JPH08252075A - Enzymatic decomposition type seasoning - Google Patents

Abstract

PURPOSE: To obtain a seasoning improved in taste by treating a product with pyroglutamyl aminopeptidase and/or 5-oxoprolinase, wherein the product is obtained by treating a protein with the culture product of Aspergillus. CONSTITUTION: A protein (wheat, deoiled soybean, etc.) is treated with the culture product of Aspergillus, and the obtained product is treated with pyroglutamyl aminopeptidase and/or 5-oxoprolinase. Pyroglutamic acid and pyroglutamyl peptide are also converted into glutamic acid and other amino acids to improve the taste.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a seasoning obtained by the action of an enzyme. The seasoning of the present invention is
(1) Not only contains amino acids and low molecular weight peptides, but (2) is a seasoning with extremely high taste, which is obtained by converting all non-tasteful pyroglutamic acid and pyroglutamyl peptides into glutamic acid and other amino acids. is there. The seasoning of the present invention is expected to have a wide range of uses as a seasoning that can be used in various foods or as a raw material for various seasonings.

[0002]

2. Description of the Related Art The most popular natural seasonings that have been known so far are so-called degradable natural seasonings, which are produced by decomposing proteins. This degradable natural seasoning is classified into an acid degrading type and an enzymatic degrading type. The acid-decomposable seasoning is produced by using vegetable protein such as soybean or wheat as a raw material.
Hyd obtained from yzed Vegetable Protein (hereinafter sometimes abbreviated as HVP) and animal proteins such as gelatin and milk casein
There is a rolled Animal Protein (hereinafter sometimes abbreviated as HAP).

However, H is generally obtained by acid hydrolysis.
The reaction conditions for obtaining VP and HAP are usually 100 ° C. and 1 to 2 days. As described above, the reaction at a high temperature for a long time consumes a large amount of energy and causes a problem of corrosion of the apparatus. Furthermore, while hydrolysis of protein with an acid is simple, it also has drawbacks such as generation of off-flavor, excessive decomposition of amino acids, and high salt content due to neutralization.

Therefore, in recent years, a production method utilizing a proteolytic enzyme has been studied in order to effectively utilize the flavor of the raw material. That is, as the enzyme-decomposable seasoning, egg white that has been enzymatically decomposed (JP-A-48-68773) and defatted soybean that has been enzymatically decomposed (JP-A-51-7085).
2), those obtained by enzymatically decomposing cheese whey as a raw material (JP-A-62-151155), those obtained by enzymatically decomposing corn gluten meal (Japanese Patent Publication No. 2-295437), and the like.

By the way, even if the same substrate protein is used, the cleavage site may differ depending on the type of proteolytic enzyme, so that the taste of the degradation product may differ. That is, the taste varies depending on the degree of free amino acid production, the molecular weight of the produced peptide, the amino acid composition, the sequence order, and the like. This causes the following problems. For example, in the case of soybean protein, bitterness is generally easily generated when an enzyme having a strong endopeptidase activity is acted (Kengo Ishida et al., Food Journal, 28, 524 (197).
6)). The production of bitterness is the most difficult point in the application of endopeptidase to food processing, and an enzymatic decomposition method that does not produce bitterness is desired.

In response to this problem, in studies with milk casein and soybean protein, various amino acids were released by further treating the bitter peptides produced by the endopeptidase treatment with exopeptidases derived from microorganisms, particularly amino-terminal It has been reported that leucine, valine, phenylalanine, leucine at the carboxyl terminus, phenylalanine, valine, etc. are liberated to significantly reduce bitterness and increase umami (food industry and enzyme, Eiji Ichishima, p102, Asakura Shoten).

[0007] In soy sauce, glutamine released from the raw material protein by endopeptidase and exopeptidase is not converted into glutamic acid by glutaminase produced by Aspergillus oryzae, so that pyroglutamic acid having no umami is produced and glutamic acid, which is the main component of umami, is produced. It has also been reported that it will decrease (Soy sauce chemistry and technology, edited by Tochikura Tatsurokuro, p181, Japan Brewing Association). Furthermore, in soy sauce, residual peptides such as glycylproline, glycylaspartic acid, glycylglutamic acid, glutamylproline, glutamylglycine, and glutamylglutamic acid were confirmed as undegraded peptides (science and technology of soy sauce, Tochikura Tatsurokuro edition, p176, Japan Brewing Association), it is also believed that their residual causes the deterioration of the taste of soy sauce.

For the above-mentioned problems, a search for soy sauce koji having a high glutaminase activity (Shoken, Vol. 8, No. 3,
1982), a method for producing thermostable glutaminase (JP-B-49-48759), and a method for producing salt-tolerant glutaminase (JP-A-63-94975, JP-A-2-26137).
9), application to miso brewing (J. Brew. Soc. Ja
pan. Vol. 86, No. 7, p529 (199
Attempts such as 1)) have been made. Furthermore, efforts have been reported to improve the glutamic acid release rate (glutamic acid concentration (weight / volume) / total nitrogen (weight / volume)) in a protein hydrolyzate by degrading the above peptide with peptidase (Japanese Patent Application No. 5- 300804).

However, as seen in the case of soy sauce brewing, although the enzymatic hydrolysis of protein requires a great deal of labor and time, the amino acid liberation rate is low, and soybean protein contains the highest amount. In addition, the release rate of glutamic acid, which is important for taste, is low. Therefore, at the present time, a technique for releasing a large amount of glutamic acid, which has an important influence on taste, has not been established.

[0010]

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a seasoning having a higher amino acid liberation rate, especially a glutamic acid liberation rate than the conventional enzyme-decomposable seasonings.

[0011]

Means for Solving the Problems The present inventors have improved the glutamic acid release rate by separating, identifying, and quantifying substances other than the above-mentioned peptides that are related to glutamic acid from protein hydrolysates and enzymatically degrading them. As a result of repeated intensive studies, the present invention has been completed. That is, in the present invention, a protein hydrolyzate obtained by allowing a protein to act on a protein degrading enzyme contained in a koji mold culture is further treated with (1) pyroglutamylaminopeptidase and / or (2) 5-oxoprolinase. It is a seasoning that can be obtained. The present invention will be described in detail below.

First, the separation, identification, and quantification of glutamic acid-related substances will be briefly described. The protein hydrolyzate of the protein was subjected to ultrafiltration membrane with a molecular weight cut of 10,000 to remove the polymer, and the external solution was subjected to strong basic anion exchange column chromatography (AG1-X8 column, manufactured by Bio-Rad), reverse Phase column chromatography (CAPCEL
LPAK C18, Shiseido Co., Ltd., mass spectrometer (JM
S-HX110 / HX110 TANDEM MASS
SPECTROMETER) and analyzed for glutamic acid-related substances. As a result, we discovered the presence of a pyroglutamyl peptide, the presence of which was unknown in protein hydrolysates. That is, the present pyroglutamyl peptide contains pyroglutamic acid at the amino group terminal and is contained in the oligo- and polypeptide fractions having a molecular weight of about 250 to 3,000. For example, pGlu-G
It was a group of polypeptides in which about 20 amino acids were bound to pGlu from dipeptides such as lu and pGlu-Asp.

A feature of the present invention is to allow these pyroglutamyl peptides to act with pyroglutamyl aminopeptidase and / or 5-oxoprolinase. As a result, the release rate of glutamic acid and other amino acids can be improved, and as a result, a protein hydrolyzate with improved taste can be obtained.

In order to obtain the seasoning of the present invention, first, a protein hydrolyzate is obtained by reacting a protein containing a protein hydrolase obtained by culturing koji mold or a fraction thereof with a protein as a raw material. start from. The type of protein applicable to the present invention is not particularly limited, but those having a high glutamic acid content are preferable. Further, the protein raw material is not limited to pure protein, and any protein containing a large amount of protein may be used. Specifically, defatted soybeans, soybean protein isolate,
Wheat and the like can be mentioned, and these can be used alone or in appropriate combination of two kinds. Of course, protein raw materials other than these may be used. Further, the pretreatment of the protein raw material before the enzyme treatment is not particularly limited, but it is preferable to degrease using a surfactant or an organic solvent.

Next, a protein hydrolyzate is obtained by reacting a culture containing a protein hydrolase obtained by culturing Aspergillus oryzae with the above protein raw material or a fraction thereof to obtain a protein hydrolyzate. The koji mold may be of any type, and those commonly used in the brewing industry may be used. Specifically, Aspergillius oryzae, Aspergillius soya, Aspergillius tamari and the like may be used. In the present invention, in order to terminate the decomposition in the shortest possible time, as a culture of Aspergillus oryzae, a modified method (Hagiwara. B. et al., J.
Biochem. 45.165 (1958)), a culture having an activity of 200 units / ml for liquid koji and 1,000 units / g · koji for solid koji is preferable. As will be described repeatedly, a fraction obtained by fractionating a culture containing a protein hydrolase obtained by culturing Aspergillus oryzae by a technique such as centrifugation, ultrafiltration or ion exchange chromatography may be used.

By reacting the enzyme group contained in the koji mold culture and the fraction thereof with such a protein, the protein is decomposed into a peptide and further an amino acid by an enzymatic reaction. In order to efficiently carry out this enzymatic reaction, it is preferable that the enzyme group or its fraction and the protein raw material coexist in an aqueous solution. At this time, the reaction may be carried out while stirring. This enzyme reaction varies depending on the enzyme concentration and the protein raw material, but is, for example, 10 to 60 ° C., preferably 30 to 50.
It may be carried out at 3 ° C. for 3 to 11 days, preferably 5 to 10 days. The aqueous solution containing the protein hydrolase and the protein raw material has a pH of 5 to 9, preferably a pH of 6 to 6 using a buffer solution or the like.
It is desirable to adjust to 8.

After the completion of the reaction, insoluble matters such as unreacted raw material proteins may be removed by a conventional separation method such as centrifugation or filtration. The produced proteolysis solution may be subjected to amino acid analysis, ninhydrin quantification, and weight decomposition rate measurement by weight comparison before and after enzyme treatment.

Next, pyroglutamylaminopeptidase and / or 5-oxoprolinase is allowed to act on the protein hydrolyzate obtained as described above. As the pyroglutamyl aminopeptidase and 5-oxoprolinase, purified products and crude purified products may be used, but normally, the culture solution of the microorganism may be used as it is. That is, a culture of Bacillus subtilis containing Bacillus subtilis containing pyroglutamyl aminopeptidase and / or a culture of baker's yeast containing Saccharomyces cerevisiae containing 5-oxoprolinase are allowed to coexist with the above protein hydrolyzate in an aqueous solution. Good. Also,
At this time, the reaction may be carried out while stirring.

In the present invention, the natto bacterium containing Bacillus subtilis containing pyroglutamylaminopeptidase may be any natto bacterium generally used in the brewing industry, regardless of its type. Also, baker's yeast containing 5-oxoprolinase (Saccharomyces cerevisiae)
The baker's yeast usually used in the brewing industry may be used regardless of the type.

In the present invention, in order to complete the decomposition in the shortest possible time, as a Bacillus subtilis culture, its pyroglutamylaminopeptidase activity is used as a substrate for pGlu-Ala. Dolittle et al.'S method (Meth. Enzymol. 19, 555 (1
5 mU when measured according to the description in 970))
Cultures that retain the above activities are preferred. Similarly, as a baker's yeast (Saccharomyces cerevisiae) culture, 5
-Oxoprolinase activity using ¹⁴ C oxoproline as a substrate (Proc. Nat
l. Acad. Sci. USA 68,2982 (19
71 m) when measured according to the description in 71))
Cultures that retain the above activities are preferred.

A culture of Bacillus subtilis and / or 5 containing Pyroglutamylaminopeptidase
The reaction conditions for the action of a baker's yeast (Saccharomyces cerevisiae) culture containing -oxoprolinase are not particularly fixed, but usually 10 to 60 ° C, preferably 3
It may be carried out at 0 to 50 ° C. for 3 to 48 hours, preferably 24 to 30 hours. The aqueous solution containing these enzymes and protein hydrolyzate is preferably adjusted to pH 5-9, preferably pH 6-8, using a buffer solution or the like. In this way, the resulting reaction solution is used as it is, or if necessary, dried to obtain a seasoning having a high glutamic acid release rate.

[0022]

EXAMPLES The present invention will be described below with reference to examples.
The present invention is not limited to the embodiments.

(Example 1): Preparation of protein hydrolyzate and separation of pyroglutamyl peptide First, the preparation of the protein hydrolyzate used first will be described. That is, 5% (weight / volume) of adipron SU
(Desaccharified and separated soybean protein, manufactured by Ajinomoto Co., Inc.) To 2 L of solution,
Adipron E3 (isolated soybean protein, manufactured by Ajinomoto Co., Inc.) 1.5% (weight / volume) and 0.5% (weight / volume) 1 potassium hydrogen phosphate in a medium containing 30 ° C. and 120 r
Aspergillus (Aspergillu) cultivated by reciprocal shaking for 48 hours.
oryzae ATCC 22788) 1 L of culture was added and 4
The enzymatic decomposition reaction was carried out at 0 ° C. for 10 days in the absence of sodium chloride. Then, the reaction solution was centrifuged at 7,000 rpm for 10 minutes to obtain 2.8 L of protein hydrolyzate.

The polymer hydrolyzate (500 ml) prepared as described above was subjected to ultrafiltration membrane having a molecular weight of 10,000 to remove the polymer, and 400 ml of an external solution was obtained. 100 ml of the obtained external liquid was subjected to strong basic anion exchange column chromatography (AG1-X8 column, 260 mmφ × 700 m).
m, manufactured by Bio-Rad). Elution after washing with water
A28 at a flow rate of 80 ml / hr with 0.05M acetic acid, 0.2M acetic acid, 2.0M acetic acid, 1.0N hydrochloric acid.
0 was measured, and 50 ml of a peak eluted with 2.0 M acetic acid and 1.0 N hydrochloric acid was collected. Then, the obtained fraction 200
Reversed phase column chromatography (CAPCELL)
PAK C18, 4.6φ × 250 mm, manufactured by Shiseido Co., Ltd.), A214 was measured, and 2 ml of its peak was collected. Further, after drying the obtained sample, a mass spectrometer (J
MS-HX110 / HX110 TANDEM MAS
Structural analysis was carried out by S SPECTROMETER.

As a result, in the protein hydrolyzate so far,
We discovered the presence of a pyroglutamyl peptide whose existence was unknown. That is, the present pyroglutamyl peptide has pyroglutamic acid at the amino group terminal,
It was contained in the oligo- and polypeptide fractions having a molecular weight of about 250 to 3,000. For example, pGlu-Glu, pGl
It was a group of polypeptides in which about 20 amino acids were bound to pGlu from a dipeptide such as u-Asp. Next, in Example 2, a method in which pyroglutamyl aminopeptidase and 5-oxoprolinase are allowed to act on the pyroglutamyl peptide to separate a large amount of glutamic acid will be described.

Example 2 Production of Seasoning Solution by Action of Pyroglutamyl Aminopeptidase and 5-Oxoprolinase 5% (weight / volume) Adipron SU (Desaccharified and separated soybean protein, manufactured by Ajinomoto Co., Inc.) solution 2L, 1.5% (weight / volume) adipron E3 (isolated soy protein, Ajinomoto Co., Inc.)
Manufactured), and 0.5% (weight / volume) medium containing 1 potassium hydrogen phosphate at 30 ° C., 120 rpm, 48 hours,
Aspergillus oryzae ATCC reciprocally shake-cultured
22788) 1 L of the culture was added, and the enzymatic degradation reaction was carried out at 40 ° C. for 10 days in the absence of salt. Then, the reaction solution was centrifuged at 7,000 rpm for 10 minutes, and then 2.
8 L of protein hydrolyzate was obtained.

To 1.0 L of the obtained protein hydrolyzate, 100 ml of a culture of Bacillus subtilis ATCC 6051 containing Bacillus subtilis ATCC 6051 having 5 mU of pyroglutamylaminopeptidase activity and 10 m of 5-oxoprolinase activity were prepared.
Baker's yeast possessed by U (Saccharomyces cerevisiae A
TCC 15248) 100 ml culture was added and
The enzyme reaction was performed at 24 ° C for 24 hours. Then, the decomposed product was centrifuged to obtain a decomposed filtrate. Table 1 shows the results of quantifying the total nitrogen, the formol nitrogen and the amount of glutamic acid in the decomposition filtrate.

[0028]

[Table 1]

As shown in the table, compared to the enzyme-free group, the decomposition rate shown as formol nitrogen / total nitrogen and the amount of glutamic acid exhibiting umami significantly increased, so that the taste was significantly improved. You can see that Therefore, the obtained decomposition filtrate can be used as it is as an excellent amino acid solution seasoning. In addition, the value of 1.22 for glutamic acid / total nitrogen is significantly higher than that of conventional soy sauce (the value is about 0.6).

[0030]

INDUSTRIAL APPLICABILITY According to the present invention, Bacillus subtilis containing Bacillus subtilis containing pyroglutamyl aminopeptidase in a protein hydrolyzate obtained by allowing a koji mold culture to act on a protein.
By reacting the culture and / or baker's yeast (Saccharomyces cerevisiae) culture containing 5-oxoprolinase, the remaining pyroglutamyl peptide is decomposed, the decomposition rate is low, and the proteolysis was weak in taste. It is possible to obtain a seasoning having a high decomposition rate and a high glutamic acid content and excellent taste.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Jiro Kataoka 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Food Research Institute

Claims (2)

[Claims] 1. A protein hydrolyzate obtained by reacting a protein as a substrate with a proteinase contained in a koji mold culture, and further comprising (1) pyroglutamylaminopeptidase and / or (2) 5-oxoprolinase. Seasoning obtained by acting on. 2. The seasoning according to claim 1, wherein the protein hydrolyzate contains a pyroglutamyl peptide.