JPH06228197A - Method for fractionating gamma-livetin from egg yolk - Google Patents

Abstract

PURPOSE:To obtain gamma-livetin from a water-soluble protein fraction lowered in the content of egg yolk lipoprotein in high recovery ratio by using an egg yolk subjected to freezing and melting treatment as a starting material and hydrolyzing the egg yolk and purify gamma-livetin. CONSTITUTION:The method for fractionating gamma-livetin is to fractionate gamma-livetin by adding water of 6 or more times based on liquid egg subjected to freezing and melting treatment to the liquid egg to precipitate egg yolk lipoprotein and then recovering the separated supernatant. Furthermore, the method for purifying gamma-livetin is to purify gamma-livetin by ethanol fraction from the supernatant or to purify gamma-livetin by ammonium sulfate fraction or sodium sulfate fraction from the supernatant or to purify gamma-livetin by an affinity chromatography from the supernatant.

Description

Detailed Description of the Invention

[0001]

The present invention relates to γ-containing egg yolk.
Gamma in egg yolk for the administration of ribetin to humans and animals
-A method for separating ribetin from egg yolk lipids, lipoproteins and water-soluble proteins.

[0002]

BACKGROUND OF THE INVENTION Egg yolk serves as a reservoir of nutrient components for embryo growth. Its composition is 48% water, 17.5% protein, 32.5% lipid, 2.0% ash,
Egg yolk contains about 50% solids. Of these, trace amounts are dialyzable, but most exist as lipoproteins, that is, giant complexes in which proteins and lipids are bound at various ratios. In addition, the protein that is not bound to lipid, that is, the water-soluble protein is about 10% in the solid content. The water-soluble proteins include ribetin, phosvitin and trace amounts of riboflavin binding protein.
Ribetin is classified into α-ribetin, β-ribetin and γ-ribetin. α-, β- and γ-ribetin have been shown to be immunologically equivalent to parental albumin serum albumin, α-glycoprotein and γ-globulin, respectively. Therefore, γ-ribetin, which acts as γ-globulin, is expected to be applied to veterinary medicine and human medicine and as a research reagent in recent years.

In the purification method of γ-ribetin, conventionally,
Several methods have been reported, and column separation, ultracentrifugation, and electrophoresis are performed at the laboratory level. However, it is difficult to obtain a large amount of γ-ribetin by these methods and it is not suitable for industrialization.

As mentioned above, egg yolk is mostly lipids and lipoproteins. Therefore, removing lipids and lipoproteins from egg yolk as the first step of purifying γ-ribetin is advantageous for facilitating the subsequent purification. To remove lipids and lipoproteins from egg yolk, Polson et al. [Polson, A. et al., Immunol. Commu
n. 9 : 495-514 (1980)], Jensenius,
JC, J. Immunol. Methods 46 : 63-68 (1981)], Hatta et al. [Hatta, H. et al., Agric. Biol. Chem. 54 : 2531-2
535 (1990), Japanese Patent Laid-Open No. 64-38098], Auricio et al.
[Aulisio, CG, Proc. Soc. Exp. Biol. 126 : 312-315
(1981)], Bade [Bade, H., J. Immunol. Methods 72:
421-426 (1984)], a method of externally adding something such as adding a polysaccharide, polyethylene glycol, an organic solvent, etc. has been reported.

On the other hand, in order to fractionate and purify γ-ribetin in egg yolk and apply it to medicines, cosmetics, foods, etc., in order to utilize it, it is necessary to mix substances which are of safety concern for the human body and their use. Incorporation of substances in question must be avoided. The methods of Polson, Jensenius and Hatta all add polyethylene glycol, dextran sulfate, carrageenan, etc. to separate egg yolk lipoprotein. Therefore, in the water-soluble protein fractions obtained by these methods, their contamination is unavoidable,
This means that substances other than egg yolk components have been added. These impede the application and use of γ-ribetin in medicines, cosmetics, foods and the like. The method using a highly toxic organic solvent such as chloroform, isopropanol, and acetone is also not preferable from the above viewpoint. Further, when chloroform having high polarity is used, the yolk water-soluble protein is denatured in the purification process, and thus the chloroform method is not suitable for purification of γ-ribetin. In addition, dextran sulfate is very expensive, and cost is a problem.

On the other hand, a method which does not use additives has been reported up to the present time, and by using this method, these drawbacks and problems can be avoided. There are the following methods.

Japanese Unexamined Patent Publication (Kokai) No. 3-204898 discloses a method of precipitating egg yolk lipid and egg yolk lipoprotein by adding 3 times or more of water, a salt solution, and a buffer solution to egg yolk and mixing them. There is. In addition, Kwan et al. Subsequently reported a method of precipitating yolk lipoprotein by similarly hydrolyzing egg yolk [Kwan, L., J. Food Sci. 56 : 153.
7-1541 (1991)]. Furthermore, Akita et al. Reported a 6-fold dilution method at pH 5.0 to 5.2 [Akita, EM.
and Nakai, S., J. Food Sci. 57 : 629-634 (1992)]. These methods are the same in that egg yolk is diluted with water to insolubilize lipids and lipoproteins. These are simple and extremely excellent methods for partially purifying the yolk antibody from egg yolk. However, since egg yolk is raw and cannot be preserved, these methods cannot be industrialized. Therefore, in the case of industrialization, a method using frozen egg yolk as a starting material is necessary.

[0008]

[Problems to be Solved by the Invention] As a method for cryopreservation, Jensenius reported a method in which egg yolk was hydrolyzed 9 times at neutral pH and then freeze-thawed [Jensenius, J.
C., J. Immunol. Methods 46 : 63-68 (1981)]. However, since this method freezes a 10-fold diluted solution of egg yolk in the end, it is completely unsuitable industrially due to space problems and freezing cost. To solve this problem, JP-A-3-204898 describes a method of using frozen egg yolk as a starting material and performing a hydration operation after thawing. However, when egg yolk subjected to freeze-preservation and thawing treatment was used as a starting material, there was a drawback that the recovery rate of γ-ribetin in the supernatant obtained by the hydrolysis treatment was very poor (Table 1). As a result of intensive studies, the present inventors have found a method for obtaining γ-ribetin in a supernatant obtained by hydrolyzing with a high recovery rate, even though it has been subjected to cryopreservation and thawing treatment, and completed the present invention. The object of the present invention is to use γ-ribetin in a water-soluble protein fraction having a reduced egg yolk lipoprotein content by hydrolyzing egg yolk that has been subjected to freeze-thaw treatment as a starting material. It is to provide a method of obtaining a high recovery rate.

[0009]

Means for Solving the Problems The present invention comprises adding 6 times or more of water to a freeze-thawed liquid egg to cause precipitation of egg yolk lipoprotein and recovering the supernatant. Method for fractionating ribetin, method for purifying γ-ribetin from the supernatant by ethanol fractionation, γ-by ammonium sulfate fractionation or sodium sulfate fractionation from the supernatant
A method for purifying rivetin and a method for purifying γ-ribetin from the supernatant by affinity chromatography.

The present invention will be described in detail below.

The liquid egg in the present invention refers to whole egg liquid after breaking eggs, raw yolk liquid from which egg white liquid has been partially removed after breaking eggs, or yolk liquid diluted with water. Liquid consisting only of egg yolk without white and yolk membrane is not included in the definition of liquid egg. The liquid egg may be prepared from eggs of birds such as chicken and turkey duck. The freezing operation in the present invention is a method of freezing the liquid egg at 0 to -200 ° C, preferably -20 ° C to -40 ° C. The time may be the time when all the liquid eggs are frozen. In addition, the thawing operation is sufficient if the frozen liquid egg can be thawed, and the temperature is 4 to 50.
℃, preferably 37 ℃. As the water in the present invention, tap water, well water, deionized water, distilled water, pure water, salt solution and the like are used. In terms of safety, the closer it is to pure water, the better. The pH of the water added to the liquid egg may be between 4 and 9, and is preferably 5 to 7. From the viewpoint of separating the yolk lipoprotein and the yolk water-soluble protein, the amount of water to be added to the liquid egg is preferably 6 times or more the amount of liquid egg, and if it is less than that, the separation is difficult. The temperature during stirring and mixing is preferably 0 ° C to 50 ° C, and is preferably room temperature from the viewpoint of operation, hygiene, and high yield of γ-ribetin. At 4 ° C, the recovery of γ-ribetin is slightly worse. The stirring and mixing time for separation is not particularly limited,
It suffices that mixing is carried out, separation is possible immediately after stirring, and it is suitably selected in about 10 minutes to 1 day. Centrifugation is a method for collecting the supernatant and removing the precipitate, and the gravity may be about 2000 xg. Further, to remove the precipitate, filtration using a filter aid such as filter paper and Celite, or filtration through a membrane can be used. In addition, since the precipitate is rapidly precipitated after stopping stirring, it is possible to recover the supernatant by decantation.

The ethanol concentration in the ethanol fractionation which can be used in the method for purifying γ-ribetin from the supernatant obtained in the present invention may be in the range of 15% to 99% as the final concentration. From the viewpoint of excluding other proteins and increasing the purity in the protein, 20% to 30% is preferable. or,
From the viewpoint of removing lipids, 50% to 80% is particularly desirable, and the precipitate suspension after ethanol precipitation is filtered to remove insoluble matter, whereby a completely defatted filtrate can be obtained. In addition, 20% to 30% ethanol fractionation treatment described above
By combining 50% to 80% ethanol fractionation treatment, a highly pure and completely defatted γ-ribetin fraction can be obtained. There is no particular problem with the order of the ethanol fractionation treatment at that time, and either treatment may be performed first.
Further, the ammonium sulfate concentration in the ammonium sulfate fractionation may be 20% to 50% in terms of the final saturated ammonium sulfate concentration, and from the viewpoint of improving the purification degree, it is preferably about 30% to 40%. The final concentration of sodium sulfate in the sodium sulfate fractionation is preferably 10% to 20%. Avid AL ^™ gel (Bioprobe) is particularly preferable as the affinity chromatography that can be used in the method for purifying γ-ribetin from the supernatant obtained in the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples.

[0014]

EXAMPLES In the examples, the antibody titer was used as an index for investigating the recovery rate of γ-ribetin, and the ELISA method was used to measure the antibody titer.

(ELISA method) First, the description of Hamada et al. From the S. mutans Ingbritt strain [Hamada, S. et al., J. Gen. Microb
iol. 135 : 335-344 (1989)], cell-associated glucosyltransferase (Cell-associated gl
ucosyltransferase, CA-GTase) was dissolved in 50 mM sodium carbonate buffer (pH 9.6) to 1 μg / ml, and the resulting solution was added to each well of a 96-well plate [Immulon 2 Dynatech]. 100 μl of each was placed in the plate and left overnight at 4 ° C. to adsorb the cells on the plate. Next, the plate was washed 6 times with PBS-T [PBS pH 7.4 containing 0.05% Tween20]. After washing, plate the plate with 3% BSA (bovine serum albumin).
After contacting with PBS pH7.4 containing PBS at 37 ° C for 1 hour to perform blocking, the plate was washed 6 times with PBS-T. Now add 100 μl of a two-step dilution of each preparation to each well,
The reaction was allowed to proceed at 37 ° C for 1 hour. After the reaction is complete,
After washing 3 times with PBS-T, add peroxidase-conjugated anti-chicken IgG antibody (100 μl of protein amount 1.67 μg / ml) to each well as a secondary antibody and incubate for 30 minutes at 25 ° C. The plate was washed 6 times with T. Next, each well of the plate was added with 50 ml of 0.2 M disodium phosphate-0.1 M citrate buffer (pH 5.0) and the substrate o-phenylenediamine 20.
Add 100 μl of the solution containing mg and hydrogen peroxide 10 μl,
The reaction was carried out at 25 ° C for 20 minutes. The reaction was stopped by adding 100 μl of 3N sulfuric acid to each well. After the reaction,
The antibody titer was measured by measuring the absorbance of each well at 492 nm. The antibody titer was the dilution ratio at which the absorbance was 0.2.

Further, in the following examples, CA-G derived from S. mutans
Chicken's pectoral muscles were immunized with Tase together with Freund's complete adjuvant (FCA), and chicken laid eggs in which anti-CA-GTase antibody was produced in blood were used as a starting material.

The total lipid amount was determined by extracting the total lipid with chloroform-methanol (2: 1), distilling off the organic solvent, and taking the weight of the residue as the total lipid amount.

The protein quantification method is carried out by the Bradford dye-binding method [Bradford, MM, Anal. Biochem. 72:
248-254 (1976)] and bovine serum albumin was used as a standard.

Example 1 An egg containing an antibody against CA-GTase (anti-CA-GTase antibody) was manually broken into eggs, and the egg white was partially separated using the shell for use in home cooking to prepare a liquid egg. Got The percentage of egg white contained in it was about 15% in the liquid egg. -40 the liquid egg
It was frozen at ℃ for 12 hours and thawed at 37 ℃ for 2 hours. Pure water (room temperature) was added to 10 ml of the freeze-thaw processed liquid egg at a volume of 4 to 12 times, that is, a final dilution ratio of 5 to 13 times, and 10
After stirring and mixing for 1 minute, the supernatant and the precipitate were separated by centrifugation at 2000 xg for 10 minutes at room temperature, and the supernatant fraction was collected.
The lipid amount and antibody titer in the supernatant fraction were measured, and the total lipid amount in the first liquid egg and the recovery rate for the antibody titer were determined. As a comparison, a supernatant fraction was obtained by the same method using an egg yolk liquid containing no white and yolk membrane as a starting material, which was obtained using a syringe. The results are shown in Table 1.

[0020]

[Table 1]

As is clear from Table 1, the recovery rate of antibody in the supernatant was higher when liquid egg was used as the starting material than when egg yolk liquid was used at any dilution ratio. Furthermore, antibody recovery was much higher than lipid recovery and protein recovery, indicating that the antibody was selectively recovered in the supernatant. If the amount of water added is less than 6 times (7 times at the final dilution ratio), the supernatant fraction cannot be obtained regardless of which starting material is used. 7
It has also been shown that a hydration amount of 2 times or more is required.

The protein components in the obtained supernatant were analyzed by SDS-polyacrylamide gel electrophoresis. As a result, most of the protein components were α-ribetin, β-ribetin and γ-ribetin.

From the above, it has been clarified that the use of liquid egg as the starting material is a better method for purifying the antibody than the case of using yolk liquid.

Example 2 Next, the effect of temperature when water was added was examined.

4 ° C was added to 4 ml of the freeze-thawed liquid egg described in Example 1.
Pure water was added up to a final magnification of 11 times, and the mixture was stirred and mixed at 4 ° C. for 10 minutes and then centrifuged at 2000 × g at 4 ° C. for 10 minutes to collect a supernatant fraction. Further, the supernatant fraction was collected in the same manner as above except that pure water at room temperature was used and the temperature of stirring and mixing was brought to room temperature. The results are shown in Table 2.

[0026]

[Table 2]

As is clear from the table, when the treatment temperature is room temperature, the antibody titer recovery rate becomes 81%, and the recovery rate at 4 ° C. is 50%.
It was found to be much higher than%, and more antibody could be recovered. Also, there was almost no difference in the recovery rate of egg yolk lipoprotein amount. Thus, by setting the operating temperature after freeze-thaw treatment to room temperature, a water-soluble protein fraction with enhanced antibody recovery was obtained at a low egg yolk lipoprotein content, which was almost the same as that obtained at 4 ° C. It has become possible to obtain.

Example 3 The supernatant obtained after hydrolyzing contains a few% of lipid. Therefore, the lipid was completely removed from the supernatant using ethanol. Freeze-thaw egg 10 as described in Example 1
00 ml was hydrolyzed 11 times at room temperature, stirred and mixed for 30 minutes, and then filtered through a filter paper to obtain a supernatant fraction as a filtrate. After cooling the supernatant fraction to 4 ° C, 99% EtOH (-20 ° C) was added to a final concentration of 60%, and the mixture was stirred at 4 ° C for 1 hour and then centrifuged (12000 x g, 4 ° C, 30 ° C). The precipitate was recovered by The precipitate was dissolved in 30 mM NaCl to prepare a homogeneous solution, and the solution was filtered using filter paper to collect the filtrate. as a result,
Γ with complete lipid removal and 61% antibody recovery
-The Rivetin fraction could be obtained.

Example 4 Next, using the freeze-thaw solution as described in Example 1 as a starting material, ethanol was used to finally purify γ-ribetin to a purity of 99% or more. That is, a hydrolyzed supernatant fraction was obtained as a filtrate in the same manner as in Example 3, and then the supernatant fraction was cooled to 4 ° C. 99% EtOH (-20 ℃) was added to the solution to a final concentration of 60%, and the mixture was stirred at 4 ℃ for 1 hour and then centrifuged (12000 x
g , 4 ° C., 30 minutes) to recover the precipitate. Precipitate 30 ° C at 4 ° C
After dissolving in mM NaCl to prepare a homogeneous solution, the solution was filtered using filter paper to collect the filtrate. 50 to the filtrate
Add 4% EtOH (-20 ° C) to a final concentration of 30%.
After stirring for 1 hour in a centrifuge (12000 x g , 4
The precipitate was collected at (° C, 30 minutes). After dissolving the precipitate in 30 mM NaCl to prepare a homogeneous solution, adjust the pH of the solution to 7.4, add 50% EtOH (-20 ℃) to a final concentration of 25%, and stir at 4 ℃ for 1 hour. After that, centrifuge (12000
The precipitate was collected at x g , 4 ° C, 30 minutes). Precipitate at 4 ° C
The product was dissolved in 30 mM NaCl to prepare a homogeneous solution, and the final purified fraction was obtained. The results are shown in Table 3. An antibody (γ-ribetin) fraction containing no lipid was obtained in the final purified fraction. or,
The purity was 99% or more as a result of gel filtration analysis.
The antibody (γ-ribetin) recovery rate was as high as 49%.

[0030]

[Table 3]

Example 5 Next, γ-ribetin was purified from the hydrolyzed supernatant obtained in Example 3 using a column. That is, 14 ml of the freeze-thawed liquid egg described in Example 1 was watered 11 times at room temperature, and after stirring and mixing for 30 minutes,
A supernatant fraction was obtained as a filtrate by filtration using filter paper.
Furthermore, the supernatant fraction was applied to an Avid AL ^™ (Bioprobe) column. The column was washed with PBS (pH 7.4) and then further washed with 0.1 M glycine-hydrochloric acid (pH 5.0). Then 0.1M glycine with 20% glycerol-
It was eluted with hydrochloric acid (pH 4.0). Collect the eluted fractions and perform Sephacry
l Apply to S-300 column (Pharmacia) and apply 280
The elution fraction absorbing the wavelength of nm was collected and used as the final standard.
The results are shown in Table 4.

The final preparation contains an antibody (γ
-Ribetin) fraction was obtained. The recovery rate was as high as 38%. In addition, this sample is SDS-PAGE [Laemmli, UK, Natu
re227: 680-685 (1970)] (7.5% gel concentration, sample treatment under non-reducing condition at 100 ° C. for 5 minutes), and a single band of γ-ribetin alone was detected.

[0033]

[Table 4]

Example 7 The hydrolyzed supernatant fraction described in Example 3 was subjected to 40% saturated ammonium sulfate salting out, and further 30% saturated ammonium sulfate salting out, followed by dialysis against PBS (pH 7.4) to obtain the final standard. I got the goods. The final preparation contains no lipids (γ-
Rivetin) fraction was obtained. This sample is SDS-PAGE
[Laemmli, UK, Nature 227: 680-685 (1970)] (7.5%
When the gel concentration and sample treatment were analyzed under non-reducing conditions at 100 ° C for 5 minutes), a single band of γ-ribetin alone was detected.

Similar .gamma.-ribetin fractions were obtained when salting out with 17% and then with 14% sodium sulfate instead of 40% and 30% saturated ammonium sulfate salting out.

[0036]

As described above, according to the present invention, egg yolk to γ
-As a first step in separating and purifying ribetin, removal of egg yolk lipids and egg yolk lipoproteins from egg yolk was performed without additives, and the safety was high, and the immunological action as γ-globulin was retained. A ribetin fraction can be obtained. The method of obtaining a supernatant containing γ-globulin by adding 6 times or more water to the freeze-thawed liquid egg of the present invention, compared to the case of using a conventional freeze-thawed egg yolk,
It is excellent in that γ-globulin can be obtained in the supernatant at a high recovery rate. Further, from these fractions, the components in egg yolk, particularly γ-ribetin, can be purified easily, inexpensively and safely, and for the first time, it can be effectively used not only in medicines and cosmetics but also in foods. Furthermore, useful materials such as phosphadylcholine can be easily collected from the egg yolk lipids and egg yolk lipoprotein fractions recovered as a precipitate after hydration,
This is an extremely excellent method in terms of production efficiency.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Mariko Saito 5-3 28, Kotobuki-cho, Odawara-shi, Kanagawa, Kanebo Co., Ltd. (72) Inventor Hideaki Yokoyama 2-chome, Suhara-Miyashiro-cho, Kakamigahara-shi, Gifu Prefecture 115 No. 2 (72) Inventor Yu Tatezawa No. 130, 1212 Hino, Gifu City

Claims (4)

[Claims] 1. A method for fractionating γ-ribetin, which comprises adding 6 times or more of water to a freeze-thawed liquid egg to cause precipitation of egg yolk lipoprotein and recovering the supernatant. 2. A method for purifying γ-ribetin from the supernatant according to claim 1 by ethanol fractionation. 3. A method for purifying γ-ribetin from the supernatant according to claim 1 by ammonium sulfate fractionation or sodium sulfate fractionation. 4. A method for purifying γ-ribetin from the supernatant according to claim 1 by affinity chromatography.