KR101406052B1 - METHOD FOR QUANTIFICATION OF γ―AMINOBUTYRIC ACID - Google Patents

Abstract

In order to exclude amino acids such as glutamic acid which are similar in structure to GABA, dehydrogenase, which requires a specific amino acid transferase and oxidized form of nicotinamide adenine dinucleotide phosphate as a coenzyme, is used to generate reduced type nicotinamide adenine dinucleotide phosphate And then measuring the water-soluble formazan dye produced in the presence of the tetrazolium salt that forms the water-soluble formazan dye in the resulting NADPH, and then measuring the resulting water-soluble formazan dye. And provides a quantitative method.

Description

METHOD FOR QUANTIFICATION OF < RTI ID = 0.0 > γ-AMINOBUTYRIC ACID &

The present invention relates to a method for quantitatively and easily quantifying γ-aminobutyric acid (hereinafter may be referred to as GABA) by colorimetric analysis without being affected by various amino acids contained in the sample.

GABA is a kind of amino acid that exists extensively in nature, and its physiological functions such as hypotensive action and mental stability action are known. In anticipation of this action, many foods containing GABA added or fortified have been produced (see Patent Document 1). In addition, although only a small amount of GABA is contained in cereal seeds, it is known that GABA content is increased by germinating seeds (see Patent Document 2).

The increase in GABA in cereal seeds depends on the post-harvest treatment and quality, and in the case of foodstuffs that have been germinated and enhanced GABA content, it is assured to the consumer that the germination process reliably increases GABA Is required.

Patent Document 3 discloses a method of dephosphorylating an oxidized nicotinamide adenine dinucleotide phosphate or an oxidized nicotinamide adenine dinucleotide phosphate with an alkali esterase to form an oxidized nicotinamide adenine dinucleotide phosphate or a reduced nicotinamide adenine dinucleotide And measuring the resulting formazan dye by reacting a reduced nicotinamide adenine dinucleotide with a tetrazolium salt in the presence of an electron transporting agent.

Patent Document 1: Japanese Patent Application Publication No. 2007-159017 Patent Document 2: Japanese Patent Application Laid-Open No. 2003-250512 Patent Document 3: Japanese Patent Application Laid-Open No. 2005-304483

However, when the method of Patent Document (3) is applied to quantitate GABA in samples containing various amino acids such as foods and beverages, the formazan dye precipitates due to the influence of mixed amino acids, and accurate measurement can not be performed . Therefore, in the quantitative analysis of GABA in which a variety of amino acids are mixed, such as in foods, a separation analysis method using an amino acid analysis system or a high-volume apparatus such as HPLC is used to exclude the influence of various amino acids to be mixed. However, such a separation assay requires a high-volume apparatus and can not simultaneously analyze multiple samples. Therefore, it has been pointed out that the GABA content displayed on the product does not guarantee the GABA content actually contained in the product.

As described above, the method of quantitating GABA in a sample containing various amino acids such as food and drink has several problems. That is, in the case of the method for measuring the formazan pigment, accurate measurement is almost impossible due to the influence of the mixed amino acid. In the case of the separation method, since a high-volume apparatus is required, bad.

An object of the present invention is to solve such a problem, and it is an object of the present invention to provide a method of analyzing a sample containing a small amount of a sample without using an amino acid analyzer or a high-volume apparatus such as HPLC and without being influenced by various amino acids contained in the sample And a method for simultaneously and rapidly quantifying GABA.

GABAse is an enzyme complex that exists in various organisms such as microorganisms and tomatoes, and has GABA amino transferase activity and succinate dehydrogenase activity. The present inventors have paid attention to quantitatively converting the oxidized nicotinamide adenine dinucleotide phosphate of coenzyme into a reduced form when the production of succinic acid semialdehyde from GABA by this enzyme and the resulting succinic acid semialdehyde are oxidized, From the resulting reduced nicotinamide adenine dinucleotide phosphate, the formation of a water-soluble hydrazone was coupled through an electron donor. As a result, the conversion of GABA to succinic acid was coupled with the formation of water-soluble hydrazone, and it became possible to quantify GABA according to the colorimetric method. Further, by using the colorimetric method, it is possible to use a microplate reader of a comparatively low cost, and at the same time, it has succeeded in reducing the reaction system, thereby completing the present invention.

Namely, the present invention provides a method for quantifying? -Aminobutyric acid in a sample containing various amino acids such as food and drink,

(1) a step of reacting a sample with γ-aminobutyric acid aminotransferase and oxidized nicotinamide adenine dinucleotide, dehydrogenase requiring as a coenzyme to produce reduced type nicotinamide adenine dinucleotide phosphate, and then deactivating the enzyme;

(2) a step of reacting a reduced type nicotinamide adenine dinucleotide phosphate with an electron transporting agent in the presence of a tetrazolium salt to form a water-soluble formazan dye, and

(3) a step of measuring water-soluble formazan.

The present invention relates to a method for producing NADPH by reacting dehydrogenase, which requires NADP as a coenzyme specific for GABA and an amino acid transferase specific to the product, to produce NADPH followed by the action of an electron carrier on NADPH in the presence of a water soluble tetrazolium salt GABA can be easily and easily quantified by measuring the produced water-soluble formazan pigment, and thus has the following effects.

The method of the present invention is economical because it does not require a high-volume apparatus such as an amino acid analysis system or HPLC, and it is also possible to obtain GABA contained in a sample of a small amount of a sample without being influenced by various amino acids contained in the sample Simultaneously, we can quantify quickly.

According to the method of the present invention, since the GABA contained in foods such as rice can be rapidly and simultaneously quantified, it is possible to measure the content of GABA added to various foods due to the physiological function being noted, It is possible to guarantee the contained GABA content.

In addition, even in a germinated brown rice product in which GABA is enhanced by producing GABA using a group of metabolic enzymes contained in rice, it becomes possible to display the GABA content more accurately, and a product having a low GABA content due to failure in germination of brown rice It can be used for inspection. In addition, according to the present invention, it is possible to easily test the difference in GABA content between germplasm by germination treatment, so that it is possible to contribute to selection of a variety against germinated brown rice.

[Fig. 1] is an explanatory view of the principle of quantitative determination of GABA.
[Fig. 2] A calibration chart using a GABA standard sample.
[Fig. 3] A comparison chart of the quantitation in the Dabsyl derivatization HPLC method and the quantification in the present invention.
Fig. 4 is a diagram for examining the GABA production ability in various varieties of Thailand.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the best mode for carrying out the present invention will be described by way of examples. However, the present invention is not limited to these examples.

The method of quantifying GABA of the present invention is a method for quantitatively determining various amino acids, particularly, amino acid such as glutamic acid having a structure similar to that of GABA, a specific amino acid transferase and an oxidized nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP (Hereinafter, referred to as NADPH) by using dehydrogenase (hereinafter, also referred to as NADP-requiring dehydrogenation oxygen) that requires a reducing form of nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADPH) And a step of reacting the resulting NADPH with an electron transporting agent in the presence of a tetrazolium salt to generate a magan pigment and measuring the resultant water-soluble formazan dye.

Here, the various amino acids are amino acids having a structure similar to GABA, such as glutamic acid, serine, glycine, and histidine in addition to the above-mentioned glutamic acid.

Examples of the amino acid transferase include a GABA amino transferase (hereinafter referred to as GABA-T) and a NADP requiring dehydrogenase include succinic acid semialdehyde dehydrogenase (hereinafter referred to as SSADH).

The electron carrier may be an artificial electron carrier such as phenazine methosulfate (hereinafter referred to as PMS), 1-methoxyphenazine methosulfate (hereinafter referred to as 1-MeO-PMS), phenazine ethosulfate , PES) can be exemplified.

The tetrazolium salt that produces the water-soluble formazan dye is, for example, sodium salt of 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -2H-tetrazolium ) Can be exemplified.

The principle of the present invention is shown in Fig. The present invention relates to a method for quantifying γ-aminobutyric acid in a sample containing various amino acids such as a food or drink, which comprises γ-aminobutyric acid aminotransferase and NADP-requiring dehydrogenase oxygen, A first step of producing NADPH specific to an amino acid transferase and then inactivating the enzyme; a step of reducing the tetrazolium salt to NADPH produced in the first step to produce a water-soluble formazan dye; And a second step of measuring the temperature. That is, the first step of converting the NADP to NADPH using GABA-T and SSADH and then inactivating the enzyme, and the second step of removing the enzyme from the NADPH via the electron transfer agent in the presence of the tetrazolium salt And a second step of measuring a color (yellow color) caused by water-soluble formazan.

In the first step, the addition of a tetrazolium salt that produces a water-soluble formazan dye and an electron donor without inactivating the enzyme leads to simultaneous reduction of the tetrazolium salt that is not specific to the enzyme reaction, and GABA can be accurately measured none.

In addition, when a tetrazolium salt that produces a water-insoluble formazan dye is used, since the reduced formazan dye is precipitated, accurate measurement becomes difficult.

Deactivation of the enzyme is achieved, for example, by making the reaction solution acidic. More specifically, an acid such as hydrochloric acid or sulfuric acid may be added to adjust the pH to 2 or less.

Hereinafter, the present invention will be described in more detail by showing a few examples.

Example  One

An example of the effect of amino acids on the quantification of GABA according to the present invention is shown below.

Reagent 1 containing GABAse, NADP as follows, Reagent 2 as a stop solution, and Reagent 3 containing 1-MeO-PMS, WST-8 were prepared.

Reagent 1

100 mM Tris buffer (pH 8.9)

10 mM? -Ketoglutaric acid

2 mM 2-mercaptoethanol

0.5 mM NADP

0.25 U / mL GABAse (derived from Pseudomonas fluorescens)

Reagent 2

1 M sulfuric acid

Reagent 3

1-MeO-PMS, WST-8 mixed reagent (Wako Pure Chemical Cell Counting Kit-8)

Using these reagents, GABA concentration was measured according to the following procedure. That is, 90 μL of reagent 1 was added to 100 μL of the sample using an aqueous solution prepared so that the concentration of GABA as a sample was 0.1 ppm, 0.5 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm, 50 ppm, and 100 ppm, Then, 10 μL of reagent 2 was mixed, 5 μL of reagent 3 was added, and the absorbance at a wavelength of 470 nm was measured by a microplate reader (Sapphire manufactured by Tekan) according to the end method. In addition, the addition of 100 ppm of glutamic acid, serine, glycine, and histidine to the sample was also measured. As shown in Fig. 2, the correlation coefficient of GABA was 0.999 up to 50 ppm, indicating high linearity and quantitatively measurable. It has also been found that it is not affected by other amino acids such as glutamic acid, serine, glycine, histidine and the like.

Example  2

GABA in food was quantified.

As shown in FIG. 3, a very high correlation coefficient of 0.999 was obtained when the GABA content in the water extracts of various pure white rice, brown rice and germinated brown rice was measured and compared according to the Dabsyl derivatization-HPLC method. From this, it was found that quantification by colorimetric analysis is possible with the same accuracy as HPLC method.

Example  3

GABA in germinated brown rice was quantified.

GABA was determined by immersing and germinating Jeongbaekmi and brown rice in water. The various brown rice samples from Thailand were immersed in water, and the GABA content before and after the treatment was measured by the method shown in Example 1. As a result, an increase in the GABA content was observed in the low temperature drying and the sun drying. On the other hand, GABA-producing enzymes were inactivated in high-temperature dried rice widely used in Thailand in the rainy season, and the increase in GABA was not shown in Fig. 4, confirming the reliability of the method.

Example  4

We quantified GABA in chocolate.

Quantitation of GABA contained in commercial chocolate was carried out by the present method and HPLC method. GABA added chocolate and ordinary chocolate were hydrothermally extracted and the GABA content was measured by the method shown in Example 1. [ In GABA added chocolate, 2895 + 84 ppm of GABA was quantified, but GABA was not detected from normal chocolate under the same conditions. In the HPLC method, GABA containing 2965 + 54 ppm was detected in chocolate containing GABA, but not in normal chocolate. From these results, it was confirmed that the method of the present invention was effective in assuring the GABA content in GABA-added foods.

Example  5

We quantified GABA in soft drinks.

In the same manner as in Example 1, GABA contained in commercially available soft drinks was quantified. Measurement of GABA content in soft drinks and amino acid-added soft drinks to which GABA was added showed that 9842 ± 95 ppm of GABA was determined in GABA-added soft drinks and in the case of amino acid-added soft drinks not containing GABA under the same conditions No GABA was detected. From these results, it was confirmed that the method of the present invention was effective in assuring GABA content in GABA-added soft drinks.

From these results, it was confirmed that it was possible to guarantee the GABA content in a wide range of foods by the method of the present invention.

Comparative Example

The methods using water-insoluble formazan MTT instead of water-soluble Formazan WST-8 were compared.

In the case of using MTT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) in various pure white rice, brown rice and germinated brown rice water extracts, Since the product precipitated and deposited on the microplate surface, it was impossible to perform the analysis. This is thought to be due to the co-precipitation of MTT formazan with protein contained in the water extract of food. In addition, when GABA in the GABA-containing chocolate water extract was analyzed, the absorption of visible light by the colored material contained in the extract interfered with the analysis.

On the other hand, when water-soluble formazan was applied, there was no problem of solubility and the problem could be solved by increasing the formazan concentration.

Basically, dehydrogenase, which requires a specific amino-transferase and an oxidized form of nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP) as a coenzyme, is used to exclude amino acids such as glutamic acid which are similar in structure to GABA , A second step of producing reduced nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADPH), and a second step of measuring an aqueous formazan dye produced by causing an electron donor to act on the resulting NADPH in the presence of a tetrazolium salt And a method for quantifying GABA in a simple and easy manner.

Specifically, dehydrogenase, which requires a specific amino transferase and an oxidized form of nicotinamide adenine dinucleotide phosphate (hereinafter referred to as NADP) as a coenzyme, is used to exclude amino acids such as glutamic acid, which are similar in structure to GABA, (Hereinafter referred to as " NADPH "), followed by the first step of adding sulfuric acid, and a step of reacting the resulting NADPH with an electron transporting agent in the presence of a tetrazolium salt to form a water- And a second step of measuring the concentration of GABA.

More specifically,

As a method for quantifying? -Aminobutyric acid in a food or drink,

(1) A method for producing reduced-type nicotinamide adenine dinucleotide phosphate by reacting a food water extract or beverage with γ-aminobutyric acid aminotransferase and dehydrogenase which requires oxidized nicotinamide adenine dinucleotide as a coenzyme, ,

(2) a step of reacting a reduced type nicotinamide adenine dinucleotide phosphate with an electron transporting agent in the presence of a tetrazolium salt to form a water-soluble formazan dye, and

(3) a step of measuring water-soluble formazan, which is a method for quantitatively determining? -Aminobutyric acid in a food or drink. Here, the tetrazolium salt is 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -2H-tetrazolium sodium salt.

The method of quantifying? -Aminobutyric acid of the present invention is not limited to the quantitative determination of? -Aminobutyric acid in the food as shown in the examples, but may be a measurement sample containing an amino acid such as glutamic acid having a structure similar to that of GABA, For example, it is apparent from the above-described principle of the present invention that it can be applied to a biological specimen sample.

Claims (8)

As a method for quantifying? -Aminobutyric acid in a sample containing an amino acid,
(1) a step of reacting a sample with a? -Aminobutyl amino cyclotransferase and a succinic acid semialdehyde dehydrogenase to produce a reduced type nicotinamide adenine dinucleotide phosphate, and then adjusting the pH of the reaction solution to 2 or less;
(2) a step of reacting the reduced type nicotinamide adenine dinucleotide phosphate with an electron transporting agent in the presence of a tetrazolium salt that produces a water-soluble formazan dye to produce a water-soluble formazan dye, and
(3) a step of measuring the resulting water-soluble formazan dye. The method according to claim 1,
Wherein the tetrazolium salt is 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -2H-tetrazolium sodium salt. 3. The method of claim 2,
Wherein the electron transport is selected from phenazine methosulfate, 1-methoxyphenazine methosulfate and phenazine ethosulfate. The method according to claim 1,
Wherein the measurement of the water-soluble formazan dye measures the absorbance at a wavelength of 470 nm. The method according to claim 1,
Wherein the step of bringing the pH of the reaction solution to 2 or less is a step of adding sulfuric acid. 6. The method according to any one of claims 1 to 5,
Wherein the sample comprises glutamic acid, serine, glycine or histidine. The method according to claim 6,
Wherein the sample is a food product. delete

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