osteopontin separation method

Abstract

This invention relates to a method for separating osteopontin, particularly a method for separating osteopontin from a milk-containing raw material. The method includes: a first separation step, adjusting the milk-containing raw material to a first pH value to cause the osteopontin to co-coagulate with at least one other protein to obtain a first precipitate; a second separation step, mixing the first precipitate with a solution of a first salt to cause at least partial decoagulation of the precipitate to release the osteopontin, and separating to obtain a solution containing osteopontin; a third separation step, adjusting the solution containing osteopontin to a second pH value to obtain a second precipitate containing osteopontin. Optionally, the third separation step is carried out in the presence of a second salt, and the first pH value is such that the osteopontin contained in the first precipitate is at least 70% by mass of the total osteopontin in the milk-containing raw material.

Description

Technical Field

[0001] This invention belongs to the field of food raw material processing and production. Specifically, it relates to a method for separating or extracting proteins, and more specifically, it relates to a method for separating, extracting or concentrating osteopontin from human or animal sources. Background Technology

[0002] Osteopontin (OPN) is a highly post-translational modified glycoprotein composed of 300 amino acids. It is synthesized by various tissues and appears in almost all body fluids, including breast milk. OPN is present in high amounts in breast milk, accounting for more than 10% of the total protein content in human milk. According to literature, it is about 138 mg / L in breast milk in the early stage of lactation and about 157 mg / L in the late stage of lactation, making it one of the five most abundant breast milk proteins.

[0003] Human milk OPN differs slightly from cow's milk OPN. Human milk OPN is composed of 298 amino acids and is an active, secretory, phosphorylated glycoprotein that binds to calcium, while cow's milk OPN is composed of 262 amino acids. Human milk OPN contains up to 34 phosphoserine and 2 phosphothreonine residues. It has been demonstrated that both human and cow's milk OPN can resist protease hydrolysis for 1 hour in neonatal gastric juice at 37°C and pH 3.0, indicating that OPN can withstand in vitro digestion by neonatal gastric juice. Furthermore, it is speculated that this ensures that breast milk-derived OPN reaches the lower digestive tract to exert its biological activity.

[0004] Opontonin (OPN), a secreted phosphorylated glycoprotein, has multiple functions, including synergistic involvement with lactoferrin in immune function development, immunomodulatory activity, tissue remodeling, bone formation, and cell adhesion, migration, proliferation, and differentiation. OPN contains RGD and non-RGD integrin-binding domains, including integrin and CD44 binding sites, and exerts various functions by binding to receptors on the cell membrane, thereby participating in various cellular signaling pathways. OPN can be expressed on various cell types, including bone cells, macrophages, smooth muscle cells, endothelial cells, and epithelial cells, playing important roles in mediating cell chemotaxis, aggregation, adhesion, proliferation, and migration, as well as bone mineralization and remodeling, immune regulation, signal transduction, and immunity against infectious diseases. It can also be expressed in certain disease states, such as atherosclerosis, nephropathy, and tumorigenesis. Transcriptional modifications of the gene encoding OPN have been observed, with altered splice transcripts leading to different forms of OPN expression in certain disease states. OPN exerts its many biological functions through interactions with integrins, including a large family of heterodimeric transmembrane receptors that mediate both cell-cell and cell-matrix interactions, and it can play a role in inflammatory diseases. OPN has been studied using cell models, animal models, and randomized clinical trials. Current research indicates that it plays important roles in intestinal proliferation and maturation, myelination of the brain, neural development, and immune development. In in vitro studies, incubating b-OPN (bovin OPN) with gastric juice from human newborns revealed that b-OPN exhibits gastric digestive resistance comparable to human OPN, and in experiments with 10-week-old mice, b-OPN intake increased plasma OPN levels. Human milk OPN is also involved in the development of neural tissues related to brain development, behavior, and cognitive abilities in infants.

[0005] As a multifunctional bioactive protein involved in many biological processes, OPN needs to be isolated and purified. It can be used as an excipient in general foods and special foods, especially infant formula. Currently, it has been added to infant formula in the form of whey protein powder.

[0006] Similar to lactoferrin, the concentration of OPN in cow's milk is much lower than that in human milk, and the concentration in milk-based infant formula is even lower. In recent years, with scientists' research on osteopontin, some new separation methods have been developed for the purification and separation of OPN.

[0007] Reference 1 discloses a method for separating osteopontin, which is as follows: osteopontin is separated from other substances in whey by adjusting the pH value, and osteopontin is separated by ion exchange or precipitation of insoluble calcium salts.

[0008] Reference 2 discloses a method for separating osteopontin from selected dairy raw materials. The separation principle is to separate osteopontin from a variety of complex dairy raw materials with high yield and high purity using anion exchange technology. The specific method is as follows: the selected dairy raw material is directly contacted with anion exchange medium; then the anion exchange medium is washed; finally, the protein bound to the anion exchange medium is recovered, thereby obtaining a mixture containing the separated osteopontin.

[0009] Reference 3 discloses a method for extracting osteopontin from cow's milk. The separation principle of this patent is to extract OPN by ion exchange chromatography, and the eluent is further purified by reversed-phase hydrophobic method. The specific separation method is as follows: First, centrifuge 1000–2000 mL of milk for 15–30 min to remove the precipitate, then mix with 200–400 mL of DEAE-Sephacel resin and stir at 3–6 °C for 12–24 h. After standing, remove the precipitate and pour the mixture into a chromatographic column. Equilibrate the column with a phosphate buffer solution with a concentration of 0.01–0.015 mol / L and a pH of 7.2–7.6 at a flow rate of 3–5 mL / min. Then, perform gradient elution at a flow rate of 3–5 mL / min and a detection wavelength of 280 nm. The gradient elution is completed in three stages, and the gradient eluents from each stage are collected separately. Next, hydrophobic chromatography is used to separate the gradient eluents separately to obtain eluents containing crude purified osteopontin. Further, hydrophobic chromatography is used to further separate the eluents, and the collected solutions are mixed. Finally, osteopontin in milk is obtained by dialysis and lyophilization.

[0010] In addition, other attempts have been reported. For example, reference 4 publishes a method for separating osteopontin from human plasma. The separation principle is to use a specific buffer system to bind total osteopontin and human serum albumin in human plasma with DEAE-Cibacron blue 3GA, while excluding immunoglobulin G. Then, osteopontin is separated from human serum albumin by different elution solutions.

[0011] Although attempts have been made to separate and extract OPN from various sources, the current processes and separation results are still insufficient.

[0012] References:

[0013] Reference 1: US 7259243B2

[0014] Reference 2: CN103492408A

[0015] Reference 3: CN101485381A

[0016] Reference 4: An antibody-free sample pretreatment method for osteopontincombined with MALDI-TOF MS / MS analysis, Yuye Zhou et al., PLoS One. 2019Mar 7; 14(3) Summary of the Invention

[0017] The problem the invention aims to solve

[0018] As mentioned earlier, given the special value of OPN proteins, attempts have been made to isolate or extract OPN from various sources. However, the following problems have been encountered in long-term practice:

[0019] Reference 1 describes the separation and extraction of OPN from whey. However, the content of OPN in whey is limited. Therefore, the efficiency of separating and extracting OPN is not high and it is difficult to meet the requirements of large-scale production.

[0020] Reference 2 describes the direct contact of milk sources with specific pH values ​​and conductivity with the packing material of anion exchange chromatography columns, followed by washing and separation. However, overall, when milk sources without separation treatment are subjected to the above treatment, there are issues with improving the efficiency of the overall process.

[0021] The method provided in reference 3 requires separation through two production line columns, which results in extremely high costs and low production efficiency.

[0022] Furthermore, according to the results published in cited reference 4, the concentration of recombinant osteopontin obtained by this method is approximately 1 μg / mL, therefore it is not suitable for industrial production.

[0023] Therefore, based on the development of the prior art, the present invention provides a method for extracting OPN protein from milk-containing raw materials. The method of the present invention can efficiently extract more OPN protein from milk-containing raw materials. In particular, for cases where animal milk (cow, sheep, etc.) is used as raw material, it can solve the problem that the prior art cannot efficiently extract all osteopontin.

[0024] Solution for solving the problem

[0025] Through long-term research, the inventors discovered that the above-mentioned technical problems can be solved by implementing the following technical solution:

[0026] [1]. This invention provides a method for isolating osteopontin from milk-containing raw materials, wherein the method includes:

[0027] In the first separation step, the milk-containing raw material is adjusted to a first pH value so that the osteopontin co-coagulates with at least one other protein to obtain a first precipitate.

[0028] In the second separation step, the first condensate is mixed with a solution of the first salt to at least partially decondense the condensate to release the osteopontin, and a solution containing osteopontin is obtained by separation.

[0029] In the third separation step, the solution containing osteopontin is adjusted to a second pH value to obtain a second precipitate containing osteopontin. Optionally, the third separation step is also carried out in the presence of a second salt.

[0030] The first pH state is such that the osteopontin contained in the precipitate is 70% or more by mass of the total osteopontin in the milk-containing raw material.

[0031] [2]. According to the method of [1], wherein the milk-containing raw material is selected from human or animal-derived milk-containing raw materials.

[0032] [3]. The method according to [1] or [2], wherein the first pH value is below 4.7.

[0033] [4]. The method according to any one of [1] to [3], wherein, in the first separation step, the milk-containing raw material is adjusted to the first pH value state by means of an organic acid.

[0034] [5]. The method according to any one of [1] to [4], wherein, in the first separation step, the at least another protein includes casein.

[0035] [6]. The method according to any one of [1] to [5], wherein, in the second separation step, the solution of the first salt is an aqueous solution of the first metal salt, and the concentration of the first metal salt in the solution of the first salt is 0.2 to 7 g / L.

[0036] [7]. The method according to any one of [1] to [6], wherein the osteopontin content in the solution containing osteopontin obtained in the second separation step is 20% by mass or more, based on total protein.

[0037] [8]. The method according to any one of [1] to [7], wherein the second separation step is performed by mechanical filtration and / or ultrafiltration to separate a solution containing osteopontin.

[0038] [9]. The method according to any one of [1] to [8], wherein, in the third separation step, the state of the second pH value is such that at least 90% by mass of osteopontin in the solution containing osteopontin coagulates.

[0039]

[10] . The method according to any one of [1] to [9], wherein the third separation step is carried out in the presence of a second salt, the second salt being a second metal salt, the second metal salt being the same as or different from the first metal salt.

[0040]

[11] . According to the method described in

[10] , the second metal salt is the same as the first metal salt, both of which are selected from calcium salts.

[0041]

[12] . The method according to any one of [1] to

[11] , wherein, in the third separation step, the solution containing osteopontin is adjusted to a second pH value by means of a buffer solution.

[0042]

[13] . The method according to any one of [1] to

[12] , wherein, after the third separation step, a step of purifying the second condensate to remove salt components is further included.

[0043]

[14] . The method according to any one of [1] to

[13] , wherein:

[0044] The milk-containing raw material is an animal-derived milk-containing raw material;

[0045] The first pH value is 4.2 to 4.4, and the second pH value is 6.0 to 8.5.

[0046]

[15] . The method according to any one of [1] to

[14] , wherein, before entering the second pH state, both the first salt and the second salt are dissolved in their respective solutions.

[0047]

[16] . According to the method described in [1] to

[15] , wherein the milk-containing raw material is cow's milk or sheep's milk.

[0048] The effects of the invention

[0049] By implementing the above technical solution, the present invention can achieve the following technical effects:

[0050] 1) Compared with the prior art, the present invention can separate osteopontin in milk raw materials more quickly and conveniently, without relying on traditional ion exchange column treatment in the main separation process;

[0051] 2) The method of the present invention can process various types of milk-containing raw materials and can separate and collect most of the osteopontin in the milk-containing raw materials, which has significantly improved efficiency compared with the prior art.

[0052] 3) The separation method of the present invention is more conducive to large-scale industrial production;

[0053] 4) In some preferred embodiments of the present invention, the final osteopontin obtained using the present invention has significantly improved purity;

[0054] 5) This invention is the first to propose the above-mentioned new method for rapid and efficient separation of osteopontin, and also provides a reference for related practices in this field. Attached Figure Description

[0055] Figure 1 Process flow diagrams of some specific embodiments of the present invention Detailed Implementation

[0056] The present invention will now be described in detail. The descriptions of the technical features described below are based on representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted that:

[0057] 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.

[0058] In this specification, the terms "substantially" or "truly" are used to indicate that the standard deviation from the theoretical model or theoretical data is within 3%, preferably 2%, more preferably 1%, and that the deviation here also includes systematic deviations.

[0059] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0060] In this manual, the use of terms such as "first..." and "second..." is merely for distinguishing the names of different components and does not represent the order or timing of use of the corresponding substances or components.

[0061] In this specification, the term "milk" is used to refer to the fluid obtained from the mammary glands of a mammal in lactation. The term "milk" should be interpreted broadly and encompasses both raw milk (i.e., the fluid obtained directly from the mammary glands) and standardized dairy products (such as skim milk or whole milk), in which the concentration of milk fat has been reduced relative to the original raw milk.

[0062] In this specification, the term "solution" is used to refer to a homogeneous or microemulsion system in which the solute is essentially or substantially dissolved under the conditions of the appropriate solvent, pH, and concentration.

[0063] In this specification, "whey" is used as a collective term referring to the watery byproduct produced during the production of cheese or casein from milk.

[0064] In this instruction manual, "normal temperature" refers to an indoor temperature of 23±2℃.

[0065] In this specification, the term "condensation" refers to the process by which a distinct solid / semi-solid phase is produced from a homogeneous system (solution or microemulsion), resulting in significant phase separation. The term "decondensation" refers to the reverse process of the aforementioned "condensation" process.

[0066] In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0067] 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.

[0068] The present invention provides a method for separating osteopontin from milk-containing raw materials, wherein the method involves separating the milk-containing raw materials three times by means of pH value and the use of salt to separate osteopontin from the milk-containing raw materials.

[0069] Furthermore, the method for isolating osteopontin from milk-containing raw materials in this invention is mainly based on the following insights:

[0070] In current known technologies, osteopontin is extracted from milk or whey and purified using ion exchange methods such as DEAE-sephace and DEAE-cibacron blue 3GA. However, the yield and purity are far below the requirements for clinical testing and industrial production. The inventors of this application have unexpectedly discovered that under specific pH conditions, milk-containing raw materials can co-coagulate with other proteins. Furthermore, the coagulated OPN-containing components, when mixed with a metal salt solution, can be re-extracted and dissolved in the solution; furthermore, adjusting the pH of the solution will cause OPN to coagulate again. Therefore, this invention overcomes the problems of low yield and high price in existing industrial production methods for extracting OPN from whey or raw milk.

[0071] The method for separating osteopontin according to the present invention may specifically include the following three separation steps:

[0072] In the first separation step, the milk-containing raw material is adjusted to a first pH value so that the osteopontin co-coagulates with at least one other protein to obtain a first precipitate.

[0073] In the second separation step, the first condensate is mixed with a solution of the first salt to at least partially decondense the condensate to release the osteopontin, and a solution containing osteopontin is obtained by separation.

[0074] In the third separation step, the solution containing osteopontin is adjusted to a second pH value to obtain a second precipitate containing osteopontin. Optionally, the third separation step is carried out in the presence of a second salt.

[0075] Furthermore, the first pH value is lower than the second pH value.

[0076] Using the separation method described above, the present invention can separate most of the osteopontin from milk-containing raw materials and obtain osteopontin products with relatively improved purity.

[0077] (Contains dairy ingredients)

[0078] There are no particular limitations in principle regarding the milk-containing raw materials of this invention. The milk-containing raw materials can be human-derived or animal-derived. Furthermore, in some preferred embodiments, the milk-containing raw materials of this invention can be animal-derived and can be optionally pretreated to obtain raw materials suitable for direct subsequent osteopontin separation.

[0079] Animal-derived milk-containing raw materials typically include milk derived from cows, sheep, camels, deer, or horses. In some preferred embodiments, the milk-containing raw material of the present invention is milk derived from cows or sheep.

[0080] Such milk can be raw milk or milk that has undergone some degree of processing. In some preferred embodiments, for ease of subsequent separation, the milk can preferably be processed through concentration, defatting, or other steps to obtain milk with reduced fat content. In some more specific embodiments, the milk-containing raw material is defatted milk, wherein the fat content, based on the total mass of the milk, is 0.5% by mass or less, preferably 0.3% by mass or less, and more preferably 0.1% by mass or less. There are no particular limitations on the defatting method; defatting can typically be performed by centrifugation.

[0081] In addition to the milk mentioned above, whey or whey concentrates may also be included as the milk-containing raw material of this invention. Examples of whey-like substances include sweet whey, acid whey, and serum whey. Sweet whey is whey obtained during a rennet-based milk coagulation process, such as that occurring during the production of yellow cheese; acid whey refers to whey obtained during a chemical or biological acidification process of milk, such as that occurring during the production of farm cheese or quark cheese, or during the production of casein / casein salts; serum whey refers to milk from which milk fat and casein micelles have been removed. However, serum whey typically contains some free casein species that have been dissociated from these native casein micelles before the micelles are removed. Serum whey can be produced, for example, by microfiltration of skim milk through a filter or membrane having a pore size of about 0.1 micrometers and collecting the resulting permeate as milk serum.

[0082] The milk-containing raw material of the present invention can be one or a mixture of milk or whey as described above. Further, for the milk-containing raw material, especially one that can be directly subjected to subsequent osteopontin separation, in some preferred embodiments, the total protein content, based on the total mass of the raw material, can be less than 5 g / 100 mL, preferably less than 4 g / 100 mL, and more than 0.2 g / 100 mL, preferably more than 0.5 g / 100 mL. In addition, the types of proteins in the milk-containing raw material include, besides osteopontin, various whey proteins, various caseins, etc. In some specific embodiments, the osteopontin content, based on the total protein content in the milk-containing raw material, is 0.01–25% by mass, preferably 0.03–15% by mass, more preferably 0.05–5% by mass.

[0083] In addition, for milk-containing raw materials, pretreatment can be performed before osteopontin separation, including sterilization and component adjustment. Sterilization processes include pasteurization, which can be carried out at 62℃~65℃ for 30 minutes or at 75℃~90℃ for 15s~16s.

[0084] Furthermore, in some preferred embodiments of the present invention, the pasteurized milk raw materials can be stored at a temperature not exceeding 10°C, preferably not exceeding 8°C, for subsequent processing.

[0085] (First separation step)

[0086] The first separation step of the present invention mainly includes adjusting the pH value of the milk-containing raw material to cause osteopontin and at least one other protein to co-coagulate, thereby obtaining a first coagulated product.

[0087] In some preferred embodiments of the present invention, for the purpose of preventing protein denaturation or for safety reasons, adjusting the pH of the milk-containing raw material to a first pH value can be achieved by adding an organic acid. Commonly listed organic acids include one or a mixture of several of the following: citric acid, malic acid, lactic acid, tartaric acid, formic acid, acetic acid, oxalic acid, succinic acid, caffeic acid, or ascorbic acid. Furthermore, there are no particular limitations on the method of using the above-mentioned organic acids; they can be added directly to the milk-containing raw material, or they can be mixed with the milk-containing raw material in the form of an aqueous solution of the organic acid.

[0088] There are no particular restrictions on the temperature conditions for adjusting the pH value of dairy raw materials. It can be carried out at the storage temperature of the dairy raw materials mentioned above, or at room temperature or close to room temperature.

[0089] Furthermore, when the milk-containing raw material system reaches the first pH value, the osteopontin in the system co-coagulates with at least one other protein to obtain the first condensate. While there are no particular limitations on the "at least one other protein," it is typically one or more caseins. Examples of caseins include α-s1 casein, α-s2 casein, β-casein, and κ-casein.

[0090] In some specific embodiments of the present invention, the first condensate is presented in the form of settleable flocs or agglomerates. Preferably, these condensates formed by co-condensation are precipitates.

[0091] In this invention, the first pH value is such that the osteopontin contained in the first condensate is at least 70% by mass, preferably at least 80% by mass, more preferably at least 90% by mass, and even more preferably at least 95% by mass, of the total osteopontin in the milk-containing raw material. Therefore, in principle, the pH value range can be adjusted according to the above principles for different types or sources of milk-containing raw materials.

[0092] In some specific embodiments, the first pH value can be below 4.7, preferably below 4.6, and can be above 3.5, preferably above 4.0, and more preferably above 4.2. Such a pH range is particularly effective when the aforementioned "at least another protein" is casein. This ensures that most of the osteopontin coagulates without causing a large amount of casein to coagulate simultaneously. Furthermore, if the pH value is too low, in some cases, other proteins besides osteopontin may undergo undesirable coagulation or denaturation; if the pH value is too high, the osteopontin content in the co-coagulate is low or co-coagulation is difficult to occur.

[0093] In some specific embodiments of the present invention, for the pH range of the first state of the present invention, considering the sufficiency of osteopontin coagulation, the pH value can be set between 4.0 and 4.6, preferably between 4.2 and 4.4. It is known that various proteins are sensitive to pH (related to isoelectric point). By adjusting to the first pH state, the present invention can efficiently coagulate more osteopontin, while the amount of other proteins co-coagulated is also appropriate; that is, co-coagulation effectively increases the osteopontin content in the coagulate. Therefore, relative to the milk-containing raw material, the first separation step of the present invention is equivalent to a first concentration / enrichment step for osteopontin.

[0094] Furthermore, the present invention does not impose any particular restrictions on other auxiliary means that can be used during the co-condensation process, such as using mechanical agitation to control the state of the condensate and accelerate the co-condensation process.

[0095] Furthermore, the obtained condensate can be separated using conventional solid-liquid separation methods. In some specific implementations, the condensate can be used with or without washing.

[0096] (Second separation step)

[0097] In this invention, a second separation step is used to release osteopontin from the first condensate obtained in the first separation step.

[0098] Specifically, a solution containing a first salt is provided. In the solution of the first salt, the salt is substantially dissolved in the solution; therefore, the concentration and pH of the solution are limited to achieve a substantially dissolved state of the salt. In some specific embodiments, the first salt can form the solution at a concentration far below saturation in a solution with a pH range of 6 to 7.5; in other specific embodiments, the first salt can form the solution at a concentration far below saturation in a solution with a pH range of 3.8 to 5.0.

[0099] Furthermore, in some preferred embodiments of the present invention, the first salt is a first metal salt. The metal salt is selected from one or more divalent metal salts, and in some more preferred embodiments, the divalent metal salt is selected from one or more calcium or magnesium salts. Additionally, the first salt can be an inorganic acid salt or an organic acid salt, such as hydrochloride, sulfate, phosphate, acetate, propionate, etc. More preferably, the first salt can be a salt of an inorganic acid, such as a phosphate and / or hydrochloride. Therefore, in a further preferred embodiment of the present invention, the first salt can be a calcium salt of an inorganic acid, such as calcium chloride or calcium phosphate.

[0100] In addition to the first metal salt of divalent metals mentioned above, other metal salts of organic or inorganic acids, such as acid salts of alkali metals, may be used to achieve the required ionic strength without affecting the technical effect of the present invention.

[0101] Regarding the concentration of the first salt in the solution, from the perspective of promoting osteopontin release, the concentration of the first salt, especially the first metal salt, can be 0.2 to 7 g / L, preferably 0.5 to 5 g / L, and more preferably 1 to 3 g / L.

[0102] When the condensate obtained in the first separation step is mixed with the solution of the first salt, the solution of the first salt has a suitable ionic strength environment and pH value, and the pH value can be adjusted using the organic acid described above whenever necessary.

[0103] Suitable ionic strength and pH value allow osteopontin to be released from the precipitate during the decoagulation process. In some preferred embodiments of the invention, the pH value of the first salt solution is 4.2 to 5.0, preferably 4.6 to 4.8. Such a pH environment is advantageous for the decoagulation of osteopontin, especially when the protein co-coagulating with osteopontin is one or more caseins, and the release of other proteins (caseins) can be easily suppressed while controlling the release of osteopontin from the precipitate.

[0104] Furthermore, a solution containing osteopontin can be obtained through separation. There are no particular limitations on the separation method; it can include mechanical filtration, ultrafiltration, electroosmosis, etc. Preferably, an ultrafiltration membrane with a suitable pore size can be used for yield processing. In the osteopontin-containing solution obtained through separation, the content of osteopontin as a percentage of total protein is further increased (especially relative to the protein in the first coagulate). Undesirable impurities or other proteins can be removed through separation.

[0105] Therefore, relative to the first precipitate, the second separation step of the present invention corresponds to a second concentration / enrichment step for osteopontin.

[0106] In some specific embodiments of the present invention, the osteopontin-containing solution obtained in the second separation step contains 20% by mass or more, preferably 30% by mass or more, and more preferably 35% by mass or more, based on total protein. Furthermore, the amount of osteopontin in the osteopontin-containing solution obtained in the second separation step, based on the total amount of osteopontin in the first precipitate, is 80% by mass or more, preferably 90% by mass or more, and more preferably 95% by mass or more.

[0107] (Third separation step)

[0108] In the third separation step of the present invention, the osteopontin-containing solution obtained in the second separation step is subjected to coagulation at a suitable second pH value to obtain a second precipitate. Optionally, the coagulation can be carried out in the presence of an additional second salt.

[0109] In some specific embodiments of the present invention, by adjusting the pH value of the solution containing osteopontin, the first salt is precipitated, and the change in the physical and charge environment of the solution also causes the coagulation of proteins such as osteopontin.

[0110] Furthermore, as long as the above-mentioned condensation conditions are met, there are generally no particular restrictions on the second pH value. In some specific embodiments, the second pH value can be 6.0–8.5, preferably 6.2–8.0, and more preferably 6.5–7.5. Additionally, the present invention does not impose any particular restrictions on the method for adjusting the second pH value. In some specific embodiments, the solution containing osteopontin can be brought to the above-mentioned pH range by adding an alkaline substance. In other specific embodiments, the above adjustment is preferably achieved using buffer components or buffer solutions commonly used in the art, such as phosphate buffer solutions with a pH of 7.

[0111] In some other specific embodiments, for the purpose of assisting coagulation, an additional second salt may be used in addition to the first salt originally present in the solution containing osteopontin. The selection range of the second salt is the same as that of the first salt described herein, and the second salt may be the same as or different from the first salt. In some preferred embodiments, the second salt may be a calcium salt of the aforementioned inorganic acid; more preferably, the second salt is calcium chloride and / or calcium phosphate.

[0112] By adjusting to the second pH value, at least 90% by mass, preferably at least 95% by mass, of the osteopontin in the solution containing osteopontin can coagulate to obtain a second precipitate.

[0113] Furthermore, in some specific embodiments of the present invention, the content of osteopontin in the second condensate obtained by the third separation process is 30% by mass or more, preferably 40% by mass or more, more preferably 50% by mass or more, and even more preferably 60% by mass or more, based on the total mass of protein.

[0114] (Post-purification process)

[0115] Optionally, during or after the third separation described above, the present invention may perform further post-purification treatment on the second condensate. In this invention, the post-purification treatment may include ion exchange and ultrafiltration.

[0116] For the post-purification method of ion exchange, for ease of operation, in a preferred embodiment, ion exchange treatment can be performed simultaneously with the third separation described above.

[0117] Furthermore, regarding ion exchange, this invention mainly refers to the use of anion exchange equipment for processing. In some specific embodiments, after the osteopontin-containing solution obtained in the second separation step is passed into the aforementioned anion exchange equipment, the pH value of the mobile phase is adjusted (for example, using a phosphate-based buffer solution as described above), so that the second precipitate condenses in the stationary phase (chromatographic column) of the anion exchange equipment. This allows for the adjustment of the mobile phase (e.g., flow rate) to discharge unwanted impurities (other protein components and salt components) from the waste outlet of the anion exchange equipment.

[0118] For the anion exchange apparatus that can be used in this invention, the present invention considers apparatus with a strongly basic stationary phase to be advantageous, as it can provide a suitable retention time for osteopontin to remove as much of the other protein and salt components as possible. Examples of such apparatus include the TOYOPEARL SuperQ-650 (the exchange column packing material has quaternary amino functional groups).

[0119] After treatment with an anion exchange apparatus, osteopontin adsorbed on the stationary phase can be separated and enriched using a low-concentration salt solution. There are no particular restrictions on the type and concentration of the salt solution that can be used, as long as it meets the usual "salt-dissolving" conditions for osteopontin.

[0120] Optionally, the enriched osteopontin described above can be further purified by mechanical filtration, ultrafiltration, or other methods to obtain a final stock solution containing osteopontin. Subsequently, it can be dried, for example, by (low-temperature) spray drying or freeze-drying, to obtain powdered osteopontin. In some specific embodiments of the present invention, the purity of the osteopontin finally obtained through the above-mentioned anion exchange treatment method can be 90% by mass or more, preferably 92% by mass or more, and more preferably 95% by mass or more.

[0121] In some other embodiments of the present invention, the post-purification process can also be performed separately after the third separation step described above. Specifically, the condensate (also referred to as precipitate) obtained in the third separation step can be treated using methods such as mechanical filtration or ultrafiltration to obtain a solution system containing osteopontin. Subsequently, osteopontin powder can be obtained by drying, for example, by (low-temperature) spray drying or freeze drying. In some specific embodiments of the present invention, the purity of the osteopontin finally obtained by such methods can be 40% by mass or more, preferably 45% by mass or more, and more preferably 50% by mass or more.

[0122] The present invention provides a highly efficient method for separating and extracting OPN protein from milk-containing raw materials, especially from milk-containing raw materials such as cow's milk and sheep's milk. The process flow of the present invention can significantly save separation and processing time, and can obtain purity and / or yield that is comparable to or improved by the prior art.

[0123] Example

[0124] The present invention will be further described below through specific embodiments.

[0125] Example 1 :

[0126] 1. Raw milk is transported to the factory via milk trucks;

[0127] 2. The fresh milk tanks are transported to the fresh raw milk storage tanks through the fresh milk receiving process and stored at 0-6℃;

[0128] 3. The raw milk is pumped to the pasteurization system, which integrates a separator. The raw milk is preheated and then fed into the separator to separate it into cream and skim milk. The cream, as a byproduct, can be processed using other processes. Skim milk is required to have a fat content of <0.1%.

[0129] 4. Skim milk is pasteurized at 75°C for 15 seconds and then cooled to 0-6°C;

[0130] 5. Pasteurized skim milk should be stored at 0–6°C;

[0131] 6. After adding the batch of fresh milk to the reaction tank, add lactic acid to acidify it to a pH of approximately 4.3;

[0132] 7. After observing the whey separation, filter the whey liquid through the inner screen of the reaction vessel and discharge it from the reaction vessel for further processing, leaving only acidified curd in the reaction vessel;

[0133] 8. Add calcium chloride to the reaction vessel to achieve a concentration of 4.5 g / L, and adjust the pH to the optimal level of approximately 4.3. OPN will bind with calcium ions and be released from the curd.

[0134] 9. The OPN filtrate containing certain impurities is discharged from the reaction vessel through the inner screen of the reaction vessel. The impurities, excess casein, whey, and ions are removed by ultrafiltration using a 10 kDa membrane to purify the OPN solution.

[0135] 10. Pass the ultrafiltration OPN filtrate through an anion exchange column (the exchange column packing principle is based on quaternary amino functional groups (TOYOPEARL SuperQ-650, 100μm), and first wash the column bed with a 200mM phosphate buffer solution (disodium hydrogen phosphate) at pH=7), allowing the chromatography column to adsorb OPN. The feed liquid passing through the chromatography column is discharged as waste liquid.

[0136] 11. After all the OPN filtrate from a batch has passed through the chromatography column, the OPN is eluted from the chromatography column using a low-concentration 800mM NaCl solution and transferred to a temporary storage tank for later use.

[0137] 12. Use a 10KD membrane for ultrafiltration to remove ions and purify the OPN solution to obtain the OPN stock solution;

[0138] 13. Using low-temperature spray drying technology to dry the OPN stock solution, 95% pure OPN powder can be obtained.

[0139] Example 2 :

[0140] 1. Raw milk is transported to the factory via milk trucks;

[0141] 2. The fresh milk tanks are transported to the fresh raw milk storage tanks through the fresh milk receiving process and stored at 0-6℃;

[0142] 3. The raw milk is pumped to the pasteurization system, which integrates a separator. The raw milk is preheated and then fed into the separator to separate it into cream and skim milk. The cream, as a byproduct, can be processed using other processes. Skim milk is required to have a fat content of <0.1%.

[0143] 4. Skim milk is pasteurized at 75°C for 15 seconds and then cooled to 0-6°C;

[0144] 5. Pasteurized skim milk should be stored at 0–6°C;

[0145] 6. After adding the batch of fresh milk to the reaction tank, add lactic acid to acidify the pH to 4.3;

[0146] 7. After observing the whey separation, filter the whey liquid through the inner screen of the reaction vessel and discharge it from the reaction vessel for further processing, leaving only acidified curd in the reaction vessel;

[0147] 8. Add calcium chloride to the reaction vessel to achieve a concentration of 4.5 g / L, and adjust the pH to the optimal level of approximately 4.3. OPN will bind with calcium ions and be released from the curd.

[0148] 9. The OPN filtrate, containing certain impurities, is discharged from the reaction vessel through the inner screen. Impurities, excess casein, whey, and ions are then removed via ultrafiltration using a 10 kDa membrane to purify the OPN solution.

[0149] 10. Adjust the pH of the ultrafiltration OPN filtrate to approximately 7 using sodium hydroxide (maintain the temperature between 8 and 10°C). After standing for 4 hours, OPN will precipitate from the filtrate.

[0150] 11. Use a 10KD membrane for ultrafiltration to remove impurities and ions, purify the OPN solution, and obtain the OPN stock solution;

[0151] 12. The obtained OPN slurry can be dried using low-temperature spray drying technology to obtain OPN powder with 50% purity.

[0152] Comparative Example 1 :

[0153] 1. Raw milk is transported to the factory via milk trucks;

[0154] 2. The fresh milk tanks are transported to the fresh raw milk storage tanks through the fresh milk receiving process and stored at 0-6℃;

[0155] 3. The raw milk is pumped to the pasteurization system, which integrates a separator. The raw milk is preheated and then fed into the separator to separate it into cream and skim milk. The cream, as a byproduct, can be processed using other processes. Skim milk is required to have a fat content of <0.1%.

[0156] 4. Skim milk is pasteurized at 75°C for 15 seconds and then cooled to 0-6°C;

[0157] 5. Pasteurized skim milk should be stored at 0–6°C;

[0158] 6. Add calcium chloride to the pasteurized milk container until the concentration reaches 4 g / L;

[0159] 7. Heat the pasteurized milk container at 70℃ for 15 minutes;

[0160] 8. After observing the whey separation, filter the whey liquid through the inner screen of the reaction vessel and discharge it from the reaction vessel for further processing, leaving only acidified curd in the reaction vessel;

[0161] 9. The OPN filtrate, containing certain impurities, is discharged from the reaction vessel through the inner screen. Impurities, excess casein, whey, and ions are then removed via ultrafiltration using a 10 kDa membrane to purify the OPN solution.

[0162] 10. Adjust the pH of the ultrafiltration OPN filtrate to approximately 7 using sodium hydroxide (maintain the temperature between 8 and 10°C). After standing for 4 hours, OPN will precipitate from the filtrate.

[0163] 11. Use a 10KD membrane for ultrafiltration to remove impurities and ions, purify the OPN solution, and obtain the OPN stock solution;

[0164] 12. The obtained OPN slurry is dried using low-temperature spray drying technology to obtain OPN powder with a purity of 23%.

[0165] Comparative Example 2 :

[0166] 1. Raw milk is transported to the factory via milk trucks;

[0167] 2. The fresh milk tanks are transported to the fresh raw milk storage tanks through the fresh milk receiving process and stored at 0-6℃;

[0168] 3. The raw milk is pumped to the pasteurization system, which integrates a separator. The raw milk is preheated and then fed into the separator to separate it into cream and skim milk. The cream, as a byproduct, can be processed using other processes. Skim milk is required to have a fat content of <0.1%.

[0169] 4. Skim milk is pasteurized at 75°C for 15 seconds and then cooled to 0-6°C;

[0170] 5. Pasteurized skim milk should be stored at 0–6°C;

[0171] 6. Add calcium chloride to the pasteurized milk container until the concentration reaches 4 g / L;

[0172] 7. Heat the pasteurized milk container at 70℃ for 15 minutes;

[0173] 8. After observing the whey separation, filter the whey liquid through the inner screen of the reaction vessel and discharge it from the reaction vessel for further processing, leaving only acidified curd in the reaction vessel;

[0174] 9. The OPN filtrate, containing certain impurities, is discharged from the reaction vessel through the inner screen. Impurities, excess casein, whey, and ions are then removed via ultrafiltration using a 10 kDa membrane to purify the OPN solution.

[0175] 10. Adjust the pH of the ultrafiltration OPN filtrate to approximately 7 using sodium hydroxide (maintain the temperature between 8 and 10°C). After standing for 4 hours, OPN will precipitate from the filtrate.

[0176] 11. Use a 10KD membrane for ultrafiltration to remove impurities and ions, purify the OPN solution, and obtain the OPN stock solution;

[0177] 12. The obtained OPN slurry is dried using low-temperature spray drying technology to obtain OPN powder with a purity of 31%.

[0178] Comparative Example 3 :

[0179] 1. Raw milk is transported to the factory via milk trucks;

[0180] 2. The fresh milk tanks are transported to the fresh raw milk storage tanks through the fresh milk receiving process and stored at 0-6℃;

[0181] 3. The raw milk is pumped to the pasteurization system, which integrates a separator. The raw milk is preheated and then fed into the separator to separate it into cream and skim milk. The cream, as a byproduct, can be processed using other processes. Skim milk is required to have a fat content of <0.1%.

[0182] 4. Skim milk is pasteurized at 75°C for 15 seconds and then cooled to 0-6°C;

[0183] 5. Pasteurized skim milk should be stored at 0–6°C;

[0184] 6. After adding the batch of fresh milk to the reaction tank, add lactic acid to acidify it to a pH of approximately 4.3;

[0185] 7. After observing the whey precipitation, filter the whey solution through the inner sieve of the reaction vessel and discharge it from the reaction vessel for OPN extraction;

[0186] 8. Pass the ultrawhey through an anion exchange column (the exchange column packing principle is quaternary amino functional groups (TOYOPEARL SuperQ-650, 100μm), and first wash the column bed with a 100mM phosphate buffer solution (disodium hydrogen phosphate) at pH=7), allowing the chromatography column to adsorb OPN, and the feed liquid passing through the chromatography column is discharged as waste liquid.

[0187] 9. After all the OPN filtrate from a batch has passed through the chromatography column, use a low-concentration 700mM NaCl solution to elute the OPN from the chromatography column and transfer it to a temporary storage tank for later use.

[0188] 10. Use a 10KD membrane for ultrafiltration to remove ions and purify the OPN solution to obtain the OPN stock solution;

[0189] 11. OPN stock solution can be dried using low-temperature spray drying technology to obtain OPN powder with a purity of 0.5%.

[0190] 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 this disclosure should not be limited thereto.

[0191] The various embodiments of this disclosure have been described above. These descriptions are exemplary and not exhaustive, and are not 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 improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

[0192] Industrial availability

[0193] The method of this invention can be used industrially for the separation or purification of OPN.

Claims

1. A method for isolating osteopontin from a milk containing raw material, characterized in that, The method comprises: a first separation step, in which the milk-containing raw material is adjusted to a first pH state so that the osteopontin and at least another protein are co-precipitated to obtain a first precipitate, wherein the first pH is 3.5-4.7, the another protein comprises casein, and in the first separation step, lactic acid is used to adjust the milk-containing raw material to the first pH state; a second separation step, which is performed after the first separation step, in which the first precipitate is mixed with a solution of a first salt so that the first precipitate is at least partially de-precipitated to release the osteopontin, and a solution containing osteopontin is separated, wherein in the second separation step, mechanical filtration and / or ultrafiltration are used to separate the solution containing osteopontin, and the first precipitate is mixed directly with the solution of the first salt or is mixed with the solution of the first salt after being washed, the solution of the first salt has a pH of 4.2-5.0, and in the second separation step, the solution of the first salt is an aqueous solution of calcium chloride, and the concentration of the aqueous solution of calcium chloride is 0.2-7 g / L; a third separation step, in which a basic substance is added to the solution containing osteopontin to adjust the solution containing osteopontin to a second pH state, and the osteopontin is precipitated from the solution containing osteopontin to obtain a second precipitate containing osteopontin, and more than 90% of the total mass of the osteopontin in the solution containing osteopontin is precipitated to obtain the second precipitate; optionally, the third separation step is performed in the presence of a second salt, and the second pH is 6.0-8.5; the first pH state is such that the osteopontin contained in the first precipitate accounts for more than 70% of the total osteopontin in the milk-containing raw material.

2. The method of claim 1, wherein, The milk-containing raw material is selected from milk-containing raw materials of human or animal origin.

3. The method according to claim 1 or 2, characterized in that, The first pH is 4.0-4.

6.

4. The method according to claim 1 or 2, characterized in that, In the first separation step, the casein is selected from α-s1 casein, α-s2 casein, β-casein or κ-casein.

5. The method according to claim 1 or 2, characterized in that, The content of the osteopontin in the solution containing osteopontin obtained in the second separation step accounts for more than 20% of the total protein.

6. The method of claim 1 or 2, wherein, In the third separation step, the second pH state is such that at least 95% of the total mass of the osteopontin in the solution containing osteopontin is precipitated.

7. The method according to claim 1 or 2, characterized in that, The third separation step is performed in the presence of a second salt, and the second salt is a second metal salt, which is the same as or different from the first metal salt.

8. The method of claim 7, wherein, The second metal salt is the same as the first metal salt.

9. The method of claim 1 or 2, wherein, In the third separation step, the solution containing osteopontin is adjusted to the second pH state by using a buffer solution.

10. The method of claim 1 or 2, wherein, After the third separation step, a step of purifying the second precipitate to remove salt components is further included.

11. The method of claim 1 or 2, wherein, Before entering the second pH state, the first salt and the second salt are dissolved in respective solutions.

12. The method of claim 1 or 2, wherein, The milk-containing raw material is cow milk or goat milk.

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