JPH05310797A - Method for separating ovomucoid - Google Patents

Abstract

PURPOSE:To separate ovomucoid without requiring many hours in operation and damaging physiological activity of ovomucoid in separating ovomucoid from egg-white protein. CONSTITUTION:Egg-white protein is dissolved in 5-10wt.% aqueous solution of trichloroacetic acid, the supernatant liquid is filtered by an ultrafilter having >=50,000 fractioned molecular weight, trichloroacetic acid is removed by dialysis method and the solution is concentrated with an ultrafilter <=50,000 fractioned molecular weight to separate ovomucoid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for separating ovomucoid. More specifically, it relates to a method for separating ovomucoid from egg white protein.

[0002]

2. Description of the Related Art Egg white contains about 40 kinds of proteins, and lysozyme having industrially easy separation and useful physiological activity is used. Ovomucoid, which is a protein having useful physiological activity, is present in the egg white of about 11%.

Ovomucoid is a glycoprotein having a molecular weight of 28,000 and an isoelectric point of 3.9 to 4.5, which is a heat-resistant trypsin inhibitor present in egg white and also acts on chymotrypsin to inactivate it. It also forms a complex with inactivated enzymes such as anhydrotrypsin (active center serine substituted with dehydroalanine), tosyllysyl chloromethyl ketone treated trypsin, and tosyl phenylalanyl chloromethyl ketone treated chymotrypsin. Let

With respect to the ovomucoid having such a physiological activity, a technique for industrially mass-separating the ovomucoid at a low cost has not been established in the past, and only the following laboratory-level separation method is available.

Typical separation methods are precipitation method and ion exchange chromatography method. The latter method uses carboxymethylcellulose or diethylaminoethylcellulose as a carrier, and although the ovomucoid separation and recovery rate are good, it takes about 25 hours for carboxymethylcellulose and about 18 hours for diethylaminoethylcellulose per operation. Therefore, it is inefficient.

On the other hand, the former method also has the advantage that a large amount of treatment can be carried out at one time, but as described below, long steps (1) to (6) are required until the ovomucoid is completely separated. Not only does it take time, but the disposal of the ammonium sulphate is expensive because of the large amount of ammonium sulfate used, and the ovomucoid is heat-treated at 100 ° C. for 5 minutes, which may impair its physiological activity. (1) Egg protein protein solution with lysozyme and avidin removed
Add an equal amount of ammonium sulfate to (pH 7.0) and collect the upper layer with a centrifuge. (2) Slowly add 0.5 M sulfuric acid until crystallization starts in the upper layer liquid. Then, add 0.5 M sulfuric acid to adjust the pH to 4.6.
Adjust to and crystallize overnight. (3) Add 8 g of ammonium sulfate per 100 ml of the upper layer liquid collected by the centrifuge. (4) Saturate the upper layer liquid collected by the centrifuge with ammonium sulfate. (5) Discard the upper layer with a centrifuge, collect the precipitate, dissolve the precipitate in water, and heat at 100 ° C. for 5 minutes. (6) The upper layer is collected with a centrifuge, and the upper layer is heated at 0 ° C under a temperature condition of
Dialyze with 65% ethanol to collect ovomucoid.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for separating ovomucoid from egg white protein that does not require a long time for operation and does not impair the physiological activity of ovomucoid. ..

[0008]

The object of the present invention is as follows.
Egg protein was dissolved in an aqueous solution of trichloroacetic acid having a concentration of 5 to 10% by weight, and the supernatant was filtered through an ultrafiltration membrane having a molecular weight cutoff of 50,000 or more, and then trichloroacetic acid was removed by dialysis. It is achieved by a method of separating ovomucoid which concentrates on the solution with an ultrafiltration membrane having a molecular weight cut-off of 50,000 or less.

As the egg protein, any of those before removal of lysozyme, avidin and the like or those after removal thereof can be used. Egg protein is diluted with about 5 to 15 times pure water, and an aqueous solution of trichloroacetic acid having a concentration of 50% by weight, for example, is added to the aqueous solution. Adjusted to be%. Since trichloroacetic acid is a strong acid, if its concentration is too high, it will denature all proteins including ovomucoid.
Its maximum concentration as an aqueous solution is limited to 10% by weight.
On the other hand, if the concentration of the aqueous solution is less than this, the desired effect cannot be obtained.

Regarding the quantitative relationship between egg protein and trichloroacetic acid, the pH of the mixed solution is preferably 0.9 to 2.0.
Is selected to be in the range. At a pH below this, the ovomucoid will be denatured, while at a pH above this it will not be possible to precipitate any protein other than ovomucoid.

In this manner, the mixed solution prepared by dissolving the egg protein in the aqueous solution of trichloroacetic acid having a concentration of 5 to 10% by weight is allowed to stand for about 1 hour, and then the supernatant is subjected to a molecular weight cut-off.
Precipitated components such as other protein components are removed by filtration with an ultrafiltration membrane of 50,000 or more, preferably 100,000 to 500000. It takes about 3 hours to reach here.

From the solution containing ovomucoid as the main component obtained by this ultrafiltration, trichloroacetic acid was removed by a dialysis method, and then the molecular weight cutoff of the solution was 5000
Concentration is performed using an ultrafiltration membrane of 0 or less, preferably 50,000 to 20,000, and the gel-like protein attached to the membrane is dried under reduced pressure to obtain ovomucoid as a white powder.

[0013]

[Effects of the Invention] When ovomucoid is separated from egg protein, other proteins etc. are efficiently removed by using trichloroacetic acid. An industrial method was established by which the

[0014]

EXAMPLES The present invention will now be described with reference to examples.

Example 1 80 g of egg white protein was diluted with pure water about 10 times, and
100 ml of 0 wt% trichloroacetic acid aqueous solution was added (concentration of trichloroacetic acid aqueous solution was about 6 wt%). Volume of this mixture (pH 1.2) is 2
After being placed in a L beaker and allowed to stand for about 1 hour, the supernatant was filtered by a cross-flow method using an ultrafiltration membrane having a cut-off molecular weight of about 200,000 to remove precipitated components. About 850 ml of the filtrate, about 1 using a dialysis membrane with a molecular weight cutoff of about 10,000.
It was dialyzed for 0 hour to remove trichloroacetic acid.

About 800 ml of the solution obtained through the above steps was concentrated by a cross-flow method using an ultrafiltration membrane having a molecular weight cut off of about 40,000, and the gel-like protein attached to the surface of the filtration membrane was concentrated. About 0.3g
Of ovomucoid was obtained as a white powder. The confirmation was performed by the HPLC method (electrophoresis method).

Claims (1)

[Claims] 1. An egg protein is dissolved in an aqueous solution of trichloroacetic acid having a concentration of 5 to 10% by weight, and the supernatant thereof is fractionated to have a molecular weight.
After filtration with an ultrafiltration membrane of 50,000 or more, trichloroacetic acid was removed by a dialysis method, and the molecular weight cutoff of the solution was 50,000.
The following method for separating ovomucoid, which comprises concentrating with an ultrafiltration membrane.