JP6931012B2 - Cooking method with high production of γ-aminobutyric acid - Google Patents
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Description
本発明は、γーアミノ酪酸高産出の調理方法に関し、特に、単一のラクトバチルス・ブレビス（Lactobacillus brevis）で、高産出γーアミノ酪酸能力の菌株CPC202を産出するものに関し、そして、含有量が29.8g/Literにも達するγーアミノ酪酸を生成できるものに関する。 The present invention relates to a high-yielding γ-aminobutyric acid cooking method, particularly to a single Lactobacillus brevis that produces a high-yielding γ-aminobutyric acid-capacity strain CPC202, and has a content of 29.8. It relates to those capable of producing γ-aminobutyric acid that reaches g / Lite.
中華民国国民健康署の2013〜2015年国民栄養健康状況変遷調査によれば、18歳以上の国民に、高血圧有病率が24.1%になる。高血圧は、心臓病、卒中、腎臓病、及び眼病等の疾病の重大リスク因子であり、世界高血圧学会は、地球全体に約18%の人口（九百四十万）の主な死亡原因が高血圧に関していることを推定する。高血圧は、通常、症候がないため、多い人が、自身に高血圧があると分かっても、人体器官に傷害を与えるまでに、何れかの血圧を制御する処置を取らない。 According to the 2013-2015 Nationality Law of the Republic of China's National Nutrition and Health Status Survey, the prevalence of hypertension is 24.1% among people aged 18 and over. Hypertension is a significant risk factor for diseases such as heart disease, stroke, kidney disease, and eye disease, and the Japanese Society of Hypertension states that hypertension is the main cause of death in about 18% of the world's population (9.4 million). Estimate that it is related. Hypertension is usually asymptomatic, so many people, even if they find themselves to have high blood pressure, do not take any action to control it before they damage human organs.
また、今の人々は、生活難が大きいため、睡眠に悪影響を与え、全台湾において、不眠症有病率が、19.3%（2013台湾睡眠医学学会）になり、2017年の調査によれば、更に、多い「年寄り」及び「女性」に、睡眠問題がある。長期の睡眠不足により、焦りが発生し、長期に、不眠や焦りに暴露されると、最後に、不安神経症に発展し、データから分かるように、抗不安薬を利用する人の数が、毎年に増加し、台湾うつ病有病率が、約7.3%（約150万人）であり、それは、症候が重大であるため、医者に助けて貰う一部であり、大部分は、軽い症候があっても、病院へ行かないため、保健食品で、不眠焦りや不安を解消するための潜在市場は、非常に大きくなり、また、世界保健機関は、うつ病と、新世紀の三大病気として見なす。 In addition, people today have a great difficulty in living, which adversely affects sleep, and the prevalence of insomnia in all Taiwan is 19.3% (2013 Taiwan Society of Sleep Medicine), according to a 2017 survey. In addition, many "elderly" and "women" have sleep problems. Long-term lack of sleep causes impatience, and long-term exposure to insomnia and impatience eventually leads to anxiety, and as the data show, the number of people using anti-anxiety medications, Increasing each year, the prevalence of depression in Taiwan is about 7.3% (about 1.5 million people), which is part of getting help from a doctor because the symptoms are serious, most of which are mild symptoms. Even if there is, the potential market for insomnia and anxiety with health foods is very large because we do not go to the hospital, and the World Health Organization has announced that depression and the three major illnesses of the new century Consider as.
γーアミノ酪酸（γ-aminobutyric acid,GABA）は、四つの炭素からなる非タンパクの組み立ての水溶性天然アミノ酸であり、それの分布が、非常に広くて、動植物にも、存在して、イオン化状態で、存於原核や真核細胞の遊離アミノ酸群に存在する。動物体内において、γーアミノ酪酸は、血液脳関門を通しなくて、神経組織だけに存在し、それは、脳みそによって自己合成されたものであり、また、脳みその含有量は、0.1〜0.6mg/gである。γーアミノ酪酸は、交感神経系ニューロトランスミッターであるドーパミンを抑制する作用があり、神経の激しやすい性質を制御でき、てんかんの発生を低下でき、また、高血圧や焦りの生成を抑えることができ、そのため、心血管機能や圧力解放及び落ち着きの効果に、大きい役目を発揮できる。また、γーアミノ酪酸の生成は、TCAサイクル（TCA cycle）にあるグルタメート（glutamate）が、Lーグルタミン酸デカルボキシラーゼ（Glutamate decarboxylase,GAD）とビタミンB6によって促進され、γーアミノ酪酸のGABAシャント（GABA shunt）を介して生成される。γーアミノ酪酸は、新規な機能性原料で、幅広く、保健食品や医薬化学工業等に応用される。 γ-aminobutyric acid (GABA) is a water-soluble natural amino acid composed of four carbons that is a non-protein assembly, and its distribution is very wide, and it is also present in animals and plants and is in an ionized state. It is present in the free amino acid group of prokaryotes and eukaryotic cells. In animals, γ-aminobutyric acid is present only in nervous tissue, not through the blood-brain barrier, it is self-synthesized by the brain, and the content of the brain is 0.1-0.6 mg / g. Is. γ-Aminobutyric acid has the effect of suppressing dopamine, which is a sympathetic nervous system neurotransmitter, can control the violent nature of nerves, can reduce the occurrence of epilepsy, and can suppress the production of hypertension and impatience. Therefore, it can play a major role in cardiovascular function, pressure release, and calming effect. In addition, the production of γ-aminobutyric acid is promoted by glutamate decarboxylase (GAD) and vitamin B6 in the TCA cycle, and GABA shunt of γ-aminobutyric acid. ) Is generated. γ-Aminobutyric acid is a novel functional raw material and is widely applied to health foods, pharmaceutical and chemical industries, and the like.
γーアミノ酪酸は、化学合成や天然物からの抽出、或いは微生物代謝によって生成される。その中、化学合成法の欠点は、コストが高く、産出が低いだけでなく、合成方法の条件が厳しく、化学物質の残存の問題があり、純粋物であっても、天然製品ではなく、また、生産過程において、リスク溶剤、そして、有毒溶剤が使用されるため、得られたγーアミノ酪酸は、主として、化学工業や医薬分野に応用されて、食品には、応用できなく、天然の食品添加剤として応用できない。天然抽出のγーアミノ酪酸は、時間やコストがかかるし、産出が低い問題がある。また、微生物発酵は、今、業界において、よく利用される、γーアミノ酪酸を生産する方法であり、その製品は、コストが低くて、含有量が高い、また、安全に食品に応用できる利点が得られる。 γ-Aminobutyric acid is produced by chemical synthesis, extraction from natural products, or microbial metabolism. Among them, the drawbacks of the chemical synthesis method are not only high cost and low yield, but also the conditions of the synthetic method are strict, there is a problem of residual chemical substances, and even if it is a pure product, it is not a natural product, and Since risk solvents and toxic solvents are used in the production process, the obtained γ-aminobutyric acid is mainly applied to the chemical industry and the pharmaceutical field, and cannot be applied to foods. It cannot be applied as an agent. Naturally extracted γ-aminobutyric acid has a problem that it is time-consuming, costly, and low in yield. In addition, microbial fermentation is now a popular method in the industry for producing γ-aminobutyric acid, which has the advantages of low cost, high content, and safe food application. can get.
微生物発酵については、既に、多い関連特許や文献が提案され、例えば、中華民国特許公告TW I460272 Bの説明書には、「コルディセプス属（Cordyceps）真菌類ライスベース（Fungus rice base）を発酵させてγーアミノ酪酸を調理する方法とその応用」が掲示され、それによると、複合菌株を利用して、所定のプロセスと菌種割合でγーアミノ酪酸を生産するものであり、また、中国特許公告CN 105482981 Aの説明書には、「血圧低下保健酢の生産方法」が掲示され、それによっても、二種類のラクトバチルスを結合してγーアミノ酪酸を製作する調理方法であり、上記らの提案と本提案は、単一のラクトバチルス・ブレビス菌株で30 g/Liter γーアミノ酪酸が得られる方式が、異なり、中国特許公告CN 103013879 Bの説明書には、「γーアミノ酪酸の高産出能力を有する菌株」が掲示され、この提案は、植物ラクトバチルスをγーアミノ酪酸生産の菌種とするが、本提案に用いられる菌株と異なり、また、その提案の特許に、γーアミノ酪酸の産出量が138 mg/Literであると掲示され、本提案に用いられた菌株によって産出されたγーアミノ酪酸の産出量（〜30 g/Liter）よりも、遥かに低く、また、Jang EKらは、2015年に、Pubmed定期刊行物に、文献（Jang EK et al., "γ-aminobutyric acid(GABA) production and angiotensinー1 converting enzyme (ACE) inhibitory activity of fermented soybean containing Sea Tangle by the coーculture of Lactobacillus brevis with Aspergillus oryzae", Journal of Microbiology and Biotechnology, 2015, 25:1315ー20.）を提案し、それは、アスペルギルス‐オリーゼにラクトバチルス・ブレビスを結合して、一同に1.9 g/kgのγーアミノ酪酸を発酵産出でき、その発酵方式と菌株の使用形態及びγーアミノ酪酸産出は、ともに、本提案との差異が極めて大きい。 Many related patents and documents have already been proposed for microbial fermentation. For example, the description of Republic of China Patent Announcement TW I460272 B states that "Cordyceps fungus rice base is fermented. "Methods for cooking γ-aminobutyric acid and its applications" was posted, which uses complex strains to produce γ-aminobutyric acid in a predetermined process and bacterial species ratio, and also in China Patent Publication CN. In the instruction manual of 105482981 A, "Production method of blood pressure lowering health vinegar" is posted, which is also a cooking method for producing γ-aminobutyric acid by combining two types of lactobacillus. This proposal differs in the method of obtaining 30 g / Lite γ-aminobutyric acid with a single Lactobacillus brevis strain, and the description of Chinese patent publication CN 103013879 B states that it has a high production capacity of γ-aminobutyric acid. "Strain" is posted, and this proposal uses plant Lactobacillus as a strain for producing γ-aminobutyric acid, but unlike the strain used in this proposal, the patent of the proposal states that the amount of γ-aminobutyric acid produced is 138. Much lower than the production of γ-aminobutyric acid (~ 30 g / Liter), which was posted as mg / Lite and produced by the strain used in this proposal, and Jang EK et al. In 2015 , Pubmed periodical, literature (Jang EK et al., "γ-aminobutyric acid (GABA) production and angiotensin-1 converting enzyme (ACE) inhibitory activity of fermented soybean containing Sea Tangle by the co-culture of Lactobacillus brevis with Aspergillus oryzae ", Journal of Microbiology and Biotechnology, 2015, 25: 1315-20.), which binds Lactobacillus brevis to Aspergillus-olise and ferments 1.9 g / kg of γ-aminobutyric acid together. It can be produced, and the fermentation method, the usage pattern of the strain, and the production of γ-aminobutyric acid are both extremely different from the present proposal.
上記の分析結果に基づくと、一般の従来ものは、単一のラクトバチルス・ブレビスでγーアミノ酪酸高産出能力を有する菌株を提供できないから、実用的とは言えない。 Based on the above analysis results, the general conventional one cannot be said to be practical because a single Lactobacillus brevis cannot provide a strain having a high production ability of γ-aminobutyric acid.
本発明者は、上記欠点を解消するため、慎重に研究し、また、学理を活用して、有効に上記欠点を解消でき、設計が合理である本発明を提案する。 In order to eliminate the above-mentioned defects, the present inventor proposes the present invention in which the above-mentioned defects can be effectively eliminated by careful research and by utilizing the theory, and the design is rational.
本発明の主な目的は、従来技術の上記問題を解消でき、単一のラクトバチルス・ブレビス（Lactobacillus brevis）でγーアミノ酪酸高産出能力を有できる、菌株CPC202のγーアミノ酪酸高産出の調理方法を提供する。 A main object of the present invention is a cooking method for producing high γ-aminobutyric acid of strain CPC202, which can solve the above-mentioned problems of the prior art and can have high production capacity of γ-aminobutyric acid with a single Lactobacillus brevis. I will provide a.
本発明の他の目的は、γーアミノ酪酸を高生成産出できる乳酸菌株CPC202を選別し、その菌株或いはその組み立てを、血圧改善や睡眠改善及び減圧の効果が得られる保健食品や栄養補充品或いは医薬組み立て物に応用できる、γーアミノ酪酸高産出の調理方法を提供する。 Another object of the present invention is to select a lactic acid strain CPC202 capable of producing γ-aminobutyric acid with high production, and to select the strain or its assembly for health foods, nutritional supplements or pharmaceuticals capable of improving blood pressure, improving sleep and reducing pressure. Provided is a cooking method with high production of γ-aminobutyric acid, which can be applied to an assembly.
本発明は、上記の目的を達成するため、γーアミノ酪酸高産出の調理方法であり、必要とする個体に、乳酸菌株CPC202或いはその組み立て物を与え、リットル含有量が20グラム（含む）以上に達するγーアミノ酪酸が得られ、単一の乳酸菌でγーアミノ酪酸の高産出が実現され、その中、当該乳酸菌株CPC202が、ラクトバチルス・ブレビスであるものと鑑定され、また、その組み立て物は、ラクトバチルス・ブレビスの成長や培養及び発酵の基質に適合する3炭素や4炭素及び5炭素の酸類、アルコール類であり、そして、必要とする個体は、グルタメート（glutamate）である。 The present invention is a cooking method for high production of γ-aminobutyric acid in order to achieve the above object, and a lactic acid bacterium strain CPC202 or an assembly thereof is given to an individual in need, and the liter content is 20 grams (including) or more. Reaching γ-aminobutyric acid is obtained, and high production of γ-aminobutyric acid is realized with a single lactic acid bacterium, in which the lactic acid bacterium strain CPC202 is identified as Lactobacillus brevis, and its assembly is Lactobacillus brevis growth, culture and fermentation substrates are compatible with 3 carbon, 4 carbon and 5 carbon acids, alcohols, and the individual in need is glutamate.
本発明の上記実施例において、当該乳酸菌株は、天然物検体から選別されるラクトバチルスの分離株である。 In the above-mentioned embodiment of the present invention, the lactic acid bacterium strain is a isolate of Lactobacillus selected from a natural product sample.
本発明の上記実施例において、当該分離株は、発酵物や野菜或いは乳幼児便検体から選別される。 In the above embodiment of the present invention, the isolate is selected from fermented products, vegetables or infant stool samples.
本発明の上記実施例において、当該ラクトバチルス・ブレビスの成長や培養及び発酵の基質は、固体或いは液体形態の培養媒質である。 In the above embodiment of the present invention, the substrate for growth, culture and fermentation of Lactobacillus brevis is a culture medium in solid or liquid form.
本発明の上記実施例において、当該乳酸菌株は、発酵培養を介して、リットル毎に含有量が29.82グラムに達するγーアミノ酪酸を産出する。 In the above embodiment of the present invention, the lactic acid bacterium strain produces γ-aminobutyric acid having a content of 29.82 grams per liter through fermentation culture.
本発明の上記実施例において、当該グルタメートの濃度は、5〜9%（w/v）の範囲内にある。 In the above embodiment of the present invention, the concentration of the glutamate is in the range of 5 to 9% (w / v).
本発明の上記実施例において、当該乳酸菌株は、グラム染色鏡でグラム陽性桿菌を観察した。 In the above-mentioned example of the present invention, the lactic acid bacterium strain was observed with a Gram-staining mirror as a Gram-positive rod.
本発明の上記実施例において、当該乳酸菌株は、カタラーゼやオキシダーゼ及び運動性を有しない。 In the above embodiment of the present invention, the lactic acid bacterium strain does not have catalase, oxidase and motility.
本発明の上記実施例において、当該乳酸菌株は、好気雰囲気及び嫌気雰囲気下で成長する。 In the above-mentioned embodiment of the present invention, the lactic acid bacterium strain grows in an aerobic atmosphere and an anaerobic atmosphere.
本発明の上記実施例において、当該乳酸菌株は、自生胞子を生成しない。 In the above embodiment of the present invention, the lactic acid bacterium strain does not produce native spores.
本発明は、また、上記のγーアミノ酪酸を有して、血圧改善や睡眠改善及び減圧の効果を有する保健食品や栄養補充品或いは医薬組み立て物を提供する。 The present invention also provides health foods, dietary supplements or pharmaceutical assemblies that have the above-mentioned γ-aminobutyric acid and have the effects of improving blood pressure, improving sleep and reducing pressure.
以下、図面を参照しながら、本発明の特徴や技術内容について、詳しく説明するが、それらの図面等は、参考や説明のためであり、本発明は、それによって制限されることが無い。 Hereinafter, the features and technical contents of the present invention will be described in detail with reference to the drawings, but those drawings and the like are for reference and explanation, and the present invention is not limited thereto.
図１〜図４は、それぞれ、本発明のGAD遺伝子発現PCR産物の電気泳動図や、本発明の乳酸菌株のGABA生成量概念図、本発明の乳酸菌株CPC202の顕微写真図及び本発明の乳酸菌株CPC202の16S rDNAシーケンス概念図である。図のように、本発明は、γーアミノ酪酸高産出の調理方法であり、単一の微生物物種を利用して、発酵培養で、高産出のγーアミノ酪酸（γ-aminobutyric acid, GABA）を産出し、今までに、微生物を利用してγーアミノ酪酸を生産する時、産出が最も高い方法である。本発明は、γーアミノ酪酸を生産できる乳酸菌株で、γーアミノ酪酸を生産し、主として、必要とする個体に、乳酸菌株CPC202或いはその組み立て物を与え、リットル含有量が20グラム（含む）以上に達するγーアミノ酪酸が得られ、単一の乳酸菌でγーアミノ酪酸の高産出が実現され、その中、当該乳酸菌株CPC202が、ラクトバチルス・ブレビス（Lactobacillus brevis）であるものと鑑定され、また、その組み立て物は、ラクトバチルス・ブレビスの成長や培養及び発酵の基質（固体や液体形態の培養媒質である）に適合する3炭素や4炭素及び5炭素の酸類、アルコール類であり、そして、必要とする個体は、濃度が5〜9%（w/v）の範囲内のグルタメート（glutamate）である。 1 to 4 are an electrophoretic diagram of the GAD gene expression PCR product of the present invention, a conceptual diagram of the amount of GABA produced by the lactic acid bacterium strain of the present invention, a microscopic photograph of the lactic acid bacterium strain CPC202 of the present invention, and a lactic acid bacterium of the present invention, respectively. It is a 16S rDNA sequence conceptual diagram of the strain CPC202. As shown in the figure, the present invention is a cooking method with high production of γ-aminobutyric acid, which produces high production of γ-aminobutyric acid (GABA) by fermentation culture using a single microbial species. However, to date, it is the highest production method when producing γ-aminobutyric acid using microorganisms. The present invention is a lactic acid bacterium strain capable of producing γ-aminobutyric acid, which produces γ-aminobutyric acid and mainly gives a lactic acid bacterium strain CPC202 or an assembly thereof to an individual in need, and has a liter content of 20 grams (including) or more. Reaching γ-aminobutyric acid is obtained, and high production of γ-aminobutyric acid is realized with a single lactic acid bacterium, in which the lactic acid bacterium strain CPC202 is identified as Lactobacillus brevis, and its The assembly is a 3-carbon, 4-carbon or 5-carbon acid, alcohol, and need that is compatible with the growth, culture and fermentation substrates of Lactobacillus brevis (which is a culture medium in solid or liquid form). Individuals are glutamate with concentrations in the range of 5-9% (w / v).
上記乳酸菌株は、天然物（例えば、発酵物や野菜或いは乳幼児便検体）からラクトバチルスの分離株を分別して、γーアミノ酪酸生産制御遺伝子のグルタミン酸脱炭酸酵素（glutamate decarboxylase, GAD）発見量及びγーアミノ酪酸生成量等を検出する。 For the above lactic acid bacterium strain, a isolate of Lactobacillus was separated from a natural product (for example, fermented product, vegetable or infant stool sample), and the amount of glutamic acid decarboxylase (GAD) found in the γ-aminobutyric acid production control gene and γ -Detect the amount of aminobutyric acid produced.
以下の実施例は、ただ、例を挙げて本発明を説明するためのものであり、本発明の特許請求の範囲は、それによって制限されない。 The following examples are merely for the purpose of explaining the present invention by way of examples, and the scope of claims of the present invention is not limited thereto.
実施例一：γーアミノ酪酸生成制御遺伝子GAD発見テスト
本実施例において、ポリメラーゼ連鎖反応（polymerase chain reaction, PCR）で、各菌株のGAD遺伝子の表現形態を検知し、始めに、GABAを分泌できる乳酸菌株を選別し、結果は、図１のようである。図に、14065がBCRCの番号で、アシドフィルス菌（Lactobacillus acidophilus）を指し、この標準菌株は、陽性対照に供され、Mは、DNA分子径の標準である。
Example 1: γ-aminobutyric acid production control gene GAD discovery test In this example, the expression form of the GAD gene of each strain is detected by the polymerase chain reaction (PCR), and first, a lactic acid bacterium capable of secreting GABA. The strains were selected and the results are shown in FIG. In the figure, 14065 is the BCRC number, which refers to Lactobacillus acidophilus, this standard strain is used as a positive control, and M is the standard for DNA molecular diameter.
実施例二：γーアミノ酪酸生成方式
本発明は、乳酸菌株を、乳酸桿菌MRS培養基において、活性化培養させ、γーアミノ酪酸を生産する時、MRS培養液に7%（w/v）グルタメートを添加して、37℃の細菌培養器において、二日間培養した後、発酵液を、4℃条件下、13,000 rpmで、15分に遠心分離させ、上澄みに対して、γーアミノ酪酸生成量を検知する。
Example 2: γ-aminobutyric acid production method In the present invention, when a lactic acid bacterium strain is activated and cultured in a lactic acid bacterium MRS culture group to produce γ-aminobutyric acid, 7% (w / v) glutamate is added to the MRS culture solution. Then, after culturing for 2 days in a bacterium incubator at 37 ° C, the fermented liquor is centrifuged at 13,000 rpm for 15 minutes under 4 ° C conditions, and the amount of γ-aminobutyric acid produced is detected in the supernatant. ..
実施例三：γーアミノ酪酸生成量検知
本テストは、液体クロマトグラフィー・タンデム質量分析（LC/MC/MC）で、菌株γーアミノ酪酸の生成量（図２のように）を分析検知し、乳酸菌珠CPC202は、発酵培養により、29.820 g/Lのγーアミノ酪酸（発酵液）が産出され、ある文献によると、高血圧病者は、毎日18 mg γーアミノ酪酸であれば、優れた血圧調節効果が得られ、そのため、本方法による産出量は、文献によって建言された高血圧病者の毎日使用量の1656倍になる。
Example 3: Detection of γ-aminobutyric acid production amount In this test, the amount of γ-aminobutyric acid produced (as shown in Fig. 2) of the strain γ-aminobutyric acid was analyzed and detected by liquid chromatography tandem mass analysis (LC / MC / MC), and lactic acid bacteria were detected. Tama CPC202 produces 29.820 g / L of γ-aminobutyric acid (fermented liquid) by fermentation culture, and according to one document, hypertensive patients can have an excellent blood pressure regulating effect with 18 mg γ-aminobutyric acid daily. Obtained and therefore the yield by this method is 1656 times the daily use of hypertensives as stated in the literature.
実施例四：乳酸菌株配列解析
本発明の乳酸菌株は、台湾中油株式会社が伝統発酵食品から選別する菌種であり、原株番号は、CPC202である。生産したγーアミノ酪酸の乳酸菌株CPC202に対して、食品工業発展研究所菌種中心によって菌種學名鑑定を行い、グラム染色鏡で、グラム陽性桿菌を観察し、結果は、図３のように、カタラーゼやオキシダーゼ及び運動性を有しなく、好気雰囲気や嫌気雰囲気下で成長するだけでなく、自生胞子を生成しない。16S rDNAシーケンスに基づいて、比較解析を行い、その菌株CPC202の菌種が、ラクトバチルス・ブレビス（Lactobacillus brevis）であると鑑定確認でき、図４から分かるように、CPC202 16S rDNA遺伝子フラグメントが、最もLactobacillus brevisに近接し、シーケンス類似性が、100%になり、CPC202が、Lactobacillus brevis菌属であることを確証できる。また、API認証システムを利用して、API 50の生化学的検討を行い、結果から分かるように、CPC202が、最もLactobacillus brevisに近接、CPC202が、Lactobacillus brevis菌属であることを確証できる。上記の16S rDNA配列アライメント及びAPI 50生化学的検討に基づいて、当該菌株CPC202の菌種が、ラクトバチルス・ブレビス（表一の菌種鑑定を参照すること）であり、また、菌株種類が、衛福部公告の食用安全菌株である。
Example 4: Lactic Acid Bacteria Strain Sequence Analysis The lactic acid bacterium strain of the present invention is a strain selected by Taiwan Chuyu Co., Ltd. from traditional fermented foods, and the original strain number is CPC202. The produced γ-aminobutyric acid lactic acid bacterium strain CPC202 was subjected to catalase analysis by the food industry development research institute center, and Gram-positive rods were observed with a Gram stain mirror. It has no catalase, oxidase and motility, and not only grows in an aerobic or anaerobic atmosphere, but also does not produce native spores. A comparative analysis was performed based on the 16S rDNA sequence, and it was confirmed that the strain of the strain CPC202 was Lactobacillus brevis. As can be seen from FIG. 4, the CPC202 16S rDNA gene fragment is the most. It is close to Lactobacillus brevis and has 100% sequence similarity, confirming that CPC202 belongs to the genus Lactobacillus brevis. In addition, the API authentication system is used to conduct a biochemical study of API 50, and as can be seen from the results, it can be confirmed that CPC202 is the closest to Lactobacillus brevis and CPC202 belongs to the genus Lactobacillus brevis. Based on the above 16S rDNA sequence alignment and API 50 biochemical examination, the strain of the strain CPC202 is Lactobacillus brevis (see the strain identification in Table 1), and the strain type is It is an edible safe strain announced by the Ministry of Health and Welfare.
本発明のCPC202 16S rDNAのシーケンス表において、位置116のrに、a或いはg（DNAシーケンスを測定する時、aとgが同時に出現すること）が表示され、これは、ヘテロ接合体（Heterozygote）で、微生物によく現す現象である。
シーケンス分析に使用されるプライマー名與プライマーシーケンス
In the sequence table of the CPC202 16S rDNA of the present invention, a or g (a and g appear at the same time when measuring the DNA sequence) is displayed at r at position 116, which is a heterozygote (Heterozygote). This is a phenomenon that often appears in microorganisms.
Primer Name Name Primer Sequence Used for Sequence Analysis
上記のように、本発明は、選別して、得られた乳酸菌株を実験で、その特徴及び機能を検知することにより、その乳酸菌株CPC202が、γーアミノ酪酸の高産出が実現され、γーアミノ酪酸は、交感神経系ニューロトランスミッターであるドーパミンを抑制する作用があり、神経の激しやすい性質を制御でき、てんかんの発生を低下でき、また、高血圧や焦りの生成を抑えることができ、心血管機能や圧力解放及び落ち着きの効果に、大きい役目を発揮できる。その菌株或いはその組み立てを、血圧改善や睡眠改善及び減圧の効果が得られる保健食品や栄養補充品或いは医薬組み立て物に応用でき、経済価値があると思う。 As described above, in the present invention, by detecting the characteristics and functions of the lactic acid bacterium strain obtained by selecting the lactic acid bacterium strain in an experiment, the lactic acid bacterium strain CPC202 realizes high production of γ-aminobutyric acid, and γ-amino Butyric acid has the effect of suppressing dopamine, which is a sympathetic nervous system neurotransmitter, can control the violent nature of nerves, reduce the occurrence of epilepsy, suppress the production of hypertension and impatience, and cardiovascular. It can play a major role in the function, pressure release, and calm effect. I think that the strain or its assembly can be applied to health foods, dietary supplements, or pharmaceutical assemblies that have the effects of improving blood pressure, improving sleep, and reducing pressure, and has economic value.
以上のように、本発明は、γーアミノ酪酸高産出の調理方法であり、有効に従来の諸欠点を解消でき、単一のラクトバチルス・ブレビス（Lactobacillus brevis）でγーアミノ酪酸（GABA）高産出能力を有する菌株を提供でき、γーアミノ酪酸に、重要な生理機能を有し、抗鬱や抗焦り或いは血圧調節の医薬と保健の重要原料として利用でき、本発明によるγーアミノ酪酸高産出の乳酸菌株CPC202は、血圧改善や睡眠改善及び減圧効果を有する保健食品や栄養補充品或いは医薬組み立て物に適用でき、そのため、本発明は、より進歩のかつより実用ので、法に従って特許請求を出願する。以上は、ただ、本発明のより良い実施例であり、本発明は、それによって制限されることが無く、本発明に係わる特許請求の範囲や明細書の内容に基づいて行った等価の変更や修正は、全てが、本発明の特許請求の範囲内に含まれる。 As described above, the present invention is a cooking method for producing high γ-aminobutyric acid, which can effectively eliminate various conventional drawbacks, and a single Lactobacillus brevis produces high γ-aminobutyric acid (GABA). A lactic acid bacterium with high γ-aminobutyric acid production according to the present invention, which can provide a strain having the ability, has an important physiological function for γ-aminobutyric acid, can be used as an important raw material for medicine and health for antidepressant, anti-irritant or blood pressure regulation. The strain CPC202 can be applied to health foods, dietary supplements or pharmaceutical assemblies having blood pressure improving, sleeping improving and depressurizing effects, and therefore the present invention is more advanced and more practical, so a patent application is filed in accordance with the law. The above is merely a better embodiment of the present invention, and the present invention is not limited thereto, and equivalent modifications made based on the scope of claims and the contents of the specification relating to the present invention. All modifications are within the scope of the claims of the present invention.
Claims (4)
単一の乳酸菌である乳酸菌株ＣＰＣ２０２或いはその混合物により、リットル含有量が２０グラム以上に達するγーアミノ酪酸（γ−ａｍｉｎｏｂｕｔｙｒｉｃ ａｃｉｄ， ＧＡＢＡ）が得られ、単一の乳酸菌でγーアミノ酪酸の高産出が実現され、
当該乳酸菌株が、天然物である発酵物、野菜あるいは乳児の便のサンプルから、ラクトバチルスの分離株を分別して、当該乳酸菌株ＣＰＣ２０２が、ラクトバチルス・ブレビス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）であるものと鑑定され、
また、その混合物は、ラクトバチルス・ブレビスの成長や培養及び発酵に適した培地として３炭素〜５炭素の酸類、アルコール類を含むものであり、そして、それは、濃度が５〜９％（ｗ／ｖ）の範囲内のグルタメート（ｇｌｕｔａｍａｔｅ）である
ことを特徴とする乳酸菌株を用いてγーアミノ酪酸を生産する方法。 A method for producing γ-aminobutyric acid using a lactic acid bacterium strain.
A single lactic acid bacterium, lactic acid bacterium strain CPC202 or a mixture thereof, gives γ-aminobutyric acid (GABA) having a liter content of 20 grams or more, and a single lactic acid bacterium produces high γ-aminobutyric acid. Realized,
The lactic acid bacterium strain separates a isolate of Lactobacillus from a sample of a natural fermented product, a vegetable or an infant's stool, and the lactic acid bacterium strain CPC202 is determined to be Lactobacillus brevis. ,
The mixture also contains 3 to 5 carbon acids, alcohols as a medium suitable for the growth, culture and fermentation of Lactobacillus brevis, which has a concentration of 5-9% (w / w /). A method for producing γ-aminobutyric acid using a lactic acid bacterium strain characterized by being a glutamate within the range of v).
当該乳酸菌株は、発酵培養を介して、リットル毎に含有量が２９．８２グラムに達するγーアミノ酪酸を産出する
ことを特徴とする乳酸菌株を用いてγーアミノ酪酸を生産する方法。 In the method for producing γ-aminobutyric acid using the lactic acid bacterium strain according to claim 1.
A method for producing γ-aminobutyric acid using a lactic acid bacterium strain, which is characterized by producing γ-aminobutyric acid having a content of 29.82 grams per liter through fermentation culture.
当該乳酸菌株は、グラム染色鏡でグラム陽性桿菌を観察した
ことを特徴とする乳酸菌株を用いてγーアミノ酪酸を生産する方法。 In the method for producing γ-aminobutyric acid using the lactic acid bacterium strain according to claim 1.
The lactic acid bacterium strain is a method for producing γ-aminobutyric acid using a lactic acid bacterium strain in which a gram-positive rod is observed with a Gram stain mirror.
当該乳酸菌株は、好気雰囲気及び嫌気雰囲気下で成長し、カタラーゼやオキシダーゼ及び運動性を有しなく、自生胞子を生成しない
ことを特徴とする乳酸菌株を用いてγーアミノ酪酸を生産する方法。 In the method for producing γ-aminobutyric acid using the lactic acid bacterium strain according to claim 1.
A method for producing γ-aminobutyric acid using a lactic acid bacterium strain that grows in an aerobic and anaerobic atmosphere, has no catalase, oxidase, and motility, and does not produce spontaneous spores.
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