JPH03204898A - Fractuation of water-soluble yolk protein - Google Patents

Abstract

PURPOSE:To inexpensively and safely purify the subject protein useful for drugs, foods, etc., by adding a specific amount of water, etc., to a yolk liquid to precipitate yolk lipoprotein. CONSTITUTION:A yolk liquid is mixed with 3-5 times volume of de-ionized water, distilled water, a salt aqueous solution prepared by adding a salt such as NaCl to the water in a concentration of <=10mM or a buffer solution such as a phosphoric acid buffer solution having a salt concentration of <=10mM and a pH of approximately 7, and stirred at 0-50 deg.C (preferably 10-25 deg.C) for 10min - approximately 1 day to precipitate yolk lipoprotein, which is centrifuged with a gravity of approximately 2000Xg to provide the objective protein.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for fractionating egg yolk into lipids, riboproteins and water-soluble proteins.

[Conventional technology]

The yolk serves as a storehouse of nutrients for embryonic development. Its composition is 48% water, 17% protein.
．． 5%, fat 32.5%, ash 2.0%, and egg yolk contains approximately 50% solids. A small amount of this is dialysable, but most of it exists as riboproteins, that is, large complexes in which proteins and lipids are combined in various proportions. In addition, proteins that are not bound to lipids, that is, water-soluble proteins, account for 10% of the solid content.
That's about it. The water-soluble proteins include livetin, phosvitin, and trace amounts of boflavin-binding protein. Lihetin includes α-libetin, β-lihetin and γ-lihetin.
It is classified as lihetin. α-β- and T-livetin are present in parental serum albumin, α-glycoprotein, and γ-
It has been shown to be immunologically equivalent to globulin. Due to its action as T-globulin, T-lihetin has recently been expected to be applied to animal and human medicine and as a research reagent.

Several methods have been reported for the purification of γ-lihetin, and separation using columns, ultracentrifugation, and electrophoresis have been used at the laboratory level. However, it is difficult to obtain large amounts of γ-lihetin by these methods, and these methods are not suitable for industrialization. Therefore, several batch methods that can process large quantities have been reported. The first step in all of these methods is to separate egg yolk lipids and yolk riboproteins from water-soluble proteins.

Conventionally, organic solvents have been used to separate egg yolk lipids and egg yolk riboproteins from water-soluble proteins. However, this method is not suitable for purifying T-livetin because egg yolk proteins, especially water-soluble proteins, are denatured by the organic solvent.

To date, the following methods have been reported as methods for separating egg yolk lipids and egg yolk riboproteins from water-soluble proteins that do not use organic solvents.

Polson precipitates egg yolk lipids and egg yolk ribon protein using polyethylene glycol, and purifies T-livetin from the supernatant (Polson, A.
, M. et al., ImmunologicalC.
Communications, 9(5): 495
-514 (1980)), and Giensenius precipitated egg yolk lipids and egg yolk ribon protein using dextran sulfate and calcium ions, and extracted γ-
Purification of livetin (Jenseniu)
s+ J, C,, Journalof I+*sun
Logical Methods, 46: 63-
68 (1981)).

Hatta states that it is possible to precipitate most of the egg yolk liver protein using sodium alginate and purify T livetin from the supernatant [Hatta.

H, et al, Journal of Food
5science, 53(2):425-427&
432 (1988), Japanese Unexamined Patent Publication No. 63-215699].

In addition, Hatta states that it is possible to precipitate most of the egg yolk liver protein using one or more selected from agar, carrageenan, furcelleran, pectin, and xanthan gum, and purify γ-livetin from the supernatant. [Unexamined Japanese Patent Publication No. 64-38098].

[Problem to be solved by the invention]

In order to fractionate and purify γ-lihetin in egg yolk and apply it to medicines, cosmetics, foods, etc., it is necessary to avoid the contamination of substances that pose safety issues for the human body and substances that pose problems in terms of their effects. Contamination must be avoided; the Polson, Jensenius, and Hatta methods all use polyethylene glycol,
Dextran sulfate, carrageenan, etc. are added. Therefore, the water-soluble protein fractions obtained by these methods are unavoidably contaminated with substances other than egg yolk components. These things hinder the application and use of T-lihetin in medicines, cosmetics, foods, etc. Also, dextran sulfate is very expensive;
Cost becomes an issue. Polyethylene glycol requires a large amount to process egg yolks, so its cost is also an issue.

[Means to solve the problem]

In order to solve these drawbacks and problems, the present inventors have proposed a method that does not use additives, or even if additives are used, can be easily and completely removed, and in an inexpensive manner. We have been conducting extensive research to find a method to separate egg yolk lipids and yolk riboproteins from water-soluble proteins from egg yolks. As a result, it is possible to precipitate the egg yolk by adding one selected from water, salt solution, and buffer solution in an amount more than three times the amount of the egg yolk to the egg yolk and mixing it, and to precipitate the egg yolk in an amount more than five times the amount of the egg yolk. The present inventors have discovered that egg yolk lipids and egg yolk lipoproteins can be precipitated by adding and mixing one selected from water and a salt aqueous buffer, and have completed the present invention.

The purpose of the present invention is to remove egg yolk lipids and egg yolk riboproteins from egg yolks without additives as the first step in separating and purifying γ-livetin from egg yolks, which is highly safe and can be used as T-globulin. γ-, which retains the immunological effects of
The aim is to provide livetin.

In particular, in the present invention, since the only external additives are water, salt aqueous solution, or buffer solution, this invention is the first time that γ-livetin can be used in pharmaceuticals, cosmetics, and three foods without considering the safety of components other than egg yolk components. Application becomes possible.

Furthermore, useful substances such as phosphatidylcholine, cholesterol, and triglycerides can be collected from the egg yolk riboproteins and egg yolk lipids precipitated in the present invention by conventional methods. This is extremely effective in terms of production efficiency, considering that it is impossible to easily obtain the above-mentioned substances using conventional methods that use carrageenan, for example, because carrageenan and lipids bond together. It's a method.

In the present invention, the egg yolk liquid refers to raw egg yolk liquid separated from egg white liquid after egg breaking, egg yolk liquid obtained by diluting it with water, egg yolk liquid prepared by adding water to egg yolk powder, or obtained by thawing frozen egg yolk. It refers to the egg yolk liquid. Egg yolk is chicken,
It may be prepared from eggs of birds such as turkeys and ducks.

The water used in the present invention includes tap water, well water, and other water commonly used in daily life that has been deionized using an ion exchange resin, distilled water, and the like. Also, from the standpoint of safety, the closer it is to pure water, the better.

The aqueous salt solution in the present invention refers to NaCj! in the above water. ,
KCj! , LiCj2, etc., with a weak ionic strength aqueous solution, preferably with a salt concentration of approximately 10 mM or less. The amount may be such that the ionic strength of the diluted egg yolk solution is equivalent to that of water.

The buffer solution used in the present invention preferably has a salt concentration of about 10 mM or less and a pH around neutrality.

For example, phosphate buffer, Tris buffer, PBS (Phosphate Buff) diluted approximately 20 times or more.
eredSaline), etc.

The amount of water, salt solution, or buffer solution added to the undiluted egg yolk solution is preferably at least three times the amount of the egg yolk solution, from the perspective of separating egg yolk rib protein and egg yolk water-soluble protein. Separation is difficult below. Furthermore, from the viewpoint of removing egg yolk lipids and egg yolk liver protein from egg yolk water-soluble protein, the amount is preferably more than 5 times the amount of egg yolk liquid, and if it is less than that, the separation will be incomplete. The temperature during stirring and mixing is preferably from O'C to 50°C.
From operational and sanitary standpoints, a temperature of 10' to 25°C is desirable. Further, the stirring/mixing time for separation may be appropriately selected as long as mixing is performed and separation is possible immediately after stirring, and is desirably about 10 minutes to one day.

A method for separating the egg yolk riboprotein precipitate is centrifugation, and the gravity may be about 2000×g. Filtration using a filter paper or membrane can also be used to separate the precipitate, and since the precipitate settles quickly after stopping stirring, it is also possible to separate the egg yolk riboprotein by decantation. Decantation can be performed more efficiently by using a filter aid such as Celite.

[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples.

In addition, in the examples, antibody titer was used as an index to examine the recovery rate of water-soluble protein, and the antibody titer was measured using E.
The LISA method was used.

(ELISA method) First, dry E. coli^987P cells were
50mM sodium carbonate buffer (p
Pour 100 pl of the solution obtained by dissolving H9,6) into each well of a 96-well plate (IMURON 2, manufactured by Guinatec), leave it at 4°C overnight, and plate the bacterial cells. was adsorbed to. Next, the plate was washed with PBS-T (PB containing 0.05% Tween-20).
S (pH 7,4)) six times. After washing, soak the plate in PBS containing 3% BSA (bovine serum albumin).
(pH 7,4) at 37°C for 1 hour to perform blocking, and then the plate was washed 6 times with PBS-T. Here, 100 μl of a two-step dilution of each sample was added to each well, and a reaction was performed at 37° C. for 1 hour. After the reaction, the plate was washed 6 times with PBS-T, and the plate was further treated with peroxidase-conjugated anti-chicken Ig as a secondary antibody.
G antibody (protein amount 1.67 μg/mjl 100
μl was added to each well, incubated at 25°C for 30 minutes, washed 6 times with PBS-T, and then added to each well of the plate with 0.2 M disodium phosphate-0.1 M citrate buffer ( 20 mg of 0-phenylenediamine as a substrate and 10 μl of hydrogen peroxide were added to 50 mffi (pH 5, 0), and the mixture was reacted at 25°C for 20 minutes.

After the reaction was completed, the antibody titer was measured by measuring the absorbance (OD4wt) of each well. The antibody titer has an absorbance of 0.
The dilution ratio was set to 2.

Example 1 The relationship between the amount of water added to undiluted egg yolk solution and the recovery rate of lipids and antibody titers in the supernatant was investigated.

As the egg yolk liquid, E. coli A987P was used as FCA.
In addition, anti-E coli obtained by immunizing chicken breast muscles
Egg yolks from eggs laid by chickens with A987P antibody produced in their blood were used, with the egg whites separated. Water was added 3 times to 11 times to 10 ml of egg yolk liquid, stirred and mixed at 100C for 10 minutes, and then centrifuged at 2000Xg for 10 minutes to separate into a precipitate fraction and a supernatant fraction. The lipid amount, protein amount, and antibody titer in both supernatants were measured, and the recovery rate was calculated based on the total lipid amount and antibody titer in the initial egg yolk solution.The antibody titer per unit protein amount was calculated, and the specific activity was determined. And so. That is, specific activity indicates the concentration of antibody in the protein.

The method for quantifying the total lipid amount is the Forte method (FolchJ
,, The Journal of Biology
cal Chemistry, 226: 497 (
After extracting the total lipids using 1957), the organic solvent was distilled off and the weight of the residue was taken as the total lipid amount.

The protein emplacement method is Blandford's dye binding method (Bradford, M., Analyt
icalBiochemistry, 72: 284
-254 (1976):1, and raw serum albumin was used as a standard. The results are shown in Figure 1.

As is clear from Figure 1, when the amount of water added to the undiluted egg yolk solution is 3 times or more (that is, the hydration ratio is 4 times), the specific activity becomes about 2 times or more compared to the undiluted egg yolk solution, especially 7 times (the hydration ratio is 4 times). 8
When the concentration was higher than 4 times, it was the highest, about 4 times, and the egg yolk ribon protein and antibody were effectively separated. At that time, the recovery rate of the antibody into the supernatant was 70% or more, and most of the antibody was recovered into the supernatant. That is, it is considered that under these conditions, the antibody was selectively recovered in the supernatant.

This also indicates that the protein components of the supernatant were subjected to 5DS-polyacrylamide gel electrophoresis (LaeIliU, K,
Nature, 227: 680-695 (1
This can be confirmed by analysis using 970)). That is, 3
After adding twice as much water, the content of γ-lihetin relative to the total protein gradually increases, and when seven times as much water is added, the supernatant contains mostly water-soluble proteins. They were α-livetin, β-livetin, and γ-lihetin.

Regarding egg yolk lipids, the amount of lipids recovered in the supernatant suddenly and drastically decreased after adding 5 times more water (6 times the hydration ratio) to the egg yolk stock solution, and Only about 20% was recovered in the supernatant at 9x magnification).

From the above, it has been shown that if the amount of water is more than three times the amount of egg yolk fluid, most of the egg yolk riboproteins will precipitate, and the egg yolk water-soluble proteins will be selectively recovered in the supernatant. Ta. In addition, if the amount of water is more than 5 times the amount of egg yolk liquid, egg yolk riboproteins and egg yolk lipids are
Most of it precipitated, and it was shown that egg yolk water-soluble protein was selectively recovered in the supernatant.

Example 2 100 mff1 of undiluted egg yolk liquid to 800 m of distilled water! ! were added, mixed and stirred at 10°C for 10 minutes, centrifuged at 2000×g and 10°C for 10 minutes, and the supernatant and precipitate were collected.

The precipitate contains 76% of the total egg yolk lipids and 33% of the antibody titer.
was recovered. Furthermore, 24% of the total egg yolk lipid and 67% of the antibody titer were recovered in the supernatant.

As a result of analyzing the protein components in the supernatant by 5DS-poIJ'7 acrylamide gel electrophoresis, it was found that most of the protein components were α-livetin, β-livetin, and γ-livetin.
- It was lihetin.

Example 3 A supernatant and precipitate were collected in the same manner as in Example 2, except that distilled water was replaced with a 3mM 111°C sodium chloride aqueous solution. The precipitate contains 73% of the total egg yolk lipids and 3% of the antibody titer.
3% was recovered. In addition, the supernatant contains 27% of total egg yolk lipids.
% and antibody titer were recovered. As a result of analyzing the protein components in the supernatant by 5DS-polyacrylamide gel electrophoresis, it was found that most of the protein components were α
-lihetin, β-lihetin and γ-lihetin.

Example 4 Distilled water was mixed with 5mM sodium phosphate buffer (pH
The supernatant and precipitate were collected in the same manner as in Example 2, except that 6 and 8) were replaced. The precipitate contains 74% of the total egg yolk lipids.
and 33% of the antibody titer was recovered. Furthermore, 26% of the total egg yolk lipid and 67% of the antibody titer were recovered in the supernatant. Furthermore, as a result of analyzing the protein components in the fluid by 5DS-polyacrylamide gel electrophoresis, most of the protein components were α-livetin, β-livetin, and T-lihetin.

Example 5 A supernatant and precipitate were collected in the same manner as in Example 2, except that distilled water was replaced with PBS diluted 50 times.

The precipitate contains 73% of the total egg yolk lipids and 33% of the antibody titer.
was recovered. Furthermore, 27% of the total egg yolk lipids and 67% of the antibody titer were recovered in the supernatant. Analysis of protein components in the supernatant by 5DS-polyacrylamide gel electrophoresis revealed that most of the protein components were α-lihetin, β-lihetin, and T-libetin.

〔Effect of the invention〕

As described above, according to the present invention, it is possible to obtain an egg yolk water-soluble fraction in which T-lihetin is not denatured and egg yolk lipids and egg yolk riboproteins are completely removed.

In addition, the components in egg yolk can be purified easily, inexpensively, and safely from these fractions, making it possible for the first time to effectively utilize them not only in pharmaceuticals and cosmetics but also in foods.

Furthermore, useful substances such as phosphatidylcholine can be easily collected from precipitated egg yolk lipids and egg yolk riboproteins, making this method extremely efficient in terms of production efficiency.

[Brief explanation of drawings]

Figure 1 shows the relationship between the antibody titer in the supernatant of Example 1, the recovery rate of lipids, and the hydration rate, as well as the specific activity and the hydration rate. 26th

Claims (2)

[Claims] (1) Adding 3 times or more of water, a salt solution, and a buffer solution to the egg yolk solution to cause precipitation of the egg yolk riboprotein and separate the aqueous solution of the egg yolk water-soluble protein. A method for fractionating egg yolk riboprotein and egg yolk water-soluble protein. (2) By adding more than 5 times the amount of one selected from water, aqueous salt solution, and buffer to the egg yolk solution, precipitation of egg yolk riboproteins and egg yolk lipids is caused, and an aqueous solution of egg yolk water-soluble proteins is separated. A method for fractionating egg yolk lipids, egg yolk riboproteins, and egg yolk water-soluble proteins, characterized in that: