JP2010193833A - Method for separating and recovering whey protein with porous membrane having functional groups interacting with protein - Google Patents

Abstract

The present invention provides an industrially useful method for effectively and rapidly separating and recovering whey proteins such as α-lactalbumin, β-lactoglobulin, and lactoferrin from whey without heat treatment. With the goal.
A method for separating and recovering whey protein using a porous membrane having a functional group that interacts with protein is provided.
[Selection figure] None

Description

  The present invention relates to a method for separating and recovering whey protein from whey using a porous membrane having a functional group that interacts with protein.

  Milk contains many useful proteins. Among these, whey protein in whey, which is a by-product of the production of cheese obtained by coagulating casein, is a variety of proteins. It has been attracting attention because it may develop immune functions. However, since whey is a by-product in the cheese production process and it is not industrially easy to effectively separate the various whey proteins contained in it, it is often used. It is not discarded.

There are three main components in whey protein, α-lactalbumin, β-lactoglobulin, and lactoferrin. Besides these, serum albumin and immunoglobulins such as IgG, IgA, and IgM are included. α-Lactalbumin has an effect of killing tumor cells, and is effective in killing bacteria in the upper respiratory tract and protecting the gastrointestinal mucosa. Since β-lactoglobulin is highly antigenic and acts as an allergen, it may be required to be removed from milk to prevent the development of allergies. On the other hand, it binds retinal (vitamin A), and retinal from the intestine. Since it has an action of promoting uptake and has high binding properties to long-chain fatty acids, it is expected to be isolated and used as a functional protein. Lactoferrin collects iron absorption from the intestinal tract and has the effect of inhibiting the growth of various bacteria. It binds to free iron released from cells that have been destroyed by neutrophils, preventing oxidation of tissues, It acts to regulate the immune response of immunocompetent cells.
Therefore, it is required to industrially collect and separate whey protein from whey, which is a by-product of milk or cheese production, and many attempts have been made.

As a method for recovering α-lactalbumin from whey, Patent Document 1 adjusts the pH of whey to 4.0 to 7.5, and then heat-treats at 85 ° C. or higher to associate and aggregate β-lactoglobulin. A method for separating and recovering α-lactalbumin using an ultrafiltration membrane or a microfiltration membrane is described. A method of separating β-lactoglobulin and α-lactalbumin aggregated by this method and recovering a solution having a high α-lactalbumin content is disclosed.
Patent Document 2 discloses a method in which whey protein is desalted, α-lactalbumin is aggregated and recovered from whey by adjusting pH and temperature, and β-lactoglobulin is recovered as a supernatant. .
Patent Document 3 discloses a method of removing β-lactoglobulin from whey by adjusting the whey to pH 4 to 6 and then bringing it into contact with an anion exchange resin.

Japanese Patent Laid-Open No. 5-236883 Japanese Patent Laid-Open No. 7-123927 Japanese Patent No. 2652260

In the method disclosed in Patent Document 1, protein denaturation may occur due to the use of heat treatment, and it is difficult to prevent a small amount of β-lactoglobulin from being mixed. In addition, the concentration of α-lactalbumin in the recovered solution is about 0.1%, which is the same as the whey of the stock solution, and cannot be recovered at a high concentration. In the method disclosed in Patent Document 2, there is no possibility of denaturation of protein because it does not involve heat treatment at high temperature, but it is quick and efficient because it requires multiple steps and a centrifugation step. Processing is difficult, and it is difficult to apply as an industrial whey protein separation and recovery application. In the method disclosed in Patent Document 3 above, β-lactoglobulin is adsorbed to the anion exchange resin, so that highly removed whey can be obtained. In order to separate them at the same time, another separation method is required.
Under such circumstances, the present invention is an industrially useful method for effectively and rapidly separating and recovering whey proteins such as α-lactalbumin, β-lactoglobulin, and lactoferrin from whey without heat treatment. Is intended to provide.

As a result of intensive investigations to solve the above problems, the present inventor has conventionally produced a by-product such as cheese production by using a porous film having a functional group that interacts with a protein, and contains useful components. However, from whey that was often processed as waste, it was efficient at an industrial level without heat treatment of whey proteins such as α-lactalbumin, β-lactoglobulin and lactoferrin having immune function. And it discovered that it could isolate | separate and collect rapidly, and came to complete this invention.
That is, the present invention is as follows.
1. A method for separating and recovering whey protein using a porous membrane having a functional group that interacts with the protein;
2. Including the following steps: 1. A method for separating and recovering whey protein according to 1.
(A) a step of allowing whey to permeate through a porous membrane having an anion exchange group or a cation exchange group to adsorb a part of the whey protein to the porous membrane and recovering the permeate;
(B) a step of eluting and recovering whey protein adsorbed on the porous membrane by passing an aqueous solution containing salt;
(C) The solution permeated through the porous membrane having a cation exchange group in the step (A) or the solution eluted and recovered from the porous membrane having an anion exchange group in the step (B) is converted into a porous membrane having a hydrophobic group. A step of allowing a portion of whey protein in the solution to adsorb to the porous membrane having the hydrophobic group by permeating and recovering the permeate;
(D) A step of eluting and recovering whey protein adsorbed on the porous membrane having a hydrophobic group.

3. The whey protein adsorbed by the porous membrane having an anion exchange group in the step (A) is α-lactalbumin and β-lactoglobulin,
The whey protein adsorbed on the porous membrane having a hydrophobic group in the step (C) is β-lactoglobulin. Or 2. A method for separating and recovering whey protein according to 1.
4). The whey protein adsorbed by the porous membrane having a cation exchange group in the step (A) is lactoferrin,
The whey protein adsorbed on the porous membrane having a hydrophobic group in the step (C) is β-lactoglobulin. Or 2. A method for separating and recovering whey protein according to 1.
5). 1. Using a porous membrane in which an anion exchange group is fixed to the surface via a graft chain ~ 3. The method for separating and recovering whey protein according to any one of the above.
6). 1. Using a porous membrane in which a cation exchange group is fixed on the surface via a graft chain 2. 4. The method for separating and recovering whey protein according to any one of the above.
7). 1. Using a porous membrane in which a hydrophobic group is fixed to the surface via a graft chain ~ 4. The method for separating and recovering whey protein according to any one of the above.

  By using the method for separating and recovering whey protein of the present invention, whey proteins such as α-lactalbumin, β-lactoglobulin, and lactoferrin are efficiently processed from whey (whey) at an industrial level without heat treatment. Can be separated and recovered efficiently and quickly.

Hereinafter, a mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail. The present invention is not limited to the following embodiment, and can be implemented with various modifications within the scope of the gist.
The present embodiment is a method for separating and recovering whey protein using a porous membrane having a functional group that interacts with protein. Porous membranes with functional groups that interact with proteins have selective adsorption and non-adsorption properties, depending on the type of protein, by controlling the pH and salt concentration. Whey protein can be separated and recovered by recovering only the protein as a non-adsorbing permeation component, or by adsorbing only a specific protein and then recovering it as an elution fraction.

The “whey” in this embodiment is also called whey, and is a byproduct obtained as a supernatant when coagulating casein from milk to produce cheese. Similarly, the supernatant produced when placed is whey and is not limited thereto.
“Whey protein” in the present embodiment means α-lactalbumin, β-lactoglobulin, lactoferrin, serum albumin, immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin contained in whey. M (IgM), lactoperoxidase, lysozyme, and the like. Among these, α-lactalbumin, β-lactoglobulin and lactoferrin having immune function are preferably used as proteins for separation and recovery, but are not limited thereto.

The term “porous membrane having a functional group that interacts with a protein” used in the present embodiment refers to an anion exchange group, a cation exchange group, which are functional groups, on the surface of a porous body serving as a base material and its pores. , A porous membrane having a hydrophobic group immobilized thereon.
A plurality of functional groups may be immobilized on the same porous membrane as long as the protein exhibits selective adsorption or non-adsorption properties by controlling conditions such as pH and salt concentration.
The substrate of the porous film is not particularly limited, but is preferably composed of a polyolefin polymer from the viewpoint of maintaining mechanical properties. Examples of polyolefin polymers include, for example, homopolymers of olefins such as ethylene, propylene, butylene and vinylidene fluoride, two or more types of copolymers of the olefins, or one or more types of the olefins. Examples thereof include copolymers with perhalogenated olefins. A mixture of two or more of these polymers may be used. Examples of perhalogenated olefins include tetrafluoroethylene and chlorotrifluoroethylene.
Among these, polyethylene or polyvinylidene fluoride is preferable as the base material of the porous film, and polyethylene is more preferable from the viewpoint that the material is particularly excellent in mechanical strength and can obtain a high adsorption capacity.

The form of the porous membrane is not particularly limited as long as the solution can be passed therethrough, and examples thereof include a flat membrane, a nonwoven fabric, a hollow fiber membrane, a monolith, a capillary, a disc, and a cylindrical shape. . Among these forms, a hollow fiber membrane is preferable from the viewpoints of ease of production, scale-up property, and packing property of the membrane when it is molded into a module.
In the present embodiment, the hollow fiber porous membrane is a cylindrical or fibrous porous membrane having a hollow portion, and the inner layer and the outer layer of the hollow fiber are continuous by pores that are through-holes. Means a porous body having a property of allowing liquid or gas to permeate from the inner layer to the outer layer or from the outer layer to the inner layer. The outer diameter and inner diameter of the hollow fiber are not particularly limited as long as the porous membrane can physically hold the shape and can be molded into a module.
In addition, when a porous membrane in which a graft chain is fixed to the surface of the porous body as a base material and its pores and a functional group that interacts with the protein is fixed to the graft chain is used, the porous membrane is adsorbed. This is more preferable because the amount of protein increases.

The method for immobilizing the anion exchange group on the porous membrane is not particularly limited, but generally, a highly reactive functional group such as epoxy or amine is introduced on the substrate surface of the porous membrane, and then the anion is added to the functional group. It can be performed by a method of bonding a compound having an exchange group.
Further, as the porous membrane having the anion exchange group fixed on the surface thereof, for example, commercially available porous membranes such as SartobindQ and SartobindD made of cellulose based on cellulose and Pall's MuslangQ made of polyethersulfone as a base. A membrane can also be used.
The method for fixing the cation exchange group to the porous membrane is not particularly limited, but a highly reactive functional group such as epoxy or amine is introduced onto the substrate surface of the porous membrane, and then the cation exchange group is added to the functional group. It can be carried out by a method of binding a compound having it.
As the porous membrane having a cation exchange group, a commercially available porous membrane such as Sartobind S manufactured by Sartorius or Mustang S manufactured by Pall can be used.
The method for fixing the hydrophobic group to the porous membrane is not particularly limited, but a highly reactive functional group such as epoxy or amine is introduced on the substrate surface of the porous membrane, and then the hydrophobic group is added to the functional group. It can carry out by the method of combining the compound which has this.
Moreover, as the porous film having the hydrophobic group, a commercially available porous film such as Sartobind Phenyl manufactured by Sartorius can also be used.

  A large number of methods for producing porous membranes having functional groups immobilized via graft chains and their properties have been reported in patent literature and non-patent literature. For example, a porous membrane in which an anion exchange group described in JP-A-2-132132 and Journal of Chromatography A, 689 (1995) 211-218 is fixed via a graft chain is a porous body and its pores. A graft chain is fixed to the surface of the resin, and an anion exchange group is fixed to the graft chain. In such a porous membrane, since the anion exchange group is immobilized on the surface of the porous membrane via a graft chain, the amount of adsorption of α-lactalbumin and β-lactoglobulin is remarkably increased. The present inventor has found that the method can be particularly preferably used in the method for separating and recovering whey protein.

Further, a porous membrane in which a cation exchange group described in JP-A-2-160845 and Biotechnol. Prog. 1994, 10, 76-81 is fixed via a graft chain has a cation exchange group having a graft chain. The porous membrane fixed through the membrane is characterized in that a graft chain is fixed to the surface of the porous body and its pores, and a cation exchange group is fixed to the graft chain. In such a porous membrane, since the amount of lactoferrin adsorbed significantly increases because the cation exchange group is immobilized on the surface of the porous membrane via the graft chain, the method for separating and recovering whey protein in the present embodiment It can be particularly preferably used.
In addition, the porous membrane in which the hydrophobic group described in Biotechnol. Prog. 1994, 13, 89-95 is immobilized via a graft chain has a graft chain immobilized on the surface of the porous body and its pores. Further, a hydrophobic group is fixed to the graft chain. In such a porous membrane, the amount of β-lactoglobulin adsorbed remarkably increases because the hydrophobic group is immobilized on the surface of the porous membrane via a graft chain. It can be particularly preferably used in the separation and recovery method.

The anion exchange group is not particularly limited as long as it has a property of adsorbing α-lactalbumin and β-lactoglobulin at pH 6.0 to 8.0. For example, diethylamino group (DEA, Et ₂ N—), quaternary ammonium Group (Q, R ₃ N ⁺ -), quaternary aminoethyl group (QAE, R ₃ N ⁺ -(CH ₂ ) _2- ), diethylaminoethyl group (DEAE, Et ₂ N- (CH ₂ ) _2- ), diethylaminopropyl group _{(DEAP, Et 2 N- (CH} 2) 3 -) , and the like. Here, R is not particularly limited, and R bonded to the same N may be the same or different, and preferably represents a hydrocarbon group such as an alkyl group, a phenyl group, or an aralkyl group. Examples of the quaternary ammonium group include a trimethylamino group (trimethylammonium group, Me ₃ N ⁺ -). DEA and Q are preferable and DEA is more preferable from the viewpoint of easy chemical fixation to the porous membrane and high adsorption capacity.

In the cation exchange group PH4.0～8.0, is not particularly limited if any property of adsorbing lactoferrin, for example, sulfopropyl group _{(SP, SO 2 - (CH} 2) 3 -), sulfone group (- SO ₃ H), carboxyl group (COOH—) and the like.
The hydrophobic group is not particularly limited as long as it has a property of adsorbing β-lactoglobulin in an aqueous solution containing a salt of 0.5 M to 1.0 M, and examples thereof include a phenyl group, an octyl group, and a butyl group.

  In this embodiment, milk such as α-lactalbumin, β-lactoglobulin, and lactoferrin is passed by passing whey containing a whey protein useful for the porous membrane having a functional group that interacts with the above protein. Separate and recover the purified protein. At this time, it is preferable to separate and collect the protein by selectively adsorbing or non-adsorbing the protein using the charge interaction and hydrophobic interaction between the functional group of the porous membrane and the protein. As a porous body having a functional group that interacts with such a protein, beads used in column chromatography can be used in addition to the porous film. It is desirable to use it.

When selective adsorption and non-adsorption of a protein are controlled by charge interaction, the conditions for separation and recovery are examined based on the isoelectric point (pI) of the protein. For example, in the case of whey proteins such as α-lactalbumin, β-lactoglobulin and lactoferrin, the respective pI is pI = 4.9 for α-lactalbumin, pI = 5.1 for β-lactoglobulin, and lactoferrin is pI = 8.2 to 8.9, from which α-lactalbumin and β-lactoglobulin are selectively adsorbed on the anion exchange membrane in the pH range of 5.0 to 8.0, Lactoferrin is selectively adsorbed on the cation exchange membrane. At the same time, lactoferrin selectively permeates through the anion exchange membrane without adsorption, and α-lactalbumin and β-lactoglobulin selectively permeate through the cation exchange membrane without adsorption. It is possible to separate only lactoferrin from α-lactalbumin and β-lactoglobulin using such selective adsorption and non-adsorption properties by charge interaction.
Although the pH of milk is almost neutral, generally, in order to solidify casein and produce cheese, the pH of whey produced as a by-product after cheese production is also in this pH range because it is performed in a weakly acidic pH range. In order to separate whey protein by charge interaction, it is preferable to adjust the whey to a pH in the range of 5.0 to 8.0. At this time, the alkali component used for adjusting the pH is not particularly limited, but it is easy to adjust with an aqueous sodium hydroxide solution.

In order to separate α-lactalbumin and β-lactoglobulin with a small difference in pI values, it is preferable to use the difference in hydrophobic interaction because it cannot be achieved by charge interaction. In general, globulin proteins are more hydrophobic than albumin proteins, and the tendency is more prominent near pI. That is, β-lactoglobulin present in an aqueous solution having a pH in the range of 4.0 to 6.0 has a stronger hydrophobicity than α-lactalbumin. In this case, when the salt concentration of the aqueous solution is high, β-Lactoglobulin adsorbs to a hydrophobic group, resulting in a state of lower free energy entropy, while α-lactalbumin becomes enthalpy of lower free energy by remaining in an aqueous solution. , Each stabilized. It is possible to separate α-lactalbumin and β-lactoglobulin by utilizing such selective adsorption and non-adsorption properties due to hydrophobic interaction.
The above properties are more pronounced in the case of a porous membrane in which the functional group is fixed to the graft chain and the graft chain is fixed to the surface of the pore than the porous membrane in which the functional group is fixed to the surface of the pore. Therefore, selective adsorption and non-adsorption tend to be easily realized. This is because the number of adsorption points between the protein and the functional group is larger in the latter case where the functional group is immobilized on the porous membrane via the graft chain than in the former case where the functional group is immobilized on the surface. The reason is considered to be.

  In the present embodiment, the amount of whey that passes through the porous membrane having a functional group that interacts with the protein is determined by the concentration of the whey protein contained in the solution and each whey protein of the porous membrane to be used. It can be appropriately determined from the amount adsorbed on.

In this embodiment, when separating and recovering whey protein using a porous membrane having an anion exchange group or a cation exchange group and a porous membrane having a hydrophobic group,
(A) a step of allowing whey to permeate through a porous membrane having an anion exchange group or a cation exchange group to adsorb a part of the whey protein to the porous membrane and recovering the permeate;
(B) A step of eluting and collecting the whey protein adsorbed on the porous membrane by passing an aqueous solution containing a salt;
(C) The solution permeated through the porous membrane having a cation exchange group in the step (A) or the solution eluted and recovered from the porous membrane having an anion exchange group in the step (B) is converted into a porous membrane having a hydrophobic group. A step of allowing a portion of whey protein in the solution to adsorb to the porous membrane having the hydrophobic group by permeating and recovering the permeate;
(D) a step of eluting and recovering whey protein adsorbed on the porous membrane having the hydrophobic group,
It is preferable to include the process of containing.
When using a porous membrane having an anion exchange group in the step (A), whey proteins adsorbed on the porous membrane are α-lactalbumin and β-lactoglobulin,
When a porous membrane having a hydrophobic group is used in the step (C), the whey protein adsorbed on the porous membrane is β-lactoglobulin.

  In this embodiment, more specifically, when separating and recovering whey protein using a porous membrane having an anion exchange group and a porous membrane having a hydrophobic group, first, (a) whey has an anion exchange group The solution is passed through the porous membrane, α-lactalbumin and β-lactoglobulin are adsorbed on the porous membrane, and a permeate containing a high content of lactoferrin is recovered. At this time, the whey is preferably adjusted to pH 5.0 to 8.0, more preferably pH 6.0 to 8.0, and still more preferably pH 6.0 to 7.0. By this step (a), α-lactalbumin and β-lactoglobulin are adsorbed and removed from the whey, and a solution containing only lactoferrin in a high content can be recovered as a permeate.

Subsequently, (b) in order to elute α-lactalbumin and β-lactoglobulin adsorbed on the anion exchange group, a solution containing a high concentration salt was passed through the porous membrane, and α-lactalbumin and β-lacto Collect the globulin mixture as the eluate. The salt used for elution is not particularly limited, but any salt selected from sodium chloride, sodium sulfate, sodium phosphate, sodium citrate, and ammonium sulfate is preferable, and sodium chloride or ammonium sulfate is more preferable. The salt concentration is preferably 0.5M to 1.0M, more preferably 0.7M to 1.0M. The pH of the solution at the time of elution is not particularly limited, but pH 4.0 to 8.5 at which protein denaturation does not occur is preferable, and pH 6.0 to 8.0 is more preferable.
In the subsequent step (c) for separating α-lactalbumin and β-lactoglobulin, the mixed solution obtained in (b) is passed through a porous membrane having a hydrophobic group. At this time, the mixed solution is adjusted to pH 4.0 to 6.0 in order to increase the hydrophobicity of β-lactoglobulin. The salt concentration is passed while maintaining the salt concentration of step (b). In this step (c), β-lactoglobulin is adsorbed to the hydrophobic group of the porous membrane, and a permeate containing a high content of α-lactalbumin is recovered.

Subsequently, in the step (d), an aqueous solution containing a salt having a pH of 6.0 to 8.5 and a low salt concentration of 0 to 0.3 M is passed through the porous membrane having a hydrophobic group to which β-lactoglobulin is adsorbed. Then, an elution fraction containing a high content of β-lactoglobulin is recovered. Here, the kind of salt is not specifically limited.
Α-lactalbumin, β-lacto which is a whey protein useful from whey using the porous membrane having an anion exchange group and the porous membrane having a hydrophobic group by the steps (a) to (d) above Globulin and lactoferrin can be separated and recovered.

  On the other hand, in the present embodiment, when separating and recovering whey protein using a porous membrane having a cation exchange group and a porous membrane having a hydrophobic group, first, (a ′) the whey is porous with a cation exchange group. The solution is passed through the membrane, lactoferrin is adsorbed on the porous membrane, and a permeate containing a high content of α-lactalbumin and β-lactoglobulin is recovered. At this time, the whey is preferably adjusted to pH 5.0 to 8.0, more preferably pH 6.0 to 8.0, and still more preferably pH 6.0 to 7.0.

Subsequently, (b ′) in order to elute the lactoferrin adsorbed on the cation exchange group, a solution containing a high-concentration salt is passed through the porous membrane, and a mixture of these proteins is recovered as an eluent. The salt used for elution is not particularly limited, but sodium chloride is preferably used. The salt concentration is preferably 0.3M to 1.0M, more preferably 0.5M to 1.0M. The pH of the solution at the time of elution is not particularly limited, but pH 4.0 to 8.5 at which protein denaturation does not occur is preferable, and pH 6.0 to 8.0 is more preferable.
In the subsequent step (c ′) for separating α-lactalbumin and β-lactoglobulin, the mixed solution obtained in (a ′) is passed through a porous membrane having a hydrophobic group. At this time, in order to increase the hydrophobicity of β-lactoglobulin, the mixed solution is adjusted to an aqueous solution containing a salt of pH 4.0 to 6.0 and 0.5 M to 1.0 M. The salt to be used is preferably any salt selected from sodium chloride, sodium sulfate, sodium phosphate, sodium citrate and ammonium sulfate, more preferably sodium sulfate or ammonium sulfate. In (c ′), β-lactoglobulin is adsorbed on the hydrophobic group of the porous membrane, and a permeate containing a high content of α-lactalbumin is recovered.

Subsequently, an aqueous solution containing a salt having a pH of 6.0 to 8.5 and a low salt concentration of 0 M to 0.3 M is passed through the porous membrane having a hydrophobic group to which β-lactoglobulin is adsorbed in the step (d ′). And elution fractions containing a high content of β-lactoglobulin are collected. Here, the kind of salt is not specifically limited.
Α-lactalbumin, β-lactalbumin, which is a whey protein useful from whey, using the porous membrane having a cation exchange group and the porous membrane having a hydrophobic group by the steps (a ′) to (d ′). -Lactoglobulin and lactoferrin can be separated and recovered.
In addition, the whey protein obtained by separation and recovery can be purified to a higher purity as required, generally using the principle of chromatography. For example, the lactoferrin recovery solution obtained in step (a) may contain a small amount of lysozyme. To remove the lysozyme, the transferrin recovery solution is adjusted to pH 9 to express adsorption to the cation exchange group. In such a condition, the solution is passed through a porous membrane having a cation exchange group or a cation exchange chromatography column to adsorb and remove only lysozyme, and the permeate is recovered to obtain more highly purified lactoferrin. Can be obtained.

  Hereinafter, the present embodiment will be described more specifically based on reference examples and examples. However, the scope of the present invention is not limited to the following examples.

[Reference Example 1] Preparation of a porous membrane module in which a functional group that interacts with a protein is fixed to the surface via a graft chain (i) Introduction of graft chain into hollow fiber porous membrane A polyethylene hollow fiber porous membrane having a maximum pore size of 0.3 μm as measured by the bubble point method described in (viii) below at 0 mm was placed in a sealed container, and the air in the container was replaced with nitrogen. Thereafter, while cooling with dry ice from the outside of the container, γ rays 200 kGy were irradiated to generate radicals. The obtained polyethylene hollow fiber porous membrane having radicals was put in a glass reaction tube and the pressure in the reaction tube was reduced to 200 Pa or less to remove oxygen in the reaction tube. A reaction solution consisting of 3 parts by volume of glycidyl methacrylate (GMA) adjusted to 40 ° C. and 97 parts by volume of methanol was injected into 20 parts by mass of the hollow fiber porous membrane, and then left to stand for 12 minutes in a sealed state to perform graft polymerization. The reaction was performed to introduce graft chains into the hollow fiber porous membrane. The reaction solution composed of GMA and methanol was previously bubbled with nitrogen to replace oxygen in the reaction solution with nitrogen.

  After the graft polymerization reaction, the reaction solution in the reaction tube was discarded. Next, dimethyl sulfoxide was placed in the reaction tube to wash the hollow fiber porous membrane, thereby removing the remaining glycidyl methacrylate, its oligomer, and the graft chain not fixed to the hollow fiber porous membrane. After discarding the washing liquid, dimethyl sulfoxide was further added and washing was performed twice. Subsequently, washing was performed three times in the same manner using methanol. The hollow fiber porous membrane after washing was dried and weighed. The weight of the hollow fiber porous membrane was 138% before graft chain introduction, and the graft ratio defined as the ratio of the weight of the graft chain to the weight of the base material Was 38%.

(Ii) Fixation of anion exchange group (tertiary amino group) to graft chain After swelled by immersing a hollow fiber porous membrane into which a dried graft chain has been introduced for 10 minutes or more, it is immersed in pure water to obtain water. Replaced. 20 parts by mass of a reaction solution composed of a mixed solution of 50 parts by volume of diethylamine and 50 parts by volume of pure water was placed in a glass reaction tube with respect to the hollow fiber porous membrane after the graft reaction, and adjusted to 30 ° C. A hollow fiber porous membrane with a graft chain introduced therein was inserted and allowed to stand for 210 minutes to replace the epoxy group of the graft chain with a diethylamino group, thereby obtaining a hollow fiber porous membrane having a diethylamino group as an anion exchange group. It was. The obtained hollow fiber porous membrane had an outer diameter of 3.3 mm and an inner diameter of 2.1 mm. In the hollow fiber porous membrane, 80% of the epoxy groups of the graft chain were substituted with diethylamino groups.
The substitution rate T was calculated using the following formula (I) as the number of moles N ₁ substituted with a diethylamino group out of the number of moles N ₀ of the epoxy group.

T = 100 × N ₁ / N ₀
= 100 × {(w ₁ −w ₀ ) / M ₁ } / {w ₀ (dg / (dg + 100)) / M ₀ }
... (I)

In formula (I), M ₁ is the molecular weight of diethylammonium (73.14), w ₀ is the weight of the hollow fiber porous membrane after the graft polymerization reaction, w ₁ is the weight of the hollow fiber porous membrane after the diethylamino group substitution reaction, dg is the graft ratio, and M ₀ is the molecular weight of GMA (142).

(Iii) Fixation of cation exchange group (sulfone group) to graft chain The hollow fiber porous membrane into which the dried graft chain was introduced was immersed in methanol for 10 minutes or more to swell, and then immersed in pure water to replace with water. . 20 parts by mass of a reaction solution comprising a mixed solution of 10 parts by mass of sodium sulfite, 15 parts by mass of 2-propanol, and 75 parts by mass of pure water is put into a glass reaction tube with respect to the hollow fiber porous membrane after the graft reaction. Adjusted. A hollow fiber porous membrane having graft chains introduced therein is inserted and allowed to stand for 10 minutes to replace the epoxy group of the graft chain with a sulfone group, thereby obtaining a hollow fiber porous membrane having a sulfone group as a cation exchange group. It was. The obtained hollow fiber porous membrane had an outer diameter of 3.3 mm and an inner diameter of 2.1 mm. In the hollow fiber porous membrane, 3% of the epoxy groups of the graft chain were substituted with sulfone groups.
The substitution rate T was calculated using the following formula (II) as the number of moles N ₂ substituted with the sulfone group out of the number of moles N ₀ of the epoxy group.

T = 100 × N ₂ / N ₀
= 100 × {(w ₂ −w ₀ ) / M ₂ } / {w ₀ (dg / (dg + 100)) / M ₀ }
... (II)

In formula (II), M ₂ is the molecular weight of sodium sulfite (103.05), w ₀ is the weight of the hollow fiber porous membrane after the graft polymerization reaction, w ₂ is the weight of the hollow fiber porous membrane after the sulfone group substitution reaction, dg is the graft ratio, and M ₀ is the molecular weight of GMA (142).

(Iv) Fixation of hydrophobic group (phenyl group) to graft chain The hollow fiber porous membrane into which the dried graft chain was introduced was immersed in methanol for 10 minutes or more to swell, and then immersed in pure water to replace with water. . 20 parts by mass of a reaction solution composed of 3 parts by mass of aniline, 0.4 parts by mass of sodium hydroxide, and 97 parts by mass of pure water is placed in a glass reaction tube with respect to the hollow fiber porous membrane after the graft reaction. Adjusted to ° C. A hollow fiber porous membrane having a phenyl group as a hydrophobic group is obtained by inserting a hollow fiber porous membrane into which a graft chain is introduced and allowing to stand for 20 hours to replace the epoxy group of the graft chain with a phenyl group. It was. The obtained hollow fiber porous membrane had an outer diameter of 3.3 mm and an inner diameter of 2.1 mm. In the hollow fiber porous membrane, 78% of the epoxy groups of the graft chain were substituted with phenyl groups.
The substitution rate T was calculated using the following formula (III) as the number of moles N ₃ substituted with a phenyl group out of the number of moles N ₀ of the epoxy group.

T = 100 × N ₃ / N ₀
= 100 × {(w ₃ −w ₀ ) / M ₃ } / {w ₀ (dg / (dg + 100)) / M ₀ }
... (III)

In formula (III), M ₃ is the molecular weight of aniline (93.13), w ₀ is the weight of the hollow fiber porous membrane after the graft polymerization reaction, w ₃ is the weight of the hollow fiber porous membrane after the phenyl group substitution reaction, dg Is the graft ratio, and M ₀ is the molecular weight of GMA (142).

(V) Production of a hollow fiber porous membrane module in which a functional group that interacts with a protein is fixed via a graft chain. Anion exchange groups, cation exchange groups, and hydrophobic groups obtained in (ii) to (iv) Bundled three hollow fiber porous membranes fixed through a graft chain, and fixed both ends to a polysulfonic acid module case with an epoxy potting agent so as not to block the hollow portion of the hollow fiber porous membrane, Three types of hollow fiber porous membrane modules were prepared in which each functional group was fixed via a graft chain. The obtained module has an inner diameter of 0.9 cm, a length of about 3.3 cm, an inner volume of the module of about 2 mL, an effective volume of the hollow fiber porous membrane in the module of 0.85 mL, and a hollow fiber excluding the hollow portion. The volume of the porous membrane alone was 0.53 mL. These obtained modules were washed by passing 200 mL of pure water, and used as an evaluation module in the following examples as anion exchange membrane module, cation exchange membrane module and hydrophobic membrane module, respectively.

(Vi) Preparation of whey (whey) 1L of commercially available raw milk was sterilized by standing at 65 ° C for 30 minutes, then cooled to room temperature, and added to lactic acid while stirring to adjust to pH 5.0 The stirring was stopped and the mixture was allowed to stand for 60 minutes. The cheese component in which milk casein coagulates is removed by filtering with a filter paper (Whatman, 40), and the whey that has passed through the filter paper is further filtered through a 0.45 μm microfiltration membrane (Sartorius, Mini Sarto Plus SW17829K). As a result, a clear whey was obtained.
(Vii) Identification and quantification of whey protein by gel filtration chromatography GE Healthcare Biosigns, AKTAexplorer100, GE Healthcare Bioscience, Hipprep 16/60 Sephacryl S-200HR as a gel filtration column, Molecular weight fractionation was carried out by passing 1 mL of sample at a flow rate of 0.5 mL / min using 50 mM sodium phosphate pH 7.0 as a buffer.

(Viii) Bubble Point Method The maximum pore diameter of the hollow fiber porous membrane as a substrate was measured using a bubble point method. One end of a hollow fiber porous membrane having a length of 8 cm was closed, and a nitrogen gas supply line was connected to the other end via a pressure gauge. In this state, nitrogen gas was supplied to replace the inside of the line with nitrogen, and then the hollow fiber porous membrane was immersed in ethanol. At this time, the hollow fiber porous membrane was immersed in a state where a slight pressure of nitrogen was applied so that ethanol did not flow back into the line. With the hollow fiber porous membrane immersed, the pressure of nitrogen gas was slowly increased, and the pressure P at which nitrogen gas bubbles started to stably emerge from the hollow fiber porous membrane was recorded. From this, the maximum pore diameter of the hollow fiber porous membrane was calculated according to the following formula (V), where d is the maximum pore diameter and γ is the surface tension.

d = C ₁ γ / P (IV)

In formula (IV), C ₁ is a constant. C ₁ γ = 0.632 (kg / cm) when ethanol was used as the immersion liquid, and the maximum pore diameter d (μm) was determined by substituting P (kg / cm ² ) into the above equation.

[Example 1]
After adjusting 200 mL of the whey obtained in (vi) to pH 7.5, the solution was passed through the anion exchange membrane module prepared in (v), and the permeate was collected (step (a)). When the recovered liquid obtained was analyzed by gel filtration chromatography according to (vii), the strongest peak appeared at the position of molecular weight 83000 corresponding to lactoferrin, and 93% of the total protein in the permeate was lactoferrin from the peak area. Was confirmed.
Subsequently, 20 mL of 50 mM sodium phosphate buffer, pH 5.0 containing 0.7 M sodium chloride was passed through the anion exchange membrane module, and the protein adsorbed on the anion exchange membrane module was eluted and collected (step (b)). The obtained eluate was passed through a hydrophobic membrane module, and the permeate was collected (step (c)). When this was analyzed by gel filtration chromatography according to (vii), it was equivalent to α-lactalbumin. The strongest peak appeared at the position of molecular weight 14200, and it was confirmed from the peak area that 95% of the total protein in the permeate was α-lactalbumin.

  Next, 20 mL of 50 mM sodium phosphate buffer, pH 7.0 was passed through the hydrophobic membrane module to elute the adsorbed protein (step (d)). When this was analyzed by gel filtration chromatography according to (vii), the strongest peak appeared at the position of molecular weight 18300 corresponding to β-lactoglobulin, and 89% of the total protein was β-lactoglobulin from the peak area. It was confirmed.

[Example 2]
200 mL of whey obtained in (vi) was adjusted to pH 7.0, and then passed through the cation exchange membrane module prepared in (v) to collect the permeate (step (a ′)). Subsequently, 20 mL of 20 mM Tris buffer containing 0.7 M sodium chloride, pH 8.0 was passed through to elute and recover the protein adsorbed on the cation exchange membrane module (step (b ′)). When the collected liquid was analyzed by gel filtration chromatography according to (vii), the strongest peak appeared at the position of molecular weight 83000 corresponding to lactoferrin, and 95% of the total protein in the permeate was lactoferrin from the peak area. Was confirmed.

Next, ammonium sulfate was added to the permeation recovery solution of the cation exchange membrane module obtained in the step (a ′) so as to have a concentration of 0.7 M, and the pH was adjusted to 5.0. This was passed through the hydrophobic membrane module, and the permeate was collected (step (c ′)). When the recovered liquid thus obtained was analyzed by gel filtration chromatography according to (vii), the strongest peak appeared at the position of molecular weight 14200 corresponding to α-lactalbumin, and 91% of the total protein in the permeate was observed from the peak area. % Was confirmed to be α-lactalbumin.
Next, 20 mL of 50 mM sodium phosphate buffer, pH 7.0 was passed through the hydrophobic membrane module to elute the adsorbed protein (step (d ′)). When this was analyzed by gel filtration chromatography according to (vii), the strongest peak appeared at a molecular weight of 18300 corresponding to β-lactoglobulin, and 87% of the total protein was β-lactoglobulin from the peak area. It was confirmed.

  By the method for separating and recovering whey protein by the porous membrane having a functional group that interacts with the protein of the present invention, the useful whey protein is efficiently and efficiently recovered from the whey after production of cheese that is usually discarded. It has industrial applicability that it is separated and recovered with accuracy.

Claims (7)

  A method for separating and recovering whey protein using a porous membrane having a functional group that interacts with protein. The method for separating and recovering whey protein according to claim 1, comprising the following steps:
(A) a step of allowing whey to permeate through a porous membrane having an anion exchange group or a cation exchange group to adsorb a part of the whey protein to the porous membrane and recovering the permeate;
(B) a step of eluting and recovering whey protein adsorbed on the porous membrane by passing an aqueous solution containing salt;
(C) The solution permeated through the porous membrane having a cation exchange group in the step (A) or the solution eluted and recovered from the porous membrane having an anion exchange group in the step (B) is converted into a porous membrane having a hydrophobic group. A step of allowing a portion of whey protein in the solution to adsorb to the porous membrane having the hydrophobic group by permeating and recovering the permeate;
(D) A step of eluting and recovering whey protein adsorbed on the porous membrane having a hydrophobic group. The whey protein adsorbed by the porous membrane having an anion exchange group in the step (A) is α-lactalbumin and β-lactoglobulin,
The method for separating and recovering whey protein according to claim 1 or 2, wherein the whey protein adsorbed on the porous membrane having a hydrophobic group in the step (C) is β-lactoglobulin. The whey protein adsorbed by the porous membrane having a cation exchange group in the step (A) is lactoferrin,
The method for separating and recovering whey protein according to claim 1 or 2, wherein the whey protein adsorbed on the porous membrane having a hydrophobic group in the step (C) is β-lactoglobulin.   The method for separating and recovering whey protein according to any one of claims 1 to 3, wherein a porous membrane having an anion exchange group fixed to the surface via a graft chain is used.   The method for separating and recovering whey protein according to any one of claims 1, 2, and 4, wherein a porous membrane having a cation exchange group immobilized on the surface via a graft chain is used.   The method for separating and recovering whey protein according to any one of claims 1 to 4, wherein a porous membrane having a hydrophobic group fixed to the surface via a graft chain is used.