TWI484178B - Method for quantification of γ-aminobutyric acid - Google Patents

Description

Quantitative method of γ-aminobutyric acid

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

GABA is one of the amino acids widely present in the natural world, and is known to have physiological functions such as blood pressure lowering action and mental stability. Many foods to which GABA is added or strengthened are produced by expecting the action (see Patent Document 1). Further, although the cereal seed contains only a small amount of GABA, it is known that the GABA content can be increased by germination of the seed (refer to Patent Document 2).

The increase in GABA content in cereal seeds varies with post-harvest handling or quality. In addition, for foods that have been enhanced by the germination treatment to enhance the GABA content, it is necessary to ensure to the consumer that the GABA content is indeed increased by the germination treatment.

Patent Document 3 discloses an assay for the activity of alkaline phosphatase (Alkaline Phosphatase), which is an oxidized nicotinamide adenine dinucleotide phosphate (NADP) or a reduced nicotinamide Adenine dinucleotide phosphate (abbreviated as NADPH) is converted to oxidized nicotine guanamine adenine dinucleotide or reduced nicotine guanamine adenine dinucleoside by dephosphorylation with alkaline phosphatase The acid is then reacted with a reduced nicotine indoleamine adenine dinucleotide and a tetrazolium salt in the presence of an electron carrier to form a formazan pigment, and the formazan pigment is measured.

[Advanced technical literature]

[Patent Document 1] Japanese Patent Laid-Open Publication No. 2007-159017

[Patent Document 2] Japanese Patent Laid-Open Publication No. 2003-250512

[Patent Document 3] Japanese Patent Laid-Open Publication No. 2005-304483

However, when the amount of GABA in a sample containing various amino acids such as foods and drinks is quantified, when the method of Patent Document 3 is used, the formazan pigment is precipitated due to the influence of the mixed amino acid, so that it cannot be correctly performed. Determination. Therefore, in the quantitative analysis of GABA in which various amino acids are mixed in foods and drinks, in order to eliminate the influence of various amino acids to be mixed, it is necessary to use a separation analysis method using an expensive instrument such as an amino acid analyzer or HPLC. However, in such separation analysis methods, not only expensive instruments but also multiple samples cannot be analyzed at the same time. Therefore, it is often accused that the GABA content indicated on the product does not necessarily guarantee the actual content of GABA in the product.

As described above, there are several problems in the method of quantifying GABA in a sample containing various amino acids such as foods and drinks. That is, in the method for measuring the formazan pigment, it is almost impossible to accurately measure due to the influence of the mixed amino acid, and in the separation analysis method, not only a high-priced instrument but also multiple tests cannot be simultaneously analyzed. Physically inefficient.

The present invention has been made in order to solve such problems, and an object of the present invention is to provide an expensive apparatus such as an amino acid analyzer or HPLC, which is not affected by various amino acids contained in the sample, and the like. At the same time, a method for quantifying GABA contained in a sample of a multi-sample is easily and quickly.

GABAse is an enzyme complex that exists in a wide variety of organisms such as microorganisms or tomatoes, and has both GABA transaminase activity and succinate dehydrogenase activity. The present inventors focused on the formation of succinate semialdehyde by the action of this enzyme on GABA, and then when the succinic semialdehyde formed is oxidized, the oxidized nicotine guanamine adenine can be used as a coenzyme. Nucleotide phosphate (NADP) is quantitatively converted to a reduced form (NADPH), and the reduced form of nicotine indoleamine adenine dinucleotide phosphate (NADPH) is produced and then hydrolyzed via a coupling reaction of an electron carrier. Sexuality. As a result, by performing a coupling reaction of "conversion from GABA to succinic acid" and "production of water-soluble hydrazine", the quantitative determination of GABA can be performed by a colorimetric method. Further, the present invention can be accomplished by using a colorimetric method to use a relatively inexpensive microplate reader while successfully reducing the reaction system.

That is, the present invention is a method for quantifying GABA in a sample containing various amino acids such as foods and drinks, and is characterized in that it comprises the following steps: (1) a sample, a γ-aminobutyric acid transaminase, and a request Step of inactivating an enzyme after oxidizing nicotine indoleamine adenine dinucleotide phosphate (NADP) is reacted as a coenzyme dehydrogenase to form reduced nicotine indoleamine adenine dinucleotide phosphate (NADPH) (2) reacting reduced nicotine indoleamine adenine dinucleotide phosphate (NADPH) with an electron carrier in the presence of a tetrazolium salt capable of producing a water-soluble formazan pigment to form a water-soluble nail a step of licking the pigment; and (3) a step of measuring the water-soluble formazan pigment.

The present invention acts on a transaminase specific for GABA, a dehydrogenase which is a coenzyme which is specific for its product, and generates NADPH, and then in the presence of a water-soluble tetrazolium salt, The electron transporter and NADPH are allowed to form a water-soluble formazan dye, and the water-soluble formazan dye produced by the measurement can be used to quantitatively quantify GABA. Therefore, the present invention has the following effects.

According to the method of the present invention, not only an expensive instrument such as an amino acid analyzer or HPLC is required, but also economical, and it is not affected by various amino acids contained in the sample, and can be simultaneously and simply and quickly The GABA contained in the sample of a small amount of multi-sample was quantified.

According to the method of the present invention, GABA contained in foods such as rice can be quantitatively and rapidly quantified, and the content of GABA added to foods in various foods of physiological physiology can be measured, thereby ensuring The GABA content contained in these products.

In addition, in the GABA-enhanced germinated black rice product which is produced by the metabolic enzyme group in the rice, the GABA content can be more accurately indicated, so that the method can be used to detect the failure of the germination treatment of the black rice. A product with a low content of GABA. Further, since the difference in the GABA content due to the germination treatment can be easily detected according to the present invention, it contributes to the selection of the germinated rice.

Hereinafter, the best mode for carrying out the invention will be described based on the embodiments. Again, of course, the invention is not limited by such embodiments.

The GABA quantification method of the present invention comprises the following steps: in order to eliminate interference of various amino acids, especially amino acids such as glutamic acid which are similar in structure to GABA, and use specific transaminase and oxidized type smoke Alkaline guanamine adenine dinucleotide phosphate (hereinafter referred to as NADP) as a coenzyme dehydrogenase (hereinafter also referred to as NADP required dehydrogenase), in the reaction to produce reduced nicotine indoleamine adenine dinucleotide Phosphoric acid (hereinafter referred to as NADPH), a step of inactivating the enzyme; and in the presence of a tetrazolium salt capable of forming a water-soluble formazan dye, causing the electron carrier to react with the generated NADPH to form a water-soluble formazan pigment And measuring the step of producing the water-soluble formazan pigment.

Here, "various amino acids" means the aforementioned glutamic acid and other amino acids having a structure similar to GABA, such as glutamic acid, serine, glycine, and histidine.

The transaminase can be exemplified by, for example, GABA transaminase (hereinafter referred to as GABA-T), and the NADP-desired dehydrogenase can be exemplified by succinic semialdehyde dehydrogenase (hereinafter referred to as SSADH).

The electronic communication body can be exemplified by an artificial electronic communication body such as a phenotype Methyl sulfate (Phenazine methosulfate, hereinafter referred to as PMS), 1-methoxyphene Methyl sulfate (hereinafter referred to as 1-MeO-PMS), pheno Ethyl sulfate (Phenazine ethosulfate, hereinafter referred to as PES).

The tetrazolium salt which can form a water-soluble formazan dye can be exemplified by, for example, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-2H-tetrazolium sodium salt. (hereinafter referred to as WST-8).

The principle of the invention is as shown in Figure 1. The present invention is a method for quantifying γ-aminobutyric acid in a sample containing various amino acids such as foods and drinks, and the method comprises the steps of using γ-aminobutyric acid transaminase and NADP required dehydrogenase. The second enzyme is a first step of inactivating the enzyme after generating NADPH specific for γ-aminobutyric acid transaminase; and reducing the tetrazolium salt by NADPH produced in the first step to form a water-soluble solution. The second step of measuring the color of water-soluble nails. That is, the first step of inactivating the enzyme after converting NADP into NADPH using GABA-T and SSADH; and the NADPH and electron transporter in the presence of a tetrazolium salt capable of producing a water-soluble formazan pigment The second step of producing a water-soluble formamidine and measuring the color (yellow) of the water-soluble formamidine.

In the first step, if the tetrazolium salt which can form a water-soluble formazan dye and the electron carrier are added without deactivating the enzyme, the reduction reaction of the tetrazolium salt of the non-specific enzyme reaction proceeds simultaneously. The measurement of GABA could not be performed correctly.

Further, when a tetrazolium salt which can form a water-insoluble ampoule pigment is used, since the formazan dye formed by the reduction precipitates, it is difficult to accurately measure it.

The inactivation 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 more specifically described by way of examples.

(Example 1)

Hereinafter, examples of the amino acid which affects the quantitative GABA of the present invention are listed.

A reagent containing GABAse and NADP, a reagent 1 belonging to a reaction stop solution, and a reagent 3 containing 1-MeO-PMS and WST-8 are prepared.

Reagent 1

100mM Tris buffer (pH 8.9)

10mM α-ketoglutaric acid

2mM 2-mercaptoethanol

0.5mM NADP

0.25 U/mL GABAse (from Pseudomonas fluorescens)

Reagent 2

1M sulfuric acid

Reagent 3

Mixed reagent of 1-MeO-PMS and WST-8 (Cell Counting Kit-8 manufactured by Wako Pure Pharmaceutical Co., Ltd.)

Using these reagents, the GABA concentration was determined by the following procedure. That is, an aqueous solution having a GABA concentration of 0.1 ppm, 0.5 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm, 50 ppm, and 100 ppm was used as a sample, and 90 μL of the reagent 1 was added to 100 μL of the sample, and then heated at 30 ° C for 15 minutes. Next, 10 μL of the reagent 2 was mixed, and 5 μL of the reagent 3 was further added, and then the absorbance at a wavelength of 470 nm was measured by a microplate reader (Sapphire, manufactured by Tekan Co., Ltd.) according to the endpoint method. In addition, 100 ppm of glutamic acid, serine, glycine, and histidine were added to the sample. As shown in Fig. 2, the correlation coefficient of GABA in the range of 50 ppm is 0.999, which shows a high linearity and can be used for quantification. In addition, it is also shown to be unaffected by other amino acids such as glutamic acid, serine, glycine, and histidine.

(Example 2)

Quantify GABA in foods.

Further, the GABA content in various water extracts of refined white rice, black rice, and germinated rice was measured by Dabsyl derivative-HPLC method, and the results were as shown in Fig. 3, and an extremely high correlation of 0.999 was obtained. From this, it can be seen that this method is a colorimetric analysis quantitative method which can obtain the same accuracy as the HPLC method.

(Example 3)

Quantify the GABA in germinated rice.

The refined white rice and the black rice were immersed in water, and the GABA was quantified at the time of germination. The various black rice samples produced in Thailand were subjected to water immersion treatment, and the GABA content before and after the treatment was measured by the method shown in Example 1. As a result, the GABA content in the low-temperature dried rice and the sun-dried rice was increased. On the other hand, rice which is widely used in high-temperature drying treatment in Thailand during the rain period is inactivated by the GABA-producing enzyme, and as shown in Fig. 4, the GABA content cannot be increased, thereby confirming the reliability of the method of the present invention. .

(Example 4)

Quantify GABA in chocolate.

The quantification of GABA contained in commercially available chocolate was carried out according to the method of the present invention and the HPLC method. The GABA-added chocolate and the usual chocolate were subjected to hot water extraction, and the GABA content was measured by the method shown in Example 1. The results showed that the GABA content of the chocolate added with GABA was 2895±84 ppm, and under the same conditions, GABA was not detected in the general chocolate. In the HPLC method, the measured value of the chocolate added with GABA was 2965 ± 54 ppm, and the average chocolate was not detected. From these results, it was confirmed that the method of the present invention is also effective for assuring the GABA content of the food to which GABA is added.

(Example 5)

Quantify GABA in a refreshing beverage.

The same procedure as in Example 1 was carried out to quantify the GABA contained in the commercially available refreshing beverage. When the GABA content in the refreshing beverage to which GABA was added and the refreshing beverage to which the amino acid was added was measured, the GABA content of the refreshing beverage to which GABA was added was 9842±95 ppm, and under the same conditions, the GABA was not added and the amine group was added. No GABA was detected in the sour refreshing drink. From these results, it was confirmed that the method of the present invention is also effective for assuring the GABA content of the refreshing beverage to which GABA is added.

These results confirm that the GABA content in a wide range of foods can be ensured in accordance with the method of the present invention.

Comparative example

A comparison was made by using a method of replacing water-soluble formazan WST-8 with MTT which is a water-insoluble formamidine.

MTT (ie 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole bromide is used in various water extracts of refined white rice, black rice and germinated rice. In the case of 鎓), even at a low concentration, the reaction product precipitates and deposits on the surface of the microplate, so that analysis cannot be performed. It is speculated that this is because the protein and the like contained in the water extract of the food co-precipitate with the MTT formazan. Further, when analyzing GABA in the water extract of GABA-containing chocolate, the absorption of the visible light portion caused by the coloring matter contained in the extract hinders the analysis.

On the other hand, when water-soluble formamidine is used, there is no problem of solubility, and the problem can be solved by increasing the concentration of formazan.

First, basically, the present invention provides a method for easily quantifying GABA, the method comprising the following steps: in order to exclude interference with an amino acid such as glutamic acid similar to GABA structure, a specific transaminating agent is used. An enzyme, and a dehydrogenase which requires oxidized nicotine indoleamine adenine dinucleotide phosphate (hereinafter referred to as NADP) as a coenzyme to form reduced nicotine indoleamine adenine dinucleotide phosphate (hereinafter referred to as NADPH) The first step; and the second step of causing the generated NADPH to act on the electron transporter in the presence of the tetrazolium salt and measuring the produced water-soluble formazan dye.

In particular, the present invention provides a method for easily quantifying GABA, the method comprising the following steps: in order to exclude interference with amino acids such as glutamic acid similar to GABA structure, specific transaminating agents are used. An enzyme, and a dehydrogenase which requires oxidized nicotine indoleamine adenine dinucleotide phosphate (hereinafter referred to as NADP) as a coenzyme to produce reduced nicotine indoleamine adenine dinucleotide phosphate (hereinafter referred to as NADPH) After that, the first step of adding sulfuric acid; and the second step of causing the generated NADPH to act on the electron transporter in the presence of the tetrazolium salt and measuring the produced water-soluble formazan dye.

More specifically, the present invention provides a method for quantifying γ-aminobutyric acid in a food or beverage, the method comprising the steps of: (1) making an aqueous extract or beverage of a food, and a γ-amine group a step of adding sulfuric acid after reacting with butyryltransaminase and a dehydrogenase which requires oxidized nicotine indoleamine adenine dinucleotide phosphate as a coenzyme to form reduced nicotine indoleamine adenine dinucleotide phosphate;

(2) a step of reacting a reduced nicotine indoleamine adenine dinucleotide phosphate with an electron carrier in the presence of a tetrazolium salt capable of producing a water-soluble formazan dye to form a water-soluble formazan dye ;as well as

(3) a step of determining a water-soluble formamidine pigment;

Among them, the tetrazolium salt is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-2H-tetrazolium sodium salt.

Moreover, it is understood from the above-described principle of the present invention that the method for quantifying γ-aminotyrosic acid of the present invention is not limited to the quantification of γ-aminotyrosic acid in the food and beverage shown in the examples, and may be applied to the determination of the content and A sample of an amino acid such as glutamic acid having a similar GABA structure, such as a sample of a biological sample.

Figure 1 is a schematic diagram of the principle of GABA quantification.

Figure 2 is a calibration curve using the GABA standard sample.

Figure 3 is a graph comparing the quantification of the present invention by the Dabsyl derivatization HPLC method.

Figure 4 is a review of the GABA production capacity of various varieties in Thailand.

Claims (8)

A method for quantifying γ-aminobutyric acid, which is a method for quantifying γ-aminobutyric acid in a sample containing an amino acid, which comprises the following steps: (1) making a sample, γ-aminobutyric acid transamination The enzyme reacts with a dehydrogenase which requires oxidized nicotine indoleamine adenine dinucleotide phosphate as a coenzyme to form reduced nicotine indoleamine adenine dinucleotide phosphate, and then makes the reaction solution acidic to pH 2 or lower. (2) reacting reduced nicotine indoleamine adenine dinucleotide phosphate with an electron carrier in the presence of a tetrazolium salt capable of producing a water-soluble formazan dye to form a water-soluble formamidine a step of dyeing; and (3) a step of measuring the produced water-soluble formamidine pigment. The method for quantifying γ-aminobutyric acid according to claim 1, wherein the tetrazolium salt is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrobenzene Base)-2H-tetrazolium sodium salt. The method for quantifying γ-aminobutyric acid according to the second aspect of the patent application, wherein the dehydrogenase which requires the oxidized nicotine indoleamine adenine dinucleotide phosphate as a coenzyme is succinic semialdehyde dehydrogenase. The method for quantifying γ-aminobutyric acid according to Item 3 of the patent application, wherein the electron carrier is selected from the group consisting of phenotype Methyl sulfate, 1-methoxyphene Methyl sulfate and pheno Ethyl sulfate. The method for quantifying γ-aminobutyric acid according to the first aspect of the patent application, wherein the measurement of the water-soluble formamidine is carried out by measuring the absorbance at a wavelength of 470 nm. The method for quantifying γ-aminobutyric acid according to the first aspect of the patent application, wherein the operation of making the reaction solution acidic to pH 2 or lower is added with sulfuric acid. The method for quantifying γ-aminobutyric acid according to any one of claims 1 to 6, wherein the sample contains glutamic acid, seric acid, glycine or histidine. A method for quantifying γ-aminobutyric acid according to item 7 of the patent application, wherein the sample is a food or drink.