Preparation method of high-content immune globulin rich in secretory immune globulin A

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of high-content immune globulin rich in secretory immune globulin A. The invention discloses a preparation method of high-content immune globulin rich in secretory immune globulin A. The preparation method comprises the following steps: (1) centrifugally degreasing bovine coloctrum to obtain degreased bovine coloctrum whey; (2) adjusting the pH value of the defatted bovine colostrum whey to 7.0-7.4, and centrifuging to obtain a supernatant; (3) performing ultrafiltration concentration and freeze drying on the supernate to obtain immune globulin; according to the immune globulin prepared by the method, the SIgA content in the immune globulin is 8-15%, and the total content of secretory immune globulin A, immune globulin M and immune globulin G in the immune globulin is greater than 60%; the preparation method provided by the invention has no complex pretreatment step, and is a simple, rapid and efficient method for preparing high-content immune globulin rich in secretory immune globulin A.

Description

Technical Field

[0001] The present application relates to the technical field of biology, and in particular to a method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A. Background Art

[0002] Bovine colostrum is the milk produced by dairy cows in the first few days after giving birth. It not only contains abundant nutrients such as protein, fat, inorganic salts, and vitamins, but also contains numerous bioactive substances such as cytokines, growth factors, and immune factors. Immunoglobulins (Igs) are the most prominent immune factors in colostrum, serving as the most important defense against pathogens such as viruses and bacteria. Among Igs, SIgA is the most important antibody in human mucosal immunity. It is highly stable and highly resistant to hydrolysis by acids, bases, and proteases. Its half-life on mucosal surfaces is three times that of IgG, and its protective effect in the exocrine tract can last for over four months. Its ability to prevent bacterial adhesion to the intestinal mucosa is 7-10 times greater than that of other immunoglobulins. Over 95% of infections occur in or through the mucosa. SIgA participates in mucosal immunity, building a defensive barrier against bacterial and viral pathogens and playing a powerful role in infection prevention. The protective effect of SIgA against infection has been confirmed in numerous clinical and research studies. Studies have shown that SIgA can block the attachment of avian influenza viruses, whereas single-chain IgA and IgG do not. The significant reduction in the incidence of gastroenteritis, diarrhea, otitis media, neonatal sepsis, and allergies in breastfed infants is attributed to the significant role played by the large amounts of SIgA in colostrum.

[0003] The immunoglobulins in bovine colostrum primarily include immunoglobulin M (IgM), secretory immunoglobulin A (SIgA), and immunoglobulin G (IgG). IgG is a monomeric molecule composed of two heavy chains and two light chains, with a molecular weight of approximately 150 kDa. IgM is a multimer composed of five monomeric molecules, with a molecular weight of approximately 950 kDa. IgA is divided into two types: serum (monomeric) and secretory (dimeric, SIgA). Secretory SIgA is predominant in bovine colostrum, with a molecular weight of 320-420 kDa. The molecular weight differences among the three immunoglobulins form one of the foundations for their separation and enrichment using molecular sieves and ultrafiltration techniques. The immunoglobulin content in bovine colostrum is 40-150 times that of normal milk. IgG is the highest in bovine colostrum, at 50-100 mg / ml, accounting for 80%-90% of the total Igs. IgM and SIgA are each approximately 3-6 mg / ml, each accounting for approximately 5-10% of the total Igs. Bovine colostrum SIgA shares the same immunogenicity as human colostrum SIgA, inhibiting bacterial proliferation, neutralizing toxins, and protecting the intestinal mucosa. It also resists the action of proteases, preventing digestion and inactivation. Scientifically processed to remove components such as casein, lactoglobulin, and lactose, and to increase SIgA content, bovine colostrum is "breast-milk-like," making it an ideal substitute for human colostrum.

[0004] Currently, the main methods for preparing immunoglobulins from animal colostrum include salting-out, molecular sieves, ion exchange, and ultrafiltration. These methods require time-consuming and labor-intensive sample pretreatment. Ultrafiltration separation technology is widely used for large-scale preparation of biomacromolecules, particularly heat-sensitive immunoglobulins, due to its low cost, high throughput, energy-saving and environmentally friendly operation, mild operating conditions, and the absence of temperature and pH fluctuations and protein inactivation. However, preparation using ultrafiltration requires sample pretreatment, making the process relatively complex.

[0005] Therefore, how to develop a method that is simple in process and can obtain high-content immunoglobulins rich in secretory immunoglobulin A has become an urgent problem to be solved. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present application provides a method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A.

[0007] The present application discloses a method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A. This method omits the sample pretreatment step and directly ultrafiltration concentrates bovine colostrum using a 200kDa membrane. The SIgA content in the immunoglobulin sample can reach 8-15%, and the total Igs content in the immunoglobulin sample (i.e., the total content of secretory immunoglobulin A, immunoglobulin M, and immunoglobulin G) can reach over 60%. This method omits the labor-intensive and time-consuming sample pretreatment step, greatly simplifying the preparation process. It is a simple, efficient, and rapid method for preparing animal Igs, suitable for industrial-scale production of bovine colostrum products rich in SIgA and high in Ig content.

[0008] In a first aspect, the present application provides a method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A, using the following technical solution: A method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A, the preparation method comprises the following steps: (1) centrifuging and defatting the bovine colostrum to obtain defatted bovine colostrum whey; (2) adjusting the pH value of the defatted bovine colostrum whey to 7.0-7.4, centrifuging, and obtaining a supernatant; (3) The supernatant is concentrated by ultrafiltration and freeze-dried to obtain immunoglobulin.

[0009] By adopting the above-mentioned technical scheme, the present application adopts the steps of centrifugal defatting, adjusting the pH value, centrifugal clarification, ultrafiltration concentration and freeze drying to prepare a high-content immunoglobulin rich in secretory immunoglobulin A. The complicated sample pretreatment steps are eliminated in the preparation method, which not only avoids the denaturation and inactivation of immunoglobulin Ig during the preparation process, but also reduces the loss of immunoglobulin Ig caused by multiple operations. At the same time, a large amount of impurities are removed, thereby increasing the immunoglobulin Ig content in the sample.

[0010] In the present application, the pH of the solution is adjusted as far away from the isoelectric point as possible by adjusting the pH, thereby effectively ensuring the extraction of immunoglobulins.

[0011] Preferably, in step (1), when the bovine colostrum is subjected to centrifugal degreasing, the conditions for centrifugal degreasing are as follows: the temperature for centrifugal degreasing is 4-8°C; the speed for centrifugal degreasing is 4000-8000 r / min; and the time for centrifugal degreasing is 10-30 min.

[0012] Preferably, in step (1), the prepared defatted bovine colostrum whey is post-processed, and the steps are as follows: filtering the defatted bovine colostrum whey through a 150-250 mesh sieve.

[0013] Further preferably, in the step (1), the prepared defatted bovine colostrum whey is post-processed, the steps of which are as follows: filtering the defatted bovine colostrum whey twice through a 200-mesh sieve.

[0014] Preferably, in step (2), the step of adjusting the pH is as follows: adding alkali to adjust the pH value of the defatted bovine colostrum whey.

[0015] Preferably, the alkali is 0.2-0.5 mol / L sodium hydroxide.

[0016] Preferably, in step (2), before adjusting the pH value of the defatted bovine colostrum whey to 7.0-7.4, the defatted bovine colostrum whey is diluted 0.5-2 times with a diluent.

[0017] More preferably, the skimmed bovine colostrum whey is diluted 0.5-1 times with a diluent.

[0018] By adopting the above technical solution, the present application uses a diluent to dilute the defatted bovine colostrum whey, which is beneficial to the centrifugal defatting of the bovine colostrum and improves the yield.

[0019] Preferably, the diluent includes at least one of phosphate buffer and physiological saline.

[0020] Preferably, the concentration of the phosphate buffer is 5-15 mmol / L.

[0021] Preferably, in the ultrafiltration concentration step (3), ultrafiltration equipment with a membrane molecular weight cut-off of 100-200 KDa is used for ultrafiltration concentration.

[0022] Further preferably, the membrane molecular weight cut-off of the ultrafiltration device is 200 KDa.

[0023] By adopting the above technical solution, the ultrafiltration equipment with a membrane cutoff molecular weight of 200KDa is selected in this application for the reaction, which can retain both SIgA and IgG, which is about 10% higher than the IgG retention rate using a 100kDa membrane. This is because the membrane also has electrical properties and has reverse selectivity. When the membrane is negatively charged, it can prevent negatively charged IgG from passing through. Therefore, although the pore size of the membrane is larger than that of IgG, it can also prevent IgG from passing through.

[0024] This application utilizes 200kDa membrane separation technology to enrich and prepare immunoglobulins, eliminating sample pretreatment steps and significantly simplifying the preparation process. It is an effective method for preparing SIgA-rich and high-Ig-content immunoglobulins. Compared with traditional methods such as casein removal and salting-out, this method offers advantages such as simplicity, efficiency, speed, low cost, and cost savings. It is particularly suitable for the preparation and industrial-scale production of heat-sensitive biological products such as immunoglobulins.

[0025] Preferably, when ultrafiltration equipment is used to ultrafilter and concentrate the supernatant, an ultraviolet spectrophotometer is used to monitor the A 280 The change of A 280 Stop adding water at 0.05, and when the ultrafiltration concentration is about 1 / 5 of the original volume, measure A 280 The reaction was terminated when the concentration was below 0.05.

[0026] By adopting the above technical solution, in the process of ultrafiltration and concentration, the permeate A is continuously monitored. 280 Change, when A 280 Stop adding water at 0.05. 280 The reaction is terminated below 0.05. In this application, the A 280 Monitoring can effectively increase the total immunoglobulin content and remove impurity proteins in the supernatant.

[0027] In a second aspect, the present application provides a high-content immunoglobulin rich in secretory immunoglobulin A, using the following technical solution: A high-content immunoglobulin rich in secretory immunoglobulin A, wherein the immunoglobulin contains secretory immunoglobulin A, immunoglobulin M and immunoglobulin G; the total content of secretory immunoglobulin A, immunoglobulin M and immunoglobulin G in the immunoglobulin is greater than 60%; wherein the content of secretory immunoglobulin A in the immunoglobulin is 8-15%.

[0028] The high-content immunoglobulin rich in secretory immunoglobulin A prepared in the present application contains at least three immunoglobulins, namely secretory immunoglobulin A, immunoglobulin M and immunoglobulin G. The content of secretory immunoglobulin A in the immunoglobulin is 8-15%, which is more than three times the content of SIgA in the control test sample. The total content of secretory immunoglobulin A, immunoglobulin M and immunoglobulin G in the immunoglobulin is greater than 60%, which is a high-content immunoglobulin product.

[0029] In summary, this application includes at least one of the following beneficial technical effects: The present application discloses a method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A. The immunoglobulin prepared by the method has a secretory immunoglobulin A content of 8-15% in the immunoglobulin, and a total content of secretory immunoglobulin A, immunoglobulin M, and immunoglobulin G in the immunoglobulin greater than 60%. The preparation method of the present application does not require complicated pre-treatment steps, greatly simplifies the operation process, and does not cause inactivation and loss of immunoglobulins during the preparation process. It is a simple, rapid and efficient method for preparing high-content immunoglobulins rich in secretory immunoglobulin A, and is suitable for industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the molecular sieve analysis spectrum of the immunoglobulin sample prepared in Example 1.

[0031] Figure 2 This is a standard curve diagram for the determination of SIgA in the immunoglobulin sample prepared in Example 1; Figure 3 is the electrophoresis pattern of the immunoglobulin sample solution; wherein, 1 is a SIgA standard solution with a concentration of 0.05 mg / ml; 2 is a SIgA standard solution with a concentration of 0.1 mg / ml; 3 is a SIgA standard solution with a concentration of 0.2 mg / ml; 4 is a SIgA standard solution with a concentration of 0.3 mg / ml; 5 is a SIgA standard solution with a concentration of 0.4 mg / ml; 6 is the bovine colostrum freeze-dried powder of Example 1; 7 is the bovine colostrum freeze-dried powder of Example 2; 8 is the bovine colostrum freeze-dried powder of Example 3; 9 is the sample of Comparative Example 1; Figure 4 This is the affinity chromatography column analysis spectrum of the immunoglobulin sample prepared in Example 1. DETAILED DESCRIPTION

[0032] The technical solution of the present application is further illustrated below through specific embodiments. The specific embodiments do not limit the scope of protection of the present application; some non-essential modifications and adjustments made by others based on the concept of the present application still fall within the scope of protection of the present application.

[0033] The raw materials involved in this application are all commercially available products, and the application is further described in detail below in conjunction with the examples and comparative examples.

[0034] The present application adopts the steps of centrifugal defatting, pH adjustment, centrifugal clarification, 200kDa membrane ultrafiltration concentration and freeze drying to prepare a high-content immunoglobulin rich in secretory immunoglobulin A. The specific steps are as follows: (1) Centrifugal degreasing: Take the bovine colostrum and centrifuge it at 4000-8000r / min for 10-30min at 4-8℃ to remove the upper fat layer and the bottom sediment. Filter the defatted bovine colostrum twice through a 150-250 mesh sieve to collect the defatted bovine colostrum whey.

[0035] The bovine colostrum used is the bovine colostrum from the cow on the first day after calving, the bovine colostrum from the second day after calving, the bovine colostrum from the third day after calving, or the mixed bovine colostrum from the first to third days after calving.

[0036] (2) Adjust pH value: After diluting the defatted bovine colostrum whey by 0.5-2 times with a diluent, alkali is added to adjust the pH of the defatted bovine colostrum whey to 7.0-7.4.

[0037] The diluent includes at least one of phosphate buffer and physiological saline; further, the diluent is 5-15 mmol / L phosphate buffer (pH 7.0) or physiological saline.

[0038] The alkali is sodium hydroxide with a concentration of 0.2-0.5 mol / L.

[0039] (3) Centrifugal clarification: The pH-adjusted skimmed bovine colostrum whey is centrifuged at 8000-12000 r / min for 10-30 min at 4-8°C, or centrifuged at 8000-12000 r / min using a disc centrifuge to remove denatured proteins or precipitates, and the supernatant is retained.

[0040] (4) Ultrafiltration concentration: The supernatant after centrifugation was ultrafiltered and concentrated using an ultrafiltration device with an ultrafiltration membrane with a molecular weight cutoff of 100-200 kDa. The pressure was adjusted to 0.25-0.35 MPa and the flow rate was 2-5 L / min. The ultrafiltration was concentrated to about half of the original volume, and deionized water was added to the original volume. This cycle was repeated and the A of the ultrafiltrate was monitored by a UV spectrophotometer. 280 The change of A 280 Stop adding deionized water at 0.05, and finally, ultrafiltration and concentration to about 1 / 5 of the original volume, and determine A 280 The filtration process ends when the concentration is below 0.05. This step effectively removes proteins with a molecular weight less than 200 kDa (such as most caseins, some IgG, BSA, lactoglobulin, lactalbumin, etc.), as well as minerals and lactose, from the colostrum. HPLC analysis shows that, in the molecular sieve profile, except for a protein peak at a retention time of 19.925 minutes, all other impurities with a molecular weight below IgG have been completely removed, and the ultrafiltration concentrate is collected.

[0041] (5) Freeze-drying The ultrafiltration concentrate is freeze-dried, and the freeze-drying parameters are set as follows: in the pre-freezing stage, pre-freeze below -40°C for 2 to 4 hours, turn on the vacuum and heating control system, maintain at -35°C for 0.5 to 1 hour, then enter the sublimation drying stage, control the temperature at -10 to -5°C, maintain for 6 to 12 hours, and in the analytical drying stage, control the temperature at 25 to 30°C, maintain for 3 to 5 hours, until the sample temperature and the shelf temperature are consistent, and then terminate the freeze-drying to obtain a high-content immunoglobulin rich in secretory immunoglobulin A.

[0042] The high-content immunoglobulin rich in secretory immunoglobulin A prepared by the method of the present application contains secretory immunoglobulin A, immunoglobulin M and immunoglobulin G, wherein the content of secretory immunoglobulin A in the immunoglobulin is 8-15%, and the total content of secretory immunoglobulin A, immunoglobulin M and immunoglobulin G in the immunoglobulin is greater than 60%.

[0043] Example 1: A method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A, comprising the following steps: (1) Centrifugal degreasing: Take 1L of bovine colostrum (cow colostrum on the first day of calving) and centrifuge it at 4000r / min at 4℃ for 15min to remove the upper fat layer and the bottom sediment. Then filter it twice through a 200-mesh sieve to collect the defatted bovine colostrum whey.

[0044] (2) Adjusting pH value: Dilute the skimmed bovine colostrum whey with 10 mmol / L phosphate buffer (pH 7.0) by 1 time, and then adjust the pH of the solution to 7.0 with 0.25 mol / L NaOH.

[0045] (3) Centrifugal clarification: The pH-adjusted skimmed bovine colostrum whey was centrifuged at 8000 rpm for 30 min at 4°C to remove denatured proteins or precipitates, and the supernatant was retained.

[0046] (4) Ultrafiltration concentration: The supernatant after centrifugation was ultrafiltered and concentrated using an ultrafiltration membrane with a molecular weight cutoff of 200 kDa. The pressure was adjusted to 0.25 MPa and the flow rate was 2.5 L / min. The ultrafiltration was concentrated to about half of the original volume. Deionized water was added to the original volume. This cycle was repeated. The A of the ultrafiltrate was monitored by a UV spectrophotometer. 280 The change of A 280 Stop adding deionized water at 0.05, and finally, ultrafiltration and concentration to about 1 / 5 of the original volume, and determine A 280 The HPLC test showed that in the molecular sieve spectrum, except for a protein peak at a retention time of 19.925 min, all impurities below the molecular weight of IgG had been completely removed to obtain an ultrafiltration concentrate.

[0047] (5) Freeze drying: The prepared ultrafiltration concentrate was freeze-dried, and the freeze-drying process parameters were as follows: Freeze dryer model: YWLG-50F freeze dryer; The freeze-drying parameters are set as follows: in the pre-freezing stage, pre-freeze below -40°C for 3 hours, turn on the vacuum and heating control system, maintain at -35°C for 0.8 hours, then enter the sublimation drying stage, control the temperature at -8°C, maintain for 9 hours, and in the desorption drying stage, control the temperature at 28°C, maintain for 4 hours, until the sample temperature and the shelf temperature are consistent, end the freeze-drying, and obtain a high-content immunoglobulin sample rich in secretory immunoglobulin A.

[0048] Example 2: A method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A, comprising the following steps: (1) Centrifugal degreasing: Take 1L of bovine colostrum (cow colostrum on the second day after calving) and centrifuge it at 4000r / min at 4°C for 30min to remove the upper fat layer and the bottom sediment. Then filter it twice through a 200-mesh sieve to collect the defatted bovine colostrum whey.

[0049] (2) Adjusting the pH value: Dilute the skimmed bovine colostrum whey 0.5 times with normal saline, and then adjust the pH of the solution to 7.0 with 0.3 mol / L NaOH.

[0050] (3) Centrifugal clarification: The centrifugal clarification step is consistent with the implementation steps in Example 1.

[0051] (4) Ultrafiltration and concentration: The ultrafiltration and concentration steps are the same as those in Example 1; (5) Freeze-drying: The freeze-drying step is consistent with the implementation steps in Example 1, and a high-content immunoglobulin sample rich in secretory immunoglobulin A is obtained.

[0052] Example 3: A method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A, comprising the following steps: (1) Centrifugal degreasing: Take 1 L of bovine colostrum (mixed bovine colostrum from cows on the 1st to 3rd day of calving) and centrifuge it at 4°C and 4000 r / min for 30 minutes to remove the upper fat layer and the bottom sediment. Then filter it twice through a 200-mesh sieve to collect the defatted bovine colostrum whey.

[0053] (2) Adjusting the pH value: Dilute the skimmed bovine colostrum whey 0.5 times with normal saline, and then adjust the pH of the solution to 7.1 with 0.5 mol / L NaOH.

[0054] (3) Centrifugal clarification: The centrifugal clarification was carried out in the same manner as in Example 1.

[0055] (4) Ultrafiltration and concentration: The ultrafiltration and concentration were carried out in the same manner as in Example 1.

[0056] (5) Freeze-drying: The freeze-drying step is consistent with the implementation steps in Example 1, and a high-content immunoglobulin sample rich in secretory immunoglobulin A is obtained.

[0057] Example 4: A method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A, comprising the following steps: (1) Centrifugal degreasing: Take 1L of bovine colostrum (cow colostrum on the first day of calving) and centrifuge it at 8°C and 8000r / min for 10 minutes to remove the upper fat layer and the bottom sediment. Then filter it twice through a 200-mesh sieve to collect the defatted bovine colostrum whey.

[0058] (2) Adjusting pH value: Dilute the skimmed bovine colostrum whey twice with 10 mmol / L phosphate buffer (pH 7.0), and then adjust the pH of the solution to 7.4 with 0.2 mol / L NaOH.

[0059] (3) Centrifugal clarification: The centrifugal clarification was carried out in the same manner as in Example 1.

[0060] (4) Ultrafiltration and concentration: The ultrafiltration and concentration were carried out in the same manner as in Example 1.

[0061] (5) Freeze-drying: The freeze-drying step is consistent with the implementation steps in Example 1, and a high-content immunoglobulin sample rich in secretory immunoglobulin A is obtained.

[0062] Comparative Example 1: Take 1 L of bovine colostrum (cow colostrum on the first day of calving) and centrifuge it at 4°C and 4000 r / min for 30 minutes to remove the upper fat layer and the bottom sediment. Then filter it twice through a 200-mesh sieve to collect the defatted bovine colostrum whey. The whey is sterilized by filtering it through a 0.2 μm filter membrane. The sterilized bovine colostrum is directly freeze-dried to prepare bovine colostrum freeze-dried powder.

[0063] Comparative Example 2: The difference between this embodiment and comparative example 1 is that ultrafiltration and concentration are not performed using an ultrafiltration membrane with a molecular weight cut-off of 200 kDa. The specific steps are as follows: (1) Centrifugal defatting: Take 1 L of bovine colostrum (cow colostrum on the first day of calving) and centrifuge it at 4000 r / min at 4°C for 15 min to remove the upper fat layer and the bottom sediment. Then filter it twice through a 200-mesh sieve to collect the defatted bovine colostrum whey. (2) Adjusting pH value: Dilute the skimmed bovine colostrum whey with 10 mmol / L phosphate buffer (pH 7.0) by 1 time, and then adjust the pH of the solution to 7.0 with 0.25 mol / L NaOH.

[0064] (3) Centrifugal clarification: The pH-adjusted skimmed bovine colostrum whey was centrifuged at 8000 rpm for 30 min at 4°C to remove denatured proteins or precipitates, and the supernatant was retained.

[0065] (4) Sterilize the supernatant by filtering through a 0.2 μm filter membrane; (5) Freeze-drying: The freeze-drying step is the same as that in Example 1 to obtain freeze-dried bovine colostrum powder.

[0066] Performance test 1: Molecular sieve analysis of TSK gel G3000pwxl The immunoglobulin content in the sample was detected using molecular sieve chromatography technology. The steps are as follows: 1. Test conditions are as follows: 1) High performance liquid chromatography; 2) Chromatographic column: TSKgel G3000pwxl (7.5 × 300 mm), Japan (TOSOH); 3) Mobile phase: 0.02 mol / L PBS-0.2 mol / L NaCl, pH 6.9; 4) Detection wavelength: 280 nm, injection volume: 15 μl, mobile phase flow rate: 0.45 ml / min, column temperature: 25°C.

[0067] 5) Elution program: Gradient elution program: Table 1 Elution program Time (min) A(％) B(％) 0 100 0 4.5 80 20 5.5 60 40 15 40 60 15.5 20 80 22.0 0 100 2. Result analysis: The relative contents of the three immunoglobulins and miscellaneous proteins in the samples prepared in the above examples and comparative examples were evaluated by area normalization method (analyzed using TSK molecular sieves). The results are shown in Table 2.

[0068] Table 2 Relative contents of IgM, SIgA, IgG and miscellaneous proteins in immunoglobulin samples *Content of foreign proteins (%) = 100 - relative content of IgM - relative content of SIgA - relative content of IgG. Combined with the test results of Examples 1-4, it can be seen that in the immunoglobulin sample prepared by the method of the present application, the total content of secretory immunoglobulin A, immunoglobulin M and immunoglobulin G in the immunoglobulin is greater than 60%; the content of secretory immunoglobulin A in the immunoglobulin is 8-15%.

[0069] Combining the test results of Example 1 and Comparative Example 1, it can be seen that the IgM, SIgA and IgG in Example 1 are increased by 78.48%, 56.36 and 108.63% respectively compared with Comparative Example 1, and the miscellaneous proteins in Example 1 are reduced by 56.83% compared with Comparative Example 1, indicating that the method of the present application effectively increases the content of IgM, SIgA and IgG in the immunoglobulin sample.

[0070] Combining the test results of Example 1 and Comparative Example 2, it can be seen that the contents of IgM, SIgA and IgG in Example 1 are higher than those in Comparative Example 2, indicating that ultrafiltration and concentration of the supernatant after centrifugation effectively increases the contents of IgM, SIgA and IgG in the immunoglobulin sample.

[0071] 3. Result analysis: The retention time of standard IgG (Sigma, purity ≥95%) was used as a reference to determine the nature of the sample peak, and the relative content of the sample peak was calculated by area normalization.

[0072] After HPLC detection, in the TSK molecular sieve analysis spectrum, the retention time of standard IgG is 16.565 (dimer) and 17.567 (monomer). In Example 1, in the sample concentrated and freeze-dried by ultrafiltration with a 200kDa membrane, except for the protein peak retained at 19.925min (accounting for 12.6%), no protein peak appeared in the spectrum after this retention time, indicating that the impurity protein after the retention time of 20min has been filtered out. The protein peaks with retention times of 16.161 and 17.391 in the sample are basically consistent with the retention times of the dimer and monomer of standard IgG, thereby determining that they are dimers and monomers of IgG. The relative percentages of dimers and monomers in the sample peaks were calculated by area normalization method to be 4.1% and 65.7%, respectively, and the relative content of IgG was 69.8% ( Figure 1 ).

[0073] Molecular sieve chromatography can determine the molecular weight of proteins, and the three Igs can be distinguished due to their molecular weight differences. The TSK gel G3000pwxl column has a molecular weight fractionation range of 10,000 to 500,000. IgM, with a molecular weight of approximately 950,000, cannot enter the gel and is eluted first, resulting in a protein peak with a retention time of 13.594 (content 2.8%), which exhibits the large molecular weight characteristics of IgM. SIgA has a molecular weight of approximately 400,000, with a retention time of 14.796 (content 14.8%). This content is consistent with the value determined by rocket electrophoresis (11.08%), confirming that this protein peak is SIgA.

[0074] It can be seen that the sample in Example 1 contains at least three types of Igs, namely, IgM, SIgA and IgG, and the relative content of the total Igs is greater than 60%.

[0075] Performance test 2: Rocket electrophoresis method to detect SIgA content Rocket electrophoresis was used to detect the SIgA content in the sample. The specific operation is as follows: 1. Preparation of agarose gel plate containing rabbit anti-bovine SIgA polyclonal antibody: Take a 1.2% agarose solution preheated at 55°C, add an appropriate amount of antiserum (volume concentration 1-2%), mix well, pour into a gel-making device, and make a gel plate with a uniform thickness of 1.5 mm. Punch holes at the designed positions with a hole diameter of 2.0 mm and a hole spacing of 2.5 mm. Use a needle to pick out the gel in the hole.

[0076] 2. Sample solution preparation: About 100.0 mg of the bovine colostrum freeze-dried powder (i.e., immunoglobulin sample) prepared in Example 1 was weighed, 5 ml of a 0.4% BSA solution was added and mixed, and then 5 ml of 0.01 mol / L glutaraldehyde was added to make the volume 10 ml. The solution was allowed to stand at room temperature for 30 min and used for determination after appropriate dilution to obtain a sample solution.

[0077] 3. Preparation of SIgA standard series solutions: Accurately weigh 1.00 mg of SIgA (purity ≥ 95%, derived from bovine colostrum), add 0.5 ml of 0.4% BSA solution and mix well, then add 0.5 ml of 0.01 mol / L glutaraldehyde solution and make up to volume in a 1.0 ml volumetric flask. The SIgA content of this solution is 1.0 mg / ml, which is used as the SIgA stock solution.

[0078] The SIgA stock solution was diluted with 0.005 mol / L glutaraldehyde and prepared into a standard series of solutions containing 0.05, 0.1, 0.2, 0.3, and 0.4 mg of SIgA per 1 ml.

[0079] 4. Sample addition and electrophoresis: (1) Add 1000ml of 0.05mol / L barbital-sodium barbital electrode buffer (pH 8.6) to the electrophoresis tank. Place the perforated gel plate on the horizontal rail of the electrophoresis tank, with the perforated end at the negative electrode. Cover both ends of the gel plate with two layers of filter paper strips. Turn on the power, add the sample, and use a microinjector to accurately draw 5μl of each of the SIgA standard series solution and sample solution, and add them to the perforated gel plate containing the antibody. After adding the sample, adjust the voltage to 50V and run the electrophoresis for 30min. Then adjust the voltage to 100-150V and run the electrophoresis for about 5-10h.

[0080] (2) Turn off the power, remove the gel plate, and soak it in saline for about 0.5-1 hour to wash away unbound antigens and antibodies.

[0081] (3) Stain and decolorize with Coomassie Brilliant Blue R250 staining solution, and take photos or scan for storage.

[0082] 5. Calculation of results: After staining and decolorization of the gel, a clear rocket peak can be seen. The distance from the upper edge of the small hole to the top of the rocket peak is accurately measured with a digital vernier caliper. The SIgA content (mg / ml) is used as the horizontal coordinate and the rocket peak height (mm) is used as the vertical coordinate to draw a regression equation curve. In the concentration range of 0.05-0.4 mg / ml, a good linear relationship is found. The regression equation is y=103.94x+4.7596, r=0.9952( Figure 2 ).

[0083] Accurately measure the rocket peak length of the sample, substitute it into the regression equation and calculate the SIgA concentration in the sample. Calculate the SIgA content in the sample according to the following formula: Where: X: SIgA content in the sample, g / 100g; m1: SIgA content in the sample calculated by regression equation, mg / ml; m: sample concentration, mg / ml; f: dilution factor of the sample.

[0084] The determination of SIgA content in the samples is shown in Table 3.

[0085] Table 3 SIgA content in bovine colostrum samples The test results in Table 3 show that the average SIgA content in the control test (i.e., Comparative Example 1) is 3.07%, and the average SIgA content in the bovine colostrum after ultrafiltration, concentration, and freeze-drying through a 200 kDa membrane (i.e., Examples 1-3) is 9.62%, which is 3.1 times the SIgA in the control test (i.e., Comparative Example 1). The electrophoretic pattern of the immunoglobulin sample solution is shown in FIG. Figure 3 shown.

[0086] Performance test 3, Protein G affinity chromatography column test analysis The SIgA content in the sample prepared in Example 1 was detected and analyzed using a Protein G affinity chromatography column. The specific steps are as follows: 1. Testing conditions and methods are as follows: 1) High performance liquid chromatography; 2) Chromatographic column: Pharmacia HI-Trap Protein G column (1 ml, USA); 3) Mobile phase A: 0.05 mol / L phosphate buffer (pH 6.5); 4) Mobile phase B: 0.05 mol / L glycine-HCl buffer (pH 2.5); 5) Detection wavelength: 280 nm; injection volume: 25 μl; flow rate: 0.5 ml / min; column temperature: 25°C.

[0087] 6) Prepare samples and perform gradient elution according to the method for determination of immunoglobulin IgG in health foods (GB / T 5009.194-20). Calculate the relative percentages of the penetration peak and elution peak using the area normalization method.

[0088] 2. Result analysis: Test results such as Figure 4 In the analytical spectrum, the elution peak at retention time 14.070 corresponds to standard IgG, and it was determined to be IgG (content 59.5%). IgM and SIgA were not adsorbed by the affinity column and were directly eluted. Due to the difference in affinity between IgM, SIgA and protein G, it can be determined that the protein peak at retention time 3.263 corresponds to IgM (content 10.7%), and the protein peak at retention time 4.039 corresponds to SIgA (content 29.82%). Because impurities such as the retention time 19.925 (content 12.6%) in the TSK column are not adsorbed by the affinity column, and the two peaks are not completely separated, the results of IgM and SIgA are quite different.

[0089] Performance test 4: IgM, SIgA, IgG and total Igs content detection in Example 1 The IgM, SIgA, IgG and total Igs contents in the sample prepared in Example 1 were detected. The specific steps are as follows: In the TSK molecular sieve method, IgM and SIgA (purity ≥95%) purified from bovine colostrum were used as standards, and the IgM and SIgA contents in the sample prepared in Example 1 were determined by a single-point calibration method. The detection steps are the same as those of performance test 1.

[0090] The IgG content in the sample prepared in Example 1 was determined using the standard curve method in affinity chromatography, and the detection steps were the same as those in performance test 3.

[0091] Table 4 IgM, SIgA, IgG and total Igs contents in Example 1 From the data results in Table 4, it can be seen that the average content of IgM in the immunoglobulin sample prepared in Example 1 is 2.31%, the average content of SIgA is 11.18%, the average content of IgG is 51.32%, and the total Igs content is 64.81%.

[0092] Example 4: In order to study the effect of different pH on immunoglobulins, the defatted bovine colostrum in step (2) of Example 1 was adjusted to different pH values to prepare immunoglobulins, and the contents of IgM, SIgA, IgG and total Igs in the immunoglobulin samples were detected.

[0093] The detection method is: in the TSK molecular sieve method, using IgM and SIgA purified from bovine colostrum (purity ≥95%) as the standard, the IgM and SIgA content in the sample is determined by the single-point calibration method. The detection steps are the same as performance test 1.

[0094] Table 5 Effects of different pH on immunoglobulin samples According to Table 5, when the pH value of skimmed bovine colostrum whey is adjusted to 7.0-7.4, the total content of secretory immunoglobulin A, immunoglobulin M and immunoglobulin G in immunoglobulins is greater than 60%; and when the pH value of skimmed bovine colostrum whey is 7.0-7.4, the content of secretory immunoglobulin A is the highest.

Claims

1. A method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A, characterized in that: The preparation method comprises the following steps: (1) Centrifuging the bovine colostrum to obtain defatted bovine colostrum whey; (2) adjusting the pH value of the defatted bovine colostrum whey to 7.0-7.4, centrifuging, and obtaining a supernatant; (3) The supernatant is concentrated by ultrafiltration and freeze-dried to obtain immunoglobulin.

2. The method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A according to claim 1, characterized in that: In the step (1), when the bovine colostrum is subjected to centrifugal degreasing, the conditions for centrifugal degreasing are as follows: the temperature for centrifugal degreasing is 4-8°C; the speed for centrifugal degreasing is 4000-8000 r / min; and the time for centrifugal degreasing is 10-30 min.

3. The method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A according to claim 1, characterized in that: In the step (1), the prepared defatted bovine colostrum whey is post-processed, and the steps are as follows: filtering the defatted bovine colostrum whey through a 150-250 mesh sieve.

4. The method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A according to claim 1, characterized in that: In the step (2), the step of adjusting the pH is as follows: adding an alkali to adjust the pH value of the defatted bovine colostrum whey; the alkali is 0.2-0.5 mol / L sodium hydroxide.

5. The method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A according to claim 1, characterized in that: In the step (2), before adjusting the pH value of the skimmed bovine colostrum whey to 7.0-7.4, the skimmed bovine colostrum whey is diluted 0.5-2 times with a diluent.

6. The method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A according to claim 5, characterized in that: The diluent includes at least one of phosphate buffer and physiological saline; the concentration of the phosphate buffer is 5-15 mmol / L.

7. The method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A according to claim 1, characterized in that: In the ultrafiltration concentration step (3), ultrafiltration equipment with a membrane molecular weight cutoff of 100-200 KDa is used for ultrafiltration concentration.

8. The method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A according to claim 7, characterized in that: The membrane molecular weight cut-off of the ultrafiltration device is 200 KDa.

9. The method for preparing a high-content immunoglobulin rich in secretory immunoglobulin A according to claim 7, characterized in that: When ultrafiltration equipment is used to ultrafilter and concentrate the supernatant, the ultraviolet spectrophotometer is used to monitor the A 280 The change of A 280 Stop adding water at 0.05, and when the ultrafiltration concentration is 1 / 5 of the original volume, measure A 280 The reaction was terminated when the concentration was below 0.

05.

10. A high-content immunoglobulin rich in secretory immunoglobulin A prepared by the method according to any one of claims 1 to 9, characterized in that: The immunoglobulin contains secretory immunoglobulin A, immunoglobulin M and immunoglobulin G; the total content of secretory immunoglobulin A, immunoglobulin M and immunoglobulin G in the immunoglobulin is greater than 60%; the content of secretory immunoglobulin A in the immunoglobulin is 8-15%.

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