JP4693487B2 - Method for producing lactic acid bacteria and tea beverage - Google Patents
Method for producing lactic acid bacteria and tea beverage Download PDFInfo
- Publication number
- JP4693487B2 JP4693487B2 JP2005137347A JP2005137347A JP4693487B2 JP 4693487 B2 JP4693487 B2 JP 4693487B2 JP 2005137347 A JP2005137347 A JP 2005137347A JP 2005137347 A JP2005137347 A JP 2005137347A JP 4693487 B2 JP4693487 B2 JP 4693487B2
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- Prior art keywords
- tea
- gaba
- lactic acid
- tea extract
- acid bacteria
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Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Tea And Coffee (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、新規な乳酸菌、これを利用した茶飲料およびその製造方法に関する。 The present invention relates to a novel lactic acid bacterium, a tea beverage using the same, and a method for producing the tea beverage.
γ-アミノ酪酸(γ-aminobutyric acid, 以下「GABA」と略記する)は、生物界に微量ながら広く存在するアミノ酸の一種である。これは野菜、果物を初め各種の食品素材に含まれており、食生活の中で摂取されている食品成分の一つである。GABAは、例えばヒトでは、脳内に局在し、抑制系の神経伝達物質として働くことが判っている。また、GABAは、その生理的作用に関して、血管拡張による血圧低下(上昇抑制)作用(非特許文献1参照)を初めとして、動脈硬化予防、肝機能改善、腎機能向上、精神安定などの作用が報告されている(非特許文献2参照)。更に、GABAは、中性脂肪増加抑制作用(肥満防止作用)；更年期障害症状緩和；記憶改善、学習能力増強などの脳機能改善作用；アルコール代謝促進などの様々な生理機能を有することも知られている(非特許文献3-5参照)。なお、生体内においてGABAは、グルタミン酸デカルボキシラーゼ(グルタミン酸脱炭酸酵素：Glutamate decarboxylase)の作用によりグルタミン酸が脱炭酸されて生成する。 γ-aminobutyric acid (hereinafter abbreviated as “GABA”) is a kind of amino acid that exists widely in the living world. This is included in various food materials such as vegetables and fruits, and is one of the food components ingested in the diet. GABA, for example, in humans has been found to localize in the brain and act as a neurotransmitter in the inhibitory system. In addition, GABA has actions such as prevention of arteriosclerosis, improvement of liver function, improvement of kidney function, and mental stability, including blood pressure reduction (inhibition suppression) action by vasodilation (see Non-Patent Document 1). Has been reported (see Non-Patent Document 2). GABA is also known to have various physiological functions such as neutral fat increase inhibitory action (anti-obesity action); alleviation of symptoms of climacteric disorder; improvement of brain function such as improvement of memory and learning ability; and promotion of alcohol metabolism. (See Non-Patent Document 3-5). In vivo, GABA is produced by decarboxylation of glutamate by the action of glutamate decarboxylase (glutamate decarboxylase).
これらの有用な生理作用から、GABAは機能性食品素材として注目されているが、現在知られている主なGABA含有食品素材のGABA含有量は非常に少ない。GABA含有量が多いと報告されている、例えば米胚芽などでもせいぜい0.02%迄である。このことから、GABAが本来有する機能乃至薬理作用を発揮し得るに必要な量を食品から摂取することは、従来困難であった。 Because of these useful physiological actions, GABA is attracting attention as a functional food material, but the GABA content of the main GABA-containing food materials currently known is very low. It is reported that the GABA content is high, for example, rice germ is up to 0.02%. For this reason, it has been difficult in the past to ingest from the food an amount necessary to exhibit the function or pharmacological action inherent to GABA.
近年、各種GABAを含有する食品素材に人為的な処理を施してその含有量を増加させる方法(強化する方法)と共に、健康食品素材としてのGABA自体の生産技術が開発、提案されている(特許文献1-3および非特許文献6-8参照)。 In recent years, the production technology of GABA itself as a health food material has been developed and proposed, along with a method of artificially treating food materials containing various GABA to increase its content (method of strengthening) (patents) Reference 1-3 and Non-patent Reference 6-8).
より詳しくは、特許文献1には、乳製品を発酵用培地として用いて、該培地中においてGABA産生能を発揮する乳酸菌(ラクトバチルス ブレビス(Lactobacillus brevis)TY414(FERM P-16910)を発酵させる方法が開示されている。 More specifically, Patent Document 1 discloses a method of fermenting a lactic acid bacterium (Lactobacillus brevis TY414 (FERM P-16910) that exhibits GABA production ability in a dairy product as a fermentation medium. Is disclosed.
特許文献2には、果実、野菜などの原料に、グルタミン酸と特定の乳酸菌とを添加して、乳酸菌発酵を行うGABA高含有乳酸菌発酵飲食品の製造法が提案されている。ここに用いられる乳酸菌は、ラクトバチルス ブレビスIFO3960株、同IFO12005株、同IFO12520株、同IFO13109株および同IFO13110株から選択される株である。 Patent Document 2 proposes a method for producing a GABA-rich lactic acid bacterium-fermented food / drink product in which glutamic acid and a specific lactic acid bacterium are added to raw materials such as fruits and vegetables to perform lactic acid bacterium fermentation. The lactic acid bacteria used here are strains selected from the Lactobacillus brevis IFO3960 strain, the IFO12005 strain, the IFO12520 strain, the IFO13109 strain and the IFO13110 strain.
特許文献3には、ポリフェノールを除去した茶抽出液中でラクトバチルス属乳酸菌を培養して、甘い花香を有するGABA高含有物を得る方法が開示されている。 Patent Document 3 discloses a method of culturing Lactobacillus lactic acid bacteria in a tea extract from which polyphenols have been removed to obtain a high GABA content having a sweet floral scent.
非特許文献6は、米焼酎粕にpH調製後にグルタミン酸脱炭酸酵素生産能の高い乳酸菌(ラクトバチルス ブレビスIFO12005)を生育させるとGABAが高収率で製造できることを報告している。 Non-Patent Document 6 reports that GABA can be produced in high yield by growing lactic acid bacteria (Lactobacillus brevis IFO12005) having a high ability to produce glutamate decarboxylase in rice shochu after pH adjustment.
非特許文献7および8は、キムチより分離した乳酸菌(ラクトバチルス ヒルガディ(Lactobacillus hilgardii)K-3株)が高いGABA生産能を有し、この菌の発酵によってGABA濃度の高い(20%以上)食品素材(ファーマギャバTM, 株式会社ファーマフーズ研究所)が得られることを報告している。 Non-patent documents 7 and 8 show that a lactic acid bacterium (Lactobacillus hilgardii K-3 strain) isolated from kimchi has a high GABA production ability, and a high GABA concentration (20% or more) by fermentation of this bacterium. It has been reported that materials (Pharma GABA TM , Pharma Foods Laboratory Co., Ltd.) can be obtained.
上記のように、GABA産生能を有する乳酸菌(ラクトバチルス属)は、幾つか知られているが、実際には、これら乳酸菌のGABA産生能は、発酵用培地(食品)の種類、組成、発酵条件などに左右され、必ずしも期待される高濃度のGABAを産生できず、しかも、場合によっては、得られる食品素材に該食品本来の香味とは異なった培地由来乃至発酵産物由来の異味臭、不快臭などを付与してしまうおそれがあった。 As described above, some lactic acid bacteria (Lactobacillus genus) having GABA production ability are known, but in fact, the GABA production ability of these lactic acid bacteria is the type, composition, fermentation of fermentation medium (food). Depending on the conditions, etc., it is not always possible to produce the expected high concentration of GABA, and in some cases, the obtained food material may have a different flavor or unpleasant odor derived from a medium or fermentation product that is different from the original flavor of the food. There was a risk of adding odor.
本発明者らも、GABAを強化した飲食品(機能性食品)の開発を目的として、以前から鋭意研究を重ねてきたが、その過程で、摘採した茶葉を嫌気的条件下におくことによって処理したギャバロン茶が多量のGABAを蓄積されており、これが高血圧性疾患の予防および治療に有効な血圧上昇抑制作用を有する旨の報告(非特許文献9参照)に着目した。この報告から、乳酸菌をお茶に作用させれば、GABAを多量に含む飲料が製造できると考え、この着想に基づいて更に研究を重ねた。尚、ギャバロン茶は、その製造過程においてグルタミン酸脱炭酸酵素の関与は推定できるが、該お茶に関連する乳酸菌の存在についての報告はなされていない。 The present inventors have also intensively studied for the purpose of developing food and drink (functional food) reinforced with GABA, but in the process, the picked tea leaves are treated under anaerobic conditions. Attention was paid to a report (see Non-Patent Document 9) that Gabalon tea has accumulated a large amount of GABA and has an effect of suppressing blood pressure rise effective in the prevention and treatment of hypertensive diseases. From this report, it was thought that beverages containing a large amount of GABA could be produced if lactic acid bacteria were allowed to act on tea, and further research was conducted based on this idea. In addition, although the involvement of glutamate decarboxylase can be estimated in the production process of Gabalon tea, there is no report about the presence of lactic acid bacteria related to the tea.
しかるに、茶葉および茶抽出物は、従来から、これらの中にカテキンなどのポリフェノール類（タンニン）が含まれ、その摂取によれば、これらの成分に基づく抗ガン作用、抗微生物作用などが期待できることが知られている。この茶抽出物中にタンニンが存在することおよびこれに基づいて茶抽出物が抗微生物作用などを有することは、該茶抽出物中で乳酸菌を培養、生育させる場合、乳酸菌自体の生育が阻害されること、また該乳酸菌に由来する酵素の活性が阻害されることを意味している。事実、本発明者らの研究によれば、GABA産生能を有することが報告されている乳酸菌、その他の微生物は、総じて、茶抽出物中では、期待されるGABA産生能を発揮しないことが確認された。
本発明の目的は、茶抽出物中でGABAを産生する乳酸菌を提供することおよび該乳酸菌を利用してGABAを強化したお茶を提供することにある。 An object of the present invention is to provide a lactic acid bacterium that produces GABA in a tea extract and to provide a tea enriched with GABA using the lactic acid bacterium.
本発明者らは、上記目的を達成するために、引き続き研究を重ねた結果、茶抽出液中で生育し、しかも該液中で著量のGABAを産生可能な、ラクトバチルス ブレビスに属する新たな乳酸菌を見いだした。また、本発明者らは、この乳酸菌を利用して、所望のGABAを強化したお茶を製造する技術を確立するに成功した。本発明はこれらの発見を基礎として、更に研究を重ねた結果、完成されたものである。 As a result of continuous research to achieve the above object, the present inventors have succeeded in developing a new extract belonging to Lactobacillus brevis that grows in tea extract and can produce a significant amount of GABA in the liquid. I found lactic acid bacteria. In addition, the present inventors have succeeded in establishing a technique for producing a desired GABA-enhanced tea using this lactic acid bacterium. The present invention has been completed as a result of further research based on these findings.
本発明は、下記項1および2に示す乳酸菌、項3-7に示す茶飲料の製造方法および項8に示す茶飲料を提供する。 The present invention provides the lactic acid bacteria shown in the following items 1 and 2, the method for producing a tea beverage shown in the item 3-7, and the tea beverage shown in the item 8.
項1. 茶抽出物中においてGABAを産生するラクトバチルス属に属する乳酸菌。 Item 1. A lactic acid bacterium belonging to the genus Lactobacillus that produces GABA in a tea extract.
項2. ラクトバチルス ブレビス (Lactobacillus brevis) mh4219 (FERM P-20327)である項1に記載の乳酸菌。 Item 2. The lactic acid bacterium according to Item 1, which is Lactobacillus brevis mh4219 (FERM P-20327).
項3. 項1または2に記載の乳酸菌を茶抽出物に作用させて、GABAを含有する茶抽出物を得る、茶飲料の製造方法。 Item 3. A method for producing a tea beverage, wherein the lactic acid bacteria according to Item 1 or 2 are allowed to act on a tea extract to obtain a tea extract containing GABA.
項4. 茶抽出物がタンニン含量0.03重量％以上のものである項3に記載の方法。 Item 4. The method according to Item 3, wherein the tea extract has a tannin content of 0.03% by weight or more.
項5. 茶抽出物が緑茶、中国茶および紅茶からなる群から選ばれる少なくとも1種の茶抽出物である項3に記載の方法。 Item 5. The method according to Item 3, wherein the tea extract is at least one tea extract selected from the group consisting of green tea, Chinese tea and black tea.
項6. 茶抽出物が、更にグルタミン酸およびその塩類から選ばれる少なくとも1種を含有するものである項3に記載の方法。 Item 6. The method according to Item 3, wherein the tea extract further contains at least one selected from glutamic acid and salts thereof.
項7. 茶抽出物が、更にグルタミン酸およびその塩類から選ばれる少なくとも1種を0.01-15w/v%含有するものである項3に記載の方法。 Item 7. The method according to Item 3, wherein the tea extract further contains 0.01-15 w / v% of at least one selected from glutamic acid and salts thereof.
項8. 項3に記載の方法により得られるGABAを含有する茶飲料。 Item 8. A tea beverage containing GABA obtained by the method according to Item 3.
本発明によれば、例えば血圧低下(上昇抑制)作用などを有し、降圧剤などとして有用な新しい茶飲料が提供され、これは特定保険食品、健康食品などとして有用である。 According to the present invention, for example, a new tea beverage having a blood pressure lowering (rising suppression) action and useful as an antihypertensive agent is provided, which is useful as a specific insurance food, health food and the like.
以下、本発明者らが新たにスクリーニングした乳酸菌につき詳述する。 Hereinafter, the lactic acid bacteria newly screened by the present inventors will be described in detail.
(1) 乳酸菌のスクリーニング
(1-1)起源
本菌株は、野菜漬け物から分離された。
(1) Screening for lactic acid bacteria
(1-1) Origin This strain was isolated from pickled vegetables.
本発明者らは、各種発酵食品、野菜などの植物を被験試料として、これらを生理食塩水にて希釈懸濁させた後、懸濁液をブロムクレゾール・パープル・プレートカウント寒天培地(BCP寒天培地)にて培養し、得られるコロニーを釣菌し、更にこれらをBCP寒天培地に画線して培養する操作を繰り返すことによって、被験試料中に存在する乳酸菌などを純粋培養した結果、その中に本菌株を見出した。 The present inventors used various fermented foods, vegetables and other plants as test samples, diluted and suspended in physiological saline, and then suspended the bromcresol, purple, plate count agar medium (BCP agar medium). ), And the resulting colonies are fished, and further, the operation of streaking these on a BCP agar medium and culturing them is repeated, and as a result, the lactic acid bacteria etc. present in the test sample are purely cultured. We found this strain.
(1-2)スクリーニング方法
本菌株のスクリーニングは、上記純粋培養された菌について、それらの茶抽出物中におけるGABA産生能を指標として、例えば以下のようにして実施される。即ち、候補菌を茶抽出物（グルコース1.0%、ペプトン1.0%、酵母エキス0.5%およびグルタミン酸ナトリウム1水和物1.0%を更に添加）に接種して、嫌気および通気攪拌培養し、培養液中のGABA量を高速液体クロマトグラフィー(High Performance Liquid Chromatography, HPLC)により測定する。乳酸菌の接種量は、一般には茶抽出物1cc中に菌体が約1×105個以上、好ましくは1×10７個前後含まれるものとなる量から選ばれるのが適当である。培養条件は、一般に、発酵温度20-45℃程度、好ましくは25-37℃程度、発酵時間5-72時間程度から選ばれる。
(1-2) Screening method Screening of the present strain is carried out for the above purely cultured fungi, for example, as follows using the GABA production ability in the tea extract as an index. That is, inoculate the candidate bacteria into a tea extract (added with 1.0% glucose, 1.0% peptone, 0.5% yeast extract and 1.0% sodium glutamate monohydrate), anaerobic and aeration stirring culture, The amount of GABA is measured by high performance liquid chromatography (HPLC). The amount of lactic acid bacteria inoculated is generally selected from the amount that will contain about 1 × 10 5 or more, preferably about 1 × 10 7 cells in 1 cc of tea extract. The culture conditions are generally selected from a fermentation temperature of about 20-45 ° C, preferably about 25-37 ° C, and a fermentation time of about 5-72 hours.
ここで、「茶抽出物」としては、酒石酸鉄比色法により測定されるタンニン（ポリフェノール）含量が0.03-15mg/mLの範囲にあるものを使用する。 Here, as the “tea extract”, one having a tannin (polyphenol) content in the range of 0.03-15 mg / mL measured by the iron tartrate colorimetric method is used.
本明細書において「茶抽出物中においてGABAを産生する」乃至「茶抽出物中におけるGABA産生能」とは、タンニン含量が4.3mg/mLの茶抽出物中、30℃で72時間培養した場合に、100mg/dL以上のGABAを生成、蓄積することをいうものとする。 In the present specification, “produces GABA in tea extract” to “GABA production ability in tea extract” means when cultivated at 30 ° C. for 72 hours in a tea extract having a tannin content of 4.3 mg / mL In addition, it means to produce and accumulate 100 mg / dL or more of GABA.
上記スクリーニング方法によって選択された菌は、常法に従い分離、精製することができる。例えば、上記培養後に、培養液を3000回転/分、4℃、10分間遠心分離して集菌することによって菌体を得ることができる。その精製は、通常の乳酸菌の精製手段に従うことができる。培養液、培養物および精製菌体は、これらを凍結乾燥することもできる。かくして得られる凍結乾燥品、凍結乾燥菌体も、本発明乳酸菌に包含される。 Bacteria selected by the screening method can be isolated and purified according to a conventional method. For example, cells can be obtained by centrifuging and collecting the culture solution after culturing at 3000 rpm for 10 minutes at 4 ° C. The refinement | purification can follow the normal refinement | purification means of lactic acid bacteria. The culture solution, culture and purified cells can be freeze-dried. The freeze-dried product and freeze-dried cells thus obtained are also included in the lactic acid bacteria of the present invention.
(2) 本発明乳酸菌
本発明乳酸菌の代表的例は、上記の如きスクリーニング方法に従って選択され、本発明者らによりラクトバチルス ブレビスmh4219 (Lactobacillus brevis mh4219)と命名された菌株を挙げることができる。その菌学的性質および他の性質は、以下の通りである。
(2) Lactic acid bacterium of the present invention A typical example of the lactic acid bacterium of the present invention includes a strain selected according to the screening method as described above and named by the present inventors as Lactobacillus brevis mh4219. Its bacteriological and other properties are as follows.
(a)肉眼的特徴
(a-1) MRS寒天培地
円形からやや不規則、低凸状隆起、平滑からやや粗造、中央部白色、周縁部白色から無色。
(a) Macroscopic features
(a-1) MRS Agar Medium Slightly irregular from a circle, low convex ridge, slightly rough from smooth, white in the center, white from the peripheral white.
(a-2) BL寒天培地
不規則, 低凸状隆起、平滑からやや粗造、中央部黄褐色, 周縁部白褐色。
(a-2) BL agar medium Irregular, low convex ridge, smooth to slightly rough, yellowish brown in the center, white brown in the periphery.
(b) 顕微鏡的特徴
桿菌で運動性を持たない。芽胞は形成しない。
(b) Microscopic characteristics It is a koji mold and has no motility. Spores do not form.
(c) 生育
通性嫌気性であり、10〜45℃、pH4〜9で良好に生育する。
(c) Growth It is facultative anaerobic and grows well at 10-45 ° C and pH 4-9.
(d) 生理学的、生化学的特徴
グラム染色性 ＋
カタラーゼ −
オキシダーゼ −
乳酸発酵形式：ヘテロ
乳酸の旋光度：DL
糖の資化性：
陽性(+)：グルコース、L-アラビノース、リボース、D-キシロース、ガラクトース、フルクトース、マルトース、α-メチル- D-グルコシド、
陰性(-)：グリセロール、エリスリトール、D-アラビノース、L-キシロース、アドニトール、β-メチル-D-キシロシド、マンノース、L-ソルボース、ラムノース、ズルシトール、イノシトール、ソルビトール、α-メチル-D-マンノシド、アミグダリン、アルブチン、エスクリン、サリシン、セロビオース、ラクトース、スクロース、トレハロース、イヌリン、メレジトース、ラフィノース、デンプン、グリコーゲン、キシリトール、ゲンチビオース、T-ツラノース、D-リキソース、D-タガトース、D-フコース、L-フコース、D-アラビトール、L-アラビトール、2-ケトグルコン酸、
擬陽性(±)：マンニトール、N-アセチル-グルコサミン、メリビオース、グルコン酸、5-ケト-グルコン酸。
(d) Physiological and biochemical characteristics Gram staining +
Catalase −
Oxidase −
Lactic acid fermentation format: Hetero Rotation of lactic acid: DL
Sugar assimilation:
Positive (+): glucose, L-arabinose, ribose, D-xylose, galactose, fructose, maltose, α-methyl-D-glucoside,
Negative (-): glycerol, erythritol, D-arabinose, L-xylose, adonitol, β-methyl-D-xyloside, mannose, L-sorbose, rhamnose, dulcitol, inositol, sorbitol, α-methyl-D-mannosid, amygdalin , Arbutin, esculin, salicin, cellobiose, lactose, sucrose, trehalose, inulin, melezitose, raffinose, starch, glycogen, xylitol, gentibiose, T-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose, D -Arabitol, L-arabitol, 2-ketogluconic acid,
False positive (±): mannitol, N-acetyl-glucosamine, melibiose, gluconic acid, 5-keto-gluconic acid.
(e) 16s rDNAの塩基配列(500塩基分)：
配列番号：1に示す通りである。
(e) 16s rDNA base sequence (500 bases):
It is as shown in SEQ ID NO: 1.
16S rRNAシークエンスに基づく分子系統の研究手順は次に示すとおりである。本菌株をMRS寒天培地に植菌し、30℃、2日培養後の培養物を供試菌体とする。菌体からゲノムの抽出には、Insta Gene Matrix (Bio Rad, CA, U.S.A.)を使用し、操作はBio Rad社のプロトコールに従う。得られたゲノムDNAを鋳型としてPCR法により16s rDNA遺伝子の内の5’末端側約500bpを増殖させる。PCRには、PCRキット(MicroSep500 16s rDNA Bacterial Identification PCR Kit, Applied Biosystems, CA, U.S.A.)を使用する。サイクルシークエンスには、シークエンシングキット(MicroSep500 16s rDNA Bacterial Identification Sequencing Kit, Applied Biosystems, CA, U.S.A.)を使用する。サーマルサイクラーにはGeneAmp PCR System 9600 (Applied Biosystems, CA, U.S.A.)を使用する。DNAシーケンサーには、ABI PRISM 3100 DNA Sequencer (Applied Biosystems, CA, U.S.A.)を使用する。各操作は、Applied Biosystems社のプロトコールに従う。 The molecular phylogenetic research procedure based on the 16S rRNA sequence is as follows. This strain is inoculated on an MRS agar medium, and the culture after 2 days of culture at 30 ° C. is used as a test cell. Insta Gene Matrix (Bio Rad, CA, U.S.A.) is used to extract the genome from the cells, and the operation follows the protocol of Bio Rad. About 500 bp of the 5 'end side of the 16s rDNA gene is propagated by PCR using the obtained genomic DNA as a template. For PCR, a PCR kit (MicroSep500 16s rDNA Bacterial Identification PCR Kit, Applied Biosystems, CA, U.S.A.) is used. For cycle sequencing, a sequencing kit (MicroSep500 16s rDNA Bacterial Identification Sequencing Kit, Applied Biosystems, CA, U.S.A.) is used. GeneAmp PCR System 9600 (Applied Biosystems, CA, U.S.A.) is used for the thermal cycler. For the DNA sequencer, ABI PRISM 3100 DNA Sequencer (Applied Biosystems, CA, U.S.A.) is used. Each operation follows the protocol of Applied Biosystems.
得られた16s rDNAの塩基配列は、MicroSep Microbial Identification System Softwareを用いて、その相同性検索を行い、また、近隣接合法(Saitou and Nei, 1987)による分子系統樹の作成を行う。相同性検索のためのデーターベースとしては、MicroSep Bacterial 500 Library v.0023 (Applied Biosystems, CA, U.S.A.)を使用する。また、得られた16s rDNAの塩基配列について、BLAST Homology Search (Altsuchul et al., 1997)を用いて、DNAデータバンク(GeneBank/EMBL/DDBJ)に対する相同性検索をも行う。 The base sequence of the obtained 16s rDNA is subjected to homology search using MicroSep Microbial Identification System Software, and a molecular phylogenetic tree is created by the neighborhood joining method (Saitou and Nei, 1987). As a database for homology search, MicroSep Bacterial 500 Library v.0023 (Applied Biosystems, CA, U.S.A.) is used. In addition, the homology search for the DNA data bank (GeneBank / EMBL / DDBJ) is also performed on the obtained 16s rDNA base sequence using BLAST Homology Search (Altsuchul et al., 1997).
本菌株の16s rDNAの502塩基配列を決定した結果は、配列番号：1に示すとおりである。この塩基配列をBLASTHomology Searchを用いてデータバンクに対する検索を行った結果、ラクトバチルス・ブレビス(Lactobacillus brevis)と99.5%の相同性を有することが確認された。 The result of determining the 502 base sequence of 16s rDNA of this strain is as shown in SEQ ID NO: 1. As a result of searching this data base using the BLASTHomology Search, the data bank was confirmed to have 99.5% homology with Lactobacillus brevis.
以上の諸性質から、本菌株の同定を行った。バージィーズ・マニュアル・オブ・システマティック・バクテリオロジー(Bergey's Manual of Systematic Bacteriology) に照らして、本菌株はLactobacillus brevisに属する菌株と同定された。 This strain was identified from the above properties. In the light of Bergey's Manual of Systematic Bacteriology, this strain was identified as belonging to Lactobacillus brevis.
本株に近似する保存株としては、NBRC12005, NBRC12520などが存在する。しかしながら、本株はこれらの保存株と対比して、茶抽出物中におけるGABA産生能において明確に相違している。従って、本発明者らは、本株を新規な株と同定した。 NBRC12005, NBRC12520, etc. exist as conserved strains similar to this strain. However, this strain clearly differs in the ability to produce GABA in the tea extract compared to these stocks. Therefore, the present inventors identified this strain as a novel strain.
該菌株は、平成16年12月16日に、日本国茨城県つくば市東1-1-1 中央第6に住所を有する独立行政法人 産業技術総合研究所 特許生物寄託センターに、寄託番号FERM P-20327として寄託されている。 The strain was founded on December 16, 2004 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST), which has an address at 1-1-1 Higashi 1-1-1, Tsukuba, Ibaraki, Japan. Deposited as 20327.
(3) 乳酸菌による発酵
本発明は、上記菌株(Lactobacillus brevis mh4219, FERM P-2-327)を初めとして、茶抽出物中においてGABAを産生するラクトバチルス属に属する乳酸菌を利用して、茶抽出物からGABAを強化された茶飲料を得る方法をも提供する。以下、この方法につき詳述する。
(3) Fermentation by lactic acid bacteria The present invention uses the above-mentioned strains (Lactobacillus brevis mh4219, FERM P-2-327), and tea extraction using lactic acid bacteria belonging to the genus Lactobacillus that produce GABA in the tea extract. There is also provided a method for obtaining a GABA-enhanced tea beverage from a product. Hereinafter, this method will be described in detail.
本発明方法は、本発明乳酸菌の培養用培地として茶抽出物を利用することを必須とする。ここで茶抽出物には、緑茶、中国茶（ウーロン茶、ジャスミン茶、プーアル茶など）および紅茶の茶葉から水または熱水で抽出して得られる抽出液が含まれる。該抽出液のJAS規格によるBrix(可溶性固形物(糖度)濃度、即ち、溶液温度20℃における溶液100g中の砂糖(ショ糖)のg数)は、通常0.01%-20%の範囲にあるのが好ましい。また、該抽出液のタンニン含量(酒石酸鉄比色法による)は、通常0.03〜15mg/mLの範囲にある。 The method of the present invention essentially requires the use of a tea extract as a culture medium for the lactic acid bacteria of the present invention. Here, the tea extract includes green tea, Chinese tea (oolong tea, jasmine tea, puer tea, etc.) and an extract obtained by extraction with tea or tea from tea leaves. Brix (soluble solids (sugar content) concentration, that is, the number of grams of sugar (sucrose) in 100 g of solution at a solution temperature of 20 ° C.) according to the JAS standard of the extract is usually in the range of 0.01% to 20%. Is preferred. The tannin content of the extract (according to the iron tartrate colorimetric method) is usually in the range of 0.03 to 15 mg / mL.
培地中には、特に必要ではないが、本発明乳酸菌の生育を助け、発酵反応を促進するための、通常の乳酸菌の培養に適した栄養成分を添加配合することもできる。該栄養成分としては、例えばグルコース、澱粉、蔗糖、乳糖、デキストリン、ソルビトール、フラクトースなどの炭素源；酵母エキス、ペプトンなどの窒素源；ビタミンB、ビタミンD、ビタミンC、ビタミンE、ビタミンKなどのビタミン類；鉄、銅、亜鉛などのミネラル類などを挙げることができる。これらの栄養成分の配合量は、特に限定されるものではないが、通常、炭素源および窒素源はそれぞれ0.01〜10.0w/v%程度、好ましくは約0.1〜2.0w/v%の範囲内とするのが好ましい。ビタミン類およびミネラル類は、それぞれ0.1w/v%以下の配合量とされるのが好ましい。 Although it is not particularly necessary in the medium, nutritional components suitable for normal cultivation of lactic acid bacteria can be added and blended to assist the growth of the lactic acid bacteria of the present invention and promote the fermentation reaction. Examples of the nutrient component include carbon sources such as glucose, starch, sucrose, lactose, dextrin, sorbitol, and fructose; nitrogen sources such as yeast extract and peptone; vitamin B, vitamin D, vitamin C, vitamin E, vitamin K and the like Vitamins; minerals such as iron, copper and zinc can be mentioned. The amount of these nutritional components is not particularly limited, but usually the carbon source and nitrogen source are each in the range of about 0.01 to 10.0 w / v%, preferably about 0.1 to 2.0 w / v%. It is preferable to do this. Vitamins and minerals are preferably blended in amounts of 0.1 w / v% or less.
また、得られる茶飲料のGABA含量を更に向上させたい場合には、培地に更にグルタミン酸脱炭酸酵素の基質であるグルタミン酸およびその塩類(グルタミン酸ナトリウム、グルタミン酸カリウムなど)並びにこれらを含む食品原料、例えば、昆布エキス、アミノ酸液(調味液)などを適宜添加することもできる。これらの内ではグルタミン酸ナトリウム・1水和物が好ましい。その添加量は、得られるGABA強化茶飲料のGABA含量および香味、風味などを考慮して適宜選択することができる。例えば、グルタミン酸ナトリウム・1水和物では約20w/v%まで、より好ましくは約0.01-10w/v%の範囲から選択することができる。この配合量に依存して、約5.5〜5500mg/dL程度のGABA含量の茶飲料を得ることができ、殊に、100mg/dLのオーダーでGABAを含有する高GABA含量の茶飲料を得ることができる。 Further, when it is desired to further improve the GABA content of the obtained tea beverage, glutamic acid and its salts (sodium glutamate, potassium glutamate, etc.) which are further glutamic acid decarboxylase substrates in the medium, and food materials containing these, for example, A kelp extract, an amino acid solution (seasoning solution), and the like can be appropriately added. Of these, sodium glutamate monohydrate is preferred. The amount of addition can be appropriately selected in consideration of the GABA content, flavor, flavor and the like of the obtained GABA-enhanced tea beverage. For example, sodium glutamate monohydrate can be selected from the range of up to about 20 w / v%, more preferably about 0.01-10 w / v%. Depending on the blending amount, a tea beverage having a GABA content of about 5.5 to 5500 mg / dL can be obtained, and in particular, a high GABA content tea beverage containing GABA on the order of 100 mg / dL can be obtained. it can.
本発明乳酸菌は、pH4〜9の範囲において充分生育するものであり、特に必要ではないが、所望により、培地中にクエン酸、乳酸などの有機酸や水酸化ナトリウムなどのアルカリを添加してそのpHを適宜調整することができる。好ましい培地pHは、一般には約4.5〜6.0の範囲である。 The lactic acid bacterium of the present invention grows sufficiently in the range of pH 4 to 9, and is not particularly necessary, but if desired, an organic acid such as citric acid or lactic acid or an alkali such as sodium hydroxide is added to the medium. The pH can be adjusted as appropriate. Preferred medium pH is generally in the range of about 4.5 to 6.0.
本発明乳酸菌の茶抽出物中における発酵反応は、利用する本発明乳酸菌の生育に適した条件下で実施することができる。より詳しくは、必要に応じてグルタミン酸ナトリウム、その他の栄養成分を添加した茶抽出物を予め適当な条件、例えば121℃、15分間などの条件で殺菌し、ここに本発明乳酸菌を接種する。該乳酸菌は予めMRS寒天培地などで培養したものから白金耳で接種することができ、また液体培地を用いて前培養したものから接種することもできる。接種する乳酸菌の菌数は、一般には、約1×104個以上、好ましくは1×106〜1×107個程度の範囲内とするのが望ましい。乳酸菌の発酵は、通常、約20〜45℃、好ましくは25〜37℃の温度下に、約24〜168時間、好ましくは約48〜96時間を要して実施することができる。この発酵には、特に通気および攪拌は必要でない。また、発酵中、脱炭酸反応により培地(茶抽出物)のpHが上昇する場合があり、この場合は、必要に応じて1〜50w/v%のクエン酸、乳酸、塩酸などを用いてpH調整を行うことができる。 The fermentation reaction in the tea extract of the lactic acid bacterium of the present invention can be carried out under conditions suitable for the growth of the lactic acid bacterium of the present invention to be used. More specifically, if necessary, a tea extract to which sodium glutamate and other nutritional components are added is sterilized in advance under appropriate conditions, for example, at 121 ° C. for 15 minutes, and inoculated with the lactic acid bacteria of the present invention. The lactic acid bacteria can be inoculated with platinum loops from those previously cultured in an MRS agar medium or the like, or can be inoculated from those previously cultured using a liquid medium. In general, the number of lactic acid bacteria to be inoculated is desirably about 1 × 10 4 or more, preferably in the range of about 1 × 10 6 to 1 × 10 7 . The fermentation of lactic acid bacteria can be carried out usually at a temperature of about 20 to 45 ° C., preferably 25 to 37 ° C., taking about 24 to 168 hours, preferably about 48 to 96 hours. This fermentation does not particularly require aeration and stirring. In addition, during fermentation, the pH of the medium (tea extract) may increase due to the decarboxylation reaction.In this case, the pH is adjusted with 1-50 w / v% citric acid, lactic acid, hydrochloric acid, etc. as necessary. Adjustments can be made.
発酵反応終了後、得られる茶抽出物は、これを透明な茶飲料とするために除菌処理することができ、かくして本発明茶飲料とすることができる。この除菌操作は、常法に従って例えば遠心分離機を用いて実施することができ、また濾過操作によることもできる。更に、プレート式殺菌機などを用いた殺菌処理によることもできる。尚、本発明茶飲料は特に透明なものに限定はされず、従って、上記除菌処理を行うことなく、得られる混濁液自体を本発明茶飲料とすることもできる。 After completion of the fermentation reaction, the resulting tea extract can be sterilized to make it a transparent tea beverage, thus making the tea beverage of the present invention. This sterilization operation can be carried out according to a conventional method using, for example, a centrifuge, or can be performed by a filtration operation. Further, sterilization treatment using a plate type sterilizer or the like may be performed. The tea beverage of the present invention is not particularly limited to a transparent one. Therefore, the obtained turbid liquid itself can be used as the tea beverage of the present invention without performing the sterilization treatment.
(4) 茶飲料
かくして、所望の茶飲料を得ることができる。このものは高いGABA含量を有することによって特徴付けられる。しかも、お茶特有の香味、風味を維持する特徴を有している。
(4) Tea drink Thus , a desired tea drink can be obtained. This is characterized by having a high GABA content. Moreover, it has the characteristic of maintaining the flavor and flavor peculiar to tea.
本発明茶飲料は、更に必要に応じて、この種の茶飲料と同様に、適当な可食性担体(食品素材)の適当量を配合することができる。該担体としては、得られる茶飲料のその飲食に適した味などを付与するための素材、例えば甘味料、香料などを挙げることができる。 The tea beverage of the present invention can be further blended with an appropriate amount of an appropriate edible carrier (food material) as required for this type of tea beverage. Examples of the carrier include materials for imparting a taste suitable for the eating and drinking of the obtained tea beverage, such as sweeteners and fragrances.
得られる本発明飲料は、適当な容器に充填して製品とすることができる。該製品は、さわやかな喉ごしで茶本来の香味、旨みを有している。 The obtained beverage of the present invention can be filled into a suitable container to obtain a product. The product has a refreshing throat and has the original flavor and taste of tea.
本発明茶飲料は、その高いGABA含量に基づいて、その摂取によって、GABA本来の生理作用である、例えば血管拡張による血圧低下(上昇抑制)作用、中性脂肪増加抑制作用(肥満防止作用)、脳機能改善作用、精神安定作用などが期待できる。更に、本発明茶飲料は、更年期障害症状の緩和、記憶改善、学習能力増強、アルコール代謝促進、肝機能改善、腎機能向上などの作用も期待できる。従って、本発明茶飲料は、これらの作用を奏し得る特定保険食品、健康食品などとして有用である。 The tea beverage of the present invention is based on its high GABA content, and by its ingestion, GABA's original physiological action, for example, blood pressure lowering (inhibition suppression) action due to vasodilation, neutral fat increase inhibition action (anti-obesity action), Expected to improve brain function and stabilize the body. Furthermore, the tea beverage of the present invention can also be expected to have effects such as alleviating climacteric symptoms, improving memory, enhancing learning ability, promoting alcohol metabolism, improving liver function, and improving kidney function. Therefore, the tea beverage of the present invention is useful as a specific insurance food, health food or the like that can exert these actions.
これらの作用を期待できる本発明茶飲料の摂取量は、これを摂取する生体の年齢、性別、体重、疾患の程度などに応じて適宜決定され、特に限定されるものではない。一般にはGABA 5〜100mg/dLを含有する茶飲料を、一日に約50〜1000mLを摂取、服用させればよい。 The intake of the tea beverage of the present invention that can be expected to have these effects is appropriately determined according to the age, sex, body weight, degree of disease, etc. of the living body ingesting it, and is not particularly limited. In general, a tea beverage containing GABA 5-100 mg / dL may be taken and taken at about 50-1000 mL per day.
実施例
以下、本発明を更に詳しく説明するため、本発明に利用する菌株の単離方法および得られる菌株を用いた茶抽出物の発酵方法を実施例として挙げるが、本発明はこれらの実施例に限定されるものではない。
Examples Hereinafter, in order to explain the present invention in more detail, the method for isolating strains used in the present invention and the method for fermenting tea extract using the obtained strains will be described as examples. The present invention is not limited to these examples. It is not limited to.
本発明乳酸菌の単離
野菜漬け物を生理食塩水にて希釈懸濁後、懸濁液をブロムクレゾール・パープル・プレートカウント寒天培地(BCP寒天培地)にて培養し、得られるコロニーを釣菌し、更にこれらをBCP寒天培地に画線して培養する操作を繰り返すことによって、被験試料中に存在する乳酸菌を純粋培養した。
Isolation of the lactic acid bacteria of the present invention After the vegetable pickles are diluted and suspended in physiological saline, the suspension is cultured on bromcresol purple plate count agar medium (BCP agar medium), and the resulting colonies are fished. Furthermore, by repeating the operation of streaking them on a BCP agar medium and culturing them, the lactic acid bacteria present in the test sample were purely cultured.
上記純粋培養された乳酸菌1×10７cfu/mLを茶抽出物（グルコース1.0%、ペプトン1.0%、酵母エキス0.5%およびグルタミン酸ナトリウム・1水和物1.0%を更に添加、タンニン含量約4.3mg/mL）1 L中に接種し、30℃で72時間、静置培養し、培養液中のGABA量を高速液体クロマトグラフィー(High Performance Liquid Chromatography, HPLC)により測定し、測定値が100mg/dLを超えるものとして、本菌株(Lactobacillus brevis mh4219)を単離した。 1 × 10 7 cfu / mL of the above pure cultured lactic acid bacteria was added to tea extract (glucose 1.0%, peptone 1.0%, yeast extract 0.5% and sodium glutamate monohydrate 1.0%, tannin content about 4.3 mg / mL) Inoculate in 1 L, incubate at 30 ° C for 72 hours, and measure the amount of GABA in the culture by high performance liquid chromatography (HPLC). The measured value is 100 mg / dL. As a surplus, this strain (Lactobacillus brevis mh4219) was isolated.
GABA産生試験
Brix 3%およびタンニン含量(酒石酸鉄比色法による、以下同じ)4.3mg/mLの緑茶エキス100mLに、0.5%グルコース、0.25% 酵母エキス、0.5%ペプトンおよび1.0%グルタミン酸ナトリウム・1水和物を加えて、本発明乳酸菌培養用茶抽出物培地を調製した。
GABA production test
Brix 3% and tannin content (according to iron tartrate colorimetric method, the same shall apply hereinafter) 4.3 mg / mL green tea extract 100 mL, 0.5% glucose, 0.25% yeast extract, 0.5% peptone and 1.0% sodium glutamate monohydrate In addition, a tea extract medium for culturing lactic acid bacteria of the present invention was prepared.
対照として、上記において緑茶エキスの替わりに蒸留水100mLを用いて同様にして、対照培地を調製した。 As a control, a control medium was prepared in the same manner using 100 mL of distilled water instead of the green tea extract.
上記各培地10mLに、実施例1で単離した本発明乳酸菌(Lactobacillus brevis mh4219)の一白金耳(約1×108個)を接種し、30℃で72時間、静置培養を行って培養液を得た。 10 mL of each medium is inoculated with one platinum loop (about 1 × 10 8 pieces) of the lactic acid bacterium of the present invention (Lactobacillus brevis mh4219) isolated in Example 1, and cultured by performing stationary culture at 30 ° C. for 72 hours. A liquid was obtained.
得られた培養液中のGABA生成量を下記条件下にHPLCにて測定した。 The amount of GABA produced in the obtained culture broth was measured by HPLC under the following conditions.
本明細書において、GABA含量は、この測定法に従って測定された値にて表示されるものとする。 In this specification, GABA content shall be displayed by the value measured according to this measuring method.
尚、同条件下のHPLCにて、供試培地中のグルタミン酸含量をも測定した。 In addition, the glutamic acid content in the test medium was also measured by HPLC under the same conditions.
＜GABA含量測定のためのHPLC条件＞
発酵液中のGABA含量は発酵液を限外濾過(東洋濾紙製ウルトラフィルターユニットUSY-1)後、ο-フタルアルデヒドによるポストカラム蛍光法を用いた高速液体クロマトグラフィー(HPLC)によって測定した。
HPLCの条件
カラム：Shim-pak VP-ODS (150mm×4.6mm I.D.)
移動相：10mM 1-ヘプタンスルフォン酸ナトリウムを含む20mMリン酸ナトリウム緩衝液pH2.5
流速：0.8 mL/min
温度：45℃
反応液：和光純薬製OPA試薬キット
流速：0.4 mL/min
温度：45℃
検出：蛍光検出器 Ex 350nm、Em 450nm
測定されたGABA含量および培地中のグルタミン酸含量から、下式に従って、GABA変換率(%)を算出した。本明細書において、GABA変換率(%)とは、この式に従って測定される値にて表示されるものとする。
<HPLC conditions for GABA content measurement>
The GABA content in the fermentation broth was measured by high performance liquid chromatography (HPLC) using a post-column fluorescence method with ο-phthalaldehyde after ultrafiltration (Ultra Filter Unit USY-1 made by Toyo Roshi).
HPLC condition column: Shim-pak VP-ODS (150mm x 4.6mm ID)
Mobile phase: 20 mM sodium phosphate buffer pH 2.5 containing 10 mM sodium 1-heptanesulfonate
Flow rate: 0.8 mL / min
Temperature: 45 ° C
Reaction solution: Wako Pure Chemical Industries OPA reagent kit Flow rate: 0.4 mL / min
Temperature: 45 ° C
Detection: Fluorescence detector Ex 350nm, Em 450nm
From the measured GABA content and the glutamic acid content in the medium, the GABA conversion rate (%) was calculated according to the following formula. In this specification, the GABA conversion rate (%) is displayed as a value measured according to this equation.
GABA変換率(%) = A / B ×100
A: GABA生成実測値(mg)
B: GABA生成理論値(mg)
尚、上記B(GABA生成理論値)は、利用した培地に含まれるグルタミン酸が全てGABAに変換された時のGABA量(mg)を算出した値である。
GABA conversion rate (%) = A / B x 100
A: GABA production measurement (mg)
B: Theoretical value of GABA generation (mg)
The B (theoretical value for GABA production) is a value obtained by calculating the amount of GABA (mg) when all glutamic acid contained in the medium used is converted to GABA.
得られた結果を表1に示す。尚、表1には、比較のため、本発明乳酸菌以外の以下の乳酸菌株を用いて同様の試験を繰り返した結果を併記する。
<比較乳酸菌>
ラクトバチルス ブレビス NBRC12005株 (NBRC保存株)
ラクトバチルス ブレビスOFC 34株 (大塚食品株式会社保存株)
ラクトバチルス ブレビスOFC 31株 (同上)
ラクトバチルス プランタラムJCM1149株 (Japan Collection of Microorganisms寄託菌、ATCC No. 14917に相当する)
ラクトコッカス ラクティスFERM P-13646株 (独立行政法人 産業技術総合研究所 特許生物寄託センター保存菌)
The results obtained are shown in Table 1. For comparison, Table 1 also shows the results of repeating the same test using the following lactic acid strains other than the lactic acid bacteria of the present invention.
<Comparison lactic acid bacteria>
Lactobacillus brevis NBRC12005 strain (NBRC stock)
Lactobacillus brevis OFC 34 (Otsuka Foods Co., Ltd. preservation stock)
Lactobacillus brevis OFC 31 (same as above)
Lactobacillus plantarum JCM1149 strain (corresponding to Japan Collection of Microorganisms, ATCC No. 14917)
Lactococcus lactis FERM P-13646 strain (National Institute of Advanced Industrial Science and Technology (AIST))
表1に示される結果から、本発明乳酸菌株Lactobacillus brevis mh4219株は、茶エキスを含む培地（茶抽出物）で培養した場合に、該茶エキスを含まない培地で培養した場合と略同等の著量のGABAを生成することが明らかである。 From the results shown in Table 1, the lactic acid strain Lactobacillus brevis mh4219 of the present invention, when cultured in a medium containing tea extract (tea extract), is substantially the same as that cultured in a medium not containing the tea extract. It is clear that it produces a quantity of GABA.
これに対して、他のラクトバチルス・ブレビス(Lactobacillus brevis)中には、茶エキスを含まない培地中で多量のGABAを産生するものも存在するが、これらは茶エキスを含む培地中では、GABA産生能を発揮できず、実質的にGABAを産生し得ないことが判る。 On the other hand, in other Lactobacillus brevis (Lactobacillus brevis), there are those that produce a large amount of GABA in a medium not containing tea extract, but these in the medium containing tea extract, It can be seen that the production ability cannot be exhibited, and GABA cannot be produced substantially.
また、他の乳酸菌(Lactobacillus plantarum, Lactococcus lactis)は、茶エキスを含まない培地中でも、実質的に産生し得ず、勿論、茶エキスを含む培地中ではGABAを産生しないことが明らかである。 In addition, it is clear that other lactic acid bacteria (Lactobacillus plantarum, Lactococcus lactis) cannot be produced substantially even in a medium not containing a tea extract, and of course, GABA is not produced in a medium containing a tea extract.
GABA含有茶飲料の製造法
緑茶抽出液(Brix 0.25%、タンニン量360μg/ml)に、グルタミン酸ナトリウム・1水和物3.0％、大豆ペプトン1％および酵母エキス0.5%を添加して原料液を調製し、121℃で15分間滅菌した。
GABA-containing tea beverage production method Prepare green tea extract (Brix 0.25%, tannin amount 360μg / ml) by adding sodium glutamate monohydrate 3.0%, soybean peptone 1% and yeast extract 0.5%. And sterilized at 121 ° C. for 15 minutes.
予め、該原料液と同じ組成の培地で前培養したmh4219株16mLを、上記原料液800mLに接種(接種後の生菌数1×107cfu/ml)し、30℃で72時間培養した。培養後、12,000回転／分で10分間遠心し、菌体を除去して培養上澄を得た。 16 mL of mh4219 strain pre-cultured in a medium having the same composition as that of the raw material solution was inoculated into 800 mL of the above raw material solution (the number of viable bacteria after inoculation was 1 × 10 7 cfu / ml) and cultured at 30 ° C. for 72 hours. After culturing, centrifugation was performed at 12,000 rpm for 10 minutes to remove the cells and obtain a culture supernatant.
この培養上澄は、良好な香気を有し、約1.5％(1580mg/dL)のGABAを含有していた(実施例1の測定法に従って測定した。表2参照)。このとき、グルタミン酸からGABAへの変換率は約94%であった。尚、利用した緑茶抽出液中に含まれるグルタミン酸含量は、約1.8mg/dLであった。 This culture supernatant had a good aroma and contained about 1.5% (1580 mg / dL) GABA (measured according to the measurement method of Example 1. See Table 2). At this time, the conversion rate from glutamic acid to GABA was about 94%. The glutamic acid content contained in the used green tea extract was about 1.8 mg / dL.
比較のため、伝統的発酵食品として知られている、茶葉を乳酸発酵させた阿波番茶および碁石茶並びにギャバロン茶について、それらのGABA含量を同様にして測定した。得られた結果を表2に併記する。 For comparison, GABA content of Awaban tea, Goishi tea, and Gabaron tea, which are known as traditional fermented foods and fermented with lactic acid, was measured in the same manner. The results obtained are also shown in Table 2.
尚、これら各お茶のGABA含量は、市販の各茶葉を購入し、飲用説明書に従い、通常の飲用濃度になるように、阿波番茶および碁石茶はそれぞれ6g/L、ギャバロン茶は12g/Lとなるように茶葉を熱水(90℃)にて3分間抽出し、得られた液を試料として測定した。 The GABA content of these teas is 6 g / L for Awaban tea and Goishi tea, and 12 g / L for Gabaron tea, respectively, so that they can be purchased at the usual drinking concentration according to the drinking instructions. The tea leaves were extracted with hot water (90 ° C.) for 3 minutes, and the obtained liquid was measured as a sample.
表2に示される結果から明らかなとおり、阿波番茶および碁石茶はいずれもGABAをほとんど含んでいない。また、ギャバロン茶は、茶葉を嫌気処理することにより茶葉中に含まれるグルタミン酸を茶葉中の酵素で脱炭酸させるものであり、そのGABA含量は茶葉に含まれるグルタミン酸量に依存しており、高濃度とすることはできない。しかも、ギャバロン茶は、上記嫌気処理によってお茶本来の旨みや味わいが損なわれてしまう不利がある。 As is clear from the results shown in Table 2, both Awabancha and Sosekicha contain almost no GABA. In addition, Gabalon tea is an anaerobic treatment of tea leaves to decarboxylate the glutamic acid contained in the tea leaves with enzymes in the tea leaves. It cannot be. Moreover, Gabalon tea has the disadvantage that the original taste and taste of tea are impaired by the anaerobic treatment.
尚、阿波番茶および碁石茶の発酵に関与する乳酸菌としてはラクトバチルス・プランタラムやラクトバチルス・バクシノステルクスが想定される（小崎道雄編著、雪印乳業健康生活研究所編、「乳酸発酵の文化譜」、1996、中央法規出版株式会社参照）が、前記表2に示すとおり、これらのお茶GABA含量は非常に少なく、このことから、これらの乳酸菌は茶抽出物中におけるGABA産生能は有していないと考えられる。 In addition, Lactobacillus plantarum and Lactobacillus baxinosterux are assumed as lactic acid bacteria involved in fermentation of Awabancha and Goishi tea (edited by Michio Kosaki, edited by Snow Brand Milk Products and Health Institute, “Culture of Lactic Acid Fermentation”) , 1996, Chuo Law & Co., Ltd.), as shown in Table 2 above, these tea GABA contents are very low. Therefore, these lactic acid bacteria have the ability to produce GABA in tea extracts. It is not considered.
グルタミン酸ナトリウム・1水和物5%、大豆ペプチド1％、グルコース1％および酵母エキス0.5%が含有されるように各成分を添加し、また、クエン酸の添加によりpHを5.0に調整した緑茶抽出液(Brix 4.0% タンニン量5.7mg/mL)1Lを、121℃で10分間滅菌し、このものに予めGYP培地(グルコース1%、ペプトン1%および酵母エキス0.5%含有)で30℃下に12時間前培養したmh4219株20mLを接種(接種後の生菌数：1×107cfu/mL)し、30℃で72時間静置培養した。 Extracted green tea with 5% sodium glutamate monohydrate, 1% soy peptide, 1% glucose and 0.5% yeast extract, and adjusted to pH 5.0 by adding citric acid 1 L of liquid (Brix 4.0% tannin amount 5.7 mg / mL) was sterilized at 121 ° C for 10 minutes, and this was preliminarily added to GYP medium (containing 1% glucose, 1% peptone and 0.5% yeast extract) at 30 ° C at 12 ° C. Inoculated with 20 mL of mh4219 strain that had been pre-cultured for an hour (the number of viable bacteria after inoculation: 1 × 10 7 cfu / mL), and then statically cultured at 30 ° C. for 72 hours.
培養終了後、菌体を遠心分離(10000回転/分、10分間)により除去し、得られた培養上澄を脱イオン水で20倍に希釈した後、121℃で15分間殺菌を行い、予め洗浄したPETボトルに200mLずつ充填して、本発明緑茶飲料を得た。この緑茶飲料中には、1本200mL当たりにGABA 272mgを含むものであった(GABA変換率：96.9%)。 After completion of the culture, the cells are removed by centrifugation (10000 rpm / minute, 10 minutes), and the obtained culture supernatant is diluted 20-fold with deionized water and then sterilized at 121 ° C for 15 minutes. The washed PET bottle was filled with 200 mL each to obtain the green tea beverage of the present invention. This green tea beverage contained 272 mg of GABA per 200 mL (GABA conversion rate: 96.9%).
グルタミン酸ナトリウム・1水和物3%、大豆ペプチド1％、グルコース1％および酵母エキス0.5%が含有されるように各成分を添加し、また、クエン酸の添加によりpHを5.0に調整した緑茶抽出液(Brix 3.0% タンニン量4.3mg/mL)4Lを、121℃で10分間滅菌し、このものに予めGYP培地で30℃下に12時間前培養したmh4219株80mLを接種し(接種後の生菌数：1×107cfu/mL)、30℃で72時間静置培養した。 Extracted green tea with 3% sodium glutamate monohydrate, 1% soy peptide, 1% glucose and 0.5% yeast extract, and adjusted to pH 5.0 by adding citric acid 4L of the liquid (Brix 3.0% tannin amount 4.3mg / mL) was sterilized at 121 ° C for 10 minutes, and this was inoculated with 80mL of mh4219 strain that had been pre-cultured in GYP medium at 30 ° C for 12 hours. Bacterial count: 1 × 10 7 cfu / mL), static culture at 30 ° C. for 72 hours.
培養後、得られた培養液を実施例3と同様に処理して菌体を除去して培養上澄を得た。この培養上澄のGABA含量は、1600mg/dLであり、グルタミン酸からGABAへの変換率は、約95%であった。 After the culture, the obtained culture solution was treated in the same manner as in Example 3 to remove the cells and obtain a culture supernatant. The culture supernatant had a GABA content of 1600 mg / dL, and the conversion rate from glutamic acid to GABA was about 95%.
この培養液1Lに、水19 Lを加え、プレート式殺菌機にて137℃で5秒間殺菌し、予め洗浄および滅菌したペットボトルに350mLずつ無菌的に充填して、本発明茶飲料を調製した。得られた茶飲料は1本(350mL)当たり280mgのGABAを含有しており、良好な風味を有していた。 19 L of water was added to 1 L of this culture solution, sterilized at 137 ° C. for 5 seconds with a plate-type sterilizer, and aseptically filled into pre-washed and sterilized PET bottles 350 mL each to prepare the tea beverage of the present invention. . The obtained tea beverage contained 280 mg of GABA per one (350 mL) and had a good flavor.
<官能試験>
無作為に選んだパネラー8名（男女同数）に、実施例4で調製した本発明茶飲料試料と、比較のため、原料として利用した茶抽出液（未発酵）を上記本発明飲料と同濃度に希釈したもの比較試料とを試飲させ、下記各項目について、各基準に従って3段階評価(3点評価)させた。
<Sensory test>
Eight randomly selected panelists (same number of men and women), the tea beverage sample of the present invention prepared in Example 4, and the tea extract (unfermented) used as a raw material at the same concentration as the beverage of the present invention for comparison. A comparative sample was diluted and was sampled, and the following items were evaluated in three stages (three-point evaluation) according to each criterion.
<評価項目>
・ こく（深み）：強い=3点, 普通=2点, 弱い=1点
・ 苦味：程よい=3点、やや弱いあるいはやや強い=2点、弱いか或いは強い=1点
・ 旨み：強い=3点、普通=2点、弱い=1点
・ 甘味：強い=3点、普通=2点、弱い=1点
各試料について得られた評価点の合計を、下記表3に示す。
<Evaluation item>
・ Body (depth): Strong = 3 points, Normal = 2 points, Weak = 1 point ・ Bitterness: Moderate = 3 points, Slightly weak or slightly strong = 2 points, Weak or strong = 1 point ・ Taste: Strong = 3 Point, normal = 2 points, weak = 1 point ・ Sweetness: strong = 3 points, normal = 2 points, weak = 1 point The total evaluation score obtained for each sample is shown in Table 3 below.
尚、表3には、実施例3において比較として示したギャバロン茶を比較例として、同様に試験して得られた結果を併記する。 Table 3 also shows the results obtained by testing in the same manner using the Gabalon tea shown as a comparison in Example 3 as a comparative example.
表3に示される結果から明らかなとおり、本発明茶飲料は、多量のGABAを含んでいるにもかかわらず、原料茶と同等もしくはこれを凌ぐ優れた官能評価を与えられるものであり、緑茶本来の味わいおよび風味を有するものであることが判る。これに対してギャバロン茶は、そのような優れた評価は得られないことが明らかである。 As is apparent from the results shown in Table 3, the tea beverage of the present invention is capable of giving an excellent sensory evaluation equivalent to or exceeding that of the raw tea, despite containing a large amount of GABA. It can be seen that it has a taste and flavor of. On the other hand, it is clear that Gavalon tea cannot obtain such excellent evaluation.
実施例5と同様にして得られた培養上澄0.1Lに、ウーロン茶エキス(Brix9%)0.5L、水17.4LおよびビタミンC 2.5gを添加し、137℃で5秒殺菌し、予め洗浄および滅菌したペットボトルに500mLずつ充填した。このようにして一本当たり約44mgのGABAを含有し、良好な風味のウーロン茶飲料を得た。 To culture supernatant 0.1L obtained in the same manner as in Example 5, oolong tea extract (Brix9%) 0.5L, water 17.4L and vitamin C 2.5g were added, sterilized at 137 ° C for 5 seconds, washed and sterilized beforehand. Each 500 ml of the bottle was filled. In this way, a oolong tea beverage having about 44 mg of GABA per bottle and having a good flavor was obtained.
実施例5と同様にして得られた培養上澄1Lに、紅茶エキス(Brix8.1%)1.1L、濃縮レモン果汁(酸度22％)100mLおよびブドウ糖果糖液糖2.7Lを加え、更に水を加えて全量を30Lとした。これを100mLずつガラス瓶に充填し、キャップを巻しめた後、85℃で30分間殺菌した。このようにして1本あたりGABAを約53mg含む紅茶系飲料を得た。 To 1 L of the culture supernatant obtained in the same manner as in Example 5, 1.1 L of black tea extract (Brix 8.1%), 100 mL of concentrated lemon juice (acidity 22%) and 2.7 L of glucose fructose liquid sugar are added, and water is further added. The total volume was 30L. 100 mL of this was filled into a glass bottle, wound with a cap, and then sterilized at 85 ° C. for 30 minutes. Thus, a tea-based beverage containing about 53 mg of GABA per bottle was obtained.
グルコース0.5%、グルタミン酸ナトリウム1.1%、大豆ペプトン0.5%および酵母エキス0.1%が含有されるように各成分を添加した紅茶エキス(Brix 3.0%、タンニン含量4.5mg/mL) 10Lを、137℃で3秒間殺菌し、同じ培地で30℃下に12時間前培養したラクトバチルス・ブレビス mh4219株500mL を接種（接種後の生菌数1×107cfu/ml）した。このものを30℃で48時間静置培養し、得られる培養液から遠心分離により菌体を除去して、透明な発酵液を得た。 10 L of black tea extract (Brix 3.0%, tannin content 4.5 mg / mL) with each component added to contain 0.5% glucose, 1.1% sodium glutamate, 0.5% soybean peptone and 0.1% yeast extract at 137 ° C Sterilized for 2 seconds and inoculated with 500 mL of Lactobacillus brevis mh4219 strain pre-cultured at 30 ° C. for 12 hours in the same medium (the number of viable bacteria after inoculation was 1 × 10 7 cfu / ml). This was statically cultured at 30 ° C. for 48 hours, and the cells were removed from the resulting culture by centrifugation to obtain a transparent fermentation broth.
この発酵液のGABA含量は、約600mg/100mLであり、GABA変換率は約95%であった。 The GABA content of this fermentation broth was about 600 mg / 100 mL, and the GABA conversion rate was about 95%.
得られた発酵液9.0Lにブドウ糖果糖液糖10.0 L、ビタミンC 30gおよび水181 Lを加え、137℃で3秒間殺菌し、350mLずつペットボトルに充填して、GABAを約30mg/100mLの濃度で含む紅茶飲料を得た。 Add 9.0 L of glucose fructose liquid sugar, 30 g of vitamin C and 181 L of water to 9.0 L of the obtained fermentation broth, sterilize at 137 ° C for 3 seconds, fill 350 mL each into a PET bottle, and GABA concentration of about 30 mg / 100 mL A black tea beverage was obtained.
本発明は、茶抽出物中でGABA産生能を発揮する新しい乳酸菌およびこれを利用してGABAを強化された茶飲料を製造する方法を提供するものであり、得られる茶飲料は、高GABA含量に基づいて、GABA本来の各種生理活性を奏し得、機能性食品、健康食品などとして有用である。 The present invention provides a novel lactic acid bacterium that exhibits GABA production ability in a tea extract and a method for producing a GABA-enhanced tea beverage using the same, and the resulting tea beverage has a high GABA content. Based on the above, it can exhibit various physiological activities inherent to GABA and is useful as a functional food, health food, and the like.
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