A method for isolating active substances from milk-derived raw materials, products and uses thereof

Abstract

The application discloses a method for separating active substances from milk raw materials, products and applications thereof, and relates to the field of dairy product processing. Through synergistic optimization of raw material pretreatment, membrane filtration and chromatography conditions, the application realizes simultaneous high-purity separation of multiple active substances such as alpha-lactalbumin, beta-lactoglobulin and bone bridge protein from whey protein liquid, and overcomes the problems of poor raw material adaptability, insufficient separation purity and difficulty in simultaneous extraction of multiple active substances in the prior art. The whole process condition is mild, the process flow is simple, economic, environmental protection and easy to industrialize, and a new way is provided for high-value comprehensive utilization of whey protein resources.

Description

Technical Field

[0001] This invention relates to the field of dairy processing, and more specifically, to a method for separating active substances from milk raw materials, as well as the products and applications thereof. Background Technology

[0002] Whey is a major byproduct of dairy product production, such as cheese and yogurt. Whey wastewater contains a large amount of whey protein, which is rich in nutrients and has high biological activity. Direct discharge of whey not only results in a serious waste of protein resources but is also a significant source of pollution for the dairy industry. Whey protein is a mixture of various functional proteins, mainly including α-lactalbumin (α-La), β-lactoglobulin (β-Lg), bovine serum albumin, lactoferrin, and immunoglobulins. α-lactalbumin accounts for 20-25% of the total protein content, and β-lactoglobulin accounts for 30-55%. In addition, whey also contains trace amounts of bioactive substances with important physiological functions, such as osteopontin (OPN).

[0003] Alpha-lactalbumin is widely used in functional health products and infant formula. Beta-lactoglobulin also has various benefits, but it is also a major allergen causing milk allergy; therefore, its effective separation from alpha-lactalbumin is of great significance. Osteopontoxin is an active substance found in both human and cow's milk, but its concentration in cow's milk is low. Infant formula based on cow's milk needs to be fortified with osteopontin to make its composition closer to breast milk. Therefore, efficiently separating high-purity versions of these active substances from whey not only effectively reduces environmental pollution from whey wastewater but also has significant economic value.

[0004] Currently, the main methods for separating and recovering whey protein from whey include precipitation, membrane separation technology, and chromatography.

[0005] Precipitation methods selectively precipitate target proteins by heating or changing the pH value. Although relatively simple to operate, these methods require large amounts of acid, alkali, and salt, which can easily lead to severe protein denaturation, and the post-processing steps are complex. For example, a two-step ammonium sulfate precipitation method is used for primary separation of whey protein, or isoelectric point precipitation is used to remove casein, but these methods are difficult to achieve high-purity separation of single protein components.

[0006] While membrane separation technologies (such as ultrafiltration and microfiltration) are energy-efficient and easily scaled up industrially, conventional membrane techniques struggle to effectively separate proteins with similar molecular weights, such as α-lactalbumin and β-lactoglobulin, due to the similar molecular weights of many proteins in whey. For example, while multi-stage ultrafiltration can achieve a certain degree of separation, the purity often falls short of the requirements for high-purity products. Although recent studies have combined membrane filtration with electrodialysis for the simultaneous concentration and desalting of whey proteins, achieving simultaneous high-purity separation of multiple target proteins remains challenging.

[0007] Traditional chromatography methods often suffer from low yields, limited scaling-up capabilities, and high energy consumption. For example, cation exchange chromatography can be used to separate α-lactalbumin from whey, or ion exchange combined with dextran gel chromatography can be used to extract basic lactic proteins. However, these methods typically target only a single protein, have lengthy processes, and are difficult to achieve simultaneous and efficient separation of multiple proteins.

[0008] For the separation of osteopontin, existing technologies mainly employ specific chromatographic processes. However, these methods are usually designed only for the single target of osteopontin, with relatively specific processes, demanding conditions, and cumbersome steps. They also have high requirements for the pretreatment of raw materials and are difficult to effectively integrate with the recovery processes of other active substances in whey (such as α-lactalbumin and β-lactoglobulin).

[0009] In view of this, the present invention is proposed. Summary of the Invention

[0010] The purpose of this invention is to provide a method for separating active substances from dairy raw materials, as well as the products and applications thereof.

[0011] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a method for separating active substances from milk-derived raw materials, comprising the following steps: pretreating a whey protein solution, the pretreatment comprising: adjusting the conductivity to a first set threshold and adjusting the pH to a second set threshold; the first set threshold being 2~10 mS / cm and the second set threshold being 6~7.2; and performing anion exchange chromatography on the pretreated protein solution to obtain a flow-through containing α-lactalbumin, a washing fraction containing β-lactoglobulin, and an elution fraction containing osteopontin.

[0012] Secondly, embodiments of the present invention provide a product prepared by a method for separating active substances from dairy raw materials as described in the foregoing embodiments.

[0013] Thirdly, embodiments of the present invention provide the application of the method or product described in the foregoing embodiments in the preparation of food or health products.

[0014] The present invention has the following beneficial effects: (1) The process flow was simplified by optimizing the pretreatment of raw materials. This application enables the high-purity extraction of α-lactalbumin, β-lactoglobulin, and osteopontin from natural membrane-separated whey protein solutions. Unexpectedly, this application discovers that, through specific pretreatment steps combined with anion exchange chromatography, multiple target proteins can be efficiently separated directly from natural membrane-separated whey protein solutions without complex pretreatment, achieving high-value utilization of dairy processing byproducts.

[0015] (2) Through optimization of process parameters, the simultaneous and high-purity separation of multiple active substances was achieved. This application achieves the orderly separation of α-lactalbumin, β-lactoglobulin and osteopontin through systematic screening and optimization of pretreatment, chromatography process parameters and membrane filtration technology, and the purity of each active substance can reach a high level.

[0016] (3) The process conditions are mild, which preserves the natural activity of the target protein and the product has good safety. The entire separation and purification process in this application is carried out in a mild aqueous system, avoiding the protein denaturation and inactivation problems caused by high temperatures or extreme pH in existing precipitation methods. The operating conditions of each chromatography step have been optimized to maintain the native conformation and biological activity of various active proteins to the greatest extent. Furthermore, the raw materials and excipients used in this application all meet food-grade or pharmaceutical-grade requirements, do not involve toxic or harmful organic solvents, and pose no risk of residual organic solvents in the products. Endotoxin levels and bioburden are controllable. The three active protein products obtained can be directly used as functional ingredients in infant formula or as protein raw materials for food and special dietary uses, demonstrating good safety and application value.

[0017] (4) Easy to scale up industrially The process route of this application is clear, the operating parameters are controllable, and it has good batch stability and reproducibility. Compared with traditional methods, the process flow of this application is relatively simple, the production cycle is short, intermediate processing steps and auxiliary material consumption are reduced, production energy consumption and waste liquid discharge are reduced, and it has good economic and environmental benefits, making it easy to realize large-scale industrial production. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1This is a schematic diagram of the process flow for Example 1; Figure 2 The image shows the SDS-PAGE degradation patterns of α-La during simulated in vitro gastrointestinal digestion in infants; where A represents group 1, B represents group 2, and C represents group 4. Figure 3 The curves show the change in the degree of hydrolysis of α-La samples over digestion time; yellow represents group 1, blue represents group 2, and red represents group 4. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] The terms “first,” “second,” “third,” etc., are used only to distinguish descriptions and should not be interpreted as indicating or implying relative importance.

[0022] The term "natural membrane-separated whey protein liquid" refers to a liquid product rich in whey protein obtained by directly removing most of the casein micelles and fat globules from fresh milk using membrane separation technology (preferably microfiltration).

[0023] On one hand, embodiments of the present invention provide a method for separating active substances from dairy raw materials, which includes the following steps: The whey protein solution is pretreated, the pretreatment including: adjusting the conductivity to a first set threshold and adjusting the pH to a second set threshold; the first set threshold is 2~10 mS / cm and the second set threshold is 6~7.2. The pretreated protein solution was subjected to anion exchange chromatography to obtain a flow-through containing α-lactalbumin, a wash fraction containing β-lactoglobulin, and an elution fraction containing osteopontin.

[0024] In some embodiments, the pretreatment includes: implementing the conductivity adjustment and pH adjustment by nanofiltration, wherein the molecular cutoff of the nanofiltration membrane is 100 Da to 800 Da, specifically any one or any two of the following: 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 350, 360, 380, 400, 450, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 760, 780, and 800 Da.

[0025] In some embodiments, the temperature for performing the conductivity and pH adjustments is 0~10°C, specifically any one or any two of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10°C.

[0026] In some embodiments, the first set threshold can be any one or a range between any two of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10 mS / cm.

[0027] In some embodiments, the second set threshold can be any one or a range between any two of 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, and 7.2.

[0028] In some embodiments, the first set threshold is 3~6 mS / cm, and the second set threshold is 6.4~7.0.

[0029] In some embodiments, the pretreatment further includes adjusting the total protein concentration of the whey protein solution to a third set threshold, wherein the third set threshold is 0.5% to 2% (w / w), specifically it can be any one or any two of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.4%, 1.6%, 1.8% and 2%.

[0030] In some embodiments, the steps of the anion exchange chromatography include washing and elution.

[0031] In some embodiments, the washing solution used for washing impurities includes a 0.2~0.6 M NaCl solution, the concentration of which can be any one or any two of 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, and 0.6 M.

[0032] In some embodiments, the eluent used for elution includes a 0.6-1 M NaCl solution, the concentration of which can be any one or any two of 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 and 1 M.

[0033] In some embodiments, the sodium chloride in the NaCl solution of the washing solution and / or eluent can be sodium chloride with a purity of 99% or higher, such as a salt solution.

[0034] In some embodiments, the amount of the washing solution is 5 to 15 column volumes, specifically any one or any two of 5, 6, 8, 10, 12, 14 and 15 column volumes.

[0035] In some embodiments, the amount of eluent used is 2 to 10 column volumes, specifically any one or any combination of 2, 3, 4, 6, 8, and 10 column volumes.

[0036] In some embodiments, the anion exchange chromatography is a strong anion exchange chromatography.

[0037] In some embodiments, the chromatography column packing for the anion exchange chromatography is agarose-based strong anion exchange packing.

[0038] In some embodiments, the chromatography column packing material for the anion exchange chromatography is cross-linked agarose with glucose chains, a cross-linking degree of 4%–8%, quaternary ammonium functional groups, a particle size distribution of 150–350 μm, an average particle size of 200–300 μm, and a substrate pore size of 10–50 nm. The cross-linking degree can be any one or any two of 4%, 5%, 6%, 7%, and 8%. The particle size distribution can be any one or any two of 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, and 350 μm. The average particle size can be any one of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 and 300 μm or any combination thereof. The pore size of the substrate spheres can be any one of 10, 20, 30, 40 and 50 nm or any combination thereof.

[0039] In some embodiments, the column height of the anion exchange chromatography is 20-30 cm, and the column diameter is 250-350 mm. The column height can be any one of 20, 22, 24, 26, 28, or 30 cm, or a range between any two. The column diameter can be any one of 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, or 350 mm, or a range between any two.

[0040] In some embodiments, the chromatography procedure of the anion exchange chromatography includes: CIP-high salt regeneration-equilibration-sample loading-washing after sample loading-impurity washing-elution.

[0041] In some embodiments, the CIP includes CIP cleaning of the chromatography column with a 0.4-0.6 M NaOH solution; the concentration of the NaOH solution can be any one or any two of 0.4, 0.45, 0.5, 0.55 and 0.6 M.

[0042] In some embodiments, during the CIP step, the amount of 0.4-0.6 M NaOH solution used is 1-10 times the column volume; specifically, the column volume can be any one or any two of 1, 2, 4, 6, 8, and 10 times.

[0043] In some embodiments, the high-salt regeneration includes rinsing the chromatography column with a 0.4-0.6 M NaCl solution; the concentration of the NaCl solution can be any one or any two of 0.4, 0.45, 0.5, 0.55, and 0.6 M.

[0044] In some embodiments, during the high-salt regeneration step, the amount of 0.4-0.6 M NaCl solution used is 1-10 times the column volume; specifically, the column volume can be any one or any two of 1, 2, 4, 6, 8, and 10 times.

[0045] In some embodiments, the equilibration solution used for equilibration includes RO water, and the amount used is 5 to 20 times the column volume; the column volume can specifically be any one or any two of 5, 6, 8, 9, 10, 11, 12, 14, 16, 18 and 20 times.

[0046] In some embodiments, the eluent used for elution after sample loading includes RO water, and the volume is 1 to 10 times the column volume. Specifically, the column volume can be any one or any combination of 1, 2, 4, 6, 8, and 10 times the column volume.

[0047] In some embodiments, the method further includes at least one of the following: separating α-lactalbumin from the flow-through liquid; separating β-lactoglobulin from the washing components; and separating osteopontin from the eluted components.

[0048] In some embodiments, the step of separating α-lactalbumin from the flow-through fluid includes: The flow-through liquid is concentrated by ultrafiltration to obtain a first permeate and a first retentate; The first permeate is concentrated by ultrafiltration and then by microfiltration to obtain the second retentate.

[0049] In some embodiments, the molecular cutoff of the filter membrane for ultrafiltration of the flow-through liquid is 40~60 kDa, specifically any one or any two of 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60 kDa; the concentration factor is 10~25 times, specifically any one or any two of 10, 12, 14, 16, 18, 20, 22, 24, and 25 times; the ultrafiltration temperature is 0~25°C, optionally 4~12°C; the washing filtrate includes RO water; and the washing is performed until the conductivity of the retentate is <1 ms / cm, for example, 0.1~0.9 ms / cm.

[0050] In some embodiments, the molecular cutoff of the filter membrane used for ultrafiltration of the first permeate is 3-15 kDa, selectable from any one or any two of 3, 4, 6, 8, 9, 10, 11, 12, 14, 15 kDa; the concentration factor can be any one or any two of 10, 12, 14, 16, 18, 20, 22, 24, 25 times; the ultrafiltration temperature can be 0-25°C, selectable from 4-12°C; the washing filtrate includes RO water; washing is performed until the conductivity of the retentate is <0.8 ms / cm, for example 0.1-0.79 ms / cm; and the concentration is performed until the solids content is >18%, for example 18.1%-30%. The pore size of the filter membrane for microfiltration is any one or any two of the following: 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 μm. Microfiltration uses ceramic membranes or capsule filters.

[0051] In some embodiments, the method further includes drying the second retentate. When the feedstock is a natural membrane-separated whey protein solution, the second retentate is α-lactalbumin.

[0052] In some embodiments, the step of separating β-lactoglobulin from the washed components includes: nanofiltration concentration, microfiltration, and / or drying of the β-lactoglobulin-containing washed components; the molecular cutoff of the nanofiltration membrane is 200-500 Da, selectable as any one or any two of 200, 250, 300, 350, 400, 450, and 500 Da; the concentration factor can be any one or any two of 1, 2, 3, 4, and 5 times; the ultrafiltration temperature can be 0-25°C, selectable as 4-12°C; the washing filtrate includes RO water; washing is performed until the conductivity of the retentate is <1 ms / cm, for example 0.1-0.9 ms / cm; and concentration is performed until the solids content is >18%, for example 18.1%-30%. The pore size of the microfiltration membrane is any one or any combination of 0.1, 0.2, 0.3, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μm. Microfiltration employs ceramic membranes or capsule filters. After drying, β-lactoglobulin powder is obtained.

[0053] In some embodiments, the step of separating osteopontin from the eluent fraction includes: ultrafiltration concentration and / or drying of the osteopontin-containing eluent fraction; the molecular cutoff of the ultrafiltration membrane is 3-15 kDa, selectable from any one or any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, and 15 kDa; the concentration factor is any one or any two of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and 40 times; the ultrafiltration membrane material can be RC material, referring to regenerated cellulose membrane; the ultrafiltration temperature can be 0-25°C, selectable from 4-12°C; the washing filtrate includes RO water; washing is performed until the conductivity of the retentate is <0.8 ms / cm, for example 0.1-0.79 ms / cm; and concentration is performed until the solids content is >18%, for example 18.1%-30%. After drying, osteopontin powder is obtained.

[0054] In some embodiments, the drying method includes freeze drying and spray drying.

[0055] In some embodiments, the whey protein solution comprises a natural membrane-separated whey protein solution.

[0056] In some embodiments, the natural membrane-separated whey protein solution is obtained from cow's milk.

[0057] In some embodiments, the method for preparing the natural membrane-separated whey protein solution includes: microfiltration and concentration of skim milk, and the permeate obtained is the natural membrane-separated whey protein solution.

[0058] In some embodiments, the pore size of the filter membrane used for microfiltration of skim milk is 0.05 to 0.2 μm, and can be selected as any one or any two of 0.05, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18 and 0.2 μm.

[0059] In some embodiments, the concentration factor of the skim milk by microfiltration is 1 to 5 times, which can be selected as any one or any two of 2, 3, 4 and 5 times.

[0060] In some embodiments, the temperature for microfiltration of skim milk is 40~55°C, specifically any one or any two of 40, 42, 44, 46, 48, 50, 52, 54 and 55°C.

[0061] In some embodiments, the active substance includes at least one of α-lactalbumin, β-lactoglobulin, and osteopontin.

[0062] In some embodiments, the casein content of the natural membrane-separated whey protein solution is ≤5% (w / w) of the total protein, for example, 0.1% to 4.9% (w / w).

[0063] In some embodiments, the β-lactoglobulin is isolated with a purity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%, or between any two of these values.

[0064] In some embodiments, the osteopontin is isolated with a purity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%, or between any two of these values.

[0065] In some embodiments, the α-lactalbumin has a separation purity of 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, and 100%, or between any two of these values.

[0066] On the other hand, embodiments of the present invention provide a product prepared by a method for separating active substances from dairy raw materials as described in any of the foregoing embodiments.

[0067] On the other hand, embodiments of the present invention also provide the application of the method or product described in any of the foregoing embodiments in the preparation of food or health products.

[0068] On the other hand, embodiments of the present invention also provide a formulation (or composition) rich in α-lactalbumin, wherein the α-lactalbumin accounts for more than 95% (≥95%) of the total protein; the α-lactalbumin is bound with calcium ions, and the amount of calcium ions bound by the α-lactalbumin is 1~1.4 mol Ca². + / mol α-La; the formulation contains no IgG or the mass of IgG accounts for less than 0.1% (≤0.1%) of the total protein.

[0069] In some embodiments, the content of α-lactalbumin (as a percentage of total protein) may be ≥95%, 96%, 97%, 98% or 99% (w / w).

[0070] In some embodiments, the calcium ion binding capacity of the α-lactalbumin is 1, 1.1, 1.2, 1.3, and 1.4 molCa². + / mol α-La, or any one of them or any two of them.

[0071] In some embodiments, the content of IgG in the formulation (as a percentage of total protein) is ≤0.02%, 0.04%, 0.06%, 0.08%, or 0.1% (w / w).

[0072] In some embodiments, the formulation satisfies at least one of the following: (1) The preparation does not contain perchlorate and chlorate, and the nitrate content is <5mg / 100g and the nitrite content is <1mg / 100g; (2) The ash content of the preparation is < (1~1.2) g / 100g; (3) The preparation contains β-lactoglobulin, and the mass of β-lactoglobulin accounts for less than 1% (≤1%) of the total protein. (4) The preparation is free of mercury, aluminum, arsenic, and lead, and the chromium content is <0.2 mg / kg; (5) The preparation is a powder with a moisture content of 5~8g / 100g, a fat content of <0.3g / 100g, and a protein content of 85~87g / 100g; (6) The preparation does not contain aflatoxin M1; (7) The impurity level of the preparation is ≤8mg / kg.

[0073] In some embodiments, the formulation is the flow-through containing α-lactalbumin described in any of the foregoing embodiments, or a product of the flow-through further purified, such as the second retentate or the dried product of the second retentate in any of the foregoing embodiments. The preparation method is the same as described in any of the foregoing embodiments and will not be repeated here. The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0074] In this embodiment, solids were detected according to Method I of GB 5009.3-2016, and the contents of α-lactalbumin and β-lactoglobulin were detected using high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-MS / MS) under the following conditions: Chromatographic column: Acquity UPLC BEH 300 C18 column (particle size 1.7μm, 2.1×100mm); Chromatographic conditions: Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile aqueous solution containing 0.1% formic acid. The injection volume was 5 μL, and the column temperature was 40°C. The LC elution gradient program was as follows: initially, 5% mobile phase B was held for 1.2 min, then the linear gradient was increased to 60% mobile phase B within 1.8 min, then to 80% mobile phase B within 1 min, and then to 100% mobile phase B within 0.2 min. The system was then equilibrated at 100% mobile phase B for 1.4 min, and then returned to 5% mobile phase B within 2.2 min. The total run time for each injection was 8 min.

[0075] Ion source: Electrospray ionization (ESI); Mass spectrometry conditions: capillary voltage 3.50 kV, cone voltage 30 V, source temperature 150°C, desolvation gas temperature 350°C, cone gas flow rate 50 L / h nitrogen, desolvation gas flow rate 900 L / h nitrogen, argon collision gas pressure 3 × 10⁻⁶ for MS / MS analysis. -3 mbar; Calculate the purity of α-La and β-Lg using the following formula: ; ; Where x represents the purity of α-La and y represents the purity of β-Lg.

[0076] Determination of rehydration properties: Instruments and Equipment: 1.1 Electronic balance: sensitivity of 0.01g; 1.2 Beaker: 200mL, clean, with a dry inner wall and an intact surface without cracks; 1.3 Glass rod: φ 6mm, complete and undamaged; 1.4 Plate: φ 90mm, clean and dry; 1.5 Sulfuric acid paper: clean; 1.6 Second chronograph.

[0077] Determination procedures: Measure 100 mL of 65℃ warm water into a 200 mL beaker (ensuring the water temperature is 65℃). Weigh 5.1 g of sample onto sulfuric acid paper. Slowly pour the weighed sample into the beaker. Use a glass rod to stir along the container wall at a constant speed of three revolutions per second (clockwise) for 30 seconds. Let it stand for 2 minutes. First, pour a small amount into a petri dish and observe for any flocculent material. Then, observe the liquid surface and bottom of the beaker for any clumps or residue adhering to the walls.

[0078] Determination of boiling (thermal stability) test: B. 1 Step Measure 200 mL of distilled water into a 250 mL beaker and heat it to 40℃~45℃. Accurately weigh 16.0 g of the sample and slowly add it while stirring at 800~1000 rpm / min until completely dissolved (approximately 5 min). Let it stand for hydration for 15 min. Pour the solution into a 250 mL Erlenmeyer flask and place the flask in a 92℃ water bath. When the solution in the flask reaches 90℃, maintain this temperature for 10 min, then place it in cooling water to stand. When the temperature cools to 25℃±1℃, slowly pour 2~3 mL into a petri dish to observe for any flocculent material. Gradually and slowly pour the upper layer of liquid into the petri dish for observation until all the liquid has been poured out, while simultaneously observing for any precipitate at the bottom of the Erlenmeyer flask.

[0079] B. 2 The results are determined as follows: If there is flocculent material or obvious sediment at the bottom of the conical flask, the heat resistance of this batch of raw materials is deemed to be non-compliant with quality requirements (obvious sediment refers to lumps or flocculent material at the bottom of the conical flask, or trace amounts of fine particles at the bottom of the conical flask, which indicates that the heat resistance of this batch of raw materials meets quality requirements).

[0080] Solubility: C. 1 Instruments and Equipment: 1.1 Electronic balance: sensitivity 0.01g; 1.2 Beakers: clean, dry inner walls, and intact surfaces without cracks; 1.3 Pipette; 1.4 Stirrer; 1.5 Centrifuge; 1.6 FT-120 Milk Component Analyzer; C. 2 Determination Procedure: 2.1 Dissolve 8g of whey protein powder in 200ml of water, stir at 600-800rpm for 10-15min until evenly dispersed. 2.2 Allow to hydrate at room temperature for 30min, then take 50ml and place it in a sample cup for later use. 2.3 Calibration of the FT120 Milk Component Analyzer Standard Curve: Prepare solutions with theoretical protein content values ​​of 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, and 3.4% using the protein powder, determine the protein content using the Kjeldahl method, and correct for it on the standard curve of skim milk. 2.4 Place the sample cup in the FT120 Milk Component Analyzer to determine the protein content in the solution, record the instrument test value, and record it as A. 2.5 Take 100g of the above solution, place it in a centrifuge tube, and centrifuge at 4000rpm for 20min. 2.6 Remove the centrifuge tube and use a pipette to take 50g of the supernatant, placing it in a sample cup for later use. 2.7 Place the sample cup into the FT120 milk component analyzer to determine the protein content in the solution, recording the instrument reading as B. 2.8 Calculate the solubility: Solubility = Protein content in supernatant (value B) / Protein content in original solution (value A).

[0081] Detection of centrifugal sedimentation rate: Instruments and equipment: 1.1 Electronic balance: sensitivity 0.001g; 1.2 Beaker: clean, dry inner wall, and intact surface without cracks; 1.3 Stirrer; 1.4 Centrifuge; 1.5 Microfiltration membrane: PES material organic spiral wound membrane of appropriate pore size; 1.6 Ultrafiltration membrane: PES material organic spiral wound membrane of appropriate pore size; Determination Procedure: 2.1 Dissolve 4g of whey protein powder in 80ml of water, stir at 600-800 rpm for 10-15 minutes until evenly dispersed. 2.2 Allow to hydrate at room temperature for 30 minutes. 2.3 Place 10mL of sample into a centrifuge tube and record the weight of the centrifuge tube (A) and the weight of the sample (B). 2.4 Centrifuge at 4000 rpm for 20 minutes. 2.5 Remove the centrifuge tube, slowly pour out the supernatant, invert the centrifuge tube onto filter paper, and let it stand for 10 minutes. 2.6 Weigh the centrifuge tube plus the precipitate and record the weight as C. 2.7 Calculate the centrifugation precipitation rate: Centrifugation precipitation rate = Precipitate weight (value C - value A) / Sample weight (value B).

[0082] Detection of bovine osteopontin: Instrument model: Waters XEVO TQ-XS; Determination Procedure: Weigh 1 g (accurate to 0.01 g) of sample and dissolve in ultrapure water to a final volume of 50 mL. Accurately transfer 200 μL of sample solution, add buffer solution, add 1 / 2 dithiothreitol, mix well, and react at 70 °C for 30 min. Remove and cool to room temperature, add iodoacetamide, mix well, and let stand in the dark for 30 min. Add trypsin solution, mix well, and incubate at 37 °C for at least 12 hours. After completion, remove and add formic acid to terminate the reaction. Add bovine osteopontin internal standard solution, vortex to mix, and filter through a 0.22 μM filter membrane for detection by liquid chromatography-mass spectrometry.

[0083] Preparation of the standard curve: Take 10 μL, 25 μL, 50 μL, 100 μL, 150 μL, and 200 μL of bovine osteopontin-specific peptide standard intermediate solution, respectively, and add the same volume (50 μL) of isotope-labeled specific peptide intermediate solution to each bottle. Dilute to 1 mL with 0.1% formic acid aqueous solution. The resulting standard working solutions in each bottle have concentrations of 10 nmol / L, 25 nmol / L, 50 nmol / L, 100 nmol / L, 150 nmol / L, and 200 nmol / L, respectively. Prepare the standard curve immediately before use. The linear correlation coefficient r should be no less than 0.995. Result: Y = 0.548889X + 0.113575, r 2 =0.9997.

[0084] Example 1 A method for separating active substances from dairy raw materials, comprising the following steps.

[0085] 1. Raw material pretreatment The natural membrane-separated whey protein solution was obtained as follows: skim milk was microfiltered using a 0.1-micron PES organic spiral wound membrane at a microfiltration temperature of 50°C; the transmembrane pressure difference was controlled at 0.3 bar, and the concentration factor was 3 times; the washing filtrate was RO water, and the washing filtrate was 1.2 times the volume of skim milk. The obtained permeate was the natural membrane-separated whey protein solution, and the casein detection result was not detected.

[0086] Natural membrane-separated whey protein solution was concentrated by 200 Da nanofiltration at 4-10°C to adjust the conductivity of the protein solution to 3.7 mS / cm. At this point, the pH was 6.6 and the total protein mass fraction in the solution was 0.9% (w / w).

[0087] 2. Anion exchange chromatography Natural membrane separation of whey protein solution was performed using anion exchange chromatography. The chromatographic column was named TA-Q-XL-6BB, and its framework consisted of 6% highly cross-linked agarose with dextran chains, with a particle size distribution ranging from 150 to 250 μm and an average particle size of 250 μm. The functional group of this column was a quaternary ammonium group (-N). + (CH3)3), with an ion loading of 230–330 μmol Cl. - The column is filled with medium at a density of approximately 30 nm, with a column height of 25 cm and a column diameter of 300 mm. The maximum pressure is 0.4 MPa, and the maximum flow rate is 900 cm / h.

[0088] The chromatography procedure is CIP-high salt regeneration-equilibrium-sample loading-washing after sample loading-impurity washing-elution, and the process flow is shown in Table 1.

[0089] Table 1 Chromatography process parameters

[0090] Obtain the flow-through solution, the washing component, and the elution component.

[0091] 3. Treatment of the flow-through fluid in step 2 The flow-through was ultrafiltered using a 50 kDa organic membrane at 10°C. It was first concentrated 20 times and then washed with RO water until the conductivity of the retentate was <1 mS / cm (in this embodiment, the conductivity after washing was 0.8 mS / cm). The permeate and retentate from the 50 kDa ultrafiltration were obtained.

[0092] 4. Process the ultrafiltration permeate from step 3. The permeate from the 50 kDa ultrafiltration was concentrated using a 10 kDa organic membrane ultrafiltration at 10°C. After a 20-fold concentration, the solution was washed with RO water until the conductivity of the retentate was <0.8 mS / cm (in this embodiment, the conductivity after washing was 0.6 mS / cm). Further concentration was then achieved until the solids content was >18% (w / w) (in this embodiment, the solids content after washing was 18.2%). Filtration was then performed using a 0.22-micron capsule filter (the entire feed solution passed through without washing).

[0093] 5. Drying Step 4: After spray drying, the material is used to obtain α-lactalbumin powder.

[0094] 6. Process the washing components from step 2. In step 2, the washed components were concentrated using a 200 Da organic membrane nanofiltration system at 10°C. After a 3-fold concentration, the filtrate was washed with RO water until the conductivity of the retentate was <1 mS / cm (in this embodiment, the conductivity after washing was 0.9 mS / cm). The solution was then concentrated until the solids content was >18% (w / w) (in this embodiment, the solids content after washing was 22%). Filtration was then performed using a 0.22-micron capsule filter (the entire feed solution passed through without washing).

[0095] 7. Drying The material from step 6 is spray-dried to obtain β-lactoglobulin powder.

[0096] 8. Process the elution fraction from step 2. The eluted fraction from step 2 was concentrated using a 10 kDa organic membrane ultrafiltration at 10°C. After being concentrated 30 times, the filtrate was washed with RO water until the conductivity of the retentate was <0.8 mS / cm (in this embodiment, the conductivity after washing was 0.5 mS / cm). The filtrate was then concentrated until the solids content was >18% (w / w) (in this embodiment, the solids content after washing was 18.3%).

[0097] 9. Drying The material from step 8 is spray-dried to obtain osteopontin powder.

[0098] Refer to the process flow diagram Figure 1 .

[0099] Experimental Example 1 The α-lactalbumin powder in Example 1 was tested, and the results are as follows.

[0100] Table 2. Detection results of α-lactalbumin powder in Example 1

[0101] Table 3. Detection results of OPN powder in Example 1

[0102] Table 4. Detection results of β-lactoglobulin powder in Example 1

[0103] Note: ALA content is the percentage of α-lactalbumin by mass relative to the total protein content. BLG content is the percentage of β-lactoglobulin by mass relative to the total protein content.

[0104] Example 2 Based on the method of Example 1, multiple experimental groups were set up. Group 1 was the same as in Example 1, while the difference between Groups 2 to 11 and Example 1 was in the pretreatment conditions, as shown in Table 5. Each group was repeated three times.

[0105] 1. Detect the purity and extraction rate of α-La, β-Lg and osteopontin.

[0106] The formula for calculating the extraction rate is: .

[0107] Table 5. Conditions and test results for each experimental group

[0108] Note: Total protein concentration is the mass fraction of total protein in the feed solution.

[0109] In Group 2, the raw material pretreatment of Example 1 was performed, and the conductivity of the natural membrane-separated whey protein solution was 1.8 mS / cm. Compared with Group 1, the process time increased by 3-4 times under the same flow rate and retention time, and the cost and the possibility of microbial contamination increased significantly.

[0110] 2. Detect the calcium binding amount of α-La in groups 1, 2, and 4.

[0111] Table 6. Calcium binding capacity of α-La

[0112] The α-La in groups 1, 2, and 4 are all characterized as native α-La, but their bound state Ca 2+ The calcium content showed significant differences between groups. Quantitative analysis indicated that the α-La prepared from group 4 had the highest calcium binding capacity, reaching 1.375 ± 0.007 mol Ca. 2+ / mol α-La; Group 2 had the lowest binding amount, only 0.836±0.003 mol Ca. 2+ / mol α-La; while the binding amount in group 1 is in the middle. Theoretically, when only the main binding site is specifically occupied, Ca 2+The molar ratio with α-La should be 1:1. Therefore, it can be inferred that the high calcium content in group 4 indicates that some of its secondary binding sites have already bound to Ca. 2+ Additional coordination occurred; the binding amount in group 1 was closer to the theoretical value of single-site binding; while group 2 showed obvious decalcification.

[0113] This significant difference in binding amounts most likely stems from the drastically different microenvironmental stress conditions in each purification process. For Group 1, the separation and purification process was consistently carried out under neutral and mild conditions, preserving the native conformation and initial metal coordination state of α-La to the greatest extent possible. Conversely, the preparation process for Group 4 required adjusting the pH of the loading solution to 5.7. In this slightly acidic environment, the spatial structure of α-La may undergo partial unfolding or conformational relaxation, exposing previously buried secondary binding sites, which then bind additionally with the participation of free calcium ions in the system. When the pH of the system is neutralized again after elution, the protein conformation renatures and re-contracts, thereby binding these additionally bound Ca2+ ions. 2+ It is firmly locked in the spatial network. The extremely low calcium content of Group 2 is likely due to the irreversible loss of naturally bound calcium ions caused by its long and complex processing.

[0114] 3. α-La in vitro simulation of infant gastrointestinal digestion This embodiment utilizes a standardized infant static in vitro digestive model (INFOGEST 2.0) to systematically track and compare the hydrolysis kinetics and peptide release patterns of high-purity α-La (groups 1, 2, and 4) from three different process sources during the simulated gastric and intestinal stages.

[0115] Figure 2 SDS-PAGE degradation profiles of α-La from three different purification processes (Groups 1, 2, and 4) during simulated in vitro gastrointestinal digestion in infants were presented. Gel electrophoresis results showed that during simulated gastric juice digestion (SGF), the characteristic bands of the three types of α-La gradually faded in color with increasing digestion time, but all exhibited significant digestive resistance overall. Even at the end of 60 min of gastric digestion, a large number of undegraded intact α-La molecules remained in the system. However, when the digestive fluid transitioned to simulated intestinal juice (SIF), the hydrolysis rate of α-La increased dramatically; after only 15 min of intestinal digestion, the intact α-La monomer bands had almost disappeared, indicating that they were rapidly degraded into low-molecular-weight peptides or free amino acids.

[0116] This characteristic of "resistance in the stomach and rapid digestion in the intestines" is mainly controlled by the specific gastrointestinal physiological environment of infants and the natural spatial conformation of α-La itself. On the one hand, the microenvironment pH of standard infant simulated gastric juice is usually set at around 5.3. This weakly acidic condition deviates from the optimal catalytic region of pepsin. In addition, the secretion concentration of pepsin in infants is much lower than that in adults, thus significantly weakening the enzymatic hydrolysis efficiency in the gastric digestion stage. On the other hand, α-La in its natural state maintains a highly compact globular folded conformation. Most of the specific cleavage sites of pepsin on its polypeptide chain are deeply buried inside the hydrophobic core, and the huge steric hindrance effectively shields the physical contact between digestive enzymes and substrates. It is worth noting that Ca 2+ The regulatory effect of coordination state on α-La digestion kinetics: Ca 2+ The specific binding of the protein can form an effective electrostatic shielding effect at the coordination center, weaken the electrostatic repulsion within the molecule, and cause the protein's tertiary conformation to contract, thereby further enhancing its physical resistance to protease hydrolysis.

[0117] During in vitro protein digestion, digestive enzymes such as pepsin and trypsin continuously cleave peptide bonds, releasing free primary amino groups. Therefore, by quantitatively measuring the increase in free amino groups relative to the total peptide bonds in the system, the degree of protein hydrolysis (DH) can be accurately calculated, thus macroscopically reflecting its digestibility.

[0118] The curves showing the change in the degree of hydrolysis of the three α-La samples (Group 1, Group 2, and Group 4) with digestion time are shown below. Figure 3 As shown, the degree of hydrolysis (DH) of all three samples showed a phased upward trend with increasing digestion time. In the initial stage of simulated gastric digestion (SGF) (0–5 min), the degree of hydrolysis showed a slight jump, followed by an extremely slow plateau between 5 and 60 min. When the system transitioned to simulated intestinal digestion (SIF) at 60 min, the degree of hydrolysis curve showed a significant and sharp increase. This characteristic of DH change perfectly corroborates the qualitative pattern of "resistance in the gastric stage and rapid hydrolysis in the intestinal stage" observed in the aforementioned SDS-PAGE degradation profile.

[0119] Although the three α-La groups showed a consistent overall trend across different digestion stages, their absolute degree of hydrolysis began to differentiate significantly upon entering the intestinal digestion stage. At the digestion endpoint (120 min), group 1 achieved the highest degree of hydrolysis (67.3%), followed by group 2 (56.4%), while group 4 exhibited the lowest degree of hydrolysis (only 49.2%). The underlying reason for this significant difference in hydrolysis degree can be traced back to the different processes that induced the Ca2+ hydrolysis. 2+ Combine state differences and the resulting tertiary conformational fine-tuning.

[0120] Example 3 Based on the method of Example 1, groups 12 and 13 are set up. Groups 12 and 13 are largely the same as those in Example 1, except that: No 0.22-micron filtration is performed in step 4 of group 12.

[0121] Group 13 omits the 50kDa ultrafiltration in step 3 and directly processes the flow-through liquid in step 4.

[0122] The α-lactalbumin levels in groups 1, 2, 4, 12, and 13 were detected, and the results are as follows.

[0123] Table 7. Detection results of α-lactalbumin powder

[0124] Note: ALA content is the percentage of α-lactalbumin by mass to total protein. BLG content is the percentage of β-lactoglobulin by mass to total protein. IgG content is the percentage of IgG by mass to total protein.

[0125] Example 4 Based on the method of Example 1, group 14 is set up, which is roughly the same as the method of Example 1, except that step 6 of group 14 omits the step of filtering with a 0.22-micron capsule filter.

[0126] The β-lactoglobulin powders in groups 1 and 14 were tested, and the results are as follows.

[0127] Table 8. Detection results of β-lactoglobulin powder

[0128] Comparative Example 1 A method for separating active substances from dairy raw materials, which is generally different from Example 1 in that the particle size of the chromatographic column packing is 100-200 μm, and other parameters are the same as in Example 1.

[0129] Using the same 300mm chromatography column as in Example 1, with a column height of 25cm, the linear flow rate was 700cm / h. The decrease in linear flow rate led to a corresponding increase in retention time, increasing the process time by 20%-25%. At the same time, due to the extended contact time between the feed solution and the packing material, incomplete flow of α-lactalbumin occurred, resulting in a decrease in the α-lactalbumin extraction rate to 70% and a decrease in the purity of β-lactoglobulin to 83%.

[0130] Comparative Example 2 A method for separating active substances from milk raw materials, largely the same as in Example 1, with the difference being: The column packing material is composed of 4% highly cross-linked agarose with dextran chains, and the pore size of the substrate is 70 nm. Other parameters of the column are the same as in Example 1.

[0131] Using the same 300mm chromatography column as in Example 1, with a column height of 25cm and a linear flow rate of 900cm / h, the column bed pressure resistance of 2.5Bar caused the linear flow rate of 900cm / h to be unsustainable in the later stages of sample loading. The linear flow rate needed to be reduced to below 600cm / h. The reduction in linear flow rate led to a corresponding increase in retention time, increasing the process time by 20%-25%. Example 1 had a pressure resistance of 4bar, and at the same time, the purity of osteopontin decreased to 65%, and the extraction rate decreased to 58%.

[0132] Comparative Example 3 A method for separating active substances from milk raw materials, largely the same as in Example 1, with the difference being: The washing solution was a 350 mmol / L NaCl solution.

[0133] The purity of β-lactoglobulin decreased to 80%, and the extraction rate decreased to 75.6%; the purity of osteopontin decreased to 72%. The purity of α-lactalbumin did not change significantly.

[0134] Comparative Example 4 A method for separating active substances from milk raw materials, largely the same as in Example 1, with the difference being: The washing solution was a 450 mmol / L NaCl solution.

[0135] The extraction rate of osteopontin decreased to 77.3%, while the purity of α-lactalbumin and β-lactoglobulin remained largely unchanged.

[0136] Comparative Example 5 A method for separating active substances from milk raw materials, largely the same as in Example 1, with the difference being: The eluent was a 700 mmol / L NaCl solution.

[0137] The extraction rate of osteopontin decreased to 64.2%, while the purity of α-lactalbumin and β-lactoglobulin remained largely unchanged.

[0138] Comparative Example 6 Based on the method of Example 1, multiple experimental groups were set up, which were roughly the same as those in Example 1, except that the anion exchange chromatography packing was different, while the parameters of the chromatography column were the same as in Example 1.

[0139] Q Sepharose Big Beads consist of large-particle-size cross-linked agarose microspheres (150~200 µm) with trimethylammonium (Q) strong anion exchange groups bonded to their surface.

[0140] Q Sepharose Fast Flow consists of a cross-linked 6% agarose framework and quaternary ammonium (Q) strong anion exchange groups, with an average particle size of 90 μm and a particle size range of 45–165 μm.

[0141] Q Purose 6 XL is an agarose-based strong anion exchange medium. The base spheres are dextran grafted with 6% highly cross-linked agarose, and the ligands are -O-CH2CHOHCH2N. + (CH3)3, with an average particle size of 90 μm.

[0142] Hedera Q BB is an agarose-based strong anion exchange medium. The base spheres are made of dextran grafted with 6% highly cross-linked agarose, and the ligands are quaternary ammonium groups. The average particle size is 200 μm.

[0143] Q Singarose BB is an agarose-based strong anion exchange medium. The base spheres are dextran-grafted with 6% highly cross-linked agarose, and the ligands are quaternary ammonium groups. The average particle size is 200 μm, and the ion loading is 180-250 μmol Cl. - / mL.

[0144] The packing information and its corresponding test results are shown in Table 9.

[0145] Table 9. Chromatography packing materials and their test results

[0146] Note: Processing capacity (%) = (V 实施例1 -V 对照 ) / V 实施例1 V represents the volume of raw material liquid processed in a single batch.

[0147] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for separating active substances from dairy raw materials, characterized in that, It includes the following steps: The whey protein solution is pretreated, the pretreatment comprising: adjusting the conductivity to a first set threshold and the pH to a second set threshold; the first set threshold being 2~10 mS / cm and the second set threshold being 6~7.2; adjusting the conductivity and pH by nanofiltration, wherein the molecular cutoff of the nanofiltration membrane is 100~300 Da; and the temperature for adjusting the conductivity and pH is 0~10℃. The pretreated protein solution was subjected to anion exchange chromatography to obtain a flow-through containing α-lactalbumin, a wash fraction containing β-lactoglobulin, and an elution fraction containing osteopontin. The anion exchange chromatography is a strong anion exchange chromatography, and the steps of the anion exchange chromatography include washing and elution; the washing solution used for washing includes 0.2~0.6 M NaCl solution; the elution solution used for elution includes 0.6~1 M NaCl solution; the chromatography column packing material of the anion exchange chromatography is cross-linked agarose with glucose chains, the degree of cross-linking is 4%~8%, the functional group is quaternary ammonium group, and the average particle size is 200~300 μm.

2. The method according to claim 1, characterized in that, The first set threshold is 3~6 mS / cm, and the second set threshold is 6.4~7.0; And / or, the pretreatment further includes adjusting the total protein concentration of the whey protein solution to a third set threshold, the third set threshold being 0.5% to 2% (w / w).

3. The method according to claim 1, characterized in that, The amount of the washing solution used is 5 to 15 times the column volume; The amount of the eluent used is 2 to 10 times the column volume; The particle size distribution of the chromatography column packing material for the anion exchange chromatography is 150~350 μm, and the pore size of the substrate spheres is 10~50 nm. And / or, the column height of the anion exchange chromatography is 20-30 cm and the column diameter is 250-350 mm.

4. The method according to claim 1, characterized in that, The anion exchange chromatography procedure includes: CIP-high salt regeneration-equilibration-sample loading-washing after sample loading-impurity washing-elution; The CIP process includes cleaning the chromatography column with 0.4-0.6 M NaOH solution. In the CIP step, the volume of 0.4–0.6 M NaOH solution used is 1–10 times the column volume; The high-salt regeneration includes washing the chromatography column with 0.4~0.6 M NaCl solution; In the high-salt regeneration step, the amount of 0.4~0.6 M NaCl solution used is 1~10 times the column volume; The equilibration solution used includes RO water, and the amount used is 5 to 20 times the column volume; The elution solution used after sample loading includes RO water, and the amount used is 1 to 10 times the column volume.

5. The method according to any one of claims 1 to 4, characterized in that, The method further includes: The flow-through liquid is concentrated by ultrafiltration to obtain a first permeate and a first retentate; The first permeate is concentrated by ultrafiltration and then microfiltration to obtain the second retentate. The filter membrane used for ultrafiltration concentration of the flow-through liquid has a molecular cutoff of 40~60kDa and a concentration factor of 10~25 times. The washing filtrate includes RO water and is washed until the conductivity of the retentate is <1ms / cm. The membrane used for ultrafiltration concentration of the first permeate has a molecular cutoff of 3~15kDa and a concentration factor of 10~25 times. The washing filtrate includes RO water. The washing is carried out until the conductivity of the retentate is <0.8 ms / cm and the concentration is carried out until the solids content is >18%. The pore size of the microfiltration membrane is 0.1~10μm.

6. The method according to claim 5, characterized in that, The method further includes drying the second retentate.

7. The method according to any one of claims 1 to 4, characterized in that, The method further includes: concentrating the β-lactoglobulin-containing washed fraction using nanofiltration, microfiltration, and / or drying; wherein the nanofiltration membrane has a molecular cutoff of 200-500 Da and a concentration factor of 1-5 times; the washing filtrate includes RO water; the washing is carried out until the conductivity of the retentate is <3 mS / cm and the solids content is >18%; and the microfiltration membrane has a pore size of 0.1-10 μm. And / or, the method further includes: ultrafiltration concentration and / or drying of the osteopontin-containing eluted fraction, wherein the ultrafiltration membrane has a molecular cutoff of 3-15 kDa, a concentration factor of 20-40 times, the washing filtrate includes RO water, the washing is carried out until the conductivity of the retentate is <0.8 ms / cm, and the concentration is carried out until the solids content is >18%.

8. The method according to any one of claims 1 to 4, characterized in that, The whey protein solution includes natural membrane-separated whey protein solution; The preparation method of the natural membrane-separated whey protein solution includes: microfiltration of skim milk, and the permeate obtained is the natural membrane-separated whey protein solution; The pore size of the microfiltration membrane is 0.05~0.2 μm; The temperature of the microfiltration is 40~55℃; And / or, the active substance includes at least one of α-lactalbumin, β-lactoglobulin and osteopontin; The casein content of the natural membrane-separated whey protein solution is ≤5% (w / w) of the total protein.

9. A formulation rich in α-lactalbumin, characterized in that, The α-lactalbumin constitutes more than 95% of the total protein; the α-lactalbumin is bound to calcium ions, and the amount of calcium ions bound to the α-lactalbumin is 1~1.4 mol Ca². + / mol α-La; the formulation contains no IgG or the mass of IgG is less than 0.1% of the total protein; The preparation method of the formulation includes the following steps: The whey protein solution is pretreated, wherein the pretreatment step is the pretreatment described in claim 1 or 2; The pretreated protein solution was subjected to anion exchange chromatography to obtain a formulation containing α-lactalbumin.

10. The formulation according to claim 9, characterized in that, The formulation satisfies at least one of the following: (1) The preparation does not contain perchlorate and chlorate, and the nitrate content is <5mg / 100g and the nitrite content is <1mg / 100g; (2) The ash content of the preparation is <1.2 g / 100g; (3) The preparation contains β-lactoglobulin, and the mass of β-lactoglobulin accounts for less than 1% of the total protein.

11. The formulation according to claim 9 or 10, characterized in that, The preparation method of the formulation adopts the method described in claim 1, 2, 3, 5 or 6.

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