Preparation method of immunoglobulin powder and application thereof
By employing cell survival technology freezing, milk fat separation, compound rennet curdling, ultrafiltration, and vacuum freeze-drying, the problem of low immunoglobulin purity in existing technologies has been solved, achieving efficient, high-purity, and high-quality immunoglobulin powder preparation suitable for pharmaceutical and food production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INNER MONGOLIA MENGNIU DAIRY IND (GROUP) CO LTD
- Filing Date
- 2024-12-19
- Publication Date
- 2026-06-23
AI Technical Summary
Existing methods for extracting immunoglobulins suffer from low purity and cumbersome procedures, lacking efficient and standardized preparation methods.
A preparation method employing cell survival technology freezing, milk fat separation, compound rennet coagulation, ultrafiltration, and vacuum freeze-drying, combined with low-temperature technology to preserve immunoglobulin activity, and using a compound enzyme of aspartic protease, papain, and bromelain for coagulation, achieves efficient and high-purity immunoglobulin powder preparation.
It achieves efficient, high-purity, and high-quality preparation of immunoglobulin powder, avoiding the introduction of harmful substances in chemical methods. It is suitable for pharmaceutical and food production, and the product has good color, taste, and flavor.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of protein extraction technology, and in particular to a method for preparing immunoglobulin powder and its application. Background Technology
[0002] Bovine colostrum's disease-fighting ability primarily derives from immunoglobulins, substances capable of resisting various pathogens with different structures. It is one of the most crucial components of the animal's immune system and possesses significant potential as an antibacterial and antiviral agent. Producing large quantities of immunoglobulins is not only beneficial to human health but also economically viable; however, it requires high costs and efficient purification processes. Currently, there is no standardized and systematic method internationally for the extraction, detection, and purification of immunoglobulins. Therefore, this area of research has broad prospects and has become a hot topic for many scholars.
[0003] CN103965353A discloses a method for obtaining immunoglobulins from colostrum, comprising: (a) heat-treating colostrum from days 0 to 7 to remove milk fat; (b) aseptically microfiltration of the resulting skim milk to produce a first residue R1 containing casein and a first permeate P1; (c) ultrafiltration of the first permeate P1 to produce a second permeate P2 containing lactose and minerals and a second residue R2, wherein the immunoglobulins are concentrated; and (d) the immunoglobulins obtained from the second residue are separated by treatment with an anion exchanger. However, the purity of the immunoglobulins obtained by this method needs to be improved.
[0004] CN115417929A discloses a method for extracting bovine colostrum immunoglobulins, comprising the following steps: centrifugation and defatting: bovine colostrum is centrifuged and defatted, then the intermediate layer is filtered to obtain colostrum whey; ammonium sulfate precipitation: the colostrum whey is diluted, then saturated ammonium sulfate solution is added, stirred, allowed to stand, centrifuged, and the supernatant A is removed. Phosphate buffer is added to precipitate A, stirred evenly, then saturated ammonium sulfate solution is added, allowed to stand, centrifuged, and precipitate B is removed. Saturated ammonium sulfate solution is added to supernatant B, centrifuged, and supernatant C is removed to obtain precipitate C; purification: precipitate C is desalted by gel column chromatography, concentrated by ultrafiltration, and separated by column chromatography to obtain bovine colostrum immunoglobulins. This method introduces ammonium sulfate for three salting-out processes, resulting in a high-purity product, but the operation is cumbersome and complex.
[0005] Therefore, it is necessary to provide a method for preparing high-efficiency, high-purity immunoglobulin powder. Summary of the Invention
[0006] To address the aforementioned technical problems, this invention provides a method for preparing immunoglobulin powder and its application. This invention employs a preparation method involving cell survival technology freezing, milk fat separation, curd precipitation, ultrafiltration, and vacuum freeze-drying. The low-temperature technology throughout the process maximizes the preservation of immunoglobulin activity, and the use of a compound rennet solves the problem of curd failure. The synergistic effect of each step improves the curdling effect, achieving the preparation of highly efficient, high-purity, and high-quality immunoglobulin powder.
[0007] To achieve this objective, the present invention adopts the following technical solution:
[0008] In a first aspect, the present invention provides a method for preparing immunoglobulin powder, the method comprising the following steps:
[0009] (1) Collect bovine colostrum and freeze it using cell survival technology (CAS);
[0010] (2) Thaw the frozen bovine colostrum and separate the milk fat to obtain skimmed bovine colostrum;
[0011] (3) The skimmed bovine colostrum and compound rennet are mixed to coagulate, and then the milk is broken to obtain whey.
[0012] The compound chymotrypsin includes aspartic protease, papain and bromelain.
[0013] (4) Ultrafiltration of whey solution to obtain immunoglobulin solution;
[0014] (5) The immunoglobulin solution is freeze-dried under vacuum to obtain the immunoglobulin powder.
[0015] This invention uses bovine colostrum as raw material and employs cell survival technology (CAS) for freeze preservation to maximize the preservation of immunoglobulin activity. A compound rennet consisting of aspartic protease, papain, and bromelain is used for coagulation; the three enzymes work synergistically to improve the coagulation effect. Simultaneously, ultrafiltration sterilization and purification are combined with vacuum freeze-drying to obtain lyophilized immunoglobulin powder. The entire preparation method is progressive, achieving the preparation of highly efficient and high-purity immunoglobulin powder. The resulting product has excellent color, taste, and flavor, and possesses high economic application value.
[0016] Preferably, the bovine colostrum in step (1) is bovine colostrum within 72 hours (e.g., 16 hours, 24 hours, 48 hours, 60 hours, or 72 hours).
[0017] Preferably, the vibration frequency of freezing in step (1) is 400 to 600 MHz (e.g., 400 MHz, 450 MHz, 500 MHz, 550 MHz, 600 MHz, etc.), and the freezing temperature is -10 to -5℃ (e.g., -10℃, -9℃, -8℃, -7℃, -6℃, -5℃, etc.).
[0018] Preferably, the thawing temperature in step (2) is 2 to 8°C (for example, it can be 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc.).
[0019] Preferably, step (2) further includes diluting the bovine colostrum with water after thawing.
[0020] Preferably, the protein content in the diluted bovine colostrum is 3.5 to 6.0 wt% (e.g., 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, etc.).
[0021] Preferably, the milk fat separation in step (2) is performed by centrifugation.
[0022] Preferably, the centrifugation speed is 6000-10000 r / min (e.g., 6000 r / min, 7000 r / min, 8000 r / min, 9000 r / min, 10000 r / min, etc.).
[0023] Preferably, the compound rennet in step (3) comprises, by mass percentage, 40-55% aspartic protease (e.g., 40%, 44%, 48%, 52%, 55%, etc.), 30-50% papain (e.g., 30%, 35%, 40%, 45%, 50%, etc.), and 15-25% bromelain (e.g., 15%, 18%, 20%, 22%, 24%, 25%, etc.).
[0024] Preferably, the ratio of compound rennet to skimmed bovine colostrum in step (3) is 2.8 to 3.0 kg / 1000L (e.g., 2.8 kg / 1000L, 2.85 kg / 1000L, 2.9 kg / 1000L, 2.95 kg / 1000L, 3.0 kg / 1000L, etc.).
[0025] Preferably, the compound rennet in step (3) is added in the form of a compound rennet aqueous solution.
[0026] Preferably, the mass ratio of the compound rennet to water in the compound rennet aqueous solution is 1:(20-50) (e.g., 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, etc.).
[0027] Preferably, the temperature of the curd in step (3) is 30-40°C (e.g., 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, etc.), and the curdling time is 40-60 min (e.g., 40 min, 45 min, 50 min, 55 min, 60 min, etc.).
[0028] Preferably, the demulsification in step (3) is carried out by stirring.
[0029] Preferably, after demulsification in step (3), a filtration operation is performed to obtain whey.
[0030] Preferably, the filter screen used for filtration has a mesh size of 60-80 mesh (e.g., 60 mesh, 65 mesh, 70 mesh, 75 mesh, 80 mesh, etc.).
[0031] Preferably, the filter membrane used in step (4) has a pore size of 20-500 kDa (for example, it can be 20 kDa, 50 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, etc.).
[0032] Preferably, the ultrafiltration in step (4) includes a combination of at least two of the following: 150-250 kDa (e.g., 150 kDa, 180 kDa, 200 kDa, 220 kDa, 240 kDa, 250 kDa, etc.), 80-120 kDa (e.g., 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, etc.) and 40-60 kDa (e.g., 40 kDa, 45 kDa, 50 kDa, 55 kDa, 60 kDa, etc.). Preferably, ultrafiltration is performed by sequentially using filter membranes with pore sizes of 150-250 kDa, 80-120 kDa and 40-60 kDa.
[0033] Preferably, the ultrafiltration pressure in step (4) is 0.1 to 0.3 MPa (for example, it can be 0.1 MPa, 0.15 MPa, 0.2 MPa, 0.25 MPa, 0.3 MPa, etc.).
[0034] Preferably, the number of ultrafiltrations in step (4) is 1 to 3 times (for example, 1 time, 2 times, or 3 times).
[0035] Preferably, the vacuum freeze drying in step (5) includes pre-freezing, sublimation drying and desorption drying.
[0036] Preferably, the pre-freezing and cooling to a temperature of -30 to -25°C (e.g., -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, etc.) and the cooling rate is 1 to 2°C / min (e.g., 1°C / min, 1.2°C / min, 1.5°C / min, 1.8°C / min, 2°C / min, etc.).
[0037] Preferably, the vacuum degree of the sublimation drying is 10. -5 ~10 -3 Torr (for example, it could be 10) -5 Torr, 5×10 - 5 Torr, 10 -4 Torr, 5×10 -4 Torr, 10 -3 Torr et al.
[0038] Preferably, the desorption drying temperature is raised to 30-40°C (e.g., 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, etc.), and the heating rate is 1-2°C / min (e.g., 1°C / min, 1.2°C / min, 1.5°C / min, 1.8°C / min, 2°C / min, etc.).
[0039] Preferably, the preparation method includes the following steps:
[0040] (1) Collect bovine colostrum within 72 hours and freeze it using cell survival technology at a vibration frequency of 400-600MHz and a temperature of -10--5℃.
[0041] (2) Thaw the frozen bovine colostrum to a temperature of 2-8℃, dilute it with water until the protein content in the bovine colostrum is 3.5-6.0 wt%, and separate the milk fat at a centrifugation speed of 6000-10000 r / min to obtain defatted bovine colostrum;
[0042] (3) The compound rennet and skimmed bovine colostrum are mixed at a dosage ratio of 2.8-3.0 kg / 1000 L, and coagulated at 30-40 °C for 40-60 min. Then, the mixture is broken up by stirring and filtered through a 60-80 mesh filter to obtain whey. The compound rennet comprises 40-55% aspartic protease, 30-50% papain and 15-25% bromelain by mass percentage. The compound rennet is added in the form of a compound rennet aqueous solution, and the mass ratio of compound rennet to water in the compound rennet aqueous solution is 1:(20-50).
[0043] (4) The whey solution was ultrafiltered 1 to 3 times at 0.1 to 0.3 MPa using a filter membrane with a pore size of 20-500 kDa to obtain an immunoglobulin solution;
[0044] (5) The immunoglobulin solution is subjected to vacuum freeze-drying to obtain the immunoglobulin powder; the vacuum freeze-drying includes pre-freezing, sublimation drying and desorption drying; the pre-freezing temperature is -30 to -25°C, the cooling rate is 1 to 2°C / min, and the vacuum degree of the sublimation drying is 10. -5 ~10 -3 Torr, wherein the desorption drying is heated to a temperature of 30-40°C at a rate of 1-2°C / min.
[0045] Secondly, the present invention provides an application of the method for preparing immunoglobulin powder according to the first aspect in the preparation of pharmaceuticals and food.
[0046] Compared with the prior art, the present invention has at least the following beneficial effects:
[0047] (1) The present invention adopts a preparation method of cell survival technology freezing, milk fat separation, curd precipitation, ultrafiltration and vacuum freeze drying. The low temperature technology throughout the process preserves the activity of immunoglobulins to the greatest extent. The use of compound rennet solves the problem of curd failure. The synergistic effect of each step improves the curd effect, and realizes the preparation of high efficiency, high purity and high quality immunoglobulin powder.
[0048] (2) The present invention uses physical methods to separate and extract immunoglobulins, avoiding the introduction of harmful chemicals in chemical methods. This method is safe and green and can be directly applied to the production of pharmaceuticals and food. Detailed Implementation
[0049] To facilitate understanding of the present invention, the following embodiments are provided. Those skilled in the art should understand that these embodiments are merely illustrative and should not be construed as limiting the scope of the invention.
[0050] Example 1
[0051] This embodiment provides a method for preparing immunoglobulin powder, including the following steps:
[0052] (1) Bovine colostrum collection: Bovine colostrum raw milk within 24 hours was collected and frozen using CAS freezing and preservation equipment, with vibration parameters of 500MHz and freezing parameters of -8℃.
[0053] (2) Defatting: Thaw the frozen bovine colostrum at 4°C, dilute it with pure water to 4.0 wt% protein, put it into a milk fat separator, set the speed to 8000 r / min, remove the milk fat, and obtain defatted bovine colostrum;
[0054] (3) Curdling: Add compound rennet to skimmed bovine colostrum at a ratio of 3.0 kg / 1000 L at 4℃. Dissolve the compound rennet in pure water at a ratio of 1:20, and then add it to skimmed bovine colostrum. Stir at 300 r / min for 5 min, and let it stand in a 36℃ water bath for 45 min.
[0055] The compound rennet comprises, by weight percentage, 55% aspartic protease, 30% papain, and 15% bromelain;
[0056] (4) Demulsification: Stir the coagulated protein and filter it using an 80-mesh filter to obtain the filtered whey.
[0057] (5) Ultrafiltration: The whey was passed through ultrafiltration membranes of 200 kDa, 100 kDa and 50 kDa in sequence. The threshing liquid was mixed with pure water at a ratio of 1:1 at 0.15 MPa. The ultrafiltration was repeated 3 times to obtain an immunoglobulin solution.
[0058] (6) Vacuum freeze-drying: The ultrafiltered immunoglobulin solution is placed in a vacuum freeze dryer and subjected to pre-freezing, sublimation drying, and desorption drying in sequence to obtain immunoglobulin powder; wherein the pre-freezing is carried out at a temperature of -25°C at a rate of 1°C / min, and the vacuum degree of the sublimation drying is 10. -4 Torr, the desorption drying is performed by increasing the temperature to 30°C at a rate of 1°C / min.
[0059] Example 2
[0060] This embodiment provides a method for preparing immunoglobulin powder, including the following steps:
[0061] (1) Bovine colostrum collection: Bovine colostrum raw milk within 48 hours was collected and frozen using CAS freezing and preservation equipment, with vibration parameters of 400MHz and freezing parameters of -10℃.
[0062] (2) Defatting: Thaw the frozen bovine colostrum at 2°C, dilute it with pure water to 3.5 wt% protein, put it into a milk fat separator, set the speed to 6000 r / min, remove the milk fat, and obtain defatted bovine colostrum;
[0063] (3) Curdling: Add compound rennet to skimmed bovine colostrum at a ratio of 2.8 kg / 1000 L at 2℃. Mix the compound rennet with pure water at a ratio of 1:30 and then add it to skimmed bovine colostrum. Stir at 200 r / min for 5 min and let it stand in a 30℃ water bath for 60 min.
[0064] The compound rennet comprises, by weight percentage, 50% aspartic protease, 30% papain, and 20% bromelain;
[0065] (4) Demulsification: Stir the coagulated protein and filter it using a 60-mesh filter to obtain the filtered whey.
[0066] (5) Ultrafiltration: The whey was passed through ultrafiltration membranes of 250 kDa, 80 kDa and 40 kDa in sequence. The retentate was mixed with pure water at a ratio of 1:1 at 0.1 MPa. The ultrafiltration was repeated 3 times to obtain an immunoglobulin solution.
[0067] (6) Vacuum freeze-drying: The ultrafiltered immunoglobulin solution is placed in a vacuum freeze dryer and subjected to pre-freezing, sublimation drying, and desorption drying in sequence to obtain immunoglobulin powder; wherein the pre-freezing is carried out at a temperature of -30℃ at a rate of 1.5℃ / min, and the vacuum degree of the sublimation drying is 10. -5 Torr, the desorption drying is performed by increasing the temperature to 35°C at a rate of 1.5°C / min.
[0068] Example 3
[0069] This embodiment provides a method for preparing immunoglobulin powder, including the following steps:
[0070] (1) Bovine colostrum collection: Bovine colostrum raw milk within 72 hours was collected and frozen using CAS freezing and preservation equipment, with vibration parameters of 600MHz and freezing parameters of -5℃.
[0071] (2) Defatting: Thaw the frozen bovine colostrum at 8°C, dilute it with pure water to 6.0 wt% protein, put it into a milk fat separator, set the speed to 10000 r / min, remove the milk fat, and obtain defatted bovine colostrum;
[0072] (3) Curdling: Add compound rennet to skimmed bovine colostrum at a ratio of 3.0 kg / 1000 L at 8℃. Mix the compound rennet with pure water at a ratio of 1:50 and then add it to skimmed bovine colostrum. Stir at 400 r / min for 3 min and let it stand in a 40℃ water bath for 40 min.
[0073] The compound rennet comprises, by weight percentage, 40% aspartic protease, 35% papain, and 25% bromelain;
[0074] (4) Demulsification: Stir the coagulated protein and filter it using a 60-mesh filter to obtain the filtered whey.
[0075] (5) Ultrafiltration: The whey was passed through ultrafiltration membranes of 150 kDa, 120 kDa and 60 kDa in sequence. The retentate was mixed with pure water at a ratio of 1:1 at 0.3 MPa. The ultrafiltration was repeated 3 times to obtain an immunoglobulin solution.
[0076] (6) Vacuum freeze-drying: The ultrafiltered immunoglobulin solution is placed in a vacuum freeze dryer and subjected to pre-freezing, sublimation drying, and desorption drying in sequence to obtain immunoglobulin powder; wherein the pre-freezing is carried out at a temperature of -30℃ at a rate of 2℃ / min, and the vacuum degree of the sublimation drying is 10. -6 Torr, the desorption drying is performed by increasing the temperature to 40°C at a rate of 2°C / min.
[0077] Example 4
[0078] This embodiment provides a method for preparing immunoglobulin powder. The only difference between this embodiment and Example 1 is that in step (3), the compound chymotrypsin includes 10% aspartic protease, 60% papain and 30% bromelain by mass percentage. Other operations are the same as in Example 1.
[0079] Example 5
[0080] This embodiment provides a method for preparing immunoglobulin powder. The only difference between this method and Example 1 is that in step (3), the compound chymotrypsin includes 20% aspartic protease, 75% papain and 5% bromelain by mass percentage. Other operations are the same as in Example 1.
[0081] Example 6
[0082] This embodiment provides a method for preparing immunoglobulin powder. The only difference between this method and Example 1 is that in step (3), the compound chymotrypsin includes 10% aspartic protease, 10% papain and 80% bromelain by mass percentage. Other operations are the same as in Example 1.
[0083] Example 7
[0084] This embodiment provides a method for preparing immunoglobulin powder. The only difference between this embodiment and Example 1 is that in step (5), only 200KDa and 100KDa ultrafiltration membranes are used for ultrafiltration. Other operations are the same as in Example 1.
[0085] Example 8
[0086] This embodiment provides a method for preparing immunoglobulin powder. The only difference between this embodiment and Example 1 is that in step (5), only 200KDa and 50KDa ultrafiltration membranes are used for ultrafiltration. Other operations are the same as in Example 1.
[0087] Comparative Example 1
[0088] This comparative example provides a method for preparing immunoglobulin powder. The only difference between this method and Example 1 is that in step (1), ordinary freezing is used at a temperature of -8°C. Other operations are the same as in Example 1.
[0089] Comparative Example 2
[0090] This comparative example provides a method for preparing immunoglobulin powder. The only difference between this method and Example 1 is that, in step (3), the compound rennet does not contain aspartic protease, the ratio of the other two components remains unchanged, the total amount of compound rennet remains unchanged, and other operations are the same as in Example 1.
[0091] Comparative Example 3
[0092] This comparative example provides a method for preparing immunoglobulin powder. The only difference between this method and Example 1 is that in step (3), the compound chymopapain does not contain papain. The ratio of the other two components remains unchanged, and the total amount of compound chymopapain remains unchanged. Other operations are the same as in Example 1.
[0093] Comparative Example 4
[0094] This comparative example provides a method for preparing immunoglobulin powder. The only difference between this method and Example 1 is that, in step (3), the compound rennet does not contain bromelain. The ratio of the other two components remains unchanged, and the total amount of compound rennet remains unchanged. Other operations are the same as in Example 1.
[0095] Test Example 1
[0096] The curd state in the preparation methods provided in the above embodiments and comparative examples was recorded, and the content of the isolated and extracted immunoglobulins was detected by heparin affinity column. The results are summarized in Table 1.
[0097] Table 1
[0098]
[0099]
[0100] The test results show that:
[0101] (1) As can be seen from Examples 1 to 8, the present invention uses CAS freezing preservation technology to preserve bovine colostrum and uses a compound rennet of aspartic protease, papain and bromelain to coagulate. The two work together to effectively improve the coagulation effect. At the same time, the preparation process of ultrafiltration and vacuum freeze drying is used to achieve the preparation of high efficiency and high purity immunoglobulin powder.
[0102] (2) By comparing Example 1 and Examples 4-6, it can be seen that the present invention can achieve better curdling effect by reasonably combining the contents of the three enzymes in the compound rennet.
[0103] A comparison of Examples 1 and 7-8 shows that the present invention can increase the content of immunoglobulins by using a combination of three pore sizes of filter membranes for ultrafiltration.
[0104] (3) As can be seen from the comparison between Example 1 and Comparative Example 1, the CAS freezing and preservation technology used in this invention can preserve the activity of immunoglobulins to a greater extent than ordinary freezing, and can also assist the subsequent curdling process.
[0105] The comparison between Example 1 and Comparative Examples 2-4 shows that the three components of the compound rennet used in this invention—aspartic protease, papain, and bromelain—are indispensable, and they have a synergistic effect, which can significantly improve the rennet effect.
[0106] Test Example 2
[0107] The color, taste, and flavor of the immunoglobulin powders obtained by the preparation methods provided in the above embodiments and comparative examples were evaluated (the scoring criteria are shown in Table 2), and the results are summarized in Table 3.
[0108] Table 2
[0109]
[0110] Table 3
[0111]
[0112]
[0113] The test results show that:
[0114] (1) As can be seen from Examples 1 to 8, the present invention uses CAS freezing preservation technology to preserve bovine colostrum, uses compound rennet for coagulation, and uses ultrafiltration and vacuum freeze drying preparation process to obtain immunoglobulin powder of high quality with excellent color, taste and flavor.
[0115] (2) By comparing Example 1 and Examples 4-6, it can be seen that by further rationally combining the contents of the three enzymes in the compound rennet, the present invention can achieve a better rennet effect, thereby further improving the color, taste and flavor quality of the obtained immunoglobulin powder.
[0116] A comparison of Examples 1 and 7-8 shows that the present invention, by using a combination of three pore sizes of filter membranes for ultrafiltration, can further improve the color, taste, and flavor quality of the obtained immunoglobulin powder.
[0117] (3) By comparing Example 1 and Comparative Examples 1, 2-4, it can be seen that the CAS freezing preservation technology and specific compound rennet used in this invention can simultaneously improve the color, taste and flavor quality of immunoglobulin powder.
[0118] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.
Claims
1. A method for preparing immunoglobulin powder, characterized in that, The preparation method includes the following steps: (1) Collect bovine colostrum and freeze it using cell survival technology; (2) Thaw the frozen bovine colostrum and separate the milk fat to obtain skimmed bovine colostrum; (3) The skimmed bovine colostrum and compound rennet are mixed to coagulate, and then the milk is broken to obtain whey. The compound chymotrypsin includes aspartic protease, papain and bromelain. (4) Ultrafiltration of whey solution to obtain immunoglobulin solution; (5) The immunoglobulin solution is freeze-dried under vacuum to obtain the immunoglobulin powder.
2. The preparation method according to claim 1, characterized in that, The bovine colostrum mentioned in step (1) is bovine colostrum within 72 hours; Preferably, the vibration frequency of freezing in step (1) is 400 to 600 MHz, and the freezing temperature is -10 to -5°C.
3. The preparation method according to claim 1 or 2, characterized in that, Step (2) involves thawing to a temperature of 2–8°C; Preferably, step (2) further includes diluting the bovine colostrum with water after thawing; Preferably, the protein content in the diluted bovine colostrum is 3.5–6.0 wt%. Preferably, the milk fat separation in step (2) is performed by centrifugation; Preferably, the centrifugation speed is 6000-10000 r / min.
4. The preparation method according to any one of claims 1-3, characterized in that, The compound rennet in step (3) comprises, by weight percentage, 40-55% aspartic protease, 30-50% papain, and 15-25% bromelain.
5. The preparation method according to any one of claims 1-4, characterized in that, The ratio of compound rennet to skimmed bovine colostrum in step (3) is 2.8–3.0 kg / 1000 L; Preferably, the compound rennet in step (3) is added in the form of a compound rennet aqueous solution; Preferably, the mass ratio of the compound rennet to water in the compound rennet aqueous solution is 1:(20-50); Preferably, the temperature of the curd in step (3) is 30-40°C, and the curdling time is 40-60 min.
6. The preparation method according to any one of claims 1-5, characterized in that, The demulsification process described in step (3) is carried out by stirring. Preferably, after demulsification in step (3), a filtration operation is performed to obtain whey; Preferably, the filter screen used for filtration has a mesh size of 60-80.
7. The preparation method according to any one of claims 1-6, characterized in that, The ultrafiltration membrane used in step (4) has a pore size of 20-500 kDa; Preferably, the ultrafiltration in step (4) includes the use of at least two of the following: filter membranes with pore sizes of 150-250 kDa, 80-120 kDa and 40-60 kDa. More preferably, ultrafiltration is performed by sequentially using filter membranes with pore sizes of 150-250 kDa, 80-120 kDa and 40-60 kDa. Preferably, the pressure of ultrafiltration in step (4) is 0.1–0.3 MPa; Preferably, the number of ultrafiltrations in step (4) is 1 to 3.
8. The preparation method according to any one of claims 1-7, characterized in that, The vacuum freeze drying in step (5) includes pre-freezing, sublimation drying, and desorption drying; Preferably, the pre-freezing and cooling to a temperature of -30 to -25°C is performed at a rate of 1 to 2°C / min. Preferably, the sublimation drying is performed at a vacuum of 10 -5 ~ 10 -3 Torr. Preferably, the desorption drying process involves heating to a temperature of 30–40°C, with a heating rate of 1–2°C / min.
9. The preparation method according to any one of claims 1-8, characterized in that, The preparation method includes the following steps: (1) Collect bovine colostrum within 72 hours and freeze it using cell survival technology at a vibration frequency of 400-600MHz and a temperature of -10--5℃. (2) Thaw the frozen bovine colostrum to a temperature of 2-8℃, dilute it with water until the protein content in the bovine colostrum is 3.5-6.0 wt%, and separate the milk fat at a centrifugation speed of 6000-10000 r / min to obtain defatted bovine colostrum; (3) The compound rennet and skimmed bovine colostrum are mixed at a dosage ratio of 2.8-3.0 kg / 1000 L, and coagulated at 30-40 °C for 40-60 min. Then, the mixture is broken up by stirring and filtered through a 60-80 mesh filter to obtain whey. The compound rennet comprises 40-55% aspartic protease, 30-50% papain and 15-25% bromelain by mass percentage. The compound rennet is added in the form of a compound rennet aqueous solution, and the mass ratio of compound rennet to water in the compound rennet aqueous solution is 1:(20-50). (4) The whey solution was ultrafiltered 1 to 3 times at 0.1 to 0.3 MPa using a filter membrane with a pore size of 20-500 kDa to obtain an immunoglobulin solution; (5) The immunoglobulin solution is subjected to vacuum freeze-drying to obtain the immunoglobulin powder; the vacuum freeze-drying includes pre-freezing, sublimation drying and desorption drying; the pre-freezing temperature is -30 to -25°C, the cooling rate is 1 to 2°C / min, and the vacuum degree of the sublimation drying is 10. -5 ~10 -3 Torr, wherein the desorption drying is heated to a temperature of 30-40°C at a rate of 1-2°C / min.
10. The application of a method for preparing immunoglobulin powder according to any one of claims 1-9 in the preparation of pharmaceuticals and food.