JPH0689025B2 - Method for recovering water-soluble protein from egg yolk - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for recovering a water-soluble protein from egg yolk, and is particularly suitable for recovering a water-soluble protein such as an IgG antibody and an IgM antibody from an immunized chicken egg. The present invention relates to a method for recovering a water-soluble protein from egg yolk.

[Prior Art] In recent years, it has become possible to generate specific antibodies using immunized chicken eggs, and the possibility of passive immunization using chicken egg antibodies instead of conventional blood antibodies is increasing. Chicken serum antibody IgG, IgM
Translocate to the offspring via the yolk, and these antibodies are present in the water-soluble protein fraction of the yolk. Therefore, in order to effectively utilize the physiological activity of the antibody, it is necessary to collect fractions of the yolk water-soluble protein and the yolk lipoprotein.

Conventionally, as a method of recovering a water-soluble protein from egg yolk, a method of extracting the egg yolk with chloroform to obtain the remaining solution (TTKramer et al. 1970 Immunology 19,157; REFa
ith et al. 1973 Immunology 25, 151) or a method in which polyethylene glycol is added to egg yolk liquid to separate the egg yolk lipoprotein by precipitation to obtain a water-soluble protein from the supernatant. Also, as a method for separating and purifying egg yolk water-soluble protein and egg yolk lipoprotein, a method has been proposed by Hatta et al. (Journal of the Japanese Society of Agricultural Chemistry, Vol. 62, No. 3 (Abstracts of 1988 Conference, 4th page, 2Aa15)).

[Problems to be Solved by the Invention] However, the method of extracting egg yolk lipoprotein with chloroform not only has a poor recovery rate of water-soluble proteins,
Since chloroform is harmful to the living body, it is necessary to completely remove the chloroform in order to use the recovered antibody, which is a water-soluble protein, in the living body, which is inconvenient for purification. Further, the method of adding carrageenan also has a problem that the recovery rate of water-soluble protein is poor and the antibody titer is lowered.
Furthermore, carrageenan is reported to be an inducer of neoplastic colitis (James L. Breeling, et al. INFECTION A
ND IMMUNITY Vol.56, No.7 1988.p.1754 ~ 1759, Onderdon
k, AB, et al 1983.Enhancement of experimental ulce
rative colitis by immunization with Bacterides vul
gatus.Infect.Immun.42: 783-788.).

Further, in order to facilitate storage and handling of the antibody as a water-soluble protein separated from egg yolk, it is preferable to make the water-soluble protein a dry solid. The method using polyethylene glycol has a good recovery rate of the water-soluble protein, but since the recovered supernatant contains polyethylene glycol, there is a problem that it is difficult to dry it.

The present invention has been made in view of the above problems,
The purpose is to separate the antibody, which is a water-soluble protein from egg yolk, by separating it from the egg yolk lipoprotein without lowering the antibody titer, and to collect it efficiently after drying. It is to provide a recovery method.

[Means and Actions for Solving the Problems] In order to achieve the above object, in the invention according to claim 1, a substance selected from a water-soluble cellulose derivative, polyvinyl alcohol, and cyclodextrin in egg yolk liquid. An aqueous solution containing at least one of the above was added and mixed, and the supernatant was collected.

Egg yolk liquid includes not only raw yolk liquid separated from egg white liquid after breaking eggs, but also a solution diluted with water, and yolk prepared not only from chicken but also from other poultry eggs such as ducks. Good.

As the water-soluble cellulose derivative, methyl cellulose, hydroxypropyl cellulose (HPC) and hydroxypropyl methyl cellulose (HPMC) are preferable. The amount of the water-soluble substance added to the yolk liquid may be in the range of 0.1 to several percent such that the concentration of the water-soluble substance in the final solution after the total amount of the aqueous solution has been added. It is preferable to use the water-soluble substance alone from the viewpoint of handling, but a plurality of types may be mixed and used.
Further, the water-soluble substance is added to the yolk liquid in the state of an aqueous solution, and the concentration thereof may be within the above-mentioned final concentration after the addition of the aqueous solution, but from the viewpoint of easy handling, the viscosity of the solution does not increase 1 to A concentration of 3% is preferred. Egg yolk can be treated with water 2 to 3 times because it is easier to handle than using raw yolk liquid as it is.
It is better to dilute 5 times.

After adding a solution of the water-soluble substance in an appropriate concentration to the yolk liquid and stirring the mixture, the yolk lipoprotein is separated from the water-soluble protein as a precipitate when left to stand. Separation of egg yolk lipoprotein precipitate is generally performed by centrifugation,
When the concentration of the egg yolk treatment liquid is low, the separation can be carried out as it is, by adding a super-auxiliary agent or by decantation.

The obtained supernatant may be stored as it is until use, but it is preferable to dry it by an appropriate method and store it in a solid state in order to facilitate storage and handling.

Further, in the second aspect of the present invention, a polymer substance which dissolves in egg yolk liquid with a copolymer of methacrylic acid and ethyl acrylate or methyl methacrylate or hydroxypropylmethyl cellulose phthalate such that it is more alkaline than pH 5.0 to 6.0. Were contacted in a non-uniform state, and the supernatant was then collected.

The copolymer of methacrylic acid and ethyl acrylate is insoluble in neutral water, but is soluble at pH 5.5, soluble in alcohol and becomes an emulsion by adding an alcohol solution to water. The copolymer of methacrylic acid and methyl methacrylate is insoluble in neutral water but soluble at pH 6.0 or above,
It dissolves in alcohol, but an emulsion cannot be obtained by adding an alcohol solution to water. Hydroxypropylmethylcellulose phthalate dissolves in alcohol, but an emulsion cannot be obtained even if an alcohol solution is added to water. That is, in the invention according to the second aspect, the polymer substance comes into contact with the yolk liquid in a non-uniform state.

In the case of a high molecular weight substance that cannot obtain an emulsion from an alcohol solution, it is contacted with the yolk liquid by adding the alcohol solution (concentration several percent) to the yolk liquid (diluted with water). The amount is preferably 0.05 to 0.3% with respect to the solution after addition. The alcohol solution may be added to the yolk liquid all at once, but it is better to add it little by little. When the emulsion is added, the same operation as in the case of the aqueous solution may be performed.

[Examples] Hereinafter, the present invention will be described more specifically.

(Collecting method of immunized chicken egg) Cilia-type toxigenic Escherichia coli (porcine diarrhea-derived Escherichia coli) 987
p, k88 and k99 respectively using Minka liquid medium 37
After culturing at ℃ by the standard method, the bacterial cells were collected by centrifugation, and the collected bacterial cells were subjected to a homogenizer to separate cilia. Mix the obtained cilia with an appropriate adjuvant and intramuscularly inject the chicken,
6 weeks after the first immunization, the second immunization 1
It went like the first time. The eggs laid by this chicken were used as immune chicken eggs.

Also, rotavirus (Shimane strain from bovine diarrhea) was added to MA-104
The cells were contacted and cultured by a standard method. The rotavirus was purified from the culture solution by ultrafiltration, concentrated, mixed with an appropriate adjuvant, intramuscularly injected into a chicken, and the first immunization was performed. Four weeks later, the second immunization was the same as the first immunization. Went to.
The eggs laid by this chicken were used as immune chicken eggs.

(Method for recovering water-soluble protein from egg yolk). When using water-soluble substances.

Egg yolk was separated from the immunized chicken egg, and water was added to dilute the yolk 2.5 times to obtain yolk liquid. Cellulose derivative,
10 ml of an aqueous solution in which polyvinyl alcohol or cyclodextrin was dissolved at a predetermined concentration was added, and the mixture was stirred at room temperature (25 ° C) and left for 30 minutes. This solution was centrifuged at 5000 g for 20 minutes, and a supernatant was obtained.

． When using a copolymer of methacrylic acid and ethyl acrylate.

Water was added to a copolymer of methacrylic acid and ethyl acrylate (Eudragit L30D (manufactured by Rhm Pharma)) to prepare an emulsion having a predetermined concentration. Add 10 ml of this emulsion to 10 ml of yolk liquid obtained in the same manner as above, and add at room temperature (25 ° C).
After stirring for 30 minutes, the mixture was left for 30 minutes. This solution was centrifuged at 5000 g for 20 minutes, and a supernatant was obtained.

When a substance insoluble in water was used A copolymer of methacrylic acid and methyl methacrylate (Eudragit L100 (manufactured by Rhm Pharma)) was dissolved in alcohol at a concentration of 5%. 2.5 as obtained above
A predetermined amount of the alcohol solution of the copolymer was added dropwise to a solution in which 10 ml of the double-diluted egg yolk solution was added with water to make the total amount approximately 2 ml. The amount of water added to the 2.5-fold diluted yolk liquid was adjusted so that the total amount with the amount of the alcohol solution added was 10 ml. This solution was centrifuged at 5000 g for 20 minutes, and a supernatant was obtained.

Hydroxypropyl methylcellulose phthalate HPMC
P) was dissolved in alcohol at a concentration of 5%. A predetermined amount of the alcohol solution of the copolymer was added dropwise to 10 ml of the 2.5-fold diluted egg yolk liquid obtained in the same manner as described above with water to make the total amount approximately 20 ml. The amount of water added to the 2.5-fold diluted yolk liquid is the sum of the amount of alcohol solution added dropwise.
It was adjusted to 10 ml. This solution was centrifuged at 5000 g for 20 minutes, and a supernatant was obtained.

(Measurement of antibody titer) The antibody titer against E. coli was measured by an agglutination test according to a standard method.

The antibody titer against rotavirus was measured by a neutralization test according to a standard method.

The table below shows the results of a test for collecting water-soluble proteins from egg yolk of chicken eggs that were immunized against various Escherichia coli or rotaviruses according to the above-described method, and the antibody titer of the collected supernatant was measured. The concentration in the table indicates the concentration of each added substance relative to the solution after the total amount of each solution was added to the yolk liquid, and the recovered amount indicates the amount of the supernatant obtained after centrifugation.

As a comparative example, the test results of the egg yolk liquid treated with an equal amount of chloroform and then treated with an equal amount of an aqueous solution of carrageenan and the test results of whole eggs are also shown.

As is clear from the table, in the conventional method of Comparative Example, the recovered amount of the supernatant was 7 ml in the case of the chloroform treatment and 5.5 ml in the case of the carrageenan aqueous solution treatment, which was as small as 5.5 ml. In any of the above collection methods, the collected amount was as large as 17 ml, and it was possible to obtain a supernatant of about 2.4 times that of the chloroform treatment and about 3 times that of the carrageenan aqueous solution treatment.
Also, when a substance that does not dissolve in neutral water is used, the amount of supernatant recovered is slightly worse than when a water-soluble substance is used, but it is almost twice as high as that of the conventional method. Qing was obtained.

The antibody titer of the obtained supernatant was the same as that of whole egg against any immunoantigen except for 877p of Escherichia coli in the case of the chloroform treatment of the comparative example, but in the case of the carrageenan aqueous solution treatment. Was lower than that of whole egg for all immune antigens. On the other hand, in the case of the present invention using a water-soluble substance, 9% of E. coli treated with 1.5% polyvinyl alcohol was used.
Except for 87p, it was equivalent to that of whole egg against all immunogens. Also, when a substance that does not dissolve in neutral water is used, it is equivalent to whole egg against immune antigens other than 987p of E. coli, and hydroxypropylmethylcellulose phthalate against 987p of E. coli. When an emulsion of a copolymer of methacrylic acid and ethyl acrylate (solution with a concentration of 0.25%) was used, it was equivalent to that of whole eggs, but in other cases, it decreased to the same extent as in the case of the conventional method.

The resulting supernatant is dried by an appropriate method to facilitate handling and storage, and the water-soluble protein is stored in a solid state, but the supernatant is not dried but stored as an aqueous solution or concentrated. You may. When a water-soluble substance is used, a large amount of the water-soluble substance is also dissolved in the supernatant. Therefore, the solid-state water-soluble protein obtained by drying contains the water-soluble substance. All the water-soluble substances used are substances that have been approved for in vivo administration, so there is no problem.

The present invention may also be applied to the recovery of water-soluble proteins other than antibodies.

[Effects of the Invention] As described in detail above, according to the present invention, an antibody which is a water-soluble protein can be efficiently separated from egg yolk by separating it from egg yolk lipoprotein with almost no decrease in antibody titer, Moreover, the dried solid-state water-soluble protein can be easily obtained from the recovered aqueous solution.

Claims (2)

[Claims] 1. An egg yolk solution is mixed with an aqueous solution containing at least one substance selected from water-soluble cellulose derivatives, polyvinyl alcohol and cyclodextrin, and the mixture is recovered. A method for recovering a water-soluble protein from egg yolk. 2. A copolymer of methacrylic acid and ethyl acrylate or methyl methacrylate, hydroxypropylmethyl cellulose phthalate, etc. in egg yolk liquid, pH 5.0 to 6.0.
A method for recovering a water-soluble protein from egg yolk, which comprises contacting a high-molecular substance which is more alkaline and contacting it in a heterogeneous state, and recovering the supernatant.