume ratio of 4:1, vortexed and mixed, adsorbed in a constant temperature shaker at 30°C for 90 min to obtain an extract, and the complex of egg yolk phosvitin and pH-sensitive composite magnetic nanogel was separated under an external magnetic field. The complex was placed in an alkaline solution, and the pH-sensitive composite magnetic nanogel was separated by removing the external magnetic field, thereby obtaining egg yolk phosvitin.

[0090] Example 9

[0091] A method for extracting egg yolk phosvitin, the specific steps are as follows:

[0092] Step 1. Take egg yolk liquid, add 2 times the mass of distilled water, stir at 4°C for 1 h, then freeze at 15000 x g for 20 min, remove the supernatant, and collect the precipitate to obtain egg yolk particles;

[0093] Step 2. The egg yolk particles were suspended in 100 g / L NaCl solution at a mass-volume ratio of 1:10, stirred overnight at 4°C, heated to 85°C and maintained for 20 min, then dialyzed with double distilled water for 24 h, frozen at 15000 x g for 15 min, the supernatant was collected, the pH was adjusted to 8.0, stirred at 4°C for 1 h and centrifuged again to collect the supernatant, which was freeze-dried to obtain the egg yolk pretreatment sample;

[0094] Step 3. The egg yolk pretreatment sample was added with acetic acid-sodium acetate buffer solution to prepare a sample solution with a pH of 4-4.5 and a concentration of 10 mg / mL;

[0095] Step 4. The sample solution was taken and added with pH-sensitive composite magnetic nanogel at a mass-volume ratio of 4:1, vortexed and mixed, adsorbed in a constant temperature shaker at 30°C for 90 min to obtain an extract, and the complex of egg yolk phosvitin and pH-sensitive composite magnetic nanogel was separated under an external magnetic field. The complex was placed in an alkaline solution, and the pH-sensitive composite magnetic nanogel was separated by removing the external magnetic field, thereby obtaining egg yolk phosvitin.

[0096] Example 10

[0097] A method for extracting egg yolk phosvitin, the specific steps are as follows:

[0098] Step 1. Take the egg yolk liquid, add 2 times the mass of distilled water, stir in a magnetic field at 4°C for 1 h, then centrifuge at 15000 x g for 20 min, remove the supernatant, and collect the precipitate to obtain egg yolk particles;

[0099] Step 2. The egg yolk particles are suspended in a 100 g / L NaCl solution with a mass to volume ratio of 1:10, stirred overnight at 4°C, heated to 85°C and kept for 20 min, then dialyzed with double distilled water for 24 h, centrifuged at 15000 x g for 15 min, the supernatant is collected, the pH is adjusted to 8.0, stirred at 4°C for 1 h and centrifuged again to collect the upper clear liquid, then freeze-dried to obtain the egg yolk pretreatment sample;

[0100] Step 3. Add acetic acid-sodium acetate buffer solution to the egg yolk pretreatment sample to prepare a sample solution with a pH of 4-4.5 and a concentration of 10 mg / mL;

[0101] Step 4. Take the sample solution and add pH-sensitive composite magnetic nanogel with a mass to volume ratio of 5:1, vortex to mix, adsorb for 100 min in a constant temperature shaker at 30°C to obtain the extract, separate the egg yolk phosvitin and the pH-sensitive composite magnetic nanogel complex under an external magnetic field, place the complex in an alkaline solution, and remove the external magnetic field to separate the pH-sensitive composite magnetic nanogel, thus obtaining the egg yolk phosvitin.

[0102] Comparative Example 3

[0103] The difference between this comparative example and Example 8 is that the pH-sensitive composite magnetic nanogel prepared in Comparative Example 1 is used, and the specific process is as follows:

[0104] A method for extracting egg yolk phosvitin, the specific steps are as follows:

[0105] Step 1. Take the egg yolk liquid, add 2 times the mass of distilled water, stir in a magnetic field at 4°C for 1 h, then centrifuge at 15000 x g for 20 min, remove the supernatant, and collect the precipitate to obtain egg yolk particles;

[0106] Step 2. The egg yolk particles are suspended in a 100 g / L NaCl solution with a mass to volume ratio of 1:10, stirred overnight at 4°C, heated to 85°C and kept for 20 min, then dialyzed with double distilled water for 24 h, centrifuged at 15000 x g for 15 min, the supernatant is collected, the pH is adjusted to 8.0, stirred at 4°C for 1 h and centrifuged again to collect the upper clear liquid, then freeze-dried to obtain the egg yolk pretreatment sample;

[0107] Step 3. Add acetic acid-sodium acetate buffer solution to the egg yolk pretreatment sample to prepare a sample solution with a pH of 4-4.5 and a concentration of 10 mg / mL;

[0108] Step 4. Take the sample solution and add the pH-sensitive composite magnetic nanogel prepared in Comparative Example 1 at a mass-to-volume ratio of 4:1, mix by vortex oscillation, adsorb for 90 min in a constant-temperature shaker at 30°C to obtain an extract, separate the complex of egg yolk phosvitin and the pH-sensitive composite magnetic nanogel under an applied magnetic field, place the complex in an alkaline solution, and remove the applied magnetic field to separate the pH-sensitive composite magnetic nanogel, thereby obtaining egg yolk phosvitin.

[0109] Comparative Example 4

[0110] This comparative example differs from Example 8 in that the pH-sensitive composite magnetic nanogel prepared in Comparative Example 2 is used, and the procedure is as follows:

[0111] A method for extracting egg yolk phosvitin, the specific steps of which are as follows:

[0112] Step 1. Take egg yolk liquid, add 2 times the mass of distilled water, magnetically stir at 4°C for 1 h, then centrifuge at 15000 x g for 20 min, remove the supernatant, and collect the precipitate to obtain egg yolk particles;

[0113] Step 2. Suspend the egg yolk particles in a 100 g / L NaCl solution at a mass-to-volume ratio of 1:10, stir overnight at 4°C, heat to 85°C and maintain for 20 min, then dialyze against double-distilled water for 24 h, centrifuge at 15000 x g for 15 min, collect the supernatant, adjust the pH to 8.0, stir at 4°C for 1 h, and again centrifuge to collect the supernatant, which is then lyophilized to obtain an egg yolk pretreatment sample;

[0114] Step 3. Add acetic acid-sodium acetate buffer solution to the egg yolk pretreatment sample to prepare a sample solution with a pH of 4-4.5 and a concentration of 10 mg / mL;

[0115] Step 4. Take the sample solution and add the pH-sensitive composite magnetic nanogel prepared in Comparative Example 2 at a mass-to-volume ratio of 4:1, mix by vortex oscillation, adsorb for 90 min in a constant-temperature shaker at 30°C to obtain an extract, separate the complex of egg yolk phosvitin and the pH-sensitive composite magnetic nanogel under an applied magnetic field, place the complex in an alkaline solution, and remove the applied magnetic field to separate the pH-sensitive composite magnetic nanogel, thereby obtaining egg yolk phosvitin.

[0116] Comparative Example 5

[0117] The comparative example is different from example 8 in that the complex is not placed in a basic solution to separate the chicken egg yolk phosvitin in step 4, and the specific process is as follows:

[0118] A method for extracting chicken egg yolk phosvitin, the specific steps are as follows:

[0119] Step 1. Take the chicken egg yolk liquid, add 2 times the mass of distilled water, stir magnetically at 4°C for 1h, then centrifuge at 15000xg for 20min, remove the supernatant, and collect the precipitate to obtain egg yolk particles;

[0120] Step 2. The egg yolk particles are suspended in a 100g / L NaCl solution with a mass to volume ratio of 1:10, stirred overnight at 4°C, heated to 85°C and kept for 20min, then dialyzed with double distilled water for 24h, centrifuged at 15000xg for 15min, the supernatant is collected, the pH is adjusted to 8.0, stirred at 4°C for 1h and centrifuged again to collect the upper clear liquid, then freeze-dried to obtain the chicken egg yolk pretreatment sample;

[0121] Step 3. Add acetic acid-sodium acetate buffer solution to the chicken egg yolk pretreatment sample to prepare a sample solution with a pH of 4-4.5 and a concentration of 10mg / mL;

[0122] Step 4. Take the sample solution and add pH-sensitive composite magnetic nanogel with a mass to volume ratio of 4:1, vortex to mix, adsorb for 90min in a constant temperature shaker at 30°C to obtain the extract, separate the chicken egg yolk phosvitin and the pH-sensitive composite magnetic nanogel complex under an external magnetic field, remove the external magnetic field, and separate the pH-sensitive composite magnetic nanogel to obtain the chicken egg yolk phosvitin.

[0123] Performance test:

[0124] (1) The ability of pH-sensitive composite magnetic nanogel to adsorb chicken egg yolk phosvitin

[0125] The ability of pH-sensitive composite magnetic nanogel to adsorb chicken egg yolk phosvitin in the extract of step 4 of examples 6-10 and comparative examples 3-4 is calculated, the mass concentration of yolk phosvitin in the solution is determined by fluorescence spectroscopy, the fluorescence intensity at 350nm wavelength is taken as the reference, and a standard curve is drawn with yolk phosvitin standard, and the calculation formula is as follows:

[0126] Qe = (C0-C e ) V / m

[0127] In the formula, Qe is the adsorption capacity of pH-sensitive composite magnetic nanogel (mg / g); C0 is the initial mass concentration of yolk phosvitin in the solution (mg / mL); C eThe mass concentration of the yolk phosvitin in the solution after adsorption equilibrium (mg / mL); m is the mass of the pH-sensitive composite magnetic nanogel (g).

[0128] Table 3 Adsorption capacity of the pH-sensitive composite magnetic nanogel in Examples 6-10 and Comparative Examples 3-4 for chicken egg yolk phosvitin

[0129]

[0130]

[0131] As shown in Table 3, the adsorption capacity of the pH-sensitive composite magnetic nanogel in Examples 6-10 was 598.33-610.25 mg / mL, and the adsorption capacity tended to be stable when the adsorption time reached 90 min. The adsorption capacity of the composite magnetic nanogel prepared by Comparative Example 1 did not change significantly. The adsorption capacity of the composite magnetic nanogel prepared by Comparative Example 2 was 537.61 mg / mL. It can be seen that, without the addition of allyl isothiocyanate, the Fe 3+ The decrease in the adsorption amount further led to a decrease in the adsorption capacity of the pH-sensitive composite magnetic nanogel for chicken egg yolk phosvitin.

[0132] (2) Yield and extraction rate of chicken egg yolk phosvitin

[0133] The phosphorus content was determined by the micro-determination of phosphorus. The yolk phosvitin contains 10% of phosphorus, and the phosphorus content accounts for 80% of the total phosphorus content of the egg yolk. Therefore, by determining the phosphorus content of the chicken egg yolk particles and the extraction liquid, the yield and extraction rate of the yolk phosvitin can be calculated, and the calculation formula is as follows:

[0134]

[0135] Table 4 Yield and extraction rate of chicken egg yolk phosvitin in Examples 6-10 and Comparative Examples 3-5

[0136] Yield (%) Extraction rate (%) Example 6 6.67 69.33 Example 7 7.05 67.84 Example 8 7.52 73.16 Example 9 6.83 71.02 Example 10 7.16 65.45 Comparative Example 3 6.54 63.12 Comparative Example 4 4.87 51.26 Comparative Example 5 5.55 54.71

[0137] As shown in Table 4, the yield of the chicken egg yolk phosvitin extracted in Examples 6-10 was 6.67-7.52%, and the extraction rate was 65.45-73.16%. The yield and extraction rate were both high. The yield and extraction rate of the chicken egg yolk phosvitin extracted in Comparative Example 3 were not significantly different from those in Examples 6-10. The yield and extraction rate of the yolk phosvitin extracted by the composite magnetic nanogel prepared by Comparative Example 4 without the addition of allyl isothiocyanate in Comparative Example 2 decreased significantly. In Comparative Example 5, the complex was not placed in an alkaline solution during desorption, and only the external magnetic field was removed. However, an alkaline environment is conducive to weakening the interaction between the yolk phosvitin and Fe 3+The binding force between them is weak, so the pH sensitivity of Comparative Example 5 cannot be achieved, and the yield and extraction rate of egg yolk phosvitin are also decreased.

[0138] The above description is only the preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application, by using the disclosed methods and technical contents. Therefore, any simple modification, equivalent replacement, equivalent change and modification of the above embodiments made according to the technical essence of the present application, without departing from the technical solutions of the present application, still belongs to the protection scope of the technical solutions of the present application.

Claims

1. A method for preparing a pH-sensitive composite magnetic nanogel, characterized in that: The specific steps include: S1. The curdlan was dissolved in 0.1 mol / L sodium hydroxide solution to obtain a concentration of 2% -3% curdlan solution, and then Fe3O4 powder was added, stirred and infiltrated, allowed to stand for 1-5h, filtered, washed, and freeze-dried to obtain a magnetic porous gel; S2. The magnetic porous gel was mixed with a polydopamine solution having a concentration of 1 mg / mL, ultrasonicated for 20-30 min, stirred at room temperature for 20-24 h, filtered, washed, and dried to obtain a polydopamine-coated magnetic porous gel; S3 horseradish was dissolved in an ethanol solution to obtain a horseradish solution, after adding the polydopamine-coated magnetic porous gel, stirring and infiltrating, filtering, washing, and drying to obtain a magnetic porous gel - polydopamine - horseradish nanogel; S4. Disperse the magnetic porous gel-polydopamine-horseradish extract nanogel in a 5 mg / mL FeCl3 solution with a pH of 5, stir and soak, filter, wash, and dry to obtain a pH-sensitive composite magnetic nanogel.

2. A method for extracting egg phosvitin, characterized in that: The method comprises the following preparation steps: Step 1. Take egg yolk liquid, add 2 times the weight of distilled water, stir magnetically at 4°C for 1 hour, and centrifuge at 15,000 × g for 15-20 minutes. Remove the supernatant and collect the precipitate to obtain egg yolk particles. Step 2. The egg yolk particles were suspended in 100 g / L NaCl solution at a mass-to-volume ratio of 1:10, stirred at 4°C overnight, heated to 85°C and maintained for 20 min, then dialyzed with double-distilled water for 24 h, and refrigerated centrifuged at 15,000 × g for 15 min. The supernatant was collected and the pH was adjusted to 8.

0. The supernatant was stirred at 4°C for 1 h and centrifuged again, and then lyophilized to obtain the egg yolk pretreatment sample. Step 3. Add acetic acid-sodium acetate buffer solution to the egg yolk pre-treatment sample to prepare a sample solution with a pH of 4-4.5 and a concentration of 10 mg / mL; Step 4. Take the sample solution and add the pH-sensitive composite magnetic nanogel described in claim 1, vortex oscillate to mix, and adsorb in a constant temperature shaker at 30°C for 80-100 minutes to obtain an extract. After applying an external magnetic field, separate the complex of egg yolk phosvitin and the pH-sensitive composite magnetic nanogel, place the complex in an alkaline solution, and remove the external magnetic field to separate the pH-sensitive composite magnetic nanogel to obtain egg yolk phosvitin; the mass volume ratio of the pH-sensitive composite magnetic nanogel to the sample solution in step 4 is (4-5):

1.

3. The method for preparing a pH-sensitive composite magnetic nanogel according to claim 1, wherein: In step S1, the mass volume ratio of Fe3O4 powder to curdlan solution is 1:(2-20).

4. The method for preparing a pH-sensitive composite magnetic nanogel according to claim 1, wherein: In step S2, the mass volume ratio of the magnetic porous gel to the polydopamine solution is 1:(1-2).

5. The method for preparing a pH-sensitive composite magnetic nanogel according to claim 1, wherein: The concentration of the horseradish solution in step S3 is 8%-10%; the mass volume ratio of the polydopamine-coated magnetic porous gel to the horseradish solution is 1:(3-5).

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