200946908 六、發明說明： 【發明所屬之技術領域】 本發明係有關使T _胺基路酸（Gama-aminobutyric acid，以下有時亦稱為GAB A)不受試料中含有之各種胺基 酸之影響，而藉由比色分析而簡單迅速地進行定量的方法。 【先前技術】 GABA為天然界廣泛存在之胺基酸之一種，已知其具有 血壓降低作用、精神安定作用等生理機能性。因期待其作 用，而製造許多經添加或強化GABA之食品（參照專利文獻 1)。此外，穀類種子中雖然僅含有少量GABA，但已知藉由 使種子發芽可增加GABA含量（參照專利文獻2)。 穀類種子中之GABA含量的增加係隨著收穫後處理或 品質而異，另外，對經由發芽處理而強化GABA含量之食 品，必須要求對消費者保證藉由該發芽處理確實增加了 GABA含量。 專利文獻3揭示一種驗性碗酸酶（Alkal ine Phosphatase)活性之測定法，其係使氧化型菸鹼醯胺腺嘌 呤二核苷酸填酸（nicotinamide adenine dinucleotide phosphate，簡稱NADP)或氧化型菸鹼醯胺腺嘌呤二核苷 酸鱗酸（簡稱NADPH)藉由驗性填酸酶進行脫磷酸化反應， 而轉換成氧化型菸鹼醯胺腺嘌呤二核苷酸或還原型菸鹼醯 胺腺嘌呤二核苷酸，然後在電子傳達體之存在下，使還原 型菸鹼醯胺腺嘌呤二楱苷酸與四唑鏽鹽（tetrazolium salt)反應而生成曱臢（formazan)色素，並測定該曱猜 4 321191 200946908 色素。 ' [先行技術文獻] - [專利文獻1]曰本特開2007-159017號公報 [專利文獻2]日本特開2003-250512號公報 [專利文獻3]日本特開2005-304483號公報 【發明内容】 (發明欲解決之課題） 然而，在定量如飲食品等含有各種胺基酸之試料中之 ® GABA時，當使用專利文獻3之方法時，會因混合存在之胺 基酸之影響而使甲臢色素沉殿，故無法進行正確之測定。 因此，在飲食品等混合存在有各種胺基酸之GABA之定量分 析中，為了排除混合之各種胺基酸之影響，必須使用利用 胺基酸分析儀或HPLC等高價儀器之分離分析法。但是，在 此等分離分析法中，不僅需要高價儀器，並且亦無法同時 分析多個檢體。因此，常被指責製品上所標示之GABA含量 〇 並不一定保證是製品中之GABA實際含量的問題。 如上所述，將飲食品等含有各種胺基酸之試料中之 GABA予以定量的方法，有數個課題。亦即，在測定曱臢色 素之方法中，因受到混合存在之胺基酸之影響而幾乎不可 能正確地測定，另外，在分離分析法中，不僅需要高價儀 器，而且無法同時分析多個檢體而效率不佳。 本發明係為了解決此等課題而研創者，其目的係提供 一種不需使用胺基酸分析儀或HPLC等高價儀器，且不受檢 體中所含之各種胺基酸等之影響，而可同時並簡易迅速地 5 321191 200946908 定量少量多檢體之試料中所含之GABA的方法。 GABAse係存在於微生物或番茄等各式各樣之生物中 之酵素複合體’同時具有GABA轉胺酶活性及破珀酸脫氫酶 ’舌性。本發明者等係著眼於藉由此酵素作用於而生成 琥珀酸半醛（succinate semialdehyde) ’然後當生成之號 ί白I半龜_被氧化時，可將輔扭之氧化型終驗醯胺腺嗓吟二 核苷酸罐酸（NADP)定量地轉換成還原型（nadph)，生成之還 原型終驗醯胺腺β票吟二核苷酸麟酸（NADPH)再經由電子供 應體之偶合反應（coupling reaction)而生成水溶性腙。其 ❹ 結果’藉由進行「從GABA轉換成琥珀酸」與「水溶性腙之 生成」之偶合反應，可利用比色法進行GABA之定量。此外， 藉由利用比色法可使用較廉價之微量盤測讀儀 (microplate reader)，同時可成功地縮小反應系，而完 成本發明。 (解決課題之方法） 亦即，本發明係將飲食品等含有各種胺基酸之試料中 ◎ 之GABA予以定量的方法，其特徵為包括下述步驟： (1) 使試料、r -胺基酪酸轉胺酶、與要求氧化型菸 鹼醯胺腺嘌呤二核苷酸（MDP)作為輔酶之脫氫酶進行反應 而生成還原型菸鹼醯胺腺嘌呤二核苷酸磷酸（NADPH)後，使 酵素失活的步驟； (2) 在可生成水溶性曱臢色素之四嗤鑌鹽之存在 下’使還原型菸鹼醯胺腺嘌呤二核苷酸磷酸（NADPH)與電子 傳達體進行反應’而生成水溶性曱臢色素的步驟；以及 321191 6 200946908 (3)測定水溶性甲臢色素的步驟。 — (發明之效果） * 本發明係使對GABA具有特異性之轉胺酶、與要求對 其生成物具有特異性之NADp作為輔酶的脫氫酶進行作 用而生成NADPH，然後在水溶性四唾鑌鹽之存在下，使 電子傳達體與勵pH而生成水溶性甲臢色素作用，藉由測 定生成之水溶性甲臢色素而可簡易地定量GABA，因此，本 發明具有以下之效果。 1% 若依據本發明方法’不僅不需要胺基酸分析儀或HpLC 等高價儀器，而且具經濟性，且不受檢體中所含之各種胺 基酸等之影響，而且可同時並簡易迅速地將少量多檢體之 試料中所含之GABA予以定量。 依據本發明方法，可同時且迅速地定量米類等食品中 所含之GABA’亦可測定生理機能性受注目之各式各樣之食 品中所添加之GABA在食品中之含量，因而可保證此等製品 H 中所含之GABA含量。 此外，在利用由米類内在之代謝酵素群而生成GABA 之GABA強化的發牙玄米製品中’亦可更正確地標示GABA 含量，故可利用此方法檢出因玄米之發芽處理失敗所導致 產生之GABA含量少之製品。此外，由於依據本發明可容易 地檢測出發芽處理所致之GABA含量之品種間差異，故對於 發芽玄米之品種選拔等有所貢獻。 【實施方式】 以下，依據實施例說明用於實施本發明之最佳狀態。 321191 7 200946908 又’當然本發明並不受此等實施例所限制。 本發明之GABA定量方法係包括下述步驟： - 為了排除各種胺基酸的干擾，尤其是與GABA結構相 - 似之麵胺酸等胺基酸，而使用特異性之轉胺酶、與要求氧 化型於驗醯胺腺嘌呤二核苷酸磷酸（以下稱為NADP)作為 輔酶之脫氫酶（以下亦稱為NADP要求性脫氫酶），在反應 生成還原型菸驗醯胺腺嘌呤二核苷酸碟酸（以下稱為 NADPH )後，使酵素失活的步驛；以及200946908 VI. Description of the Invention: [Technical Field] The present invention relates to the fact that G-aminobutyric acid (hereinafter sometimes referred to as GAB A) is not affected by various amino acids contained in a sample. A method of quantifying simply and quickly by colorimetric analysis. [Prior Art] 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 their effects (refer to 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 Alkal ine Phosphatase, which is an oxidized nicotinamide adenine dinucleotide phosphate (NADP) or an oxidized tobacco. Alkaline indole adenine dinucleotide squaric acid (abbreviated as NADPH) is converted to oxidized nicotine indoleamine adenine dinucleotide or reduced nicotinamide by dephosphorylation by a virulence enzymease Adenine dinucleotide, which is then reacted with a tetrazolium salt to form a ruthenium (formazan) pigment in the presence of an electron transporter. The 曱 guess 4 321191 200946908 pigment. [Patent Document 1] [Patent Document 1] JP-A-2007-159017 [Patent Document 2] JP-A-2003-250512 [Patent Document 3] JP-A-2005-304483 (The subject to be solved by the invention) However, when the method of the method of Patent Document 3 is used to quantify the ? GABA in a sample containing various amino acids such as foods and drinks, it is caused by the influence of the mixed amino acid. The hyperthyroid pigmentation sinks the temple, so it is impossible to make the correct measurement. 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 a high-priced 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 ruthenium 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. Simultaneously and quickly and easily 5 321191 200946908 quantify the method of GABA contained in a small amount of multi-sample sample. GABAse is an enzyme complex present in a wide variety of organisms such as microorganisms or tomatoes, and has both GABA transaminase activity and spearic acid dehydrogenase' tongue. The inventors of the present invention focused on the formation of succinate semialdehyde by the action of this enzyme, and then the oxidized final test amine of the succinated succinimide Adenine dinucleotide canister acid (NADP) is quantitatively converted to a reduced form (nadph), and the resulting reduced form of the final testin amine gland beta-salt dinucleotide (NADPH) is coupled via an electron donor. A reaction reaction produces a water-soluble hydrazine. The ❹ result can be quantified by the colorimetric method by performing a coupling reaction of "conversion from GABA to succinic acid" to "production of water-soluble hydrazine". In addition, by using a colorimetric method, a relatively inexpensive microplate reader can be used, and at the same time, the reaction system can be successfully narrowed down, and the invention is completed. (Means for Solving the Problem) 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, an r-amine group After reacting a butyryltransaminase with a dehydrogenase that requires oxidized nicotine indoleamine adenine dinucleotide (MDP) as a coenzyme to form reduced nicotine indoleamine adenine dinucleotide phosphate (NADPH), a step of inactivating an enzyme; (2) reacting a reduced nicotine indoleamine adenine dinucleotide phosphate (NADPH) with an electron carrier in the presence of a tetrahydropalpium salt capable of producing a water-soluble anthraquinone pigment 'Step of producing a water-soluble enamel pigment; and 321191 6 200946908 (3) A step of measuring a water-soluble formazan pigment. - (Effect of the invention) * The present invention acts to produce a NADPH by a transaminase specific for GABA and a dehydrogenase which is a coenzyme which is required to be a product thereof, and then is in a water-soluble four-saliva In the presence of a sulfonium salt, the electron transporter is allowed to react with the pH to form a water-soluble formazan dye, and the water-soluble formazan dye produced by the measurement can be easily quantified. Therefore, the present invention has the following effects. 1% If the method according to the present invention does not require not only an expensive instrument such as an amino acid analyzer or HpLC, but also economical, and is not affected by various amino acids contained in the sample, and can be simultaneously and easily The GABA contained in the sample of a small amount of multiple samples was quantified. According to the method of the present invention, the GABA' contained in foods such as rice can be simultaneously and rapidly quantified, and the content of GABA added in various foods of physiological functions can be measured, thereby ensuring the content of the GABA in the food. The GABA content contained in these products H. In addition, in the use of GABA-enhanced hair-cutting rice products that produce GABA from the inner metabolic enzyme group of rice, the GABA content can be more accurately indicated, so this 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. [Embodiment] Hereinafter, the best mode for carrying out the invention will be described based on the embodiments. 321191 7 200946908 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 the interference of various amino acids, especially amino acids such as facial acid, which are similar to GABA structure, and use specific transaminase, and requirements The oxidized form is used as a coenzyme dehydrogenase (hereinafter also referred to as NADP required dehydrogenase) as a coenzyme adenine dinucleotide phosphate (hereinafter referred to as NADP), and a reduced type of guanamine adenine is produced in the reaction. a step of inactivating an enzyme after nucleotide acid (hereinafter referred to as NADPH);
在可生成水溶性甲臢色素之四唾鏽鹽之存在下，使電 Q 子傳達體與生成之NADPH作用而生成水溶性甲臢色素，並 測定生成之水溶性曱臢色素的步驟。 在此’「各種胺基酸」係指前述之楚胺酸以及其他具 有與GABA相似結構的胺基酸，例如麵胺酸、絲胺酸、甘胺 酸、組胺酸。 轉胺酶可例示如GABA轉胺酶（以下稱為GABA-T), NADP要求性脫鼠酶可例不如玻拍酸半醒·脫氮酶（以下稱為 電子傳達體可例示如人工電子傳達體，例如吩哄曱基 硫酸鹽（Phenazinemethosulfate，以下稱為 PMS)、1-甲 氧基吩畊曱基硫酸鹽（以下稱為卜Me〇-PMS)、吩明:乙基硫 酸鹽（Phenazine ethosulfate，以下稱為 PES)。 可生成水溶性甲臢色素之四唑鑌鹽可例示如2-(2-甲 氧基硝’基苯基）-3-( 4-麟基苯基）-2H-四唾鐵納鹽（以下 稱為 WST-8)。 321191 8 200946908 本發明之原理係如第1圖所示。本發明係將飲食品等 ' 含有各種胺基酸之試料中之7-胺基酪酸予以定量的方 - 法，該方法係包括下述步驟：使用r -胺基酪酸轉胺酶與 NADP要求性脫氫酶之二酵素系，於生成對7-胺基路酸轉 胺酶具有特異性之NADPH後，使酵素失活的第1步驟；以 及將四唑鑌鹽以第1步驟生成之NADPH進行還原而生成水 溶性甲臢色素，並測定水溶性曱臢之顏色的第2步驟。亦 即，使用GABA-T及SSADH將NADP轉換成NADPH後，使酵 ® 素失活的第1步驟；以及在可生成水溶性曱臢色素之四唑 鑌鹽之存在下，藉由NADPH與電子傳達體作用而生成水溶 性甲臢，並測定該水溶性甲臢之顏色（黃色）的第2步驟。 在第1步驟中，若未使酵素失活而添加可生成水溶性 甲臢色素之四唑鑌鹽與電子傳達體，則非特異性酵素反應 之四唑鏽鹽的還原反應會同時進行，因而無法正確地進行 GABA之測定。 φ 此外，當使用可生成水不溶性曱臢色素之四σ坐鏽鹽 時，由於經還原而生成之曱臢色素會沉澱，故難以正確測 定。 酵素之失活係藉由例如使反應溶液成為酸性而達 成。更具體言之，係添加鹽酸或硫酸等酸而使pH成為2 以下即可。 以下，藉由實施例更具體說明本發明。 (實施例1) 以下，列示會影響本發明定量GABA之胺基酸的例子。 9 321191 200946908 調製含有GABAse、NADP之如下述之試劑卜屬於反應 停止液之試劑2、以及含有1-MeO-PMS、WST-8之試劑3。 試劑1 100mM Tris 緩衝液（pH8. 9)In the presence of a tetra-salt salt capable of producing a water-soluble formazan dye, a step of reacting the generated Q-substance with the generated NADPH to form a water-soluble formazan dye, and measuring the produced water-soluble quinone pigment. Here, "various amino acids" means the aforementioned sulphate and other amino acids having a structure similar to GABA, such as face acid, serine, glycine, and histidine. The transaminase can be exemplified by, for example, GABA transaminase (hereinafter referred to as GABA-T), and the NADP-required deratrating enzyme can be inferior to the glassy acid half-awake deaminase (hereinafter referred to as an electron-transmitting body, such as artificial electron communication). Body, such as Phenazinemethosulfate (hereinafter referred to as PMS), 1-methoxyphene sulfhydryl sulfate (hereinafter referred to as Bu Me〇-PMS), and phenotype: 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-methoxynitro-phenyl)-3-(4-linylphenyl)-2H- Tetrapropionate salt (hereinafter referred to as WST-8). 321191 8 200946908 The principle of the present invention is shown in Fig. 1. The present invention is a 7-amino group in a sample containing various amino acids, such as foods and drinks. A method for quantifying butyric acid, the method comprising the steps of: using a r-aminobutyric acid transaminase and a NADP-required dehydrogenase dienzyme system to produce a 7-amino basal acid transaminase The first step of inactivating the enzyme after the specific NADPH; and the reduction of the tetrazolium salt by the NADPH produced in the first step to form a water-soluble solution The second step of measuring the color of the water-soluble quinone pigment, that is, the first step of inactivating the enzyme, converting the NADP into NADPH using GABA-T and SSADH; In the presence of a tetrazolium salt of an anthraquinone dye, a second step of producing a water-soluble formamidine by the action of NADPH and an electron transporter, 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 an electron carrier are added without deactivating the enzyme, the reduction reaction of the tetrazolium rust salt of the non-specific enzyme reaction proceeds simultaneously, and thus the GABA cannot be correctly performed. In addition, when a tetrasodium sulphate salt which can form a water-insoluble bismuth pigment is used, it is difficult to accurately measure the ruthenium dye which is formed by reduction, and the enzyme is inactivated by, for example, a reaction solution. 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, the influence will be described. Invent An example of an amino acid of GABA. 9 321191 200946908 A reagent containing the following reagents including GABAse and NADP, a reagent 2 as a reaction stop solution, and a reagent containing 1-MeO-PMS and WST-8 3. Reagent 1 100 mM Tris buffer Liquid (pH 8. 9)
10mM α-酮戊二酸 2mM 2-巯基乙醇 0.5mM NADP 0. 25U/mL GABAse (源自 Pseudomonas fluorescens (螢光假單胞菌）） 試劑2 1M硫酸 試劑3 1-Me0-PMS、WST-8之混合試劑（和光純製藥公司製之 Cell Counting Kit-8) 使用此等試劑，依以下之步驟測定GABA濃度。亦即， 使用 GABA 浪度為 0. lppm、〇·5ρρπι、lppm、5ppm、l〇ppm、 20ppm、50ppm、lOOppm之水溶液作為試料，在試料1〇〇//L 中添加90//L之試劑1，然後將其於3(rc加溫15分鐘，接 著，混合10//L之試劑2，再添加5/zL之試劑3，然後以 微I盤測讀儀（Tekan公司製之sapphire)依據終點法測 定波長470nm之吸光度。此外，亦測定在試料中添加1〇卟卵 之麩胺酸、絲胺酸、甘胺酸、組胺酸者。如第2圖所示， GABA在50ppm範圍内之相關係數為〇 999，顯示很高之直 線性，而可用於定量。此外，亦顯示不會受麩胺酸、絲胺 321191 10 200946908 酸、甘胺酸、組胺酸等其他胺基酸之影響。 (實施例2) - 定量食品中之GABA。 另外，依據Dabsyl衍生物化-HPLC法測定各種精製白 米 '玄米、發芽玄米之水萃取物中之GABA含量並予以比 較，結果如第3圖所示，獲得〇· 999之極高相關性。由此 可知’此方法為可獲得與HPLC法同等精確度之比色分析定 量法。 ® (實施例3) 定量發芽玄米中之GABA。 將精製白米、玄米浸潰於水中，於發芽時進行GABA 之定量。將泰國產之各種玄米試料進行水浸漬處理，以實 施例1所示之方法測定處理前後之GABA含量，結果顯示低 溫乾燥米與曰曬乾燥米中之GABA含量有增加。另一方面， 在泰國於雨期時經廣泛使用高溫乾燥處理之米，因其GABA ❹生成酵素失活’如第4圖所示，無法使GABA之含量增加， 由此可確認本發明方法之可靠性。 (實施例4) 定量巧克力中之GABA。 依據本發明之方法及HPLC法進行市售巧克力中含有 之GABA的定量。將添加有GABA之巧克力及通常之巧克力 進行熱水萃取’以實施例1所示之方法測定GABA含量。結 果顯不添加GABA之巧克力之GABA含量為2895±84ppm，在 同一條件下’一般巧克力則未檢測到GABA。在HPLC法中， 11 321191 200946908 添加GABA之巧克力測定值為2965±54ppm，一般巧克力則 未檢測到。由此等結果可確認，本發明之方法亦有效於保 - 證添加有GABA之食品的GABA含量。 · (實施例5) 定量清涼飲料中之GABA。 與實施例1同樣地操作，進行市售清涼飲料所含GABA 的定量。當測定添加有GABA之清涼飲料及添加有胺基酸之 清涼飲料中的GABA含量時，添加有GABA之清涼飲料之 GABA定量為9842±95ppm，在同一條件下，不含GABA而添 〇 加有胺基酸之清涼飲料則未檢測到GABA。由此等結果可確 認’本發明之方法亦有效於保證添加有GABA之清涼飲料的 GABA含量。 此等結果可確認，依據本發明之方法，可保證廣範圍 之食品中之GABA含量。 比較例 以使用屬於非水溶性曱臢之MTT替代水溶性曱臢 〇 WST-8的方法來進行比較。 在各種精製白米、玄米、發芽玄米之水萃取物中使用 MTT (亦即溴化3-(4,5-二曱基-2-噻唑基）-2,5-二苯基 -2H-四唑鏽）時，即使於低濃度，反應生成物亦會沉澱而 沉積附著於微量盤表面，因而無法進行分析。推測此係由 於食品之水萃取物所含之蛋白質等與MTT甲臢會共同沉澱 之故。再者，當分析含有GABA之巧克力之水萃取物中的 GABA時，由萃取物中所含有色物質所導致之可見光部分之 12 321191 200946908 吸收會妨礙分析。 ' 另一方面，當使用水溶性甲臢時，則無溶解性之問 - 題，藉由增加甲臢濃度而可解決問題。 首先，基本上，本發明係提供一種簡易地定量GABA 之方法，該方法之特徵係包括下述步驟：為了排除與GABA 結構相似之麩胺酸等胺基酸之干擾，而使用特異性轉胺 酶、與要求氧化型菸鹼醯胺腺嘌呤二核苷酸磷酸（以下稱 為MDP)作為輔酶之脫氫酶，生成還原型菸鹼醯胺腺嘌呤 ® 二核苷酸磷酸（以下稱為NADPH)的第1步驟；以及在四 唑鑌鹽之存在下，使生成之NADPH與電子傳達體作用，並 測定生成之水溶性曱臢色素的第2步驟。 詳而言之，本發明係提供一種簡易地定量GABA之方 法，該方法之特徵係包括下述步驟：為了排除與GABA結構 相似之麵胺酸等胺基酸之干擾，而使用特異性轉胺酶、與 要求氧化型菸鹼醯胺腺嘌呤二核苷酸磷酸（以下稱為 Q NADP)作為輔酶之脫氫酶：在生成還原型於驗醯胺腺嗓呤 二核苷酸磷酸（以下稱為NADPH)後，添加硫酸的第1步 驟；以及在四唑鏽鹽之存在下，使生成之NADPH與電子傳 達體作用，並測定生成之水溶性曱臢色素的第2步驟。 更詳細言之，本發明係提供一種飲食品中之T -胺基 酪酸之定量方法，該方法之特徵係包括下述步驟： (1 )使食品之水萃取液或飲料、與7 -胺基酷·酸轉胺 酶、與要求氧化型於驗醢胺腺σ票呤二核苷酸作為輔酶之脫 氫酶進行反應而生成還原型菸鹼醯胺腺嘌呤二核苷酸磷酸 13 321191 200946908 後，添加硫酸的步驟； (2 )在可生成水溶性甲臢色素之四唑鑌鹽之存在 下，使還原型菸鹼醯胺腺嘌呤二核苷酸磷酸與電子傳達體 進行反應，而生成水溶性曱臢色素的步驟；以及 (3)測定水溶性甲臢色素的步驟； 其中，四唑鏽鹽為2-(2-甲氧基-4-硝基苯基）-3-(4-石肖基苯基）-2H-四唾鑌納鹽。 又，由前述之本發明之原理可知，本發明之7—胺基 酿酉欠之疋罝方法並不限定於實施例所示之飲食物中之τ — 胺基酪酸之定量，亦可適用於測定含有與GABA結構相似之 麩胺酸等胺基酸的試料，例如生體檢體試料。 【圖式簡單說明】 第1圖係GABA定量之原理說明圖。 第2圖係使用GABA標準試料之檢量線圖。 第3圖係藉由Dabsy 1衍生化HPLC法定量與本發明定 量的比較線圖。 第4圖係泰國之各種品種之GABA生成能力的檢討圖。 【主要元件符號說明】 無。 14 32119110 mM α-ketoglutaric acid 2 mM 2-mercaptoethanol 0.5 mM NADP 0. 25 U/mL GABAse (from Pseudomonas fluorescens) Reagent 2 1M sulfuric acid reagent 3 1-Me0-PMS, WST-8 The mixed reagent (Cell Counting Kit-8 manufactured by Wako Pure Chemical Co., Ltd.) Using these reagents, the GABA concentration was measured by the following procedure. That is, an aqueous solution having a GABA wave width of 0.1 ppm, 〇·5ρρπι, 1 ppm, 5 ppm, 10 ppm, 20 ppm, 50 ppm, and 100 ppm is used as a sample, and a reagent of 90//L is added to the sample 1 〇〇//L. 1, then warm it at 3 (rc for 15 minutes, then, mix 10 / / L of reagent 2, then add 5 / zL of reagent 3, and then use a micro I disk tester (Sapphire made by Tekan) based The end point method measures the absorbance at a wavelength of 470 nm. In addition, the glutamic acid, serine, glycine, and histidine added to the sample are also measured. As shown in Fig. 2, the GABA is in the range of 50 ppm. The correlation coefficient is 〇999, which shows a high linearity and can be used for quantification. In addition, it also shows that it is not subject to other amino acids such as glutamic acid, silkamine 321191 10 200946908 acid, glycine acid, histidine and the like. (Example 2) - Quantification of GABA in foods. Further, the GABA content in various water extracts of various refined white rice 'myster rice and germinated rice was determined according to Dabsyl Derivatization-HPLC method, and the results are as shown in Fig. 3. Show that the extremely high correlation of 〇· 999 is obtained. It can be seen that 'this method is available Colorimetric analysis quantitative method with the same accuracy as the HPLC method. ® (Example 3) Quantification of GABA in germinated rice. The refined white rice and black rice were immersed in water, and the GABA was quantified at the time of germination. The sample was subjected to water immersion treatment, and the GABA content before and after the treatment was measured by the method shown in Example 1. The results showed that the GABA content in the low-temperature dry rice and the dried rice was increased. On the other hand, it was widely used in Thailand during the rain period. The high-temperature-dried rice was inactivated by the GABA ❹-producing enzyme. As shown in Fig. 4, the GABA content could not be increased, and the reliability of the method of the present invention was confirmed. (Example 4) Quantification of GABA in chocolate Quantification of GABA contained in commercially available chocolate was carried out according to the method of the present invention and HPLC method. The GABA content was measured by the method shown in Example 1 by subjecting the GABA-added chocolate and the usual chocolate to hot water extraction. The GABA content of the chocolate added with GABA was 2895±84 ppm. Under the same conditions, GABA was not detected in the general chocolate. In the HPLC method, 11 321191 200946908 GABA was added. The measured value of the chocolate was 2965±54 ppm, and the average chocolate was not detected. From the results, it was confirmed that the method of the present invention is also effective for ensuring the GABA content of the GABA-added food. (Example 5) Quantifying the refreshing beverage GABA. The amount of GABA contained in a commercially available refreshing beverage was measured in the same manner as in Example 1. When the GABA content in a refreshing beverage to which GABA was added and a refreshing beverage to which an amino acid was added was measured, GABA was added. The GABA content of the beverage was 9842±95 ppm. Under the same conditions, GABA was not detected in the refreshing beverage without GABA and added with amino acid. 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 曱臜 〇 WST-8 with MTT which is a water-insoluble hydrazine. MTT (ie 3-(4,5-dimercapto-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 rust, 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 presumed that this is due to the co-precipitation of the protein contained in the water extract of the food and 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 formazan 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 having a similar GABA structure, a specific transaminating agent is used. An enzyme, and a dehydrogenase which requires oxidized nicotine guanamine adenine dinucleotide phosphate (hereinafter referred to as MDP) as a coenzyme to form reduced nicotine guanamine guanidine dinucleotide phosphate (hereinafter referred to as NADPH) The first step of the method; 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 water-soluble quinone pigment produced. In particular, the present invention provides a method for easily quantifying GABA, the method comprising the steps of: using a specific transaminating agent in order to exclude interference with an amino acid such as a face acid similar to GABA structure An enzyme, and a dehydrogenase that requires oxidized nicotine indoleamine adenine dinucleotide phosphate (hereinafter referred to as Q NADP) as a coenzyme: in the production of a reduced form of adenine dinucleotide phosphate (hereinafter referred to as After NADPH), the first step of adding sulfuric acid; and the second step of reacting the produced NADPH with the electron transporter in the presence of a tetrazolium rust salt, and measuring the water-soluble anthraquinone pigment produced. More specifically, the present invention provides a method for quantifying T-aminobutyric acid in a food or beverage, the method comprising the following steps: (1) making an aqueous extract or beverage of a food, and a 7-amino group Cool acid transaminase, reacting with a dehydrogenase that requires an oxidized form to detect amidoxime genomic DNA as a coenzyme to form reduced nicotine indoleamine adenine dinucleotide phosphate 13 321191 200946908 a step of adding sulfuric acid; (2) reacting the reduced nicotine indoleamine adenine dinucleotide phosphate with an electron carrier in the presence of a tetrazolium salt capable of forming a water-soluble formazan dye to form a water-soluble solution a step of measuring a pigment; and (3) a step of determining a water-soluble formamidine pigment; wherein the tetrazolium rust salt is 2-(2-methoxy-4-nitrophenyl)-3-(4-stone base Phenyl)-2H-tetrapyrene salt. Further, it is understood from the above-described principle of the present invention that the method for the 7-amino-based brewing of the present invention is not limited to the quantification of τ-aminobutyric acid in the food and beverage shown in the examples, and is also applicable to A sample containing an amino acid such as glutamic acid similar to the GABA structure, for example, a sample of a biological sample, was measured. [Simple description of the diagram] Figure 1 is a schematic diagram of the principle of quantitative GABA. Figure 2 is a calibration curve using the GABA standard sample. Figure 3 is a graph comparing the quantitation with the present invention by the Dabsy 1 derivatization HPLC method. Figure 4 is a review of the GABA production capacity of various varieties in Thailand. [Main component symbol description] None. 14 321191