Chromatographic packing and purification method for purifying alpha-lactalbumin and beta-lactoglobulin

Abstract

The present invention belongs to the field of food processing and relates to a chromatography packing and a purification method for purifying alpha-lactalbumin and beta-lactoglobulin. Specifically, it relates to a chromatography packing for purifying alpha-lactalbumin and beta-lactoglobulin, which is obtained by treating whey protein for said purifying alpha-lactalbumin and beta-lactoglobulin, wherein the packing has a porous spherical structure, the spherical structure comprises a base sphere and a modifier, the modifier is at least partially present on the surface of the base sphere, the base sphere comprises a natural polysaccharide substance, the natural polysaccharide substance contains agarose and cellulose crosslinked with each other, and the base sphere is derivatized with dextran, the modifier comprises an ion exchange ligand, and the ion exchange ligand is connected with the dextran and the base sphere via a covalent bond, the ion exchange ligand contains a quaternary amine group or a salt thereof, and the coupling density of the ion exchange ligand of the packing is 150 μmol / mL-250 μmol / mL.

Description

Technical Field

[0001] This invention relates to a chromatographic packing material and purification method for purifying α-lactalbumin and β-lactoglobulin, belonging to the field of food processing. Background Technology

[0002] Whey is the supernatant obtained from fresh cow's milk after acid precipitation or filtration to remove most of the casein. Cow's whey contains various whey proteins, of which approximately 50% is β-lactoglobulin (β-Ig) and 20% is α-lactalbumin (α-Ia). In human breast milk, the α-lactalbumin content in whey protein can reach 40%, and it contains virtually no β-lactoglobulin. α-lactalbumin is an important component of infant formula, playing a role in promoting infant development, regulating immune function, and improving sleep. While β-lactoglobulin provides essential amino acids, aids in the absorption of fat-soluble substances, and participates in mineral transport, it is a common allergen and can trigger allergic reactions in individuals with cow's milk allergies. Therefore, in the formulation of infant formula, a whey protein raw material with high α-lactalbumin content and low (or even no) β-lactoglobulin content is needed to make the product's nutritional structure closer to breast milk.

[0003] Currently, the domestic market for α-lactalbumin powder largely relies on imports, lacking domestically developed and controllable high-efficiency separation technology. Therefore, developing a technical method for efficiently separating α-lactalbumin and β-lactoglobulin from whey has significant practical implications and application value.

[0004] Existing processes for separating and extracting α-lactalbumin and β-lactoglobulin mainly include membrane separation and chromatography. While membrane separation is simple to operate, it suffers from problems such as low separation purity (usually below 40%), high β-lactoglobulin residue, low yield, and poor batch-to-batch product consistency, which limits its application in industrial production.

[0005] With the development and widespread adoption of chromatography technology and related equipment and consumables, many industries have begun using chromatography to prepare protein raw materials. Chromatography offers advantages such as high purity, high yield, high reproducibility, and suitability for large-scale production. In the field of dairy product processing, lactoferrin is a typical example of production using chromatography, often processing hundreds of tons of milk daily. After purification, the lactoferrin achieves a purity of over 95%, which is unmatched by other methods. Therefore, chromatography, as a highly efficient separation method, is the preferred method for large-scale separation of α-lactalbumin and β-lactoglobulin.

[0006] Chromatographic packing materials are a key consumable in chromatographic purification processes, typically packed into chromatographic columns. These packing materials generally have a porous spherical structure with numerous micropores on their surface and interior for material entry and exit. Depending on the type of packing material, grafting modifications and ligand coupling may be performed to achieve different functions. Chromatographic packing materials separate target proteins from other impurities through specific interactions (such as size exclusion, ion exchange, hydrophobicity, and affinity), thus purifying the target protein. Currently, common materials for chromatographic packing materials include natural polysaccharides, polymers, and inorganic materials. Polysaccharide-based packing materials, represented by agarose, are characterized by good biocompatibility, a wide particle size distribution, and numerous hydroxyl groups on the surface and interior of the spheres for ligand modification and coupling, allowing for high protein binding capacity and thus enabling the processing of large batches of samples. Polymer-based packing materials, represented by polymethyl methacrylate (PMMA), are characterized by high pressure resistance, good mechanical strength, and relatively uniform pore size, allowing for higher flow rates, but their loading capacity is generally limited.

[0007] Among the existing chromatographic methods and packing materials for separating α-lactalbumin and β-lactoglobulin, reference 1 discloses a method for separating α-lactalbumin and β-lactoglobulin based on sulfonic acid-based strong cation exchange polymer packing material, and points out that β-lactoglobulin can be flow-through while α-lactalbumin is adsorbed within the whey pH range of 4.5–4.8; reference 2 discloses a technical solution for separating β-lactoglobulin and α-lactalbumin using quaternary ammonium-based strong anion exchange packing material, which discloses a whey pH range of 6.0–7.5 and a single batch processing capacity of 60 times the column bed volume of whey.

[0008] However, further development of a high-efficiency chromatographic packing material for separating α-lactalbumin and β-lactoglobulin with high sample throughput and high sample processing speed is of great practical significance and application value.

[0009] References:

[0010] Reference 1: CN106793797A

[0011] Reference 2: CN120607599A Summary of the Invention

[0012] The problem the invention aims to solve

[0013] Although the above techniques have achieved the separation of α-lactalbumin and β-lactoglobulin to some extent, in practice, it has been found that the existing techniques still have the following shortcomings, such as:

[0014] The separation effect of reference 1 is greatly affected by the quality of raw materials. It requires that the impurities in the sample (such as casein, IgG, glycomacropeptide, etc.) be as few as possible, and that parameters such as conductivity and pH be strictly controlled. It has poor compatibility with existing mature processes and is not conducive to industrial scale-up.

[0015] Reference 2 uses a quaternary ammonium-based strong anion exchange resin for separation. Although it has a certain selectivity under specific pH conditions, the processing capacity is limited and the processing speed is low, making it difficult to meet the efficiency requirements of large-scale production.

[0016] It is evident that some of the existing chromatographic packing materials used for the separation of β-lactoglobulin and α-lactalbumin have high sample requirements and complex pretreatment, while others have too small a sample throughput and slow processing speed. In contrast, dairy processing involves large-scale processing, simple processing requirements, short shelf life of materials, and is also sensitive to cost.

[0017] In view of the above problems, the present invention aims to provide a chromatography packing material and method for efficiently separating α-lactalbumin and β-lactoglobulin from whey, which has the following advantages:

[0018] (1) Large sample volume per batch and fast processing speed;

[0019] (2) The processing method is simple and the process has good repeatability;

[0020] (3) The packing has excellent mechanical properties, good durability and low production cost.

[0021] Solution for solving the problem

[0022] [1]. A chromatography packing material for purifying and separating α-lactalbumin and β-lactoglobulin, characterized in that the packing material is used to purify and separate α-lactalbumin and β-lactoglobulin by treating whey protein.

[0023] in,

[0024] The filler has a porous spherical structure, the spherical structure including a base sphere modifier, the modifier being at least partially present on the surface of the base sphere.

[0025] The base sphere comprises a natural polysaccharide containing cross-linked agarose and cellulose, and the base sphere is dextran-derived.

[0026] The modifier includes an ion-exchange ligand, and the ion-exchange ligand is covalently linked to the dextran and the spheroid.

[0027] The ion exchange ligand comprises a quaternary ammonium group or its salt, and the coupling density of the ion exchange ligand of the packing material is 150 μmol / mL-250 μmol / mL.

[0028] [2]. The chromatography packing material according to [1] is characterized in that the particle size distribution range of the packing material is 100μm-350μm.

[0029] [3]. The chromatography packing material according to [1] or [2] is characterized in that the mass ratio of cellulose to agarose in the base sphere can be 1:1 to 1:10; the base sphere is substantially composed of cross-linked natural polysaccharides.

[0030] [4]. The chromatography packing material according to any one of [1] to [3] is characterized in that the molecular weight of the dextran is 10kDa-300kDa.

[0031] [5]. The chromatography packing material according to any one of [1] to [4] is characterized in that the surface pore size of the packing material having a porous spherical structure is set to be sufficient to allow protein molecules with a molecular weight of 10kDa-2000kDa to diffuse into the interior of the sphere.

[0032] [6]. The method for preparing chromatographic packing material according to any one of [1] to [5] is characterized in that the method comprises the following steps:

[0033] The emulsification and crosslinking steps involve emulsifying and crosslinking agarose and cellulose to form the matrix spheres;

[0034] The grafting derivatization step involves grafting the dextran onto the base sphere;

[0035] The ligand coupling step involves reacting the grafted and derivatized base spheres with a compound containing a ligand so that the ion-exchange ligand is covalently linked to the dextran (branched chain) and the base spheres.

[0036] [7]. The chromatography packing material according to [6] is characterized in that, in the emulsification and crosslinking step, the emulsification and crosslinking is carried out under alkaline conditions and in the presence of a crosslinking agent, wherein the crosslinking agent is selected from one or more of an epoxy compound or an aldehyde compound.

[0037] [8]. The preparation method according to [6] or [7] is characterized in that the compound containing the ligand is an epoxy compound containing the ligand.

[0038] [9]. A purification and separation method using the chromatography packing material described in any one of [1] to [5], characterized in that the method includes the following steps:

[0039] (1) After defatting the milk raw material, it is filtered through a membrane to obtain whey protein with casein removed;

[0040] (2) The whey obtained in step (1) is loaded onto a chromatography column containing the chromatography packing material according to any one of claims 1 to 5 for chromatography. Afterwards, pure water and inorganic salt are used as mobile phases for rebalancing and elution, respectively.

[0041] (3) Collect the flow-through and eluent from step (2) respectively to obtain the flow-through with α-lactalbumin as the main component and the eluent with β-lactoglobulin as the main component.

[0042]

[10] . According to the purification and separation method described in [9], the packing material has a dynamic binding capacity of 150 g / L to 200 g / L for β-lactoglobulin at a linear flow rate of whey protein below 1000 cm / h, and the back pressure of the chromatography column is less than 2 bar.

[0043] The effects of the invention

[0044] By implementing the above technical solution, the present invention achieves the following beneficial effects:

[0045] The chromatographic packing material of this invention has a dense internal structure, which means that the packing material has stronger mechanical properties and also provides more sites for ligand coupling and protein binding. This invention demonstrates the advantages of composite agarose-cellulose spheres from a microscopic perspective. At the same time, the chromatographic packing material of this invention has a high loading capacity and a large sample throughput.

[0046] The chromatography packing material according to the present invention has a higher loading flow rate, can process more samples in the same time and has a higher number of times the packing material can be reused in the chromatography process of lactalbumin, which is beneficial for large-scale production applications.

[0047] This invention demonstrates that by screening composite matrix sphere materials, modifying them with dextran grafting, and optimizing ligand density, the following was achieved:

[0048] 1) Improved packing performance, enabling the chromatography packing to have both high sample throughput and high sample processing speed, with a throughput increase of at least 20% compared to existing methods, while maintaining high purity and yield.

[0049] 2) This packing material has the advantages of high pressure resistance and wide pH range, good biocompatibility and long service life, which can meet the industrial purification needs of α-lactalbumin and solve the shortcomings of existing packing materials in terms of pressure resistance and processing capacity.

[0050] 3) This chromatography process is simple and easy to perform, highly compatible, requires no additional sample adjustments, and is less expensive than existing processes.

[0051] In actual production, the chromatographic packing material and purification method of the present invention for purifying and separating α-lactalbumin and β-lactoglobulin can be used to separate α-lactalbumin and β-lactoglobulin from whey protein after obtaining whey by filtering fresh milk through a defatted membrane. This can be achieved simply by loading and eluting the whey protein through packing material chromatography.

[0052] In some preferred embodiments, by collecting the flow-through from the chromatography, a whey flow-through that is essentially free of β-lactoglobulin and has an α-lactalbumin content >45% can be obtained; by collecting the eluent from the chromatography, β-lactoglobulin with a purity greater than 80% can be obtained. Using this process, 1000L of this packing material can process at least 300 tons of whey separated from fresh milk per day, featuring high throughput and high efficiency. It can be used for large-scale production of α-lactalbumin extraction from whey, greatly reducing production costs. Attached Figure Description

[0053] Figure 1 Flowchart of whey protein extraction process in a specific embodiment of the present invention

[0054] Figure 2 A flow chart of the chromatographic packing material production process in a specific embodiment of the present invention.

[0055] Figure 3 Pressure-flow rate curves of the chromatography packing material in Example 1 and Comparative Example 1 of this invention.

[0056] Figure 4 Electron micrograph of cross-section of the chromatography packing material in Example 2 of this invention.

[0057] Figure 5 Electron micrograph of the cross-sectional section of the chromatography packing material of Comparative Example 1 of this invention.

[0058] Figure 6 Chromatographic patterns of microfiltration whey separation and purification using the chromatographic packing materials of Example 1 and Comparative Example 1 of this invention.

[0059] Figure 7 Electrophoretic patterns of the chromatographic packing materials used in Example 1 and Comparative Example 1 of this invention for the separation and purification of whey via microfiltration.

[0060] Figure 8 Electrophoretic patterns of the chromatographic packing materials used in Example 2 and Comparative Example 2 of this invention for the separation and purification of whey via microfiltration.

[0061] Figure 9 Example 1 of this invention: Capillary electrophoresis results of the sample before purification.

[0062] Figure 10 Example 1 of this invention: Capillary electrophoresis results of purified sample. Detailed Implementation

[0063] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0064] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0065] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0066] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0067] In this specification, the terms "substantially" or "basically" are used to indicate that the actual error is less than 1%.

[0068] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0069] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0070] In this manual, unless otherwise specified, "%" indicates a percentage by mass.

[0071] In this specification, "w / v" indicates the weight / volume percentage concentration.

[0072] <Chromatography Packing Material>

[0073] The chromatographic packing material for purifying α-lactalbumin and β-lactoglobulin involved in this invention has a porous spherical structure. The packing material can separate and purify the α-lactalbumin and β-lactoglobulin contained in whey protein by treating whey protein.

[0074] The filler includes a base ball and a modification formed on the surface of the base ball.

[0075] Baseball

[0076] The base sphere of the present invention comprises natural polysaccharides, and the natural polysaccharides necessarily include agarose and cellulose.

[0077] In some specific implementations, the base sphere may contain other materials besides the aforementioned natural polysaccharides, such as silicone, styrene copolymers, etc.

[0078] In some preferred embodiments, for the base spheres of the present invention, the content of the natural polysaccharides is 90% by mass or more, more preferably 95% by mass or more, based on the total mass of the base spheres (excluding any crosslinking agents that may be used). More preferably, the base spheres are substantially composed of the aforementioned natural polysaccharides through crosslinking.

[0079] In addition, there are no particular restrictions on the agarose and cellulose in the natural polysaccharide of the present invention. From the perspective of improving separation efficiency, the mass ratio of cellulose to agarose can be 1:1-1:10, preferably 1:1-1:8, more preferably 1:2-1:6, and more preferably 1:5.

[0080] Furthermore, in some specific embodiments, various esters or phenylcarbamate functional group derivatives of microcrystalline cellulose can be used for the cellulose used in the base spheres of the present invention.

[0081] The base spheres of this invention can be formed by cross-linking the aforementioned natural polysaccharides. There are no particular limitations on the method of cross-linking, but a cross-linking agent is preferably used. There are no particular limitations on the type of cross-linking agent, but examples include one or more of epoxides, aldehydes, etc.

[0082] This invention adds cellulose (within the above-mentioned proportion range) to a traditional single agarose filler. By utilizing the high biocompatibility between cellulose and agarose, the agarose and cellulose complement each other when cross-linking into spheres, resulting in a denser and more uniform porous structure with superior mechanical properties.

[0083] Modifications on the surface of the base sphere

[0084] Modifications to the surface of the base spheres of the present invention include ion-exchange ligands for ion exchange.

[0085] In order to increase the reaction sites between the base sphere and the modifier, the base sphere is derivatized with dextran and then coupled with the ion exchange ligand.

[0086] The aforementioned dextran can be grafted onto the base sphere, and then the dextran-derived base sphere is coupled with a compound containing the aforementioned ion-exchange ligand. The dextran-derived base sphere (especially after activation treatment) can generate more activation sites in the dextran branches, on the surface of the base sphere, and inside. Subsequent addition of a ligand-containing compound can covalently react with these activation sites, thereby coupling with the base sphere. In other words, the modifier of the present invention is at least formed on the surface of the base sphere, and optionally, is also partially present inside the base sphere.

[0087] In some preferred embodiments, the molecular weight of the dextran used for grafting, from the viewpoint of improving protein loading and recovery, can be, for example, 10kDa-500kDa, preferably 10kDa-300kDa, more preferably 15kDa-200kDa, and even more preferably 20kDa-150kDa.

[0088] The ion-exchange ligands of the present invention necessarily include a quaternary ammonium group or a salt thereof. There are no particular limitations in principle on the compounds containing such ligands; however, in some preferred embodiments, for ease of coupling, epoxy compounds containing a quaternary ammonium group or a salt thereof may be used, typically 2,3-epoxypropyltrimethylammonium chloride (GTA).

[0089] Packing properties

[0090] The ligand coupling density of the filler material described in this invention is 150 μmol / mL-250 μmol / mL, preferably 180 μmol / mL-220 μmol / mL, and also includes 160 μmol / mL, 170 μmol / mL, 190 μmol / mL, 200 μmol / mL, 210 μmol / mL, 230 μmol / mL, 240 μmol / mL, etc.

[0091] By controlling the ligand coupling density of the packing material within the above range, the packing material can achieve both high protein binding capacity and high recovery rate.

[0092] If the ligand coupling density of the packing material is higher than the above range, some proteins will bind too tightly to the packing material. Although the protein loading capacity is high, the protein recovery rate is low, resulting in loss due to non-recovery. If the ligand coupling density of the packing material is lower than the above range, the protein loading capacity may be too low.

[0093] Furthermore, in some embodiments, the particle size distribution range (Dv10-Dv95) of the filler is 100μm-350μm, preferably 120μm-310μm, and more preferably 150μm-300μm. The surface pore size of the filler with the porous spherical structure allows protein molecules with a molecular weight of 10kDa-2000kDa to diffuse into the interior of the spheres.

[0094] <Preparation of Chromatography Packing Material>

[0095] The preparation method of the chromatography packing material described in this invention is not particularly limited in principle. For example, it may include an emulsification and crosslinking step of agarose and cellulose, a grafting and derivatization step, and a ligand coupling step.

[0096] Emulsification and crosslinking steps

[0097] The present invention uses emulsification crosslinking to crosslink agarose and cellulose to form the base spheres of the present invention.

[0098] In some embodiments, the emulsification and crosslinking step of agarose and cellulose involves dissolving a polysaccharide raw material containing cellulose and agarose in water to form an aqueous phase, dispersing the aqueous phase in an oil phase containing a surfactant, and adding a crosslinking agent under alkaline conditions to carry out an emulsification and crosslinking reaction to form composite polysaccharide spheres.

[0099] In some embodiments, the conditions for the emulsification crosslinking reaction are not particularly limited in principle. For example, in some specific embodiments, the oil phase containing the surfactant is prepared by dissolving the polymeric surfactant in an organic solvent to prepare an oil phase solution with a concentration of 0.5%-3.0% (w / v), and maintaining a stirring speed of 200 rpm-600 rpm at 55°C-65°C. In some specific embodiments, the polymeric surfactant is preferably Span 80, the organic solvent is preferably toluene, the preferred concentration is 2.0% (w / v), and the preferred stirring speed is 250 rpm-350 rpm.

[0100] In some specific embodiments, the aqueous phase is prepared by placing cellulose powder and agarose powder in an aqueous solution to prepare a 4%-8% (w / v) suspension, for example, a 6.0% (w / v) suspension. The powders are then completely dissolved under heating and stirring (60℃-95℃). The mass ratio of the cellulose powder to the agarose powder can be 1:1-1:10, preferably 1:1-1:8, more preferably 1:2-1:6, and even more preferably 1:5.

[0101] In some embodiments, the resulting dissolved aqueous solution is transferred to a constant-pressure dropping funnel and added dropwise to a preheated oil phase containing a surfactant in a first reactor under continuous stirring.

[0102] Furthermore, the alkaline conditions for crosslinking can be achieved by using an alkaline regulator, such as sodium hydroxide. Additionally, in some specific embodiments, the crosslinking agent may be, for example, epichlorohydrin or glutaraldehyde.

[0103] In some specific embodiments, the amount of the crosslinking agent added is 30%-100% of the dry weight of the mixed polysaccharide powder, preferably 40%-80%, for example, 50%. The amount of the alkaline regulator added is, for example, 1 mol / L-3 mol / L. The activation treatment step is carried out at, for example, 50°C-65°C for 2-6 hours, preferably 3-4 hours.

[0104] In some embodiments, the system obtained from the above reaction is de-oiled to obtain cross-linked microspheres. Preferably, the subsequent grading and sieving process can be performed to obtain microspheres of suitable particle size.

[0105] In some specific embodiments, the microspheres obtained after removing the oil phase are collected, for example, by wet sieving, to obtain highly cross-linked agarose-cellulose-based sphere activation intermediate I with a particle size range of 100 μm-350 μm. In some embodiments, the particle size range of the collected microspheres is preferably 120 μm-310 μm, more preferably 150 μm-300 μm, and examples include 130 μm, 140 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 260 μm, 270 μm, etc.

[0106] The above processing can yield intermediate I (base ball) that satisfies the present invention.

[0107] Steps of grafting

[0108] After obtaining intermediate I, it can be grafted and derivatized to graft dextran onto intermediate I. In some specific embodiments, the grafting and derivatization can be carried out under alkaline conditions.

[0109] There are no particular restrictions on the dextran that can be used in the grafting process. In some specific embodiments, the molecular weight range of the dextran is preferably 10 kDa to 300 kDa, more preferably 15 kDa to 200 kDa, and even more preferably 20 kDa to 150 kDa.

[0110] The alkaline conditions required for graft derivatization reactions can be achieved by using an alkaline regulator. An example alkaline regulator is sodium hydroxide, and the amount added is, for example, 1 mol / L to 3 mol / L.

[0111] The grafting and derivatization step of the dextran is carried out at, for example, 50°C-65°C, for a reaction time of, for example, 6-12 hours, preferably 8-10 hours. After the reaction, a neutral wet bulb can be obtained by washing with deionized water.

[0112] Through the above grafting process, intermediate II of the present invention (base sphere derived via dextran grafting) can be obtained.

[0113] Steps of ligand coupling

[0114] The above intermediate II (grafted derivative base sphere) is reacted with a compound containing a ligand to couple the ligand to the filler.

[0115] In some embodiments, the above reaction involves a coupling reaction step between intermediate II and a compound containing an ion-exchange ligand. Preferably, intermediate II may be activated prior to the coupling step to increase the efficiency of the coupling reaction.

[0116] There are no particular restrictions as long as the ion exchange ligand contains a quaternary ammonium group or its salt, such as a quaternary ammonium salt, preferably 2,3-epoxypropyltrimethylammonium chloride (GTA).

[0117] In some embodiments, in the coupling reaction step, the feed ratio of the compound containing the ion-exchange ligand is, for example, 40 g / L-160 g / L relative to the wet bulb (1 L) of the aforementioned intermediate II, preferably 50 g / L-150 g / L, more preferably 60 g / L-120 g / L, and even more preferably 80 g / L-100 g / L; the feed ratio of the base is 50 g / L-150 g / L relative to the wet bulb (1 L) of the aforementioned intermediate II, preferably 70 g / L-150 g / L, and even more preferably 80 g / L-120 g / L. The coupling reaction step of the ion-exchange ligand is carried out at, for example, 50°C-65°C, for a reaction time of, for example, 10-15 hours, preferably 10-12 hours.

[0118] Furthermore, after the reaction is complete, unreacted compounds can be removed through post-treatment. Post-treatment can be performed using buffer solutions, deionized water, etc.

[0119] <Purification and Separation Methods Using Chromatographic Packing Materials>

[0120] The present invention also relates to a purification and separation method for separating α-lactalbumin and β-lactoglobulin using the chromatographic packing material described herein.

[0121] After obtaining the packing material of the present invention, a chromatography apparatus using these packing materials as the stationary phase can be fabricated in accordance with the usual practices in the art; typically, it can be a chromatography column.

[0122] Furthermore, the purification method includes, for example, the following steps:

[0123] (1) After defatting the milk raw material, it was filtered through a membrane to obtain a whey protein sample with casein removed;

[0124] (2) Load the whey sample obtained in step (1) onto a chromatography column packed with the above-mentioned chromatography packing material for chromatography, and use pure water and inorganic salt as mobile phases for rebalancing and elution respectively.

[0125] (3) Collect the flow-through and eluent from step (2) respectively to obtain the flow-through with α-lactalbumin as the main component and the eluent with β-lactoglobulin as the main component.

[0126] The collected flow-through and eluents were enriched with α-lactalbumin and β-lactoglobulin, respectively, which can be further purified to obtain high-purity protein products.

[0127] There are no particular limitations in principle regarding the milk raw material of this invention. The milk raw material can be a human-derived or animal-derived milk-containing raw material. And in some preferred embodiments, the milk-containing raw material of this invention can be an animal-derived milk raw material.

[0128] Animal-derived milk sources typically include milk from cows, sheep, camels, deer, or horses. In some preferred embodiments, the milk source of the present invention is milk from cows or sheep.

[0129] Such milk raw materials can be raw milk or milk raw materials that have undergone a certain degree of processing (milk powder, skimmed milk powder, etc.). In some specific implementation schemes, from the perspective of facilitating subsequent separation, in addition to using raw milk, it is preferable to process the above-mentioned milk through processes such as concentration and skimming to obtain milk with reduced fat content.

[0130] In some implementations, there are no particular restrictions on the method of defatting, but preferably it is carried out without the use of external chemical reagents, typically by methods such as centrifugation. The membrane filtration is used to remove casein components, and includes treating the defatted milk with a membrane filter to separate casein from whey.

[0131] In some embodiments, the whey sample obtained after defatting and membrane filtration is purified by chromatography. In some specific embodiments, the whey sample is loaded onto the chromatography column at a linear flow rate of 300 cm / h-1000 cm / h, preferably 500 cm / h-800 cm / h, more preferably 600 cm / h-800 cm / h. The UV absorption of the flow-through is monitored, and the flow-through is collected when the UV absorption begins to rise until the loading is completed. The resulting flow-through contains α-lactalbumin as the main component.

[0132] In some embodiments, after sample loading, the chromatography column is washed with, for example, 5-10 column volumes of pure water to remove residual unbound impurities. Then, the column is eluted with, for example, 5-10 column volumes of eluent, and the eluent is collected based on the UV absorption peak. The UV absorption of the eluent is monitored, and collection begins when the UV absorption starts to rise until elution is complete. The resulting eluent contains β-lactoglobulin as the main component. In some specific embodiments, such elution can be performed once or multiple times.

[0133] In some specific embodiments, the eluent can be one or more types. In some specific embodiments, the inorganic salt in the eluent can be a sodium salt or a potassium salt, such as sodium chloride or potassium chloride. The concentration of the inorganic salt can be 0.5M-2M, for example, 1M.

[0134] In some embodiments, the chromatography packing material of the present invention has a dynamic binding capacity of 150 g / L to 200 g / L for β-lactoglobulin at a linear flow rate of less than 1000 cm / h, and the back pressure of the chromatography column is less than 2 bar, preferably less than 1.5 bar, and further less than 1 bar.

[0135] In some embodiments, according to the purification method using the chromatographic packing material described above, the α-lactalbumin content in the collected flow-through is greater than 40%, preferably greater than 45% (based on the total protein in the flow-through); and the β-lactoglobulin content in the collected eluent is greater than 75%, preferably greater than 80% (based on the total protein in the eluent).

[0136] In some embodiments, according to the purification method using the above-described chromatographic packing material, the protein recovery rate is 70% or more, preferably 75% or more, further preferably 80% or more, and more preferably 85% or more.

[0137] Example

[0138] 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 in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0139] Example 1:

[0140] (1) Dissolve the polymeric surfactant Span 80 in toluene to prepare an oil phase solution with a concentration of 2.0% (w / v) and maintain a stirring speed of 300 rpm at 60°C.

[0141] (2) Place cellulose powder and agarose powder (mass ratio 1:5) in an aqueous solution to prepare a 6% (w / v) suspension. Dissolve the powder completely by heating and stirring (95°C).

[0142] (3) Transfer the dissolved mixed sugar solution obtained in step (2) to a constant pressure dropping funnel, and add it dropwise to the preheated oil phase in step (1) under continuous stirring in the first reactor. At 60°C, add the crosslinking agent epichlorohydrin, the amount of which is 50% of the dry weight of the mixed sugar powder, and add 1 mol / L sodium hydroxide, and continue the reaction for 3 hours.

[0143] (4) After the reaction is complete, the system is allowed to cool naturally to room temperature. Stirring is stopped and the upper oil phase is removed. The obtained microspheres are washed repeatedly with acetone, 0.5 mol / L hydrochloric acid, deionized water and 0.5 mol / L sodium hydroxide solution. Finally, microspheres with a particle size range of 100 μm-350 μm are collected by wet sieving to obtain highly cross-linked composite agarose-cellulose-based sphere activation intermediate I.

[0144] (5) Transfer 1 L of activated intermediate I obtained in (4) to the second reactor, add 500 mL of dextran aqueous solution with a molecular weight of 150 kDa and a concentration of 500 mg / mL, and then add 100 g of sodium hydroxide. React at 50 °C for 12 hours. After the reaction, wash with deionized water until neutral to obtain dextran-grafted modified spherical intermediate II (Dv10 is about 138 μm, Dv95 is about 333 μm).

[0145] (6) Take 1 L of the wet bulb of intermediate II and place it in the third reactor. Add 200 mL of deionized water, 100 g of sodium hydroxide and 100 g of 2,3-epoxypropyltrimethylammonium chloride (GTA). React at 50 °C for 12 hours. After the reaction is complete, wash the solution sequentially with deionized water, 1 M sodium chloride solution, 0.1 M acetate buffer (pH 4.0) and 0.1 M Tris-HCl buffer (pH 8.0) until the conductivity and pH of the effluent are constant to completely remove unreacted reagents and ions.

[0146] Example 2:

[0147] (1) Dissolve the polymeric surfactant Span 80 in toluene to prepare an oil phase solution with a concentration of 2.0% (w / v) and maintain a stirring speed of 300 rpm at 60°C.

[0148] (2) Place cellulose powder and agarose powder (mass ratio of 0.5:5.5) in an aqueous solution to prepare a 6% (w / v) suspension. Dissolve the powder completely at 95°C with heating and stirring.

[0149] (3) Transfer the dissolved mixed sugar solution obtained in step (2) to a constant pressure dropping funnel, and add it dropwise to the preheated oil phase in step (1) under continuous stirring in the first reactor. At 60°C, add the crosslinking agent epichlorohydrin, the amount of which is 50% of the dry weight of the mixed sugar powder, and add 1 mol / L sodium hydroxide, and continue the reaction for 3 hours.

[0150] (4) After the reaction is complete, the system is allowed to cool naturally to room temperature. Stirring is stopped and the upper oil phase is removed. The obtained microspheres are washed repeatedly with acetone, 0.5 mol / L hydrochloric acid, deionized water and 0.5 mol / L sodium hydroxide solution. Finally, microspheres with a particle size range of 100 μm-350 μm are collected by wet sieving to obtain highly cross-linked composite agarose-cellulose-based sphere activation intermediate I.

[0151] (5) Transfer 1 L of activated intermediate I obtained in (4) to the second reactor, add 500 mL of dextran aqueous solution with a molecular weight of 150 kDa and a concentration of 500 mg / mL, and then add 100 g of sodium hydroxide. React at 50 °C for 12 hours, and wash with deionized water until neutral to obtain dextran-grafted modified spherical intermediate II.

[0152] (6) Take 1 L of the wet bulb of intermediate II and place it in the third reactor. Add 200 mL of deionized water, 100 g of sodium hydroxide and 100 g of 2,3-epoxypropyltrimethylammonium chloride (GTA). React at 50 °C for 12 hours. After the reaction is complete, wash the solution sequentially with deionized water, 1 M sodium chloride solution, 0.1 M acetate buffer (pH 4.0) and 0.1 M Tris-HCl buffer (pH 8.0) until the conductivity and pH of the effluent are constant to completely remove unreacted reagents and ions.

[0153] Example 3:

[0154] (1) Dissolve the polymeric surfactant Span 80 in toluene to prepare an oil phase solution with a concentration of 2.0% (w / v) and maintain a stirring speed of 300 rpm at 60°C.

[0155] (2) Place cellulose powder and agarose powder (mass ratio 1:5) in an aqueous solution to prepare a 6% (w / v) suspension. Dissolve the powder completely at 95°C with heating and stirring.

[0156] (3) Transfer the dissolved mixed sugar solution obtained in step (2) to a constant pressure dropping funnel, and add it dropwise to the preheated oil phase in step (1) under continuous stirring in the first reactor. At 60°C, add the crosslinking agent epichlorohydrin, the amount of which is 50% of the dry weight of the mixed sugar powder, and add 1 mol / L sodium hydroxide, and continue the reaction for 3 hours.

[0157] (4) After the reaction is complete, the system is allowed to cool naturally to room temperature. Stirring is stopped and the upper oil phase is removed. The obtained microspheres are washed repeatedly with acetone, 0.5 mol / L hydrochloric acid, deionized water and 0.5 mol / L sodium hydroxide solution. Finally, microspheres with a particle size range of 100 μm-350 μm are collected by wet sieving to obtain highly cross-linked composite agarose-cellulose-based sphere activation intermediate I.

[0158] (5) Transfer 1 L of activated intermediate I obtained in (4) to the second reactor, add 500 mL of dextran aqueous solution with a molecular weight of 150 kDa and a concentration of 500 mg / mL, and then add 100 g of sodium hydroxide. React at 50 °C for 12 hours, and wash with deionized water until neutral to obtain dextran-grafted modified spherical intermediate II.

[0159] (6) Take 1 L of the wet bulb of intermediate II and place it in the third reactor. Add 200 mL of deionized water, 100 g of sodium hydroxide and 80 g of 2,3-epoxypropyltrimethylammonium chloride (GTA). React at 50 °C for 12 hours. After the reaction is complete, wash the solution sequentially with deionized water, 1 M sodium chloride solution, 0.1 M acetate buffer (pH 4.0) and 0.1 M Tris-HCl buffer (pH 8.0) until the conductivity and pH of the effluent are constant to completely remove unreacted reagents and ions.

[0160] Comparative Example 1:

[0161] (1) Dissolve the polymeric surfactant Span 80 in toluene to prepare an oil phase solution with a concentration of 2.0% (w / v) and maintain a stirring speed of 300 rpm at 60°C.

[0162] (2) Place the agarose powder in an aqueous solution to prepare a 6% (w / v) suspension. Dissolve the powder completely at 95°C with heating and stirring.

[0163] (3) Transfer the dissolved sugar solution obtained in step (2) to a constant pressure dropping funnel, and add it dropwise to the preheated oil phase in step (1) under continuous stirring in the first reactor. At 60°C, add the crosslinking agent epichlorohydrin at 50% of the dry weight of the mixed sugar powder, and add 1 mol / L sodium hydroxide, and continue the reaction for 3 hours.

[0164] (4) After the reaction is complete, the system is allowed to cool naturally to room temperature. Stirring is stopped and the upper oil phase is removed. The obtained microspheres are washed repeatedly with acetone, 0.5 mol / L hydrochloric acid, deionized water and 0.5 mol / L sodium hydroxide solution. Finally, microspheres with a particle size range of 100 μm-350 μm are collected by wet sieving to obtain highly cross-linked agarose-based activated intermediate I.

[0165] (5) Transfer 1 L of activated intermediate I obtained in (4) to the second reactor, add 500 mL of dextran aqueous solution with a molecular weight of 150 kDa and a concentration of 500 mg / mL, and then add 100 g of sodium hydroxide. React at 50 °C for 12 hours, and wash with deionized water until neutral to obtain dextran-grafted modified spherical intermediate II.

[0166] (6) Take 1 L of the wet bulb of intermediate II and place it in the third reactor. Add 200 mL of deionized water, 100 g of sodium hydroxide and 100 g of 2,3-epoxypropyltrimethylammonium chloride (GTA). React at 50 °C for 12 hours. After the reaction is complete, wash the solution sequentially with deionized water, 1 M sodium chloride solution, 0.1 M acetate buffer (pH 4.0) and 0.1 M Tris-HCl buffer (pH 8.0) until the conductivity and pH of the effluent are constant to completely remove unreacted reagents and ions.

[0167] Comparative Example 2:

[0168] (1) Dissolve the polymeric surfactant Span 80 in toluene to prepare an oil phase solution with a concentration of 2.0% (w / v) and maintain a stirring speed of 300 rpm at 60°C.

[0169] (2) Place cellulose powder and agarose powder (mass ratio of 0.5:5.5) in an aqueous solution to prepare a 6% (w / v) suspension. Dissolve the powder completely at 95°C with heating and stirring.

[0170] The dissolved mixed sugar solution obtained in step (2) was transferred to a constant pressure dropping funnel and added dropwise to the preheated oil phase of step (1) in the first reactor under continuous stirring. At 60°C, epichlorohydrin, a crosslinking agent, was added at an amount equal to 50% of the dry weight of the mixed sugar powder, and 1 mol / L sodium hydroxide was added. The reaction was continued for 3 hours.

[0171] (4) After the reaction is complete, the system is allowed to cool naturally to room temperature. Stirring is stopped and the upper oil phase is removed. The obtained microspheres are washed repeatedly with acetone, 0.5 mol / L hydrochloric acid, deionized water and 0.5 mol / L sodium hydroxide solution. Finally, microspheres with a particle size range of 150 μm-250 μm are collected by wet sieving to obtain highly cross-linked composite agarose-cellulose-based sphere activation intermediate I.

[0172] (5) Transfer 1 L of activated intermediate I obtained in (4) to the second reactor, and add 200 mL of deionized water, 100 g of sodium hydroxide and 100 g of 2,3-epoxypropyltrimethylammonium chloride (GTA). React at 50 °C for 12 hours. After the reaction is complete, the solution is circulated and washed sequentially with deionized water, 1 M sodium chloride solution, 0.1 M acetate buffer (pH 4.0) and 0.1 M Tris-HCl buffer (pH 8.0) until the conductivity and pH of the effluent are constant, so as to completely remove unreacted reagents and ions.

[0173] Comparative Example 3:

[0174] (1) Dissolve the polymeric surfactant Span 80 in toluene to prepare an oil phase solution with a concentration of 2.0% (w / v) and maintain a stirring speed of 300 rpm at 60°C.

[0175] (2) Place cellulose powder and agarose powder (mass ratio 1:5) in an aqueous solution to prepare a 6% (w / v) suspension. Dissolve the powder completely at 95°C with heating and stirring.

[0176] (3) Transfer the dissolved mixed sugar solution obtained in step (2) to a constant pressure dropping funnel, and add it dropwise to the preheated oil phase in step (1) under continuous stirring in the first reactor. At 60°C, add the crosslinking agent epichlorohydrin, the amount of which is 50% of the dry weight of the mixed sugar powder, and add 1 mol / L sodium hydroxide, and continue the reaction for 3 hours.

[0177] (4) After the reaction is complete, the system is allowed to cool naturally to room temperature. Stirring is stopped and the upper oil phase is removed. The obtained microspheres are washed repeatedly with acetone, 0.5 mol / L hydrochloric acid, deionized water and 0.5 mol / L sodium hydroxide solution. Finally, microspheres with a particle size range of 150 μm-250 μm are collected by wet sieving to obtain highly cross-linked composite agarose-cellulose-based sphere activation intermediate I.

[0178] (5) Transfer 1 L of activated intermediate I obtained in (4) to the second reactor, add 500 mL of dextran aqueous solution with a molecular weight of 150 kDa and a concentration of 500 mg / mL, and then add 100 g of sodium hydroxide. React at 50 °C for 12 hours, and wash with deionized water until neutral to obtain dextran-grafted modified spherical intermediate II.

[0179] (6) Take 1 L of the wet bulb of intermediate II and place it in the third reactor. Add 200 mL of deionized water, 100 g of sodium hydroxide and 160 g of 2,3-epoxypropyltrimethylammonium chloride (GTA). React at 50 °C for 12 hours. After the reaction is complete, wash the solution sequentially with deionized water, 1 M sodium chloride solution, 0.1 M acetate buffer (pH 4.0) and 0.1 M Tris-HCl buffer (pH 8.0) until the conductivity and pH of the effluent are constant to completely remove unreacted reagents and ions.

[0180] Experimental Example 1

[0181] The chromatography packing materials obtained in Example 1 and Comparative Example 1 were subjected to pressure-flow rate tests. The pressure-flow rate curve testing conditions were: 25°C using purified water, 300 mm column, and 20 cm column height.

[0182] according to Figure 3The pressure-flow-rate curves of the chromatography packing material shown are as follows: Comparative Example 1 uses a single-material agarose-based spheres with a maximum linear flow rate of 2000 cm / h, while Example 1 uses composite agarose-cellulose-based spheres with a maximum linear flow rate of 2500 cm / h. The latter exhibits better mechanical properties and lower back pressure at the same flow rate. Lower back pressure means that it can withstand higher loading flow rates, process more samples in the same time, and has a higher number of reusable packing materials, which is beneficial for large-scale production applications.

[0183] Experiment Example 2

[0184] Cross sections of the chromatography packing materials obtained in Example 2 and Comparative Example 1 were scanned using electron microscopy. The electron microscopy parameters are as follows: Figure 4 , Figure 5 As shown.

[0185] according to Figure 4 , Figure 5 The cross-sectional electron micrographs shown show that, compared to Comparative Example 1, the internal structure of the packing material in Example 2 is more compact. This means that the packing material has stronger support and pressure resistance, and also provides more sites for ligand coupling and protein binding, demonstrating the advantages of the composite agarose-cellulose spheres from a microscopic perspective.

[0186] Experimental Example 3

[0187] The ligand density and standard protein loading of the chromatography packing materials obtained in Examples 1, 2, 3 and Comparative Example 3 were determined. Specifically, the determinations were performed according to the following methods.

[0188] <Determination of Ligand Density (Ion Loading) by Titration>

[0189] 1) The packing material to be tested is packed into a pre-packed column of fixed volume, with a column bed volume of 5 ml;

[0190] 2) Wash the chromatography column with 30 mL of 1 M NaCl;

[0191] 3) Rinse with 20mL of purified water;

[0192] 4) Rinse with 20 mL of 0.5 M sodium acetate for ion exchange, and collect the effluent into a 250 mL Erlenmeyer flask;

[0193] 5) Rinse with 20mL of purified water;

[0194] 6) The collected effluent was diluted to 100 mL with purified water and 5 drops of potassium chromate indicator were added.

[0195] 7) Titrate the sample collection solution with 0.1M AgNO3 standard solution. The titration endpoint is when the solution changes from yellow to orange-red and remains unchanged for 30 seconds. Record the volume of standard solution used in the titration, V1.

[0196] Formula for calculating ion loading:

[0197] Cl ion loading per mL of packing material (mmol / mL) = (C AgNO3 ×V AgNO3 )÷Vm

[0198] C AgNO3 Concentration (M) of AgNO3 standard solution

[0199] V AgNO3 Volume (mL) of AgNO3 consumed during sample titration.

[0200] Vm: Column volume of chromatography (mL)

[0201] <10% flow-through assay for protein loading (dynamic protein loading)>

[0202] Chromatography column: Pack the test packing material into a 5 ml column volume chromatography column, with a column bed height of 10 cm.

[0203] Mobile phase A (Buffer A): 20 mM PB, pH 6.5;

[0204] Mobile phase B (Buffer B): 20 mM PB + 0.5 M sodium chloride, pH 6.5;

[0205] Retention time: 2 minutes

[0206] Sample: β-lactoglobulin dissolved in Buffer A, concentration 4 mg / ml

[0207] The experiment was conducted using a chromatography system programmed with a detection wavelength of 280 nm. The chromatography column was connected to the system, and the flow rate was set to 2.5 ml / min. The column was washed with mobile phase B for 3 cv and equilibrated with mobile phase A for 10 cv. The sample was loaded using a sample pump until the absorbance at 280 nm reached 10% of the peak height for complete flow-through, at which point loading was stopped, and the loading volume V1 was recorded. The column was washed with mobile phase A for 10 cv and eluted with mobile phase B for 10 cv. The eluent was collected, and the elution volume V2 was recorded. The protein concentration C1 was then determined.

[0208] Formula for calculating dynamic protein load:

[0209] Dynamic protein loading per mL of filler = Sample loading volume V1 × Sample concentration / Column bed volume

[0210] Formula for calculating protein recovery rate:

[0211] Protein recovery rate = (elution volume V2 × protein concentration C1) / (sample loading volume V1 × sample concentration) × 100%

[0212] Table 1. Ligand density and standard protein loading of chromatography packing materials

[0213]

[0214] According to Table 1, the chromatography packing material of Comparative Example 3, which had the highest ligand density in the comparative tests, had the highest protein loading capacity, but its protein recovery rate was low. This is presumably because the excessively high ligand density caused some proteins to bind too tightly to the packing material, resulting in loss and inability to be recovered. Therefore, selecting an appropriate ligand density that balances high protein binding capacity and high recovery rate is crucial. Besides the significant correlation with ligand density, protein loading capacity can be further optimized by adjusting factors such as the pore size of the packing material and the molecular weight of the grafted dextran.

[0215] Experiment Example 4

[0216] The chromatographic packing materials obtained in Examples 1 and 2 and Comparative Examples 1 and 2 were packed with samples according to the following steps, and the microfiltered whey was separated and purified by chromatography.

[0217] 1) Take 4L of chromatography packing material and pack it into a 140mm manual chromatography column. The column height is 23cm and the column volume is 3.54L.

[0218] 2) Obtain 500L of microfiltered whey from fresh skim milk via membrane separation;

[0219] 3) Rinse the chromatography column with 10 column volumes of RO water or a solution containing inorganic salts.

[0220] 4) The whey described in 2) is loaded onto the chromatography column at a linear velocity of 600 cm / h;

[0221] 5) Start collecting the column eluent when the UV absorption rises, and continue collecting until the sample loading is complete;

[0222] 6) Rinse the chromatography column with 5 column volumes of RO water;

[0223] 7) Elute the chromatography column with 5 column volumes of 1M NaCl solution and collect the eluent based on the UV absorption peak.

[0224] comprehensive Figure 6 , Figure 7 , Figure 8The chromatographic and electrophoretic patterns shown indicate that the chromatographic packing material of Comparative Example 1 exhibited β-lactoglobulin flow-through overloading at 100 CV (left red box), while the chromatographic packing material of Example 1 exhibited β-lactoglobulin flow-through overloading at 120 CV (right red box). This demonstrates that the composite agarose-cellulose packing material has a higher loading capacity and a larger sample throughput. The chromatographic packing material of Comparative Example 2, lacking dextran grafting, had a sample throughput of only 70 CV, while the chromatographic packing material of Example 2, which was modified with dextran grafting, had a sample throughput of 110 CV.

[0225] according to Figure 9 , Figure 10 The capillary electrophoresis results of the samples before and after purification are shown. The samples were separated and purified using the chromatographic packing material obtained in Example 1. After purification, the peak area ratio of α-lactalbumin in the sample (elution) increased from 39.62% before purification to 81.35%, which shows that the separation and purification of α-lactalbumin and β-lactoglobulin is significant.

[0226] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0227] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A chromatographic packing material for separating and purifying α-lactalbumin and β-lactoglobulin, characterized in that, The packing material is used to separate and purify α-lactalbumin and β-lactoglobulin by treating whey protein. in, The filler has a porous spherical structure, the spherical structure comprising a base sphere and a modifier, the modifier being at least partially present on the surface of the base sphere. The base sphere comprises a natural polysaccharide containing cross-linked agarose and cellulose, and the base sphere is dextran-derived. The modifier includes an ion-exchange ligand, and the ion-exchange ligand is covalently linked to the dextran and the spheroid. The ion exchange ligand comprises a quaternary ammonium group or its salt, and the coupling density of the ion exchange ligand of the packing material is 150 μmol / mL-250 μmol / mL.

2. The chromatography packing material according to claim 1, characterized in that, The particle size distribution range of the filler is 100μm-350μm.

3. The chromatography packing material according to claim 1 or 2, characterized in that, In the base sphere, the mass ratio of cellulose to agarose can be 1:1 to 1:10; the base sphere is essentially composed of cross-linked natural polysaccharides.

4. The chromatography packing material according to any one of claims 1 to 3, characterized in that, The molecular weight of the dextran is 10kDa-300kDa.

5. The chromatography packing material according to any one of claims 1 to 4, characterized in that, The surface pore size of the filler with the porous spherical structure is set to be sufficient to allow protein molecules with a molecular weight of 10kDa-2000kDa to diffuse into the interior of the sphere.

6. The method for preparing the chromatography packing material according to any one of claims 1 to 5, characterized in that, The method includes the following steps: The emulsification and crosslinking steps involve emulsifying and crosslinking agarose and cellulose to form the matrix spheres; The grafting derivatization step involves grafting the dextran onto the base sphere; The ligand coupling step involves reacting the grafted and derivatized base spheres with a compound containing a ligand so that the ligand is covalently bonded to the dextran and the base spheres.

7. The chromatography packing material according to claim 6, characterized in that, In the emulsification and crosslinking step, the emulsification and crosslinking are carried out under alkaline conditions and in the presence of a crosslinking agent, wherein the crosslinking agent is selected from one or more epoxy compounds or aldehyde compounds.

8. The preparation method according to claim 6 or 7, characterized in that, The compound containing a ligand is an epoxy compound containing a ligand.

9. A purification and separation method using the chromatographic packing material according to any one of claims 1 to 5, characterized in that, The method includes the following steps: (1) After defatting the milk raw material, it is filtered through a membrane to obtain whey protein with casein removed; (2) The whey obtained in step (1) is loaded onto a chromatography column containing the chromatography packing material according to any one of claims 1 to 5 for chromatography. Afterwards, pure water and inorganic salt are used as mobile phases for rebalancing and elution, respectively. (3) Collect the flow-through and eluent from step (2) respectively to obtain the flow-through with α-lactalbumin as the main component and the eluent with β-lactoglobulin as the main component.

10. The purification and separation method according to claim 9, wherein the packing material has a dynamic binding capacity of 150 g / L to 200 g / L for β-lactoglobulin at a linear flow rate of whey protein below 1000 cm / h, and the back pressure of the chromatography column is less than 2 bar.

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