A method for preparing highly active natural immunoglobulin freeze-dried powder with high antioxidant properties

Abstract

This application relates to the technical field of solid extraction of highly active immunoglobulins, and in particular to a method for preparing highly active natural immunoglobulin freeze-dried powder with high antioxidant properties. The method comprises the following steps: defatting, enzymatic hydrolysis, filtration, membrane concentration, and freeze-drying bovine colostrum to obtain highly active immunoglobulin freeze-dried powder; the total immunoglobulin content in the highly active immunoglobulin freeze-dried powder is 90%-99.5%; the lactose content is less than 0.01%. This application discloses a method for preparing highly active natural immunoglobulin freeze-dried powder with high antioxidant properties, wherein the total immunoglobulin content in the freeze-dried natural immunoglobulin prepared by this method is 90%-99.5%; the lactose content is less than 0.01%. The preparation method of this application is a simple, rapid, and efficient method for preparing highly active natural immunoglobulin freeze-dried powder with high antioxidant properties, suitable for large-scale industrial production.

Description

Technical Field

[0001] This application relates to the technical field of solid extraction of highly active immunoglobulins, and in particular to a method for preparing highly active natural immunoglobulin freeze-dried powder with high antioxidant properties. Background Technology

[0002] Since the beginning of this century, the global public health field has continuously faced the severe challenge of the spread of novel and mutated viruses. The human immune system maintains the body's health through complex mechanisms. When pathogens invade, B lymphocytes are activated and secrete specific immunoglobulins, among which IgG is the most abundant antibody component in serum, accounting for more than 75% of the total antibodies in the secondary immune response. By specifically recognizing and binding antigens, it plays a role in neutralizing toxins, regulating phagocytosis, and activating complement, and is the core effector molecule of the body's adaptive immunity. In addition, the natural mechanism by which newborns acquire passive immunity through the ingestion of maternal colostrum fully verifies the key role of exogenous immunoglobulins in local intestinal defense. Orally administered IgG can directly bind pathogens in the intestine, preventing their colonization and invasion through immune rejection.

[0003] However, the market is currently flooded with products claiming to boost immunity, but their actual efficacy varies greatly. Many products contain only common nutrients or non-specific immune stimulants, lacking key antibody components that can directly participate in antigen neutralization and thus failing to establish a targeted immune barrier in the body. In contrast, looking back at the history of medical development, bovine colostrum, rich in active ingredients such as immunoglobulins, has been used to assist in fighting infection. Its mechanism of action lies in the fact that when the body is stimulated by specific antigens, immunoglobulins can specifically recognize and neutralize pathogens. Modern research further shows that immunoglobulins play a crucial role in antiviral and anti-infection processes by binding to pathogens, activating the complement system, and regulating macrophage phagocytosis. Among them, IgG, as the most abundant immunoglobulin subtype in serum and colostrum, has the most significant passive immunization effect. Therefore, bovine colostrum and its derivatives, rich in highly active IgG, are widely recognized as effective functional materials for enhancing the body's defense capabilities.

[0004] Currently, there are no mature, industrially produced high-purity natural antibody products available. This is primarily because the harsh conditions such as high temperatures, extreme pH levels, or high shear forces not only cause direct loss of active factors like IgG and SIgA, but more importantly, may lead to conformational changes in immunoglobulin molecules or inactivation of active fragments. Furthermore, existing technologies often face a trade-off between yield and purity in large-scale production, making it difficult to simultaneously meet industrial cost control and high-quality pharmaceutical / food standards.

[0005] Therefore, developing a process that can maintain the natural activity of antibodies, achieve high-purity extraction, and is suitable for industrial-scale production has become an urgent problem to be solved. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this application provides a method for preparing highly active natural immunoglobulin freeze-dried powder with high antioxidant properties.

[0007] In a first aspect, this application provides a method for preparing highly active natural immunoglobulin lyophilized powder with high antioxidant properties, using the following technical solution: A method for preparing highly active natural immunoglobulin freeze-dried powder with high antioxidant properties includes the following preparation steps: bovine colostrum is defatted, enzymatically hydrolyzed, filtered, concentrated by membrane and freeze-dried to obtain highly active immunoglobulin freeze-dried powder; The total immunoglobulin content in the highly active immunoglobulin freeze-dried powder is 90%-99.5%, and the lactose content is less than 0.01%.

[0008] Preferably, the method for preparing the highly active natural immunoglobulin lyophilized powder with high antioxidant properties includes the following preparation steps: (1) The bovine colostrum was centrifuged to remove fat, and the result was defatted bovine colostrum; (2) After adding enzymes to skimmed bovine colostrum and reacting, casein is removed by solid-liquid separation to obtain whey; (3) Filter the whey to obtain bovine colostrum whey; (4) Bovine colostrum whey is concentrated using a membrane separation device, and then lactose is removed by rinsing to obtain immunoglobulin concentrate; (5) After freeze-drying the immunoglobulin concentrate, highly active immunoglobulin freeze-dried powder is obtained.

[0009] Preferably, in step (1), the temperature for centrifuging and defatting bovine colostrum is 40-50℃.

[0010] More preferably, during centrifugal defatting, the centrifugation speed is 3000-6000 rpm, the temperature of bovine colostrum is controlled at 40-50℃ during centrifugation, and the current setting of the centrifugal defatting machine is 7.5-8.5A.

[0011] In one specific feasible implementation, the bovine colostrum is processed. When the bovine colostrum is frozen, it is placed in a slicer to slice and pulverize. After pulverization, it is placed in a thawing tank for heating and thawing. After thawing, the temperature of the bovine colostrum is ≤25℃. The thawed bovine colostrum is then transported to a centrifugal defatting machine for defatting.

[0012] In one specific feasible implementation, when the bovine colostrum is fresh, the colostrum is milked from the cow and collected, immediately placed in a refrigerated container at 4-10°C, and then transported to a centrifugal defatting machine for defatting.

[0013] Preferably, in step (2), the temperature at which the enzyme is added for reaction is 36-38°C and the reaction time is 4-10 minutes.

[0014] Preferably, in step (2), the enzyme includes at least one of abomasalase, pepsin, trypsin, microbial protease, and plant protease.

[0015] Preferably, in step (2), the enzyme is abomasal enzyme.

[0016] More preferably, the abomasal enzyme includes at least one of animal-derived abomasal enzyme, microbial-derived abomasal enzyme, or recombinant abomasal enzyme.

[0017] More preferably, the activity of the abomasal enzyme is 880-890 IMCU / g.

[0018] Preferably, in step (3), the sieve used in the filtration process is 400-600 mesh, which can filter out casein.

[0019] Preferably, in step (4), the membrane used in the membrane separation device has a molecular weight cutoff of 1-10 kDa.

[0020] By adopting the above technical solution, a membrane separation device with a molecular weight cutoff of 1-10 kDa is used for targeted concentration and lactose removal.

[0021] Preferably, in step (4), during the process of rinsing to remove lactose, pure water or buffer solution is used for rinsing 1-7 times.

[0022] By adopting the above technical solution, enzymes are added to skimmed bovine colostrum to induce the directional aggregation of micelle protein components. After solid-liquid separation to retain the protein aggregates, whey is obtained, thereby achieving efficient extraction of the target active ingredients from skimmed bovine colostrum.

[0023] The temperature of the skimmed bovine colostrum is adjusted to 36-38℃, and enzymes are added according to the process ratio. The κ-type stable peptides are hydrolyzed to promote the complexation and flocculation of the α / β type main proteins. After the insoluble protein phase is precipitated, the whey is sieved to retain the highly allergenic protein components and obtain clear whey.

[0024] This application uses natural bovine colostrum as raw material and prepares a high-concentration, high-activity, high-antioxidant, lactose-free lyophilized immunoglobulin powder through a specific process. The prepared lyophilized immunoglobulin powder is rich in various immunoglobulins (IgG, SIgA, IgM, etc.), lactoferrin and other bioactive components, and has unique immune support and antiviral and antibacterial effects. The lyophilized immunoglobulin powder prepared by the method of this application not only retains high-quality protein nutrition, but also significantly enhances the immune effect based on highly active immunoglobulins, helping the body resist health threats such as viruses and bacteria.

[0025] Secondly, this application provides a highly active natural immunoglobulin freeze-dried powder with high antioxidant properties, using the following technical solution: A highly active immunoglobulin lyophilized powder prepared by the above method, wherein the immunoglobulin lyophilized powder comprises lactoferrin at a content of 0.8±0.04wt%, lactose at a content of 0wt%, and immunoglobulins at a total content of 95.43±3.54wt%; wherein the immunoglobulins comprise IgG at a content of 85.76±4.45wt% and SIgA at a content of 9.98±2.23wt%.

[0026] Thirdly, this application provides an application of highly active immunoglobulin lyophilized powder in the preparation of food, health products, or pharmaceuticals, employing the following technical solution: The application of a highly active immunoglobulin lyophilized powder prepared by the above method or the above-mentioned highly active immunoglobulin lyophilized powder in the preparation of food, health products or pharmaceuticals.

[0027] In summary, this application includes at least one of the following beneficial technical effects: This application discloses a method for preparing highly active natural immunoglobulin freeze-dried powder with high antioxidant properties. The total immunoglobulin content of the natural immunoglobulin freeze-dried powder prepared by this method is 90%-99.5%, and the lactose content is less than 0.01%. The preparation method described in this application is a simple, rapid, and efficient method for preparing highly active natural immunoglobulin freeze-dried powder with high antioxidant properties, which is suitable for large-scale industrial production. Attached Figure Description

[0028] Figure 1 The results of SDS-PAGE electrophoresis analysis of the lyophilized immunoglobulin powder; Figure 2 Fourier transform infrared spectra of defatted bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder. Figure 3 X-ray diffraction patterns of defatted bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder; Figure 4 Comparison of ABTS free radical scavenging activities of skimmed bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder and immunoglobulin lyophilized powder; Figure 5 Comparison of DPPH free radical scavenging activities of defatted bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder. Detailed Implementation

[0029] The technical solutions of this application are further illustrated by specific embodiments below. These specific embodiments do not represent a limitation on the scope of protection of this application. Any non-essential modifications and adjustments made by others based on the concept of this application still fall within the scope of protection of this application.

[0030] All raw materials used in this application are commercially available products. Trypsin, enzyme activity 300,000 U / g, Shanghai Yien Biotechnology Co., Ltd. Pepsin, enzyme activity 300,000 U / g, Shenzhen Fusheng Biotechnology Co., Ltd. Abomasal enzyme, animal-derived abomasal enzyme (calf), enzyme activity 890 IMCU / g, Shanghai Yien Biotechnology Co., Ltd. Acidic protease, enzyme activity 300,000 U / g, Shanghai Yien Biotechnology Co., Ltd. Neutral protease, enzyme activity 300,000 U / g, Shanghai Yien Biotechnology Co., Ltd. Papain, enzyme activity 300,000 U / g, Shenzhen Fusheng Biotechnology Co., Ltd. The present application will be further described in detail below with reference to the embodiments.

[0031] A method for preparing highly active natural immunoglobulin lyophilized powder with high antioxidant properties, the method comprising the following steps: (1) Collection and centrifugation defatting: S1. Collection of bovine colostrum: Bovine colostrum can be either frozen or fresh. When the bovine colostrum is frozen, the frozen bovine colostrum (derived from the milk of the cow within 12 hours before lactation) is placed in a slicer to be sliced ​​and pulverized. After pulverization, it is placed in a thawing tank for heating and thawing. The temperature of the bovine colostrum after thawing is ≤25℃, thus obtaining the raw bovine colostrum.

[0032] When bovine colostrum is fresh, it is milked from the cow and collected, then immediately refrigerated at 4-10℃ to obtain raw bovine colostrum.

[0033] S2, Centrifugal degreasing The bovine colostrum is transported to a centrifugal defatting machine for defatting, as follows: 110-112 kg of collected bovine colostrum is transported through pipeline to the centrifugal defatting machine for centrifugal defatting. The speed is 3000-6000 rpm, the current is set to 7.5-8.5A, and the temperature of the bovine colostrum is controlled at 40-50℃ during the operation.

[0034] (2) Enzymatic hydrolysis An enzyme was added to 100 kg of skimmed bovine colostrum to carry out a reaction, and casein was removed by solid-liquid separation to obtain whey; Preferably, the temperature for adding the enzyme to carry out the reaction is 36-38℃, and the reaction time is 4-10 minutes.

[0035] Preferably, the enzyme includes at least one of abomasalase, pepsin, trypsin, microbial protease, and plant protease.

[0036] Preferably, in step (2), the enzyme is abomasal enzyme.

[0037] More preferably, the abomasal enzyme includes at least one of animal-derived abomasal enzyme, microbial-derived abomasal enzyme, or recombinant abomasal enzyme.

[0038] More preferably, the activity of the abomasal enzyme is 880-890 IMCU / g.

[0039] (3) Filtration The whey is filtered to remove impurities, yielding bovine colostrum whey; Preferably, the sieve used in the filtration process is 400-600 mesh.

[0040] (4) Membrane concentration Bovine colostrum whey is placed in a membrane separation device and concentrated using a membrane separation device with a molecular weight cutoff of 1-10 kDa. Then, it is washed 1-7 times with pure water or buffer solution to remove lactose and obtain immunoglobulin concentrate. (5) Drying After freeze-drying the immunoglobulin concentrate at -40°C for 24-48 hours, highly active immunoglobulin lyophilized powder is obtained.

[0041] Example 1:

[0042] A method for preparing highly active natural immunoglobulin lyophilized powder with high antioxidant properties, comprising the following steps: (1) Preparation of skimmed bovine colostrum Frozen bovine colostrum (derived from milk produced by cows within 12 hours before lactation) is taken out of the cold storage, sliced ​​and pulverized in a slicer; the pulverized bovine colostrum is then placed in a thawing tank for heating and thawing. The temperature of the thawed bovine colostrum is ≤25℃, and it is continuously stirred during the thawing process.

[0043] 111 kg of thawed bovine colostrum was transported via pipeline to a centrifugal defatting machine for centrifugation at 4000 rpm and 8 A current. The temperature of the bovine colostrum was controlled at 45°C during the operation to obtain defatted bovine colostrum.

[0044] (2) Retention of highly allergenic protein aggregates Take 100 kg of skimmed bovine colostrum, add abomasal enzyme with an activity of 890 IMCU / g, stir and mix at 37℃ for 8 minutes to induce micelle protein phase separation; after solid-liquid separation to retain protein aggregates, collect the whey.

[0045] (3) Removal of denatured proteins The whey is filtered through a 500-mesh sieve to remove impurities, yielding bovine colostrum whey.

[0046] (4) Membrane concentration Bovine colostrum whey was added to a membrane separation device for sieving and concentration. The membrane used in the separation device had a molecular weight cutoff of 10 kDa. Twice the volume of ultrapure water was added to the concentrated solution for rinsing. The solution was then processed through a 10 kDa separation membrane to the original volume. The retentate was recovered, and then twice the original volume of ultrapure water was added again for membrane separation. This process was repeated 7 times until the absorbance of the permeate measured at 280 nm using a UV spectrophotometer was less than 0.05. The solution was then concentrated to 1 / 4 of its original volume to obtain the immunoglobulin concentrate.

[0047] (5) Drying The immunoglobulin concentrate was freeze-dried at -40°C for 36 hours to obtain highly active immunoglobulin freeze-dried powder.

[0048] Performance testing:

[0049] The defatted bovine colostrum prepared in step 1 of Example 1 was freeze-dried at -40°C for 36 hours to obtain defatted bovine colostrum freeze-dried powder; the bovine colostrum whey prepared in step 3 of Example 1 was freeze-dried at -40°C for 36 hours to obtain bovine colostrum whey freeze-dried powder.

[0050] The lyophilized powders of skimmed bovine colostrum, bovine colostrum whey, and immunoglobulin prepared in step 5 of Example 1 were tested. The specific steps are as follows: 1. Determination by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) The SDS-PAGE electrophoresis analysis results of the immunoglobulin lyophilized powder are as follows: Figure 1 As shown. Band 4 is an IgG standard, used as a reference. Band 2 is a lyophilized skimmed bovine colostrum powder, with low band intensities for IgG, SIgA, and IgM, indicating a relatively low immunoglobulin content. Band 3 is a lyophilized immunoglobulin powder, with significantly stronger bands for IgG, SIgA, and IgM, proving that the method disclosed in this application successfully extracted high-purity, high-content lyophilized immunoglobulin powder.

[0051] 2. Quantitative analysis of the components of skimmed bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder. Quantitative analysis of the components of defatted bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder was performed, and the results are shown in Table 1. Table 1. Quantitative analysis of components in skimmed bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder.

[0052] As shown in Table 1, the process described in this application achieves highly efficient targeted enrichment of immunoglobulins and deep selective removal of impurities in skimmed bovine colostrum. The IgG content increased from 22.8% in the raw material to 85.76% in the final product, an enrichment factor of 3.76 times; the SIgA content increased from 4.0% to 9.98%, an enrichment factor of 2.50 times; the total immunoglobulin content increased from 26.80% to 95.43%; the lactose content decreased from 30.64% to 0; and the ash content decreased from 7.0% to 0.75%, with a removal rate of 89.3%. The various component indicators exhibit a clear gradient optimization pattern, providing quantitative data support for the product's high nutritional value, high activity, and non-allergenic properties.

[0053] The high-purity natural immunoglobulin freeze-dried powder prepared in this application, with its high-quality formula containing ≥85.76% IgG, ≥9.98% SIgA, ≥0.80% lactoferrin, and ≥95.43% total immunoglobulin, demonstrates significant application value in nutritional support and immune regulation. Immunoglobulins, as core effector molecules of human humoral immunity, can specifically recognize and bind to foreign antigens such as viruses, bacteria, and toxins to form antigen-antibody complexes. These complexes then block pathogen invasion through neutralization, promote immune cell clearance through opsonization, and amplify the immune response by activating the complement system, thus constructing a multi-layered defense barrier for the body. This product is rich in natural polyclonal immunoglobulins that can recognize multiple antigenic epitopes, possessing the natural advantages of broad-spectrum pathogen neutralization and low likelihood of developing drug resistance, making it suitable for daily immune enhancement and broad-spectrum protection scenarios.

[0054] In terms of nutritional support, this product contains ≥95.43% total immunoglobulins, with a complete amino acid composition, including all eight essential amino acids and various functional peptides, making it a high-quality protein source to supplement daily dietary nutrition. Essential trace elements such as Ca, Fe, and Zn are retained in the product in a protein-bound state, with higher bioavailability than their inorganic salt forms, helping to promote bone development, hemoglobin synthesis, and maintain immune cell function, achieving dual support for nutrition and immunity. Complete lactose removal significantly reduces product osmotic pressure and the risk of sensitization, making it especially suitable for individuals with lactose intolerance.

[0055] For infants and young children, whose immune systems are not yet fully developed and whose intestinal barrier function is weak, they are susceptible to invasion by respiratory and digestive pathogens. The SIgA in this product can form a local immune barrier in the intestinal mucosa, preventing pathogen adhesion and invasion; IgG provides systemic passive immune support, helping to neutralize pathogens in the blood and tissue fluid; lactoferrin inhibits the colonization of harmful bacteria and promotes the growth of beneficial bacteria, synergistically helping to reduce the risk of infection, support the development of the immune system, and promote nutrient absorption. At the same time, the product is lactose-free, low in ash, high in purity, gentle, and easily absorbed, and can be flexibly added to infant formula and nutritional supplements to provide natural immune protection for children's healthy growth.

[0056] 3. Fourier transform infrared spectroscopy detection of skimmed bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder. Fourier transform infrared spectra of defatted bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder were analyzed, and the results are as follows: Figure 2 As shown, Figure 2 Fourier transform infrared spectra of defatted bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder, wavenumber range 400-4000 cm⁻¹. -1 Transmittance mode detection.

[0057] In 3200-3400cm -1 Within the wavenumber range, all three samples exhibited broad absorption peaks, attributed to OH and NH stretching vibrations, reflecting intermolecular hydrogen bonding and residual moisture. The peak intensity of the immunoglobulin lyophilized powder in this range was higher than that of the skimmed bovine colostrum lyophilized powder and bovine colostrum whey lyophilized powder, indicating increased exposure of polar groups and enhanced hydrogen bond network after purification. The peak intensity was highest in the 2850-2950 cm⁻¹ range. -1 Within the wavenumber range, defatted bovine colostrum lyophilized powder exhibited a distinct absorption peak, attributed to the CH stretching vibrations of the fat and protein side chains. The peak intensity decreased in bovine colostrum whey lyophilized powder, with the weakest peak in immunoglobulin lyophilized powder. This trend is consistent with the fluctuation of fat content from 1.3% to 2.48% in the component quantitative analysis in Table 1, reflecting the effective control of lipid components by the defatting and purification process. The peak intensity was highest in the 1630-1650 cm⁻¹ range. -1 Within the wavenumber range, all three samples exhibited characteristic absorption peaks, attributed to the C=O stretching vibration of the amide I band, reflecting the secondary structure of proteins. The peak position of the defatted bovine colostrum lyophilized powder was 1647 cm⁻¹. -1 Bovine colostrum whey freeze-dried powder peak position 1648cm -1 The peak position of the lyophilized immunoglobulin powder was 1649 cm⁻¹. -1 Peak position shift less than 2cm -1 This indicates that the α-helix structure of immunoglobulins remained stable during the process, without significant denaturation. (At 1530-1550 cm⁻¹) -1Within the wavenumber range, all three samples exhibited characteristic peaks of the amide II band, attributed to the coupling of NH bending vibration and CN stretching vibration. The peak position of the defatted bovine colostrum lyophilized powder was 1540 cm⁻¹. -1 Bovine colostrum whey freeze-dried powder peak position 1541cm -1 The peak position of the lyophilized immunoglobulin powder was 1542 cm⁻¹. -1 The peak position was stable, further verifying the integrity of the protein conformation. (Peak position was between 1230-1240 cm⁻¹) -1 Within the wavenumber range, all three samples exhibited characteristic peaks of the amide III band, attributed to CN stretching and NH bending vibrations. The peak shape of the immunoglobulin lyophilized powder was sharper, reflecting the characteristics of high-purity proteins. (1030-1080 cm⁻¹) -1 Within the wavenumber range, the defatted bovine colostrum freeze-dried powder exhibits a strong absorption peak, with a peak position of 1076 cm⁻¹. -1 This peak intensity is attributed to the lactose COC stretching vibration. The intensity of this peak is significantly reduced in bovine colostrum whey lyophilized powder, and almost disappears in immunoglobulin lyophilized powder, consistent with the quantitative data in Table 1 showing a decrease in lactose content from 30.64% to 0, verifying the effectiveness of nanoscale sieving in deep lactose removal. (At 1450-1470 cm⁻¹) -1 Within the wavenumber range, defatted bovine colostrum lyophilized powder exhibits a distinct absorption peak, attributed to the CH bending vibrations of protein side chains and fats. As the process progresses, the intensity of this peak decreases sequentially in bovine colostrum whey lyophilized powder and immunoglobulin lyophilized powder, reflecting the effective removal of non-immunoglobulin-like proteins and lipid components.

[0058] Calculate the amide I band at 1649 cm⁻¹ -1 The characteristic peak of lactose is 1076 cm⁻¹. -1 The transmittance ratios were approximately 0.45 for skimmed bovine colostrum lyophilized powder, approximately 0.78 for bovine colostrum whey lyophilized powder, and greater than 3.5 for immunoglobulin lyophilized powder. The increasing trend in these ratios quantitatively reflects the increase in relative protein content and the effectiveness of lactose removal, and is highly consistent with the results of quantitative component analysis.

[0059] The overall baseline of the spectrum was stable with no obvious interference from stray peaks, indicating high sample purity. The immunoglobulin lyophilized powder showed a peak density of 400-1500 cm⁻¹ in the fingerprint region. -1 The peaks are clear and the characteristic peaks have high resolution, reflecting the spectral characteristics of high-purity protein samples.

[0060] 4. X-ray diffraction pattern detection of skimmed bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder X-ray diffraction patterns of defatted bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder were analyzed, and the results are as follows: Figure 3 As shown.

[0061] Figure 3X-ray diffraction patterns of defatted bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder were obtained, with a scanning range of 2θ from 5° to 60°. Characteristic diffraction peaks appeared in all three samples near 2θ 9°, with the immunoglobulin lyophilized powder showing higher diffraction intensity at this position than the defatted bovine colostrum and bovine colostrum whey lyophilized powders, indicating an increase in the content of target protein structural units after purification. The strongest diffraction peak appeared near 2θ 20°, corresponding to the characteristic interplanar spacing of the protein. The peak shape of the immunoglobulin lyophilized powder was relatively broad, exhibiting typical amorphous or semi-crystalline structure characteristics, indicating that the freeze-drying process did not lead to excessive crystallization or denaturation aggregation of the protein. In the 2θ range of 30° to 40°, the diffraction intensity gradually decreased, the baseline was stable, and there was no obvious interference from other peaks, reflecting high sample purity. Comparing the diffraction patterns of the three samples, the immunoglobulin lyophilized powder showed a significant increase in diffraction intensity at characteristic angles, with consistent peak positions, indicating that the extraction and purification process effectively enriched immunoglobulins while preserving their natural spatial conformation. The broadening of the diffraction peaks is consistent with the structural characteristics of lyophilized biomolecules, which is beneficial for product resolubility and bioactivity retention. No sharp inorganic salt crystallization peaks were observed throughout the scanning range, consistent with the decrease in ash content in the component analysis, further verifying the selective permeation and removal effect of the 10kDa molecular weight cutoff separation membrane process on small molecule inorganic salt impurities. In the low-angle region of 2θ (5° to 10°), the immunoglobulin lyophilized powder showed a strong scattering signal, reflecting the integrity of its macromolecular structure. In the main peak region of 2θ (15° to 25°), all three samples showed similar peak profiles, indicating that the process did not change the basic crystal structure type of the protein. In the high-angle region of 2θ (greater than 30°), the diffraction signal gradually weakened and became flat, consistent with the diffraction rules of protein-like biomolecules. The overall signal-to-noise ratio of the spectrum is good, and the characteristic peaks are clearly distinguishable, providing reliable physical evidence for product structure characterization.

[0062] 5. Comparison of ABTS free radical scavenging activity and DPPH free radical scavenging activity of skimmed bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder. The ABTS free radical scavenging activity and DPPH free radical scavenging activity of skimmed bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder were compared.

[0063] Figure 4 Comparison of ABTS free radical scavenging activities of defatted bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder. Figure 5 A comparative graph showing the DPPH free radical scavenging activity of skimmed bovine colostrum lyophilized powder, bovine colostrum whey lyophilized powder, and immunoglobulin lyophilized powder. The test concentration gradient was set from 5 mg / mL to 25 mg / mL. Figure 4It was found that as the sample concentration increased from 5 mg / mL to 25 mg / mL, the ABTS free radical scavenging activity of all three samples showed a significant upward trend. At the highest concentration of 25 mg / mL, the scavenging rate of skimmed bovine colostrum lyophilized powder reached 92.18%, immunoglobulin powder reached 77.59%, and bovine colostrum whey lyophilized powder reached 59.94%. Figure 5 It was found that the DPPH free radical scavenging activity also showed a concentration-dependent increase. At a concentration of 25 mg / mL, the scavenging rate of skimmed bovine colostrum lyophilized powder was 81.95%, immunoglobulin lyophilized powder was 61.50%, and bovine colostrum whey lyophilized powder was 45.28%. Overall, skimmed bovine colostrum lyophilized powder showed the highest activity, followed by immunoglobulin lyophilized powder, while bovine colostrum whey lyophilized powder had relatively lower activity. This difference in antioxidant activity mainly stems from the different contents and types of bioactive components in each sample. Skimmed bovine colostrum retains the most complete nutritional components and is rich in vitamin C, lactoferrin, lactoperoxidase, superoxide dismutase, and other natural antioxidants. Among them, vitamin C, as a potent water-soluble antioxidant, directly contributes to the significant free radical scavenging ability. Combined with the data in the aforementioned component analysis table that the iron content of skimmed bovine colostrum is as high as 16.81 mg / 100g, it verifies that it is rich in lactoferrin. Lactoferrin inhibits the generation of free radicals by chelating iron ions, thereby enhancing the antioxidant effect. After purification and enrichment, the immunoglobulin powder retained a high concentration of lactoferrin, although some water-soluble vitamins were lost. The aforementioned component analysis showed that its iron content was 3.26 mg / 100g, significantly higher than the 1.47 mg / 100g of bovine colostrum whey. This is consistent with the result that the antioxidant activity of the immunoglobulin powder was superior to that of whey, indicating that iron-binding proteins such as lactoferrin were effectively preserved during the purification process. Furthermore, the immunoglobulin powder still contains high levels of enzymes such as lactoperoxidase and superoxide dismutase. These enzyme systems work synergistically with immunoglobulins to form an effective antioxidant defense system. Bovine colostrum whey, due to the removal of most casein and some binding proteins, has a relatively diluted concentration of active ingredients, resulting in relatively low antioxidant activity. The immunoglobulin powder prepared in this application not only possesses high immunomodulatory activity but also retains significant antioxidant function. Thanks to the synergistic effect of lactoferrin, enzymes, and trace vitamins, a scientific basis is provided for the stability and functional application of the product under high oxidative stress environments.

[0064] Example 2:

[0065] The optimization of membrane separation purification process parameters was studied: To investigate the comprehensive effects of different membrane separation times on the total immunoglobulin content, IgG content, lactose content, and product yield in the final product, lyophilized immunoglobulin powder, twice the original volume of ultrapure water was added to step (4) of Example 1, and membrane separation was performed again. This process was repeated 1, 2, 3, 4, 5, 6, and 7 times, and the effects of different membrane separation times on the components in the final product, lyophilized immunoglobulin powder, were detected. The key indicator detection results and calculation analysis are shown in Table 2.

[0066] Table 2. Effect of different membrane separation times on the final product: immunoglobulin lyophilized powder.

[0067] As shown in Table 2, the comparison and analysis of the data reveals that as the number of membrane separations increased from 1 to 7, the product yield gradually decreased from 6.25% to 2.89%, the total immunoglobulin content significantly increased from 46.01% to 95.43%, the lactose content decreased from 20.19% to 0, and the IgG content steadily increased from 39.6% to 85.76%, exhibiting a clear synergistic optimization pattern of "yield-purity-IgG enrichment". In the first two membrane separation stages, the lactose removal rate reached 48.7%, purity increased by 19.30%, IgG enrichment factor reached 1.47, and yield loss was 28.6%, indicating that the initial membrane separation can quickly remove large-molecule lactose and some impurities, while achieving preliminary enrichment of target IgG, resulting in high purification efficiency. In the second and third membrane separation stages, the lactose removal rate jumped to 96.2%, purity continued to increase by 8.27%, IgG enrichment factor increased to 1.66, and yield loss was 13.3%, which is the critical window period for purification. In the third and fourth membrane separation stages, lactose was completely removed, the purity growth rate slowed to 7.38%, IgG enrichment factor reached 1.83, and yield loss was 10.4%, indicating that marginal benefits began to decrease. In the fourth to seventh membrane separation stages, lactose remained at 0 residue, purity only increased by 14.94%, IgG enrichment factor increased from 1.83 to 2.17, and yield loss was 16.2% cumulatively, reducing the economics of deep purification. Considering yield, purity, IgG enrichment, and impurity removal efficiency, the optimal process equilibrium point is 3-4 membrane separations. At this point, the product yield is ≥3.45%, immunoglobulin purity is ≥80.96%, IgG content is ≥72.4%, and lactose residue is ≤0.76%, which can balance industrial production efficiency with high product quality requirements.

[0068] Regarding the trend of IgG content changes, the IgG content of 39.6% in Group 1 is the background value of colostrum, reflecting the initial enrichment level of IgG in whey after abomasal enzyme hydrolysis. With the increase of membrane separation times, the IgG content steadily increased from 58.4% in Group 2 to 85.76% in Group 7, and the IgG enrichment factor increased from 1.00 to 2.17, showing a continuous and controllable gradient enrichment characteristic. Among them, the rapid enrichment range was from 1 to 4 membrane separations (IgG content 39.6%→72.4%), and the fine enrichment range was from 4 to 7 membrane separations (72.4%→85.76%), indicating that this process has a continuous and stable selective retention effect on the target immunoglobulin. Data analysis showed that IgG content was significantly positively correlated with protein purity (R²>0.99), verifying that this process can efficiently enrich the target IgG while removing impurities, achieving the targeted purification goal of "removing impurities and retaining purity".

[0069] The lactose removal kinetics showed that the rapid removal range occurred after 1-3 membrane separations, with the removal rate jumping from 0% to 96.2%, consistent with the initial rapid diffusion pattern of small molecule impurities. After ≥4 membrane separations, the lactose content stabilized at 0%, confirming that the membrane selected in this process effectively blocked lactose (10000 Da) permeation, achieving complete removal. Complete elimination of lactose significantly reduces the risk of product sensitization, expanding the applicable population, especially suitable for lactose-intolerant individuals and infant formula applications.

[0070] Based on the above data comparison, the number of membrane separations in this application can be flexibly adjusted according to different product positioning: if high yield and cost control are pursued, two membrane separations are recommended, with a product yield of 4.44%, IgG content of 58.4%, and purity of 65.31%, suitable for bulk raw materials or basic functional foods; if high purity, high IgG enrichment, and low allergenicity are emphasized, four membrane separations are recommended, with a product yield of 3.45%, IgG content of 72.4%, purity of 80.96%, and zero lactose residue, suitable for high-end functional foods or special medical foods; if ultra-high purity IgG enrichment is required, six to seven membrane separations can be selected, with product purity ≥95%, IgG content ≥85%, and enrichment factor ≥2.15, suitable for scientific research reagents or high value-added preparations. In summary, this application achieves efficient extraction and targeted enrichment of immunoglobulins from skimmed bovine colostrum through a gradient membrane separation process coupled with abomasal enzyme hydrolysis. This effectively solves the technical bottlenecks of existing products, such as "low IgG enrichment, high lactose residue, and insufficient immunoglobulin purity," and provides a reliable process route for the large-scale production of high-purity, low-sensitivity, and highly active immunoglobulin products.

[0071] Example 3:

[0072] To investigate the effects of different enzymes on the composition and taste of bovine colostrum whey, the abomasal enzyme added in step (2) of Example 1 was replaced, and the effects of different enzymes on the composition and taste of the prepared bovine colostrum whey were detected. The key indicator detection results and calculation analysis are shown in Table 3.

[0073] When testing the bovine colostrum whey prepared in this embodiment, the bovine colostrum whey was freeze-dried at -40°C for 36 hours to obtain bovine colostrum whey freeze-dried powder, and then the components and taste of the bovine colostrum whey freeze-dried powder were tested.

[0074] Table 3. Effects of different enzymes on the composition and taste of bovine colostrum whey freeze-dried powder

[0075] Table 3 shows the effects of different enzymatic hydrolysis treatments on taste scores: 1-5 are poor, 6-7 are generally acceptable, and 8-9 are good.

[0076] According to the test results in Table 3, the addition of different enzymes has different effects on the content of immunoglobulins and IgG in the prepared bovine colostrum whey freeze-dried powder. This indicates that the choice of enzyme has a certain impact on the activity of immunoglobulins. When abomasal enzyme is used, the content of immunoglobulins and IgG is the best. This is because the added abomasal enzyme does not destroy immunoglobulins, thus achieving the enrichment of immunoglobulins. Secondly, after adding abomasal enzyme for reaction, the final product, immunoglobulin freeze-dried powder, has the best taste and is more suitable for use.

[0077] Example 4:

[0078] To investigate the effects of different centrifugation defatting temperatures on the composition of defatted bovine colostrum, the reaction temperature of bovine colostrum during the centrifugation defatting process in step (1) of Example 1 was adjusted. The effects of different temperatures on the composition of the prepared defatted bovine colostrum were detected. The key indicator detection results and calculation analysis are shown in Table 4.

[0079] When testing the defatted bovine colostrum prepared in the example, the defatted bovine colostrum was freeze-dried at -40°C for 36 hours to obtain defatted bovine colostrum freeze-dried powder, and then the components in the defatted bovine colostrum freeze-dried powder were tested.

[0080] Table 4. Effects of different centrifugal defatting temperatures on the composition of defatted bovine colostrum freeze-dried powder

[0081] As shown in Table 4, different centrifugation defatting temperatures affect the component content in the final product, immunoglobulin lyophilized powder. Furthermore, the final product exhibits the optimal total immunoglobulin content, IgG content, and fat content when the centrifugation defatting temperature is 40-50℃.

Claims

1. A method for preparing a highly active natural immunoglobulin freeze-dried powder with high antioxidant properties, characterized in that: The preparation process includes the following steps: Bovine colostrum is defatted, enzymatically hydrolyzed, filtered, concentrated through a membrane, and then freeze-dried to obtain highly active immunoglobulin freeze-dried powder. The total immunoglobulin content in the highly active immunoglobulin freeze-dried powder is 90%-99.5%, and the lactose content is less than 0.01%.

2. The method for preparing a highly active natural immunoglobulin freeze-dried powder with high antioxidant properties according to claim 1, characterized in that: The preparation steps include the following: (1) The bovine colostrum was centrifuged to remove fat, and the result was defatted bovine colostrum; (2) After adding enzymes to skimmed bovine colostrum and reacting, the mixture is separated to obtain whey; (3) Filter the whey to obtain bovine colostrum whey; (4) Bovine colostrum whey is concentrated using a membrane separation device and then washed to obtain immunoglobulin concentrate; (5) After freeze-drying the immunoglobulin concentrate, highly active immunoglobulin freeze-dried powder is obtained.

3. The method for preparing a highly active natural immunoglobulin freeze-dried powder with high antioxidant properties according to claim 2, characterized in that: In step (1), the temperature for centrifuging and defatting bovine colostrum is 40-50℃.

4. The method for preparing a highly active natural immunoglobulin freeze-dried powder with high antioxidant properties according to claim 2, characterized in that: In step (2), the temperature for adding the enzyme to react is 36-38℃, and the reaction time is 4-10 minutes.

5. The method for preparing a highly active natural immunoglobulin freeze-dried powder with high antioxidant properties according to claim 2, characterized in that: In step (2), the enzyme includes at least one of abomasalase, pepsin, trypsin, microbial protease, and plant protease.

6. The method for preparing a highly active natural immunoglobulin freeze-dried powder with high antioxidant properties according to claim 5, characterized in that: In step (2), the enzyme is abomasal enzyme; the abomasal enzyme includes at least one of animal-derived abomasal enzyme, microbial-derived abomasal enzyme or recombinant abomasal enzyme; the activity of the abomasal enzyme is 880-890 IMCU / g.

7. The method for preparing a highly active natural immunoglobulin freeze-dried powder with high antioxidant properties according to claim 2, characterized in that: In step (3), the sieve used in the filtration process is 400-600 mesh; in step (4), the membrane used in the membrane separation equipment has a molecular weight cutoff of 1-10 kDa.

8. The method for preparing a highly active natural immunoglobulin freeze-dried powder with high antioxidant properties according to claim 2, characterized in that: In step (4), during the rinsing process, pure water or buffer solution is used for rinsing 1-7 times.

9. A highly active immunoglobulin lyophilized powder prepared by the method according to any one of claims 1-8, characterized in that: The lyophilized immunoglobulin powder comprises 0.8±0.04wt% lactoferrin, 0wt% lactose, and a total immunoglobulin content of 95.43±3.54wt%; wherein the immunoglobulin comprises 85.76±4.45wt% IgG and 9.98±2.23wt% SIgA.

10. The use of the highly active immunoglobulin lyophilized powder prepared by the method according to any one of claims 1-8 or the highly active immunoglobulin lyophilized powder according to claim 9 in the preparation of food, health products or pharmaceuticals.

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