Preparation method for extracting high-activity immune globulin liquid from bovine colostrum

Abstract

The invention relates to the technical field of high-activity immune globulin liquid extraction, in particular to a preparation method for extracting high-activity immune globulin liquid from bovine colostrum. The invention discloses a preparation method for extracting high-activity immune globulin liquid from bovine coloctrum, which comprises the following steps: performing primary separation, secondary separation, filtration, ultrafiltration concentration and sterilization on degreased bovine coloctrum to obtain the high-activity immune globulin liquid, the primary separation comprises the following steps: adjusting the pH value of the defatted bovine colostrum to 4.6-4.7, and sieving to remove casein, so as to obtain whey; and the secondary separation comprises the following steps: adjusting the pH value of the whey to 3.8-4.0, uniformly mixing, and sieving to remove denatured protein to obtain an immunoglobulin liquid. The high-activity immune globulin liquid containing IgG, IgA, IgM, lactoferrin, lactoperoxidase, IGF-beta, lysozyme, lactose and protein, which is prepared by adopting the method disclosed by the invention, can be used for effectively improving the immunity of a human body.

Description

Technical Field

[0001] This application relates to the technical field of high - activity immunoglobulin liquid extraction, and particularly to a preparation method for extracting high - activity immunoglobulin liquid from bovine colostrum. Background Art

[0002] In recent years, the prevention and control of viral infections globally have become important public health issues. Newly emerging and mutated viruses have posed severe challenges to the global medical system. Although vaccination has played an important role in preventing and controlling the epidemic, effective antiviral drugs against mutated viruses are still very limited, some drugs have problems with drug resistance, and the widespread use of traditional antibiotics has further exacerbated the emergence of superbugs. In 2024, the intensification of biosecurity issues and the spread of new virus variants (such as monkeypox, etc.) have led to a continuous increase in the global demand for enhancing the body's immune capacity. Against this background, it is particularly important to find natural solutions that can effectively respond to viral infections and enhance immunity.

[0003] Before the advent of sulfonamides and antibiotics, bovine colostrum was widely used for the prevention and treatment of infectious diseases due to its unique antibacterial properties. When the host comes into contact with foreign substances (antigens), antibodies will bind to, recognize, and destroy viruses, bacteria, and toxins. When the antigen enters the human body again, it will stimulate the production of the same antibodies to clear the infection. The first polio vaccine in the history of modern medicine was prepared using the antibodies in bovine colostrum, which verified the great value of bovine colostrum in the fields of medicine, nutrition, and health. Immunoglobulin liquid products, as important functional foods for enhancing the body's immunity, have received extensive attention. Immunoglobulins (IgG, IgA, IgM) play a crucial role in the prevention and treatment of viral infections, and can improve the body's anti - infectious ability by binding to pathogens, promoting phagocytosis, and regulating immune responses; among them, extracting high - activity immunoglobulins from bovine colostrum has become an important direction for research and industrial development; however, the existing extraction technologies still have certain limitations, including low extraction efficiency, large losses of active ingredients during the purification process, and limited stability of key functional factors. For example, active factors such as lactoferrin, lactoperoxidase, and IGF - β are easily degraded by factors such as temperature and pH value during extraction and storage, resulting in a decrease in the immune - enhancing effect of the product.

[0004] Therefore, how to optimize the preparation method of immunoglobulin liquid products to improve the content and stability of their active ingredients is an important direction of current research. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, this application provides a preparation method for extracting high - activity immunoglobulin liquid from bovine colostrum.

[0006] In a first aspect, the present application provides a preparation method for extracting a highly active immunoglobulin liquid from bovine colostrum, adopting the following technical solutions: A preparation method for extracting a highly active immunoglobulin liquid from bovine colostrum, the steps of the preparation method are as follows: Subject the defatted bovine colostrum to primary separation, secondary separation, filtration, ultrafiltration concentration, and sterilization to obtain a highly active immunoglobulin liquid; Among them, the steps of the primary separation are as follows: adjust the pH of the defatted bovine colostrum to 4.6 - 4.7, and remove casein by sieving to obtain whey; the steps of the secondary separation are as follows: adjust the pH of the whey to 3.8 - 4.0, mix well, and remove denatured proteins by sieving to obtain an immunoglobulin liquid.

[0007] By adopting the above technical solutions, first adjust the pH of the defatted bovine colostrum to precipitate casein, remove casein by sieving to obtain whey; then adjust the pH of the whey to precipitate denatured proteins, remove denatured proteins by sieving to obtain an immunoglobulin liquid, thereby realizing the extraction of the effective components in the defatted bovine colostrum.

[0008] The present application uses natural bovine colostrum as the raw material, and obtains highly concentrated and highly active immunoglobulins through advanced extraction technologies, which can provide strong immune support for the human body and play a preventive and therapeutic role against health threats such as viruses and bacteria. The present application uses natural bovine colostrum extract, which is rich in high concentrations of immunoglobulins (IgA, IgG, IgM, IGF-β, etc.), other bioactive components (such as lactoferrin, lactoperoxidase, lysozyme, etc.), as well as lactose and proteins, providing unique immune support and strong antiviral and antibacterial effects. The immunoglobulin liquid prepared by the method of the present application not only provides high protein for the human body, but also enhances the immune effect on the basis of highly active immunoglobulins.

[0009] Preferably, before the primary separation of the defatted bovine colostrum, heat treatment is performed on the defatted bovine colostrum, and the steps are as follows: heat the defatted bovine colostrum to 40 - 50 °C.

[0010] Preferably, in the primary separation step, the sieve used for sieving is 30 - 50 mesh.

[0011] By adopting the above technical solutions, a sieve of 30 - 50 mesh can be selected to filter out casein.

[0012] Preferably, in the secondary separation step, the sieve used for sieving is 180 - 200 mesh.

[0013] By adopting the above technical solutions, a sieve of 180 - 200 mesh can be selected to filter out denatured proteins.

[0014] Preferably, in the secondary separation step, the temperature of the whey is 40 - 50 °C.

[0015] Preferably, in the secondary separation step, after adjusting the pH of the whey to 3.8 - 4, the mixing time is 3 - 7 minutes; during the mixing process, the temperature of the whey is 40 - 50 °C.

[0016] Preferably, in the primary separation step, the solution used to adjust the pH of the defatted bovine colostrum is an acid solution.

[0017] Preferably, in the secondary separation step, the solution used to adjust the pH of the whey is an acid solution.

[0018] Preferably, the acid in the acid solution is citric acid, lactic acid, vitamin C or acetic acid.

[0019] Preferably, the filtration step is as follows: The immunoglobulin liquid obtained after secondary separation is filtered through a microporous filtration membrane with a pore size of 0.22 μm.

[0020] By adopting the above technical solution, filtering with a microporous filtration membrane with a pore size of 0.22 μm can effectively remove microorganisms and other fine particles in the liquid, and further improve the purity and safety of the immunoglobulin liquid.

[0021] In this application, through the filtration step, microorganisms in the immunoglobulin liquid can be removed. Additionally, a ceramic membrane separation device with a membrane pore size of 0.22 μm or other filtration devices can be used for filtration.

[0022] Preferably, in the ultrafiltration concentration step, an ultrafiltration device with a membrane retention molecular weight of 10 - 100 KDa is used for ultrafiltration concentration.

[0023] Preferably, in the sterilization step, the sterilization temperature is 120 - 150 °C, and the sterilization time is 2 - 10 seconds.

[0024] In a specific feasible implementation, an ultra-high temperature sterilization (UHT) device can be used to sterilize the obtained ultrafiltration concentrate; specifically, the sterilization treatment is carried out at 150 °C, and the time is controlled at 5 seconds.

[0025] Preferably, the preparation step of the defatted bovine colostrum is as follows: The bovine colostrum is centrifuged to remove fat to obtain defatted bovine colostrum.

[0026] Preferably, during centrifugal defatting, the centrifugation speed is 3000 - 6000 rpm, the temperature of the bovine colostrum is controlled ≤ 25 °C during centrifugation, and the current setting value of the centrifugal defatting machine is 7.5 - 8.5 A.

[0027] In a specific feasible implementation, the raw bovine colostrum is processed. When the raw bovine colostrum is frozen raw bovine colostrum, the frozen raw bovine colostrum is put into a slicing machine for slicing and crushing, and after crushing, it is put into a thawing pool for heating and thawing. After thawing, the temperature of the raw bovine colostrum ≤ 25°C, and the thawed raw bovine colostrum is then transported to a centrifugal defatting machine for defatting treatment.

[0028] In a specific feasible implementation, when the raw bovine colostrum is fresh raw bovine colostrum, the milk is milked from the dairy cow and the raw bovine colostrum is collected, and immediately refrigerated at 4 - 10°C, and the raw bovine colostrum is transported into a centrifugal defatting machine for defatting.

[0029] Preferably, the preparation method of the high - activity immunoglobulin liquid extracted from raw bovine colostrum further includes a filling step: filling the obtained sterilized liquid.

[0030] In the second aspect, the present application provides a high - activity immunoglobulin liquid, adopting the following technical solution: A high - activity immunoglobulin liquid, wherein the high - activity immunoglobulin liquid includes IgG content of 80.1 ± 6.31 wt%, IgA content of 5.61 ± 0.45 wt%, IgM content of 5.79 ± 0.36 wt%, lactoferrin content of 0.84 ± 0.01 wt%, lactoperoxidase content of 48450 ± 237.7 ng / g, IGF - β content of 1553.4 ± 52.64 ng / g, lysozyme content of 842 ± 12.54 ng / g, lactose content of 0.01 - 6.99 wt% and protein content of 89.47 - 96.45 wt%.

[0031] In the third aspect, the present application provides an application of a high - activity immunoglobulin liquid in the preparation of food, health products or drugs, adopting the following technical solution: An application of the high - activity immunoglobulin liquid prepared by the above - mentioned method or the above - mentioned high - activity immunoglobulin liquid in the preparation of food, health products or drugs.

[0032] In summary, the present application includes at least one of the following beneficial technical effects: 1. The present application discloses a preparation method of a high - activity immunoglobulin liquid extracted from raw bovine colostrum. The defatted raw bovine colostrum is subjected to primary separation, secondary separation, filtration, ultrafiltration concentration and sterilization to prepare a high - activity immunoglobulin liquid containing IgG, IgA, IgM, lactoferrin, lactoperoxidase, IGF - β, lysozyme, lactose and protein, with high stability; 2. The present application uses the acid precipitation method to remove casein and denatured proteins from defatted raw bovine colostrum. Compared with the rennet precipitation method, the fermentation step is omitted, which is simple, efficient, time - saving and lower in cost. Description of the Drawings

[0033] Figure 1 Schematic process flow diagram of extracting highly active immunoglobulin liquid from bovine colostrum in this application; Figure 2 SDS-PAGE electrophoresis analysis of defatted bovine colostrum and immunoglobulin powder samples; among them, band 1 is IgG standard; band 2 is defatted bovine colostrum; band 3 is immunoglobulin powder sample; Figure 3 Stability test results of defatted bovine colostrum and immunoglobulin powder samples; Figure 4 Results of antioxidant ABTS free radical scavenging experiment of defatted bovine colostrum and immunoglobulin powder samples; Figure 5 Results of antioxidant DPPH free radical scavenging experiment of defatted bovine colostrum and immunoglobulin powder samples; Figure 6 Rocket immunoelectrophoresis activity determination of immunoglobulin powder samples. Specific implementation mode

[0034] The technical solution of this application is further illustrated by the following specific examples. The specific examples do not represent the limitation of the protection scope of this application; some non-essential modifications and adjustments made by others according to the concept of this application still fall within the protection scope of this application.

[0035] All raw materials involved in this application are commercially available products. The following further details this application in combination with examples.

[0036] A method for preparing an immunoglobulin liquid beverage, the method comprising the following steps: (1) Collection and centrifugal defatting: S1. Collection of bovine colostrum: Frozen bovine colostrum or fresh bovine colostrum is used for bovine colostrum: When the bovine colostrum is frozen bovine colostrum, the frozen bovine colostrum (milk from the cow within 12 hours before milking) is put into a slicing machine for slicing and pulverizing, and after pulverization, it is put into a thawing tank for heating and thawing. The temperature of the thawed bovine colostrum is ≤25°C, and the thawed bovine colostrum is then transported to a centrifugal defatting machine for defatting treatment.

[0037] When the bovine colostrum is fresh bovine colostrum, milk is squeezed from the cow and the bovine colostrum is collected, immediately refrigerated at 4 - 10°C, and directly transported to the centrifugal defatting machine for defatting.

[0038] S2. Centrifugal defatting Transport the colostrum to a centrifugal defatting machine for defatting as follows: Transport the collected colostrum to the centrifugal defatting machine through a pipeline for centrifugal defatting at a rotational speed of 3000 - 6000 rpm, with the current set at 7.5 - 8.5 A, and control the temperature of the colostrum during the operation at ≤25°C.

[0039] (2) Separation I: After heat treatment (40 - 50°C), use an acid solution to adjust the pH of the defatted colostrum to 4.6 - 4.7 to precipitate casein, and remove the casein by sieving to obtain whey.

[0040] Among them, the sieve used for sieving is 30 - 50 mesh.

[0041] The acid in the acid solution is citric acid, lactic acid, vitamin C or acetic acid.

[0042] (3) Separation II: Use an acid solution to adjust the pH of the whey to 3.8 - 4, mix and stir the whey at 40 - 50°C for 3 - 7 minutes to precipitate denatured proteins, and remove the denatured proteins by sieving to obtain immunoglobulin liquid.

[0043] Among them, the temperature of the whey during the process of Separation II is 40 - 50°C.

[0044] The sieve used for sieving is 180 - 200 mesh.

[0045] The acid in the acid solution is citric acid, lactic acid, vitamin C or acetic acid.

[0046] (4) Filtration: Use a 0.22um microporous filter membrane to remove microorganisms in the immunoglobulin liquid.

[0047] Specifically, this application can use a ceramic membrane separation device with a membrane pore size of 0.22μm or other filtration equipment for filtration.

[0048] (5) Ultrafiltration concentration: Put the filtered immunoglobulin liquid into a membrane separation device for ultrafiltration concentration.

[0049] Use an ultrafiltration device with a membrane retention molecular weight of 10 - 100KDa for ultrafiltration concentration.

[0050] (6) Sterilization: Perform UHT sterilization on the obtained ultrafiltration concentrate.

[0051] The sterilization temperature is 120 - 150°C, and the sterilization time is 2 - 10 seconds.

[0052] Specifically, an ultra-high temperature sterilization (UHT) device can be used to sterilize the obtained ultrafiltration concentrate; specifically, perform the sterilization treatment at 150°C for 5 seconds.

[0053] (7) Filling: The sterilized liquid obtained is filled to obtain a high - activity immunoglobulin liquid.

[0054] Specifically, the filling container used is a 6 - mL glass or plastic container.

[0055] The ultra - filtered and concentrated liquid is transported through a pipeline to an ultra - high - temperature sterilization (UHT) device for sterilization treatment. After sterilization, it is quickly cooled to a safe temperature to ensure the quality and stability of the product. The cooled immunoglobulin liquid solution is filled under aseptic conditions, quantitatively filled into bottles, 6 mL per bottle; after filling, the inner packaging is sealed to ensure the safety and stability of the product during storage and transportation.

[0056] Example 1: An industrial method for separating and purifying immunoglobulin from defatted bovine colostrum An industrial method for separating and purifying immunoglobulin from defatted bovine colostrum, the preparation process is as Figure 1 shown, and the specific steps are as follows: (1) Crushing, thawing and centrifugal defatting Take frozen bovine colostrum (from the milk within 12 hours before a cow gives birth) out of the cold storage, put it into a slicing machine for slicing and crushing; the crushed bovine colostrum is put into a thawing tank for heating, and the temperature of the thawed bovine colostrum is ≤25°C, and continuous stirring is carried out during the thawing process.

[0057] The thawed bovine colostrum is transported through a pipeline to a centrifugal defatting machine for centrifugal defatting, the rotation speed is 4000 rpm, the current is set at 8 A, and the temperature of the bovine colostrum during the operation process is controlled at ≤25°C to obtain defatted bovine colostrum.

[0058] (2) Removal of casein Dissolve food - grade citric acid powder in purified water, stir until completely dissolved to obtain a 0.1 M citric acid solution for standby.

[0059] Heat 100 kg of defatted bovine colostrum to 45°C, add the prepared citric acid solution to the defatted bovine colostrum while stirring until the pH value of the bovine colostrum is measured to be 4.6, and filter through a 30 - mesh sieve to remove casein to obtain whey.

[0060] (3) Removal of denatured proteins Maintain the whey temperature at 45°C, continue to slowly add the citric acid solution to the whey while stirring until the pH value of the immunoglobulin solution is measured to be 3.8; after completing the pH adjustment, maintain the whey temperature at 45°C and continue stirring for 5 minutes to ensure uniform reaction. Subsequently, the immunoglobulin liquid is filtered through a 200 - mesh sieve to remove denatured proteins to obtain the immunoglobulin liquid.

[0061] (4) Filtration Transport the immunoglobulin liquid through a pipeline to a ceramic membrane separation device for filtration. The membrane pore size is 0.22 μm.

[0062] Use pressure filtration technology for separation to effectively remove microorganisms and other fine particles in the liquid, and further improve the purity and safety of the immunoglobulin liquid. During the separation process, the operating temperature and pressure need to be strictly controlled to ensure that the separation efficiency and the functional activity of the immunoglobulin are not affected.

[0063] (5) Ultrafiltration concentration Add the filtered immunoglobulin liquid to an ultrafiltration device for ultrafiltration. The membrane retention molecular weight used in the ultrafiltration device is 10 kDa.

[0064] The specific steps are as follows: Add 2 volumes of ultrapure water to the solution obtained in step (4), ultrafilter through a 10 kDa ultrafiltration membrane to the original volume, recover the retentate, add 2 original volumes of ultrapure water, and then perform ultrafiltration again. This process is repeated 3 times until the absorbance measured at 280 nm by an ultraviolet spectrophotometer for the permeate is less than 0.05, and then concentrate it to 1 / 3 of the original volume.

[0065] (6) Sterilization and filling Transport the ultrafiltered and concentrated immunoglobulin liquid through a pipeline to an ultra-high temperature sterilization (UHT) device, perform sterilization treatment at 150 °C for 5 seconds; after sterilization, quickly cool it to a safe temperature; the cooled immunoglobulin liquid solution undergoes filling treatment in a sterile environment, quantitatively filled into bottles, 6 mL per bottle; after filling, seal the inner packaging to obtain the immunoglobulin liquid.

[0066] Performance detection: In order to detect the components in the immunoglobulin liquid prepared in Example 1, the immunoglobulin liquid prepared in Example 1 was dried by freeze-drying for 24 hours to obtain a dried immunoglobulin powder sample, and the immunoglobulin powder sample was detected.

[0067] 1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) determination The SDS-PAGE electrophoresis analysis results of the immunoglobulin powder sample are as Figure 2 shown. Band 1 is the IgG standard as a reference. Band 2 is defatted bovine colostrum, and the band intensities of its IgG, IgA, and IgM are relatively low, indicating a relatively low immunoglobulin content. Band 3 is the immunoglobulin powder sample, and the bands of its IgG, IgA, IgM, lactoferrin, lactoperoxidase, IGF-β, and lysozyme are significantly stronger, proving that the method disclosed in this application successfully extracted a high-purity and high-content immunoglobulin powder sample.

[0068] 2. Quantitative Analysis of the Composition of Defatted Colostrum and Immunoglobulin Powder Samples The quantitative analysis of the composition of the immunoglobulin powder sample and the defatted colostrum prepared in Step 1 of Example 1 is shown in Table 1 as follows: Table 1 Quantitative Analysis of the Composition of Defatted Colostrum and Immunoglobulin Powder Samples As shown in Table 1, Table 1 further verifies Figure 2 the quantitative analysis results of the band intensity in []. It shows that the IgG content in the immunoglobulin powder reaches 80.1 ± 6.31 wt%, much higher than 31.66 ± 4.52 wt% in defatted colostrum. The contents of IgA and IgM also increase significantly, being 5.61 ± 0.45 wt% and 5.79 ± 0.36 wt% respectively, while the contents in defatted colostrum are only 2.15 ± 0.3 wt% and 2.22 ± 0.3 wt%. In addition, the contents of bioactive components such as lactoferrin, lactoperoxidase, IGF-β, and lysozyme in the immunoglobulin powder sample are significantly higher than those in defatted colostrum. These bioactive substances have various functions such as immune enhancement, antibacterial, and antioxidant, further verifying the high functionality of the immunoglobulin powder sample.

[0069] 3. Quantitative Analysis of the Composition of Defatted Colostrum and Immunoglobulin Powder Samples The quantitative analysis of the composition of the immunoglobulin powder sample and the defatted colostrum prepared in Step 1 of Example 1 is shown in Table 2 as follows: Table 2 Quantitative Analysis of the Composition of Defatted Colostrum and Immunoglobulin Powder Samples As shown in Table 2, Table 2 shows the composition of defatted colostrum, indicating that lactose is the main component of defatted colostrum, with a content as high as 20.3 - 35.2 wt%, while lactose in the immunoglobulin powder sample has been basically removed. The protein content increases from 56.5 - 71.4 wt% in defatted colostrum to 89.47 - 96.45 wt% in the immunoglobulin powder sample. This result shows that by removing lactose and other impurities, the purity and applicability of the immunoglobulin powder sample are greatly improved, making it more suitable for lactose-intolerant people and consumers with high protein requirements.

[0070] Figure 2, Tables 1 and 2 together demonstrate the significant advantages of the application in extracting high-purity immunoglobulins. The present application has achieved the technology of efficiently extracting high-purity immunoglobulins from bovine colostrum, significantly increasing the content of bioactive components of immunoglobulins, while reducing unnecessary impurities and lactose content. This research result not only shows the great potential of immunoglobulin powder in the field of functional foods, but also provides a new solution for lactose-intolerant people and the high-protein demand market.

[0071] 4. Microbiological stability assessment of immunoglobulin powder samples during the shelf life According to the detection standard of "GB7101-2022 National Food Safety Standard Beverages", the microbiological stability of immunoglobulin powder samples during the shelf life was tested. The detection method was: detecting in an accelerated stability test chamber at a temperature of 37°C and a humidity of 74.8% RH. The detection results are shown in Table 3: Table 3 Microbiological stability assessment of immunoglobulin powder samples during the shelf life As shown in Table 3, Table 3 shows the microbiological stability of the immunoglobulin liquid product within 48 months. The results show that within the first month, the total number of colonies was less than 1 CFU / ml, slightly increased to 1 CFU / ml in the 3rd month, and increased to 7 CFU / ml in the 6th month, meeting the detection standard, indicating that no significant microbial growth was observed during the entire 48-month test period, proving the long-term stability of the product and minimal microbial contamination. In addition, during the entire shelf life, the growth of coliforms, molds, and yeasts was not detected, further confirming the natural antimicrobial properties of the product. This ensures the microbiological safety of the product for 48 months without using chemical preservatives or additives, reflecting its inherent preservation ability.

[0072] 5. Stability test of defatted bovine colostrum and immunoglobulin powder samples The stability of the immunoglobulin powder samples and the defatted bovine colostrum prepared in Step 1 of Example 1 was tested. The detection results are as Figure 3 shown.

[0073] As Figure 3 shown, Figure 3It shows the absorbance changes of defatted bovine colostrum and immunoglobulin powder samples at room temperature (20 °C) under different storage periods. The left figure is defatted bovine colostrum, and the right figure is the immunoglobulin powder sample. The absorbance of defatted bovine colostrum gradually increases with the extension of storage time, especially significantly in the wavelength range of 250 nm to 300 nm. This indicates that during the storage of defatted bovine colostrum, chemical reactions may occur to its lactose, minerals, and proteins, such as lactose degradation to form lactic acid, the Maillard reaction between lactose and proteins, and the oxidation reaction promoted by minerals, resulting in a significant reduction in the system stability. These reactions cause the gradual degradation of the active components in defatted bovine colostrum, affecting the product quality. The change range of the absorbance of the immunoglobulin powder sample is significantly lower than that of defatted bovine colostrum, especially in the wavelength range of 250 nm to 300 nm, and its absorbance basically remains stable. This shows that during the long-term storage of the immunoglobulin powder sample, its active components and structure both exhibit excellent stability. This stability is closely related to the high-purity immunoglobulins (such as IgG, IgA, IgM) and extremely low lactose and mineral contents in the immunoglobulin powder sample.

[0074] The lactose content of the immunoglobulin powder sample is only 20.3 - 35.2 wt%, much lower than that of defatted bovine colostrum at 20.3 - 35.2 wt%. The removal of lactose effectively avoids the instability caused by lactose degradation and lactose-protein reactions during storage. The significant reduction in the mineral content reduces its oxidative catalytic effect on proteins during storage, thereby further enhancing the system stability. In addition, the protein purity in the immunoglobulin powder sample is as high as 89.47 - 96.45 wt%, significantly higher than that of defatted bovine colostrum at 56.5 - 71.4 wt%. The rich immunoglobulins (such as IgG) have a natural protective effect in antioxidant and antibacterial aspects, further reducing the component degradation during storage. Figure 3 The absorbance stability of the immunoglobulin powder sample in [the text] proves that its structure is not significantly damaged during storage. This high stability benefits from the significant reduction in lactose and mineral contents, the improvement of protein purity, and the protective effect of highly active immunoglobulins. Components such as IgG in the immunoglobulin powder sample not only enhance the biological activity of the product but also play an important role in the antioxidant process, making it better able to maintain its function and quality than defatted bovine colostrum during storage.

[0075] 6. Determination of the antioxidant ABTS radical scavenging ability of defatted bovine colostrum and immunoglobulin powder samples The antioxidant ABTS radical scavenging ability of the defatted bovine colostrum and immunoglobulin powder samples prepared in step 1 of Example 1 was determined, and the test results are as Figure 4 shown.

[0076] As Figure 4 shown, Figure 4The ABTS radical scavenging ability of defatted bovine colostrum and immunoglobulin powder samples at different concentrations (5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL) was demonstrated. The ABTS radical scavenging ability of the immunoglobulin powder samples was significantly higher than that of defatted bovine colostrum. At 5 mg / mL, the scavenging rate of the immunoglobulin powder sample was 28.86%, while that of defatted bovine colostrum was only 12.60%. As the concentration increased to 20 mg / mL, the scavenging rate of the immunoglobulin powder sample reached 81.01%, and that of defatted bovine colostrum was 55.73%. The immunoglobulin powder samples showed better scavenging ability at each concentration, especially at high concentrations, where the radical scavenging efficiency increased significantly.

[0077] This enhancement of antioxidant capacity benefits from the abundant lactoferrin and lactoperoxidase in the immunoglobulin powder samples. Lactoferrin has the ability to directly scavenge free radicals and, at the same time, protects other active ingredients from oxidative damage by reacting with reactive oxygen species. Lactoperoxidase, as a natural antioxidant enzyme, can decompose peroxides and reduce the generation of free radicals, thus synergistically enhancing the antioxidant performance of the immunoglobulin powder samples.

[0078] 7. Determination of the antioxidant DPPH radical scavenging ability of defatted bovine colostrum and immunoglobulin powder samples The antioxidant DPPH radical scavenging ability of the defatted bovine colostrum and immunoglobulin powder samples prepared in step 1 of Example 1 was determined, and the test results are as Figure 5 shown.

[0079] As Figure 5 shown, Figure 5 the DPPH radical scavenging ability of defatted bovine colostrum and immunoglobulin powder samples at the same concentrations (5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL) was demonstrated. The DPPH radical scavenging ability of the immunoglobulin powder samples was also significantly better than that of defatted bovine colostrum. At 5 mg / mL, the scavenging rate of the immunoglobulin powder sample was 7.82%, while that of defatted bovine colostrum was 5.75%; when the concentration increased to 20 mg / mL, the scavenging rate of the immunoglobulin powder sample increased to 27.27%, and that of defatted bovine colostrum was 22.98%.

[0080] The results of the DPPH experiment were consistent with those of the ABTS experiment, further demonstrating the excellent antioxidant capacity of the immunoglobulin powder sample in different free radical systems. The antioxidant capacity of the immunoglobulin powder sample is closely related to its high content of immunoglobulins and active ingredients. Immunoglobulins such as IgG directly interact with free radicals, reducing the oxidative damage of free radicals to biomolecules. In addition, lactoferrin and lactoperoxidase have a synergistic effect in stabilizing the system, reducing the accumulation of free radicals and protecting active ingredients, significantly enhancing the antioxidant capacity of the immunoglobulin powder sample.

[0081] 8. Rocket Immunoelectrophoresis Activity Assay of Immunoglobulin Powder Sample The rocket immunoelectrophoresis activity of the immunoglobulin powder sample was assayed, and the test results are as Figure 6 shown.

[0082] As Figure 6 shown, Figure 6 The immunological activity of the immunoglobulin powder sample maintained by rocket immunoelectrophoresis technology is presented, highlighting its continuous effectiveness over time. According to the antibody capacity data at different antibody concentrations, the antibody capacity corresponding to an antibody concentration of 0.1 mg / ml is 56%, 78% for 0.2 mg / ml, 87% for 0.3 mg / ml, 91% for 0.4 mg / ml, and 100% for 0.5 mg / ml. As the immunoglobulin concentration increases, the activity of the antibody gradually enhances. Especially at a concentration of 0.5 mg / ml, the antibody activity reaches 100%, indicating that the immunopotentiating effect of immunoglobulin reaches the maximum at this concentration; as the immunoglobulin concentration increases, its immunopotentiating effect is significantly strengthened; this finding shows that highly active immunoglobulin can greatly activate the body's immune system and provide strong immune support; especially at a concentration of 0.5 mg / ml, the activity of immunoglobulin shows a 100% immune support effect, and this concentration can provide the most effective immune protection, suitable for various applications such as antiviral and antibacterial; especially its significant effect in antiviral. Immunoglobulin can effectively help the immune system recognize and combat viral infections by enhancing the antibody response in the body. The high concentration of this immunoglobulin can not only increase the reaction speed of immune cells but also improve the neutralizing ability of immunoglobulin against viruses, preventing the spread and invasion of viruses in the body; especially in the face of viral infections such as influenza and coronavirus, immunoglobulin can provide effective immune support, help the body resist viral attacks, and reduce the risk of infection.

[0083] In addition, the high activity of immunoglobulin can also combat bacteria and other pathogens, with a wide range of antibacterial effects; its powerful immune-enhancing effect provides comprehensive immune protection for the human body, helping to improve the disease resistance ability. Especially during the epidemic of viruses, immunoglobulin can provide a stronger protection barrier in the body; verified by rocket immunoelectrophoresis technology, the innovation of the product prepared by the method of this application in enhancing immune activity has been fully demonstrated. This technology maximizes the natural biological activity of immunoglobulin, making it show extremely high effects in antiviral, antibacterial and other aspects. Compared with traditional immune-enhancing products, this technology greatly improves the biological efficacy of immunoglobulin, representing a breakthrough in the field of immunity.

[0084] Example 2: To study the effects of different pH values on the extraction of high-activity immunoglobulin liquid products, the defatted bovine colostrum in step (2) of Example 1 was adjusted to different pH values to prepare immunoglobulin liquids; and the prepared immunoglobulin liquids were dried by freeze-drying for 24 hours to obtain dry immunoglobulin powder samples, and the immunoglobulin powder samples were detected, as shown in Table 4 specifically.

[0085] Among them, the extraction rate = the mass of immunoglobulin liquid / the mass of defatted bovine colostrum.

[0086] Table 4 Effects of different pH values on the extraction of high-activity immunoglobulin liquid products from bovine colostrum Conclusion Under different pH conditions, the IgG content of defatted bovine colostrum showed significant changes. At pH 4.5, the IgG content after removing casein solids was 0.1%; at pH 4.6 and pH 4.7, the extraction effect of IgG was the most significant, and the IgG content was 0.24%; this indicates that at pH 4.6 and pH 4.7, the extraction efficiency of IgG after removing casein solids was the highest; while above pH 4.8, the IgG content increased significantly, exceeding 3%, but the extraction effect was worse than that at pH 4.6 and pH 4.7. Generally speaking, the extraction effects at pH 4.6 and pH 4.7 were the most ideal.

[0087] Example 3: To study the effects of different pH values on the extraction of high-activity immunoglobulin liquid products, the whey in step (3) of Example 1 was adjusted to different pH values to prepare immunoglobulin liquids; and the prepared immunoglobulin liquids were dried by freeze-drying for 24 hours to obtain dry immunoglobulin powder samples, and the immunoglobulin powder samples were detected, as shown in Table 5 specifically.

[0088] Table 5 Effects of Different pH Values on the Extraction of High-Activity Immunoglobulin Liquid Products from Bovine Colostrum pH Denatured solids removed / kg Immunoglobulin solids / kg IgG content / % 3.7 0.05 2.65 77.95 3.8 0.1 2.6 79.45 3.9 0.2 2.5 80.11 4.0 0.3 2.4 80.14 4.1 0.4 2.3 80.01 Conclusion: In the range of pH 3.8 to pH 4.0, the extraction effects of immunoglobulin solids and the purity of IgG are the most significant and stable. Specifically, as the pH increases from 3.7 to 4.0, the extraction amount and purity of immunoglobulin gradually increase. At pH 3.8, the IgG content is 79.45%, and it reaches 80.11% at pH 3.9, and at pH 4.0, the IgG content is 80.14%, maintaining a high and stable extraction effect. Between pH 3.8 and pH 4.0, the extraction amount and purity of immunoglobulin solids show an ideal balance, indicating that this pH range has an efficient extraction effect on IgG.

[0089] The immunoglobulin liquid prepared by the method of the present application is a liquid product with a high immunoglobulin content and high activity, which can provide more efficient immune support for the human body. This product retains the most active immune components in bovine colostrum through a unique extraction technology, and can improve the body's ability to resist viruses, bacteria and pathogens while enhancing the immune function. Immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) in the product can, through their synergistic effects, improve the agility of the body's immune response and the neutralization ability of viruses, especially showing powerful effects in aspects such as antiviral and antibacterial.

[0090] Bovine colostrum has been used as a natural food to enhance immunity since ancient times. Modern research shows that components such as immunoglobulin, lactoferrin, and lactoperoxidase in bovine colostrum have powerful immune-enhancing effects and can help the body quickly recognize and fight against foreign pathogens. Different from traditional drugs and synthetic immune enhancers, the immunoglobulin liquid product disclosed in the present application contains no chemical components and no preservatives, can be directly consumed as a beverage, is completely natural and safe, and is suitable for various populations, especially those with weak immunity, such as the elderly, children and immunodeficient individuals.

Claims

1. A method for preparing a highly active immunoglobulin liquid extracted from bovine colostrum, characterized in that: The preparation method steps are as follows: The defatted bovine colostrum is subjected to primary separation, secondary separation, filtration, ultrafiltration concentration and sterilization to obtain a highly active immunoglobulin liquid; The steps of the first separation are as follows: adjusting the pH of the defatted bovine colostrum to 4.6-4.7, sieving to remove casein, and obtaining whey; The steps of secondary separation are as follows: adjust the pH of whey to 3.8-4.0, mix well, sieve to remove denatured protein, and obtain immunoglobulin liquid.

2. The method for preparing a highly active immunoglobulin liquid extracted from bovine colostrum according to claim 1, characterized in that: Before the skimmed bovine colostrum is separated once, the skimmed bovine colostrum is heat treated, and the steps are as follows: the skimmed bovine colostrum is heated to 40-50°C.

3. The method for preparing a highly active immunoglobulin liquid extracted from bovine colostrum according to claim 1, characterized in that: In the first separation step, the sieve used for sieving is 30-50 mesh; In the secondary separation step, the screen used for sieving is 180-200 mesh.

4. The method for preparing a highly active immunoglobulin liquid extracted from bovine colostrum according to claim 1, characterized in that: In the secondary separation step, the temperature of the whey is 40-50°C; In the secondary separation step, after adjusting the pH of the whey to 3.8-4, the mixing time is 3-7 minutes; during the mixing process, the temperature of the whey is 40-50°C.

5. The method for preparing a highly active immunoglobulin liquid extracted from bovine colostrum according to claim 1, characterized in that: In the first separation step, the solution used to adjust the pH of the defatted bovine colostrum is an acid solution; In the secondary separation step, the solution used to adjust the pH of the whey is an acid solution.

6. The method for preparing a highly active immunoglobulin liquid extracted from bovine colostrum according to claim 5, characterized in that: The acid in the acid solution is citric acid, lactic acid, vitamin C or acetic acid.

7. The method for preparing a highly active immunoglobulin liquid extracted from bovine colostrum according to claim 1, characterized in that: The filtering steps are as follows: filtering the immunoglobulin liquid obtained after the secondary separation through a microporous filter membrane with a pore size of 0.22 um; In the ultrafiltration and concentration step, ultrafiltration and concentration are performed using an ultrafiltration device with a membrane molecular weight cutoff of 10-100 KDa; In the sterilization step, the sterilization temperature is 120-150° C. and the sterilization time is 2-10 seconds.

8. The method for preparing a highly active immunoglobulin liquid extracted from bovine colostrum according to claim 1, characterized in that: The preparation steps of the defatted bovine colostrum are as follows: the bovine colostrum is subjected to centrifugal defatting to obtain the defatted bovine colostrum.

9. A highly active immunoglobulin liquid prepared by the method according to any one of claims 1 to 8, characterized in that: The highly active immunoglobulin liquid comprises an IgG content of 80.1±6.31wt%, an IgA content of 5.61±0.45wt%, an IgM content of 5.79±0.36wt%, a lactoferrin content of 0.84±0.01wt%, a lactoperoxidase content of 48450±237.7ng / g, an IGF-β content of 1553.4±52.64ng / g, a lysozyme content of 842±12.54ng / g, a lactose content of 0.01-6.99wt% and a protein content of 89.47-96.45wt%.

10. Use of the highly active immunoglobulin liquid prepared by the method according to any one of claims 1 to 8 or the highly active immunoglobulin liquid according to claim 9 in the preparation of foods, health products or medicines.

Images