Extraction method of vitelline globulin

Abstract

The invention provides a method for extracting vitelline globulin, which comprises the following steps: freezing yolk liquid for 4-24 hours at the temperature of (-6 DEG C)-(-18 DEG C), then returning the temperature for 12 hours at the temperature of 4 DEG C, adding deionized water for diluting, uniformly stirring under the condition of 8000-12000 rpm, then carrying out solid-liquid separation to obtain lipoprotein and supernate, and filtering the supernate by adopting an ultrafiltration membrane to obtain the vitelline globulin. And filtering the solid, and freeze-drying to obtain the IgY vitelline globulin. The method for extracting the vitelline globulin has the advantages of simplicity and easiness in operation, and is suitable for industrial application.

Description

Technical Field

[0001] The invention relates to an extraction method, in particular to a method for extracting yolk globulin. Background Art

[0002] Yolk globulin, also known as yolk antibody, is an antibody present in the yolk of immunized hens. It is transferred from the immunoglobulin in the serum of the hen and is widely used in the field of animal disease prevention and immunodiagnosis. Yolk fluid is a highly emulsified body of oil-in-water, consisting of about 48.7% water, 16.6% protein and 32.6% lipids and other components. The development and utilization of yolk globulin first requires water-oil separation to extract yolk globulin. At present, there are many methods for extracting yolk globulin from yolk fluid, including a method for extracting IgY using the principle of protein liquid-liquid phase separation disclosed in Chinese patent CN117417439A, a method for rapidly extracting yolk antibodies disclosed in Chinese patent CN108623679A, and a method for extracting yolk antibodies IgY disclosed in Chinese patent CN117603345A. However, in the extraction process, these methods require a large amount of chemical reagents, which are easily left in the yolk globulin, affecting the quality of the yolk globulin.

[0003] In order to solve the above problems, people have been seeking an ideal technical solution. Summary of the invention

[0004] The purpose of the present invention is to provide a method for extracting yolk globulin in view of the deficiencies of the prior art.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for extracting yolk globulin comprises the following steps: freezing yolk liquid at (-6°C)-(-18°C) for 4-24 hours, then warming it up at 4°C for 12 hours, adding deionized water for dilution, stirring evenly at 8000-12000 rpm, then performing solid-liquid separation to obtain lipoprotein and supernatant, filtering the supernatant with an ultrafiltration membrane, and obtaining IgY yolk globulin after filtering the solid and freeze-drying it.

[0007] The mass ratio of deionized water to egg yolk liquid is (3-5):1. After adding deionized water, stir evenly and perform solid-liquid separation by standing or centrifugal separation.

[0008] The deionized water is crushed ice or an ice-water mixture, which is added to the egg yolk liquid while stirring. After the crushed ice or ice-water mixture is added, continue stirring for 3-10 seconds until the stirring is completed.

[0009] Supercritical CO2 is used to extract lipoprotein to obtain egg yolk oil and defatted protein. The extraction temperature is 35-45°C, the extraction pressure is 30-40MPa, the extraction time is 4-6h, and the CO2 flow rate is 20-30L / h.

[0010] A sodium chloride solution with a concentration of 5% to 10% is added to the defatted protein, the pH is adjusted to 5 to 7, the mixture is heated to 80 to 100° C., the mixture is mixed evenly, centrifuged, the supernatant is taken, dialyzed with deionized water, centrifuged again, the supernatant is taken, and freeze-dried to obtain phosvitin.

[0011] The eggs of the immunized laying hens are sterilized and then the egg white and the egg yolk are separated to obtain the egg yolk liquid.

[0012] The present invention has outstanding substantial features and significant progress compared to the prior art. Specifically, the present invention provides a method for extracting yolk globulin, which extracts yolk antibody protein by combining freezing and melting. Specifically, after freezing, the macromolecular boundary water in the yolk crystallizes, causing the hydrophobic groups inside the protein to be exposed to the surface, and the intermolecular hydrophobic interaction is enhanced, so that low-density lipoprotein and / or high-density lipoprotein aggregate to form a dense gel network, and finally form yolk gel. After adding water to the frozen-thawed yolk gel, it is crushed, homogenized, and centrifuged. Most of the lipoproteins and yolk particles that are difficult to dissolve in water form a dense gel network and settle at the bottom after centrifugation, while the yolk antibody protein that is easily soluble in water is released into the aqueous phase; in particular, crushed ice of deionized water is added after freezing and thawing, and then stirred, so that the yolk antibody protein is fully dissolved in water, which is convenient for subsequent purification. DETAILED DESCRIPTION

[0013] The technical solution of the present invention is further described in detail below through specific implementation methods.

[0014] Example

[0015] A method for extracting yolk globulin comprises the following steps: sterilizing eggs of immunized laying hens, separating egg white and yolk to obtain egg yolk liquid; freezing the egg yolk liquid at (-6°C)-(-18°C) for 4-24 hours, then warming it up at 4°C for 12 hours, adding deionized water to dilute it, stirring it evenly at 8000-12000rpm, and then performing solid-liquid separation to obtain lipoprotein and supernatant, filtering the supernatant with an ultrafiltration membrane, retaining proteins with a molecular weight of 100-300kD, and freeze-drying to obtain IgY yolk globulin. The mass ratio of deionized water to egg yolk liquid is (3-5):1, stirring evenly after adding deionized water, and performing solid-liquid separation by standing or centrifugal separation. The deionized water is crushed ice or an ice-water mixture, and the crushed ice or ice-water mixture is added to the egg yolk liquid while stirring. After the crushed ice or ice-water mixture is added, stirring is continued for 3-10s to complete the stirring.

[0016] Supercritical CO2 is used to extract lipoprotein to obtain egg yolk oil and defatted protein. The extraction temperature is 35-45°C, the extraction pressure is 30-40MPa, the extraction time is 4-6h, and the CO2 flow rate is 20-30L / h.

[0017] A sodium chloride solution with a concentration of 5% to 10% is added to the defatted protein, the pH is adjusted to 5 to 7, the mixture is heated to 80 to 100° C., the mixture is mixed evenly, centrifuged, the supernatant is taken, dialyzed with deionized water, centrifuged again, the supernatant is taken, and freeze-dried to obtain phosvitin.

[0018] Immunization of laying hens: Two batches of laying hens were immunized with formaldehyde-inactivated quadrivalent influenza virus vaccine and bacterial vaccine respectively; the quadrivalent inactivated influenza virus vaccine was a mixed vaccine of H1N1, H3N2, B (Victoria) and B (Yamagata) in a concentration ratio of 1:1:1:1, and the bacterial vaccine was a mixed vaccine of Escherichia coli and Staphylococcus aureus in a concentration ratio of 1:1.

[0019] Example 1

[0020] A method for extracting yolk globulin comprises the following steps:

[0021] After sterilizing the eggs of the immunized laying hens, the egg whites and egg yolks were separated to obtain egg yolk liquid; the egg yolk liquid was frozen at -18°C for 8 hours, then warmed up at 4°C for 12 hours, and crushed ice or an ice-water mixture was added to the egg yolk liquid while stirring at 10,000 rpm. After the crushed ice or the ice-water mixture was added, stirring was continued for 3 seconds. After stirring was completed, the mixture was centrifuged for solid-liquid separation to obtain lipoprotein and supernatant.

[0022] The results showed that the ratio of the mass of immunoglobulin IgY in the supernatant to the mass of the egg yolk liquid was 10.8 mg / g.

[0023] The supernatant is filtered by an ultrafiltration membrane to retain proteins with a molecular weight of 100-300 kD, and IgY yolk globulin is obtained after freeze-drying. The crushed ice or ice-water mixture is obtained by freezing and crushing deionized water, and the mass ratio of deionized water to egg yolk liquid is 3:1.

[0024] Supercritical CO2 was used to extract lipoprotein to obtain egg yolk oil and defatted protein. The extraction temperature was 37°C, the extraction pressure was 40 MPa, the extraction time was 5 h, and the CO2 flow rate was 30 L / h.

[0025] A 10% sodium chloride solution is added to the defatted protein, the pH is adjusted to 6, heated to 100°C, mixed evenly, centrifuged, the supernatant is taken, dialyzed with deionized water, centrifuged again, the supernatant is taken, and freeze-dried to obtain phosvitin.

[0026] Comparative Example

[0027] A method for extracting yolk globulin comprises the following steps:

[0028] After sterilizing the eggs of immunized laying hens, the egg whites and egg yolks were separated to obtain egg yolk liquid; the egg yolk liquid was frozen at -18°C for 8 hours, then warmed up at 4°C for 12 hours, and deionized water at room temperature (25°C) was added to the egg yolk liquid while stirring at 10,000 rpm. After the addition was completed, stirring was continued for 3 seconds, and then centrifuged for solid-liquid separation to obtain lipoprotein and supernatant.

[0029] The results showed that the ratio of the mass of immunoglobulin IgY in the supernatant to the mass of the egg yolk liquid was 6.4 mg / g.

[0030] The supernatant was filtered by ultrafiltration membrane to retain proteins with a molecular weight of 100-300 kD, and IgY yolk globulin was obtained after freeze-drying. The mass ratio of deionized water to egg yolk liquid was 3:1.

[0031] Example 2

[0032] A method for extracting yolk globulin comprises the following steps:

[0033] After sterilizing the eggs of immunized laying hens, separate the egg white and egg yolk to obtain egg yolk liquid; freeze the egg yolk liquid at -18°C for 8 hours, then warm it up at 4°C for 12 hours, add deionized water to dilute it, stir it evenly at 8000rpm, and then separate the solid and liquid to obtain lipoprotein and supernatant, filter the supernatant with an ultrafiltration membrane, retain proteins with a molecular weight of 100-300kD, and obtain IgY yolk globulin after freeze-drying. The mass ratio of deionized water to egg yolk liquid is 5:1, stir evenly after adding deionized water, and perform solid-liquid separation by standing or centrifugation; the deionized water is crushed ice or an ice-water mixture, and crushed ice or an ice-water mixture is added to the egg yolk liquid while stirring. After the crushed ice or the ice-water mixture is added, continue stirring for 5s to complete the stirring;

[0034] Supercritical CO2 was used to extract lipoprotein to obtain egg yolk oil and defatted protein. The extraction temperature was 35°C, the extraction pressure was 30 MPa, the extraction time was 6 h, and the CO2 flow rate was 30 L / h.

[0035] A 5% sodium chloride solution is added to the defatted protein, the pH is adjusted to , the mixture is heated to 100°C, the mixture is mixed evenly, the mixture is centrifuged, the supernatant is taken, the mixture is dialyzed with deionized water, the mixture is centrifuged again, the supernatant is taken, and the mixture is freeze-dried to obtain phosvitin.

[0036] Example 3

[0037] A method for extracting yolk globulin comprises the following steps:

[0038] After sterilizing the eggs of immunized laying hens, separate the egg white and egg yolk to obtain egg yolk liquid; freeze the egg yolk liquid at -18°C for 6 hours, then warm it up at 4°C for 12 hours, add deionized water to dilute it, stir it evenly at 12000rpm, and then separate the solid and liquid to obtain lipoprotein and supernatant, filter the supernatant with an ultrafiltration membrane, retain proteins with a molecular weight of 100-300kD, and obtain IgY yolk globulin after freeze-drying. The mass ratio of deionized water to egg yolk liquid is 4:1, stir evenly after adding deionized water, and perform solid-liquid separation by centrifugation; the deionized water is crushed ice or an ice-water mixture, and crushed ice or an ice-water mixture is added to the egg yolk liquid while stirring. After the crushed ice or the ice-water mixture is added, continue stirring for 5s to complete the stirring;

[0039] Supercritical CO2 was used to extract lipoprotein to obtain egg yolk oil and defatted protein. The extraction temperature was 35-45°C, the extraction pressure was 40MPa, the extraction time was 4h, and the CO2 flow rate was 30L / h.

[0040] A 10% sodium chloride solution is added to the defatted protein, the pH is adjusted to 6, heated to 100°C, mixed evenly, centrifuged, the supernatant is taken, dialyzed with deionized water, centrifuged again, the supernatant is taken, and freeze-dried to obtain phosvitin.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or some technical features can be replaced by equivalents without departing from the spirit of the technical solution of the present invention, which should be included in the scope of the technical solution for protection of the present invention.

Claims

1. A method for extracting yolk globulin, comprising the following steps: The egg yolk liquid was frozen at (-6°C) - (-18°C) for 4-24 hours, then warmed to 4°C for 12 hours, diluted with deionized water, stirred evenly at 8000-12000 rpm, and then separated from the solid and liquid to obtain lipoprotein and supernatant. The supernatant was filtered using an ultrafiltration membrane, and the filtered solid was freeze-dried to obtain IgY yolk globulin.

2. The method for extracting yolk globulin according to claim 1, characterized in that: The mass ratio of deionized water to egg yolk liquid is (3-5):

1. After adding deionized water, stir evenly and perform solid-liquid separation by standing or centrifugal separation.

3. The method for extracting yolk globulin according to claim 2, characterized in that: The deionized water is crushed ice or an ice-water mixture, which is added to the egg yolk liquid while stirring. After the crushed ice or ice-water mixture is added, continue stirring for 3-10 seconds until the stirring is completed.

4. The method for extracting yolk globulin according to claim 3, characterized in that: Supercritical CO2 is used to extract lipoprotein to obtain egg yolk oil and defatted protein. The extraction temperature is 35-45℃, the extraction pressure is 30-40 MPa, the extraction time is 4-6 h, and the CO2 flow rate is 20-30 L / h.

5. The method for extracting yolk globulin according to claim 4, characterized in that: A sodium chloride solution with a concentration of 5%-10% is added to the defatted protein, the pH is adjusted to 5-7, the mixture is heated to 80-100°C, mixed evenly, centrifuged, the supernatant is taken, dialyzed with deionized water, centrifuged again, the supernatant is taken, and freeze-dried to obtain phosvitin.

6. The method for extracting yolk globulin according to claim 5, characterized in that: The eggs of the immunized laying hens are sterilized and then the egg white and the egg yolk are separated to obtain the egg yolk liquid.