Extraction method and application of immune globulin in pig blood

Abstract

The invention discloses a method for extracting immune globulin in pig blood and application of the method, and relates to the technical field of fresh pig blood extraction.The method comprises the following steps that S1, after fresh pig blood and an anticoagulant are evenly mixed, low-temperature standing and centrifugation are conducted, plasma liquid is separated out and stored, and the plasma liquid is obtained; s2, extracting the plasma liquid through three times of ethanol reaction to obtain an immune globulin crude product; s3, carrying out three-stage purification on the immune globulin crude product, namely precipitation treatment, hollow fiber ultrafiltration column dealcoholization treatment and affinity chromatography treatment, and finally, dialyzing and freeze-drying to obtain an immune globulin IgG finished product. According to the method for extracting the immune globulin in the pig blood, the extraction rate and the purity of the immune globulin IgG can be effectively improved.

Description

Technical Field

[0001] This application relates to the field of fresh pig blood extraction technology, and in particular to a method for extracting immunoglobulins from pig blood and its application. Background Technology

[0002] Blood is the carrier of substances in the body, containing various nutrients such as proteins, amino acids, and immune substances. Blood is composed of plasma and blood cells. Plasma contains a complex mixture of proteins, mainly albumin, globulins, and fibrinogen. Pig blood is rich in immunoglobulin G (IgG), the main immunoglobulin responsible for immune function in mammals, accounting for 75% of total blood immunoglobulins. The application of immunoglobulin IgG extracted from pig blood in functional foods has significant health benefits and market potential. Immunoglobulin IgG can enhance the body's metabolic function, improve physical strength and endurance, and reduce fatigue.

[0003] Currently, the main methods for separating IgG include ammonium sulfate fractionation precipitation, ethanol separation, ion exchange, coagulation filtration, affinity chromatography, and ultrafiltration. Ethanol separation, by adjusting the ethanol concentration and pH value, can effectively precipitate immunoglobulins, especially IgG, while effectively removing other proteins and impurities, thus improving the purity of the target protein. However, existing ethanol separation methods still suffer from low recovery rates and insufficient purity, hindering their application in functional foods. Summary of the Invention

[0004] To improve the extraction rate and purity of immunoglobulins from pig blood, this application provides a method for extracting immunoglobulins from pig blood and its application.

[0005] This application provides a method for extracting immunoglobulins from porcine blood, employing the following technical solution:

[0006] A method for extracting immunoglobulins from pig blood, comprising the following steps:

[0007] S1. Mix fresh pig blood and anticoagulant, let stand at 3-5℃ for 30-40 min; centrifuge at 1000-1500 rpm at 3-5℃ for 30-50 min; collect the supernatant; centrifuge again at 3000-4000 rpm at 3-5℃ for 30-50 min, collect the supernatant; the plasma is obtained; store at -20℃.

[0008] S2. Extract the plasma liquid through three ethanol reactions to obtain crude immunoglobulin product;

[0009] S3. Dissolve the crude immunoglobulin product in water at 2-4℃ and adjust the pH to 4-4.5. After clarification, filter and collect the precipitate. Dissolve the precipitate in water and ultrafilter it through a hollow fiber ultrafiltration column to remove alcohol. Then dissolve the precipitate in buffer solution and perform affinity chromatography. Subsequently, adjust the protein concentration to 6-8% with water and dialyze it several times with equal volumes of water at 7-8℃. Collect the precipitate and freeze-dry it to obtain the finished immunoglobulin IgG product.

[0010] Preferably, the anticoagulant is a composition of trisodium citrate, glucose, and disodium EDTA with a mass fraction of 3.8%, in a mass ratio of [missing value]. .

[0011] Preferably, the volume ratio of the fresh pig blood to the anticoagulant is: .

[0012] Preferably, the three-stage ethanol extraction includes the following steps:

[0013] First ethanol reaction: Dilute the plasma with sodium chloride solution to a protein content of 5-6%, adjust the pH of the liquid to 6.8-7.1, and cool to -1.5-0.5℃; add ethanol at -15-(-10)℃ under stirring, the amount of ethanol added being 0.25-0.28 of the plasma liquid; adjust the pH to 6.8-7.1 again, and stir the reaction at -5-(-4.5)℃ for 1-2 hours, then centrifuge at -5-(-3)℃ for 30-50 minutes, and collect the precipitate;

[0014] Second ethanol reaction: Dissolve the precipitate in sodium chloride at -2 to 0℃ and bring the volume up to 12 to 14 times the volume of the precipitate; after the precipitate is fully dissolved, adjust the pH of the solution to 5.4 to 5.6 and cool it to -1 to (-0.5)℃; add ethanol at -25 to (-20)℃ under stirring, the amount of ethanol added being 0.14 to 0.15 of the plasma liquid; adjust the pH to 5.4 to 5.6 again and stir the reaction at -5.3 to (-4.8)℃ for 1 to 2 hours; then centrifuge at -5 to (-3)℃ for 30 to 50 minutes and take the supernatant.

[0015] Third ethanol reaction: Sodium chloride is added to the supernatant, and ethanol at -15-(-10)℃ is added under stirring; the amount of ethanol added is 0.18-0.2 of the plasma liquid; the pH is adjusted to 6.9-7.1; the reaction is stirred at -5-(-4)℃ for 1-2 hours, and then centrifuged at -5-(-4)℃ for 30-40 minutes. The precipitate is the crude globulin.

[0016] Preferably, the concentration of the ethanol is 95-98%.

[0017] Preferably, the buffer solution in S3 is a phosphate buffer solution with a pH of 7.4-7.5.

[0018] Preferably, the affinity chromatography medium in S3 is Staphylococcus aureus protein A affinity chromatography medium.

[0019] Preferably, the method for preparing the Staphylococcus aureus protein A affinity chromatography medium includes the following steps:

[0020] Add Staphylococcus aureus protein A to a pH of 8-10. Dissolve the 15-20 mg / mL Staphylococcus aureus protein A solution in buffer solution; suspend the Rigose HF matrix in sodium hydroxide solution containing epichlorohydrin and react with shaking at 30-40℃ for 4-6 h to obtain an epoxy-activated matrix; repeatedly wash the activated matrix with water and dry it; add the Staphylococcus aureus protein A solution to the dried matrix and react with shaking in a constant temperature water bath at 35-45℃ for 12-15 h; after the reaction, wash it with pure water and add ethanol; store at 4-5℃.

[0021] Preferably, the solid-liquid ratio of the dried matrix and the Staphylococcus aureus protein A solution is 1g:1-1.5mL.

[0022] This application provides an application of immunoglobulins in porcine blood, employing the following technical solution:

[0023] An application of an immunoglobulin from pig blood, and the application of said immunoglobulin in functional foods.

[0024] In summary, this application includes at least one of the following beneficial technical effects:

[0025] 1. This application employs a three-stage ethanol extraction process, gradually adjusting pH and ethanol concentration to effectively remove impurities and progressively enrich immunoglobulins, thereby improving the extraction rate and purity of immunoglobulin IgG. Furthermore, this application combines a three-stage purification process: precipitation, hollow fiber ultrafiltration column de-ethanolification, and affinity chromatography, significantly enhancing the extraction rate and purity. Precipitation: By adjusting pH and temperature, complete precipitation of IgG is ensured, reducing interference from impurities and improving initial purity. Precipitation is performed at low temperatures to prevent protein denaturation and maintain its biological activity. Hollow fiber ultrafiltration column de-ethanolification: Removes residual ethanol and other small molecule impurities while concentrating IgG, improving the purity and concentration of the solution. Affinity chromatography: Using Staphylococcus aureus protein A affinity chromatography medium, IgG is specifically bound, further improving purity and removing non-specifically bound impurities, ensuring high purity and high quality of the final product.

[0026] 2. The method for extracting immunoglobulins from pig blood provided in this application yields immunoglobulins with high purity, reaching over 97%, which can be effectively applied in functional foods. Attached Figure Description

[0027] Figure 1 This is a physical image of the immunoglobulin obtained in this application. Detailed Implementation

[0028] The present application will be further described in detail below with reference to the embodiments.

[0029] The chemical reagents used in the preparation examples, embodiments, and comparative examples provided in this invention are all commercially available products.

[0030] Example 1

[0031] S1. Mix 90 mL of fresh pig blood with 10 mL of anticoagulant, let stand at 3°C ​​for 30 min, then centrifuge at 3°C ​​and 1000 rpm for 30 min; collect the supernatant; centrifuge again at 3°C ​​and 3000 rpm for 30 min, collect the supernatant; this yields plasma; store at -20°C; the anticoagulant is a composition of 3.8% by mass of trisodium citrate, glucose, and disodium EDTA, in a mass ratio of 6:3:1;

[0032] S2. Extract the plasma liquid through a three-stage ethanol reaction to obtain the crude immunoglobulin product; the specific operation is as follows:

[0033] First ethanol reaction: Dilute the plasma with 0.154 mol / L sodium chloride solution to a protein content of 5%, adjust the pH of the solution to 6.8 with 1 mol / L citric acid, and cool to -1.5℃; add 95% ethanol at -15℃ under stirring, the amount of ethanol added being 0.25% of the plasma volume; adjust the pH to 6.8 again, stir and react at -5℃ for 1 h, then centrifuge at -5℃ for 30 min, and collect the precipitate;

[0034] Second ethanol reaction: The precipitate was dissolved in 0.01 mol / L sodium chloride solution at -2℃, and the volume was adjusted to 12 times the volume of the precipitate. After the precipitate was fully dissolved, the pH of the solution was adjusted to 5.4 with an acetate-sodium acetate buffer solution at pH 4, and the temperature was lowered to -1℃. 95% ethanol at -25℃ was added under stirring, with the amount of ethanol added being 0.14 times the volume of the plasma. The pH was adjusted to 5.4 again, and the reaction was stirred at -5.3℃ for 1 hour. The mixture was then centrifuged at -5℃ for 30 minutes, and the supernatant was collected.

[0035] Third ethanol reaction: Add 5g of sodium chloride to each 1L of supernatant, and add 95% ethanol at -15℃ under stirring conditions; the amount of ethanol added is 0.18 of the plasma volume; adjust the pH to 6.9 with 0.5mol / L sodium hydroxide; stir and react at -5℃ for 1h, then centrifuge at -5℃ for 30min, and the precipitate is the crude globulin.

[0036] S3. Dissolve the crude immunoglobulin product in deionized water at 2°C, adjust the pH to 4 with 1 mol / L acetic acid, clarify, filter, and collect the precipitate; dissolve the precipitate in deionized water at 2°C, and ultrafilter through a hollow fiber ultrafiltration column to remove alcohol; then dissolve the precipitate in phosphate buffer at pH 7.4 and perform affinity chromatography; subsequently adjust the protein concentration to 6% with deionized water at 2°C, dialyze several times with equal volumes of water at 7°C, collect the precipitate, and freeze-dry to obtain the finished immunoglobulin IgG product;

[0037] The affinity chromatography medium is Staphylococcus aureus protein A affinity chromatography medium;

[0038] The method for preparing the Staphylococcus aureus protein A affinity chromatography medium includes the following steps:

[0039] Add Staphylococcus aureus protein A to a pH of 8 Dissolve in buffer solution to obtain a 15 mg / mL Staphylococcus aureus protein A solution; suspend Rigose HF matrix in a 2 mol / L sodium hydroxide solution containing epichlorohydrin, with a solid-liquid ratio of 1 g: 0.1 mL for the Rigose HF matrix to epichlorohydrin, and react with shaking at 30 °C for 6 h to obtain an epoxy-activated matrix. Wash the activated matrix repeatedly with water and dry it. Add 1 mL of Staphylococcus aureus protein A solution to 1 g of the dried matrix and react with shaking in a water bath at 35 °C for 15 h. After the reaction, wash with pure water, add 20% ethanol (v / v), and store at 4 °C.

[0040] Example 2

[0041] S1. Mix 90 mL of fresh pig blood with 10 mL of anticoagulant, let stand at 4°C for 35 min, then centrifuge at 1250 rpm at 4°C for 40 min; collect the supernatant; centrifuge again at 3500 rpm at 4°C for 40 min, collect the supernatant; this yields plasma; store at -20°C; the anticoagulant is a composition of 3.8% by mass of trisodium citrate, glucose, and disodium EDTA, in a mass ratio of 6:3:1;

[0042] S2. Extract the plasma liquid through a three-stage ethanol reaction to obtain the crude immunoglobulin product; the specific operation is as follows:

[0043] First ethanol reaction: Dilute the plasma with 0.154 mol / L sodium chloride solution to a protein content of 5.5%, adjust the pH of the solution to 7 with 1 mol / L citric acid, and cool to 0°C; add 96.5% ethanol at -12.5°C under stirring, the amount of ethanol added being 0.27 of the plasma volume; adjust the pH to 7 again, stir and react at -5°C for 1.5 h, then centrifuge at -4°C for 40 min, and collect the precipitate;

[0044] Second ethanol reaction: The precipitate was dissolved in 0.01 mol / L sodium chloride solution at -1℃, and the volume was adjusted to 13 times the volume of the precipitate. After the precipitate was fully dissolved, the pH of the solution was adjusted to 5.5 with an acetate-sodium acetate buffer solution at pH 4, and the temperature was lowered to -1℃. 96.5% ethanol at -22.55℃ was added under stirring, and the amount of ethanol added was 0.145 times the volume of the plasma. The pH was adjusted to 5.5 again, and the reaction was stirred at -5℃ for 1.5 h. The mixture was then centrifuged at -4℃ for 40 min, and the supernatant was collected.

[0045] Third ethanol reaction: Add 5.5g sodium chloride to each 1L of supernatant, and add 96.5% ethanol at -12.5℃ under stirring conditions; the amount of ethanol added is 0.19 of the plasma volume; adjust the pH to 7 with 0.5mol / L sodium hydroxide; stir and react at -4.5℃ for 1.5h, and then centrifuge at -4.5℃ for 35min. The precipitate is the crude globulin.

[0046] S3. Dissolve the crude immunoglobulin product in deionized water at 3°C, adjust the pH to 4.3 with 1 mol / L acetic acid, clarify, filter, and collect the precipitate; dissolve the precipitate in deionized water at 3°C, and ultrafilter through a hollow fiber ultrafiltration column to remove alcohol; then dissolve the precipitate in phosphate buffer at pH 7.4, and perform affinity chromatography; subsequently adjust the protein concentration to 7% with deionized water at 3°C, dialyze several times with equal volumes of water at 7°C, collect the precipitate, and freeze-dry to obtain the finished immunoglobulin IgG product;

[0047] The affinity chromatography medium is Staphylococcus aureus protein A affinity chromatography medium;

[0048] The method for preparing the Staphylococcus aureus protein A affinity chromatography medium includes the following steps:

[0049] Staphylococcus aureus protein A was dissolved in a Na₂CO₃ / NaHCO₃ buffer solution at pH 9 to obtain a Staphylococcus aureus protein A solution with a concentration of 17.5 mg / mL. Rigose HF matrix was suspended in a 2 mol / L sodium hydroxide solution containing epichlorohydrin, with a solid-liquid ratio of 1 g: 0.15 mL. The mixture was shaken at 35 °C for 5 h to obtain an epoxy-activated matrix. The activated matrix was repeatedly washed with water and dried. 1 g of the dried matrix was added to 1 mL of Staphylococcus aureus protein A solution, and the mixture was shaken in a constant temperature water bath at 40 °C for 13.5 h. After the reaction, the matrix was washed with pure water, and 20% ethanol was added. The matrix was then stored at 4.5 °C.

[0050] Example 3

[0051] S1. Mix 90 mL of fresh pig blood with 10 mL of anticoagulant, let stand at 5°C for 50 min, then centrifuge at 1500 rpm at 5°C for 50 min; collect the supernatant; then centrifuge at 4000 rpm at 5°C for 50 min, collect the supernatant; this yields plasma; store at -20°C; the anticoagulant is a composition of 3.8% by mass of trisodium citrate, glucose, and disodium EDTA, in a mass ratio of 6:3:1;

[0052] S2. Extract the plasma liquid through a three-stage ethanol reaction to obtain the crude immunoglobulin product; the specific operation is as follows:

[0053] First ethanol reaction: Dilute the plasma with 0.154 mol / L sodium chloride solution to a protein content of 6%, adjust the pH of the solution to 7.1 with 1 mol / L citric acid, and cool to -0.5℃; add 98% ethanol at -10℃ under stirring, the amount of ethanol added being 0.28% of the plasma volume; adjust the pH to 7.1 again, stir and react at -4.5℃ for 2 hours, then centrifuge at -3℃ for 30 minutes, and collect the precipitate;

[0054] Second ethanol reaction: The precipitate was dissolved in 0.01 mol / L sodium chloride solution at 0℃, and the volume was adjusted to 14 times the volume of the precipitate. After the precipitate was fully dissolved, the pH of the solution was adjusted to 5.6 with an acetate-sodium acetate buffer solution at pH 4, and the temperature was lowered to -0.5℃. 98% ethanol at -20℃ was added under stirring, and the amount of ethanol added was 0.15 times the volume of the plasma. The pH was adjusted to 5.6 again, and the reaction was stirred at -4.8℃ for 2 hours. The mixture was then centrifuged at -3℃ for 50 minutes, and the supernatant was collected.

[0055] Third ethanol reaction: Add 6g of sodium chloride to each 1L of supernatant, and add 98% ethanol at -10℃ under stirring conditions; the amount of ethanol added is 0.2 times the volume of plasma; adjust the pH to 7.1 with 0.5mol / L sodium hydroxide; stir and react at -4℃ for 2h, and then centrifuge at -4℃ for 40min. The precipitate is the crude globulin.

[0056] S3. Dissolve the crude immunoglobulin product in deionized water at 4°C, adjust the pH to 4.5 with 1 mol / L acetic acid, clarify, filter, and collect the precipitate; dissolve the precipitate in deionized water at 4°C, and ultrafilter through a hollow fiber ultrafiltration column to remove alcohol; then dissolve the precipitate in phosphate buffer at pH 7.5 and perform affinity chromatography; subsequently adjust the protein concentration to 8% with deionized water at 4°C, dialyze several times with equal volumes of water at 8°C, collect the precipitate, and freeze-dry to obtain the finished immunoglobulin IgG product;

[0057] The affinity chromatography medium is Staphylococcus aureus protein A affinity chromatography medium;

[0058] The method for preparing the Staphylococcus aureus protein A affinity chromatography medium includes the following steps:

[0059] Add Staphylococcus aureus protein A to a pH of 10 Dissolve in buffer solution to obtain a 20 mg / mL Staphylococcus aureus protein A solution; suspend Rigose HF matrix in a 2 mol / L sodium hydroxide solution containing epichlorohydrin, with a solid-liquid ratio of 1 g: 0.2 mL for the Rigose HF matrix to epichlorohydrin, and react with shaking at 40 °C for 4 h to obtain an epoxy-activated matrix. Wash the activated matrix repeatedly with water and dry it. Add 1 mL of Staphylococcus aureus protein A solution to 1 g of the dried matrix and react with shaking in a water bath at 45 °C for 12 h. After the reaction, wash with pure water, add 20% ethanol (v / v), and store at 5 °C.

[0060] Example 4

[0061] The difference between Example 4 and Example 1 is that the anticoagulant used in Example 4 is a composition of trisodium citrate, glucose and disodium EDTA with a mass fraction of 3.8% and a mass ratio of 6.5:2.5:1.

[0062] Example 5

[0063] The difference between Example 5 and Example 1 is that the anticoagulant used in Example 5 is a composition of trisodium citrate, glucose, and disodium EDTA with a mass fraction of 3.8% and a mass ratio of 7:2:1.

[0064] Example 6

[0065] The difference between Example 6 and Example 1 is that the anticoagulant used in Example 6 only includes 3.8% trisodium citrate.

[0066] Example 7

[0067] The difference between Example 7 and Example 1 is that Example 7 uses 100 mL of fresh pig blood and 10 mL of anticoagulant.

[0068] Example 8

[0069] The difference between Example 8 and Example 1 is that Example 8 uses 110 mL of fresh pig blood and 10 mL of anticoagulant.

[0070] Example 9

[0071] The difference between Example 9 and Example 1 is that Example 9 uses 80 mL of fresh pig blood and 10 mL of anticoagulant.

[0072] Example 10

[0073] The difference between Example 10 and Example 1 is that Example 10 uses 120 mL of fresh pig blood and 10 mL of anticoagulant.

[0074] Example 11

[0075] The difference between Example 11 and Example 1 is that the dried matrix used in Example 11 is 1g, and the Staphylococcus aureus protein A solution is 1.25mL.

[0076] Example 12

[0077] The difference between Example 12 and Example 1 is that the dried matrix used in Example 12 is 1g, and the Staphylococcus aureus protein A solution is 1.5mL.

[0078] Example 13

[0079] The difference between Example 13 and Example 1 is that the dried matrix used in Example 13 is 1g, and the Staphylococcus aureus protein A solution is 0.5mL.

[0080] Example 14

[0081] The difference between Example 14 and Example 1 is that the dried matrix used in Example 14 is 1g, and the Staphylococcus aureus protein A solution is 2mL.

[0082] Comparative Example 1

[0083] The difference between Comparative Example 1 and Example 1 is that the crude immunoglobulin product in Comparative Example 1 was not subjected to precipitation treatment.

[0084] Comparative Example 2

[0085] The difference between Comparative Example 2 and Example 1 is that the crude immunoglobulin product in Comparative Example 3 was not subjected to ultrafiltration and alcohol removal treatment using a hollow fiber ultrafiltration column.

[0086] Comparative Example 3

[0087] The difference between Comparative Example 3 and Example 1 is that the crude immunoglobulin product in Comparative Example 3 was not subjected to affinity chromatography.

[0088] Performance testing

[0089] Immunoglobulin IgG in fresh pig blood before the experiments in Examples 1-14 and Comparative Examples 1-3 was detected using SDS-PAGE, and the result was recorded as the theoretical maximum extraction amount. After the experiments, the amount and purity of the extracted immunoglobulin IgG were detected again using SDS-PAGE, and the extraction rate of immunoglobulin was calculated according to the following formula:

[0090]

[0091] The results are shown in Table 1.

[0092] The specific test results are as follows:

[0093]

[0094] As can be seen from the test results in Table 1, the method for extracting immunoglobulins from boar blood provided in this application embodiment can better extract immunoglobulin IgG components from boar plasma, and has a high extraction rate and product purity.

[0095] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for extracting immunoglobulins from pig blood, characterized in that: Includes the following steps: S1. Mix fresh pig blood and anticoagulant, let stand at 3-5℃ for 30-40 min; centrifuge at 1000-1500 rpm at 3-5℃ for 30-50 min; collect the supernatant; centrifuge again at 3000-4000 rpm at 3-5℃ for 30-50 min, collect the supernatant; the plasma is obtained; store at -20℃. S2. Extract the plasma liquid through three ethanol reactions to obtain crude immunoglobulin product; S3. Dissolve the crude immunoglobulin product in water at 2-4℃, adjust the pH to 4-4.5, clarify, filter, and collect the precipitate; The precipitate was dissolved in water and then ultrafiltered through a hollow fiber ultrafiltration column to remove alcohol. The precipitate was then dissolved in a buffer solution and subjected to affinity chromatography. Subsequently, the protein concentration was adjusted to 6-8% with water, and the mixture was dialyzed several times with equal volumes of water at 7-8°C. The precipitate was collected and freeze-dried to obtain the immunoglobulin IgG product.

2. The method for extracting immunoglobulins from porcine blood according to claim 1, characterized in that: The anticoagulant is a composition of trisodium citrate, glucose, and disodium EDTA with a mass fraction of 3.8%, in a mass ratio of 6-7:2-3:

1.

3. The method for extracting immunoglobulins from porcine blood according to claim 2, characterized in that: The volume ratio of the fresh pig blood to the anticoagulant is 9-11:

1.

4. The method for extracting immunoglobulins from porcine blood according to claim 1, characterized in that: The three-stage ethanol extraction includes the following steps: First ethanol reaction: Dilute the plasma with sodium chloride solution to a protein content of 5-6%, adjust the pH of the liquid to 6.8-7.1, and cool to -1.5-0.5℃; add ethanol at -15-(-10)℃ under stirring, the amount of ethanol added being 0.25-0.28 of the plasma liquid; adjust the pH to 6.8-7.1 again, and stir the reaction at -5-(-4.5)℃ for 1-2 hours, then centrifuge at -5-(-3)℃ for 30-50 minutes, and collect the precipitate; Second ethanol reaction: Dissolve the precipitate in sodium chloride at -2 to 0℃ and bring the volume up to 12 to 14 times the volume of the precipitate; after the precipitate is fully dissolved, adjust the pH of the solution to 5.4 to 5.6 and cool it to -1 to (-0.5)℃; add ethanol at -25 to (-20)℃ under stirring, the amount of ethanol added being 0.14 to 0.15 of the plasma liquid; adjust the pH to 5.4 to 5.6 again and stir the reaction at -5.3 to (-4.8)℃ for 1 to 2 hours; then centrifuge at -5 to (-3)℃ for 30 to 50 minutes and take the supernatant. Third ethanol reaction: Sodium chloride is added to the supernatant, and ethanol at -15-(-10)℃ is added under stirring; the amount of ethanol added is 0.18-0.2 of the plasma liquid; the pH is adjusted to 6.9-7.1; the reaction is stirred at -5-(-4)℃ for 1-2 hours, and then centrifuged at -5-(-4)℃ for 30-40 minutes. The precipitate is the crude globulin.

5. The method for extracting immunoglobulins from porcine blood according to claim 4, characterized in that: The concentration of the ethanol is 95-98%.

6. The method for extracting immunoglobulins from porcine blood according to claim 1, characterized in that: The buffer solution described in S3 is a phosphate buffer solution with a pH of 7.4-7.

5.

7. The method for extracting immunoglobulins from porcine blood according to claim 1, characterized in that: The affinity chromatography medium described in S3 is Staphylococcus aureus protein A affinity chromatography medium.

8. The method for extracting immunoglobulins from porcine blood according to claim 1, characterized in that: The method for preparing the Staphylococcus aureus protein A affinity chromatography medium includes the following steps: Staphylococcus aureus protein A was dissolved in a Na₂CO₃ / NaHCO₃ buffer solution with a pH of 8-10 to obtain a Staphylococcus aureus protein A solution with a concentration of 15-20 mg / mL. Rigose HF matrix was suspended in a sodium hydroxide solution containing epichlorohydrin and reacted with shaking at 30-40℃ for 4-6 h to obtain an epoxy-activated matrix. The activated matrix was repeatedly washed with water and dried. Staphylococcus aureus protein A solution was added to the dried matrix and reacted with shaking in a constant temperature water bath at 35-45℃ for 12-15 h. After the reaction, the matrix was washed with pure water and ethanol was added. The matrix was then stored at 4-5℃.

9. The method for extracting immunoglobulins from porcine blood according to claim 8, characterized in that: The solid-liquid ratio of the dried matrix and the Staphylococcus aureus protein A solution is 1g:1-1.5mL.

10. The application of an immunoglobulin in porcine blood according to any one of claims 1-9, characterized in that: The application of the immunoglobulins in functional foods.

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