Separation method of bioactive protein in bovine coloctrum and preparation method of composite bovine coloctrum product

Abstract

The invention relates to the technical field of dairy product separation and extraction, in particular to a separation method of bioactive protein in bovine coloctrum and a preparation method of a composite bovine coloctrum product. The separation method of the bioactive protein in the bovine coloctrum comprises the following steps: S1, pretreating raw milk to obtain sterilized bovine coloctrum; s2, carrying out first ultrafiltration treatment on the sterilized bovine coloctrum so as to obtain a first permeate and a first trapped fluid containing IgG; s3, performing second ultrafiltration treatment on the first permeate to obtain second permeate and second trapped fluid containing lactoferrin; and S4, carrying out third ultrafiltration treatment on the second permeate so as to obtain third trapped fluid containing basic protein. The separation method not only can efficiently extract IgG, but also can keep high activity of lactoferrin and basic protein. Meanwhile, the bovine coloctrum compound protein product rich in various high-activity proteins can be prepared through the process.

Description

Technical Field

[0001] This invention relates to the field of dairy product separation and extraction technology, specifically to a method for separating bioactive proteins from bovine colostrum and a method for preparing compound bovine colostrum products. Background Technology

[0002] Bovine colostrum is rich in various bioactive proteins, among which immunoglobulins (especially IgG), lactoferrin, and basic proteins are the most important. These proteins possess unique value in enhancing immunity, antibacterial and antiviral activity, promoting growth, and regulating physiological functions.

[0003] Currently, the main processes for isolating specific proteins from bovine colostrum are as follows: Single IgG extraction processes: Common methods include salting out, ultrafiltration, and ion exchange chromatography. These methods typically aim to achieve high-purity IgG extraction, but they often neglect or damage other bioactive proteins during the process. For example, drastic pH changes or high-salt environments can lead to irreversible denaturation of lactoferrin and basic proteins.

[0004] Mixed active protein powder process: This typically involves low-temperature pasteurization and spray drying, aiming to "retain" all active ingredients. However, this method cannot increase the relative content of specific proteins, and due to the different heat sensitivities of different proteins, it is difficult to achieve optimal activity retention of all components.

[0005] Chromatographic purification process: Although it can obtain high-purity single proteins, the process is long, costly, and requires complex equipment. In addition, there is a significant loss of activity during the process, making it completely unsuitable for the large-scale production of functional food or health product raw materials.

[0006] However, the existing separation process has the following defects: (1) Poor compatibility: The existing process is difficult to simultaneously achieve a high extraction rate of IgG and a high activity retention rate of lactoferrin and basic protein in one process route. Often, the extraction of IgG wastes other valuable components; (2) Large loss of activity: Multiple pH adjustment, phase transition, heating and other steps will produce a cumulative effect, causing serious damage to heat-sensitive and shear-sensitive lactoferrin and basic protein; (3) Complex process and high cost: The route of extracting and compounding multiple proteins separately is extremely expensive and cannot be industrialized; (4) Single product form: The final product is usually a single IgG powder, which fails to form a compound functional ingredient with synergistic enhancement effect.

[0007] Therefore, there is an urgent need to develop a new process that can extract immunoglobulins from bovine colostrum in an integrated, efficient, and gentle manner, while preserving the activity of lactoferrin and basic proteins. Summary of the Invention

[0008] This invention aims to at least partially solve one of the technical problems in related technologies. Therefore, one object of this invention is to provide a method for isolating bioactive proteins from bovine colostrum and a method for preparing a compound bovine colostrum product. This isolation method not only efficiently extracts IgG but also retains lactoferrin and basic proteins with high activity. Simultaneously, this invention provides a simple, mild, and scalable process for the co-enrichment of multiple active proteins. This process can produce a bovine colostrum compound protein product rich in various highly active proteins.

[0009] Therefore, the first aspect of the present invention provides a method for isolating bioactive proteins from bovine colostrum. According to an embodiment of the present invention, the isolation method includes: S1: Pre-treat the raw milk to obtain sterilized bovine colostrum; S2: The sterilized bovine colostrum is subjected to a first ultrafiltration process to obtain a first permeate and a first retentate containing IgG; S3: Perform a second ultrafiltration on the first permeate to obtain a second permeate and a second retentate containing lactoferrin; S4: Perform a third ultrafiltration on the second permeate to obtain a third retentate containing basic proteins.

[0010] The method for separating bioactive proteins from bovine colostrum provided by this invention sets the goal of "synergistic enrichment". Through a single process chain, IgG is extracted and lactoferrin and basic proteins are enriched simultaneously and efficiently, realizing the integrated utilization of multiple high-value active ingredients in bovine colostrum.

[0011] According to an embodiment of the present invention, the first ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 100-120 kDa for filtration and concentration.

[0012] According to an embodiment of the present invention, the second ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 30-50 kDa for filtration and concentration.

[0013] According to an embodiment of the present invention, the third ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa for filtration and concentration.

[0014] According to an embodiment of the present invention, in step S1, the sterilization treatment includes cross-flow microfiltration using a microfiltration membrane at 5-10°C.

[0015] According to an embodiment of the present invention, the pore size of the microfiltration membrane is 0.2-0.45 μm.

[0016] A second aspect of this invention provides a method for preparing a compound bovine colostrum product. According to an embodiment of the invention, the method includes: (1) The first retentate was enriched and concentrated by ultrafiltration to obtain a concentrate containing IgG. Wherein, the first retentate is the first retentate obtained by the method for separating bioactive proteins in bovine colostrum described in the first aspect; (2) The concentrated solution containing IgG is mixed with a second retentate containing lactoferrin and a third retentate containing basic protein to obtain a compound bovine colostrum liquid product. The second and third retentates are obtained by the method for separating bioactive proteins from bovine colostrum described in the first aspect.

[0017] According to an embodiment of the present invention, step (1) further includes: 1) Adjust the pH of the first retentate to 4.4-4.6 to obtain the first supernatant enriched with IgG; 2) The supernatant is centrifuged to obtain a second supernatant enriched with IgG; 3) The second supernatant is subjected to a fourth ultrafiltration process to obtain the concentrated solution containing IgG.

[0018] According to an embodiment of the present invention, the centrifugation process is carried out at a temperature of 4-8°C and a rotation speed of 5000-7000 rpm.

[0019] According to an embodiment of the present invention, the fourth ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 50-100 kDa for filtration and concentration.

[0020] According to an embodiment of the present invention, the preparation method further includes: The compound bovine colostrum liquid product is subjected to sterilization treatment.

[0021] According to an embodiment of the present invention, the preparation method further includes freeze-drying the sterilized compound bovine colostrum liquid product to obtain a compound bovine colostrum solid product.

[0022] A third aspect of this invention provides a compound bovine colostrum product. According to an embodiment of the invention, the compound bovine colostrum product is obtained by the preparation method of the compound bovine colostrum product described in the second aspect.

[0023] Existing methods for preparing bovine colostrum products primarily aim to obtain a single high-purity IgG (sacrificing other proteins) or to prepare low-purity, full-component colostrum powder. The core innovation of this invention lies in setting a "synergistic enrichment" objective. Through a single process chain, it simultaneously and efficiently extracts IgG and enriches lactoferrin and basic proteins, achieving integrated utilization of multiple high-value active ingredients in bovine colostrum. Existing technologies often rely on mild end-stage treatments (such as low-temperature drying) or the addition of protective agents after purification to retain bioactive proteins in bovine colostrum. Activity loss during purification is difficult to avoid. This invention prioritizes activity protection throughout the entire process, from initial microfiltration and cold sterilization to gentle pH-selective precipitation, low-temperature membrane separation, and a gentle end-stage sterilization tailored to the complex solution. Each step is proactively designed to protect the activity of multiple proteins, achieving systematic high-activity retention. Existing technologies produce bovine colostrum products that are either single IgG products, full-fat powder with limited active ingredient content, or physically mixed compound products (with varying sources of ingredients, potentially leading to compromised activity). The product obtained using the method of this invention is a natural complex protein powder derived from the same raw material and produced through a synergistic process. Its core value lies in the fact that the product not only has a high IgG content, but also retains high activity rates of lactoferrin and basic protein, forming an "all-in-one" product with synergistic enhancement functions.

[0024] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Detailed Implementation

[0025] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0027] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0028] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0029] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0030] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0031] According to the group standard T / WXYYXH 001-2025, the traditional process for freeze-drying bovine colostrum powder includes: milk purification, fat separation, sterilization, concentration, and freeze-drying. This process removes moisture and fat through physical methods, aiming to maximize the retention of the natural active ingredients in bovine colostrum. However, the process's purpose is merely "retention" rather than "enrichment." The resulting product generally has low IgG content and does not specifically address the loss of activity of lactoferrin and basic proteins during processing.

[0032] Existing literature mentions several methods for extracting single immunoglobulin (IgG), including: salting out (using salts such as ammonium sulfate to precipitate and separate IgG); ultrafiltration (using ultrafiltration membranes with specific molecular weight cutoffs to separate IgG based on molecular size); and ion exchange chromatography (purifying IgG through a chromatography column based on charge differences). The primary goal is to obtain high-purity, high-content IgG. While these methods typically aim for high-purity IgG extraction, they often neglect or damage other bioactive proteins during the process. For example, drastic pH changes or high-salt environments can lead to irreversible denaturation of lactoferrin and basic proteins.

[0033] Patent CN119791196A discloses the preparation of a hypoallergenic bovine colostrum complex for pets, which directly mixes bovine colostrum, hydrolyzed protein, IgY, and other ingredients. Guoyao Kangyang's "Tianbeili" special dietary powder integrates bovine colostrum powder, egg yolk globulin, and other active ingredients. By physically compounding multiple functional raw materials, it constructs a "cocktail-like" nutritional supplement to meet the market's demand for multifunctional products. However, it also has the following problems: Non-integrated extraction: Core active ingredients (such as IgG and lactoferrin) are purchased as independent raw materials and simply mixed, rather than being extracted from the same bovine colostrum raw material through a single process. Cost and activity challenges: High-quality raw materials are expensive, and the optimal activity retention conditions for each component are different; the compounding process may cause secondary damage.

[0034] The inventors have provided the following technical solutions to address the aforementioned problems.

[0035] According to a specific embodiment of the present invention, the present invention provides a method for isolating bioactive proteins from bovine colostrum, the isolation method comprising: S1: Pre-treat the raw milk to obtain sterilized bovine colostrum; S2: The sterilized bovine colostrum is subjected to a first ultrafiltration process to obtain a first permeate and a first retentate containing IgG; S3: Perform a second ultrafiltration on the first permeate to obtain a second permeate and a second retentate containing lactoferrin; S4: Perform a third ultrafiltration on the second permeate to obtain a third retentate containing basic proteins.

[0036] It should be noted that there is no particular limitation on the molecular weight cutoff of the ultrafiltration membrane used in the first ultrafiltration process, as long as IgG with a molecular weight of approximately 150 kDa is retained and enriched. According to a specific embodiment of the present invention, the first ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 100-120 kDa for filtration and concentration. Most of the smaller molecular weight lactoferrin (approximately 80 kDa) and basic proteins (approximately 15-30 kDa) permeate through the membrane into the permeate. According to a more specific embodiment of the present invention, the first ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, 102 kDa, 104 kDa, 106 kDa, 108 kDa, 110 kDa, 112 kDa, 114 kDa, 116 kDa, 118 kDa, or 120 kDa for filtration and concentration.

[0037] It should be noted that there is no particular limitation on the molecular weight cutoff of the ultrafiltration membrane used in the second ultrafiltration treatment, as long as lactoferrin is retained and enriched. According to a specific embodiment of the present invention, the second ultrafiltration treatment uses an ultrafiltration membrane with a molecular weight cutoff of 30-50 kDa for filtration and concentration. In this case, lactoferrin is retained and enriched, while basic proteins permeate through the membrane into the permeate. According to a more specific embodiment of the present invention, the second ultrafiltration treatment uses an ultrafiltration membrane with a molecular weight cutoff of 30 kD, 31 kD, 32 kD, 33 kD, 34 kD, 35 kD, 36 kD, 37 kD, 38 kD, 39 kD, 40 kD, 41 kD, 42 kD, 43 kD, 44 kD, 45 kD, 46 kD, 47 kD, 48 kD, 49 kD, or 50 kD for filtration and concentration.

[0038] It should be noted that there is no particular limitation on the molecular weight cutoff of the ultrafiltration membrane used in the third ultrafiltration treatment, as long as it retains and enriches basic proteins. According to a specific embodiment of the present invention, the third ultrafiltration treatment uses an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa for filtration and concentration. Smaller peptides, lactose, etc., are removed. The basic protein-rich solution is collected. According to a more specific embodiment of the present invention, the third ultrafiltration treatment uses ultrafiltration membranes with molecular weight cutoffs of 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, and 10 kDa for filtration and concentration.

[0039] According to a specific embodiment of the present invention, in step S1, the sterilization treatment includes cross-flow microfiltration using a microfiltration membrane at 5-10°C. For example, in step S1, the sterilization treatment includes cross-flow microfiltration using a microfiltration membrane at 5°C, 6°C, 7°C, 8°C, 9°C, and 10°C.

[0040] According to a specific embodiment of the present invention, the pore size of the microfiltration membrane is 0.2-0.45 μm. For example, the pore size of the microfiltration membrane is 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, or 0.45 μm.

[0041] According to a specific embodiment of the present invention, the present invention also provides a method for preparing a compound bovine colostrum product, the method comprising: (1) The first retentate was enriched and concentrated by ultrafiltration to obtain a concentrate containing IgG. Wherein, the first retentate is the first retentate obtained by the method for separating bioactive proteins in bovine colostrum described above; (2) The concentrated solution containing IgG is mixed with a second retentate containing lactoferrin and a third retentate containing basic protein to obtain a compound bovine colostrum liquid product. The second and third retentates are the second and third retentates obtained by the aforementioned method for separating bioactive proteins from bovine colostrum.

[0042] It should be noted that there are no particular restrictions on the ratio of the IgG-containing concentrate to the second retentate containing lactoferrin and the third retentate containing basic protein in step (2). The ratio can be adjusted appropriately according to the specifications of the compound bovine colostrum product to be prepared. According to a specific embodiment of the present invention, when the IgG-containing concentrate is mixed with the second retentate containing lactoferrin and the third retentate containing basic protein, it is compounded in a specific ratio (e.g., based on protein content 1:(0.5-1):(0.5-1)) to obtain a compound protein solution.

[0043] According to a specific embodiment of the present invention, step (1) further includes: 1) Adjust the pH of the first retentate to 4.4-4.6 to obtain the first supernatant enriched with IgG; 2) The supernatant is centrifuged to obtain a second supernatant enriched with IgG; 3) The second supernatant is subjected to a fourth ultrafiltration process to obtain the concentrated solution containing IgG.

[0044] According to one specific embodiment of the present invention, the centrifugation temperature is 4-8℃ and the rotation speed is 5000-7000 rpm. According to another specific embodiment of the present invention, the centrifugation temperature can be 4℃, 5℃, 6℃, 7℃, or 8℃, and the rotation speed can be 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, or 7000 rpm.

[0045] According to a specific embodiment of the present invention, the fourth ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 50-100 kDa for filtration and concentration. For example, the fourth ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or 100 kDa for filtration and concentration.

[0046] According to a specific embodiment of the present invention, the preparation method further includes: The compound bovine colostrum liquid product is subjected to sterilization treatment.

[0047] It should be noted that there are no particular limitations on the sterilization method. Any low-temperature sterilization method known in the art, or the method of sterilization using a filter membrane, is covered within the scope of protection of this invention.

[0048] According to a specific embodiment of the present invention, the sterilization treatment may be low-temperature pasteurization, for example, maintaining at 65~70°C for 15~30 seconds.

[0049] According to a specific embodiment of the present invention, the sterilization treatment may be terminal sterilization using sterilization-grade ultrafiltration with a membrane pore size of <0.22 μm.

[0050] According to a specific embodiment of the present invention, the preparation method further includes freeze-drying the sterilized compound bovine colostrum liquid product to obtain a compound bovine colostrum solid product.

[0051] It should be noted that there are no particular limitations on the conditions and equipment for freeze-drying. All conditions and equipment that can freeze-dry the sterilized compound bovine colostrum liquid product into protein powder are covered within the scope of protection of this invention.

[0052] According to a specific embodiment of the present invention, the present invention also provides a compound bovine colostrum product, which is obtained by the preparation method of the compound bovine colostrum product described above.

[0053] According to a more specific embodiment of the present invention, the present invention provides a method for preparing a compound bovine colostrum product, the preparation method comprising: (1) Raw material pretreatment and microfiltration sterilization: Take fresh or frozen-thawed bovine colostrum and degrease it by centrifugation. Then, use a 0.2~0.45 μm microfiltration membrane at 8~10℃ to perform cross-flow microfiltration to remove bacteria and somatic cells and achieve cold sterilization.

[0054] (2) Three-step ultrafiltration fractionation enrichment: a. First step ultrafiltration (enrichment of macromolecules): The skimmed colostrum obtained in step 1 is concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 100-120 kDa. IgG with a molecular weight of approximately 150 kDa is retained and enriched, while most of the smaller molecular weight lactoferrin (approximately 80 kDa) and basic proteins (approximately 15-30 kDa) permeate through the membrane into the permeate. The IgG enrichment solution is collected. b. Second-step ultrafiltration (enrichment of small molecules): The permeate from the first-step ultrafiltration is immediately concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 30-50 kDa. At this stage, lactoferrin is retained and enriched, while basic proteins permeate through the membrane into the permeate. c. Third step ultrafiltration: The permeate from the second step ultrafiltration is concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa, removing smaller peptides, lactose, etc. The basic protein enrichment solution is then collected.

[0055] (3) Selective acid precipitation and IgG re-enrichment: a. Under gentle stirring, precisely adjust the pH of the retentate (IgG enrichment solution) obtained in step (2) a to a narrow range of 4.4-4.6 using food-grade acid (such as citric acid or lactic acid solution). Under this pH condition, most of the casein precipitates, while IgG remains in the supernatant; b. Centrifuge at low temperature (4℃, 5000~7000 rpm, 15 min) and collect the supernatant (i.e., whey phase).

[0056] (4) Ultrafiltration concentration of IgG enrichment solution: The supernatant obtained in step (3) b was further concentrated using a 100 kDa ultrafiltration membrane system to obtain high concentrations of IgG.

[0057] (5) Compound and mild sterilization: a. Combine the IgG enrichment solution obtained in step (4) with the lactoferrin enrichment solution obtained in step (2) b and the basic protein enrichment solution obtained in step (2) c in a specific ratio (e.g., based on protein content, from 1:0.5:0.5 to 1:1:1) to obtain a composite protein solution; b. Perform low-temperature pasteurization on the composite protein solution under the following conditions: maintain at 65~70℃ for 15~30 seconds, or perform terminal sterilization using sterilization-grade ultrafiltration with a membrane pore size <0.22 μm.

[0058] (6) Low-temperature drying: The sterilized compound protein liquid is freeze-dried to obtain highly active compound bovine colostrum protein powder.

[0059] According to a more preferred embodiment of the present invention, the present invention provides a method for preparing a compound bovine colostrum product, the preparation method comprising: (1) Raw material pretreatment and microfiltration sterilization: Fresh or frozen-thawed bovine colostrum was taken and defatted by centrifugation. Then, a 0.2 μm microfiltration membrane was used at 8°C for cross-flow microfiltration to remove bacteria and somatic cells, thus achieving cold sterilization.

[0060] (2) Three-step ultrafiltration fractionation enrichment: a. First step ultrafiltration (enrichment of macromolecules): The skimmed colostrum obtained in step (1) was concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 100 kDa. IgG with a molecular weight of approximately 150 kDa was retained and enriched, while most of the smaller molecular weight lactoferrin (approximately 80 kDa) and basic proteins (approximately 15-30 kDa) permeated through the membrane into the permeate. The IgG enrichment solution was collected; b. Second-step ultrafiltration (enrichment of small molecules): The permeate from the first-step ultrafiltration is immediately concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 30 kDa. At this point, lactoferrin is retained and enriched, while basic proteins permeate through the membrane into the permeate. c. Third step ultrafiltration: The permeate from the second step ultrafiltration is concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa, removing smaller peptides, lactose, etc. The basic protein enrichment solution is then collected.

[0061] (3) Selective acid precipitation and IgG re-enrichment: a. Under gentle stirring, precisely adjust the pH of the retentate (IgG enrichment solution) obtained in step (2) a to 4.6 using food-grade acid (such as citric acid or lactic acid solution). Under this pH condition, most of the casein precipitates, while the IgG remains in the supernatant; b. Centrifuge at low temperature (4℃, 5000 rpm, 15 min) and collect the supernatant (i.e., whey phase).

[0062] (4) Ultrafiltration concentration of IgG enrichment solution: The supernatant obtained in step (3) b was further concentrated using a 100 kDa ultrafiltration membrane system to obtain high concentrations of IgG.

[0063] (5) Compound and mild sterilization: a. Combine the IgG enrichment solution obtained in step (4), the lactoferrin enrichment solution obtained in step (2) b, and the basic protein enrichment solution obtained in step (2) c in a specific ratio (e.g., based on protein content 1:0.5:0.5) to obtain a composite protein solution; b. Perform low-temperature pasteurization on the composite protein solution under the following conditions: maintain at 68°C for 15 seconds for terminal sterilization.

[0064] (6) Low-temperature drying: The sterilized compound protein liquid is freeze-dried to obtain highly active compound bovine colostrum protein powder.

[0065] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0066] Example 1 (1) Raw material pretreatment and microfiltration sterilization: Fresh or frozen-thawed bovine colostrum was taken and defatted by centrifugation. Then, a 0.2 μm microfiltration membrane was used at 8°C for cross-flow microfiltration to remove bacteria and somatic cells, thus achieving cold sterilization.

[0067] (2) Three-step ultrafiltration fractionation enrichment: a. First step ultrafiltration (enrichment of macromolecules): The skimmed colostrum obtained in step (1) was concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 100 kDa. IgG with a molecular weight of approximately 150 kDa was retained and enriched, while most of the smaller molecular weight lactoferrin (approximately 80 kDa) and basic proteins (approximately 15-30 kDa) permeated through the membrane into the permeate. The IgG enrichment solution was collected; b. Second-step ultrafiltration (enrichment of small molecules): The permeate from the first-step ultrafiltration is immediately concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 30 kDa. At this point, lactoferrin is retained and enriched, while basic proteins permeate through the membrane into the permeate. c. Third step ultrafiltration: The permeate from the second step ultrafiltration is concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa, removing smaller peptides, lactose, etc. The basic protein enrichment solution is then collected.

[0068] (3) Selective acid precipitation and IgG re-enrichment: a. Under gentle stirring, precisely adjust the pH of the retentate (IgG enrichment solution) obtained in step (2) a to 4.6 using food-grade acid (such as citric acid or lactic acid solution). Under this pH condition, most of the casein precipitates, while the IgG remains in the supernatant; b. Centrifuge at low temperature (4℃, 5000 rpm, 15 min) and collect the supernatant (i.e., whey phase).

[0069] (4) Ultrafiltration concentration of IgG enrichment solution: The supernatant obtained in step (3) b was further concentrated using a 100 kDa ultrafiltration membrane system to obtain high concentrations of IgG.

[0070] (5) Compound and mild sterilization: a. Combine the IgG enrichment solution obtained in step (4), the lactoferrin enrichment solution obtained in step (2) b, and the basic protein enrichment solution obtained in step (2) c in a specific ratio (based on protein content 1:0.5:0.5) to obtain a composite protein solution; b. Perform low-temperature pasteurization on the composite protein solution under the following conditions: maintain at 68°C for 15 seconds for terminal sterilization.

[0071] (6) Low-temperature drying: The sterilized compound protein liquid is freeze-dried to obtain highly active compound bovine colostrum protein powder.

[0072] Example 2 (1) Raw material pretreatment and microfiltration sterilization: Fresh or frozen-thawed bovine colostrum was taken and defatted by centrifugation. Then, a 0.2 μm microfiltration membrane was used at 8°C for cross-flow microfiltration to remove bacteria and somatic cells, thus achieving cold sterilization.

[0073] (2) Three-step ultrafiltration fractionation enrichment: a. First step ultrafiltration (enrichment of macromolecules): The skimmed colostrum obtained in step (1) was concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 100 kDa. IgG with a molecular weight of approximately 150 kDa was retained and enriched, while most of the smaller molecular weight lactoferrin (approximately 80 kDa) and basic proteins (approximately 15-30 kDa) permeated through the membrane into the permeate. The IgG enrichment solution was collected; b. Second-step ultrafiltration (enrichment of small molecules): The permeate from the first-step ultrafiltration is immediately concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 30 kDa. At this point, lactoferrin is retained and enriched, while basic proteins permeate through the membrane into the permeate. c. Third step ultrafiltration: The permeate from the second step ultrafiltration is concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 5 kDa, removing smaller peptides, lactose, etc. The basic protein enrichment solution is then collected.

[0074] (3) Selective acid precipitation and IgG re-enrichment: a. Under gentle stirring, precisely adjust the pH of the retentate (IgG enrichment solution) obtained in step (2) a to 4.4 using food-grade acid (such as citric acid or lactic acid solution). Under this pH condition, most of the casein precipitates, while the IgG remains in the supernatant; b. Centrifuge at low temperature (4℃, 5000 rpm, 15 min) and collect the supernatant (i.e., whey phase).

[0075] (4) Ultrafiltration concentration of IgG enrichment solution: The supernatant obtained in step (3) b was further concentrated using a 100 kDa ultrafiltration membrane system to obtain high concentrations of IgG.

[0076] (5) Compound and mild sterilization: a. Combine the IgG enrichment solution obtained in step (4), the lactoferrin enrichment solution obtained in step (2) b, and the basic protein enrichment solution obtained in step (2) c in a specific ratio (based on protein content 1:0.5:0.5) to obtain a composite protein solution; b. Perform low-temperature pasteurization on the composite protein solution under the following conditions: maintain at 65°C for 15 seconds, or perform terminal sterilization using sterilization-grade ultrafiltration with a membrane pore size <0.22 μm.

[0077] (6) Low-temperature drying: The sterilized compound protein liquid is freeze-dried to obtain highly active compound bovine colostrum protein powder.

[0078] Example 3 (1) Raw material pretreatment and microfiltration sterilization: Fresh or frozen-thawed bovine colostrum was centrifuged to remove fat. Then, a 0.45 μm microfiltration membrane was used at 10°C for cross-flow microfiltration to remove bacteria and somatic cells, achieving cold sterilization.

[0079] (2) Three-step ultrafiltration fractionation enrichment: a. First step ultrafiltration (enrichment of macromolecules): The skimmed colostrum obtained in step (1) was concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 120 kDa. IgG with a molecular weight of approximately 150 kDa was retained and enriched, while most of the smaller molecular weight lactoferrin (approximately 80 kDa) and basic proteins (approximately 15-30 kDa) permeated through the membrane into the permeate. The IgG enrichment solution was collected; b. Second-step ultrafiltration (enrichment of small molecules): The permeate from the first-step ultrafiltration is immediately concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 50 kDa. At this point, lactoferrin is retained and enriched, while basic proteins permeate through the membrane into the permeate. c. Third step ultrafiltration: The permeate from the second step ultrafiltration is concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 10 kDa, removing smaller peptides, lactose, etc. The basic protein enrichment solution is then collected.

[0080] (3) Selective acid precipitation and IgG re-enrichment: a. Under gentle stirring, precisely adjust the pH of the retentate (IgG enrichment solution) obtained in step (2) a to 4.6 using food-grade acid (such as citric acid or lactic acid solution). Under this pH condition, most of the casein precipitates, while the IgG remains in the supernatant; b. Centrifuge at low temperature (4℃, 7000 rpm, 15 min) and collect the supernatant (i.e., whey phase).

[0081] (4) Ultrafiltration concentration of IgG enrichment solution: The supernatant obtained in step (3) b was further concentrated using a 100 kDa ultrafiltration membrane system to obtain high concentrations of IgG.

[0082] (5) Compound and mild sterilization: a. Combine the IgG enrichment solution obtained in step (4), the lactoferrin enrichment solution obtained in step (2) b, and the basic protein enrichment solution obtained in step (2) c in a specific ratio (based on protein content 1:1:1) to obtain a composite protein solution; b. Perform low-temperature pasteurization on the composite protein solution under the following conditions: maintain at 70°C for 30 seconds, or perform terminal sterilization using sterilization-grade ultrafiltration with a membrane pore size <0.22 μm.

[0083] (6) Low-temperature drying: The sterilized compound protein liquid is freeze-dried to obtain highly active compound bovine colostrum protein powder.

[0084] Comparative Example 1 The highly active composite bovine colostrum protein powder was prepared according to the method in Example 1, except that step (2) ultrafiltration fractionation and enrichment was changed to two steps: (2) Ultrafiltration fractionation and enrichment: a. First step ultrafiltration (enrichment of macromolecules): The defatted colostrum obtained in step (1) is concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 100 kDa. IgG with a molecular weight of approximately 150 kDa is retained and enriched, while most of the smaller molecular weight lactoferrin (approximately 80 kDa) and basic proteins (approximately 15-30 kDa) permeate through the membrane into the permeate. The IgG enrichment solution is collected; b. Second-step ultrafiltration (enrichment of small molecules): The permeate from the first-step ultrafiltration is immediately concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 30 kDa. At this point, lactoferrin is retained and enriched, while basic proteins permeate through the membrane into the permeate.

[0085] Comparative Example 2 The highly active composite bovine colostrum protein powder was prepared according to the method in Example 1, except that step (2) ultrafiltration fractionation and enrichment was changed to a single step: (2) Ultrafiltration enrichment: The defatted colostrum obtained in step (1) was concentrated using an ultrafiltration membrane system with a molecular weight cutoff of 100 kDa. IgG with a molecular weight of approximately 150 kDa was retained and enriched, while most of the smaller molecular weight lactoferrin (approximately 80 kDa) and basic proteins (approximately 15-30 kDa) permeated through the membrane into the permeate.

[0086] Comparative Example 3 Highly active composite bovine colostrum protein powder was prepared according to the method in Example 1, except that the ultrafiltration fractionation enrichment in step (2) was not performed.

[0087] Test of compound bovine colostrum protein powder indicators Table 1 below shows the various indicators of the compound bovine colostrum protein powders prepared in Examples 1-3 and Comparative Examples 1-3.

[0088] Table 1 Content of various indicators in bovine colostrum protein powder

[0089] Note: The method for calculating the activity retention rate is: Activity retention rate (%) = (C1× V1) / (C0× V0) × 100%.

[0090] C0 and C1 represent the concentrations of lactoferrin or basic protein in the raw materials and the final product, respectively; V0 and V1 represent the total volumes of the raw materials and the final product, respectively.

[0091] The results in Table 1 above show that, compared to Comparative Examples 1-3, the high-activity composite bovine colostrum protein powder obtained by three-step ultrafiltration fractionation and enrichment in Examples 1-3 has a higher total protein content, and the content of immunoglobulins, the retention rate of lactoferrin activity, and the retention rate of basic protein activity are all higher than those in the comparative examples. The overall microbial content of the process meets national standards.

[0092] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0093] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for isolating bioactive proteins from bovine colostrum, characterized in that, include: S1: Pre-treat the raw milk to obtain sterilized bovine colostrum; S2: The sterilized bovine colostrum is subjected to a first ultrafiltration process to obtain a first permeate and a first retentate containing IgG; S3: Perform a second ultrafiltration on the first permeate to obtain a second permeate and a second retentate containing lactoferrin; S4: Perform a third ultrafiltration on the second permeate to obtain a third retentate containing basic proteins.

2. The separation method according to claim 1, characterized in that, The first ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 100-120 kDa for filtration and concentration.

3. The separation method according to claim 1, characterized in that, The second ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 30-50 kDa for filtration and concentration.

4. The separation method according to claim 1, characterized in that, The third ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 5-10 kDa for filtration and concentration.

5. The separation method according to claim 1, characterized in that, In step S1, the sterilization treatment includes cross-flow microfiltration using a microfiltration membrane at 5-10°C; Optionally, the pore size of the microfiltration membrane is 0.2-0.45 μm.

6. A method for preparing a compound bovine colostrum product, characterized in that, include: (1) The first retentate was enriched and concentrated by ultrafiltration to obtain a concentrate containing IgG. Wherein, the first retentate is the first retentate obtained by the method for separating bioactive proteins in bovine colostrum according to any one of claims 1-5; (2) The concentrated solution containing IgG is mixed with a second retentate containing lactoferrin and a third retentate containing basic protein to obtain a compound bovine colostrum liquid product. The second and third retentates are obtained by the method for separating bioactive proteins from bovine colostrum according to any one of claims 1-5.

7. The preparation method according to claim 6, characterized in that, Step (1) further includes: 1) Adjust the pH of the first retentate to 4.4-4.6 to obtain the first supernatant enriched with IgG; 2) The supernatant is centrifuged to obtain a second supernatant enriched with IgG; 3) The second supernatant is subjected to a fourth ultrafiltration process to obtain the concentrated solution containing IgG; Optionally, the centrifugation process is carried out at a temperature of 4-8°C and a rotation speed of 5000-7000 rpm. Optionally, the fourth ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 50-100 kDa for filtration and concentration.

8. The preparation method according to claim 6, characterized in that, The preparation method further includes: The compound bovine colostrum liquid product is subjected to sterilization treatment; Optionally, the preparation method further includes freeze-drying the sterilized compound bovine colostrum liquid product to obtain a compound bovine colostrum solid product.

9. A compound bovine colostrum product, characterized in that, The compound bovine colostrum product is obtained by the preparation method of the compound bovine colostrum product according to any one of claims 6-8.

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