JPH0515389A - Production of protein-like composition - Google Patents

Abstract

PURPOSE:To surely and efficiently introduce an amino acid to a protein and to obtain the tile composition by adding an organic acid to a solution containing a protein, an amino acid ester and a protease and subjecting to plastein reaction. CONSTITUTION:A raw material protein such as whey powder, at least one amino acid ester of leucine ethyl ester, isoleucine ethyl ester, valine ethyl ester, etc., and a protease such as papain are dissolved in water, mixed with an organic acid such as citric acid, adjusted to pH8.0 with sodium hydroxide, subjected to plastein reaction at 40 deg.C for 20 hours while stirring, an amino acid in the amino acid ester is introduced into the protein, which is deesterified with 1N sodium hydroxide at 40 deg.C for 1 hour while stirring, dialyzed with an ion exchange membrane, desalted and freeze-dried to give the objective protein-like composition.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a proteinaceous composition in which an amino acid is introduced into a raw material protein by utilizing a plastein reaction.

[0002]

2. Description of the Related Art Conventionally, there have been considered two methods for enzymatically hydrolyzing a protein and introducing a specific amino acid into the protein. The first method is to hydrolyze a protein into a low molecular weight peptide, and then to introduce an amino acid by utilizing an enzymatic reverse reaction to synthesize a relatively high molecular weight polypeptide. The second method is to react a protein outside the optimum conditions of the enzyme and decompose it while introducing an amino acid to obtain a low molecular weight peptide.

The so-called plastein reaction was originally attached to the former phenomenon, and the latter should be called aminolysis, to be precise. All of the latter are collectively called the plastein reaction. Recently, since the raw material protein can be used as it is and the operation method is simpler, the plasteine reaction mainly utilizing the latter aminolysis is used for the purpose of introducing amino acids and increasing the nutritional value of the raw material protein. Is often used.

Therefore, the applicant of the present invention has previously provided a method for producing a mixture of low molecular weight peptides using the plastane reaction. (JP-B-62-17520) In the method disclosed in the above-mentioned publication, soybean protein is deficient in methionine as an essential amino acid, and soybean protein is branched peptide amino acid such as valine, leucine and isoleucine in peptide form. It is introduced to improve the nutritional value of the raw material protein and further decompose it into low molecular weight peptides such as di and avian to improve the absorbability.

[0005]

The method of the above publication has a sufficient effect in improving the nutritional value of the raw material protein. However, in recent years, when supplying a nitrogen source, it has been studied to make an excess state of a certain specific amino acid and utilize the nutritional specificity, that is, the so-called amino acid imbalance effect. In particular, an excess nitrogen source of branched chain amino acids (valine, leucine, isoleucine) may be effective in improving severe heat disease, conditions such as trauma in which muscle proteins are easily degraded, or mental disorders such as liver failure. It became clear.

[0006] However, in order to utilize the amino acid imbalance effect, it is necessary to add a sufficient amount of amino acid to the starting protein, and the method disclosed in the above publication introduces sufficient amino acid. Was difficult. Also, using papain hydrolyzate of ovalbumin,
When various amino acid ethyl esters were reacted with this in the presence of papain and the introduction ratio of various amino acids was examined, the introduction ratios of valine, isoleucine, glycine, and alanine were 10
It has been reported that it was about 30% lower than that of other amino acids (Nutrition and Foods, Vol. 29, Item 295, 1).
976). According to the document, if such an amino acid is esterified with an alcohol having a high hydrophobicity, such as hexyl alcohol, the introduction rate is increased, but there is a problem in terms of safety for the human body.

[0007]

The object of the present invention is to introduce a sufficient amount of amino acid into a raw material protein,
Further, the present invention provides a method for producing a protein-like composition, which can reliably and efficiently introduce an amino acid, which is said to have a low introduction rate, into a raw material protein.

What achieves the above-mentioned object is to add a raw material protein, at least one kind of amino acid ester, and a proteolytic enzyme to a solution, and add an organic acid to the solution to add the amino acid to the protein. It is a method for producing a proteinaceous composition in which an amino acid in an ester is introduced by a plastane reaction.

The organic acid to be added is preferably citric acid, tartaric acid or malic acid. The amount of the organic acid added is 1 to 50 with respect to the solution.
It is preferably W / V%. Furthermore, the amino acid ester is preferably an amino acid ethyl ester.

The method for producing the proteinaceous composition of the present invention will be described in detail below. The method for producing a protein-like composition of the present invention comprises adding a raw material protein, at least one amino acid ester, and a proteolytic enzyme to a solution, adding an organic acid to the solution, and adding the organic acid to the protein. The amino acid in the amino acid ester is introduced by the plastein reaction. According to this method, it is possible to introduce a sufficient amount of amino acid into the raw material protein, and it is possible to reliably and efficiently introduce the amino acid, which is said to have a low introduction rate, into the raw material protein. It is possible to obtain a protein-like composition in which amino acids are introduced into a protein at a higher ratio than in the past.

In the production method of the present invention, at least one branched chain amino acid ester, an organic acid, a starting protein and a proteolytic enzyme are added to a buffer solution (reaction solution) adjusted to a pH suitable for the plastein reaction. Then, the amino acid in the branched chain amino acid ester is introduced into the raw material protein by the plastane reaction. The order of addition is as follows:
A raw material protein is added to an aqueous solution adjusted to a predetermined pH with an organic acid and a reducing agent if necessary, and stirred to prepare a raw material protein solution. Then, a liquid obtained by adding a proteolytic enzyme to water is added to the above raw material protein solution and stirred, and then a predetermined temperature, for example, 40 to 50 ° C.
Then, the plastein reaction is performed for a predetermined time of 8 to 24 hours. The order of addition is not limited to the order described above, and for example, a predetermined amount of water may be added with a previously prepared amino acid ester, an organic acid, and a reducing agent if necessary, and a predetermined pH may be added.
And the raw material protein was added to a predetermined amount of water to prepare a stirred raw material protein solution,
The above-mentioned raw material protein solution was added to the above aqueous solution, and the mixture was sufficiently stirred and, if necessary, the pH was readjusted to prepare a raw material protein solution, and a liquid obtained by adding a proteolytic enzyme to water was added to this solution. You may.

The raw material protein used in the method for producing the proteinaceous composition of the present invention may be any one as long as it can be used as a food. For example, whey, milk protein such as casein, egg white, gelatin, wheat protein, soybean protein, fish meat protein, and the like, and degradation products thereof can be used.

Further, considering that the plastin reaction (introduction of amino acid) as a raw material protein is carried out on a slightly alkaline side and in a high-concentration raw material protein solution, denaturation and hard gelation occur in the reaction solution. Those that do not are preferable, and specifically, whey, casein, and decomposition products thereof are preferable. Considering the amino acid composition of the protein-like composition to be formed, it is preferable to select a protein raw material having a high ratio of amino acids to be introduced as the raw material protein.

Further, when the proteinaceous composition produced is used as a main component of a nutritional supplement for liver diseases, gelatin and its decomposition products are preferable. When developing liver disease,
It is said that branched-chain amino acids in blood decrease and aromatic amino acids (eg, tyrosine, tryptophan, phenylalanine) increase. Gelatin and its decomposed products have relatively less aromatic amino acids than other source proteins, and thus are particularly preferable as the source protein.

The concentration of the starting protein (substrate concentration) in the starting protein solution is preferably higher than that when the protein is hydrolyzed. Since the optimum concentration for the plastein reaction may vary depending on the type of raw material protein, it varies depending on the raw material and is not a constant rate, but it is generally preferably about 20 to 60% by weight. The enzyme added to the reaction solution is
The optimum one varies depending on the type of raw material protein and the type of amino acid to be introduced, but it is preferable to use endopeptidase, and specifically, papain, trypsin, bromelain, chymotrypsin, thermolysin and the like are preferable. Further, when the above-mentioned thiol protease such as papain or bromelain is used as an enzyme, it is preferable to add cysteine hydrochloride, ascorbic acid or the like as a reducing agent.

The pH of the raw material protein solution varies depending on the type of the raw material protein and the type of amino acid to be introduced, but it is preferable that the pH is slightly more alkaline than the optimum value at the time of hydrolysis of the added enzyme. .

As the amino acids to be introduced, branched chain amino acids such as valine, leucine and isoleucine, as well as methionine, lysine and arginine can be considered. Then, these amino acids to be introduced are added to the raw material protein solution as a transparent conductor such as alcohol ester (eg, methyl ester, ethyl ester). From the viewpoint of biosafety, it is preferable to add the amino acid ester in the form of ethyl ester.

In the method for producing a proteinaceous composition of the present invention, since an organic acid is added to a raw material protein solution, a larger amount of amino acid than the conventional one, specifically, 0.3 to 1 of the raw material protein is used. 0.2 times the amount of amino acid can be efficiently introduced into the raw material protein. The present inventors have found that the effect of introducing an amino acid into a raw material protein is improved by adding an organic acid to the raw material protein solution.
The amount to be added is preferably 1 to 50 W / V%

As the organic acid, citric acid, tartaric acid, malic acid, etc. can be used. Since the organic acid as described above is added to the raw material protein solution, it is possible to introduce the amino acid into the raw material protein at an introduction rate of 50% or more (the ratio of the amino acid introduced in a peptide form to the added amino acid amount). it can.

Since the reaction solution of the proteinaceous composition into which the amino acid has been introduced as described above contains the inorganic salt formed by neutralization, those impurities are removed. This removal operation can be performed by dialysis treatment such as electrodialysis, and the proteinaceous composition can be obtained as an insoluble component. Then, the obtained proteinaceous composition may be dried to give a powdery or granular form, or may be added to water without being dried to give a suspension.

Then, the proteinaceous composition obtained by the present invention is used as a solution, to which a seasoning, spice, etc. are added, if necessary, as a main component of a supplemental drink of a nitrogen source, particularly a branched chain amino acid. You can It can also be powdered and added to ordinary meals. Furthermore, an appropriate amount of sugars, lipids, vitamins and minerals can be added to produce a comprehensive tube
It can be used as an oral nutritional supplement.

[0022]

EXAMPLES Examples of the present invention will be specifically described below. (Example 1) [Preparation of branched chain amino acid ethyl ester] L-valine 6
0 g, L-leucine 60 g, and L-isoleucine 60 g were each dissolved in concentrated sulfuric acid 40 ml, ethyl alcohol 190 ml was added, and each was independently heated and refluxed at 90 ° C. for 4 hours to carry out an esterification reaction. After completion of the reaction, sodium carbonate was added and dissolved until the pH of the reaction solution reached 6.5 to 7.5, and neutralized. Then, the formed sodium sulfate precipitate was removed by centrifugation to obtain respective amino acid ethyl ester solutions. The esterification rate calculated by comparison with the deesterified product by alkali treatment was L-valine ethyl ester 94%,
It was L-leucine ethyl ester 93% and L-isoleucine ethyl ester 91%. In the examples shown below, these amino acid esters were used so that the solution was weighed so that the indicated amount of amino acid was contained, and ethyl alcohol was removed by an evaporator as much as possible.

[Synthesis of Protein-Like Composition] Leucine ethyl ester, isoleucine ethyl ester and valine ethyl ester synthesized as described above are used as amino acid amounts of 1610 mg, 735 mg and 1019, respectively.
Transfer mg to a 100 ml Erlenmeyer flask, and
Add cysteine hydrochloride 0.15g, citric acid 3g,
After dissolving everything in water, add sodium hydroxide to pH
Was adjusted to 8. Next, 10 g of whey powder (trade name SEIBO PRO 75, manufactured by Golden California Co., Ltd.) was added and mixed to obtain 50 ml of the whole (citric acid addition concentration 6 W
/ V%). 1m as a sample with a reaction time of 0 hours
1 was taken. Then, 0.2 g of papain (Amano Pharmaceutical Co., Ltd.) dissolved in a small amount of water was added to the above beaker, mixed and stirred, and the plastin reaction was carried out at 40 ° C. for 20 hours. 1m as a sample for 20 hours of reaction
1 aliquot was taken, alkali deesterification was performed together with the sample at 0 hour reaction, diluted with citrate buffer (pH 2.2), and introduced from the amount of decrease measured using an amino acid analyzer (Hitachi 835 type). When the rate was calculated, it was valine 61
%, Isoleucine 63%, and leucine 78%.

Next, after completion of the reaction, about 50 ml of the solution was added.
After adding 150 ml of 1N sodium hydroxide and performing a deesterification reaction at 40 ° C. for 1 hour while stirring, 2N hydrochloric acid was added until pH became 6. About 300 ml thus obtained
Solution of ion exchange membrane dialysis device (TS-2-10 type,
Tokuyama Soda Co., Ltd.), desalting treatment was performed, and the process was terminated when the conductivity reached 2 ms / cm (initially 59 ms / cm). The solution after desalting was freeze-dried to obtain about 11 g of a powder of the proteinaceous composition. This powder was almost tasteless and odorless, and the protein content determined from the total nitrogen content was 78%, and the free amino acid content determined by an amino acid analyzer was 8%. When 5 mg of the powder of the present invention was completely hydrolyzed with 6N hydrochloric acid and the amino acid composition was examined by an amino acid analyzer, the results shown in Table 1 were obtained. In addition, the amino acid composition of whey, which is a raw material protein, is shown in Table 2.

[0025]

[Table 1]

[0026]

[Table 2]

As shown in Table 1, in the protein-like composition of Example 1, the amount of branched chain amino acids in the amino acid composition (total amount of valine, leucine and isoleucine) was 43% by weight, and the total free amino acids were , 8%.

Example 2 Citric acid 3 in Example 1
The same procedure as in Example 1 was performed except that 3 g of tartaric acid (addition concentration: 6 W / V%) was added instead of g. When the introduction rate of the obtained proteinaceous composition was determined, valine 55
%, Isoleucine 58%, and leucine 75%. The powder was almost tasteless and odorless, the protein content determined from the total nitrogen content was 76%, and the free amino acid content determined by an amino acid analyzer was 9%.

(Example 3) Citric acid 3 in Example 1
The same procedure as in Example 1 was performed except that 3 g of malic acid (addition concentration: 6 W / V%) was added instead of g. When the introduction rate of the obtained proteinaceous composition was determined, valine 56
%, Isoleucine 59%, and leucine 74%. The powder was almost tasteless and odorless, the protein content determined from the total nitrogen content was 76%, and the free amino acid content determined by an amino acid analyzer was 9%.

Comparative Example 1 Citric acid 3 in Example 1
Example 1 was repeated except that g was not added. When the introduction rate of the obtained proteinaceous composition was determined, valine 27%, isoleucine 30%, leucine 69
%Met. This powder was almost tasteless and odorless, and the protein content determined from the total nitrogen content was 76%, and the free amino acid content determined by an amino acid analyzer was 15%. Table 3 shows the amino acid introduction rates in Examples 1 to 3 and Comparative Example 1.

[0031]

[Table 3]

As shown in Table 3, in each of the examples, the introduction rate of all three amino acids was higher than that in the comparative example in which no organic acid was added. In particular, in the example, the introduction rate was poor in the comparative example. Valine and isoleucine also had an introduction rate of 50% or more.

[0033]

The method for producing a proteinaceous composition of the present invention comprises: a raw material protein; at least one amino acid ester;
Since an organic acid is added to the solution and the amino acid in the amino acid ester is introduced into the protein by the plastein reaction, a sufficient amount of the amino acid can be introduced into the starting protein, and It is possible to reliably and efficiently introduce an amino acid, which is said to have a low rate, into a raw material protein, and thus it is possible to obtain a protein-like composition in which the amino acid has been introduced into the raw material protein at a higher ratio than in the past. Therefore, the method for producing a proteinaceous composition according to the present invention is suitable when it is desired to add a large amount of at least one amino acid to a reaction solution and introduce it. By introducing it in the form of a peptide, it can be expected to improve the unpleasant taste and smell of the amino acid itself, or the poor solubility / dispersibility.

Claims (4)

[Claims] 1. An organic acid is added to a solution containing a raw material protein, at least one amino acid ester, and a protease, and the amino acid in the amino acid ester is added to the protein. A method for producing a proteinaceous composition, which comprises introducing the protein by a Thein reaction. 2. The method for producing a proteinaceous composition according to claim 1, wherein the organic acid is citric acid, tartaric acid, or malic acid. 3. The method for producing a proteinaceous composition according to claim 1, wherein the amount of the organic acid added is 1 to 50 W / V% with respect to the solution. 4. The method for producing a proteinaceous composition according to claim 1, wherein the amino acid ester is an amino acid ethyl ester.