Chromatography medium, preparation method thereof and application of chromatography medium in extraction of alpha-lactalbumin

Abstract

The invention belongs to the technical field of protein purification, and particularly relates to a chromatography medium, a preparation method of the chromatography medium and application of the chromatography medium to extraction of alpha-lactalbumin. The chromatography medium provided by the invention takes agarose as a matrix, and the surface of the chromatography medium is modified by a ligand through a glucan spacer arm; wherein the molecular weight of the glucan is 20 kDa to 500 kDa, the ligand comprises propylene diamine, and the density of the ligand in the chromatography medium is 200 [mu] mol / mL to 300 [mu] mol / mL. The invention also provides a preparation method of the material, and the material can be obtained by sequentially carrying out epoxy activation, glucan modification, secondary activation and ligand bonding on the matrix. When the chromatography medium provided by the invention is used for extracting alpha-lactalbumin from whey, the chromatography medium has the advantages of high loading capacity, high loading flow rate and efficient regeneration, and meets the requirements of industrial production.

Description

Technical Field

[0001] This invention belongs to the field of protein purification technology, specifically relating to chromatography media, their preparation methods, and their use in the extraction of α-lactalbumin. Background Technology

[0002] Alpha-lactalbumin (ALB) is a natural whey protein composed of 123 amino acids found in mammalian milk, playing important biological functions in infant growth, immune regulation, and nervous system development. Studies have shown that ALB in cow's milk and human milk has similar molecular weights and an amino acid sequence homology of approximately 74%, indicating similar functions. Based on this high degree of structural and functional conservation, incorporating ALB derived from cow's milk into infant formula is an effective strategy to make its composition closer to that of breast milk. Therefore, developing efficient and high-purity ALB separation and purification processes is crucial for improving the quality of infant formula, narrowing the gap with breast milk, and enhancing its market competitiveness.

[0003] Existing extraction techniques for α-lactalbumin include non-chromatographic and chromatographic methods.

[0004] Reference 1 discloses a method for extracting α-lactalbumin from fresh milk via thermal agglomeration combined with rennet treatment. By optimizing parameters, whey rich in α-lactalbumin is obtained, and the concentration ratio of α-lactalbumin to β-lactoglobulin after purification is 3:1. The main process flow of this technology is as follows: Figure 4 As shown, the specific implementation method is as follows: (1) Defatting: The raw milk is centrifuged by a defatting machine to obtain defatted milk; (2) Heat treatment: The defatted milk is heated in a water bath and then quickly placed in ice water to cool to room temperature. This step utilizes the difference in denaturation rate between β-lactoglobulin and α-lactalbumin under specific conditions during the heat denaturation process. β-lactoglobulin has a higher aggregation rate with casein, so some β-lactoglobulin can be removed; (3) Enzymatic coagulation: 0.01% of rennet with an activity of 20000 U / g and 0.1% of calcium chloride are added to the heat-treated defatted milk. After enzyme coagulation for 30 minutes, the whey and curd are separated by gauze filtration.

[0005] Reference 2 discloses a method for extracting α-lactalbumin via membrane-filtered whey acidification followed by cation exchange chromatography, achieving a purity of up to 90% for α-lactalbumin. The main process flow of this technology is as follows: Figure 5As shown, the specific implementation method is as follows: (1) Whey protein and casein are separated by spiral wound membrane microfiltration. By optimizing the conditions, the casein content in the whey protein solution of the vegetation is extremely low and the clarity is high. Moreover, the shear force generated by the microfiltration operation will not cause significant damage to the protein activity; (2) The obtained membrane-filtered whey is further precipitated by acetic acid at pH 4.5 to remove some residual casein from the whey; (3) Clarify by deep filter; (4) Obtain high-purity α-lactalbumin solution by cation exchange chromatography; (5) Use a 10KD ultrafiltration membrane for ultrafiltration and liquid replacement, spray dry and store.

[0006] With the increasing demands for product purity, activity, recovery rate, and continuous production from the biopharmaceutical and functional food industries, traditional non-chromatographic separation techniques have been gradually phased out. Their main drawbacks include: difficulty in meeting product quality standards; poor batch-to-batch consistency; low recovery and yield rates; and difficulty in achieving continuous production. In contrast, chromatography, with its high resolution, scalability, and GMP compatibility, has become the mainstream technology for the purification of proteins and other macromolecules. Chromatographic packing material is the core of the chromatographic process, and its performance directly determines the efficiency and cost of the process.

[0007] Currently, common chromatography packing materials on the market include agarose, dextran, synthetic polymers, and hydroxyapatite. Representative matrices for agarose chromatography include Sepharose and Magose, whose main advantages are good biocompatibility, adjustable pore size, and high loading capacity. However, they have limitations such as low pressure resistance and susceptibility to collapse at high flow rates. Representative matrices for dextran chromatography include Sephadex and DEAE Sephadex, whose main advantages are that they are natural polysaccharides, allowing for flexible chemical modification and moderate cost. However, they have limitations such as extremely poor pressure resistance, wide pore size distribution, and susceptibility to enzyme degradation. Representative matrices for synthetic polymer chromatography include polyacrylates, polystyrene, and polyacrylamide (such as TSKgel), whose main advantages are high pressure resistance, fast flow rate, and uniform pore size, making them suitable for large-scale continuous operations. However, they have limitations such as slightly poor biocompatibility and some effects on protein activity. Representative matrices for hydroxyapatite chromatography packing materials include BARONHAP and HAP type I / II. Their main advantages are that they are inorganic materials, resistant to acids and alkalis, specifically bind to acidic proteins or nucleic acids, and have adjustable loading capacity. However, they have limitations such as high particle hardness, high column pressure, high flow rate requirements, relatively high cost, and immature technology.

[0008] In addition, chromatography modes mainly include several categories such as gel filtration chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and mixed-mode chromatography. Gel filtration chromatography separates molecules based on their different migration rates within the pores of the packing material due to differences in molecular size; ion exchange chromatography relies on the electrostatic adsorption differences between proteins and charged groups on the packing material to achieve separation; hydrophobic chromatography utilizes the hydrophobic interaction between hydrophobic ligands and the target analyte, achieving elution by changing the salt concentration; affinity chromatography achieves highly selective purification based on the reversible binding of specific ligands on the packing material to biomolecules.

[0009] Hybrid chromatography combines two or more separation mechanisms (such as ion exchange and hydrophobic interactions, affinity and metal ligands) into the same packing material via covalent or copolymerization, forming a novel "one-column-multiple-effects" chromatography mode. Its advantages include: simplified process flow, allowing multiple purification steps to be completed in a single column packing, reducing equipment investment and operation time; improved resolution, with multiple mechanisms enhancing the ability to distinguish structurally similar proteins; reduced overall process cost by approximately 15%-30%, decreasing the number of column replacements and eluent types; and easy integration with continuous flow systems (such as SMB and MCSGP), supporting continuous production throughout the entire process.

[0010] Although chromatography has become the mainstream technology for purifying proteins and other macromolecules due to its various advantages, it still cannot meet the ever-increasing production demands. For example: 1) Traditional agarose / dextran packing materials have insufficient pressure resistance and cannot withstand high flow rates, making it difficult to meet the requirements of industrial production for column pressure (≥3MPa) and high flow rate (≥100 BV / h); 2) Synthetic polymer packing materials have non-specific adsorption problems, affecting the purity and recovery rate of the target product; 3) Hydroxyapatite packing materials are difficult to pack, have high hardness leading to increased column pressure, and are also costly, with insufficient technological maturity; 4) Single-principle chromatography media such as ion exchange or affinity packing materials can only achieve one separation mode and cannot complete multiple steps such as capture, desalting, and purification in a single operation.

[0011] References:

[0012] Reference 1: Yan Xudong, Wang Caiyun, Yun Zhanyou, et al. Study on extraction process of α-lactalbumin and evaluation of functional properties of by-products [J]. Science and Technology of Food Industry, 2011, 32(06):253-256. DOI:10.13386 / j.issn1002-0306.2011.06.045.

[0013] Reference 2: CN119320445A. Summary of the Invention

[0014] The problem the invention aims to solve

[0015] Although there are chromatographic extraction methods for α-lactalbumin, such as the one cited in reference 2 above, such research is still insufficient, and there is still a widespread need for efficient extraction of α-lactalbumin.

[0016] This invention, through extensive research, provides a chromatography medium for separating α-lactalbumin from whey and its preparation method. This medium can perform mixed-mode chromatography, combining two separation mechanisms: ion exchange and hydrophobic interaction. Furthermore, this medium has the advantages of high pressure resistance, wide pH range, and strong specific binding ability, which can meet the industrial purification needs of α-lactalbumin and effectively solve the shortcomings of existing media in terms of pressure resistance, multifunctional integration, and non-specific adsorption.

[0017] Solution for solving the problem

[0018] [1]. A chromatography medium for separating α-lactalbumin from whey, wherein the chromatography medium is based on agarose and the surface of the matrix is ​​modified with ligands via dextran spacer arms;

[0019] The dextran has a molecular weight of 20kDa-500kDa, and the ligand includes propylenediamine.

[0020] Furthermore, in the chromatography medium, the density of the ligand is 200 μmol / mL to 300 μmol / mL.

[0021] [2]. According to the chromatography medium described in [1], wherein the matrix is ​​porous spherical and the particle size of the spherical is 100μm-200μm.

[0022] [3]. According to the chromatography medium described in [1] or [2], wherein the matrix is ​​activated by a first activating reagent and then linked to the dextran; the dextran is activated by a second activating reagent and then linked to the ligand;

[0023] The first activating agent includes an epoxy compound; the second activating agent includes a halogenated compound with a double bond structure or an epoxy compound, and a brominating agent.

[0024] [4]. According to the chromatography medium described in [3], wherein the epoxy compound includes epichlorohydrin or 1,4-butanediol diglycidyl ether; the halogenated compound containing a double bond structure includes allyl halogenated hydrocarbons; and the brominating reagent includes N-bromosuccinimide.

[0025] [5]. A method for preparing a chromatography medium according to any one of [1]-[4], wherein the preparation method comprises the following steps:

[0026] The first activation step is as follows: the matrix is ​​mixed with water, a first activation reagent is added, and the matrix is ​​activated under alkaline conditions to obtain a first activated matrix;

[0027] The steps of dextran spacer modification are as follows: the first activated matrix is ​​mixed with a dextran aqueous solution, and the first activated matrix is ​​modified with dextran spacer under alkaline conditions to obtain a dextran spacer modified matrix.

[0028] The second activation step involves reacting the dextran spacer arm modified matrix with a second activation reagent to obtain a brominated dextran spacer arm modified matrix.

[0029] The steps of ligand modification are as follows: under alkaline conditions, the ligand is reacted with the brominated dextran spacer arm modified matrix in an aqueous solution to obtain the chromatography medium;

[0030] The first activating agent includes an epoxy compound; the second activating agent includes a halogenated compound containing a double bond or an epoxy compound, and a brominating agent.

[0031] [6]. According to the preparation method described in [5], in the second activation step, the dextran spacer-modified matrix is ​​first reacted with the halogenated compound containing a double bond structure or an epoxy compound in an aqueous solution under alkaline conditions, optionally in the presence of a stabilizer, and then the product is further reacted with a bromide reagent in an organic solution.

[0032] [7]. The preparation method according to [5] or [6], wherein the epoxy compound includes epichlorohydrin or 1,4-butanediol diglycidyl ether.

[0033] [8]. The preparation method according to any one of [5]-[7], wherein the halogenated compound containing a double bond structure includes allyl halogenated hydrocarbons; and the brominating agent includes N-bromosuccinimide.

[0034] [9]. The preparation method according to any one of [5]-[8], wherein the alkaline conditions are formed using sodium hydroxide.

[0035]

[10] . A method for extracting α-lactalbumin from whey, wherein the method comprises the following steps:

[0036] The steps of chromatography are as follows: the chromatography medium prepared according to any one of [1]-[4] or the preparation method according to any one of [5]-[9] is packed into the chromatography column, the whey is loaded into the chromatography column, and the flow-through is collected to obtain α-lactalbumin.

[0037] The effects of the invention

[0038] This invention provides a chromatography medium with unique ligands that can fully utilize ion exchange and hydrophobic interactions to adsorb proteins in whey, maximizing the binding of β-lactoglobulin, casein, immunoglobulins, etc., thus facilitating the easy separation of the high-purity target protein α-lactalbumin. When used for extracting α-lactalbumin from whey, it offers advantages such as high loading capacity, ability to handle high flow rates, and efficient regeneration, meeting the needs of industrial production. Furthermore, this invention also provides a method for preparing the above-mentioned chromatography medium. This method uses agarose as a matrix, sequentially subjecting it to epoxy activation, dextran spacer modification, secondary activation, and ligand coupling. The overall process is not complex and is suitable for large-scale industrial production.

[0039] Furthermore, this invention provides a method for extracting α-lactalbumin based on the aforementioned chromatographic medium. Whey is subjected to chromatography, and β-lactoglobulin, casein, immunoglobulins, etc., are bound to the medium, allowing high-purity α-lactalbumin to be obtained through sample collection and flow-through. This method has a large sample throughput (up to 100-130 CV), and due to the reasonable microsphere size distribution, it can also support ultra-high flow rate sample loading (linear flow rate up to 600 cm / h), meeting the needs of industrial production. Simultaneously, the purification process temperature is 10-20℃, and the mobile phase involved includes RO water, sodium chloride solution dissolved in RO water, and sodium hydroxide solution. The components are simple, with no potentially problematic impurities introduced, and the whey does not require high-temperature or acid treatment, thus preserving the natural active functions of whey protein to a great extent. This method can overcome the commercial production difficulties of existing processes and accelerate the commercial production process of α-lactalbumin. Attached Figure Description

[0040] Figure 1A SDS-PAGE results of purifying α-lactalbumin from whey using the media prepared in Examples 1-3 and the comparative examples in Experiment 1.

[0041] Figure 1B HPLC results of purifying α-lactalbumin from whey using the medium prepared in Example 1.

[0042] Figure 2 The results of microscopic observation of the media prepared in Examples 1-3 and the comparative example in Experiment 2.

[0043] Figure 3 The results of microbial residue detection of the media prepared in Examples 1-3 and the comparative example in Experimental Example 3.

[0044] Figure 4 The background section cites a flowchart of the extraction process for α-lactalbumin disclosed in reference 1.

[0045] Figure 5 The background section cites a flowchart of the extraction process for α-lactalbumin disclosed in reference 2.

[0046] Figure 6 The present invention provides a process flow diagram for the extraction of α-lactalbumin. Detailed Implementation

[0047] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0048] In this invention, the terms "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to the described embodiment that are included in at least one embodiment described herein and may or may not exist in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0049] In this invention, the numerical range expressed as "value A ~ value B" or "value A - value B" refers to the range that includes the endpoints values ​​A and B. In this invention, the numerical range expressed as "above value A" and "below value A" includes value A.

[0050] In this invention, the word "may" has both the meaning of performing a certain process and the meaning of not performing a certain process. In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the case where the event occurs and the case where the event does not occur.

[0051] In this invention, the term "about" can mean that a value includes the standard deviation of the error of the apparatus or method used to determine that value. The numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains a standard deviation due to the aforementioned testing apparatus or method. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this invention are modified with "about". Here, "about" generally means that the actual value is within ±5%, ±1%, or ±0.5% of a particular value or range.

[0052] In this invention, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that comprises a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such processes, methods, products, or apparatus.

[0053] In this invention, the term "normal temperature" or "room temperature" refers to an indoor ambient temperature of "23±2℃".

[0054] In this invention, "animal milk" refers to the liquid obtained from the mammary glands of a mammal in the process of lactation. The term "animal milk" should be interpreted broadly and encompasses both raw milk (i.e., the liquid obtained directly from the mammary glands) and standardized dairy products (e.g., whole milk).

[0055] Unless otherwise defined, other technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0056] I. Chromatography media

[0057] This invention provides a chromatography medium for separating α-lactalbumin from whey, the chromatography medium using agarose as a matrix, the surface of which is modified with ligands via dextran spacer arms; wherein the molecular weight of the dextran is 20kDa-500kDa, the ligands include propylenediamine, and the density of the ligands in the chromatography medium is 200μmol / mL-300μmol / mL.

[0058] In some embodiments, the ligand is propylenediamine, which has the following structure:

[0059] .

[0060] In some embodiments, the whey is derived from animal milk, such as cow's milk, sheep's milk, camel's milk, etc., preferably cow's milk. The present invention does not particularly limit the method of whey preparation; for example, animal milk can be defatted first, and then casein in the defatted milk can be removed by acidification followed by solid-liquid separation or microfiltration to obtain whey.

[0061] In some embodiments, the matrix is ​​porous spherical with a particle size of 100 μm-200 μm; preferably 150 μm-200 μm. Further, in some preferred embodiments, the matrix is ​​agarose porous microspheres with a surface rich in hydroxyl groups.

[0062] In some embodiments, the molecular weight of the dextran is 20kDa-500kDa; preferably 100kDa-500kDa.

[0063] In some embodiments, the matrix is ​​activated by a first activating agent and then linked to the dextran; the dextran is activated by a second activating agent and then linked to the ligand; wherein the first activating agent comprises an epoxy compound; the second activating agent comprises a halogenated compound containing a double bond or an epoxy compound, and a brominating agent. Further, in some embodiments, the epoxy compound comprises epichlorohydrin or 1,4-butanediol diglycidyl ether; the halogenated compound containing a double bond comprises an allyl halide; and the brominating agent comprises N-bromosuccinimide.

[0064] The chromatography medium of this invention, due to its unique ligand structure, can fully utilize ion exchange and hydrophobic interactions to adsorb proteins in whey, and can bind β-lactoglobulin, casein, immunoglobulins, etc. to the maximum extent, so that the high-purity target protein α-lactalbumin can be easily separated.

[0065] II. Preparation method of chromatography media

[0066] The present invention provides a method for preparing the above-mentioned chromatography medium, comprising the steps of first activating the matrix to obtain a first activated matrix, modifying the first activated matrix with a dextran spacer arm to obtain a dextran spacer arm modified matrix, second activating the dextran spacer arm modified matrix to obtain a brominated dextran spacer arm modified matrix, and bonding the brominated dextran spacer arm modified matrix with a ligand.

[0067] (The first activation step)

[0068] In some embodiments, the first activation step includes the following operations: mixing the matrix with water, adding a first activation reagent, and performing a first activation on the matrix under alkaline conditions to obtain a first activated matrix.

[0069] In some embodiments, the first activating agent comprises an epoxy compound; the epoxy compound comprises epichlorohydrin or 1,4-butanediol diglycidyl ether; preferably epichlorohydrin.

[0070] In some embodiments, the alkaline conditions are formed using sodium hydroxide; preferably, 10 g of 50% (w / v) sodium hydroxide is used per 100 mL of matrix.

[0071] In some specific implementations, the first activation step includes the following operations: mixing the matrix with water, adding an aqueous solution of an epoxy compound and an alkaline substance, and performing epoxy activation on the matrix to obtain a first activated matrix.

[0072] In some specific embodiments, the first activation step includes the following operations: mixing the matrix with water, adding epichlorohydrin, stirring, and then adding an aqueous sodium hydroxide solution to perform epoxy activation on the matrix to obtain a first activated matrix.

[0073] In some more specific embodiments, the first activation step includes the following operations: mixing the matrix with water, adding epichlorohydrin, stirring at 30±1℃ and 200±50 rpm for 5 min, then adding an aqueous sodium hydroxide solution, reacting for 1-2 h, and epoxy activating the matrix to obtain the first activated matrix.

[0074] In some embodiments, the volume ratio of the matrix to the mass of the epoxy compound is 100 mL: 10 g.

[0075] In some embodiments, after epoxy activation of the matrix, a step of washing it with deionized water is also included.

[0076] (Steps for modifying the dextran spacer arms)

[0077] In some embodiments, the dextran spacer arm modification step includes the following operations: mixing the first activated matrix with a dextran aqueous solution, and modifying the first activated matrix with dextran spacer arms under alkaline conditions to obtain a dextran spacer arm modified matrix.

[0078] In some embodiments, the molecular weight of the dextran is 20kDa-500kDa; preferably 100kDa-500kDa.

[0079] In some embodiments, the alkaline conditions are formed using sodium hydroxide; preferably, 10 g of sodium hydroxide is used for every 100 mL of matrix.

[0080] In some specific embodiments, the dextran spacer arm modification step includes the following operations: mixing the first activated matrix with a dextran aqueous solution, adding an alkaline substance, and modifying the first activated matrix with dextran spacer arms to obtain a dextran spacer arm modified matrix.

[0081] In some specific embodiments, the dextran spacer arm modification step includes the following operations: adding the first activated matrix to a dextran aqueous solution, then adding sodium hydroxide to modify the first activated matrix with dextran spacer arms, thereby obtaining a dextran spacer arm modified matrix.

[0082] In some more specific embodiments, the dextran spacer arm modification step includes the following operations: adding the first activated matrix to a dextran aqueous solution, then adding sodium hydroxide, reacting at 30±1℃ and 300±50rpm for 18-20h to modify the first activated matrix with dextran spacer arms, thereby obtaining a dextran spacer arm modified matrix.

[0083] In some embodiments, the volume ratio of the matrix to the mass of the dextran is 100 mL: 4 g.

[0084] In some embodiments, the dextran aqueous solution contains 4 g / 100 mL of dextran.

[0085] In some embodiments, the method for obtaining the dextran aqueous solution is as follows: weigh the dextran, add water, and stir at room temperature to obtain the solution.

[0086] In some embodiments, after modifying the first activated matrix with dextran spacer arms, the step of washing it with pure water is further included.

[0087] (Second activation step)

[0088] In some embodiments, the second activation step includes reacting the dextran spacer-modified matrix with a second activating agent to obtain a brominated dextran spacer-modified matrix.

[0089] In some embodiments, the second activating agent comprises a halogenated compound containing a double bond or an epoxy compound, and a brominating agent; the halogenated compound containing a double bond comprises an allyl halide, preferably allyl bromide; the epoxy compound comprises epichlorohydrin or 1,4-butanediol diglycidyl ether, preferably epichlorohydrin; and the brominating agent comprises N-bromosuccinimide.

[0090] In some embodiments, the second activating agent includes a halogenated compound containing a double bond and a brominating agent.

[0091] In some specific embodiments, in the second activation step, the dextran spacer-modified matrix is ​​first reacted with the halogenated compound containing a double bond or an epoxy compound in an aqueous solution under alkaline conditions, optionally in the presence of a stabilizer, and then the product is further reacted with a brominizing agent in an organic solution.

[0092] In some embodiments, the alkaline conditions are formed using sodium hydroxide; preferably, 100 mL of 1M sodium hydroxide is used for every 100 mL of matrix.

[0093] In some embodiments, the stabilizer includes sodium sulfate and / or sodium borohydride.

[0094] In some specific embodiments, the second activation step includes the following operations: mixing the dextran spacer arm modified matrix with an aqueous solution of an alkaline substance, adding a stabilizer, and then adding an epoxy compound or an allyl halide to activate the dextran spacer arm modified matrix, thereby obtaining an activated dextran spacer arm modified matrix; then mixing the activated dextran spacer arm modified matrix with a brominating reagent and an organic solution to perform a bromination reaction on the activated dextran spacer arm modified matrix, thereby obtaining a brominated dextran spacer arm modified matrix.

[0095] In some specific embodiments, the second activation step includes the following operations: adding sodium hydroxide aqueous solution, sodium sulfate and sodium borohydride sequentially to the dextran spacer arm modified matrix, stirring, and then adding allyl bromide to activate the dextran spacer arm modified matrix to obtain an activated dextran spacer arm modified matrix; adding N-bromosuccinimide and acetone aqueous solution to the activated dextran spacer arm modified matrix, reacting and filtering to obtain a brominated dextran spacer arm modified matrix.

[0096] In some more specific embodiments, the second activation step includes the following operations: adding sodium hydroxide aqueous solution, sodium sulfate and sodium borohydride sequentially to the dextran spacer arm modified matrix, stirring at 50±1℃ and 180±20rpm for 1-2h, then adding allyl bromide, reacting at 50±1℃ and 180-200rpm for 6-8h to activate the dextran spacer arm modified matrix, obtaining an activated dextran spacer arm modified matrix, adding N-bromosuccinimide and acetone aqueous solution to the activated dextran spacer arm modified matrix, reacting at 25±1℃ and 150±30rpm for 3-4h, filtering to obtain a brominated dextran spacer arm modified matrix.

[0097] In some embodiments, the volume ratio of the matrix to the mass of the sodium sulfate is 100 mL: 10 g.

[0098] In some embodiments, the volume ratio of the matrix to the mass of the sodium borohydride is 100 mL: 0.4 g.

[0099] In some embodiments, the volume ratio of the matrix to the allyl bromide is 100 mL: 40 mL.

[0100] In some embodiments, after activating the dextran spacer arm modified matrix, a step of washing it with pure water is also included.

[0101] In some embodiments, the volume ratio of the matrix to the mass of the brominated reagent is 100 mL: 30 g.

[0102] In some embodiments, the volume ratio of the matrix to the acetone aqueous solution is 100 mL:150 mL; preferably, the concentration of the acetone aqueous solution is 50% (v / v).

[0103] In some embodiments, after brominating the activated dextran spacer-modified matrix, a step of washing it with deionized water is further included.

[0104] (Steps for ligand modification)

[0105] In some embodiments, the ligand modification step includes reacting the ligand with the brominated dextran spacer arm modified matrix in an aqueous solution under alkaline conditions to obtain the chromatography medium.

[0106] In some embodiments, the alkaline conditions are formed using sodium hydroxide; preferably, 100 L of 1 M sodium hydroxide is used for every 100 mL of matrix.

[0107] In some specific embodiments, the ligand modification step includes the following operations: dissolving the ligand in an aqueous alkaline solution, adding the brominated dextran spacer arm to modify the matrix, and obtaining the chromatography medium.

[0108] In some specific embodiments, the ligand modification step includes the following operations: dissolving propylenediamine in an aqueous sodium hydroxide solution, adding the brominated dextran spacer arm to modify the matrix, and reacting to obtain the chromatography medium.

[0109] In some more specific embodiments, the ligand modification step includes the following operations: dissolving propylenediamine in an aqueous sodium hydroxide solution, adding the brominated dextran spacer arm modified matrix, reacting at 180±20 rpm for 22-24 h to obtain the chromatography medium.

[0110] In some embodiments, the volume ratio of the matrix to the mass ratio of the ligand is 100 mL: 10 g.

[0111] In some implementations, after ligand modification is completed, a step of washing with deionized water is also included.

[0112] III. Methods for extracting α-lactalbumin from animal milk

[0113] This invention provides a method for extracting α-lactalbumin from whey, comprising the following steps:

[0114] The steps of chromatography are as follows: the above-mentioned chromatography medium is packed into the chromatography column, the whey is loaded into the chromatography column, and the flow-through is collected to obtain α-lactalbumin.

[0115] In some embodiments, the chromatography column has a diameter of 100 mm, a height of 55 cm, is packed with a compression ratio of 1.15, a packing height of 20 cm, and a column bed volume of 1.57 L.

[0116] In some embodiments, the whey is derived from animal milk, such as cow's milk, sheep's milk, camel's milk, etc., preferably cow's milk. The present invention does not particularly limit the method of whey preparation; for example, animal milk can be defatted first, and then casein in the defatted milk can be removed by acidification followed by solid-liquid separation or microfiltration to obtain whey.

[0117] In some embodiments, during the chromatography step, the sample loading flow rate is 47±3 L / h.

[0118] In some embodiments, after the chromatography step, the resulting product is further subjected to ultrafiltration and spray drying.

[0119] In some embodiments, after the chromatography step, a step of washing and regenerating the chromatography column using an aqueous sodium chloride solution, an aqueous sodium hydroxide solution, or RO water is further included.

[0120] The flowchart of the method for extracting α-lactalbumin from animal milk provided by this invention is as follows: Figure 6 As shown.

[0121] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all materials and instruments used are commercially available conventional products.

[0122] Example 1

[0123] (1) Weigh 4g of dextran T100 into a reaction flask, add 100ml of water, and stir at room temperature and 150rpm for 12h.

[0124] (2) Take 100ml of agarose microspheres and 100mL of water into a reaction flask, then add 10g of epichlorohydrin (ECH), stir at 30℃ and 200rpm for 5min, then add 10g of 50% (w / v) sodium hydroxide, and continue the reaction for 1h. After the reaction is complete, wash the microspheres with a large amount of deionized water.

[0125] (3) Add the activated microspheres to the dissolved dextran solution, then add 10g NaOH, and react at 30℃ and 300rpm for 18h. After the reaction is complete, wash the microspheres with plenty of pure water.

[0126] (4) Transfer the reacted microspheres to a reaction flask, add 100 mL of 1 M NaOH solution, 10 g of sodium sulfate, and 0.4 g of sodium borohydride in sequence, stir at 50 °C and 180 rpm for 1 h, add 40 mL of allyl bromide, and react at 50 °C and 180 rpm for 6 h. After the reaction is complete, wash the microspheres with pure water in sequence.

[0127] (5) Add 30g of N-bromosuccinimide and 150ml of 50% (v / v) acetone, place at 25℃ and 150rpm for 3 hours, filter, and wash with deionized water.

[0128] (6) Weigh 10g of propylenediamine and dissolve it in 100L of 1M NaOH. Then transfer it to a reaction flask, add the microspheres, and react at 180rpm for 24 hours. After the reaction is complete, wash it with plenty of deionized water.

[0129] Example 2

[0130] (1) Weigh 4g of dextran T300 into a reaction flask, add 100ml of water, and stir at room temperature and 150rpm for 24h.

[0131] (2) Take 100ml of agarose microspheres and 100mL of water into a reaction flask, then add 10g of ECH, stir at 30℃ and 200rpm for 5min, then add 10g of 50% (w / v) sodium hydroxide, and continue the reaction for 1h. After the reaction is complete, wash the microspheres with a large amount of deionized water.

[0132] (3) Add the activated microspheres to the dissolved dextran solution, then add 10g NaOH, and react at 30℃ and 300rpm for 18h. After the reaction is complete, wash the microspheres with plenty of pure water.

[0133] (4) Transfer the reacted microspheres to a reaction flask, add 100 mL of 1 M NaOH solution, 10 g of sodium sulfate, and 0.4 g of sodium borohydride in sequence, stir at 50 °C and 180 rpm for 1 h, add 40 mL of allyl bromide, and react at 50 °C and 180 rpm for 6 h. After the reaction is complete, wash the microspheres with pure water in sequence.

[0134] (5) Add 30g of N-bromosuccinimide and 150ml of 50% (v / v) acetone, place at 25℃ and 150rpm for 3 hours, filter, and wash with deionized water.

[0135] (6) Weigh 10g of propylenediamine and dissolve it in 100L of 1M NaOH. Then transfer it to a reaction flask, add the microspheres, and react at 180rpm for 24 hours. After the reaction is complete, wash it with plenty of deionized water.

[0136] Example 3

[0137] (1) Weigh 4g of dextran T500 into a reaction flask, add 100ml of water, and stir at room temperature and 150rpm for 24h.

[0138] (2) Take 100ml of agarose microspheres and 100mL of water into a reaction flask, then add 10g of ECH, stir at 30℃ and 200rpm for 5min, then add 10g of 50% (w / v) sodium hydroxide, and continue the reaction for 1h. After the reaction is complete, wash the microspheres with a large amount of deionized water.

[0139] (3) Add the activated microspheres to the dissolved dextran solution, then add 10g NaOH, and react at 30℃ and 300rpm for 18h. After the reaction is complete, wash the microspheres with plenty of pure water.

[0140] (4) Transfer the reacted microspheres to a reaction flask, add 100 mL of 1 M NaOH solution, 10 g of sodium sulfate, and 0.4 g of sodium borohydride in sequence, stir at 50 °C and 180 rpm for 1 h, add 40 mL of allyl bromide, and react at 50 °C and 180 rpm for 6 h. After the reaction is complete, wash the microspheres with pure water in sequence.

[0141] (5) Add 30g of N-bromosuccinimide and 150ml of 50% (v / v) acetone, place at 25℃ and 150rpm for 3 hours, filter, and wash with deionized water.

[0142] (6) Weigh 10g of propylenediamine and dissolve it in 100L of 1M NaOH. Then transfer it to a reaction flask, add the microspheres, and react at 180rpm for 24 hours. After the reaction is complete, wash it with plenty of deionized water.

[0143] Comparative Example

[0144] (1) Weigh 4g of dextran T600 into a reaction flask, add 100ml of water, and stir at room temperature and 150rpm for 12h.

[0145] (2) Take 100ml of agarose microspheres and 100ml of water into a reaction flask, then add 10g of ECH, stir at 30℃ and 200rpm for 5min, then add 10g of 50% (w / v) sodium hydroxide, and continue the reaction for 1h. After the reaction is complete, wash the microspheres with a large amount of deionized water.

[0146] (3) Add the activated microspheres to the dissolved dextran solution, then add 10g NaOH, and react at 30℃ and 300rpm for 18h. After the reaction is complete, wash the microspheres with plenty of pure water.

[0147] (4) Transfer the reacted microspheres to a reaction flask, add 100 mL of 1 M NaOH solution, 10 g of sodium sulfate, and 0.4 g of sodium borohydride in sequence, stir at 50 °C and 180 rpm for 1 h, add 40 mL of allyl bromide, and react at 50 °C and 180 rpm for 6 h. After the reaction is complete, wash the microspheres with pure water in sequence.

[0148] (5) Add 30g of N-bromosuccinimide and 150ml of 50% (v / v) acetone, place at 25℃ and 150rpm for 3 hours, filter, and wash with deionized water.

[0149] (6) Weigh 10g of propylenediamine and dissolve it in 100L of 1M NaOH. Then transfer it to a reaction flask, add the microspheres, and react at 180rpm for 24 hours. After the reaction is complete, wash it with plenty of deionized water.

[0150] Experimental Example 1

[0151] This experimental example uses the media prepared in Examples 1-3 and the comparative example above to purify α-lactalbumin in whey.

[0152] 1) Process 160L of fresh milk using a skimming machine to obtain skimmed milk;

[0153] 2) Use a spiral wound membrane microfiltration system to process skim milk to separate casein and obtain a clear whey protein solution with a volume of about 160 L. Dilute it 1.5 times and load it onto the sample. The final sample volume is 240 L.

[0154] 3) Take 1.8L of the medium prepared in Examples 1-3 and the comparative example, mix it well, and replace the preservation solution with water to avoid the introduction of air bubbles. Slowly pour it into the chromatography column. The chromatography column has a diameter of 100mm and a height of 55cm. It is packed according to a compression ratio of 1.15, with a packing height of 20cm and a column bed volume of 1.57L.

[0155] 4) Rinse the chromatography column with RO water at a volume of 15 L and maintain a flow rate of 47 L / h until the loading is complete. Load 240 L of membrane-filtered whey onto the chromatography column. During the loading process, collect the flow-through peak component until the peak ends (samples are taken at different time points during the loading process, and the samples are named as CVs according to the multiple of the flow-through volume relative to the column volume. At the same time, samples from different time points are mixed and named as unmixed / mixed samples). This component is the target protein α-lactalbumin solution. After the loading is complete, continue to rinse the chromatography column with 8 L of pure water until the UV monitoring baseline drops to the initial value and stabilizes.

[0156] 5) Wash and regenerate the chromatography column with 10 L of 0.5 M sodium chloride solution dissolved in RO water at a flow rate of 23 L / h, and collect the protein fraction of the elution peak (the sample is recorded as elution).

[0157] 6) Use 5L of 0.5M sodium hydroxide prepared with RO water to wash the chromatography column at a flow rate of 23L / h to remove stubborn impurities that are still adsorbed on the chromatography column.

[0158] 7) Replace the alkali in the chromatography column with RO water until the pH at the outlet is neutral.

[0159] Experimental results:

[0160] SDS-PAGE analysis was performed on the membrane-filtered whey stock solution and the sample flow-through solution. The results are as follows: Figure 1A As shown in the figure, the purity of the target protein in the comparative example is low, and the separation effect is poor.

[0161] The whey was purified using the media prepared in Examples 1-3, and the flow-through was analyzed by HPLC. The detection method referred to NY / T 4630-2025 (Determination of α-lactalbumin and β-lactoglobulin in cow's milk and its products by high performance liquid chromatography). The detection results are as follows. Figure 1B (Example 1) and Table 1 (Average values ​​of Examples) are shown.

[0162] Table 1

[0163]

[0164] Experiment Example 2

[0165] This experimental example shows the media prepared in Examples 1-3 and the comparative example above under a microscope.

[0166] Microscopic observation results are as follows Figure 2 As shown in Table 2, the particle sizes of the media prepared in Examples 1-3 and the comparative examples are shown in the average values.

[0167] Table 2

[0168]

[0169] Experimental Example 3

[0170] This experimental example tests the microbial residues in the media prepared in Examples 1-3 and the comparative example above.

[0171] Experimental methods:

[0172] Microbial residue detection was performed according to the methods described in GB / T 38170-2019.

[0173] Experimental results:

[0174] Microbial residue test results as follows Figure 3 As shown in Table 3.

[0175] Table 3

[0176]

[0177] Experiment Example 4

[0178] This experimental example measures the ligand density and dynamic loading of the media prepared in Examples 1-3 and the comparative example.

[0179] Experimental methods:

[0180] Methods for detecting ligand density:

[0181] Sample preparation: Take an appropriate amount of medium into a vacuum filtration funnel, wash with deionized water 3-5 times, and then dry for 5 minutes. Weigh about 20 g of the dried medium into a 100 mL beaker, add about 50 mL of 1 M NaOH solution, and soak for 20 minutes, shaking well every 5 minutes. Then, use a vacuum filtration device to wash the soaked medium with plenty of deionized water and dry for 5 minutes. Accurately weigh 2.00 g of the dried medium into a 50 mL centrifuge tube, and add 20 mL of 0.1 M HCl solution to each tube using a pipette. React for 20 minutes, shaking well every 10 minutes, and then let stand for about 30 minutes until sedimentation is complete.

[0182] Titration: After complete sedimentation, take 10 mL of supernatant, add 2 drops of 1% phenolphthalein indicator, and titrate with 0.1 M NaOH solution. At the same time, perform a blank control experiment.

[0183] Results Calculation: Ion exchange capacity is expressed as the amount of exchangeable acidic groups in a unit sedimentation volume of medium. It can be calculated using the following formula. The average of two independent experiments is taken as the ion exchange capacity determination result. Under repeatability conditions, the absolute difference between two independent determination results should not exceed 10% of the arithmetic mean.

[0184] q = ((V2-V1)×0.1) / m×K

[0185] In the formula: q is the ion exchange capacity, mmol / mL;

[0186] V2 represents the volume of NaOH solution consumed by the blank control, in mL;

[0187] V1 is the volume of NaOH solution consumed by the sample solution, in mL;

[0188] m is the sample mass in g; (A total of 20 mL of 0.1 M HCl was added, and 10 mL of the supernatant was titrated, so m = 1 g)

[0189] K is the mass / volume conversion factor for the medium. Different series have different conversion factors. In this invention, K is 0.8.

[0190] Dynamic load measurement method:

[0191] Chromatography requirements:

[0192] Chromatography column: QCXKC 5 / 100;

[0193] Equilibration buffer: 20 mM PB, 0.15 M NaCl, pH 7.3;

[0194] Protein solution: 1 mg / mL bovine serum albumin (BSA);

[0195] Flow rate: 0.5 mL / min.

[0196] Chromatography process:

[0197] Equilibration: Equilibrate with equilibration buffer at a flow rate of v = 0.5 mL / min for at least 5 CV until the UV and conductivity baselines are stable.

[0198] Sample loading: Replace (bypass) the SDL chromatography system tubing with 1 mg / mL protein sample solution, and then pump it into the chromatography column at a flow rate of v = 0.5 mL / min. Start recording the UV values. Stop loading when the baseline rises steadily and then stabilizes. Record the highest UV value and calculate UVDBC10%. Determine the UVDBC10% time T.

[0199] Reequilibration: Wash the column again with equilibration buffer at 0.5 mL / min until the UV light decreases and the column reaches equilibrium.

[0200] Elution: Rinse the chromatography column with elution buffer, collect the eluted liquid, and measure the absorbance at OD280.

[0201] Result calculation:

[0202] Using the flow-through load at 10% of the background value as the dynamic adsorption load of the medium, the calculation formula is as follows:

[0203] Q_(10%)=(c×v×T) / CV

[0204] in,

[0205] Q_(10%)——Dynamic adsorption capacity, in milligrams per milliliter (mg / mL).

[0206] c — the concentration of the tagged protein sample, in milligrams per milliliter (mg / mL).

[0207] v—volume flow rate, in milliliters per minute (mL / min);

[0208] T — the time it takes to reach 10% of the maximum UV value, in minutes (min).

[0209] CV—Column volume of chromatography, in milliliters (mL).

[0210] Experimental results:

[0211] The results of ligand density testing and dynamic loading measurement are shown in Table 4.

[0212] Table 4

[0213]

Claims

1. A chromatography medium for separating α-lactalbumin from whey, characterized in that, The chromatography medium is based on agarose, and the surface of the matrix is ​​modified with ligands via dextran spacers. The dextran has a molecular weight of 20kDa-500kDa, and the ligand includes propylenediamine. Furthermore, in the chromatography medium, the density of the ligand is 200 μmol / mL to 300 μmol / mL.

2. The chromatography medium according to claim 1, characterized in that, The matrix is ​​porous spherical with a particle size of 100μm-200μm.

3. The chromatography medium according to claim 1 or 2, characterized in that, The matrix is ​​activated by a first activating agent and then linked to dextran; the dextran is activated by a second activating agent and then linked to the ligand. The first activating agent includes an epoxy compound; the second activating agent includes a halogenated compound with a double bond structure or an epoxy compound, and a brominating agent.

4. The chromatography medium according to claim 3, characterized in that, The epoxy compounds include epichlorohydrin or 1,4-butanediol diglycidyl ether; the halogenated compounds with double bond structures include allyl halogenated hydrocarbons; and the brominating reagent includes N-bromosuccinimide.

5. The method for preparing the chromatographic medium according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: The first activation step is as follows: the matrix is ​​mixed with water, a first activation reagent is added, and the matrix is ​​activated under alkaline conditions to obtain a first activated matrix; The steps of dextran spacer modification are as follows: the first activated matrix is ​​mixed with a dextran aqueous solution, and the first activated matrix is ​​modified with dextran spacers under alkaline conditions to obtain a dextran spacer modified matrix. The second activation step involves reacting the dextran spacer arm modified matrix with a second activation reagent to obtain a brominated dextran spacer arm modified matrix. The steps of ligand modification are as follows: under alkaline conditions, the ligand is reacted with the brominated dextran spacer arm modified matrix in an aqueous solution to obtain the chromatography medium; The first activating agent includes an epoxy compound; the second activating agent includes a halogenated compound containing a double bond or an epoxy compound, and a brominating agent.

6. The preparation method according to claim 5, characterized in that, In the second activation step, the dextran spacer-modified matrix is ​​first reacted with the halogenated compound containing a double bond or an epoxy compound in an aqueous solution under alkaline conditions, optionally in the presence of a stabilizer, and then the product is further reacted with a brominizing agent in an organic solution.

7. The preparation method according to claim 5 or 6, characterized in that, The epoxy compounds include epichlorohydrin or 1,4-butanediol diglycidyl ether.

8. The preparation method according to any one of claims 5-7, characterized in that, The halogenated compound containing a double bond includes allyl halogenated hydrocarbons; the brominating reagent includes N-bromosuccinimide.

9. The preparation method according to any one of claims 5-8, characterized in that, The alkaline conditions are formed using sodium hydroxide.

10. A method for extracting α-lactalbumin from whey, characterized in that, The method includes the following steps: The steps of chromatography are as follows: the chromatography medium according to any one of claims 1-4 or the chromatography medium prepared by the preparation method according to any one of claims 5-9 is packed into the chromatography column, the whey is loaded into the chromatography column, and the flow-through is collected to obtain α-lactalbumin.

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