A method for preparing an anti-aging, allergy-free lyophilized bovine colostrum immunoglobulin powder rich in natural immune factors.

Abstract

This application relates to the technical field of solid extraction of highly active immunoglobulins, and in particular to a method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors; the method comprises the following steps: bovine colostrum is subjected to a first enzymatic hydrolysis, centrifugal defatting, a second enzymatic hydrolysis, solid-liquid separation, filtration, ultrafiltration concentration, and freeze-drying to obtain a freeze-dried bovine colostrum immunoglobulin powder; the prepared freeze-dried bovine colostrum immunoglobulin powder is rich in IgG, SIgA, lactoferrin, and growth factors. The product has a complete natural conformation, good resolubility, and high storage stability. The content of the main allergens β-lactoglobulin, α-lactalbumin, α-casein, β-casein, and lactose is low. The prepared freeze-dried bovine colostrum immunoglobulin powder effectively maintains the natural activity of immune factors and enhances the anti-allergic properties of the product.

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 a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors. Background Technology

[0002] In recent years, an increasing number of studies have shown that heat treatment during dairy processing can not only denature common nutritional proteins, but also easily inactivate some natural active ingredients with anti-allergic protective potential and immunomodulatory effects. Cow's milk and colostrum naturally contain various immunomodulatory factors such as immunoglobulins, lactoferrin, and growth factors. These active and anti-allergic components help support the body's immune defense, maintain intestinal barrier function, regulate inflammatory responses, and may participate in the formation of immune tolerance. Related studies show that when dairy products are heated to approximately 64 to 65 degrees Celsius or higher for a certain period, especially under heat treatment conditions of about 30 minutes or longer, some heat-sensitive whey proteins, immunoglobulins, lactoferrin, and related enzymes are prone to conformational changes, aggregation, or decreased activity, weakening their original immunomodulatory, anti-allergic protective, and anti-aging nutritional value.

[0003] Existing nutritional supplements made from skim milk, whey protein, lactoglobulin, and casein typically undergo pasteurization, high-temperature sterilization, spray drying, or high-temperature concentration processes. While these processes improve microbial safety and extend shelf life, high-temperature treatment can also inactivate natural active proteins and anti-allergy components, reducing the active factors in dairy products that originally help boost immunity, regulate allergic reactions, and protect gut health. Furthermore, high temperatures can denature whey proteins such as β-lactoglobulin and α-lactalbumin, causing them to form complexes with casein or other proteins, altering their digestibility, absorption, and immune recognition characteristics. For individuals with insufficient digestive enzyme secretion, weak intestinal barrier function, or poor immune system regulation, these denatured aggregated proteins are not easily hydrolyzed and absorbed, potentially increasing the body's immune burden on foreign proteins and thus inducing or exacerbating IgE-mediated and non-IgE-mediated allergic reactions.

[0004] Regular cow's milk and some bovine colostrum products still contain lactose. For individuals with insufficient lactase activity, weak intestinal digestion and absorption, or poor lactose tolerance, insufficient lactose breakdown and absorption can lead to bloating, diarrhea, flatulence, and abdominal discomfort. Traditional products often improve tolerance by reducing or eliminating lactose, but this primarily reduces lactose-related discomfort and doesn't fully utilize lactose's value as a precursor to functional sugars. Therefore, it's necessary to employ novel enzymatic conversion technologies to target and convert lactose in bovine colostrum, transforming it into more easily absorbed monosaccharides and lactoligosaccharides, thereby improving lactose tolerance, providing nutritional support for beneficial gut bacteria, promoting gut microbiota balance, and further assisting in immune regulation.

[0005] In recent years, allergy-related problems have become increasingly common in the population. Irregular eating habits can also affect gut microbiota and immune regulation. Furthermore, long-term consumption of heat-denatured proteins, difficult-to-digest proteins, or overly processed foods may disrupt gut microbiota balance, intestinal barrier integrity, and the formation of immune tolerance, leading to an abnormally sensitive or inefficiently regulated immune system. Therefore, traditional methods of nutritional fortification using skim milk powder, whey protein powder, lactoglobulin, or casein are no longer sufficient to meet the needs of children, young adults, the elderly, and individuals with sensitive constitutions for low-allergenicity, highly active, easily absorbed, gut-friendly, and immune-protective nutritional products. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this application provides a method for preparing anti-aging, allergy-free bovine colostrum rich in natural immune factors.

[0007] This application provides a bovine colostrum nutritional product prepared using processes such as low-temperature separation, novel enzymatic lactose conversion, casein removal, active component enrichment, and freeze-drying. This technology does not simply remove lactose; instead, it utilizes novel enzymatic technology to convert lactose into monosaccharides and lactooligosaccharides, transforming lactose resources into functional carbohydrates that are more easily absorbed and provide intestinal nutritional support. By avoiding prolonged high-temperature processing, this technology maximizes the protection of immunoglobulins, lactoferrin, growth factors, and anti-allergic components in bovine colostrum, reduces the inactivation of heat-sensitive active ingredients and anti-allergic protective components, lowers the content of casein and other potentially allergenic components, and improves the food friendliness for lactose-intolerant individuals.

[0008] Compared to regular skim milk, lactoglobulin, casein, or UHT milk protein products, the bovine colostrum nutritional products prepared using this technology do not contain any added allergenic milk proteins. This reduces the risk of UHT-denatured proteins stimulating the body's immune system. Furthermore, by retaining natural immune factors, anti-allergy active ingredients, and other functional nutrients, the product's comprehensive value in terms of immune support, gut health maintenance, low-allergenic nutritional supplementation, and anti-aging nutrition is enhanced.

[0009] In a first aspect, this application provides a method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors, using the following technical solution: A method for preparing an anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder rich in natural immune factors includes the following steps: Bovine colostrum is subjected to a first enzymatic hydrolysis, centrifugal defatting, a second enzymatic hydrolysis, solid-liquid separation, filtration, ultrafiltration concentration, and freeze-drying to obtain an anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder.

[0010] Preferably, the preparation method comprises the following steps: (1) Add lactase to bovine colostrum to carry out hydrolysis reaction to obtain zero-lactose bovine colostrum liquid; (2) The lactose-free bovine colostrum was centrifuged to remove fat, and the resulting liquid was defatted. (3) Add abomasal enzyme to the defatted bovine colostrum for enzymatic hydrolysis, separate the solid and liquid, and then filter to obtain bovine colostrum whey liquid; (4) The bovine colostrum whey solution is concentrated by ultrafiltration and washed twice with pure water or buffer solution. The whey and unhydrolyzed lactose are removed by filtration and then freeze-dried to obtain anti-aging and non-allergenic bovine colostrum immunoglobulin freeze-dried powder.

[0011] Preferably, the amount of lactase added in step (1) is 0.5-2.5% of the lactose content in the cow's milk.

[0012] Preferably, the enzyme activity of lactase in step (1) is 50-250 U / g.

[0013] Preferably, in the hydrolysis reaction of step (1), the reaction temperature is 38-40℃ and the reaction time is 0.5-1 hour.

[0014] Preferably, during the hydrolysis reaction in step (1), the pH of the bovine colostrum solution is 6.5-6.8.

[0015] By adopting the above technical solution, lactase is added to bovine colostrum during the hydrolysis reaction to catalyze the directional hydrolysis of lactose into glucose, galactose and lactoligosaccharides, thereby achieving the functional conversion and zero lactose saccharification of lactose.

[0016] Preferably, before adding lactase for hydrolysis in step (1), the bovine colostrum is heated to 38-40°C.

[0017] By adopting the above technical solution, before adding lactase to bovine colostrum for enzymatic hydrolysis, the bovine colostrum is preheated to 38-40℃ and kept warm to provide a suitable reaction temperature for lactase and initiate the directional hydrolysis of lactose.

[0018] Preferably, in the process of centrifugation and degreasing in step (2), centrifugation is performed using a centrifuge with a rotation speed of 3000-6000 rpm.

[0019] By adopting the above technical solution, the lactose-free bovine colostrum is centrifuged to remove fat globules and milk fat layer, thus obtaining defatted bovine colostrum, which ensures fat separation efficiency and reduces the loss of heat-sensitive active ingredients.

[0020] Preferably, the amount of abomasal enzyme added in step (3) is 0.00006-0.0001% of the mass of skimmed bovine colostrum.

[0021] Preferably, the enzyme activity of the abomasalase in step (3) is 0.0000005-0.0000008 IMCU / g.

[0022] Preferably, in step (3), the enzymatic hydrolysis time is 8-15 minutes and the enzymatic hydrolysis temperature is 36-38℃.

[0023] Preferably, the abomasin in step (3) is one of animal-derived abomasin, microbial-derived abomasin, or recombinant abomasin.

[0024] Preferably, before adding abomasal enzyme for enzymatic hydrolysis in step (3), the skimmed bovine colostrum is heated to 36-38°C.

[0025] By adopting the above technical solution, before adding abomasal enzyme to the defatted bovine colostrum for enzymatic hydrolysis, the bovine colostrum liquid is subjected to mild heat treatment. The bovine colostrum liquid is heated to 36-38℃ and kept at this temperature to optimize the viscosity of the system and the state of protein micelles, thereby promoting the selective precipitation of casein and the efficiency of solid-liquid separation.

[0026] Preferably, the sieve used for filtration in step (3) is 400-600 mesh, which can filter out casein.

[0027] Preferably, in step (4), an ultrafiltration device with a membrane molecular weight cutoff of 1-20 kDa is used for ultrafiltration concentration.

[0028] Preferably, the freeze-drying temperature in step (4) is -50℃ to -40℃.

[0029] By adopting the above technical solution, this application improves the preparation method by sequentially subjecting bovine colostrum to one enzymatic hydrolysis, centrifugal defatting, a second enzymatic hydrolysis, solid-liquid separation, and filtration to remove whey allergenic proteins. The retentate is then collected for ultrafiltration concentration and freeze-drying to obtain anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder. One-step enzymatic hydrolysis: Bovine colostrum is heat-treated, and lactase is added to carry out the enzymatic hydrolysis reaction. After the lactose is fully hydrolyzed, a lactose-free bovine colostrum feed solution is obtained.

[0030] Secondary enzymatic hydrolysis involves adjusting the defatted bovine colostrum to suitable conditions for the action of abomasal enzymes, adding an appropriate amount of abomasal enzymes to induce enzymatic hydrolysis, inducing casein precipitation, and after curd formation, removing casein curds through solid-liquid separation, followed by filtration to obtain defatted, casein-free, and lactose-hydrolyzed bovine colostrum whey solution.

[0031] The steps of ultrafiltration concentration are as follows: Bovine colostrum whey solution is subjected to cross-flow filtration through an ultrafiltration membrane with a molecular weight cutoff of 1-20 kDa, and then washed twice by dialysis with pure water or buffer solution to remove unhydrolyzed lactose, β-lactoglobulin and α-lactalbumin through the ultrafiltration membrane. The retentate is collected to obtain bovine colostrum immunoglobulin solution rich in natural immune factors.

[0032] Bovine colostrum immunoglobulin solution was freeze-dried to obtain anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder.

[0033] The bovine colostrum immunoglobulin freeze-dried powder prepared by the method described in this application is rich in IgG, SIgA, lactoferrin and growth factors. The content of the main allergens β-lactoglobulin, α-lactalbumin, α-casein, β-casein and lactose is significantly reduced, making it suitable for preparing nutritional products with immunomodulatory and anti-aging functions.

[0034] This application utilizes natural bovine colostrum as raw material to prepare a high-concentration, highly active, anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder through a specific process. This product is rich in various natural bioactive components such as immunoglobulins (IgG, SIgA, etc.), lactoferrin, and growth factors, exhibiting clear immune support, antiviral, antibacterial, and synergistic anti-aging effects. The solid formulation prepared using the method described in this application, while retaining high-quality protein nutrition, leverages the complete conformation and bioavailability of highly active immune factors and growth factors. It not only significantly enhances the body's immune defense function and resists pathogenic microorganism invasion but also exerts anti-aging effects at the nutritional intervention level by delaying immune aging, reducing oxidative stress, and promoting mucosal and tissue repair, meeting the clinical and market demand for low-allergen, highly active, and anti-aging nutritional supplements.

[0035] Secondly, this application provides a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors, using the following technical solution: A freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors, comprising the following components: galactose content of 4.25±0.87wt%, glucose content of 3.10±0.94wt%, lactose content of 0%, α-casein content of 0%, β-casein content of 0%, and total protein content of 81.06±2.87wt%. The protein comprises the following components: total natural immune factors of 54.11±2.88wt%, β-lactoglobulin of 1.24±0.07wt%, and α-lactalbumin of 1.10±0.08wt%; the natural immune factors comprise the following components: immunoglobulin G of 41.7±2.68wt%, secretory immunoglobulin A of 4.93±1.05wt%, lactoferrin of 0.83±0.03wt%, epidermal growth factor (EGF) of 239.45±0.05ng / g, transforming growth factor β (TGF-β1) of 22.93±0.05ng / g, insulin-like growth factor-1 (IGF-1) of 744.89±0.05ng / g, and other components of 6.65±0.05wt%. The other components include immunoglobulin M, immunoglobulin E, immunoglobulin D, cytokines, lysozyme, and peptides.

[0036] Thirdly, this application provides an application of freeze-dried bovine colostrum immunoglobulin rich in natural immune factors, which is used for anti-aging and hypoallergenic, in the preparation of food, health products, or pharmaceuticals, using the following technical solution: The anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder prepared by the above method, or the above-mentioned anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder, can be used in the preparation of food, health products, or pharmaceuticals.

[0037] In summary, this application includes at least one of the following beneficial technical effects: This application discloses a method for preparing anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder rich in natural immune factors. The method involves subjecting bovine colostrum to a series of gentle processes, including one enzymatic hydrolysis, centrifugal defatting, a second enzymatic hydrolysis, solid-liquid separation, filtration, ultrafiltration concentration, and freeze-drying. The process avoids irreversible protein inactivation caused by temperatures above 40°C throughout the entire process. This yields a highly active immunoglobulin freeze-dried powder rich in IgG, SIgA, lactoferrin, and growth factors, effectively maintaining the natural activity of immune factors and enhancing the product's anti-allergic properties. The prepared bovine colostrum immunoglobulin freeze-dried powder contained β-casein and α-casein (the main allergens) with residual amounts of <0.1wt%, β-lactoglobulin and α-lactalbumin with residual amounts of <1.4wt%, and lactose content of 0. The product has superior purity and hypoallergenic safety. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the preparation process of a freeze-dried bovine colostrum immunoglobulin powder rich in natural immune factors that is anti-aging and hypoallergenic, according to this application. Figure 2SDS-PAGE electrophoresis analysis of skim milk powder, whey protein powder and bovine colostrum immunoglobulin lyophilized powder; Figure 3 This is a thin-layer chromatography (TLC) chromatogram of the lactose hydrolysis process products; Figure 4 A comparison of Fourier transform infrared (FTIR) spectra of skim milk powder, whey protein powder, and lyophilized bovine colostrum immunoglobulin powder; Figure 5 Figure 1 shows the results of an antioxidant ABTS free radical scavenging experiment on skim milk powder, whey protein powder, and bovine colostrum immunoglobulin lyophilized powder at different concentrations. Figure 6 The effect of bovine colostrum immunoglobulin lyophilized powder on cell viability (CCK-8 assay). Figure 7 A bar chart showing the effect of lyophilized bovine colostrum immunoglobulin powder on MIP-1 enzyme activity; Figure 8 A bar chart showing the effect of lyophilized bovine colostrum immunoglobulin powder on MIP-3 enzyme activity; Figure 9 A graph showing the relationship between bovine colostrum immunoglobulin freeze-dried powder and LH1 enzyme activity; Figure 10 A bar chart showing the effect of lyophilized bovine colostrum immunoglobulin powder on the cellular collagen synthesis metabolic pathway (TGF-β1 / Smad); Detailed Implementation

[0039] 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.

[0040] All raw materials involved in this application are commercially available products, among which, Abomasal enzyme, animal-derived abomasal enzyme (calf), enzyme activity 890 IMCU / g, Shanghai Yien Biotechnology Co., Ltd. Lactase was purchased from Xi'an Mixianer Biotechnology Co., Ltd.; Food Additive Production License No. SC10661042200511.

[0041] Skim milk powder was purchased from Fonterra, SKIMMILK POWDER Regular; The whey protein powder was purchased from Fonterra, WHEY PROTEIN CONCENTRATE 392; The present application will be further described in detail below with reference to embodiments and comparative examples.

[0042] Example 1:

[0043] A method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors, comprising the following steps: Step 1: Collection of bovine colostrum When bovine colostrum is fresh, it is collected within 72 hours after calving from healthy dairy cows and immediately stored at 4-10℃. It is then transported to the processing plant via cold chain for later use.

[0044] Step 2: One-time enzymatic hydrolysis 400 kg of bovine colostrum was heated to 39°C, and lactase was added at 1.5% of the lactose content in the colostrum. The enzyme activity of lactase was 150 U / g. Under the condition that the natural pH of the bovine colostrum was 6.6, the hydrolysis reaction was started by stirring at a constant speed of 150 rpm. During the enzymatic hydrolysis, lactose was directionally hydrolyzed into glucose and galactose, achieving 100% conversion of lactose and obtaining lactose-free bovine colostrum solution.

[0045] During the hydrolysis reaction, the reaction temperature was 39℃ and the reaction time was 0.8 hours.

[0046] Step 3: Centrifugation for degreasing Under the condition of maintaining an enzymatic hydrolysis temperature of 39℃, the lactose-free bovine colostrum was transported to a centrifugal defatting machine for centrifugal separation. The centrifugal speed was set to 4500 rpm and the equipment operating current was 8A. After separating and removing the milk fat layer, defatted bovine colostrum was obtained.

[0047] Lactase remains active in subsequent processes to ensure complete conversion of residual lactose.

[0048] Step 4: Secondary enzymatic hydrolysis 363.63 kg of defatted bovine colostrum was heated to 37°C, and abomasin was added at 0.00008% of the bovine colostrum feed weight for enzymatic hydrolysis. The abomasin activity was 0.000000712 IMCU / g, which induced specific cleavage and micelle aggregation and precipitation of κ-casein. The casein clots were removed by solid-liquid separation equipment, and the supernatant whey was collected to obtain bovine colostrum whey feed.

[0049] During the enzymatic hydrolysis process, the hydrolysis time is 8 minutes and the hydrolysis temperature is 37℃.

[0050] Step 5: Clarification and Filtration Maintain the temperature of the bovine colostrum whey solution at 37°C and continue stirring for 13 minutes to allow the remaining insoluble proteins to fully aggregate. Then, perform fine filtration through a 500-mesh sieve to remove solid impurities and obtain a clear and transparent bovine colostrum whey solution.

[0051] Step Six: Ultrafiltration Concentration The clear and transparent bovine colostrum whey solution was introduced into an ultrafiltration membrane module with a molecular weight cutoff of 10 kDa for ultrafiltration treatment. An intermittent dialysis-washing process was adopted: twice the volume of ultrapure water was added to the solution, and the solution was concentrated to the original volume by ultrafiltration, and the retentate was collected. The above "water addition-ultrafiltration concentration" cycle was repeated 5 times until the absorbance (A280) of the permeate at a wavelength of 280 nm on a UV spectrophotometer was <0.05, indicating that small molecule impurities had been fully removed. Finally, the retentate was concentrated to 1 / 4 of the initial volume to obtain the bovine colostrum immunoglobulin solution.

[0052] Step 7: Freeze-drying Bovine colostrum immunoglobulin solution was dispensed into freeze-drying trays and pre-frozen at -40°C for 3 hours. Then it was transferred to a vacuum freeze dryer and sublimated under vacuum ≤50Pa and plate temperature gradually increased to 30°C for a total drying time of 24 hours. After pulverization, 28.57 kg of highly active, lactose-free, low-allergenic, anti-aging, and non-allergenic bovine colostrum immunoglobulin freeze-dried powder was obtained.

[0053] Testing revealed that the prepared bovine colostrum immunoglobulin freeze-dried powder contained the following components: galactose content of 4.25 wt%, glucose content of 3.10 wt%, lactose content of 0%, α-casein content of 0%, β-casein content of 0%, and total protein content of 81.06 wt%. The protein comprises the following components: a total of 54.1101 wt% natural immune factors, 1.24 wt% β-lactoglobulin, and 1.10 wt% α-lactalbumin; the natural immune factors comprise the following components: 41.7 wt% immunoglobulin G, 4.93 wt% secretory immunoglobulin A, 0.83 wt% lactoferrin, 239.45 ng / g epidermal growth factor (EGF), 22.93 ng / g transforming growth factor β (TGF-β1), 744.89 ng / g insulin-like growth factor-1 (IGF-1), and 6.65 wt% other components. The other components include immunoglobulin M, immunoglobulin E, immunoglobulin D, cytokines, lysozyme, and peptides.

[0054] Example 2:

[0055] A method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors, comprising the following steps: Step 1: Collection of bovine colostrum When bovine colostrum is fresh, it is collected within 72 hours after calving from healthy dairy cows and immediately stored at 4-10℃. It is then transported to the processing plant via cold chain for later use.

[0056] Step 2: One-time enzymatic hydrolysis 400 kg of bovine colostrum was heated to 38°C, and lactase was added at 0.5% of the lactose content in the colostrum. The enzyme activity of lactase was 50 U / g. Under the condition that the natural pH of the bovine colostrum was 6.7, the hydrolysis reaction was started by stirring at a constant speed of 150 rpm. During the enzymatic hydrolysis, lactose was directionally hydrolyzed into glucose and galactose, achieving 100% conversion of lactose and obtaining lactose-free bovine colostrum solution.

[0057] During the hydrolysis reaction, the reaction temperature was 38℃ and the reaction time was 0.5 hours.

[0058] Step 3: Centrifugation for degreasing Under the condition of maintaining an enzymatic hydrolysis temperature of 38℃, the lactose-free bovine colostrum was transported to a centrifugal defatting machine for centrifugal separation. The centrifugal speed was set to 3000 rpm and the equipment operating current was 7.5A. After separating and removing the milk fat layer, defatted bovine colostrum was obtained.

[0059] Lactase remains active in subsequent processes to ensure complete conversion of residual lactose.

[0060] Step 4: Secondary enzymatic hydrolysis 350.43 kg of defatted bovine colostrum was heated to 36°C, and abomasin was added at 0.00006% of the bovine colostrum liquid mass for enzymatic hydrolysis. The abomasin enzyme activity was 0.000000534 IMCU / g, which induced specific cleavage and micelle aggregation and precipitation of κ-casein. The casein clots were removed by solid-liquid separation equipment, and the supernatant whey was collected to obtain bovine colostrum whey liquid.

[0061] During the enzymatic hydrolysis process, the hydrolysis time is 15 minutes and the hydrolysis temperature is 38℃.

[0062] Step 5: Clarification and Filtration Maintain the temperature of the bovine colostrum whey solution at 36°C and continue stirring for 10 minutes to allow the remaining insoluble proteins to fully aggregate. Then, perform fine filtration through a 400-mesh sieve to remove solid impurities and obtain a clear and transparent bovine colostrum whey solution.

[0063] Step Six: Ultrafiltration Concentration The clear and transparent bovine colostrum whey solution was introduced into an ultrafiltration membrane module with a molecular weight cutoff of 1 kDa for ultrafiltration treatment. An intermittent dialysis-washing process was adopted: twice the volume of ultrapure water was added to the solution, and the solution was concentrated to the original volume by ultrafiltration, and the retentate was collected. The above "water addition-ultrafiltration concentration" cycle was repeated 5 times until the absorbance (A280) of the permeate at a wavelength of 280 nm on a UV spectrophotometer was <0.05, indicating that small molecule impurities had been fully removed. Finally, the retentate was concentrated to 1 / 4 of the initial volume to obtain the bovine colostrum immunoglobulin solution.

[0064] Step 7: Freeze-drying Bovine colostrum immunoglobulin solution was dispensed into freeze-drying trays and pre-frozen at -40°C for 2 hours. Then it was transferred to a vacuum freeze dryer and sublimated under vacuum ≤50Pa and plate temperature gradually increased to 30°C for a total drying time of 24 hours. After pulverization, 22.23 kg of highly active, lactose-free, low-allergenic, anti-aging, and non-allergenic bovine colostrum immunoglobulin freeze-dried powder was obtained.

[0065] Testing revealed that the prepared anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder contained the following components: galactose content of 3.38 wt%, glucose content of 2.16 wt%, lactose content of 0%, α-casein content of 0%, β-casein content of 0%, and total protein content of 78.19 wt%. The protein comprises the following components: a total of 51.2301 wt% natural immune factors, 1.17 wt% β-lactoglobulin, and 1.02 wt% α-lactalbumin; the natural immune factors comprise the following components: 39.52 wt% immunoglobulin G, 4.31 wt% secretory immunoglobulin A, 0.8 wt% lactoferrin, 239.425 ng / g epidermal growth factor (EGF), 22.905 ng / g transforming growth factor β (TGF-β1), 744.865 ng / g insulin-like growth factor-1 (IGF-1), and 6.6 wt% other components. The other components include immunoglobulin M, immunoglobulin E, immunoglobulin D, cytokines, lysozyme, and peptides.

[0066] Example 3:

[0067] A method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors, comprising the following steps: Step 1: Collection of bovine colostrum When bovine colostrum is fresh, it is collected within 72 hours after calving from healthy dairy cows and immediately stored at 4-10℃. It is then transported to the processing plant via cold chain for later use.

[0068] Step 2: One-time enzymatic hydrolysis 400 kg of bovine colostrum was heated to 40°C, and lactase was added at 2.5% of the lactose content in the colostrum. The enzyme activity of lactase was 250 U / g. Under the condition that the natural pH of the bovine colostrum was 6.7, the hydrolysis reaction was started by stirring at a constant speed of 150 rpm. During the enzymatic hydrolysis, lactose was directionally hydrolyzed into glucose and galactose, achieving 100% conversion of lactose and obtaining lactose-free bovine colostrum solution.

[0069] During the hydrolysis reaction, the reaction temperature was 40℃ and the reaction time was 1 hour.

[0070] Step 3: Centrifugation for degreasing Under the condition of maintaining an enzymatic hydrolysis temperature of 40℃, the lactose-free bovine colostrum was transported to a centrifugal defatting machine for centrifugal separation. The centrifugal speed was set to 6000 rpm and the equipment operating current was 8.5A. After separating and removing the milk fat layer, defatted bovine colostrum was obtained.

[0071] Lactase remains active in subsequent processes to ensure complete conversion of residual lactose.

[0072] Step 4: Secondary enzymatic hydrolysis 353.63 kg of skimmed bovine colostrum was heated to 38°C, and abomasin was added at 0.00008% of the bovine colostrum liquid mass for enzymatic hydrolysis. The abomasin enzyme activity was 0.000000712 IMCU / g, which induced specific cleavage and micelle aggregation and precipitation of κ-casein. The casein clots were removed by solid-liquid separation equipment, and the supernatant whey was collected to obtain bovine colostrum whey liquid.

[0073] During the enzymatic hydrolysis process, the hydrolysis time is 10 minutes and the hydrolysis temperature is 38℃.

[0074] Step 5: Clarification and Filtration Maintain the temperature of the bovine colostrum whey solution at 38°C and continue stirring for 15 minutes to allow the remaining insoluble proteins to fully aggregate. Then, perform fine filtration through a 600-mesh sieve to remove solid impurities and obtain a clear and transparent bovine colostrum whey solution.

[0075] Step Six: Ultrafiltration Concentration The clear and transparent bovine colostrum whey solution was introduced into an ultrafiltration membrane module with a molecular weight cutoff of 10 kDa for ultrafiltration treatment. An intermittent dialysis-washing process was adopted: twice the volume of ultrapure water was added to the solution, and the solution was concentrated to the original volume by ultrafiltration, and the retentate was collected. The above "water addition-ultrafiltration concentration" cycle was repeated 5 times until the absorbance (A280) of the permeate at a wavelength of 280 nm on a UV spectrophotometer was <0.05, indicating that small molecule impurities had been fully removed. Finally, the retentate was concentrated to 1 / 4 of the initial volume to obtain the bovine colostrum immunoglobulin solution.

[0076] Step 7: Freeze-drying Bovine colostrum immunoglobulin solution was dispensed into freeze-drying trays and pre-frozen at -40°C for 4 hours. Then it was transferred to a vacuum freeze dryer and sublimated under vacuum ≤50Pa and plate temperature gradually increased to 30°C for a total drying time of 24 hours. After pulverization, 25.78 kg of highly active, lactose-free, low-allergenic, anti-aging, and non-allergenic bovine colostrum immunoglobulin freeze-dried powder was obtained.

[0077] The anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder comprises the following components: galactose content of 3.78 wt%, glucose content of 2.66 wt%, lactose content of 0%, α-casein content of 0%, β-casein content of 0%, and total protein content of 79.59 wt%. The protein comprises the following components: a total of 52.6701 wt% natural immune factors, 1.205 wt% β-lactoglobulin, and 1.06 wt% α-lactalbumin. The natural immune factors include the following components: 40.35 wt% immunoglobulin G, 4.88 wt% secretory immunoglobulin A, 0.815 wt% lactoferrin, 239.4 ng / g epidermal growth factor (EGF), 22.88 ng / g transforming growth factor β (TGF-β1), 744.84 ng / g insulin-like growth factor-1 (IGF-1), and 6.625 wt% other components. The other components include immunoglobulin M, immunoglobulin E, immunoglobulin D, cytokines, lysozyme, and peptides.

[0078] Performance testing: The components of the anti-aging, non-allergenic bovine colostrum immunoglobulin lyophilized powder prepared in Example 1 were analyzed, as follows: 1. Determination by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) SDS-PAGE electrophoresis analysis results of anti-aging, non-allergenic bovine colostrum immunoglobulin lyophilized powder are as follows: Figure 2 As shown in the figure, from left to right, the following are the bands: band M is the maker; band 1 is the IgG standard; band 2 is the β-lactoglobulin standard; band 3 is the casein standard; band 4 is the skim milk powder sample; band 5 is the whey protein powder sample; and band 6 is the bovine colostrum immunoglobulin lyophilized powder sample prepared in Example 1.

[0079] Depend on Figure 2Electrophoresis results showed that band 1 (IgG standard) exhibited clear characteristic bands of IgG heavy and light chains at approximately 50-55 kDa and approximately 25 kDa, serving as a positive reference for immunoglobulin identification. Band 2 (β-lactoglobulin standard) showed a single strong band at approximately 18.4 kDa, representing the characteristic peak position of β-lactoglobulin. Band 3 (casein standard) showed multiple bands in the approximately 19-25 kDa region, corresponding to the mixed characteristics of α-casein, β-casein, and κ-casein. Band 4 (skim milk powder sample) showed a distinct β-lactoglobulin band at approximately 18.4 kDa, and complex casein bands were visible in the approximately 19-25 kDa region, indicating that skim milk contained a high content of the main allergenic protein. Band 5 (whey protein powder sample) shows a strong β-lactoglobulin band at approximately 18.4 kDa and an α-lactalbumin band at approximately 14.2 kDa, but the IgG heavy chain band is weaker in the approximately 50-55 kDa region. Band 6 (bovine colostrum immunoglobulin lyophilized powder) shows strong bands at approximately 50-55 kDa and approximately 25 kDa, completely consistent with the positions of Band 1 (IgG standard), proving that the product of this application is rich in high-purity IgG; at the same time, three SIgA characteristic bands are visible, namely secretory component (70-75 kDa), heavy chain (55-60 kDa), and light chain (25 kDa), confirming that the secretory immunoglobulin structure is intact and successfully enriched; while the β-lactoglobulin band at approximately 18.4 kDa and the casein band in the approximately 19-25 kDa region are significantly weakened or almost disappeared.

[0080] Regarding electrophoretic evidence of anti-allergic properties, compared with band 2 (β-lactoglobulin standard) and band 3 (casein standard), the band intensity at the corresponding position in band 6 is extremely low, indicating that the content of major allergens such as β-lactoglobulin and casein is significantly reduced. This is consistent with the data in Table 1, proving that the allergen content of the product in this application is extremely low, which can significantly reduce the risk of IgE-mediated allergic reactions and is suitable for people with allergies and people with sensitive immune systems.

[0081] Regarding electrophoretic evidence of anti-aging properties, band 6 showed that the IgG heavy and light chain bands and the three SIgA subunit bands were highly consistent with the standard and exhibited strong signals. This indicates that the native conformation of immunoglobulins and secreted immunoglobulins remained intact, without thermal denaturation, aggregation, or peptide chain breakage. This is directly related to the ≤40℃ low-temperature process used throughout this application. Immunoglobulins with intact conformation can perform biological functions such as neutralizing free radicals, clearing pathogenic microorganisms, and reducing chronic inflammation, thus delaying the "inflammatory aging" process at the molecular level.

[0082] 2. Quantitative analysis of components 2.1 Quantitative Analysis of Proteins and Allergen Components The components of skim milk powder, whey protein powder, and the lyophilized bovine colostrum immunoglobulin powder prepared in Example 1 were quantitatively analyzed, and the results are shown in Table 1.

[0083] Table 1. Quantitative analysis of protein and allergen components in skim milk powder, whey protein powder, and lyophilized bovine colostrum immunoglobulin powder.

[0084] As shown in Table 1, the bovine colostrum immunoglobulin lyophilized powder prepared in this application achieved significant effects in both immunoglobulin enrichment and allergen removal. Regarding immunoglobulin content, the IgG content increased from 0.12 g / 100 g in skim milk powder to 41.7 g / 100 g, an enrichment factor of 347.5 times; the SIgA content increased from undetectable in skim milk powder to 4.93 g / 100 g, demonstrating that the process in this application achieved highly efficient and targeted enrichment of immunoglobulins. The total protein content increased from 32.9 g / 100 g in skim milk powder to 81.06 g / 100 g, with a purity increase of 2.46 times.

[0085] Regarding allergen control, the β-casein content decreased from 8.6g / 100g in skim milk powder to 0; the α-casein content decreased from 13.5g / 100g to 0; the β-lactoglobulin content decreased from 3.18g / 100g in skim milk powder to 1.24g / 100g, with a removal rate of 61.0%; and the α-lactalbumin content decreased from 1.13g / 100g to 1.10g / 100g. The significant reduction in the main allergenic proteins provides quantitative evidence for the product's "non-allergenic" characteristics.

[0086] Compared with whey protein products, the IgG content of the bovine colostrum immunoglobulin freeze-dried powder prepared in this application is 41.7g / 100g, which is much higher than that of whey protein powder (2.31g / 100g). The SIgA content of 4.93g / 100g is not present in whey protein powder, and the β-lactoglobulin content of 1.24g / 100g is significantly lower than that of whey protein powder (51.63g / 100g). This indicates that the product of this application effectively reduces the content of major whey allergens while retaining highly active immunoglobulins.

[0087] 2.2 Analysis of Carbohydrates and Physicochemical Indicators The sugar and physicochemical properties of skim milk powder, whey protein powder, and the lyophilized bovine colostrum immunoglobulin powder prepared in Example 1 were analyzed, and the results are shown in Table 2.

[0088] Table 2. Analysis of carbohydrates and physicochemical properties of skim milk powder, whey protein powder, and bovine colostrum immunoglobulin freeze-dried powder.

[0089] As shown in Table 2, regarding lactose conversion, the lactose content decreased from 54.5g / 100g in skim milk powder to 0, while generating 4.25g / 100g of galactose and 3g / 100g of glucose. This proves that the lactose enzymatic hydrolysis process of this application achieves 100% lactose conversion, completely eliminating the risk of lactose intolerance. Whey protein powder products have a lactose content of 7g / 100g, which may still cause adverse reactions in lactose-intolerant individuals, such as… Figure 3 As shown, Figure 3 The sample includes a standard control, an unhydrolyzed sample (i.e., bovine colostrum without lactase), and the hydrolysis product (i.e., the lactose-free bovine colostrum solution prepared in step two of Example 1). Furthermore, a small amount of lactooligosaccharides (such as galactosyl-lactose and other functional oligosaccharides) may be generated during lactose hydrolysis. These substances have prebiotic properties, selectively promoting the proliferation of beneficial intestinal bacteria such as Bifidobacteria and Lactobacillus, regulating the intestinal microecological balance, and indirectly supporting the intestinal mucosal immune barrier function and anti-aging-related metabolic pathways.

[0090] Regarding moisture and ash control, the moisture content of the bovine colostrum immunoglobulin freeze-dried powder is 1.8g / 100g, which is lower than that of skim milk powder (3.8g / 100g) and whey protein powder (3.7g / 100g), thus contributing to the product's long-term storage stability. The ash content is 4.7g / 100g, falling between that of skim milk powder (8.1g / 100g) and whey protein powder (2.8g / 100g), indicating that inorganic salt impurities are effectively controlled.

[0091] In terms of solubility and taste, the lyophilized bovine colostrum immunoglobulin powder dissolved in 1.18 minutes (0.2g / 100ml), significantly faster than skim milk powder (52.35 minutes) and whey protein powder (16.18 minutes), indicating excellent rapid dissolution and absorption. Simultaneously, the product achieved a taste score of 9, better than skim milk powder (7) and whey protein powder (6), demonstrating good sensory acceptance. Its lactose-free formula, rapid dissolution, and mild taste make it ideal as a functional carrier, suitable for combination with probiotics, plant extracts, or other prebiotics to develop immune-regulating and anti-aging nutritional intervention products for different populations.

[0092] Combining the results in Tables 1 and 2, it can be seen that in terms of low allergenicity, the contents of α-casein, β-casein, and lactose are all 0, the contents of β-lactoglobulin are 1.24 g / 100 g, and the contents of α-lactalbumin are 1.10 g / 100 g. The simultaneous elimination of multiple allergenic factors can significantly reduce the risk of IgE-mediated allergic reactions.

[0093] In terms of high immune activity, the IgG content is 41.7g / 100g and the SIgA content is 4.93g / 100g, which provides a material basis for the repair of the intestinal mucosal immune barrier and systemic immune support. It can play a role in neutralizing pathogens, scavenging free radicals, and regulating inflammatory factors.

[0094] In terms of preserving the natural conformation, the low-temperature process (-40℃) avoids thermal denaturation, allowing immunoglobulins to maintain their complete spatial structure and delaying the "inflammatory aging" process at the molecular level.

[0095] In terms of gut-friendly properties, the zero lactose content, combined with galactose and glucose conversion products, not only eliminates intolerance symptoms but also provides a carbon source for beneficial gut bacteria, promotes gut microbiome balance, and indirectly supports immune aging regulation.

[0096] In summary, the quantitative data in Tables 1 and 2 fully demonstrate that the bovine colostrum immunoglobulin freeze-dried powder prepared in this application has achieved the expected technical effects in terms of allergen removal, immunoglobulin enrichment, lactose conversion and activity retention, providing a solid scientific basis for the product's functional claims of "high activity, low allergens, and anti-aging".

[0097] 3. Infrared spectral analysis of skim milk powder, whey protein powder, and lyophilized bovine colostrum immunoglobulin powder Infrared spectroscopy analysis was performed on skim milk powder, whey protein powder, and bovine colostrum immunoglobulin lyophilized powder (prepared in Example 1).

[0098] Depend on Figure 4 The corresponding spectral data show that, in the characteristic absorption regions of proteins, near 1650 cm⁻¹ (Amide I band, mainly reflecting C=O stretching vibration and secondary structure) and near 1540 cm⁻¹ (Amide II band, reflecting NH bending vibration and CN stretching vibration), the lyophilized bovine colostrum immunoglobulin powder exhibits sharp peaks with stable transmittance changes, highly consistent with the standard conformation of natural immunoglobulins. This indicates that its secondary structures, such as α-helices and β-sheets, are intact, and no thermal denaturation, peptide chain breakage, or intermolecular cross-linking aggregation has occurred. In contrast, the skim milk powder shows a significantly broadened peak in this region with complex shoulders, suggesting strong conformational heterogeneity in the multi-protein system.

[0099] Near 3300 cm⁻¹ (Amide A band, characteristic region of NH stretching vibration and hydrogen bond network), the transmittance curve of bovine colostrum immunoglobulin lyophilized powder is smooth and continuous, indicating stable hydrogen bonding, further confirming the effective preservation of the natural spatial conformation and intramolecular forces of the active protein. Combined with the casein removal and ultrafiltration purification process of this application, the significant weakening or disappearance of the characteristic absorption peaks of whey protein powder in the spectrum directly confirms the efficient removal of major allergens such as α-casein, β-casein, and β-lactoglobulin, providing direct structural evidence for the product's "low-allergenic" characteristics at the molecular vibrational level.

[0100] Regarding the structural support for its anti-aging properties, the preservation of the intact secondary structure of immunoglobulins in the lyophilized bovine colostrum immunoglobulin powder is the material basis for its antioxidant, free radical scavenging, immunomodulatory, and intestinal barrier function-maintaining effects. The absence of peak shift and increased peak width in the Amide I / II bands of the infrared spectrum indicates that the low-temperature and mild processing method (-40℃) effectively avoids protein conformational damage during processing, ensuring that immune factors and growth factors maintain high biological efficacy, thereby supporting its anti-aging functions of delaying inflammatory aging and promoting tissue repair at the molecular level.

[0101] 4. Scavenging activity of skim milk powder, whey protein powder, and bovine colostrum immunoglobulin lyophilized powder against ABTS free radicals at different concentrations. The scavenging activity of skim milk powder, whey protein powder, and lyophilized bovine colostrum immunoglobulin powder (prepared in Example 1) on ABTS free radicals at different concentrations was tested, and the results are as follows: Figure 5 As shown.

[0102] Combination Figure 5 The experimental data show that at the three test concentrations of 5, 10, and 15 mg / mL, the free radical scavenging activity of the three groups of samples all showed a concentration-dependent increasing trend. The specific data are as follows: At a concentration of 5 mg / mL, the clearance rate of bovine colostrum immunoglobulin lyophilized powder was 13.27±0.18%, significantly higher than that of whey protein powder (3.03±0.15%) and skim milk powder (3.23±0.77%); at a concentration of 10 mg / mL, the clearance rate of bovine colostrum immunoglobulin lyophilized powder increased to 26.40±1.31%, approximately 4.04 times that of whey protein powder (6.53±1.34%) and 2.49 times that of skim milk powder (10.62±0.55%); at a concentration of 15 mg / mL, the immunoglobulin clearance rate reached 37.71±0.87%, still significantly higher than that of whey protein powder (11.73±1.00%) and skim milk powder (17.38±0.93%).

[0103] The bovine colostrum immunoglobulin lyophilized powder prepared in this application exhibits a significantly higher ABTS free radical scavenging capacity than whey protein powder and skim milk powder at the same concentration. This result is directly attributed to the mild process chain of this application, which involves "low-temperature enzymatic hydrolysis, abomasal enzyme decaseation, and ultrafiltration retention." This process fully preserves the natural spatial conformation of active components such as immunoglobulins (IgG, SIgA), lactoferrin, and growth factors, ensuring that their active sites are not damaged by thermal denaturation and can efficiently provide electrons or hydrogen atoms to neutralize ABTS free radicals. The decaseinization and gradient dialysis washing steps remove a large number of competitive interfering proteins, free metal ions, and oxidation-promoting factors, resulting in a high enrichment of highly active immune factors and endogenous antioxidant peptides, producing a multi-component synergistic antioxidant effect of "immunoglobulin-lactoferrin-growth factors." Aging is closely related to oxidative stress and "inflammatory aging." Excessive free radicals attack cell membrane lipids, structural proteins, and nucleic acids, accelerating cellular aging and immune function decline. The product exhibits a high ABTS free radical scavenging capacity, which can effectively neutralize excess reactive oxygen species in the body, reduce oxidative damage, and downregulate the activity of chronic inflammatory pathways such as NF-κB, thereby delaying the immune aging process at the molecular and cellular levels. This data provides direct in vitro experimental evidence for the "anti-aging" function claim of this application.

[0104] 5. Effects of bovine colostrum immunoglobulin lyophilized powder on cell viability The effect of bovine colostrum immunoglobulin lyophilized powder (prepared in Example 1) on cell viability is shown in the graph below. Figure 6 As shown, Figure 6 The horizontal axis represents the concentration of bovine colostrum immunoglobulins (mg / mL), and the vertical axis represents cell viability (%). Samples were prepared using serially diluted DMEM complete medium, and cell proliferation and metabolic activity at different concentrations were determined using CCK-8 cytotoxicology assays.

[0105] Experimental data showed that bovine colostrum immunoglobulin concentrations within the 0-2.4 mg / mL test range did not exhibit cytotoxicity, and cell viability in all concentration groups was significantly higher than that in the control group. Peak cell viability was observed at concentrations of 0.3 mg / mL and 0.6 mg / mL, at 136.10±1.92% and 139.20±2.88%, respectively, indicating that immunoglobulin significantly promoted cell metabolism and proliferation within this concentration range. When the concentration was further increased to 1.2 mg / mL and 2.4 mg / mL, although cell viability showed a slow decreasing trend, it remained at 126.56±1.92% and 111.36±0.96%, respectively, consistently higher than the baseline control level, confirming that the product maintains excellent biocompatibility and safety even at high doses.

[0106] The enhanced cell viability effect is closely related to the multi-component active ingredients of this product, including IgG, SIgA, lactoferrin, and growth factors (TGF-β1, EGF, IGF-1), which retain their intact natural conformation. Intact immunoglobulins can specifically bind to cell surface receptors, activating downstream signaling pathways and promoting cell cycle progression and energy metabolism. Growth factors TGF-β1, EGF, and IGF-1 can synergistically activate cell proliferation-related signaling pathways, promoting the proliferation and differentiation of epithelial cells, fibroblasts, and immune cells. Lactoferrin can inhibit oxidative stress-induced apoptosis, maintaining cell membrane integrity and mitochondrial function. The significant improvement in cell proliferation and viability directly reflects the product's functional activation effect on immune cells and tissue repair cells, providing empirical support at the cellular level for delaying cell aging and maintaining tissue homeostasis. Based on previous in vitro antioxidant data, the bovine colostrum immunoglobulin freeze-dried powder prepared in this application achieves synergistic unity of immune regulation and anti-aging functions through a multidimensional mechanism of scavenging free radicals, activating cell metabolism and inhibiting inflammatory damage. Moreover, it has no toxic reactions across the entire concentration range, meeting the safety requirements for long-term use of food and health products.

[0107] 6. Effects of bovine colostrum immunoglobulin lyophilized powder on MMP-1 enzyme activity The effect of bovine colostrum immunoglobulin lyophilized powder (prepared in Example 1) on MMP-1 enzyme activity is as follows: Figure 7 As shown, Figure 7The horizontal axis represents the concentration of bovine colostrum immunoglobulins (mg / mL), and the vertical axis represents MMP-1 enzyme activity (U / cell number). A gradient concentration of samples was prepared using DMEM complete medium to determine its regulatory effect on cellular MMP-1 secretion activity. Data showed that MMP-1 enzyme activity decreased significantly with increasing bovine colostrum immunoglobulin concentration. In the control group (0 mg / mL), MMP-1 enzyme activity was 91±4 U / cell number. After adding 0.3 mg / mL of the sample, the enzyme activity decreased to 74.52±4.84; in the 0.6 mg / mL group, it was 73.55±2.90; in the 1.2 mg / mL group, it was 70.08±5.76; and in the 2.4 mg / mL group, it further decreased to 59.52±5.76. These results indicate that the lyophilized bovine colostrum immunoglobulin powder prepared in this application has a clear concentration-dependent inhibitory effect on MMP-1. MMP-1, a core member of the matrix metalloproteinase family, is primarily responsible for degrading type I and type III collagen. Its abnormally high expression is a key pathological mechanism leading to skin photoaging, elastic fiber breakage, and tissue matrix loss. The bovine colostrum immunoglobulin lyophilized powder prepared in this application, processed at ≤40℃ throughout, fully retains active components such as transforming growth factor β (TGF-β1), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1). TGF-β1 can directly inhibit MMP-1 promoter activity and promote tissue inhibitor of matrix metalloproteinases (TIMP-1) expression through the Smad signaling pathway, synergistically acting with lactoferrin's anti-inflammatory regulatory effects to effectively block the collagen degradation cascade. This in vitro enzyme activity inhibition data directly validates the product's biological efficacy in maintaining extracellular matrix stability and delaying tissue aging. This, along with the aforementioned cell viability promotion and free radical scavenging results, further solidifies the mechanistic basis and data support for the anti-aging function claims in this application.

[0108] 7. Effect of bovine colostrum immunoglobulin lyophilized powder on MMP-3 enzyme activity The effect of bovine colostrum immunoglobulin lyophilized powder (prepared in Example 1) on MMP-3 enzyme activity is as follows: Figure 8 As shown. Figure 8The horizontal axis represents the concentration of bovine colostrum immunoglobulins (mg / mL), and the vertical axis represents MMP-3 enzyme activity (U / cell number). The experiment used DMEM complete medium to prepare samples with varying concentrations, and determined their regulatory effect on cellular MMP-3 secretion activity. Experimental data showed that MMP-3 enzyme activity decreased significantly with increasing sample concentration. In the control group (0 mg / mL), MMP-3 enzyme activity was 56.00 ± 3.00 U / cell number. After adding 0.3 mg / mL of sample, the enzyme activity decreased to 46.65 ± 4.84; in the 0.6 mg / mL group, it was 41.61 ± 0.97; in the 1.2 mg / mL group, it was 38.40 ± 3.84; and in the 2.4 mg / mL group, it further decreased to 34.56 ± 1.92. These results indicate that the product of this application has a clear concentration-dependent inhibitory effect on MMP-3.

[0109] MMP-3, also known as matrix lysozyme-1, is a core hydrolytic enzyme in extracellular matrix remodeling and inflammatory responses. It can degrade proteoglycans, fibronectin, and laminin, and can activate precursors MMP-1 and MMP-9 to form a cascade amplification effect. Abnormally high expression of MMP-3 accelerates dermal matrix degradation, disrupts tissue structure, and promotes the formation of a chronic inflammatory microenvironment, making it an important driver of tissue photoaging and immunosenescence. The inhibitory effect of the bovine colostrum immunoglobulin lyophilized powder prepared in this application on MMP-3 originates from the synergistic regulation of multiple active components. The product retains intact growth factors such as TGF-β1 and EGF, which can directly downregulate MMP-3 gene transcription levels through receptor-mediated signal transduction. Lactoferrin blocks the Fenton reaction by binding free iron ions, effectively scavenging intracellular reactive oxygen species and disrupting the ROS-dependent MMP-3 activation pathway. Simultaneously, IgG and SIgA eliminate exogenous antigens and damage-related molecular patterns, blocking Toll-like receptors and the NF-κB inflammatory signaling axis, reducing the release of pro-inflammatory cytokines such as IL-1β and TNF-α, thereby eliminating inflammatory stimuli that induce MMP-3 hyperexpression. This in vitro enzyme activity inhibition data complements previous MMP-1 inhibition results, jointly verifying the product's biological efficacy in blocking matrix degradation cascade reactions and maintaining extracellular matrix integrity. Combined with cell viability enhancement and free radical scavenging experiments, this further confirms that the bovine colostrum immunoglobulin lyophilized powder prepared in this application achieves the technical effects of delaying tissue aging and maintaining immune homeostasis by regulating matrix metabolism and the oxidative inflammatory microenvironment through multiple targets.

[0110] 8. Effect of bovine colostrum immunoglobulin lyophilized powder on LH1 enzyme activity The effect of bovine colostrum immunoglobulin lyophilized powder (prepared in Example 1) on LH1 enzyme activity, such as Figure 9 As shown; Figure 9The horizontal axis represents the concentration of bovine colostrum immunoglobulins (mg / mL), and the vertical axis represents LH1 enzyme activity (U / cell number). Experimental data showed that LH1 enzyme activity increased significantly with increasing sample concentration. The baseline LH1 activity in the control group (0 mg / mL) was 8.50 ± 1.00 U / cell number. After adding 0.3 mg / mL and 0.6 mg / mL samples, the enzyme activity steadily increased to 9.19 ± 1.94 and 10.23 ± 0.97, respectively. When the concentration reached 1.2 mg / mL and 2.4 mg / mL, the enzyme activity significantly increased to 13.44 ± 2.88 and 14.78 ± 1.54, respectively, an increase of approximately 73.9% compared to the control group, demonstrating a clear dose-dependent promoting effect.

[0111] Unlike the aforementioned defensive anti-aging pathways that inhibit matrix-degrading enzymes (MMP-1 / 3), the upregulation of LH1 (lysine hydroxylase 1) activity indicates a constructive promoting effect of the product on collagen synthesis. LH1 is a core modifying enzyme in collagen biosynthesis, responsible for catalyzing the hydroxylation of lysine residues in the procollagen peptide chain. This biochemical step directly determines the density, thermal stability, and mechanical strength of newly formed collagen fiber crosslinks. The TGF-β1, IGF-1, and EGF growth factors completely preserved in the bovine colostrum immunoglobulin lyophilized powder prepared in this application can directly activate LH1 gene transcription in fibroblasts through the Smad and PI3K / Akt signaling pathways. Simultaneously, lactoferrin improves intracellular redox homeostasis and relieves the inhibition of collagen synthesis pathways by chronic low-grade inflammation. The dose-dependent enhancement of LH1 activity indicates that this product can not only block collagen loss but also promote the maturation and crosslinking of newly formed collagen from the source, restoring the structural support and tissue elasticity of the dermis.

[0112] This data, together with the MMP inhibition experiment, forms a two-way regulatory evidence chain of promoting synthesis and inhibiting degradation. Combined with the results of enhanced cell viability and free radical scavenging, it comprehensively verifies the integrated technical advantages of bovine colostrum immunoglobulin lyophilized powder in maintaining the dynamic balance of the extracellular matrix and reversing structural aging of tissues. The product exhibits no toxic reactions across the entire concentration range and significantly activates key synthetic enzymes, further solidifying the scientific basis and application potential of the product as an anti-aging functional material.

[0113] 9. Effects of lyophilized bovine colostrum immunoglobulin powder on the relative expression level of TGF-β1 mRNA in cells. The effect of bovine colostrum immunoglobulin lyophilized powder on the relative expression level of TGF-β1 mRNA in cells, such as Figure 10 As shown, Figure 10The horizontal axis represents relative mRNA expression (with the 0 mg / mL control group as the baseline of 1.00), and the vertical axis represents bovine colostrum immunoglobulin concentration (mg / mL). Quantitative data showed that the TGF-β1 gene transcription level was significantly upregulated in a dose-dependent manner with increasing sample concentration. The baseline expression level in the control group was 1.00±0.05; after adding 0.3 mg / mL and 0.6 mg / mL samples, the expression level steadily increased to 1.10±0.05 and 1.55±0.05, respectively; when the concentration reached 1.2 mg / mL and 2.4 mg / mL, the relative expression level of TGF-β1 mRNA further increased significantly to 1.73±0.19 and 1.92±0.05, respectively, an increase of nearly 92% compared with the control group, confirming that the product of this application has a clear activating effect on the endogenous TGF-β1 synthesis pathway in target cells.

[0114] TGF-β1 is a core upstream cytokine regulating extracellular matrix remodeling and tissue repair. The dose-dependent increase in its mRNA expression level directly reflects the initiation effect of the bioactive components in the lyophilized powder on the nuclear transcriptional program. After secretion, the upregulated TGF-β1 protein can phosphorylate the Smad2 / 3 complex and translocate into the nucleus, strongly driving the transcriptional expression of type I and type III collagen genes (COL1A1 / COL3A1), while simultaneously upregulating the synthesis of lysine hydroxylase (LH1) and prolyl hydroxylase, ensuring the yield and cross-linking maturity of newly formed collagen from the gene source. More importantly, the TGF-β1 signaling pathway can feedback-inhibit AP-1 transcription factor activity, directly blocking the promoters of MMP-1 and MMP-3 genes. This, along with the previously observed decrease in matrix-degrading enzyme activity, forms a tight "synthesis-degradation inhibition" molecular regulatory loop.

[0115] The transcriptional data are highly consistent with previous results on LH1 enzyme activity enhancement, MMP inhibition, and cell proliferation, confirming that the bovine colostrum immunoglobulin lyophilized powder prepared in this application does not rely solely on physical supplementation of exogenous proteins, but rather triggers an endogenous anti-aging signaling network through the synergistic action of multiple active ingredients. The sustained activation of the TGF-β1 pathway not only provides a lasting impetus for the reconstruction of the dermal and connective tissue matrix structure, but its immune homeostasis regulatory properties also help alleviate the inhibition of fibroblast function by chronic microinflammation. These experimental results confirm, at the molecular transcriptional level, the core mechanism of action of the bovine colostrum immunoglobulin lyophilized powder prepared in this application in reversing structural aging and promoting collagen regeneration, providing complete upstream mechanism evidence and data loop support for the product's claims of high bioactivity and anti-aging function.

Claims

1. A method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors, characterized in that: The preparation method involves the following steps: bovine colostrum undergoes a first enzymatic hydrolysis, centrifugal defatting, a second enzymatic hydrolysis, solid-liquid separation, filtration, ultrafiltration concentration, and freeze-drying to obtain anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder.

2. The method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors according to claim 1, characterized in that: The preparation method steps are as follows: (1) Add lactase to bovine colostrum to carry out hydrolysis reaction to obtain zero-lactose bovine colostrum liquid; (2) The lactose-free bovine colostrum was centrifuged to remove fat, and the resulting liquid was defatted. (3) Add abomasal enzyme to the defatted bovine colostrum for enzymatic hydrolysis, separate the solid and liquid, and then filter to obtain bovine colostrum whey liquid; (4) After ultrafiltration and concentration of bovine colostrum whey, freeze-dry the liquid to obtain anti-aging and non-allergenic bovine colostrum immunoglobulin freeze-dried powder.

3. The method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors according to claim 2, characterized in that: In step (1), the amount of lactase added is 0.5-2.5% of the lactose content in the initial milk; the enzyme activity of the lactase is 50-250 U / g. In the hydrolysis reaction of step (1), the reaction temperature is 38-40℃ and the reaction time is 0.5-1 hour.

4. The method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors according to claim 2, characterized in that: In step (2), centrifugation is performed using a centrifuge with a rotation speed of 3000-6000 rpm.

5. The method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors according to claim 2, characterized in that: In step (3), the amount of abomasal enzyme added is 0.00006-0.0001% of the mass of skimmed bovine colostrum; the enzyme activity of the abomasal enzyme is 0.0000005-0.0000008 IMCU / g; In step (3), the enzymatic hydrolysis time is 8-15 minutes and the enzymatic hydrolysis temperature is 36-38℃. In step (3), the abomasin is one of animal-derived abomasin, microbial-derived abomasin, or recombinant abomasin.

6. The method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors according to claim 2, characterized in that: Before adding lactase for hydrolysis in step (1), the bovine colostrum is heated to 38-40°C; before adding abomasalase for enzymatic hydrolysis in step (3), the skimmed bovine colostrum is heated to 36-38°C.

7. The method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors according to claim 2, characterized in that: In step (4), an ultrafiltration device with a membrane molecular weight cutoff of 1-20 kDa is used for ultrafiltration concentration.

8. The method for preparing a freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin rich in natural immune factors according to claim 2, characterized in that: The freeze-drying temperature in step (4) is -50℃ to -40℃.

9. A freeze-dried powder of anti-aging, non-allergenic bovine colostrum immunoglobulin prepared by the method according to any one of claims 1-8, characterized in that: The anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder comprises the following components: galactose content of 4.25±0.87wt%, glucose content of 3.10±0.94wt%, lactose content of 0%, α-casein content of 0%, β-casein content of 0%, and total protein content of 81.06±2.87wt%. The protein comprises a total of 54.11±2.88wt% natural immune factors, 1.24±0.07wt% β-lactoglobulin, and 1.10±0.08wt% α-lactalbumin. The natural immune factors include the following components: immunoglobulin G (41.7±2.68wt%), secretory immunoglobulin A (4.93±1.05wt%), lactoferrin (0.83±0.03wt%), epidermal growth factor (239.45±0.05ng / g), transforming growth factor β (22.93±0.05ng / g), insulin-like growth factor-1 (744.89±0.05ng / g), and other components (6.65±0.05wt%). The other components include immunoglobulin M, immunoglobulin E, immunoglobulin D, cytokines, lysozyme, and peptides.

10. The use of the anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder prepared by the method according to any one of claims 1-8, or the anti-aging, non-allergenic bovine colostrum immunoglobulin freeze-dried powder according to claim 9 or 10, in the preparation of food, health products, or pharmaceuticals.

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