JP5252412B2 - Efficient production method of γ-aminobutyric acid - Google Patents
Efficient production method of γ-aminobutyric acid Download PDFInfo
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- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims description 96
- 238000004519 manufacturing process Methods 0.000 title claims description 41
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- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 52
- 239000004220 glutamic acid Substances 0.000 claims description 51
- 235000013922 glutamic acid Nutrition 0.000 claims description 51
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 36
- 102000008214 Glutamate decarboxylase Human genes 0.000 claims description 21
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- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
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- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明は、γ−アミノ酪酸の効率的生産方法に関する。 The present invention relates to an efficient method for producing γ-aminobutyric acid.
生活習慣病とも呼ばれる高血圧症は、現在通院患者原因の第１位を占めており、心疾患の基礎疾患として医療上大きな問題になっている。また、高血圧患者は高齢化社会の進行とともに益々増加する傾向にあり、予備軍を含めると現在でも１／３の日本人が高血圧患者であると言われている。医療費の急激な膨張が問題になっている現在、疾病治療に加え、病気にならない予防法の確立が益々重要性になってきている。こうした状況下、食品のもつ生体調節機能に着目し、これを疾病の治療や予防に利用しようとする動きが積極的に行われている。高血圧症もその重要なターゲットの一つになっている。 Hypertension, also called lifestyle-related disease, currently occupies the first place as a cause of outpatients and has become a major medical problem as a basic disease of heart disease. In addition, the number of hypertensive patients tends to increase with the progress of an aging society, and it is said that 1/3 of Japanese people are hypertensive patients even now including the reserve army. At present, the rapid expansion of medical expenses is a problem, and in addition to disease treatment, establishment of preventive methods that do not cause disease is becoming more and more important. Under such circumstances, attention has been paid to the bioregulatory function of food, and there is an active movement to use it for the treatment and prevention of diseases. Hypertension is one of the important targets.
γ−アミノ酪酸は神経伝達物質作用を示す非タンパク質性のアミノ酸であり、生体内ではグルタミン酸脱炭酸酵素によってグルタミン酸から生合成される。このγ−アミノ酪酸には血圧降下作用（非特許文献１、非特許文献２）、精神の安定化作用（非特許文献３、非特許文献４）、成長ホルモン分泌促進作用（非特許文献５、非特許文献６）等の生理作用があることが判っている。特に、血圧降下作用については、必要量以上摂取しても正常血圧以下になることはなく、安全性の面でも非常に優れた機能性成分と考えられている。そのため、既にこの特性を生かした製品も「ギャバロン茶」、「乳酸菌発酵飲料」等が上市されている。 γ-Aminobutyric acid is a non-protein amino acid that exhibits neurotransmitter action, and is biosynthesized from glutamic acid by glutamic acid decarboxylase in vivo. This γ-aminobutyric acid has a blood pressure lowering action (Non-Patent Document 1, Non-Patent Document 2), a mental stabilizing action (Non-Patent Document 3, Non-Patent Document 4), a growth hormone secretion promoting action (Non-Patent Document 5, It is known that there are physiological effects such as Non-Patent Document 6). In particular, the blood pressure lowering effect is considered to be an excellent functional component in terms of safety because it does not drop below the normal blood pressure even if it is ingested more than the necessary amount. For this reason, “Gabalon tea”, “lactic acid bacteria fermented beverage”, etc. are already on the market.
このγ−アミノ酪酸の機能性を食品に応用した例は「ギャバロン茶」「乳酸菌発酵飲料」以外にも多くの例がある。例えば、米胚芽の水浸漬によるγ−アミノ酪酸の蓄積・生産（特許文献１）、植物由来グルタミン酸脱炭酸酵素によるγ−アミノ酪酸の生産（特許文献２）、乳酸菌によるγ−アミノ酪酸の生産（特許文献３）若しくは麹菌によるγ−アミノ酪酸の生産（特許文献４）などである。また、上記の開示された特許のγ−アミノ酪酸の生産性を改善し、多量の米胚芽を用いたγ−アミノ酪酸の生成法として、γ−アミノ酪酸の生成法（特許文献５）が開示される。 There are many examples in which the functionality of γ-aminobutyric acid is applied to foods other than “Gabalon tea” and “lactic acid bacteria fermented beverage”. For example, accumulation and production of γ-aminobutyric acid by water immersion of rice germ (Patent Document 1), production of γ-aminobutyric acid by plant-derived glutamic acid decarboxylase (Patent Document 2), production of γ-aminobutyric acid by lactic acid bacteria ( Patent Document 3) or production of γ-aminobutyric acid by Aspergillus oryzae (Patent Document 4). Moreover, the production method of (gamma) -aminobutyric acid (patent document 5) is disclosed as a production method of (gamma) -aminobutyric acid which improved the productivity of (gamma) -aminobutyric acid of the said disclosed patent, and used a lot of rice germ. Is done.
しかしながら、上記特許においては、広範な食品に利用可能な非常に安価なγ−アミノ酪酸を多量に安定的に製造するという目的からするとその生成、生産法として、（１）生産工程が複雑或いは生産にまだ時間かかるため、生産性が十分でなく微生物の繁殖による反応液の変色、腐敗等の問題がある。（２）得られるγ−アミノ酪酸の濃度が比較的低濃度であるためγ−アミノ酪酸を十分低コストで生産することができない。（３）γ−アミノ酪酸の生産に利用する各種素材の風味がγ−アミノ酪酸溶液に残り、γ−アミノ酪酸溶液の幅広い食品への応用が困難である。等の欠点がありいずれも十分なものとは言えない。 However, in the above-mentioned patent, for the purpose of stably producing a large amount of very inexpensive γ-aminobutyric acid that can be used for a wide range of foods, as its production and production method, (1) production process is complicated or production However, it takes time, so that the productivity is not sufficient, and there are problems such as discoloration of the reaction solution due to the propagation of microorganisms and decay. (2) Since the concentration of γ-aminobutyric acid obtained is relatively low, γ-aminobutyric acid cannot be produced at a sufficiently low cost. (3) The flavors of various materials used for the production of γ-aminobutyric acid remain in the γ-aminobutyric acid solution, and it is difficult to apply the γ-aminobutyric acid solution to a wide range of foods. However, none of them are sufficient.
また、上記方法の内、特許文献６に開示された技術においては、γ−アミノ酪酸を米胚芽等のグルタミン酸脱炭酸酵素を用い添加グルタミン酸及び米胚芽等に含まれるグルタミン酸からγ−アミノ酪酸を効率良く生産する方法が開示されているが、実施例を見る限り、（１）最大でも得られたγ−アミノ酪酸溶液の濃度は反応時間６時間で３６ｇ／ｌ程度であり、十分な高濃度のγ−アミノ酪酸溶液が生産できているとは言えない。（２）反応時間が６時間と長く反応温度が高いため微生物汚染のリスクが高い。（３）米胚芽等を用いた場合米胚芽や米糠特有の風味が反応終了溶液に残るため、広範な食品への利用が困難である。等の欠点があり、より安価で広範な食品への利用可能なγ−アミノ酪酸溶液の生産法が求められていた。
本発明は、上記状況に鑑みてなされたもので、γ−アミノ酪酸の血圧上昇抑制作用等の各種生理機能を幅広い食品に応用するため、大量の高品質のγ−アミノ酪酸溶液を短時間に効率よくより高濃度、安価、簡便に製造することができる実用性に秀れた技術を提供することを目的とするものである。 The present invention has been made in view of the above circumstances, and in order to apply various physiological functions such as blood pressure increase inhibitory action of γ-aminobutyric acid to a wide range of foods, a large amount of high-quality γ-aminobutyric acid solution can be applied in a short time. An object of the present invention is to provide a technique excellent in practicality that can be efficiently produced at a higher concentration, at a lower cost, and simply.
本発明者らは、上記問題について鋭意研究を行った結果、グルタミン酸或いはグルタミン酸塩を含む溶液及び懸濁液に適当量の小麦胚芽とピリドキサルリン酸を添加して反応させ、至適温度、ｐＨで前期溶液中のグルタミン酸と小麦胚芽中のグルタミン酸を小麦胚芽中のグルタミン酸脱炭酸酵素の酵素作用によりγ−アミノ酪酸に変換し、高濃度のγ−アミノ酪酸を簡便、安価、効率的に生産する方法を完成した。 As a result of intensive studies on the above problems, the present inventors have added and reacted an appropriate amount of wheat germ and pyridoxal phosphate to a solution and suspension containing glutamic acid or glutamate, and at the optimum temperature and pH, the previous period. A method for converting glutamic acid in solution and glutamic acid in wheat germ to γ-aminobutyric acid by the enzymatic action of glutamic acid decarboxylase in wheat germ to produce high-concentration γ-aminobutyric acid simply, inexpensively and efficiently. completed.
即ち、本発明は、
１．グルタミン酸或いはグルタミン酸塩を含む溶液及び懸濁液に小麦胚芽とピリドキサルリン酸を添加して反応させ、前期溶液中のグルタミン酸と小麦胚芽中のグルタミン酸を小麦胚芽中のグルタミン酸脱炭酸酵素の酵素作用によりγ−アミノ酪酸とすることを特徴とするγ−アミノ酪酸の効率的生産方法、
２．γ−アミノ酪酸の生産のための反応時間が４時間以内であり、且つ、反応終了時のグルタミン酸からのγ−アミノ酪酸の生成収率が９５％以上である請求項１記載のγ−アミノ酪酸の効率的生産方法、
３．γ−アミノ酪酸の生産に使用する小麦胚芽が製粉メーカーで小麦のコマーシャルミル製粉時に生産される市販小麦胚芽である請求項１又は２記載のγ−アミノ酪酸の効率的生産方法、
４．γ−アミノ酪酸の生産に使用する小麦胚芽添加前のグルタミン酸溶液及び懸濁液のグルタミン酸濃度が５０ｇ／ｌ以上である請求項１、２及び３のいずれか一項記載のγ−アミノ酪酸の効率的生産方法、
に関する。
That is, the present invention
1. Glutamate or glutamate is added to and reacted with wheat germ and pyridoxal phosphate, and glutamate in the previous solution and glutamate in the wheat germ are converted to γ- by the enzymatic action of glutamate decarboxylase in the wheat germ. An efficient production method of γ-aminobutyric acid, characterized in that it is aminobutyric acid,
2. The reaction time for production of γ-aminobutyric acid is within 4 hours, and the production yield of γ-aminobutyric acid from glutamic acid at the end of the reaction is 95% or more. Efficient production method,
3. The method for efficiently producing γ-aminobutyric acid according to claim 1 or 2, wherein the wheat germ used for production of γ-aminobutyric acid is a commercial wheat germ produced at the time of milling a commercial mill of wheat by a milling maker.
4). The efficiency of γ-aminobutyric acid according to any one of claims 1, 2 and 3, wherein the glutamic acid solution and suspension before addition of wheat germ used for the production of γ-aminobutyric acid have a glutamic acid concentration of 50 g / l or more. Production method,
About.
本発明は、高濃度のグルタミン酸若しくはグルタミン酸の塩(グルタミン酸ナトリウム等)を含む溶液に一定量以上のグルタミン酸脱炭酸酵素を含む小麦胚芽及びピリドキサルリン酸を添加して至適温度、ｐＨで反応させ、該添加溶液中のグルタミン酸と小麦胚芽中のグルタミン酸とを、小麦胚芽中のグルタミン酸脱炭酸酵素の酵素作用により短時間、効率的にγ−アミノ酪酸に変換し、安定的に安価に高濃度γ−アミノ酪酸溶液を調製する方法を提供するものである。 The present invention adds a wheat germ containing a certain amount of glutamate decarboxylase and pyridoxal phosphate to a solution containing a high concentration of glutamic acid or a glutamic acid salt (such as sodium glutamate) and reacts at an optimum temperature and pH, The glutamic acid in the added solution and the glutamic acid in the wheat germ are efficiently converted into γ-aminobutyric acid in a short time by the enzymatic action of glutamic acid decarboxylase in the wheat germ, and stably and inexpensively high concentration γ-amino A method for preparing a butyric acid solution is provided.
本発明によるγ−アミノ酪酸溶液の調製法は、非常に反応効率が良いため適切な条件を選択することによって、４時間以内の反応時間内に反応終了時のグルタミン酸からのγ−アミノ酪酸の生成収率が９５％以上で高濃度のγ−アミノ酪酸溶液を調製することが容易に達成できる。γ−アミノ酪酸の生成収率を高めることは、γ−アミノ酪酸溶液をクルードな溶液として食品等に使用する場合特に重要である。その理由は、残存グルタミン酸が溶液中に多い場合、添加した食品の風味に影響するだけでなく、グルタミン酸がγ−アミノ酪酸とは逆の興奮性の神経伝達物質（野口ら：FOOD Style 21, 10(5), 1 (2006)）で、溶液中のγ−アミノ酪酸の精神安定化効果を阻害する可能性があるからである。 Since the method for preparing a γ-aminobutyric acid solution according to the present invention is very efficient in reaction, the production of γ-aminobutyric acid from glutamic acid at the end of the reaction is completed within 4 hours by selecting appropriate conditions. It can be easily achieved to prepare a high concentration γ-aminobutyric acid solution with a yield of 95% or more. Increasing the production yield of γ-aminobutyric acid is particularly important when the γ-aminobutyric acid solution is used as a crude solution in foods and the like. The reason is that when the amount of residual glutamic acid is high in the solution, not only does it affect the flavor of the added food, but also the excitatory neurotransmitter that glutamate is opposite to γ-aminobutyric acid (Noguchi et al .: FOOD Style 21, 10 (5), 1 (2006)), there is a possibility of inhibiting the tranquilizing effect of γ-aminobutyric acid in the solution.
グルタミン酸脱炭酸酵素源としては小麦胚芽を使用するが、小麦胚芽の小麦の品種、銘柄には特に限定は無くいずれの品種、銘柄の小麦胚芽も使用することができる。また、好ましくは製粉メーカーのコマーシャルミルにおける小麦の製粉過程で生成する市販小麦胚芽がより好ましい。その理由は、市販小麦胚芽のグルタミン酸脱炭酸酵素の活性が非常に高く、活性値がロット内で安定しているからである。更に、小麦胚芽中のグルタミン酸脱炭酸酵素は抽出或いは精製し、これを懸濁・溶解または固定化したものを用いることも可能である。 As a glutamate decarboxylase source, wheat germ is used, but there are no particular limitations on the wheat cultivar and brand, and any cultivar and brand of wheat germ can be used. Moreover, the commercially available wheat germ produced | generated in the milling process of the wheat in the commercial mill of a milling maker is more preferable. This is because the activity of glutamic acid decarboxylase in commercial wheat germ is very high and the activity value is stable in the lot. Furthermore, glutamic acid decarboxylase in wheat germ can be extracted or purified, and suspended, dissolved or immobilized.
反応溶液中のグルタミン酸或いはグルタミン酸の塩の量は、γ−アミノ酪酸の希望生産量や触媒源となる小麦胚芽の使用量に基づいて決定すればよいが、より生産性を高めるためには好ましくは小麦胚芽添加前の反応液のグルタミン酸としての濃度を５０ｇ／ｌ以上に設定することが好ましく、反応の至適温度４０℃、至適ｐＨ５．７でグルタミン酸或いはグルタミン酸の塩が完全に溶解しない量添加して多量のγ−アミノ酪酸を生産することも可能である。γ−アミノ酪酸の溶解度は非常に高いため、反応の初期に溶解しない量の多量のグルタミン酸或いはグルタミン酸の塩を添加しても本願発明の方法によれば、反応の進行と共に溶解していなかったグルタミン酸或いはグルタミン酸の塩が溶解し、高濃度のγ−アミノ酪酸溶液の生産を短時間に行うことができる。 The amount of glutamic acid or glutamic acid salt in the reaction solution may be determined based on the desired production amount of γ-aminobutyric acid or the amount of wheat germ used as a catalyst source. It is preferable to set the concentration of glutamic acid in the reaction solution before addition of wheat germ to 50 g / l or more, and the addition of an amount that does not completely dissolve glutamic acid or glutamic acid salt at an optimal temperature of reaction of 40 ° C. and an optimal pH of 5.7 Thus, a large amount of γ-aminobutyric acid can be produced. Since the solubility of γ-aminobutyric acid is very high, even if a large amount of glutamic acid or a salt of glutamic acid that does not dissolve in the initial stage of the reaction is added, according to the method of the present invention, glutamic acid that was not dissolved with the progress of the reaction Or the salt of glutamic acid melt | dissolves and production of a high concentration (gamma) -aminobutyric acid solution can be performed in a short time.
添加するピリドキサルリン酸の量は生産したいγ−アミノ酪酸の量や希望とする基質転換率（反応開始時に反応液に存在するグルタミン酸量のうち、γ−アミノ酪酸に転換されたものの量比）に基づいて決定すればよいが、好ましくは反応液中のグルタミン酸量（グルタミン酸塩はクルタミン酸として換算）とピリドキサルリン酸の重量比が、２０（グルタミン酸量）:１（ピリドキサルリン酸量）〜５０００（グルタミン酸量）：１（ピリドキサルリン酸量）になるようにするのが好ましく、更に好ましくは、２０（グルタミン酸量）:１（ピリドキサルリン酸量）〜１０００（グルタミン酸量）：１（ピリドキサルリン酸量）になるようにするのがより良好である。ピリドキサルリン酸は精製或いは抽出標品が利用可能である。また、ピリドキサルリン酸はビタミンＢ６の活性成分の１つとして広範な生物材料に含まれるので、これらをピリドキサルリン酸源として利用することもできる。また、ピリドキサルリン酸はピリドキサルをリン酸化することでも合成できるので、ピリドキサルからの合成系を併用してもよい。 The amount of pyridoxal phosphate to be added is based on the amount of γ-aminobutyric acid to be produced 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 to the amount converted to γ-aminobutyric acid). Preferably, the weight ratio of glutamic acid in the reaction solution (glutamate is converted to glutamic acid) and pyridoxalphosphoric acid is 20 (glutamic acid content): 1 (pyridoxallic acid content) to 5000 (glutamic acid content) : 1 (pyridoxal phosphate amount) is preferable, and more preferably, 20 (glutamate amount): 1 (pyridoxal phosphate amount) to 1000 (glutamate amount): 1 (pyridoxal phosphate amount). Is better. For pyridoxal phosphate, purified or extracted preparations can be used. Moreover, since pyridoxal phosphate is contained in a wide range of biological materials as one of the active ingredients of vitamin B6, it can also be used as a source of pyridoxal phosphate. In addition, since pyridoxal phosphate can be synthesized by phosphorylating pyridoxal, a synthesis system from pyridoxal may be used in combination.
反応条件は、ｐＨは４．０〜７．０、好ましくは５．２〜６.０とすると良い。反応温度は３０〜５０℃、好ましくは３５〜４５℃とすると良い。この反応条件を外れるとγ−アミノ酪酸の生成速度が極端に遅くなったり、グルタミン酸脱炭酸酵素が失活して反応が停止したりする弊害がある。ｐＨ調整に用いる酸は無機酸と有機酸のいずれも使用可能であるが、反応速度を高めるためには、無機酸の塩酸、硫酸、硝酸等を用いるのが好ましく、ｐＨ調整用アルカリとしては、水酸化ナトリウム、水酸化カリウム、水酸化カルシウム等が用いられる。当然、これらのｐＨ調整用の酸、アルカリはこれらに限定されるものではない。 The reaction conditions are such that the pH is 4.0 to 7.0, preferably 5.2 to 6.0. The reaction temperature is 30-50 ° C, preferably 35-45 ° C. If the reaction conditions are not met, the production rate of γ-aminobutyric acid becomes extremely slow, or glutamate decarboxylase is inactivated and the reaction is stopped. As the acid used for pH adjustment, either an inorganic acid or an organic acid can be used. In order to increase the reaction rate, it is preferable to use inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, etc. Sodium hydroxide, potassium hydroxide, calcium hydroxide, etc. are used. Of course, these acids and alkalis for pH adjustment are not limited to these.
上記したように本願発明のγ−アミノ酪酸溶液の生産方法は非常に簡便で効率が良いため、４時間以内の反応時間で４０ｇ／ｌ以上の高濃度のγ−アミノ酪酸溶液を多量に容易に調製することが可能である。また、反応効率が非常に高いため、風味に影響しγ−アミノ酪酸とは逆の生理作用を示すグルタミン酸の残存量を非常に低くすることが可能である。更に、生産に使用する材料は小麦胚芽、アミノ酸であるグルタミン酸やその塩、ビタミンＢ６の活性成分であるピリドキサルリン酸、ｐＨ調整用の酸やアルカリであり、いずれの成分も食品に用いた場合に有害作用を及ぼすものでないため、反応液そのもの、反応液からの懸濁物除去、脱色、必要に応じて脱塩等の簡単な調製操作を行うのみで食品素材等として供給することができる。 As described above, since the production method of the γ-aminobutyric acid solution of the present invention is very simple and efficient, a large amount of γ-aminobutyric acid solution having a high concentration of 40 g / l or more can be easily produced in a reaction time within 4 hours. It is possible to prepare. Moreover, since the reaction efficiency is very high, it is possible to reduce the residual amount of glutamic acid that affects the flavor and exhibits a physiological action opposite to that of γ-aminobutyric acid. In addition, the materials used for production are wheat germ, amino acid glutamic acid and salts thereof, pyridoxalphosphoric acid, which is an active ingredient of vitamin B6, and pH-adjusting acids and alkalis, all of which are harmful when used in foods. Since it does not act, it can be supplied as a food material or the like only by performing simple preparation operations such as reaction solution itself, suspension removal from the reaction solution, decolorization, and desalting as necessary.
また、高度な精製標品が必要な場合は、イオン交換クロマトグラフィーなどの常法を用いることでγ−アミノ酪酸の簡易精製が可能である。得られた標品は高純度のγ−アミノ酪酸を含むため、少量の標品を摂取、或いは食品中に混合するだけでγ−アミノ酪酸の血圧上昇抑制作用等の生理機能が期待できる。このように、本実施例によって生産したγ−アミノ酪酸液は食品素材及び栄養強化食品としても有効であると考えられる。 In addition, when a highly purified preparation is required, γ-aminobutyric acid can be simply purified by using a conventional method such as ion exchange chromatography. Since the obtained preparation contains high-purity γ-aminobutyric acid, physiological functions such as an antihypertensive action of γ-aminobutyric acid can be expected by ingesting a small amount of preparation or mixing it with food. Thus, it is considered that the γ-aminobutyric acid solution produced by this example is effective as a food material and a nutrition-enriched food.
次に、以下に示す実施例（比較例を含む）に基づいて、本発明を更に詳細に説明するが、本発明はこれらの実施例に何ら限定されるものではない。 Next, the present invention will be described in more detail based on the following examples (including comparative examples), but the present invention is not limited to these examples.
［実施例１］
キタノカオリの製粉過程で得られた市販小麦胚芽（江別製粉製）（以下市販小麦胚芽）、キタノカオリのビューラー製粉時に得られコブスマより特開平８−２８０３８４の方法により調製した小麦胚芽及び米糠（市販品）それぞれ２５０ｇを、１ｌのグルタミン酸６０ｇ、ピリドキサルリン酸３００ｍｇを含有する溶液１ｌに添加し、４０℃、ｐＨ５．７±０．１で４時間反応を行った。ピリドキサルリン酸は１時間毎に３００ｍｇ追加添加した。ｐＨはｐＨコントローラーを用いて主に塩酸を適時添加することによって制御した。４時間後に７０％になるようにエタノールを反応液に添加し反応を停止させた。反応溶液上清中のグルタミン酸とγ−アミノ酪酸の濃度は、ο−フタルアルデヒドで蛍光標識した後ＨＰＬＣで分析することにより定量し、表１の結果を得た。
[Example 1]
Commercial wheat germ (produced by Ebetsu Flour Milling) obtained during the milling process of Kitanokaori (hereinafter, commercially available wheat germ), wheat germ and rice bran prepared from Kobuma obtained at the time of milling Kitanokaori by the method of JP-A-8-280384 (commercial product) 250 g of each was added to 1 l of a solution containing 1 g of glutamic acid 60 g and pyridoxalphosphoric acid 300 mg, and reacted at 40 ° C. and pH 5.7 ± 0.1 for 4 hours. An additional 300 mg of pyridoxal phosphate was added every hour. The pH was controlled mainly by adding hydrochloric acid in a timely manner using a pH controller. After 4 hours, ethanol was added to the reaction solution to 70% to stop the reaction. The concentrations of glutamic acid and γ-aminobutyric acid in the reaction solution supernatant were quantified by fluorescent labeling with o-phthalaldehyde and analysis by HPLC, and the results shown in Table 1 were obtained.
表１より、グルタミン酸脱炭酸酵素源の酵素活性は市販小麦胚芽が最も高く、コブスマから調製した小麦胚芽が市販小麦胚芽の９０．８％、市販米糠の活性が６３．２％であった。なお、酵素活性は実施例４と同様の方法で測定した。反応に市販小麦胚芽を用いた場合γ−アミノ酪酸の生成収率は試験例２、比較例１に比べ非常に高く、ほぼ１００％の収率でグルタミン酸からγ−アミノ酪酸が生成し、その濃度は約４５ｇ／ｌと非常に高濃度であった。また、小麦胚芽を用いた場合米糠に比べ反応終了時の反応液の色、風味が非常に良好であった。 From Table 1, the enzyme activity of the glutamic acid decarboxylase source was highest in commercial wheat germ, with wheat germ prepared from cobsum 90.8% of commercial wheat germ and commercial rice bran activity 63.2%. The enzyme activity was measured by the same method as in Example 4. When commercially available wheat germ is used for the reaction, the production yield of γ-aminobutyric acid is very high compared to Test Example 2 and Comparative Example 1, and γ-aminobutyric acid is produced from glutamic acid at a yield of almost 100%. Was a very high concentration of about 45 g / l. In addition, when wheat germ was used, the color and flavor of the reaction solution at the end of the reaction were very good compared to rice bran.
以上の結果から、γ−アミノ酪酸の生産のためのグルタミン酸脱炭酸酵素源としては市販小麦胚芽が最適であり、適当量の市販小麦胚芽を用いることにより、上記の至適反応条件で４時間γ−アミノ酪酸の生成反応を行うことによって、高濃度のγ−アミノ酪酸溶液を簡易に生産できることが判る。 From the above results, commercially available wheat germ is optimal as a glutamic acid decarboxylase source for the production of γ-aminobutyric acid. By using an appropriate amount of commercially available wheat germ, γ for 4 hours under the above optimal reaction conditions -It turns out that a high concentration (gamma) -aminobutyric acid solution can be produced easily by performing the production | generation reaction of aminobutyric acid.
［実施例２］
キタノカオリの製粉過程で得られた市販小麦胚芽（江別製粉製）２５０ｇを、グルタミン酸６０ｇ、種々の量のピリドキサルリン酸を含有する溶液１ｌに添加し、４０℃、ｐＨ５．７±０．１で４時間反応を行った。ピリドキサルリン酸は４回に分けて１時間毎に追添加した。ｐＨはｐＨコントローラーを用いて主に塩酸を適時添加することによって制御した。反応液の反応停止とグルタミン酸及びγ−アミノ酪酸の定量は実施例１と同様行い、表２の結果を得た。
[Example 2]
250 g of commercially available wheat germ (made by Ebetsu Flour Milling) obtained during the milling process of Kitanokaori was added to 1 l of a solution containing 60 g of glutamic acid and various amounts of pyridoxal phosphoric acid, and 4 hours at 40 ° C. and pH 5.7 ± 0.1. Reaction was performed. Pyridoxalphosphoric acid was added in increments of 4 times every hour. The pH was controlled mainly by adding hydrochloric acid in a timely manner using a pH controller. The reaction was stopped and the amounts of glutamic acid and γ-aminobutyric acid were determined in the same manner as in Example 1, and the results shown in Table 2 were obtained.
表２より、γ−アミノ酪酸の生成量はピリドキサルリン酸濃度に依存することが判明した。生成γ−アミノ酪酸量はピリドキサルリン酸無添加では、７．７ｇ／ｌと非常に少ないのに対し、ピリドキサルリン酸添加によりその生成量は飛躍的に向上し、１００ｍｇ／ｌ以上添加することによって、９７％以上の収率でグルタミン酸からγ−アミノ酪酸が生成し、その濃度は約４５ｇ／ｌと非常に高濃度であった。 From Table 2, it was found that the amount of γ-aminobutyric acid produced depends on the pyridoxal phosphate concentration. The amount of γ-aminobutyric acid produced is very low at 7.7 g / l without the addition of pyridoxal phosphoric acid, whereas the amount produced is greatly improved by the addition of pyridoxal phosphoric acid. Γ-aminobutyric acid was produced from glutamic acid in a yield of at least%, and its concentration was very high at about 45 g / l.
以上の結果から、適当量のピリドキサルリン酸を用い、上記の至適反応条件で４時間γ−アミノ酪酸の生成反応を行うことによって、高濃度のγ−アミノ酪酸溶液を簡易に生産できることが判る。 From the above results, it can be seen that a high-concentration γ-aminobutyric acid solution can be easily produced by carrying out a γ-aminobutyric acid production reaction for 4 hours under the above optimal reaction conditions using an appropriate amount of pyridoxalphosphoric acid.
［実施例３］
市販小麦胚芽２５０ｇを、種々の濃度のグルタミン酸とピリドキサルリン酸１００ｍｇを含有する溶液１ｌに添加し、４０℃、ｐＨ５．７±０．１で４時間反応を行った。ピリドキサルリン酸は１００ｍｇを４回に分けて１時間毎に添加した。ｐＨはｐＨコントローラーを用いて主に塩酸を適時添加することによって制御した。反応液の反応停止とグルタミン酸とγ−アミノ酪酸の定量は実施例１と同様行い、表３の結果を得た。
[Example 3]
250 g of commercially available wheat germ was added to 1 liter of a solution containing various concentrations of glutamic acid and 100 mg of pyridoxal phosphoric acid, and reacted at 40 ° C. and pH 5.7 ± 0.1 for 4 hours. 100 mg of pyridoxal phosphate was added in 4 portions every hour. The pH was controlled mainly by adding hydrochloric acid in a timely manner using a pH controller. The reaction was stopped and the amounts of glutamic acid and γ-aminobutyric acid were determined in the same manner as in Example 1, and the results shown in Table 3 were obtained.
表３より、γ−アミノ酪酸の転換収率はいずれの試験例でも９５％以上であり、生成γ−アミノ酪酸量は添加したグルタミン酸量に比例して増加することが判る。 From Table 3, it can be seen that the conversion yield of γ-aminobutyric acid is 95% or more in all the test examples, and the amount of produced γ-aminobutyric acid increases in proportion to the amount of added glutamic acid.
以上の結果から、適当量のピリドキサルリン酸を用い、上記の至適反応条件で４時間γ−アミノ酪酸の生成反応を行うことによって、非常に高濃度のγ−アミノ酪酸溶液を簡易、短時間に９５％以上の高収率で生産できることが判る。特に、試験例９の１００ｇ／ｌのグルタミン酸溶液を用いた場合には、４時間の反応で６６ｇ／ｌ以上のγ−アミノ酪酸の高濃度溶液が得られた。この結果から、本発明の技術を用い、高濃度のグルタミン酸溶液、懸濁液を用いることにより、小さいスケールで多量のγ−アミノ酪酸を安価に簡易に製造できることが明らかになった。 From the above results, a very high concentration γ-aminobutyric acid solution can be obtained easily and in a short time by carrying out a γ-aminobutyric acid production reaction for 4 hours under the above optimal reaction conditions using an appropriate amount of pyridoxalphosphoric acid. It can be seen that it can be produced with a high yield of 95% or more. In particular, when the 100 g / l glutamic acid solution of Test Example 9 was used, a high concentration solution of γ-aminobutyric acid of 66 g / l or more was obtained after 4 hours of reaction. From this result, it became clear that a large amount of γ-aminobutyric acid can be easily and inexpensively produced on a small scale by using the technology of the present invention and using a high concentration glutamic acid solution and suspension.
［実施例４］
０．３Ｍのグルタミン酸と０．５ｍｇのピリドキサルリン酸を含む種々のｐＨの１Ｍリン酸バッファー溶液５ｍｌに市販小麦胚芽１０ｇより１０倍量の蒸留水で抽出したグルタミン酸脱炭酸酵素粗酵素０．１ｍｌを添加し、４０℃で適当な時間反応させた。反応を停止させた後、反応液のγ−アミノ酪酸の濃度を実施例１と同様に定量し、種々のｐＨ溶液中でのグルタミン酸脱炭酸酵素粗酵素の活性を測定し、表４の結果を得た。
[Example 4]
0.1 ml of glutamic acid decarboxylase crude enzyme extracted with 10-fold amount of distilled water from 10 g of commercial wheat germ was added to 5 ml of 1M phosphate buffer solution containing 0.3 M glutamic acid and 0.5 mg pyridoxal phosphate. And allowed to react at 40 ° C. for an appropriate time. After stopping the reaction, the concentration of γ-aminobutyric acid in the reaction solution was quantified in the same manner as in Example 1, and the activity of glutamate decarboxylase crude enzyme in various pH solutions was measured. Obtained.
表４より、小麦胚芽中のグルタミン酸脱炭酸酵素の至適ｐＨはｐＨ５．２〜６．０の間にあり、特に５．６〜５．８の間で高い活性を示した。 From Table 4, the optimum pH of glutamic acid decarboxylase in wheat germ is between pH 5.2 and 6.0, and particularly shows high activity between 5.6 and 5.8.
以上の結果から、なるべく少ない小麦胚芽を用い効率的に短時間で多量のγ−アミノ酪酸を得るためには、ｐＨ５．６〜５．８の範囲でγ−アミノ酪酸の生成反応を行うのが最適であることが明らかになった。 From the above results, in order to efficiently obtain a large amount of γ-aminobutyric acid in a short time using as little wheat germ as possible, the production reaction of γ-aminobutyric acid is carried out in the range of pH 5.6 to 5.8. It turned out to be optimal.
［実施例５］
０．３Ｍのグルタミン酸と０．５ｍｇのピリドキサルリン酸を含むｐＨ５．５の１Ｍリン酸バッファー溶液５ｍｌに市販小麦胚芽１０ｇより１０倍量の蒸留水で抽出したグルタミン酸脱炭酸酵素粗酵素０．１ｍｌを添加し、表５に示す種々の温度で１時間反応を行い、反応を停止させた後、反応液のγ−アミノ酪酸の濃度を実施例１と同様に定量し、種々の温度でのグルタミン酸脱炭酸酵素粗酵素の活性を測定し、表５の結果を得た。
[Example 5]
0.1 ml of glutamic acid decarboxylase crude enzyme extracted with 10-fold amount of distilled water from 10 g of commercially available wheat germ was added to 5 ml of 1M phosphate buffer solution of pH 5.5 containing 0.3 M glutamic acid and 0.5 mg pyridoxal phosphate. Then, after reacting at various temperatures shown in Table 5 for 1 hour and stopping the reaction, the concentration of γ-aminobutyric acid in the reaction solution was quantified in the same manner as in Example 1, and glutamic acid decarboxylation at various temperatures. The activity of the crude enzyme was measured and the results shown in Table 5 were obtained.
表５より、小麦胚芽中のグルタミン酸脱炭酸酵素の至適温度は４０℃前後にあり、特に４０℃の時高い活性を示した。 From Table 5, the optimum temperature of glutamate decarboxylase in wheat germ is around 40 ° C., and particularly shows high activity at 40 ° C.
以上の結果から、なるべく少ない小麦胚芽を用い効率的に短時間で多量のγ−アミノ酪酸を得るためには、４０℃前後でγ−アミノ酪酸の生成反応を行うのが最適であることが明らかになった。 From the above results, it is clear that in order to efficiently obtain a large amount of γ-aminobutyric acid in as short a time as possible using as little wheat germ as possible, it is optimal to perform a γ-aminobutyric acid production reaction at around 40 ° C. Became.
［実施例６］
市販小麦胚芽２５０ｇを、グルタミン酸６０ｇ、ピリドキサルリン酸１００ｍｇを含有する溶液に添加し、４０℃、ｐＨ５．７±０．１で８時間目まで反応を行った。ピリドキサルリン酸は１００ｍｇを１時間毎に追添加した。ｐＨはｐＨコントローラーを用いて主に塩酸を適時添加することによって制御した。反応液は１時間毎にサンプリングし、反応液の反応停止とグルタミン酸とγ−アミノ酪酸の定量を実施例１と同様に行い、表６の結果を得た。
[Example 6]
250 g of commercially available wheat germ was added to a solution containing 60 g of glutamic acid and 100 mg of pyridoxal phosphoric acid and reacted at 40 ° C. and pH 5.7 ± 0.1 for up to 8 hours. 100 mg of pyridoxal phosphoric acid was added every hour. The pH was controlled mainly by adding hydrochloric acid in a timely manner using a pH controller. The reaction solution was sampled every hour, and the reaction was stopped and glutamic acid and γ-aminobutyric acid were quantified in the same manner as in Example 1. The results shown in Table 6 were obtained.
表６より、γ−アミノ酪酸の転換収率は反応３時間で９５％以上と非常に高くなり、３時間以降ではほぼ１００％で約４５ｇ／ｌのγ−アミノ酪酸が得られた。しかし、反応７時間以上になると反応液の酸化、雑菌の増殖等による反応液の色、風味の劣化が起こった。 From Table 6, the conversion yield of γ-aminobutyric acid was very high at 95% or more after 3 hours of reaction, and about 45 g / l of γ-aminobutyric acid was obtained at almost 100% after 3 hours. However, when the reaction time was 7 hours or longer, the reaction solution was deteriorated in color and flavor due to oxidation of the reaction solution and proliferation of various bacteria.
以上の結果から、食品等に安全、好適に利用できる品質良好なγ−アミノ酪酸溶液を本発明の方法により調製するためには、上記の至適反応条件下で４時間以内に反応が終了するような条件でγ−アミノ酪酸の生成反応を行うことが重要であることが判る。 From the above results, in order to prepare a γ-aminobutyric acid solution of good quality that can be safely and suitably used for foods and the like by the method of the present invention, the reaction is completed within 4 hours under the above optimal reaction conditions. It turns out that it is important to perform the production | generation reaction of (gamma) -aminobutyric acid on such conditions.
〔発明の効果〕
本願発明によれば、小麦の製粉過程で安定的に排出され安価に入手が可能である小麦胚芽をグルタミン酸脱炭酸酵素源とし、このグルタミン酸脱炭酸酵素により高濃度のグルタミン酸と適当量のピリドキサルリン酸を含有する溶液中で適当な反応条件でγ−アミノ酪酸の生成反応を行うことにより、大量の高濃度γ−アミノ酪酸溶液を簡便、短時間に生産することが可能になる。
〔Effect of the invention〕
According to the present invention, wheat germ that is stably discharged during wheat milling and can be obtained at low cost is used as a glutamic acid decarboxylase source. With this glutamic acid decarboxylase, a high concentration of glutamic acid and an appropriate amount of pyridoxal phosphate can be obtained. By carrying out the reaction for producing γ-aminobutyric acid in the contained solution under suitable reaction conditions, a large amount of high-concentration γ-aminobutyric acid solution can be produced easily and in a short time.
具体的には、本願発明の方法を用いることによって、４０ｇ／ｌ以上の高濃度γ−アミノ酪酸溶液を４時間以内の短時間の反応時間で簡便に生産することが可能になり、生成収率（グルタミン酸からのγ−アミノ酪酸の生成収率）も９５％以上で、風味に影響しγ−アミノ酪酸と逆の生理作用を示すグルタミン酸の残存量の非常に少ないγ−アミノ酪酸溶液を調製することができる。 Specifically, by using the method of the present invention, it becomes possible to easily produce a high-concentration γ-aminobutyric acid solution of 40 g / l or more in a short reaction time within 4 hours, and the production yield. (Production yield of γ-aminobutyric acid from glutamic acid) is also 95% or more, and prepares a γ-aminobutyric acid solution with a very small residual amount of glutamic acid that affects the flavor and exhibits a physiological action opposite to that of γ-aminobutyric acid. be able to.
このように本願発明の技術により、従来のγ−アミノ酪酸の生産方法に比べ飛躍的に高収率で、簡便、効率的に高濃度のγ−アミノ酪酸溶液を生産することが可能になる。これにより、大量のγ−アミノ酪酸を安全、安価、容易に製造することができ、需要の莫大なγ−アミノ酪酸の安価での安定的供給に多大な寄与が期待できる。 As described above, the technology of the present invention makes it possible to produce a highly concentrated γ-aminobutyric acid solution easily and efficiently at a significantly higher yield than conventional γ-aminobutyric acid production methods. As a result, a large amount of γ-aminobutyric acid can be produced safely, inexpensively and easily, and a great contribution can be expected to a stable and inexpensive supply of γ-aminobutyric acid which is in great demand.
更に、安価に安定的に供給されるγ−アミノ酪酸を用いて種々の食品素材や栄養機能食品等を安価に簡便かつ効率的に製造することができ、γ−アミノ酪酸の生理機能を利用した食品の安価、安定供給にも大きな寄与が期待できる。 Furthermore, various food materials, nutritional functional foods, etc. can be easily and efficiently produced at low cost using γ-aminobutyric acid that is stably supplied at low cost, and the physiological function of γ-aminobutyric acid is utilized. It can be expected to contribute greatly to the low cost and stable supply of food.
また、小麦胚芽は製粉過程の副産物として大量に生じており、その処分に苦慮している面があるが、本願発明により、小麦胚芽の有効利用技術が確立されるため、これまで十分に利用されていなかった小麦胚芽の利用にも多大な貢献が期待できる。 In addition, wheat germ is produced in a large amount as a by-product of the milling process, and there are aspects that are difficult to dispose of. However, since the present invention establishes an effective technique for using wheat germ, it has been fully utilized so far. A great contribution can also be expected to the use of wheat germ that has not been used.
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