Method for separating and purifying osteopontin from human milk

Abstract

The invention relates to a method for separating and purifying osteopontin from human milk, which comprises the following steps: (1) pretreatment: collecting human milk and pretreating the human milk to remove fat and cell debris impurities to obtain pretreated emulsion; (2) osteopontin precipitation: adding a precipitator into the pretreated emulsion to precipitate osteopontin, and collecting the precipitate; (3) osteopontin dissolution: adding a solvent capable of dissolving osteopontin into the obtained precipitate, fully mixing, and then removing insoluble substances to obtain an osteopontin crude solution; (4) dialyzing and concentrating: dialyzing and concentrating the osteopontin crude solution to obtain an osteopontin concentrated solution; (5) chromatography: purifying the osteopontin concentrated solution in a chromatography manner to obtain an osteopontin purified solution; and (6) drying: drying the osteopontin purified liquid to obtain osteopontin powder. By adopting the method disclosed by the invention, the osteopontin purity of 95% or more by mass can be efficiently realized.

Description

Technical Field

[0001] This invention relates generally to the food industry, and more specifically to a method for isolating and purifying osteopontin (OPN) from human milk. Background Technology

[0002] Osteopontin (OPN) is a highly phosphorylated glycoprotein with important biological functions, widely distributed in various biological fluids and tissues. It plays multiple key roles in human physiological activities: In the skeletal system, OPN directly participates in and regulates the formation, mineralization, and remodeling of bone tissue. By regulating the activity of osteoblasts and osteoclasts, it promotes the absorption and deposition of calcium ions, which is crucial for the normal development and healthy maintenance of bones in infants and young children. In terms of immune regulation, OPN can affect the function of various immune cells (such as T cells and macrophages), promote the secretion of specific cytokines, thereby enhancing the body's innate immune defense and adaptive immune response, helping infants and young children effectively resist pathogen invasion. Furthermore, OPN also plays a role in cell adhesion, migration, signal transduction, and tissue repair, making it a bioactive substance with comprehensive benefits for early life development.

[0003] Human milk is one of the natural sources of OPN, with relatively high levels, especially in colostrum. This endogenous OPN is believed to exist in its natural conformation and may possess optimal biocompatibility and functional activity. Therefore, OPN in human milk not only provides important nutritional support for newborns but is also a key component of their passive immune protection system. However, current infant formula products face significant challenges in mimicking the OPN composition of human milk. The high temperatures and sterilization processes typically involved in production can easily lead to protein denaturation and inactivation, and exogenously added OPN has limitations in its source, content, and active form. This results in the effective content and bioactivity of OPN in the final product being far lower than in human milk, making it difficult to fully meet the comprehensive needs of infant growth and development.

[0004] Currently, OPN isolation and purification technologies mostly focus on obtaining it from raw materials such as animal tissues (e.g., cow's milk) or recombinant cell culture systems. While these methods provide a foundation for OPN research and application, efficient and large-scale preparation processes specifically for human milk remain lacking. Existing methods for extracting OPN from human milk often suffer from problems such as cumbersome procedures, multiple separation steps, low target protein recovery rates, unsatisfactory purity, and high costs. Insufficient purification efficiency and purity have become the core technical bottleneck restricting the large-scale preparation of highly active human milk-derived OPN and its application in high-end nutritional products.

[0005] Therefore, it is desirable to develop a method that can efficiently and effectively separate and purify OPN from human milk. Summary of the Invention

[0006] This invention was made in view of the aforementioned problems existing in the prior art.

[0007] Specifically, the present invention is achieved by the following:

[0008] 1. A method for isolating and purifying osteopontin from human milk, comprising the following steps:

[0009] (1) Pretreatment: Human milk is collected and pretreated to remove fat and cell debris impurities to obtain pretreated emulsion;

[0010] (2) Osteopontoprotein precipitation: Add a precipitant to the pretreated emulsion to precipitate osteopontin, and collect the precipitate;

[0011] (3) Osteopontin dissolution: Add a solvent that can dissolve osteopontin to the obtained precipitate, mix thoroughly, and then remove the insoluble matter to obtain a crude osteopontin solution;

[0012] (4) Dialysis and concentration: The crude osteopontin solution was dialyzed and concentrated to obtain osteopontin concentrate;

[0013] (5) Chromatography: The osteopontin concentrate was purified by chromatography to obtain a purified osteopontin solution; and

[0014] (6) Drying: The osteopontin purification solution is dried to obtain osteopontin powder.

[0015] 2. The method as described in Project 1, wherein the human milk is colostrum or mature milk.

[0016] 3. The method as described in any one of items 1-2, wherein in step (1), fat and cell debris impurities are removed by centrifugation;

[0017] Preferably, the centrifugation is carried out at a temperature below room temperature, for example, 0-10°C, at a speed of 3000-20000 rpm for 5-60 minutes.

[0018] 4. The method of any one of items 1-3, wherein in step (1), the human milk is sterilized, for example, by pasteurization, before removing fat and cell debris impurities.

[0019] 5. The method of any one of items 1-4, wherein in step (2), the precipitant is one or more selected from: ammonium sulfate or ethanol;

[0020] Preferably, in step (2):

[0021] A precipitant is gradually added while stirring the pretreated emulsion.

[0022] After adding the precipitant, allow the resulting solution to stand, for example, for 1-3 hours.

[0023] Step (2) is performed at room temperature or below room temperature, for example, 0-10°C, and / or

[0024] The precipitate is collected by centrifugation.

[0025] 6. The method as described in any one of items 1-5, wherein in step (3), the solvent is a phosphate buffer solution;

[0026] Preferably, where:

[0027] The conjugate acid and base in the phosphate buffer solution are sodium dihydrogen phosphate and disodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

[0028] The concentration of the phosphate buffer is 0.005-0.05 mol / L, where the concentration refers to the total concentration of the conjugate acid and base in the buffer; and / or

[0029] The pH value of the phosphate buffer solution is 5.8-7.8, preferably 6.5-7.5, for example 7.4.

[0030] 7. The method described in any one of items 1-6, wherein insoluble matter is removed by centrifugation or filtration in step (3).

[0031] 8. The method described in any one of items 1-7, wherein in step (4), dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500-5000 Da;

[0032] Concentration is achieved through ultrafiltration.

[0033] 9. The method of any one of items 1-8, wherein in step (5), the chromatography sequentially comprises: ion exchange chromatography, gel filtration chromatography, and affinity chromatography; preferably, the ion exchange chromatography uses 50 mM, pH 8.0 Tris-HCl buffer as equilibration buffer and elution buffer, the gel filtration chromatography uses 20 mM, pH 7.4 phosphate buffer as elution buffer, and the affinity chromatography uses 0.1 M, pH 8.5 Tris-HCl buffer as elution buffer.

[0034] 10. The method of any one of items 1-9, wherein in step (6), the drying is freeze drying.

[0035] The method of this invention enables efficient separation of OPN, yielding high-purity OPN that meets the quality requirements for infant formula additives. Furthermore, the method of this invention maximizes the preservation of OPN's bioactivity, thus allowing for its effective use in infant formula production. Attached Figure Description

[0036] Figure 1 A flowchart illustrating an embodiment of the method according to the present invention is shown schematically.

[0037] Figure 2 An embodiment of chromatography employed in the method according to the present invention is illustrated schematically. Detailed Implementation

[0038] Unless otherwise specified, the technical terms in this specification have the same meaning as those generally understood by those skilled in the art; however, in case of any conflict, the definitions in this specification shall prevail.

[0039] Furthermore, in the context of this invention, the terms "comprising" or "including" do not exclude other possible elements. The methods of this invention (including the various embodiments described herein) may comprise, consist of, or substantially consist of the following elements: the essential elements and necessary limitations of the invention as described herein, and any other or optional ingredients, components, steps, or limitations as described herein or as otherwise desired.

[0040] In this application, unless otherwise stated, when referring to the percentage composition of the eluent, it refers to the volume percentage; when referring to other percentage compositions, it refers to the mass composition.

[0041] In this application, unless otherwise stated, when referring to the concentration of a solid solute in a solution of a liquid solvent, it refers to the mass concentration; when referring to the concentration of a liquid solute in a solution of a liquid solvent, it refers to the volume concentration.

[0042] Unless otherwise specified, temperature in this application refers to room temperature (25°C).

[0043] It should be noted that the various aspects, features, implementation methods, experimental examples, and advantages described in this application are compatible and / or can be combined together.

[0044] This invention relates to a method for isolating and purifying osteopontin from human milk, comprising the following steps:

[0045] (1) Pretreatment: Human milk is collected and pretreated to remove fat and cell debris impurities to obtain pretreated emulsion;

[0046] (2) Osteopontoprotein precipitation: Add a precipitant to the pretreated emulsion to precipitate osteopontin, and collect the precipitate;

[0047] (3) Osteopontin dissolution: Add a solvent that can dissolve osteopontin to the obtained precipitate, mix thoroughly, and then remove the insoluble matter to obtain a crude osteopontin solution;

[0048] (4) Dialysis and concentration: The crude osteopontin solution was dialyzed and concentrated to obtain osteopontin concentrate;

[0049] (5) Chromatography: The osteopontin concentrate was purified by chromatography to obtain a purified osteopontin solution; and

[0050] (6) Drying: The osteopontin purification solution is dried to obtain osteopontin powder.

[0051] Figure 1 A flowchart illustrating the method according to the present invention is shown schematically. Reference will be made below. Figure 1 The method will be described in detail.

[0052] Step (1): Preprocessing

[0053] In step (1), human milk is collected and pretreated to remove fat and cell debris impurities, resulting in a pretreated emulsion. That is, the pretreatment includes the removal of fat and cell debris impurities.

[0054] The human milk may be colostrum or mature milk. The terms "colostrum" and "mature milk" have their well-known meanings in the art. "Colostrum" refers to the milk secreted in the first few days after childbirth (usually 2-5 days postpartum). "Mature milk" refers to the milk secreted after the colostrum period, approximately 10-15 days postpartum, when its composition has entered a relatively stable stage.

[0055] Fat and cell debris impurities can be removed by any means known in the art. In some embodiments, fat and cell debris impurities can be removed by centrifugation.

[0056] The pretreatment / centrifugation may be carried out, for example, at a temperature below room temperature (e.g., 0-10°C, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C, or any range thereof) to reduce the solubility of fat, thereby achieving better separation of fat. The centrifugation may be carried out, for example, at a speed of 3000-20000 rpm (e.g., 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 rpm, or any range thereof). The centrifugation can be performed for, for example, 5-60 minutes (e.g., 5, 10, 15, 20, 25, 30, 40, 45, 50, 60 minutes, or a range defined by any two thereof). Fat, with its relatively low density, floats to the surface under centrifugation, forming a fat layer (cream layer). Cell debris, with its relatively high density, settles under centrifugation, forming a precipitate. By discarding the upper fat layer and collecting the intermediate aqueous phase, both fat and cell debris are removed.

[0057] By using centrifugation, fat and cell debris impurities can be effectively removed, while protein denaturation can be effectively prevented and the quality of the final product can be improved.

[0058] In some embodiments, the human milk may be sterilized before removing fat and cellular debris impurities.

[0059] The sterilization treatment can be carried out by any suitable method known in the art. In some embodiments, the sterilization treatment is pasteurization. The pasteurization treatment can be carried out, for example, at a temperature of 62-65°C (e.g., 62, 63, 64, 65°C, or within the range defined by any two thereof) for, for example, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 minutes, or within the range defined by any two thereof.

[0060] (2) Osteopontin precipitation

[0061] In step (2), a precipitant is added to the pretreated emulsion obtained in step (1) to precipitate osteopontin and the precipitate is collected.

[0062] In some embodiments, the precipitant is one or more selected from ammonium sulfate or ethanol.

[0063] In some embodiments, a precipitant may be gradually added while the pretreated emulsion is being stirred.

[0064] In some embodiments, the resulting solution is allowed to stand, for example, for 1-3 hours, after the addition of the precipitant is completed, to allow for sufficient precipitation.

[0065] This step can be performed at room temperature. Alternatively, this step can be performed at a temperature below room temperature (e.g., 0-10°C, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C, or any two thereof) to reduce the solubility of osteopontin, thereby increasing the recovery / purity of osteopontin.

[0066] After pretreatment, the resulting emulsion contains not only osteopontin but also other proteins such as whey proteins (e.g., α-lactalbumin, lactoferrin), immunoglobulins (IgA, IgG, etc.), lysozyme, casein and casein macropeptide, some lipid-related substances such as membrane proteins, lipoproteins, or proteins related to the fat globule membrane, as well as polysaccharide complexes or nucleic acid fragments. By adding a precipitating agent, osteopontin co-precipitates with other substances, while a large number of water-soluble small molecule impurities remain in solution. This achieves preliminary enrichment and concentration of OPN, which helps improve the purity of the final obtained osteopontin.

[0067] The precipitate is then collected. This precipitate can be collected by centrifugation. Centrifugation promotes the separation of the precipitate from the liquid phase. The centrifugation can be performed, for example, at a temperature below room temperature (e.g., 0-10°C, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C, or any range defined by both). The centrifugation can be performed, for example, at a speed of 3000-20000 rpm (e.g., 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 rpm, or any range defined by both). The centrifugation may be performed for, for example, 5-60 minutes (e.g., 5, 10, 15, 20, 25, 30, 40, 45, 50, 60 minutes, or a range defined by any two thereof).

[0068] (3) Osteopontoprotein dissolution

[0069] In step (3), a solvent capable of dissolving osteopontin is added to the precipitate obtained in step (2), and the mixture is thoroughly mixed. Then, the insoluble matter is removed to obtain a crude osteopontin solution.

[0070] In some embodiments, the solvent is a solvent that at least partially and selectively dissolves osteopontin, such as phosphate buffer.

[0071] The conjugate acid and base in the phosphate buffer solution may be sodium dihydrogen phosphate and disodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate; the concentration of the phosphate buffer solution may be, for example, 0.005-0.05 mol / L, such as 0.005, 0.010, 0.015, 0.02, 0.03, 0.04, 0.05 mol / L, or within the range defined by any two thereof; more preferably 0.01 mol / L; wherein the concentration refers to the total concentration of the conjugate acid and base in the buffer solution.

[0072] The pH value of the phosphate buffer solution may be, for example, 5.8-7.8, preferably 6.5-7.5, such as 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, or within the range defined by any two thereof; more preferably 7.4.

[0073] After adding the solvent, mix thoroughly to ensure that osteopontin is fully dissolved.

[0074] Dissolving the precipitate with a solvent such as phosphate buffer can dissolve osteopontin while leaving at least some other substances in the precipitate undissolved. Removing these insoluble substances helps to improve the purity of the final osteopontin product.

[0075] Insoluble matter can be removed by any suitable method known in the art. For example, it can be removed by centrifugation or filtration.

[0076] (4) Dialysis and Concentration

[0077] In step (4), the crude osteopontin solution obtained in step (3) is dialyzed and concentrated to obtain osteopontin concentrate.

[0078] The dialysis can be performed by any suitable method known in the art. The dialysis conditions can be readily determined by those skilled in the art based on factors such as the molecular weight of osteopontin. For example, the dialysis can be performed using a dialysis bag with a molecular weight cutoff of 3500-5000 Da; during dialysis, the dialysis fluid (e.g., phosphate buffer) can be changed every, for example, 2-8 hours (e.g., 2, 3, 4, 5, 6, 7, 8 hours, or within any two of these ranges), and dialysis can be continued for, for example, 12-48 hours (e.g., 12, 18, 24, 30, 36, 42, 48 hours, or within any two of these ranges). Dialysis effectively / sufficiently removes small molecule impurities and salts.

[0079] After dialysis, the postdialysis solution can be concentrated to obtain osteopontin concentrate.

[0080] The concentration can be performed by any means known in the art. In some embodiments, the concentration can be performed by ultrafiltration.

[0081] (5) Chromatography

[0082] In step (5), the osteopontin concentrate obtained in step (4) is purified by chromatography to obtain osteopontin purified solution.

[0083] Figure 2 An embodiment of chromatography employed in the method according to the present invention is illustrated schematically.

[0084] Reference Figure 2 In some embodiments, the chromatography may sequentially include: ion exchange chromatography, gel filtration chromatography, and affinity chromatography.

[0085] Ion exchange chromatography may include the following steps: equilibrating the ion exchange chromatography column with equilibration buffer, loading osteopontin purification solution onto the column, washing the column, eluting with elution buffer, and collecting the peak containing OPN.

[0086] Gel filtration chromatography may include the following steps: loading the OPN solution after ion exchange chromatography onto a gel filtration chromatography column, eluting with elution buffer and collecting the peak containing OPN;

[0087] Affinity chromatography may include the following steps: equilibrating the affinity chromatography column with equilibration buffer, loading the OPN solution after gel filtration chromatography onto the column, washing, eluting with elution buffer, and collecting the peak containing OPN.

[0088] In some implementations, for ion exchange chromatography, the elution buffer may be the same as the equilibration buffer.

[0089] In some embodiments, ion exchange chromatography may use Q Sepharose Fast Flow as the medium. In some embodiments, ion exchange chromatography may use 50 mM, pH 8.0 Tris-HCl buffer as both the equilibration and elution buffers. In some embodiments, the flow rate of the elution buffer in ion exchange chromatography is 2.5 ml / min.

[0090] In some embodiments, gel filtration chromatography may use Sephacryl S-200 HR as the medium. In some embodiments, gel filtration chromatography may use phosphate-buffered saline (PBS) (conjugate acid-base pair such as sodium dihydrogen phosphate and disodium hydrogen phosphate) (e.g., the phosphate-buffered saline described above in step "(3) osteopontin dissolution"), for example, 20 mM, pH 7.4 phosphate-buffered saline (PBS) (conjugate acid-base pair such as sodium dihydrogen phosphate and disodium hydrogen phosphate) as the eluent. In some embodiments, the flow rate of the eluent in gel filtration chromatography is 1 ml / min.

[0091] In some embodiments, the affinity chromatography conditions may use Sepharose 4B as the medium. In some embodiments, affinity chromatography may use 0.1 M, pH 8.5 Tris-HCl buffer as the elution buffer. In some embodiments, the flow rate of the elution buffer in affinity chromatography is 1 ml / min. In some embodiments, neutralization with 1 M, pH 9.0 Tris-HCl buffer is performed immediately after elution.

[0092] The protein was initially separated by ion exchange chromatography, further purified by gel filtration chromatography, and finally obtained high-purity osteopontin (OPN) by affinity chromatography.

[0093] By employing the method of the present invention, the purity of osteopontin in the purified osteopontin solution obtained by chromatography can reach more than 95% by mass, which is significantly higher than the level achievable by existing technologies.

[0094] (6) Drying

[0095] In step (6), the osteopontin purified solution obtained in step (5) is dried to obtain osteopontin powder.

[0096] In some embodiments, the drying may be freeze-drying.

[0097] In some embodiments, the freeze-drying is performed using a gradient cooling procedure. For example, it can be performed by pre-freezing at -40°C for 2 hours, followed by freeze-drying at -80°C for 48 hours.

[0098] By employing freeze-drying, the bioactivity of osteopontin can be preserved to the greatest extent.

[0099] By employing the method of this invention, OPN can be efficiently separated to obtain high-purity OPN, meeting the quality requirements for infant formula additives. Furthermore, the method of this invention can also maximize the preservation of OPN's bioactivity, allowing it to exert its physiological functions in infant formula, such as promoting infant immunity and bone development, thus enabling its effective use in infant formula production.

[0100] Experimental Example

[0101] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to experimental examples. It should be understood that the specific experimental examples described herein are merely illustrative and not intended to limit the invention. Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional in the art.

[0102] Example 1

[0103] Colostrum from healthy lactating women was collected and immediately stored at 4°C and pasteurized within 6 hours, wherein pasteurization was performed by holding at 62°C for 30 minutes. The pasteurized colostrum was immediately used for the next step. 100 mL of the pasteurized colostrum sample was placed in a centrifuge and centrifuged at 4000 rpm for 15 minutes at 4°C to remove the upper fat layer. The intermediate supernatant was collected, and the lower precipitate (containing cell debris) was discarded.

[0104] Slowly add solid ammonium sulfate to the resulting clear liquid while stirring until the concentration of ammonium sulfate reaches 30% (w / v). Let the resulting mixture stand at 4°C for 2 hours, then centrifuge at 8000 r / min for 20 minutes and collect the precipitate.

[0105] Add 10 mL of phosphate-buffered saline (PBS) pre-cooled to 4°C (pH 7.4, 0.01 mol / L, conjugate acid-base pair of sodium dihydrogen phosphate-disodium hydrogen phosphate) to the precipitate and stir thoroughly to dissolve the precipitate completely. Then filter through a 0.45 μm filter membrane and collect the filtrate.

[0106] The filtrate was dialyzed using a 3500 Da molecular weight cutoff MD45 dialysis bag from the United States in approximately 100 times the volume of the solution in the bag in PBS buffer as the dialysate. The dialysate was changed every 4 hours during the dialysis process, and the dialysis was continued for 24 hours to remove small molecule impurities and salts. Subsequently, the solution was concentrated to 1 / 10 of its original volume using ultrafiltration technology.

[0107] The ultrafiltration concentrated sample was further purified by sequentially passing it through a DEAE-Sepharose ion exchange chromatography column (HiPrep DEAE FF 16 / 10, HiPrep Biotech (Hangzhou) Co., Ltd.), a Sephadex G-75 gel filtration chromatography column (Beijing Biosen Biotechnology Co., Ltd., D08006), and an anti-OPN antibody affinity chromatography column (prepared in-house using the following method: anti-human OPN polyclonal antibody (Thermo Fisher Scientific, PA1-86649) was covalently coupled to CNBr-activated agarose gel 4B (HiPrep Biotech (Hangzhou) Co., Ltd.) as a ligand and packed into a chromatography column (Beijing Solarbio Technology Co., Ltd.) for further purification). The conditions for ion exchange chromatography were as follows: Q Sepharose Fast Flow was used as the medium; 50 mM, pH 8.0 Tris-HCl was used as the equilibration buffer and elution buffer, and the flow rate was 2.5 ml / min. The conditions for gel filtration chromatography were as follows: Sephacryl S-200 HR was used as the medium; 20 mM, pH 7.4 phosphate-buffered saline (PBS) was used as the elution buffer, and the flow rate was 1 ml / min. The conditions for affinity chromatography were as follows: Sepharose 4B was used as the medium; 0.1 M, pH 8.5 Tris-HCl buffer was used as the elution buffer, and the flow rate was 1 ml / min. Immediately after elution, the protein was neutralized with 1 M, pH 9.0 Tris-HCl buffer. During purification, an automated chromatography system was used to precisely control the elution conditions to ensure optimal separation. Proteins were initially separated by ion exchange chromatography, further purified by gel filtration chromatography, and finally, high-purity OPN was obtained by affinity chromatography. The purified OPN solution was analyzed by Agilent 1290 ultra-high pressure liquid chromatography (UPLC) and the purity reached 97.3%.

[0108] The purified OPN solution was freeze-dried using a gradient cooling process, first pre-freezing at -40℃ for 2 hours, and then freeze-drying at -80℃ for 48 hours to obtain OPN powder.

[0109] Example 2

[0110] Mature milk from healthy lactating women was collected, stored at 4°C, and pasteurized within 6 hours by holding at 62°C for 30 minutes. The pasteurized colostrum was immediately used for the next step. 100 mL of the pasteurized mature milk sample was placed in a centrifuge and centrifuged at 4000 rpm for 15 minutes at 4°C to remove the upper fat layer. The clear liquid was collected, and the lower precipitate (containing cell debris) was discarded.

[0111] Slowly add ethanol to the resulting clear liquid while stirring until the final ethanol concentration reaches 70% (v / v). Let the resulting mixture stand at 4°C for 3 hours, then centrifuge at 8000 r / min for 20 minutes and collect the precipitate.

[0112] The obtained precipitate was subjected to osteopontin dissolution, dialysis and concentration, chromatography and drying steps in the same manner as in Example 1; thereby obtaining OPN powder. The OPN solution purified by chromatography (before drying) was analyzed by Agilent 1290 ultra-high pressure liquid chromatography (UPLC) and the purity reached 95% by mass.

[0113] The above description is merely an exemplary embodiment of the present invention. It should be noted that those skilled in the art can make improvements to the present invention without departing from the inventive concept, and all such improvements fall within the scope of protection of the present invention.

Claims

1. A method for isolating and purifying osteopontin from human milk, comprising the following steps: (1) Pretreatment: collecting human milk and subjecting it to pretreatment to remove fat and cell debris impurities to obtain pretreated milk; (2) Osteopontin precipitation: adding a precipitant to the pretreated milk to precipitate osteopontin, and collecting the precipitate; (3) Osteopontin dissolution: adding a solvent capable of dissolving osteopontin to the obtained precipitate, mixing thoroughly, and then removing insoluble matter to obtain a crude osteopontin solution; (4) Dialysis and concentration: subjecting the crude osteopontin solution to dialysis and concentration to obtain an osteopontin concentrate; (5) Chromatography: purifying the osteopontin concentrate by chromatography to obtain an osteopontin purified solution; and (6) Drying: drying the osteopontin purified solution to obtain an osteopontin powder.

2. The method of claim 1, wherein the human milk is colostrum or mature milk.

3. The method of any one of claims 1-2, wherein in step (1), the fat and cell debris impurities are removed by centrifugation; preferably the centrifugation is performed at a speed of 3000-20000 rpm for 5-60 minutes at a temperature lower than room temperature, such as 0-10°C.

4. The method of any one of claims 1-3, wherein in step (1), the human milk is subjected to sterilization treatment before the fat and cell debris impurities are removed.

5. The method of any one of claims 1-4, wherein in step (2), the precipitant is one or more selected from the group consisting of ammonium sulfate or ethanol; preferably wherein in step (2): the precipitant is gradually added while stirring the pretreated milk, the obtained solution is allowed to stand after the addition of the precipitant is completed, step (2) is performed at room temperature or a temperature lower than room temperature, such as 0-10°C, and / or the precipitate is collected by centrifugation.

6. The method of any one of claims 1-5, wherein in step (3), the solvent is a phosphate buffer; preferably wherein: the conjugate acid-base in the phosphate buffer is sodium dihydrogen phosphate and disodium hydrogen phosphate, or potassium dihydrogen phosphate and potassium hydrogen phosphate; the concentration of the phosphate buffer is 0.005-0.05 mol / L, wherein the concentration refers to the total concentration of the conjugate acid-base in the buffer; and / or the pH value of the phosphate buffer is 5.8-7.8, preferably 6.5-7.

5.

7. The method of any one of claims 1-6, wherein in step (3), the insoluble matter is removed by centrifugation or filtration.

8. The method of any one of claims 1-7, wherein in step (4), the dialysis is performed using a dialysis bag with a molecular weight cut-off of 3500-5000 Da; the concentration is performed by ultrafiltration. ​ 9. The method of any one of claims 1-8, wherein in step (5), the chromatography comprises sequentially: ion exchange chromatography, gel filtration chromatography, and affinity chromatography; preferably, ion exchange chromatography employs 50 mM Tris-HCl buffer, pH 8.0, as equilibration and elution buffer, gel filtration chromatography employs 20 mM phosphate buffer, pH 7.4, as eluent, and affinity chromatography employs 0.1 M Tris-HCl buffer, pH 8.5, as eluent.

10. The method of any one of claims 1-9, wherein in step (6), the drying is freeze-drying.

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