JP3299726B2 - Method for producing γ-aminobutyric acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to the production of gamma-aminobutyric acid.
It relates to a generation method .

[0002]

2. Description of the Related Art Hypertension, also called adult disease or lifestyle-related disease, is the leading cause of outpatients ("Vital Statistics" in fiscal 1995) and the basis of heart disease. It is a major problem in health and medical care as a disease. It is thought that the number of hypertensive patients tends to increase with the progress of the aging society. Is also very important. Under these circumstances, there has been an increasing trend in recent years to pay attention to the biological regulation function of foods and actively use it for treatment and prevention of diseases, and hypertension is one of the targets.

[0003] By the way, γ-aminobutyric acid is a non-protein amino acid that acts as a neurotransmitter, and is biosynthesized from glutamic acid in vivo by glutamate decarboxylase. The blood pressure lowering effect (Ta
kahasi, et al., Jap. J. Physiol. (1955)) and that it is effective in controlling blood pressure in hypertension (Chal
mers, et al., J. Hypertens. (1992)) are already widely known from the results of animal experiments. This blood pressure lowering effect of γ-aminobutyric acid has a certain limitation, and it does not fall below normal blood pressure even if it is administered in a required amount or more, so it is considered to be a very excellent functional component also in terms of safety. Can be Therefore,
γ-aminobutyric acid is an amino acid having a physiological function that should be called an antihypertensive effect rather than an antihypertensive effect,
Products that take advantage of this property are already on the market as "Gabalon tea" and the like.

[0004] In order to apply γ-aminobutyric acid to foods, there have been many examples of attempts to find natural γ-aminobutyric acid besides the above-mentioned “Gabalon tea”. For example, accumulation and production of γ-aminobutyric acid by immersing rice germ in water (Japanese Unexamined Patent Publication No.
No. 21252), extraction of γ-aminobutyric acid from citrus fruits (JP-A-10-215812), production of γ-aminobutyric acid by plant-derived glutamate decarboxylase (Japanese Patent Publication No.
-12296), production of γ-aminobutyric acid by lactic acid bacteria (JP-A-7-227245) or γ-aminobutyric acid by
Production of aminobutyric acid (JP-A-10-165191). However, from the viewpoint of obtaining a functional food material that can be used for a wide range of foods, the above-mentioned methods include: (1) the production process is complicated or takes a long time; (2) the obtained γ-aminobutyric acid is relatively Due to its low concentration, it is necessary to consume a large amount to exert its functionality, which puts a heavy burden on users. (3) It is difficult to apply widely because of its high relevance to specific foods. All of these are not satisfactory.

Further, of the above methods, Japanese Patent Application Laid-Open No. 7-2132
In the technique disclosed in No. 52, since γ-aminobutyric acid is produced from glutamate in rice germ by the action of glutamate decarboxylase, the amount of γ-aminobutyric acid produced is regulated by the amount of glutamic acid in the germ. Would. Glutamic acid is produced from the protein in the embryo by the action of proteolytic enzymes, and γ-
Although aminobutyric acid is produced, the production amount of γ-aminobutyric acid is still about 400 mg per 100 g of embryos.

The present invention has been made in view of the above-mentioned problems. In order to apply the effect of suppressing the increase in blood pressure of γ-aminobutyric acid to a wide range of foods, a large amount of γ-aminobutyric acid is safe, inexpensive, and simple. It is intended to provide a technology with excellent practicality that can be manufactured.

[0007]

The gist of the present invention will be described.

A solution containing glutamic acid or a salt of glutamic acid and 2-morpholinoethanesulfonic acid are added to at least one of rice germ, rice bran containing germ, and germ rice to cause a reaction, and the glutamic acid in the added solution is reacted with glutamic acid in the added solution. Γ-aminobutyric acid is obtained by converting the rice germ, rice bran containing germ or glutamic acid in germ rice into γ-aminobutyric acid by the enzymatic action of glutamate decarboxylase in the rice germ, rice bran containing germ or germ rice. -It relates to a method for producing aminobutyric acid.

[0009] The method of producing γ-aminobutyric acid according to claim 1 is characterized in that the glutamic acid concentration is set to 50 mmol / L or more.

A method for producing γ-aminobutyric acid according to any one of claims 1 and 2, wherein pyridoxalic acid is added to the reaction system. is there.

[0011]

The present invention summarizes the effects obtained by repeated experiments as claims.

Rice germ, rice bran or germ rice including germ, which is discharged in large quantities during the rice polishing process and is available at a low cost, is used as a source of glutamate decarboxylase. When glutamic acid in rice germ, rice bran containing germ or germ rice is reacted, a large amount of γ-aminobutyric acid is obtained. That is, since glutamic acid as a source of γ-aminobutyric acid is supplied not only from glutamic acid in the rice germ, rice bran containing germ or germ rice but also from glutamic acid in a solution to be added, sufficient γ-aminobutyric acid is obtained. Butyric acid can be produced.

The reaction system is the same as that described in JP-A-7-21325.
It is better to adjust the pH as disclosed in No. 2, but when 2-morpholinoethanesulfonic acid (hereinafter referred to as MES) is used as the acid for adjusting the pH, γ is used. It was found by experiments that the production of aminobutyric acid increased (FIG. 3).
reference). Therefore, it was confirmed that MES was optimal as the organic acid for pH adjustment.

Further, when the concentration of glutamic acid in the solution to be added is sequentially increased, γ is γ at 50 mmol / L or less.
It has been found by experiments that the rate of formation of -aminobutyric acid is sharp, and that the rate of formation of γ-aminobutyric acid is gentle above 50 mmol / L (see FIG. 1). Therefore, it was confirmed that the critical value of the glutamic acid concentration for efficiently increasing the production rate of γ-aminobutyric acid was 50 mmol / liter.

Further, it was found by experiments that addition of pyridoxal phosphoric acid to the reaction system increased the amount of γ-aminobutyric acid produced (see FIG. 2).

Since the present invention is as described above, a large amount of γ
-It is a highly practical technology that can produce aminobutyric acid at high concentration, safely, at low cost, and easily.

Furthermore, food materials containing γ-aminobutyric acid and foods such as nutritional foods can be easily and efficiently manufactured, and the manufactured food materials and nutritional foods can be used not only in the diet therapy of hypertensive patients but also in hypertensive patients. This is a highly practical technology that can be used as a nutritional food for prevention.

In addition, rice germ and rice bran containing germ are produced in large quantities as a by-product of the rice milling process, and it is currently difficult to dispose of the rice germ and rice bran. Therefore, the present invention can also contribute to waste treatment as a method that can effectively utilize unused resources such as rice germ and rice bran.

[0019]

Embodiments of the present invention will be described below.

In this embodiment, natural products containing glutamic acid decarboxylase were added to glutamic acid or a salt of glutamic acid.
A solution containing (such as sodium glutamate) is added and reacted, and glutamic acid in the added solution and glutamic acid in the natural product are converted into γ-aminobutyric acid by the enzymatic action of glutamate decarboxylase in the natural product. Things.

Rice germ is used as a natural product containing glutamate decarboxylase. In addition, rice varieties of rice germ are not limited. It is also preferable to use rice germ alone,
Rice bran and germ rice including germ can be used similarly to rice germ. These rice germ, rice bran, and germ rice can be easily prepared by using an ordinary rice mill and a suitable sieve. Further, glutamate decarboxylase may be extracted or purified from rice germ, rice bran, or germ rice, and then suspended, dissolved or immobilized.

The amount of glutamic acid or a salt of glutamic acid in the reaction solution may be determined based on the desired production amount of γ-aminobutyric acid and the amount of rice germ, rice bran including germ or germ rice used as a catalyst source. , Preferably 50 mmol /
It is set to liter or more (see Experimental Example 1 and FIG. 1 described later). Glutamic acid or a salt of glutamic acid may be purified or extracted, but can also be produced from various proteins (such as soybean proteins) by enzymatic degradation. Therefore, a system using both a protein and a protease can be used. .

The reaction conditions are such that the pH is 3.0 to 7.0, preferably
A good value is 5.5 to 6.5. The temperature is preferably 10 to 50 ° C, preferably 35 to 40 ° C.

As the acid used for adjusting the pH, either an inorganic acid or an organic acid can be used. Examples of the inorganic acid include hydrochloric acid and sulfuric acid. Organic acids include citric acid, malic acid, 2-
Morpholinoethanesulfonic acid (2-morpholinoethanes
ulfonic acid (MES) and the like can be used, but as shown in Experimental Example 3 and FIG.
MES is preferably used in the case of organic acids, since the production of aminobutyric acid varies greatly.

As the pH adjusting alkali, sodium hydroxide, potassium hydroxide, calcium hydroxide or the like is used, but is not limited thereto.

Even if the reaction is carried out under these conditions, a certain amount of γ-aminobutyric acid can be produced (about 3 g / 100 g of embryo).
g of .gamma.-aminobutyric acid), and in order to obtain the maximum production efficiency by increasing the rate of .gamma.-aminobutyric acid, pyridoxalic acid is added as shown in Experimental Example 2 and FIG. Is more desirable.

The amount of pyridoxal phosphate is determined by the amount of γ-
It may be determined based on the amount of aminobutyric acid and the desired substrate conversion rate (the ratio of the amount of glutamic acid present in the reaction solution at the start of the reaction converted to γ-aminobutyric acid), but is preferably glutamic acid or The molar ratio of glutamic acid salt to pyridoxalic acid is from 100 (glutamic acid or glutamic acid salt): 1 (pyridoxalic acid)
2,000 (glutamic acid or glutamic acid salt): 1
(Pyridoxal phosphate).

Pyridoxal phosphate can be used as a purified or extracted preparation, and since pyridoxal phosphate is contained in a wide range of biological materials as one of the active ingredients of vitamin B6, it can be used as a pyridoxal phosphate source. it can. Further, since pyridoxal phosphate can be synthesized by phosphorylating pyridoxal, a synthesis system from pyridoxal may be used in combination.

Since this embodiment is as described above, a large amount of γ-aminobutyric acid can be easily and inexpensively produced. The materials used are rice germ, rice bran or germ rice including germ, amino acid glutamic acid and its salts,
Pyridoxal phosphate, an active ingredient of vitamin B6, p
H is an acid or alkali for the preparation of H. Since none of the components has any harmful effects when used in foods, simple methods such as removal of suspended matter from the reaction solution, decolorization, and desalting as necessary It can be supplied as a food material only by performing an adjustment operation.

When a highly purified preparation is required, γ-aminobutyric acid can be easily purified by using a conventional method such as ion exchange chromatography. Since the obtained sample contains a high concentration of γ-aminobutyric acid, the effect of suppressing blood pressure increase can be expected only by ingesting a small amount of the sample or mixing it with food. Thus, γ produced according to this example
-The aminobutyric acid solution is effective as a food material and as a nutritional food.

Hereinafter, an experimental example supporting the operation and effect of this embodiment will be described.

EXPERIMENTAL EXAMPLE 1 0.2 g of germ prepared from Koshihikari was suspended in 3.0 ml of a mixed solution of sodium glutamate-20 μmol / liter pyridoxalic acid-0.1 mol / liter MES (pH 5.5) and reacted at 40 ° C. Was. Glutamic acid concentration was adjusted to the concentration shown in FIG.
After 30 minutes, the reaction was stopped by adding an aqueous solution of trichloroacetic acid to a concentration of 8%. The concentrations of glutamic acid and γ-aminobutyric acid in the reaction solution were fluorescently labeled with o-phthalaldehyde and then quantified by HPLC analysis to obtain FIG.

From FIG. 1, it was found that the production rate of γ-aminobutyric acid depends on the glutamic acid concentration. In addition, when the concentration of glutamic acid becomes a certain level or more, the rate of production of γ-aminobutyric acid increases, so it was found that the condition for producing γ-aminobutyric acid is desirably a glutamic acid concentration of 50 mmol / L or more. .

EXPERIMENTAL EXAMPLE 2 3.0 g of 100 g / l sodium glutamate-0.2 g of germ prepared from Koshihikari was used.
The mixture was suspended in a 1 mol / liter MES mixed solution (pH 5.5), and pyridoxal phosphoric acid was added to the mixture at 50 μmol / liter as needed, and reacted at 40 ° C. After a predetermined time, the reaction was stopped by adding an aqueous solution of trichloroacetic acid to a concentration of 8%. The concentrations of glutamic acid and γ-aminobutyric acid in the reaction solution were quantified by fluorescently labeling with o-phthalaldehyde and then analyzing by HPLC,
FIG. 2 is obtained.

FIG. 2 shows that the production rate of γ-aminobutyric acid can be increased by pyridoxal phosphate.

EXPERIMENTAL EXAMPLE 3 0.2 g of germ prepared from Koshihikari was mixed with 3.0 ml of 100 mmol / l sodium glutamate-50.
Micromol / liter pyridoxal phosphate and Figure 3
Suspended in a mixed solution (pH 5.5) containing each organic acid shown in
The reaction was performed at 0 ° C. After a predetermined time, the reaction was stopped by adding an aqueous solution of trichloroacetic acid to a concentration of 8%. The concentrations of glutamic acid and γ-aminobutyric acid in the reaction solution were
After fluorescent labeling with phthalaldehyde, it was quantified by HPLC analysis to obtain FIG.

From FIG. 3, it was found that the production rate of γ-aminobutyric acid depends on the type of the organic acid. Also, MES was found to be the most suitable.

As described above, according to the results of Experimental Examples 1 to 3, when γ-aminobutyric acid is efficiently produced, the concentration of glutamic acid is adjusted to 50 mmol / L or more, pyridoxal phosphoric acid is added to the reaction system, and the pH is adjusted. It has been found that each method of selecting MES as an organic acid for use is preferably employed.

Hereinafter, a specific experimental example of the present embodiment based on the results of Experimental Examples 1 to 3 will be described.

Experimental Example 4 11 g of germ prepared from Koshihikari was added to 100 ml of
0.36 mol / liter potassium glutamate-0.3 mol /
The suspension was suspended in a mixed solution of 1 MES (pH 5.5), 1.7 mg of pyridoxal phosphoric acid was added, and the mixture was reacted at 40 ° C. for 6 hours under light shielding. An additional 1.7 mg of pyridoxal phosphoric acid was added every hour from the start of the reaction. The pH was measured every hour, and 0.1 mol / liter hydrochloric acid was added so as to be in the range of 5.5 to 6.0. The concentrations of glutamic acid and γ-aminobutyric acid were quantified by fluorescently labeling with o-phthalaldehyde and then analyzing by HPLC.

FIG. 4 shows the experimental results.

From FIG. 4, it was found that by reacting for 6 hours, more than 82% of the glutamic acid initially added can be converted to γ-aminobutyric acid. The amount of γ-aminobutyric acid produced at this time is 3.1 g, which is 28.3 g of γ per 100 g of germ.
-Production of aminobutyric acid was confirmed.

It has been reported that the amount of γ-aminobutyric acid produced by immersing rice germ in water is about 0.4 to 0.5 g / 100 g germ (Japanese Patent Application Laid-Open No. 7-213252). The production efficiency can be estimated to be 50 times or more of the conventional method.

In addition, the amount of γ-aminobutyric acid required for suppressing blood pressure elevation is estimated to be
It is considered to be about 0.5 to 1 g (Y. Abe, et al., Am. J.
Hypertens., (1995)), which is about 16 to 32 ml when converted to the amount of the reaction solution in Experimental Example 4, indicating that the effect of suppressing blood pressure elevation can be expected by ingesting a small amount.

Experimental Example 5 125 g of embryos prepared from Koshihikari were suspended in 1 liter of a 0.4 mol / l sodium glutamate (pH 5.5) solution, 36.3 mg of pyridoxal phosphoric acid was added, and the mixture was stirred at 250 rpm at 40 ° C. for 6 hours at 250 rpm. Reacted. Every hour after the start of the reaction, 36.3 mg of pyridoxal phosphoric acid was additionally added. The reaction was carried out using a bioreactor TBR-2 system (Chiyoda Seisakusho), and the reaction pH was adjusted to 1 using a pH controller (Chiyoda Seisakusho TBC-pH-β).
It was controlled at 5.5 to 5.6 with N-HCl. The concentrations of glutamic acid and γ-aminobutyric acid were quantified by fluorescently labeling with o-phthalaldehyde and then analyzing by HPLC.

FIG. 5 shows the experimental results.

From FIG. 5, it was found that 87.9% of the glutamic acid initially added can be converted to γ-aminobutyric acid by the reaction for 6 hours. At this time, the production amount of γ-aminobutyric acid is 36.3 g, which is 29.0 g of γ per 100 g of germ.
-Production of aminobutyric acid was confirmed.

[Brief description of the drawings]

FIG. 1 is a graph showing the influence of glutamic acid concentration in Experimental Example 1.

FIG. 2 is a graph showing the effect of pyridoxal phosphate in Experimental Example 2.

FIG. 3 is a graph showing the influence of an organic acid in Experimental Example 3.

FIG. 4 shows the production of γ-aminobutyric acid and a decrease in glutamic acid in Experimental Example 4, and the production of γ-aminobutyric acid from added glutamic acid.
-It is the graph which showed the conversion rate to aminobutyric acid.

FIG. 5: Production of γ-aminobutyric acid and reduction of glutamic acid in Experimental Example 5, and γ-aminobutyric acid from added glutamic acid
-It is the graph which showed the conversion rate to aminobutyric acid.

Continuation of the front page (72) Inventor Kazunori Egawa 17-35 Akaya, Kamo-shi, Niigata (56) References JP-A-7-213252 (JP, A) JP-A-9-238650 (JP, A) JP-A-11 -155508 (JP, A) Journal of Neurochemistry, Vol. 39, No. 3 (1982), p. 842-849 (58) Fields surveyed (Int. Cl. ⁷ , DB name) A23L 1/30-1/305 A23L 1/10 A23L 1/172 BIOSIS (DIALOG) JICST file (JOIS) JAFIC file (JOIS)

Claims (3)

(57) [Claims] 1. A method comprising adding a solution containing glutamic acid or a salt of glutamic acid and 2-morpholinoethanesulfonic acid to at least one kind of rice germ, rice bran containing germ or germ rice, and reacting the mixture. Glutamic acid and glutamic acid in the rice germ, rice bran containing germ or germ rice are converted into γ- by enzymatic action of glutamate decarboxylase in the rice germ, rice bran containing germ or germ rice.
A method for producing γ-aminobutyric acid, which is aminobutyric acid. 2. The method for producing γ-aminobutyric acid according to claim 1, wherein the glutamic acid concentration is set to 50 mmol / L or more. 3. The γ- according to claim 1, wherein
A method for producing γ-aminobutyric acid, which comprises adding pyridoxal phosphoric acid to a reaction system.