CN116836257A

CN116836257A - Osteopontin purification method

Info

Publication number: CN116836257A
Application number: CN202310789087.4A
Authority: CN
Inventors: 罗述博; 张永久; 解庆刚; 冷友斌; 耿驰; 林丹; 周鑫; 刘洋; 关海舟; 陆思宇
Original assignee: Heilongjiang Moseidong Technology Co ltd; Heilongjiang Feihe Dairy Co Ltd
Current assignee: Heilongjiang Moseidong Technology Co ltd; Heilongjiang Feihe Dairy Co Ltd
Priority date: 2023-06-29
Filing date: 2023-06-29
Publication date: 2023-10-03

Abstract

The present invention relates to a method for isolating osteopontin, the method comprising: a step of anion exchange resin treatment, wherein the anion exchange resin is used for separating emulsion containing osteopontin, and the component (A) enriched with the osteopontin is obtained under the action of eluent; a concentration step of concentrating the component (a) using a concentration membrane; and optionally one or more of a desalting step and a drying step, wherein in the step of treating the anion exchange resin, the working temperature of the anion exchange resin is 55 ℃ or lower; the anion exchange resin is a particulate resin, and the particle size of the particles is greater than 100 μm.

Description

Osteopontin purification method

Technical Field

The invention belongs to the field of food raw material processing and production, and particularly relates to a method for separating and purifying protein, and more particularly relates to a method for separating, extracting or concentrating human-derived or animal-derived osteopontin.

Background

Osteopontin (OPN) is a highly post-translationally modified glycoprotein synthesized by a variety of tissues and found in almost all body fluids, and is present in breast milk in an amount of over 10% of the total protein content in human milk, and is reported in the literature to be about 138mg/L in early lactation and about 157mg/L in later lactation, making it one of the five most abundant breast milk proteins.

The main research direction of the infant formula is mother emulsification, so that the main research field of the dairy industry is how to make the product more similar to the breast milk by taking the breast milk as a gold standard. Osteopontin is an important component of human milk protein, and is contained in cow milk in a relatively low amount. Apart from the differences in content, there are also certain differences in structure, and breast milk osteopontin is specifically composed of 298 amino acids, is an active secretory phosphorylated glycoprotein capable of binding with calcium, while cow milk OPN is specifically composed of 262 amino acids, and contains up to 34 phosphoserine and 2 phosphothreonine in human milk OPN. Current research indicates that it plays an important role in intestinal proliferation and maturation, brain medullary sheath formation, neural development, and immune development. In vitro studies, b-OPN was found to have gastric juice digestion resistance comparable to human OPN by incubating b-OPN (bovin OPN) with human neonatal gastric juice, whereas intake of b-OPN was found to increase plasma OPN levels in experiments with 10 week old mice. Human milk OPN is also involved in brain development, behavioral and cognitive ability-related neural tissue development in fed infants.

To prevent formula fed infants from taking lower osteopontin and thereby having a negative effect on their growth and development, immune response, galactose metabolism and cytoskeletal remodeling, infant formula manufacturers aim to increase the concentration of osteopontin in their infant formulas in order to address this gap. How to separate and extract the protein from the cow milk by the process technology is a difficulty in the prior art, and the infant food formula can be further optimized to be more similar to the breast milk by using the high-purity protein raw material.

Similar to lactoferrin, the OPN concentration in cow milk is much lower than in human milk, and even lower in milk-based infant formulas. In recent years, as scientists have studied osteopontin, a number of new isolation methods have been developed for the purification and isolation of OPN. For example:

the cited document 1 discloses a method for extracting osteopontin from cow milk, which solves the problem of low extraction purity in the prior art, and prepares the osteopontin by ion exchange chromatography and two-step hydrophobic chromatography, wherein the purity is about 40%.

Reference 2 discloses a method for separating osteopontin by using concentrated feed, wherein the pH of milk raw material (whey) is controlled to be acidic at normal temperature, the conductance is 4-10mS/cm, and the adsorbed protein is collected by passing through an anion exchange medium.

Reference 3 discloses an extract and its use, which extract osteopontin from an alpha-lactalbumin-rich whey protein extract, by acidifying the whey protein, decalcifying, and then diluting to collect the precipitate as the final product.

Reference 4 discloses a method for separating and analyzing osteopontin, which comprises subjecting an object to be tested containing osteopontin to HPLC separation by using a strong anion exchange chromatographic column to obtain an osteopontin sample, wherein a filler material in the strong anion exchange chromatographic column comprises surface porous silica gel bonded with trimethyl aminopropyl or microporous ethylvinylbenzene crosslinked 55% divinylbenzene polymer bonded with trimethyl aminopropyl.

Reference 5 discloses a method for separating osteopontin, wherein partial proteins including osteopontin are precipitated by adjusting the pH value, the precipitate is dissolved by adding a salt solution to obtain a solution rich in osteopontin, and the osteopontin is precipitated by adjusting the pH value to obtain a yield of over 70%.

In reference 6, a method for isolating osteopontin using a feed containing CMP or casein species is disclosed, which enables good isolation of osteopontin using pH and specific conductance at a narrow window of the milk material. The specific conditions are as follows: at a temperature below 25 ℃, the pH=3.6-6.5, and the specific conductance is 4-10 mS/cm. Proteins having isoelectric points less than 5, including osteopontin, are collected by anion exchange media.

In addition, some of the following studies have been conducted on the use of osteopontin. For example:

reference 7 discloses a lactoferrin-osteopontin-iron complex, which is used for treating or preventing iron deficiency.

Reference 8 discloses a neurodevelopmental nutritional composition, and preparation and application thereof, comprising 2' -fucosyllactose and osteopontin, and optionally casein.

In addition, from the commercial market, the high-purity product on the market at present has the protein content of more than 78%, wherein the osteopontin accounts for more than 95% of the total protein; meanwhile, the product with low purity is obtained, and the purity is more than 20%. The basic process used is that cow's milk is acid whey or sweet whey (ph=4-5) obtained by acid coagulation or enzymatic coagulation, the sample is ultrafiltered, the osteopontin is adsorbed by anion exchange resin, then eluted with salt, ultrafiltered and CaCl is added ₂ The retentate is preserved, then sterilized and spray dried.

Although the above prior art has been studied to some extent for the isolation and application of osteopontin, it still cannot be said to be sufficient for the efficient isolation of osteopontin and for the industrial mass production, and there is still room for further investigation.

Citation literature:

citation 1: CN101485381A

Citation 2: CN103476787B

Citation 3: CN111447842A

Citation 4: CN113567573A

Citation 5: CN115073580A

Citation 6: CN103492408A

Citation 7: CN105377059A

Citation 8: CN114586983A

Disclosure of Invention

Problems to be solved by the invention

Although as described above, various attempts have been made to isolate osteopontin. It has been common that chemical precipitation is relatively complex, generally limited in product purity, and new chemicals are introduced. However, ion exchange resin-based chromatography is advantageous in terms of purity but is not efficient, and therefore, is limited in scale and is also affected by the increase in cost.

Further, in long-term industrial practice, the applicant has further found the following problems, for example:

the extraction method of osteopontin in cow milk in cited document 1 solves the problem of low extraction purity in the prior art, and prepares the osteopontin by ion exchange chromatography and two-step hydrophobic chromatography, wherein the purity is about 40%. In the ion exchange chromatography separation process, the method of stirring and standing is adopted for adsorption, the adopted resin is DEAE-Sephacel anion exchange resin, the adsorption effect can be achieved only at low temperature for a long time, and the ion exchange chromatography is followed by hydrophobic chromatography, so that the steps are complex, and a large amount of salt solution is introduced, so that the extraction efficiency and effect are greatly limited.

In reference 3, whey protein is acidified, pH is adjusted to decalcify, and then diluted and pH is adjusted to be high, and the precipitate is collected as the final product. It has been found that, firstly, the acidified protein can cause denaturation of partial protein, affect the use characteristics of related products, introduce other chemical reagents, which is unfavorable for the later application of the products, and meanwhile, the osteopontin extracted by utilizing a chemical precipitation mode has low purity and cannot meet the requirements of high extraction rate and high purity.

The use of a narrow window of pH and specific conductance of milk material in cited document 6 enables good separation of osteopontin. Proteins having isoelectric points of less than 5, including osteopontin, can be collected by anion exchange media at a pH of 3.6 to 6.5 at a specific conductance of 4 to 10mS/cm at a temperature of less than 25 ℃. The process has low flow rate and small treatment capacity, and simultaneously, the pH and specific conductance are strictly limited, the difficulty in the operation process is increased, and the control in the industrialization process is extremely difficult.

In addition, the above-mentioned scheme still belongs to the extraction technology which stays in the small-scale laboratory stage basically, and meanwhile, no further discussion is made on factors such as the working condition of the ion exchange resin. Furthermore, the existing HPLC detection method for osteopontin should not be confused with the industrial separation and purification mode of the present invention, the former focus is on separation and concentration detection, not the purity and purification efficiency of the final product.

Therefore, the technical problem to be solved by the invention is to provide a separation and purification technology of osteopontin based on ion exchange resin, which is suitable for industrial mass production, and by adjusting the characteristics and working conditions of anion exchange resin, the retention time of osteopontin can be shortened while the activity of protein is not affected, and the increase of lactose, whey protein or casein content in the product can be inhibited.

Solution for solving the problem

Through long-term researches of the inventor, the technical problems can be solved through implementation of the following technical scheme:

[1] the present invention provides a method for isolating osteopontin, wherein the method comprises:

a step of anion exchange resin treatment, wherein the anion exchange resin is used for separating emulsion containing osteopontin, and the component (A) enriched with the osteopontin is obtained under the action of eluent;

a concentration step of concentrating the component (a) using a concentration membrane;

and optionally one or more of a desalting step, a drying step,

wherein,,

in the step of treating the anion exchange resin, the working temperature of the anion exchange resin is below 55 ℃;

the anion exchange resin is a particulate resin, and the particle size of the particles is greater than 100 μm.

[2] The method according to [1], wherein the emulsion containing osteopontin has a solid content of 6 to 20 mass% and a fat content of 1 mass% or less.

[3] The method according to [1] or [2], wherein the emulsion particle diameter in the emulsion containing osteopontin is 10 μm or less.

[4] The method according to any one of [1] to [3], wherein the emulsion containing osteopontin is subjected to a heat treatment at 25 to 60 ℃ before being contacted with an anion exchange resin.

[5] The method according to any one of [1] to [4], wherein in the step of treating the anion exchange resin, the eluent is an aqueous solution of halogenated monovalent metal salt.

[6] The method according to [5], wherein an aqueous solution of halogenated monovalent metal salt as the first eluent is used to elute the carbohydrate and whey protein components; an aqueous solution of halogenated monovalent metal salt is used as a second eluent having an ionic strength greater than that of the first eluent to elute osteopontin components.

[7] The method according to any one of [1] to [6], wherein in the step of treating the anion exchange resin, the operating temperature of the anion exchange resin is 4 to 15℃or 42 to 55 ℃.

[8] The method according to any one of [1] to [7], wherein in the concentrating step, the pore diameter of the concentration membrane is 30kDa or less.

[9] The method according to any one of [1] to [8], wherein in the concentrating step, the component (A) is concentrated to 1/20 to 1/50 of the volume by a concentration membrane.

[10] The method according to any one of [1] to [9], wherein the concentrating step is followed by a desalting step in which the conductivity of the desalted system is less than 2mS/cm, a sterilizing step and a drying step in this order.

ADVANTAGEOUS EFFECTS OF INVENTION

By implementing the technical scheme, the invention can obtain the following technical effects:

1) Through optimizing the characteristics and working conditions of the anion exchange resin, the osteopontin component can be separated on the premise of not damaging the activity of the protein, and meanwhile, the retention time of the osteopontin is reduced, so that the treatment efficiency is remarkably improved;

2) The purity of the osteopontin solid can be adjusted within a wide range, and the purity of the osteopontin solid can be up to more than 80 percent by optimizing the characteristics of anion exchange resin and working conditions, so that the content of impurity components in the osteopontin can be reduced, and the purity of a final product is improved;

3) Through optimizing the characteristics of anion exchange resin and working conditions, the purity of osteopontin is improved, so that the use of subsequent purification chromatography (hydrophobic chromatography) is not needed, and the overall efficiency of the purification process is further improved.

4) The method for purifying osteopontin of the present invention is not particularly limited, and may be an untreated emulsion of human or animal, or may be a dairy product which has been processed, such as powdered milk.

5) The purification method of the invention has the advantages of simple and efficient operation on the premise of improved separation purity, is more beneficial to industrialized mass production, and can obtain high-purity whey protein component while obtaining good-purity osteopontin.

Drawings

Fig. 1: chromatograms of osteopontin extraction procedure in example 1;

fig. 2: the lyophilized sample and sterile liquid of example 1;

fig. 3: osteopontin purity detection map (red standard sample map)

Fig. 4: a standard curve.

Detailed Description

Various exemplary embodiments, features and aspects of the invention are described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, well known methods, procedures, means, equipment and steps have not been described in detail so as not to obscure the present invention.

Unless otherwise indicated, all units used in this specification are units of international standard, and numerical values, ranges of values, etc. appearing in the present invention are understood to include systematic errors unavoidable in industrial production.

In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

In the present specification, the term "particle diameter" of the anion exchange resin or "particle diameter" is used to describe the anion exchange resin, and means an average particle diameter.

In the present specification, the numerical range indicated by the term "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present invention, a numerical range indicated by "above" or "below" is a numerical range including the present number.

In the present invention, the use of "optionally" or "optionally" means that certain substances, components, steps performed, conditions applied, etc. are used or not used.

In the present invention, "Da" is used to denote the unit "Dalton" of molecular weight, i.e. "daltons".

In the present invention, unless otherwise specified, "normal temperature" as used herein refers generally to a temperature of 23.+ -. 2 ℃.

In the present invention, the unit names used are all international standard unit names, and "%" used represent weight or mass% unless otherwise specified.

In the present specification, the use of "infant" means a group of humans aged 3 years or less.

In this specification, the terms "about" or "substantially", "essentially" may mean: one value includes the standard deviation of the error of the device or method used to determine the value. The numerical ranges and parameters set forth herein are approximations that may vary depending upon the particular application. However, any numerical value inherently contains certain standard deviations found in their respective testing apparatus or methods. Accordingly, unless expressly stated otherwise, it is to be understood that all ranges, amounts, values and percentages used herein are modified by "about". Herein, "about" generally means that the standard deviation of the actual numerical value from the theoretical model or theoretical data is within 2%, more preferably within 1%.

Reference in the present disclosure to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," etc., means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.

The invention mainly provides a separation method of osteopontin which is suitable for industrial mass production and has obviously improved production efficiency and is based on anion exchange resin.

The present invention is mainly based on the following findings:

in the prior art, the anion exchange resin-based osteopontin purification method generally uses a granular resin material with a chromatography layer material of about tens of micrometers or less than 100 micrometers, which mainly considers that the sufficient adsorption effect is enough to retain the osteopontin so as to improve the yield and purity of the osteopontin, but greatly influences the working efficiency, prolongs the retention time of the osteopontin, and simultaneously, even the component separated by the anion exchange resin still needs to be treated by, for example, a hydrophobic chromatography layer so as to obtain the osteopontin product with acceptable purity, which further reduces the production efficiency. In addition, the prior art, both for the osteopontin-containing component to be treated and for the anion exchange resin during operation, is controlled at a lower temperature below about 10 ℃ to avoid denaturation of the protein component.

The present inventors have surprisingly found that increasing the particle size of the anion resin particles as the stationary phase (while optimizing the temperature conditions at which the anion resin works) when concentrating osteopontin using anion exchange resins not only greatly reduces the retention time of osteopontin, but also allows better separation from lactose, whey protein and casein. Although the mechanism is not completely understood, it is believed that in the above operation, a better balance is obtained between the surface adsorption of the stationary phase, the charge effect of the protein component and the stationary phase, and the weakening of the interaction between the protein components at the operating temperature.

(object of treatment)

The treatment object of the present invention is not particularly limited in principle, as long as it is a component containing osteopontin.

Such a treatment object may be a milk-containing component derived from a human or animal. Preferably, the milk-containing component may be derived from cattle, sheep, horses, camels, etc., and more preferably, the milk-containing component may be derived from cattle.

For such a treatment object, the purification process of the present invention may be performed in the form of an emulsion. Such an emulsion may be used as it is or may be partially treated, or may be solid emulsion, for example, emulsion obtained by mixing water with powdered milk or the like.

In some specific embodiments, the osteopontin-containing emulsion of the present invention is a defatted emulsion from the standpoint of facilitating subsequent anion exchange resin treatment.

The manner of degreasing is not particularly limited in principle, and a conventional degreasing manner such as membrane filtration or centrifugal separation, preferably, centrifugal separation is used to remove the grease from the emulsion may be used. In some preferred embodiments, the content of the oil or fat is 1 mass% or less, preferably 0.8 mass% or less, more preferably 0.6 mass% or less, based on the total mass of the emulsion, with respect to the emulsion which has been subjected to degreasing treatment and which can be directly subjected to anion exchange resin treatment.

(pretreatment)

Further, it has been found that subsequent anion exchange resin treatments can be made more efficiently by suitable pretreatment.

Pretreatment of the emulsion containing osteopontin may include adjustment of solid content, adjustment of emulsion particle size, and heat treatment.

For the adjustment of the solid content, controlling the emulsion containing osteopontin to a proper solid content is advantageous for improving the treatment efficiency, and preferably, the solid content of the emulsion is 6 to 20 mass%, more preferably, 8 to 16 mass%. Also, the content of the oil or fat may be 1 mass% or less, preferably 0.8 mass% or less, more preferably 0.6 mass% or less, for example, 0.3 mass% or less, 0.1 mass% or less, or the like, based on the total mass of the emulsion.

For the adjustment of the particle size of the emulsion, it has been found that the adsorption of the large particle size anionic resin particles can be better adapted by controlling the particle size range in the emulsion, on the one hand, to complement the specific surface area of the large particle size particles, and on the other hand, to reduce the interaction between the protein components. In some preferred embodiments, the emulsion maximum particle size of the osteopontin-containing emulsion is 10 μm or less, more preferably 8 μm or less.

For the heat treatment, although it is not suggested in the prior art that the treatment object is heat-treated before the anion exchange resin treatment to avoid denaturation of the protein, the present invention has found that the proper heat treatment does not cause the protein denaturation concerned, and also can weaken the mutual (charge) effect between the protein components in the emulsion and the adsorption effect between the carbohydrate and the protein, especially, can separate the whey protein and other components in the subsequent process better in cooperation with the proper emulsion particle size. Thus, in some preferred embodiments of the invention, the osteopontin-containing emulsion may be heated to 25-60 ℃, preferably 23-50 ℃, before the anion exchange resin treatment is performed, and additionally, the heating time may be controlled within 1 hour, for example, 0.4-0.8 hour, from the viewpoint of inhibiting protein denaturation.

(step of anion exchange resin treatment)

The solution containing osteopontin (obtained by the treatment) was treated with an anion exchange resin.

The anion exchange resin for the present invention may comprise a matrix and cationic groups.

In some specific embodiments, the matrix may, for example, comprise, or even consist essentially of, a polysaccharide. Particularly preferred are crosslinked polysaccharides. Examples of useful polysaccharides are cellulose, agarose, and/or dextran. In addition, the matrix may also be a polymer comprising or even consist essentially of a non-carbohydrate polymer. Examples of useful non-carbohydrate polymers are methyl methacrylate, polystyrene, and/or styrene-divinylbenzene.

As cationic groups, amino groups may be exemplified, or even consist essentially of. Tertiary amino groups are particularly preferred and produce quaternary ammonium groups under suitable pH conditions. The quaternary ammonium groups provide strong anion exchange characteristics to the anion exchange medium. Or, alternatively, these cationic groups may comprise one or more primary or secondary amino groups. The large number of primary or secondary amino groups typically provides the anion exchange media with weak anion exchange characteristics.

For the anion exchange resin of the present invention, it is possible to use in the form of particles having a particle diameter of more than 100. Mu.m, preferably 110 to 200. Mu.m, for example 120. Mu.m, 150. Mu.m, 180. Mu.m, etc. It has been found that increasing the particle size can significantly improve the purification separation efficiency, increase the treatment throughput, and also not cause excessive elution of casein components. In addition, under the cooperation of the working temperature and other conditions of the anion exchange resin, the carbohydrate, the whey protein and the casein can be effectively separated from the osteopontin, so that the high-purity osteopontin product can be obtained.

In some preferred embodiments of the invention, useful anion exchange resins are commercially available, for example, from the brands of mofeton, sitovan, bailinaceae, and the like.

The carbohydrate (e.g. lactose), whey protein and casein components are mainly separated from the water by anion exchange resin treatment. Wherein the carbohydrate has no charge, adsorption of whey protein and weak charge effect, is more easily eluted from the anion exchange resin than osteopontin, and binding of casein to the anion exchange resin is stronger than that of osteopontin and anion exchange resin, so that osteopontin is eluted more preferentially than casein.

Thus, the eluents usable as the purification/separation treatment may include a first eluent and a second eluent, wherein both may be aqueous solutions of halogenated monovalent metal salts, preferably chlorides of the first main group metals, such as sodium chloride, potassium chloride, and the like.

Further, the first and second eluents have a difference in ionic strength to perform different elution functions, wherein the second eluent has a higher ionic strength than the first eluent. In some specific embodiments, the concentration of the first eluent may be 2.8% (m/V%) or less, preferably 1% to 2.5% (m/V%), more preferably 1.5% to 2.2% (m/V%); the concentration of the second eluent may be greater than 2.8% (m/V%), preferably 3% to 12% (m/V%), more preferably 4% to 10% (m/V%). Wherein the first eluent is used for eluting components of the carbohydrate, whey protein and the like which are weakly bound to the anion exchange resin, and the second eluent is used for eluting osteopontin components while preventing elution of casein (beta-casein). In addition, other components that can be used in the eluent, such as a buffer component or a pH adjusting component, are not particularly limited, and may be selected and used with reference to the modes of operation that are conventional in the art.

Further, for the temperature of the anion exchange resin at the time of the purification/separation treatment of the present invention, although the principle of low temperature favorable for adsorption in the prior art, it is generally required to control the operating temperature of the anion exchange resin to, for example, 6 ℃ or less, or at most, not to exceed 40 ℃. In contrast, in the case of using the resin having the above particle diameter characteristics, the present invention is considered to prevent the denaturation of osteopontin and the increase of impurities due to the shortened retention time even if the anion exchange resin is operated at a wide range of not more than 55℃and preferably 4 to 55 ℃.

Although the principle is not completely clear, there is the following presumption:

the working temperature has an effect on substances such as proteins in the resin and the emulsion at the same time, wherein the temperature is adjusted so that the conformation and interaction of different proteins in the emulsion are changed when the emulsion contacts the resin, and higher thermal motion causes some proteins which are mutually physically or electrostatically adsorbed to be activated, for example, the electrostatic interaction between lactoferrin and osteopontin is reduced, and the interaction between casein and osteopontin is also reduced. Further, the elution of the carbohydrate component and the whey protein component can be faster by the eluent, and further, the adsorptivity of the resin particles is appropriately reduced at a proper temperature, so that the osteopontin can be more quickly separated from the anionic resin without causing dissolution of casein, thereby improving the treatment efficiency and the purity of the product.

Thus, in some embodiments of the present invention, the anion exchange resin preferably operates at a temperature in the range of 4 to 15 ℃ or 42 to 55 ℃ to avoid the optimum temperature range for microbial growth to the greatest extent, wherein the resin particle size is believed to contribute primarily to the efficiency of the process, and at the latter temperature, the temperature and particle size contribute primarily to the efficiency of the process. Therefore, more preferably, the anion exchange resin has an operating temperature of 42℃to 55 ℃.

Under the above conditions the eluted fraction of the first eluent is removed or the fraction (A') is otherwise collected to isolate the whey protein therein, whereas the eluted fraction of the second eluent is a fraction enriched with osteopontin, i.e. fraction (A).

The treatment time in the step of treating the anion exchange resin may be usually not more than 10 minutes, preferably not more than 6 minutes, more preferably 1 to 3 minutes. The process can be adjusted by varying the solids content of the emulsion containing osteopontin and the flow rate and pressure of the eluent.

(step of concentrating)

In the present invention, the component (A) may be further subjected to membrane treatment for further concentration or purification.

From the viewpoint of balance of efficiency and concentration effect, the pore diameter of the concentration membrane may be 30kDa or less, preferably 5 to 30kDa, for example, 10kDa, 15kDa, etc., to further remove low molecular weight components and thereby to improve the purity of osteopontin.

Compared with the method that the components treated by anion exchange resin in the prior art need to be further treated by hydrophobic chromatography to achieve satisfactory purity, the treatment method of the invention which uses membrane concentration after anion exchange treatment can further improve the treatment capacity and the treatment efficiency.

Further, in some preferred embodiments of the present invention, the component (A) is concentrated to 1/50 to 1/20 of the original volume, for example, 1/30, 1/40, etc., by a concentration membrane to obtain the concentrated component (B).

(other steps)

After the concentrated component (B) described above is obtained, an optional post-treatment may be used to obtain the final osteopontin product.

These post-treatment methods include one or more of a desalting step, a sterilizing step, a drying step, and the like.

In some specific embodiments of the present invention, the concentrated component (B) is treated with a step of desalting, a step of sterilizing, and a step of drying in this order.

For the step of desalting, there may be exemplified a treatment using an ultrafiltration membrane, preferably, the conductivity of the desalted system is less than 2mS/cm.

As the sterilization step, there may be exemplified a pasteurization treatment at a temperature of 60 to 80℃for 15s to 30min. In some specific embodiments, 20-30 mass% of the aseptic liquid rich in osteopontin can be obtained after sterilization treatment, and the total colony count is less than 50000cfu/g.

For the drying step, freeze drying, spray drying, etc. may be included until a desalted concentrate is produced.

In addition, as for the above-mentioned whey protein-enriched component (A'), optionally, the purified whey protein may be further obtained by concentration and post-treatment steps. The present invention is not particularly limited in principle with respect to these processing steps.

The purity of the osteopontin finally obtained by the method provided by the invention can be adjusted within a wide range, for example, the working conditions of the step of anion exchange resin treatment and the step of concentration can be adjusted, so that the purity can be realized within a wide range of more than 5%, the purity can be realized to be more than 80% under the condition of considering the treatment efficiency, the purity can be adjusted between 50 and 85% in some preferred embodiments, and the total yield can be more than 40%.

Examples

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

< quantitative determination of osteopontin content >:

(Experimental materials)

Water, GB/T6682, first order. Sodium chloride (NaCl). Trifluoroacetic acid (TFA, CF) ₃ CO ₂ H) A. The invention relates to a method for producing a fibre-reinforced plastic composite Lactic acid (CH) ₃ CH (OH) COOH). Calcium chloride (CaCl) ₂ ). Tris (hydroxymethyl) aminomethane solution (1 mol/L, ph=8.0). TFA in water (0.1%); lactic acid solution (10%): calcium chloride solution (500 mM); mobile phase a (ph=8.0): buffer solution of ph=8.0 consisting of 20mM tris and 10mM NaCl. Mobile phase B (ph=8): buffer solution with ph=8.0 consisting of 20mM tris and 800mM NaCl.

(Instrument and device)

High performance liquid chromatography: an ultraviolet detector or a diode array detector is arranged; and (3) a balance: the sensing amount is 0.1mg and 0.01g; a water bath kettle; pH meter: the precision is 0.01; a vortex mixer; an ultrasonic oscillator.

(sample extraction)

Liquid sample extraction

30g (accurate to 0.1 mg) of the sample was weighed accurately, 4mL of calcium chloride solution was added thereto, and after shaking, it was subjected to water bath at 70℃for 20 minutes. Taking out from the water bath, cooling to room temperature, regulating pH to 4.3 with lactic acid solution, fixing volume to 50mL with pure water, shaking, filtering with filter paper, filtering the filtrate with 0.22 μm filter membrane into sample bottle, and measuring on the machine.

Solid sample extraction

Accurately weighing 5g (accurate to 0.1 mg), dissolving with 30mL of water at 40-50 ℃, and vortex shaking for 5min. 4mL of calcium chloride solution was added, and after shaking, the mixture was subjected to water bath at 70℃for 20min. Taking out from the water bath, cooling to room temperature, regulating pH to 4.3 with lactic acid solution, fixing volume to 50mL with pure water, shaking, filtering with filter paper, filtering the filtrate with 0.22 μm filter membrane into sample bottle, and measuring on the machine. Whey powder with high osteopontin content is diluted 2-10 times by mobile phase A after constant volume filtration, and is filtered into a sample bottle by a filter membrane with the thickness of 0.22 mu m, and is measured by a machine.

(liquid chromatography reference conditions)

Chromatographic column: multilayer network porous polymethacrylate type quaternary ammonium salt anion exchange chromatographic column 5 μm,4.6 x 100mm;

column temperature: 40 ℃;

detection wavelength: 220nm;

sample injection amount: 10. Mu.L;

needle washing liquid: 0.1% TFA in water.

The gradient elution conditions are shown in Table 2.

TABLE 2 Mobile phase elution conditions

(Standard Curve preparation)

Taking standard series solution, taking the standard peak area of osteopontin as an ordinate, taking the concentration of the standard solution as an abscissa,

and drawing a standard curve to obtain a standard curve regression equation, see fig. 4.

(measurement of sample solution)

And (3) injecting the sample solution into a high performance liquid chromatograph, measuring the peak area, and obtaining the concentration of the osteopontin in the liquid to be measured according to a standard curve.

(test data processing)

The mass fraction X of osteopontin in the sample is expressed in grams per hundred grams (mg/100 g) and is calculated according to the following formula:

wherein:

c-calculating the concentration of the measured component curve in micrograms per liter (mg/L);

v-final constant volume of sample solution in milliliters (mL);

m-sample mass in grams (g);

f-dilution factor;

when the content of the osteopontin is more than or equal to 100mg/100g, the calculated result keeps four valid figures; when the osteopontin content is less than 100mg/100g, the calculation result keeps three significant digits.

Example 1

The osteopontin was prepared as follows:

taking skimmed milk emulsion with fat content less than 0.1% and maximum particle size less than 8 μm, ion-exchanging the skimmed milk at 4-10deg.C by using anion exchange resin (average particle size 120 μm) of mofeton to adsorb protein with anionic charge onto the ion exchange resin, keeping milk in resin column for 3min, and ejecting the emulsion with water.

The first step of elution adopts about 4 columns of 1.5 percent sodium chloride solution to carry out elution to obtain a hetero protein solution;

the second step of elution adopts 3 percent sodium chloride solution with the volume of about 4 columns to carry out elution, so as to obtain the osteopontin salt solution;

concentrating the eluate I and eluate II by 10 times with membrane with pore size of 10kDa, washing, filtering, and desalting to obtain purified whey protein and osteopontin, wherein the solid content of desalted solution is 5% and the conductivity is 1.5mS/cm.

Freeze-drying or low temperature spray-drying or membrane sterilization: respectively freeze-drying the whey protein and the osteopontin to obtain freeze-dried protein powder; or respectively carrying out low-temperature spray drying on the whey protein and the osteopontin to obtain spray-dried protein powder; or respectively performing membrane filtration sterilization or pasteurization at 72-75 ℃ for 15 seconds on the whey protein and the osteopontin to prepare the sterile whey protein and the osteopontin liquid package.

Fig. 1 is a chromatogram of the extraction process in the example, fig. 2 is a lyophilized sample and sterile liquid prepared in the example 1, and fig. 3 is a osteopontin purity detection spectrum.

Example 2

The osteopontin was prepared as follows:

skim milk emulsion with fat content less than 0.5% and maximum particle size less than 8 microns is taken, ion exchange treatment is carried out on the emulsion at 50-55 ℃ by using anion exchange resin which is the same as that of example 1 so as to enable whey protein containing osteopontin to be adsorbed on the ion exchange resin, the retention time of milk in a resin column is 1.5min, and the emulsion is ejected by water after loading.

The first step of elution adopts about 4 columns of 2% sodium chloride solution to carry out elution to obtain a mixed protein solution;

the second step of elution adopts 6 percent sodium chloride solution with the volume of about 4 columns to elute so as to obtain the osteopontin solution;

concentrating the solution of the hetero protein and the solution of the osteopontin by using a membrane with the pore diameter of 5kDa for 30 times, and then washing, filtering and desalting to obtain the purified osteopontin, wherein the solid content of the desalted solution is 12%, and the conductivity is less than 1.8mS/cm.

Freeze-drying or low temperature spray-drying or membrane sterilization: freeze-drying the product to obtain freeze-dried protein powder; or the product is subjected to low-temperature spray drying to obtain spray-dried protein powder; or the products are subjected to membrane filtration sterilization or pasteurization at 72-75 ℃ for 15 seconds to prepare the sterile liquid package.

Example 3

The osteopontin was prepared as follows:

The first step of elution adopts about 4 columns of 2.2 percent sodium chloride solution to carry out elution to obtain a mixed protein solution;

Example 4

The osteopontin was prepared as follows:

the skim milk emulsion having a fat content of less than 0.1% and a maximum particle size of less than 10 μm was ion-exchanged with the same anion exchange resin as in example 1 at a temperature of 50 to 55 ℃ to adsorb the whey protein containing osteopontin onto the ion exchange resin, the retention time of milk in the resin column was 2min, and the emulsion was ejected with water after the completion of the loading.

The first step of elution adopts about 5 columns of 2.5 percent sodium chloride solution to carry out elution to obtain a mixed protein solution;

concentrating the solution of the hetero protein and the solution of the osteopontin by using a membrane with the pore diameter of 5kDa for 30 times, and then washing, filtering and desalting to obtain the purified osteopontin, wherein the solid content of the desalted solution is 12%, and the conductivity is less than 0.5mS/cm.

Comparative example 1

The extraction and separation were carried out using an anion exchange resin filler having a particle size of 70 μm and the same material as the resin of example 1.

The osteopontin was prepared as follows:

taking skim milk emulsion with fat content less than 0.1% and maximum particle size less than 10 microns, carrying out ion exchange treatment on the emulsion at 50-55 ℃ by utilizing anion exchange resin so as to enable whey protein containing osteopontin to be adsorbed on the ion exchange resin, keeping the retention time of milk in a resin column for 5min (only reducing the flow rate and increasing the retention time in order to ensure the operating pressure of equipment to be in a proper range due to smaller pore size), and ejecting the emulsion by water after the application.

Table 1 below shows the purity and yield of the osteopontin powder obtained in examples and comparative examples, wherein the purity test was performed according to the above < quantitative determination of osteopontin content > method:

TABLE 1

	Purity/%	Yield/%
			Example 1	51.05	43.01
Example 2	70.69	74.11
			Example 3	81.29	65.83
Example 4	80.26	66.41
			Comparative example 1	42.11	40.76

It should be noted that, although the technical solution of the present invention is described in specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the technical improvements in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method of isolating osteopontin, the method comprising:

and optionally one or more of a desalting step, a drying step,

wherein,,

2. The method according to claim 1, wherein the emulsion containing osteopontin has a solid content of 6 to 20 mass% and a fat content of 1 mass% or less.

3. The method according to claim 1 or 2, wherein the emulsion maximum particle size in the emulsion containing osteopontin is below 10 μm.

4. A method according to any one of claims 1 to 3, wherein the osteopontin-containing emulsion is subjected to a heating treatment prior to contact with the anion exchange resin, the heating temperature being 25 to 60 ℃.

5. The method according to any one of claims 1 to 4, wherein in the step of treating the anion exchange resin, the eluent is an aqueous solution of halogenated monovalent metal salt.

6. The method according to claim 5, characterized in that an aqueous solution of halogenated monovalent metal salts is used as the first eluent to elute the carbohydrate and whey protein components; an aqueous solution of halogenated monovalent metal salt is used as a second eluent having an ionic strength greater than that of the first eluent to elute osteopontin components.

7. The method according to any one of claims 1 to 6, wherein in the step of treating the anion exchange resin, the operating temperature of the anion exchange resin is 4 to 15 ℃ or 42 to 55 ℃.

8. The method according to any one of claims 1 to 7, wherein in the concentrating step, the pore size of the concentrating membrane is 30kDa or less.

9. The method according to any one of claims 1 to 8, wherein in the concentrating step, the component (a) is concentrated to 1/20 to 1/50 of the volume by a concentration membrane.

10. The method according to any one of claims 1 to 9, characterized in that it comprises, in order after the concentrating step, a desalting step in which the conductivity of the desalted system is less than 2mS/cm, a sterilizing step and a drying step.