JPH1129600A - Separation and purification of lactoferrin from milk - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily and safely separate high-purity lactoferrin having physiological activities such as antimicrobial activity, lipid peroxide formation suppression and immunological function control by contacting a lactoferrin-contg. material with κ-casein followed by leaching the adsorbate (the aimed lactoferrin). SOLUTION: A lactoferrin-contg. material such as milk, defatted milk, whey, tear, saliva, cell-cultured product, microorganism-cultured product, concentrate thereof or extract therefrom, is contacted with κ-casein such as a carrier immobilized with κ-casein at pH 5.0-8.0 under an ionic strength of <=0.3 to effect adsorbing the lactoferrin to the carrier. Subsequently, the lactoferrin adsorbed is leached with a solution of salt(s) selected from sodium chloride, potassium chloride, calcium chloride, magnesium chloride and a mixture thereof (the ionic strength of the solution being 0.4-1.5) followed by desalination by e.g. electrodialysis, thus easily, safely separating and purifying the objective high- purity lactoferrin exhibiting various physiological activities from the lactoferrin- contg. material.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for easily and safely separating and purifying lactoferrin having various physiological actions as described below.

[0002]

2. Description of the Related Art Lactoferrin is an iron-binding glycoprotein having a molecular weight of about 80,000 contained in exocrine fluids such as milk, tears and saliva. Its content is high in colostrum, and about 5-
10 mg / ml, but about 0.1-
It is as low as 0.5 mg / ml. In particular, it is considered to play an important role in maintaining the healthy growth of infants because it is contained a lot in colostrum. In support of this, in recent years, many studies in vitro and in vivo have shown that lactoferrin has an antibacterial action, an inhibitory action on lipid peroxide production, a regulation action on iron absorption, an immune function regulation action, a cell proliferation action, and a bacterial infection. It has been reported to have useful physiological effects such as protective effects.

Since lactoferrin is expected to have a wide variety of physiological activities, it is an important substance in nutritional physiology and pharmacologically. As a raw material for separating lactoferrin, milk and cheese whey by-produced during cheese production are used. Lactoferrin is a safe source of natural products because it is nutritionally superior and is separated from safe milk ingredients. Also,
Recently, the use of recombinant lactoferrin has been studied.

Conventionally, as a method of separating and purifying lactoferrin from milk, a whey concentrate is passed through a cation exchanger, washed with a phosphate buffer, and then washed with the same buffer containing 0.3 M sodium chloride. There is a method of eluting lactoferrin (Japanese Patent Application Laid-Open No. 62-19523) or a method of mixing skim milk with a cation exchanger, stirring, packing the resin in a column, washing with water, and eluting with 5% sodium chloride. 63-152400). In addition, there is a method in which a substance having a strong affinity for lactoferrin is immobilized on a column and separated by affinity chromatography. For example, heparin (JP-A-63-255299), a monoclonal antibody against lactoferrin (JP-A-61-14520).
0), sulfated products (JP-A-63-25530)
0), DNA (T. William Hutchens et al., Biochimica e
Biophysica Acta , 999 (1989) 323-329), and dyes (Mariano Grasselli et al., Netherlands Milk & Dairy
J. , 50 (1996) 551-561) or the like is immobilized on a support for chromatography, brought into contact with a lactoferrin-containing milk material, and the adsorbed lactoferrin is eluted with 0.5 to 1.0 M sodium chloride. and so on.

[0005]

SUMMARY OF THE INVENTION In view of the above-mentioned state of the art, the present invention has been made for the purpose of developing a method for safely and simply and efficiently separating and / or purifying lactoferrin having many physiological actions. It is a thing.

[0006]

Means for Solving the Problems The present invention has been made to achieve the above-mentioned object, and as a result of investigations from various aspects, it has been found that a method using affinity chromatography is:
Since the specific adsorption reaction with lactoferrin is used, attention has been paid to the advantage that a high-purity product can be easily obtained without a complicated step as compared with the ion exchange chromatography method. However, this method is certainly an excellent method, but heparin, a monoclonal antibody against lactoferrin, a sulfated product, DNA, etc., which have been conventionally used for the affinity column, are available in large quantities. Is difficult. Further, there is a possibility that the immobilized substance may fall off and be mixed into lactoferrin, and there is a problem in safety for food use, and further improvement is awaited.

Accordingly, the present inventors have conducted intensive studies from various angles and have found a new fact that lactoferrin has a strong affinity for κ-casein, and immobilized κ-casein by applying this principle. When a column was prepared and the affinity for lactoferrin was examined, it was found that lactoferrin was specifically adsorbed and eluted. Furthermore, κ-
Casein is a component present in milk and has the advantage of being extremely safe and inexpensive as compared with conventionally used heparin, monoclonal antibodies and the like.

The present invention has been completed as a result of further research based on these useful new findings. Specifically, lactoferrin-containing raw material is brought into contact with κ-casein to specifically adsorb lactoferrin and then adsorbed lactoferrin. And a method for separating and / or purifying lactoferrin by eluting lactoferrin.

The present invention relates to the use of κ-casein to produce exocrine fluids such as milk, tears, saliva, etc., cultures of microorganisms and cells, genetically modified cultures, concentrated liquids and extracts thereof. The present invention relates to a method for easily separating / purifying a physiologically important lactoferrin contained in lactoferrin with high purity and easily. Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is based on a new principle discovered for the first time by the present inventors that lactoferrin has a specific and selective affinity for κ-casein. Is treated with κ-casein to specifically bind both, and then lactoferrin is separated.

Therefore, to implement the present invention, κ-
Casein is immobilized on a carrier, and a lactoferrin-containing material is brought into contact with the carrier to specifically convert lactoferrin to κ.
A so-called industrial method of adsorbing casein and then eluting the adsorbed lactoferrin, or
κ-casein is left in a free state without being immobilized on a carrier, and is contacted with a lactoferrin-containing raw material to form a conjugate or complex with lactoferrin,
Any of chemical and biological methods for separating the two after removal can be used.

For the immobilization of κ-casein, conventional techniques for immobilizing enzymes, various proteins and microorganisms on carriers can be appropriately used, that is, inorganic or organic, natural or synthetic, macromolecular or low-molecular-weight. Using various carriers such as molecules, biological systems and non-biological systems, conventionally known immobilization techniques such as a covalent bonding method, an ion bonding method, and a physical adsorption method can be appropriately used.

Examples of the carrier include polysaccharides (derivatives) such as agarose, cellulose, dextran, sepharose, and Sephadex; synthetic polymer substances such as polyacrylamide gel and polystyrene; (porous) glass; and (porous) ceramics. Inorganic or ceramic materials such as activated carbon, silica gel, alumina and clay minerals are appropriately used.

These carriers may be treated as they are, or treated with cyanogen bromide, cyanoborohydride, or the like, or treated with a cross-linking agent or a condensing reagent to further immobilize them with κ-casein according to a conventional method. May be promoted.

In order to carry out the present invention, the κ-casein thus immobilized is brought into contact with a lactoferrin-containing raw material. Examples of the lactoferrin-containing raw material include milk, skim milk, whey, etc. Milk of mammals including humans such as milk by-products of humans; various exocrine secretions such as tears and saliva; cultures of microorganisms in cells, genetically modified cultures; substances containing lactoferrin such as extracts and concentrates of these All included.

At the time of contact between κ-casein and lactoferrin, the pH is 4.5 to 8.5, preferably 5.0 to 8.5.
The ionic strength is preferably set to 8.0 and the ionic strength is set to 0.4 or less, preferably 0.3 or less.

Next, the adsorbed lactoferrin is eluted with a salt solution. At this time, the salt solution has an ionic strength of 0.3 to 1.6, preferably 0.4 to 1.5. After elution, desalting is performed according to a conventional method such as electrodialysis to obtain lactoferrin.

As the salt solution used for eluting the adsorbed lactoferrin, a salt solution selected from sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and a mixture of one or more of these is used. Hereinafter, examples of the present invention will be described.

[0019]

Example 1 Immobilization of kappa-casein: 10 ml of commercially available actigel (agarose gel, manufactured by Sterogen Bioseparation) was sufficiently washed with deionized water, filtered, and then 20 mg of kappa-casein was buffered with 10 mM phosphate buffer. dissolved in the liquid (pH 7.0) 3 ml, the sodium cyanoborohydride in a mixture (NaCNBH ₃₎ was added 130 mg, was obtained κ- casein immobilized Akuchigeru.

Separation and purification of lactoferrin: κ-casein-immobilized agarose gel prepared by the above procedure
The column was packed in a 10 cm long column of 10 cm and equilibrated with 10 mM phosphate buffer (pH 7.0). 1 ml of bovine lactoferrin (Sigma) (1 mg / ml) was added to this column.
Then, the column was washed with a 10 mM phosphate buffer (pH 7.0). Next, bovine lactoferrin adsorbed on the column was eluted by passing 20 ml of 0.5 M NaCl.

[0021]

Example 2 1 ml of human lactoferrin (Sigma) (1 mg / ml) was passed through a κ-casein-immobilized agarose column (1 cm in diameter × 10 cm in length) prepared in Example 1, and a 10 mM phosphate buffer was added. The column was washed with (pH 7.0). Next, 20 ml of 0.5 M NaCl was passed through to elute human lactoferrin adsorbed on the column.

[0022]

Example 3 κ-casein immobilized column (diameter 1 cm ×
(Length 10 cm) was prepared in the same procedure as in Example 1.
In order to confirm the specific adsorptivity of lactoferrin to the κ-casein-immobilized column in the presence of milk protein, the main proteins in whey were bovine serum albumin (BSA) (0.7 mg) and immunoglobulin (1.4).
mg), α-lactalbumin (2.8 mg), and β
Lactoglobulin (7.0 mg) was dissolved to the same level as the concentration in whey, in which bovine lactoferrin 1.0
mg was added.

The whole amount (2 ml) of the whey model solution thus prepared was passed through the above-mentioned κ-casein-immobilized column, and the column was washed with a 10 mM phosphate buffer (pH 7.0). Then 20 ml of 1.0 M NaCl
Then, the bovine lactoferrin adsorbed on the column was eluted. The amount of eluted bovine lactoferrin was determined by ELISA.
As a result, about 0.95 mg of bovine lactoferrin was recovered, confirming that the recovery of bovine lactoferrin was 96%. The purity of the recovered bovine lactoferrin was determined to be 95% or more by SDS-polyacrylamide gel electrophoresis (PAGE).

The drawings show the separation of bovine lactoferrin by affinity chromatography immobilized on κ-casein, FIG. 1 shows the relationship between the column number eluted and the absorbance (A ₂₈₀ ), and FIG. 2 shows the SDS-polyacrylamide gel An electrophoresis (PAGE) diagram is shown.

[0025]

Example 4 κ-casein immobilized column (diameter 1 cm ×
(Length 10 cm) was prepared in the same procedure as in Example 1.
In skim milk in which fat is separated from human colostrum, p with hydrochloric acid
By adjusting H4.5, casein was subjected to isoelectric point precipitation to obtain a whey fraction. The pH of the supernatant was adjusted to pH 7.0 with sodium hydroxide. 5 ml of the human colostrum whey thus prepared was passed through a κ-casein-immobilized column. After washing the column with 10 mM phosphate buffer (pH 7.0), 20 ml of 1.0 M NaCl was passed through to elute the components adsorbed on the column. This component is SDS
-Analysis by polyacrylamide gel electrophoresis (PAGE) confirmed that it was human lactoferrin. The purity of the recovered human lactoferrin was 95% or more.

[0026]

Example 5 A κ-casein fixed column (1 cm in diameter × 10 cm in length) was prepared in the same procedure as in Example 1. 10 ml of cheese whey obtained in the process of producing Gouda cheese was passed through a κ-casein-immobilized column. After washing the column with 10 mM phosphate buffer (pH 7.0), 1.0 M N
The components adsorbed on the column were eluted by passing 30 ml of aCl. Analysis of this component by SDS-polyacrylamide gel electrophoresis (PAGE) confirmed that it was bovine lactoferrin. The purity of the recovered bovine lactoferrin was 95% or more.

[0027]

As described above, lactoferrin contained in lactoferrin-containing raw materials such as milk can be separated safely and simply and / or with high purity.

[Brief description of the drawings]

FIG. 1 shows the relationship between the column number and the absorbance of eluted bovine lactoferrin.

FIG. 2 is an electrophoresis photograph showing the purity of recovered bovine lactoferrin.

 ──────────────────────────────────────────────────の Continued on the front page (72) Inventor Tomoko Ueda 1-21-3 Sakaemachi, Higashimurayama-shi, Tokyo Meiji Dairies Co., Ltd.

Claims (7)

[Claims] 1. A method for separating and purifying lactoferrin from a lactoferrin-containing raw material, comprising contacting a lactoferrin-containing raw material with κ-casein to adsorb lactoferrin, and then eluting the adsorbed lactoferrin. 2. Using a carrier on which κ-casein is immobilized,
The method according to claim 1, wherein lactoferrin is separated and purified by affinity chromatography. 3. The lactoferrin-containing raw material is at least one selected from milk, skim milk, whey, tears, saliva, cell culture, microbial culture, concentrate, and extract. Or the method of 2. 4. The method according to claim 1, wherein the pH at the time of contacting κ-casein with lactoferrin is 5.0 to 8.0. 5. The method according to claim 1, wherein the ionic strength at the time of contact between κ-casein and lactoferrin is 0.3 or less. 6. An adsorbed lactoferrin having an ionic strength of 0.
The method according to any one of claims 1 to 5, wherein the salt is eluted with a salt solution of 4 to 1.5, and then desalted by electrodialysis or the like. 7. The salt solution used for the elution is a solution of a salt selected from sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and a mixture thereof. The method according to any one of claims 1 to 4.