JP6333509B2 - Process for producing fractions containing reduced cinnamic acid analogues - Google Patents
Process for producing fractions containing reduced cinnamic acid analogues Download PDFInfo
- Publication number
- JP6333509B2 JP6333509B2 JP2012140776A JP2012140776A JP6333509B2 JP 6333509 B2 JP6333509 B2 JP 6333509B2 JP 2012140776 A JP2012140776 A JP 2012140776A JP 2012140776 A JP2012140776 A JP 2012140776A JP 6333509 B2 JP6333509 B2 JP 6333509B2
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- JP
- Japan
- Prior art keywords
- acid
- lactobacillus
- lactic acid
- dihydroferulic
- reduced
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Description
本発明は、フェルラ酸（Ｆｅｒｕｌｉｃ ａｃｉｄ）などの桂皮酸類縁化合物を産生する能力を有し、かつフェルラ酸などの桂皮酸類縁化合物を還元する能力を有する乳酸菌を用いて植物素材を処理する工程を含むことを特徴とする還元された桂皮酸類縁化合物含有画分の製造方法に関する。また、上記能力を有する乳酸菌及び還元された桂皮酸類縁化合物を含有する植物素材由来の乳酸菌発酵飲食品、上記能力を有する乳酸菌のスクリーニング方法等に関する。 The present invention includes a step of treating a plant material using a lactic acid bacterium having an ability to produce a cinnamic acid analog such as ferulic acid and an ability to reduce a cinnamic acid analog such as ferulic acid. The present invention relates to a method for producing a fraction containing a reduced cinnamic acid-related compound. Moreover, it is related with the lactic acid bacteria fermented food-drinks derived from the plant raw material containing the lactic acid bacteria which have the said capability, and the reduced cinnamic acid analog, the screening method of the lactic acid bacteria which have the said capability, etc.
近年、生活習慣病や癌、老化などを促進する要因として、生体内における酸化ストレスが重要な因子であることが明らかとなりつつある。酸化ストレスとは、様々な要因（紫外線、喫煙、食品添加物、病原菌、精神的ストレス、過激な運動、虚血、炎症など）により生体内で活性酸素種（ROS）・フリーラジカルが発生する状態であり、それによるダメージが細胞や組織に蓄積することで、最終的に生活習慣病や癌、老化を促進する。生体内には元来、このような酸化ストレスに対する防御機構が備わっており、ROSの発生を抑制したり、ダメージを受けた細胞・組織を修復したりする作用がある。更にヒトは、抗酸化物質を体内で合成したり外部から摂取したりすることにより、体内で発生するフリーラジカルを消去している。ビタミンA・C・E、コエンザイムQ10、αリポ酸、グルタチオンは代表的な抗酸化物質であるが、食品由来の抗酸化成分としては更に、トマトのリコピン、緑茶のカテキン、赤ワインのポリフェノール、ブルーベリーのアントシアニンなども知られている。また、広く植物体内に存在する抗酸化物質として、フェルラ酸やパラクマル酸等のフェノール性化合物が知られており、特に米ぬかや果物、野菜の皮等に多く含まれていることが報告されている。
フェルラ酸やパラクマル酸は、食物中では主に食物繊維（ヘミセルロース）や糖等とエステルの形態で結合しており、そのままの状態では生体内での吸収性が悪い。結合型のフェルラ酸は腸内細菌等により分解を受けることでその一部が遊離のフェルラ酸となり、腸管より吸収されるとされているが、個々人の腸内細菌叢によらず、植物素材から機能性の高いフェルラ酸等を効果的に摂取するためには、事前にフェルラ酸を遊離の形に変換しておくことが重要であると考えられる。一方で、遊離フェルラ酸でも100％が吸収されるわけではなく、また吸収後速やかに消費されてしまうため、遊離フェルラ酸の吸収性と血中での安定性が改善されれば、ヒトが摂取した際の抗酸化能力がより高まることが期待される。
植物体からフェルラ酸類を遊離する方法として、物理化学的処理、酵素処理、微生物処理等による方法についての報告がある。物理化学的処理法としては、米ぬかから米サラダ油、脂肪酸を抽出した残渣をアルカリ処理してフェルラ酸を生成する方法（特許文献１）があるが、高温でアルカリ処理し、その後抽出操作が必要となるため、工程が煩雑といった課題がある。リグニンを、水蒸気を含む気体で高温高圧処理することにより、フェルラ酸等の化合物量を増加させる方法（特許文献２）も知られているが、高温高圧処理のための設備が必要となる。
酵素を用いる方法としては、米ぬかのエタノール抽出物を酵素処理することにより、フェルラ酸を遊離する方法（特許文献３）が知られているが、エタノール抽出とその後の乾燥工程があり、作業が煩雑でエネルギーコストも高い。フェルラ酸エステラーゼやキシラナーゼ、セルラーゼ活性を有する酵素を用いて、遊離フェルラ酸を高含有する発酵アルコール飲料を製造する方法（特許文献４）も報告があるが、実際の工程を考えると、酵素の添加・反応工程が煩雑となり、酵素のコストの問題もある。
微生物を用いる方法としては、ペニシリウム・クリソゲナムの培養液とシュガービートパルプを反応させることによりフェルラ酸を遊離する方法（特許文献５）や、フェルラ酸エステラーゼ活性を有する麹菌を使用すること特徴とする高香味穀類蒸留酒に関する特許（特許文献６）があるが、これらの微生物はプロテアーゼ等の他の酵素活性も強いため、植物素材の風味を大きく変えてしまう可能性がある。
また、遊離のフェルラ酸を合成する方法として、シュードモナス・フルオレッセンス（Ｐｓｅｕｄｏｍｏｎａｓ ｆｌｕｏｒｅｓｃｅｎｓ）を用いて、オイゲノールからフェルラ酸を産生する方法（特許文献７、８）が知られているが、オイゲノールは、過剰摂取により血尿・痙攣・下痢・吐き気・意識喪失・めまい・動悸等の症状があらわれるほか、皮膚に触れるとアレルギー反応により皮膚炎を起こすことがある。そのため、食品に残存した場合の安全性に課題がある。ベンズアルデヒド誘導体を触媒の存在下、マロン酸または無水酢酸と反応させることによりフェルラ酸を誘導させる方法（特許文献９）も知られているが、１３５℃程度に加熱処理する必要があり、そのための設備が必要となる。
In recent years, it is becoming clear that oxidative stress in vivo is an important factor as a factor promoting lifestyle-related diseases, cancer, and aging. Oxidative stress is a state in which reactive oxygen species (ROS) and free radicals are generated in the body due to various factors (ultraviolet rays, smoking, food additives, pathogens, mental stress, extreme exercise, ischemia, inflammation, etc.) And the damage caused by it accumulates in cells and tissues, ultimately promoting lifestyle-related diseases, cancer, and aging. The living body originally has a defense mechanism against such oxidative stress, and has the effect of suppressing the generation of ROS and repairing damaged cells and tissues. Furthermore, humans eliminate free radicals generated in the body by synthesizing antioxidant substances in the body or taking them from the outside. Vitamin A / C / E, coenzyme Q10, alpha lipoic acid, and glutathione are typical antioxidants, but as antioxidants derived from food, lycopene from tomato, catechin from green tea, polyphenol from red wine, blueberry Anthocyanins are also known. In addition, phenolic compounds such as ferulic acid and paracoumaric acid are widely known as antioxidants present in plants, and it has been reported that they are particularly contained in rice bran, fruits, and vegetable peels. .
Ferulic acid and paracoumaric acid are mainly bound to dietary fiber (hemicellulose), sugar, and the like in the form of esters in food, and are not easily absorbed in vivo. It is said that bound ferulic acid is decomposed by intestinal bacteria, etc., and part of it becomes free ferulic acid and is absorbed from the intestinal tract. In order to effectively ingest ferulic acid having high functionality, it is considered important to convert ferulic acid into a free form in advance. On the other hand, 100% of free ferulic acid is not absorbed, and it is consumed quickly after absorption. Therefore, if the absorption of free ferulic acid and the stability in blood are improved, humans ingest it. It is expected that the antioxidant capacity will increase further.
As methods for releasing ferulic acids from plants, there are reports on methods such as physicochemical treatment, enzyme treatment, and microbial treatment. As a physicochemical treatment method, there is a method of producing ferulic acid by subjecting a residue obtained by extracting rice salad oil and fatty acids from rice bran to produce ferulic acid (Patent Document 1). Therefore, there is a problem that the process is complicated. A method for increasing the amount of a compound such as ferulic acid by treating lignin with a gas containing water vapor at a high temperature and pressure (Patent Document 2) is also known, but equipment for high temperature and pressure treatment is required.
As a method using an enzyme, a method of releasing ferulic acid by enzymatic treatment of an ethanol extract of rice bran (Patent Document 3) is known, but there are ethanol extraction and a subsequent drying step, and the work is complicated. And energy costs are high. There is also a method for producing a fermented alcoholic beverage containing a high amount of free ferulic acid using an enzyme having ferulic acid esterase, xylanase, or cellulase activity (Patent Document 4), but considering the actual process, addition of the enzyme -The reaction process becomes complicated and there is a problem of the cost of the enzyme.
As a method using a microorganism, a method of releasing ferulic acid by reacting a culture solution of penicillium chrysogenum with sugar beet pulp (Patent Document 5), or using a koji mold having ferulic acid esterase activity is high. Although there is a patent (Patent Document 6) relating to flavored cereal distilled liquor, these microorganisms also have strong other enzyme activities such as proteases, which may greatly change the flavor of plant materials.
Further, as a method for synthesizing free ferulic acid, a method of producing ferulic acid from eugenol using Pseudomonas fluorescens (Patent Documents 7 and 8) is known, but eugenol is excessive. Ingestion may cause symptoms such as hematuria, convulsions, diarrhea, nausea, loss of consciousness, dizziness, palpitation, and contact with the skin may cause dermatitis due to an allergic reaction. Therefore, there is a problem in safety when left in food. A method of inducing ferulic acid by reacting a benzaldehyde derivative with malonic acid or acetic anhydride in the presence of a catalyst is also known (Patent Document 9). Is required.
本発明は、植物素材及び乳酸菌を用いた還元された桂皮酸類縁化合物含有画分の製造方法、乳酸菌及び還元された桂皮酸類縁化合物を含有する植物素材由来の乳酸菌発酵飲食品、フェルラ酸などの桂皮酸類縁化合物を産生する能力を有し、かつフェルラ酸などの桂皮酸類縁化合物をより生体への移行性や安定性が高い還元体に変換する能力を有する乳酸菌のスクリーニング方法を提供すること等を課題とする。 The present invention relates to a method for producing a fraction containing a reduced cinnamate related compound using a plant material and lactic acid bacteria, a lactic acid bacteria fermented food or drink derived from a plant material containing lactic acid bacteria and a reduced cinnamate related compound, ferulic acid, etc. Providing a screening method for lactic acid bacteria that has the ability to produce cinnamic acid analogs and has the ability to convert cinnamic acid analogs such as ferulic acid into reduced forms with higher transferability and stability to living bodies, etc. Is an issue.
上記課題を解決するため、本発明者らは、食経験があり、安全に食することができる乳酸菌をターゲットとして、まずフェルラ酸を植物素材から遊離する乳酸菌を探索した結果、一部の乳酸菌において、フェルラ酸を遊離するフェルラ酸エステラーゼ活性を見出した。ところが、そのうちのある種の乳酸菌はフェルラ酸をジヒドロフェルラ酸（３−（４−Ｈｙｄｒｏｘｙ−３−ｍｅｔｈｏｘｙｐｈｅｎｙｌ）ｐｒｏｐｉｏｎｉｃ ａｃｉｄ）に代謝するフェルラ酸リダクターゼ活性も保有していることが明らかとなった。本発明者らの鋭意検討の結果、このジヒドロフェルラ酸がフェルラ酸よりも生体への移行性・安定性が向上していることを見出した。以上の知見から、今回見出されたフェルラ酸エステラーゼ活性を持ち、且つフェルラ酸リダクターゼ活性を持つ乳酸菌で植物素材を発酵させることにより、フェルラ酸よりも生体への移行性・安定性が向上した遊離のジヒドロフェルラ酸を生成・蓄積することができ、摂食した際の生体内での抗酸化性を高めた飲食品を提供することができる。さらに、本発明者らは、フェルラ酸エステラーゼ活性を持ち、且つフェルラ酸リダクターゼ活性を持つ乳酸菌につき、フェルラ酸以外の桂皮酸類縁化合物としてコーヒー酸（Ｃａｆｆｅｉｃ ａｃｉｄ）やパラクマル酸（ｐ−Ｃｏｕｍａｒｉｃ ａｃｉｄ）のエステルからの遊離活性・還元活性を調べたところ、これらの乳酸菌がジヒドロコーヒー酸（３−（３、４−Ｄｉｈｙｄｒｏｘｙｐｈｅｎｙｌ）ｐｒｏｐｉｏｎｉｃ ａｃｉｄ）、ジヒドロパラクマル酸（３−（ｐ−Ｈｙｄｒｏｘｙｐｈｅｎｙｌ）ｐｒｏｐｉｏｎｉｃ ａｃｉｄ）の産生能を有することを見出した。したがって、本発明は多様な還元された桂皮酸類縁化合物の製造方法等を提供する。さらに、本発明は、ジヒドロフェルラ酸等を遊離、蓄積する乳酸菌を取得するための方法として、フェルラ酸などの桂皮酸類縁化合物を産生する能力を有し、かつフェルラ酸などの桂皮酸類縁化合物をより生体への移行性が高い還元体に変換する能力を有する乳酸菌のスクリーニング方法を提供する。
すなわち、本発明は下記（１）から（１２）を提供する。
（１）還元された桂皮酸類縁化合物産生能を有する乳酸菌を植物素材中で培養する工程を含むことを特徴とする、還元された桂皮酸類縁化合物含有画分の製造方法。
（２）前記還元された桂皮酸類縁化合物産生能を有する乳酸菌が下記性質を有するものである、上記（１）に記載の還元された桂皮酸類縁化合物含有画分の製造方法；
性質：フェルラ酸エチルを０．０５重量％添加した培地において、前記乳酸菌の至適温度で４８時間培養したとき、前記フェルラ酸エチルの３０％以上をジヒドロフェルラ酸として遊離する性質。
（３）前記還元された桂皮酸類縁化合物産生能を有する乳酸菌がラクトバチルス属の乳酸菌である、上記（１）又は（２）に記載の製造方法。
（４）前記還元された桂皮酸類縁化合物産生能を有する乳酸菌が下記乳酸菌群から選択される、上記（１）から（３）に記載の還元された桂皮酸類縁化合物含有画分の製造方法；
乳酸菌群：ラクトバチルス・アシドフィルス、ラクトバチルス・ガセリ、ラクトバチルス・クリスパタス、ラクトバチルス・アミロボラス、ラクトバチルス・ジョンソニー、ラクトバチルス・ケフィラノファシエンス、ラクトバチルス・ガリナラム、ラクトバチルス・ファーメンタム、ラクトバチルス・デルブルイッキー、ラクトバチルス・ブクネリ、エンテロコッカス・フェカリス。
（５）前記還元された桂皮酸類縁化合物が、ジヒドロフェルラ酸、ジヒドロパラクマル酸又はジヒドロコーヒー酸である、上記（１）から（４）のいずれか１項に記載の還元された桂皮酸類縁化合物含有画分の製造方法。
（６）還元された桂皮酸類縁化合物産生能を有する乳酸菌及び還元された桂皮酸類縁化合物を少なくとも含有する、植物素材由来の乳酸菌発酵飲食品。
（７）前記還元された桂皮酸類縁化合物産生能を有する乳酸菌が下記性質を有するものである、上記（６）に記載の乳酸菌発酵飲食品；
性質：フェルラ酸エチルを０．０５重量％添加した培地において、前記乳酸菌の至適温度で４８時間培養したとき、前記フェルラ酸エチルの３０％以上をジヒドロフェルラ酸として遊離する性質。
（８）前記還元された桂皮酸類縁化合物産生能を有する乳酸菌がラクトバチルス属の乳酸菌である、上記（６）又は（７）に記載の乳酸菌発酵飲食品。
（９）前記還元された桂皮酸類縁化合物産生能を有する乳酸菌が下記乳酸菌群から選択される乳酸菌である、上記（６）から（８）のいずれか１項に記載の乳酸菌発酵飲食品；
乳酸菌群：ラクトバチルス・アシドフィルス、ラクトバチルス・ガセリ、ラクトバチルス・クリスパタス、ラクトバチルス・アミロボラス、ラクトバチルス・ジョンソニー、ラクトバチルス・ケフィラノファシエンス、ラクトバチルス・ガリナラム、ラクトバチルス・ファーメンタム、ラクトバチルス・デルブルイッキー、ラクトバチルス・ブクネリ、エンテロコッカス・フェカリス。
（１０）前記還元された桂皮酸類縁化合物が、ジヒドロフェルラ酸、ジヒドロパラクマル酸又はジヒドロコーヒー酸である、上記（６）から（９）のいずれか１項に記載の乳酸菌発酵飲食品。
（１１）下記ステップを少なくとも含むことを特徴とする、還元された桂皮酸類縁化合物産生能を有する乳酸菌のスクリーニング方法；
ステップ１：フェルラ酸エチルを含有する培地で検体乳酸菌を培養し、産生されたジヒドロフェルラ酸を定量するステップ
ステップ２：ステップ１において、フェルラ酸エチルに対して一定割合以上のジヒドロフェルラ酸を産生する性質を有する乳酸菌を判定するステップ。
（１２）上記（１１）に記載のスクリーニング方法によって還元された桂皮酸類縁化合物産生能を有すると判定された乳酸菌であって、下記性質を有する乳酸菌；
性質：フェルラ酸エチルを０．０５重量％添加した培地において、前記乳酸菌の至適温度で４８時間培養したとき、前記フェルラ酸エチルの３０％以上をジヒドロフェルラ酸として遊離する性質。
In order to solve the above-mentioned problems, the present inventors searched for a lactic acid bacterium that releases ferulic acid from a plant material, targeting lactic acid bacteria that have food experience and can be eaten safely. The ferulic acid esterase activity which releases ferulic acid was discovered. However, it has been clarified that certain types of lactic acid bacteria also possess ferulic acid reductase activity that metabolizes ferulic acid to dihydroferulic acid (3- (4-Hydroxy-3-methoxyphenyl) propionic acid). As a result of intensive studies by the present inventors, it has been found that this dihydroferulic acid has improved migration and stability to a living body compared to ferulic acid. Based on the above knowledge, fertilic acid esterase activity found this time and fermenting plant material with lactic acid bacteria having ferulic acid reductase activity, release that improved migration and stability to the living body than ferulic acid It is possible to produce and accumulate dihydroferulic acid, and to provide a food or drink with enhanced in vivo antioxidant properties when consumed. Furthermore, the present inventors have found that lactic acid bacteria having ferulic acid esterase activity and ferulic acid reductase activity include caffeic acid and p-coumaric acid as cinnamate-related compounds other than ferulic acid. When the free activity / reduction activity from the ester was examined, these lactic acid bacteria were found to be dihydrocaffeic acid (3- (3,4-hydroxyphenyl) propionic acid) and dihydroparacoumaric acid (3- (p-hydroxyphenyl) propionic acid). It has been found that it has productivity. Accordingly, the present invention provides various methods for producing reduced cinnamic acid analogs. Furthermore, the present invention provides a method for obtaining a lactic acid bacterium that liberates and accumulates dihydroferulic acid and the like, and has the ability to produce a cinnamic acid analog such as ferulic acid, and a cinnamic acid analog such as ferulic acid. Provided is a screening method for lactic acid bacteria having the ability to convert to a reduced form having a higher transferability to a living body.
That is, the present invention provides the following (1) to (12).
(1) A method for producing a reduced cinnamic acid-related compound-containing fraction, comprising a step of culturing a lactic acid bacterium having reduced cinnamic acid-related compound-producing ability in a plant material.
(2) The method for producing a fraction containing a reduced cinnamic acid analog compound according to (1) above, wherein the lactic acid bacterium having the ability to produce the reduced cinnamic acid analog compound has the following properties;
Properties: A property in which 30% or more of the ethyl ferulate is liberated as dihydroferulic acid when cultured for 48 hours at an optimum temperature of the lactic acid bacteria in a medium supplemented with 0.05% by weight of ethyl ferulate.
(3) The production method according to (1) or (2) above, wherein the reduced cinnamate-related compound-producing lactic acid bacterium is a Lactobacillus lactic acid bacterium.
(4) The method for producing a fraction containing a reduced cinnamic acid analog compound according to (1) to (3) above, wherein the lactic acid bacterium having the ability to produce the reduced cinnamic acid analog compound is selected from the following group of lactic acid bacteria;
Lactic acid bacteria group: Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus crispatas, Lactobacillus amylovorus, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus gallinarum, Lactobacillus fermentum, Lactobacillus・ Delbrücky, Lactobacillus bukuneri, Enterococcus faecalis.
(5) The reduced cinnamic acid analog according to any one of (1) to (4), wherein the reduced cinnamic acid analog is dihydroferulic acid, dihydroparacoumaric acid or dihydrocaffeic acid. A method for producing a compound-containing fraction.
(6) A plant material-derived lactic acid bacteria fermented food or drink containing at least lactic acid bacteria having the ability to produce reduced cinnamic acid analogs and reduced cinnamic acid analogs.
(7) The lactic acid bacteria fermented food or drink according to (6) above, wherein the reduced lactic acid bacteria having the ability to produce a cinnamic acid analog compound have the following properties;
Properties: A property in which 30% or more of the ethyl ferulate is liberated as dihydroferulic acid when cultured for 48 hours at an optimum temperature of the lactic acid bacteria in a medium supplemented with 0.05% by weight of ethyl ferulate.
(8) The lactic acid bacteria fermented food or drink according to (6) or (7) above, wherein the lactic acid bacteria having the ability to produce reduced cinnamic acid analogs are Lactobacillus lactic acid bacteria.
(9) The lactic acid bacteria fermented food or drink according to any one of (6) to (8) above, wherein the lactic acid bacteria having the ability to produce reduced cinnamic acid analogs are lactic acid bacteria selected from the following group of lactic acid bacteria;
Lactic acid bacteria group: Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus gallinarum, Lactobacillus fermentum, Lactobacillus・ Delbrücky, Lactobacillus bukuneri, Enterococcus faecalis.
(10) The lactic acid bacteria fermented food or drink according to any one of (6) to (9) above, wherein the reduced cinnamic acid-related compound is dihydroferulic acid, dihydroparacoumaric acid or dihydrocaffeic acid.
(11) A screening method for lactic acid bacteria having the ability to produce reduced cinnamic acid analogs, comprising at least the following steps;
Step 1: culturing specimen lactic acid bacteria in a medium containing ethyl ferulate and quantifying the produced dihydroferulic acid Step 2: producing dihydroferulic acid at a certain ratio or more with respect to ethyl ferulate in
(12) A lactic acid bacterium determined to have the ability to produce cinnamic acid analogs reduced by the screening method described in (11) above, and having the following properties;
Properties: A property in which 30% or more of the ethyl ferulate is liberated as dihydroferulic acid when cultured for 48 hours at an optimum temperature of the lactic acid bacteria in a medium supplemented with 0.05% by weight of ethyl ferulate.
本発明の製造方法によれば、非常に簡便、効率的、安価な方法で、還元された桂皮酸類縁化合物含有画分を製造することができる。特に、本発明の製造方法は乳酸菌発酵を利用するため、特別な設備を必要とせず、１０℃から５０℃といったコントロールしやすい温度帯で野菜素材中の還元された桂皮酸類縁化合物量を増加させることができる。また、原料として植物素材を用いるため、得られる還元された桂皮酸類縁化合物含有画分は、安全性、栄養価が非常に高く、乳酸菌による発酵のため風味にも優れ、特に飲食品の用途に好適である。還元された桂皮酸類縁化合物含有画分中の植物由来成分は、還元された桂皮酸類縁化合物が生体内で吸収しやすい形態となっていると同時に、桂皮酸類縁化合物よりも安定性が向上しているため、摂取した際に高い機能性が期待される。
本発明はさらに、還元された桂皮酸類縁化合物産生能を有する乳酸菌のスクリーニング方法を提供する。本発明スクリーニング方法によれば、本発明製造方法等に好適に利用できる乳酸菌を効率よくスクリーニングすることができる。また、このようにして得られた乳酸菌を定期的に摂取することにより、消化管内においてもこれらの乳酸菌が桂皮酸類縁化合物の還元反応を行なうため、摂取した食品中の桂皮酸類縁化合物の効果がより高まることが期待される。更に、これらの乳酸菌のうち、消化液耐性があり、腸管内でも生存が可能な菌株を用いることで、よりいっそう桂皮酸類縁化合物の効果が高まることが期待される。
According to the production method of the present invention, a reduced cinnamic acid analog-containing fraction can be produced by a very simple, efficient and inexpensive method. In particular, since the production method of the present invention uses lactic acid bacteria fermentation, no special equipment is required, and the amount of reduced cinnamic acid analogs in vegetable materials is increased in a temperature range that is easy to control, such as 10 ° C. to 50 ° C. be able to. In addition, because the plant material is used as a raw material, the obtained fraction containing cinnamic acid-related compounds has a very high safety and nutritional value, and is excellent in flavor due to fermentation by lactic acid bacteria. Is preferred. The plant-derived component in the fraction containing the reduced cinnamic acid analog is in a form that the reduced cinnamic acid analog is easily absorbed in vivo, and at the same time, the stability is improved compared to the cinnamic acid analog. Therefore, high functionality is expected when ingested.
The present invention further provides a method for screening lactic acid bacteria having the ability to produce reduced cinnamic acid analogs. According to the screening method of the present invention, lactic acid bacteria that can be suitably used in the production method of the present invention can be efficiently screened. In addition, by periodically ingesting the lactic acid bacteria obtained in this way, these lactic acid bacteria also carry out the reduction reaction of cinnamic acid analogs in the digestive tract, so that the effects of cinnamic acid analogs in the ingested food are effective. Expected to increase further. Further, among these lactic acid bacteria, the effect of cinnamic acid-related compounds is expected to be further enhanced by using strains that are resistant to digestive juice and can survive in the intestinal tract.
（１）本発明の還元された桂皮酸類縁化合物含有画分の製造方法
本発明の還元された桂皮酸類縁化合物含有画分の製造方法は、還元された桂皮酸類縁化合物産生能を有する乳酸菌を植物素材中で培養する工程を少なくとも含むことを特徴とする、還元された桂皮酸類縁化合物含有画分の製造方法である。
還元された桂皮酸類縁化合物産生能を有する乳酸菌は、植物素材中で遊離の還元された桂皮酸類縁化合物の産生に好適な条件で培養したときに、還元された桂皮酸類縁化合物を遊離できる乳酸菌である限りにおいて特に限定されない。
具体的には、植物素材より桂皮酸類縁化合物を遊離する活性（桂皮酸類縁化合物エステラーゼ活性）を持ち、且つその桂皮酸類縁化合物を一定以上還元された桂皮酸類縁化合物に還元する活性（桂皮酸類縁化合物リダクターゼ活性）を持つことを指標として選択することができる。このように選択される乳酸菌は、植物等に含まれるフェノール性化合物が有するエステル結合を切断する活性を持ち、植物素材より遊離したフェルラ酸等の桂皮酸類縁化合物を血中への移行性・安定性の良い還元体に変換することができる。
還元された桂皮酸類縁化合物産生能を有する乳酸菌としては、下記の性質を有するものが例示される。
性質：フェルラ酸エチルを０．０５重量％添加した培地において、前記乳酸菌の至適温度で４８時間培養したとき、前記フェルラ酸エチルの３０％以上をジヒドロフェルラ酸として遊離する性質。
上記性質の有無の試験において、フェルラ酸エチルとしては、例えば東京化成工業株式会社などの公知のものを用いることができる。ジヒドロフェルラ酸としては、例えばＡｌｆａ Ａｅｓａｒなどの公知のものを用いることが出来る。培地としては、乳酸菌の生育に好適なものを用いることができ、例えばＭＲＳ−Ｂｒｏｔｈ培地や脱脂乳培地など通常用いる培地の中から、最も対象の乳酸菌の生育に適した培地を適宜選択して使用できる。乳酸菌の生育に好適な培養条件を採用し、これにより培養４８時間で乳酸菌数が十分に増え、フェルラ酸又はジヒドロフェルラ酸の産生能を適切に評価できる。遊離したフェルラ酸又はジヒドロフェルラ酸の割合は、培養上清を高速液体クロマトグラフィー（ＨＰＬＣ）法に供し、フェルラ酸エチル、フェルラ酸又はジヒドロフェルラ酸のピーク面積を検出することにより算出できる。培地の種類、至適温度等の培養条件、高速液体クロマトグラフィー法の条件等、より具体的には後述の実施例に記載の方法を採用することができる。「前記フェルラ酸エチルの３０％以上をジヒドロフェルラ酸として遊離する性質」における「３０％以上」とは、３０％から１００％であればよいが、４０％以上、５０％以上、６０％以上、７０％以上と割合が高い程好ましい。本性質を持つ乳酸菌については、フェルラ酸と構造が近似しているコーヒー酸やパラクマル酸などの桂皮酸類縁化合物のエステル体に対しても同様の活性を有し、ジヒドロコーヒー酸やジヒドロパラクマル酸を生成する。
還元された桂皮酸類縁化合物産生能を有する乳酸菌の具体例としては、ラクトバチルス属、ビフィドバクテリウム属、ストレプトコッカス属、ラクトコッカス属、ロイコノストック属、オエノコッカス属、エンテロコッカス属に属する乳酸菌が例示される。ラクトバチルス属としては、ラクトバチルス・アシドフィルス、ラクトバチルス・ガセリ、ラクトバチルス・クリスパタス、ラクトバチルス・アミロボラス、ラクトバチルス・ジョンソニー、ラクトバチルス・ケフィラノファシエンス、ラクトバチルス・ガリナラム、ラクトバチルス・ファーメンタム、ラクトバチルス・デルブルイッキー、ラクトバチルス・ブクネリ等が例示され、エンテロコッカス属としてはエンテロコッカス・フェカリス等が例示される。なかでも、ラクトバチルス・アシドフィルス、ラクトバチルス・ガセリ、ラクトバチルス・クリスパタス、ラクトバチルス・アミロボラス、ラクトバチルス・ジョンソニー、ラクトバチルス・ケフィラノファシエンス、ラクトバチルス・ガリナラム、ラクトバチルス・ファーメンタム、ラクトバチルス・デルブルイッキー、ラクトバチルス・ブクネリ、及び、エンテロコッカス・フェカリスが好ましく、ラクトバチルス・ガセリＦＥＲＭ−ＢＰ１０９５３、ＪＣＭ１０２５、ＪＣＭ１０３１、ＪＣＭ１１３０、ＪＣＭ１１３１、ＪＣＭ８７８７、ＪＣＭ８７８８、ラクトバチルス・アシドフィルスＪＣＭ１１３２、ラクトバチルス・クリスパタスＪＣＭ１０３７、ＪＣＭ１１８５、ＪＣＭ５８０６、ラクトバチルス・アミロボラスＪＣＭ１０３３、ＪＣＭ１１２６、ＪＣＭ１１２７、ラクトバチルス・ジョンソニーＪＣＭ２０１０、ＪＣＭ２１２２、ラクトバチルス・ケフィラノファシエンスＪＣＭ６９８５、ＳＢＴ１１２２３、ラクトバチルス・ガリナラムＪＣＭ１０３６、ラクトバチルス・ファーメンタムＳＢＴ１８４６、ＳＢＴ２５３７、ラクトバチルス・デルブルイッキーＳＢＴ０８０３、ＳＢＴ１３７１、及び、ラクトバチルス・ブクネリＳＢＴ１１４１１、エンテロコッカス・フェカリスＳＢＴ１１２０がより好ましく、ラクトバチルス・ガセリＦＥＲＭ−ＢＰ１０９５３が最も好ましい。
なお、フェルラ酸、コーヒー酸及びパラクマル酸の産生能が確認された乳酸菌は、桂皮酸類縁化合物産生能を有する乳酸菌を植物素材中で培養する工程を少なくとも含むことを特徴とする、桂皮酸類縁化合物含有画分の製造方法において利用可能である。そのような乳酸菌は具体的には後述する実施例でフェルラ酸、コーヒー酸及びパラクマル酸の産生能が確認された乳酸菌を例示することができる。
植物素材とは、結合型、エステル型の桂皮酸類縁化合物が少量でも含まれるものであれば特に限定されないが、野菜、穀物、果物、草類、豆類、芋類、種子、米ぬか、ふすま等の素材に由来するものが挙げられる。植物素材とは、上記に例示された植物素材等そのものであってもよいが、植物素材を適宜加工等して得られるものであってもよい。加工方法としては、ミキサー等による粉砕等が例示されるが、特に限定されるものではない。乳酸菌が生育するための十分な水分があることが好ましいが、植物素材を調整後に水分を添加などして調整することもできる。
より効率的に植物素材から桂皮酸類縁化合物を遊離するため、食物繊維分解酵素により植物素材を事前に酵素処理してもよい。使用する酵素としては、キシラナーゼ、セルラーゼ等が例示されるが特に限定されるものではない。例えば、野菜汁に上記酵素を添加し、上記酵素の至適温度で数時間から２日程度酵素反応させて得られる植物素材を用いることにより、より効率的に桂皮酸類縁化合物を遊離することができる。
還元された桂皮酸類縁化合物産生能を有する乳酸菌を植物素材中で培養する方法は、植物素材中で乳酸菌が生育し、植物素材から還元された桂皮酸類縁化合物を産生できる条件である限り特に限定されない。例えば、植物素材に乳酸菌培養物を０．５から１０％程度添加し、乳酸菌が生育可能な温度範囲で1日から７日発酵させることにより培養できるが、これに限定されない。
本発明還元された桂皮酸類縁化合物含有画分の製造方法によって得られる還元された桂皮酸類縁化合物含有画分は、植物素材中で乳酸菌を培養して得られる培養物そのものであってもよいが、培養物に添加物等を添加し、又は不純物を除去する等して得られるものであってもよい。また、培養物から還元された桂皮酸類縁化合物を分画、精製する等して、還元された桂皮酸類縁化合物濃度がより高い還元された桂皮酸類縁化合物含有画分とすることもできる。
（２）本発明乳酸菌発酵飲食品
本発明乳酸菌発酵飲食品は、還元された桂皮酸類縁化合物産生能を有する乳酸菌、還元された桂皮酸類縁化合物を少なくとも含有する、植物素材由来の乳酸菌発酵飲食品である。
還元された桂皮酸類縁化合物産生能を有する乳酸菌としては、植物素材中で遊離の還元された桂皮酸類縁化合物の産生に好適な条件で培養したときに、還元された桂皮酸類縁化合物を遊離できる乳酸菌である限りにおいて特に限定されず、具体的には上記（１）本発明の還元された桂皮酸類縁化合物含有画分製造方法で説明したものを用いることができる。
還元された桂皮酸類縁化合物としては、ジヒドロフェルラ酸、ジヒドロコーヒー酸、ジヒドロパラクマル酸が例示される。
植物素材としては、上記（１）本発明還元された桂皮酸類縁化合物含有画分製造方法で説明したものを用いることができる。例えば、野菜汁、果汁等を用いた場合、風味、栄養価が優れた乳酸菌発酵飲料を提供することができる。
本発明乳酸菌発酵飲食品中の桂皮酸類縁化合物濃度は、植物素材中にどの程度含まれているかによるが、本発明の乳酸菌により、素材中の還元された桂皮酸類縁化合物量を増加させることができる。
本発明乳酸菌発酵飲食品の製造方法は特に限定されないが、本発明還元された桂皮酸類縁化合物含有画分の製造方法により製造することができる。
本発明の植物素材由来の乳酸菌発酵飲食品は、乳や発酵乳などの食品と混合することにより、飲料や発酵乳、デザートなどの形態で提供することができる。
（３）本発明スクリーニング方法
本発明スクリーニング方法は、下記ステップを少なくとも含むことを特徴とする、還元された桂皮酸類縁化合物産生能を有する乳酸菌のスクリーニング方法である；
ステップ１：フェルラ酸エチルを含有する培地で検体乳酸菌を培養し、産生されたジヒドロフェルラ酸の化合物を定量するステップ、
ステップ２：ステップ１において、フェルラ酸エチルに対して一定割合以上のジヒドロフェルラ酸を産生する性質を有する乳酸菌を判定するステップ。
本発明スクリーニング方法によれば、コーヒー酸やパラクマル酸などのエステル体に対しても作用することができ、かつ還元された桂皮酸類縁化合物産生能に優れた乳酸菌をスクリーニングすることができる。
ステップ１において、検体乳酸菌とは還元された桂皮酸類縁化合物産生能の有無の判定対象となる乳酸菌である。ステップ１における培養条件は乳酸菌が好適に生育できる限りにおいて特に限定されず、例えば検体乳酸菌の生育に好適な培地にて至適温度で、乳酸菌数が十分に達する適当な時間（例えば１２から４８時間程度）培養することが例示される。含有するフェルラ酸エチル、ジヒドロフェルラ酸の培地中の濃度も特に限定されないが、例えば０．０１から１重量％、好ましくは０．０５重量％程度が例示される。フェルラ酸、ジヒドロフェルラ酸の定量方法は、例えば高速液体クロマトグラフィー法を採用することができ、例えば上記（１）本発明の還元された桂皮酸類縁化合物酸含有画分の製造方法で説明した方法を採用することができる。ステップ２における一定割合とは、所望の還元された桂皮酸類縁化合物産生能に応じて適宜設定することができ、例えば１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％又は９０％等と設定することができる。
このようにして所望の還元された桂皮酸類縁化合物産生能を有する乳酸菌をスクリーニングすることができ、例えば下記性質を有する乳酸菌を、還元された桂皮酸類縁化合物産生能を有する乳酸菌として得ることができる。
性質：フェルラ酸エチルを０．０５重量％添加した培地において、前記乳酸菌の至適温度で４８時間培養したとき、前記フェルラ酸エチルの３０％以上をジヒドロフェルラ酸として遊離する性質。
以下、本発明を実施例によりさらに具体的に説明するが、本発明の範囲は下記の実施例
に限定されるものではない。
(1) Method for Producing Reduced Cinnamic Acid Related Compound-Containing Fraction of the Present Invention The method for producing a reduced cinnamic acid analog-containing fraction of the present invention comprises a method for producing a reduced cinnamic acid analog-containing compound. A method for producing a fraction containing a reduced cinnamic acid analog compound, comprising at least a step of culturing in a plant material.
Lactic acid bacteria having the ability to produce reduced cinnamic acid analogs are lactic acid bacteria capable of releasing reduced cinnamic acid analogs when cultured under conditions suitable for the production of free reduced cinnamic acid analogs in plant materials. As long as it is, it is not particularly limited.
Specifically, it has an activity to release cinnamic acid analogs from plant materials (cinnamate analog compound esterase activity), and an activity to reduce the cinnamic acid analog to a cinnamic acid analog that has been reduced to a certain extent (cinnamate) Having an analogous compound reductase activity) can be selected as an index. Lactic acid bacteria selected in this way have the activity of cleaving ester bonds of phenolic compounds contained in plants, etc., and transfer and stability of cinnamic acid analogues such as ferulic acid released from plant materials into the blood It can be converted into a reductant with good properties.
Examples of lactic acid bacteria having the ability to produce reduced cinnamic acid analogs include those having the following properties.
Properties: A property in which 30% or more of the ethyl ferulate is liberated as dihydroferulic acid when cultured for 48 hours at an optimum temperature of the lactic acid bacteria in a medium supplemented with 0.05% by weight of ethyl ferulate.
In the test for the presence or absence of the above properties, as the ferulic acid ethyl, for example, a known one such as Tokyo Chemical Industry Co., Ltd. can be used. As the dihydroferulic acid, known ones such as Alfa Aesar can be used. As the medium, a medium suitable for the growth of lactic acid bacteria can be used. For example, a medium most suitable for the growth of the target lactic acid bacteria is appropriately selected from, for example, MRS-Broth medium and skim milk medium. it can. By adopting culture conditions suitable for the growth of lactic acid bacteria, the number of lactic acid bacteria can be increased sufficiently in 48 hours of culture, and ferulic acid or dihydroferulic acid production ability can be appropriately evaluated. The ratio of the released ferulic acid or dihydroferulic acid can be calculated by subjecting the culture supernatant to high performance liquid chromatography (HPLC) and detecting the peak area of ethyl ferulate, ferulic acid or dihydroferulic acid. More specifically, the methods described in the examples described later can be employed, such as the type of medium, the culture conditions such as the optimum temperature, the conditions of the high performance liquid chromatography method and the like. “30% or more” in “the property of liberating 30% or more of the ethyl ferulate as dihydroferulic acid” may be 30% to 100%, but 40% or more, 50% or more, 60% or more, A higher ratio of 70% or more is preferable. Lactic acid bacteria with this property have the same activity against esters of cinnamic acid analogues such as caffeic acid and paracoumaric acid, which have structures similar to ferulic acid, and dihydrocaffeic acid and dihydroparacoumaric acid. Is generated.
Specific examples of lactic acid bacteria having the ability to produce reduced cinnamic acid analogs include lactic acid bacteria belonging to the genus Lactobacillus, Bifidobacterium, Streptococcus, Lactococcus, Leuconostoc, Oenococcus, Enterococcus Is done. The Lactobacillus genus includes Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus crispatas, Lactobacillus amylovorus, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus gallinaram, Lactobacillus fermentum , Lactobacillus delbruicki, Lactobacillus bukuneri etc. are exemplified, and Enterococcus faecalis is exemplified as the genus Enterococcus. Among them, Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus johnsonii, Lactobacillus kefiranofaciens, Lactobacillus gallinarum, Lactobacillus fermentum, Lactobacillus Delbluky, Lactobacillus bukuneri and Enterococcus faecalis are preferred, Lactobacillus gasseri FERM-BP10953, JCM1025, JCM1031, JCM1130, JCM1131, JCM8787, JCM8788, Lactobacillus acidophilus JCM1132, Lactobacillus JCS1132 JCM1185, JCM5806, Lactobacillus amylobolus JCM10 3, JCM1126, JCM1127, Lactobacillus johnsony JCM2010, JCM2122, Lactobacillus kefiranofaciens JCM6985, SBT11223, Lactobacillus gallinarum JCM1036, Lactobacillus fermentum SBT1847, SBT2537, Lactobacillus der T And Lactobacillus bukuneri SBT11411 and Enterococcus faecalis SBT1120 are more preferred, and Lactobacillus gasseri FERM-BP10953 is most preferred.
The lactic acid bacterium confirmed to produce ferulic acid, caffeic acid and paracoumaric acid includes at least a step of culturing lactic acid bacteria having cinnamate-related compound-producing ability in a plant material. It can utilize in the manufacturing method of a content fraction. Specific examples of such lactic acid bacteria include lactic acid bacteria that have been confirmed to be capable of producing ferulic acid, caffeic acid, and paracoumaric acid in the examples described below.
The plant material is not particularly limited as long as it contains a small amount of bound or ester-type cinnamic acid-related compounds, but vegetables, grains, fruits, grasses, beans, potatoes, seeds, rice bran, bran, etc. The thing derived from a raw material is mentioned. The plant material may be the plant material or the like exemplified above, but may be obtained by appropriately processing the plant material. Examples of the processing method include pulverization with a mixer and the like, but are not particularly limited. It is preferable that there is sufficient water for lactic acid bacteria to grow, but it can also be adjusted by adding water after adjusting the plant material.
In order to release cinnamic acid-related compounds from plant materials more efficiently, the plant materials may be pre-enzymed with dietary fiber degrading enzymes. Examples of the enzyme to be used include xylanase and cellulase, but are not particularly limited. For example, by using a plant material obtained by adding the enzyme to vegetable juice and allowing the enzyme reaction at the optimal temperature of the enzyme for several hours to 2 days, the cinnamic acid analog can be released more efficiently. it can.
The method of cultivating lactic acid bacteria having the ability to produce reduced cinnamic acid analogs in plant materials is particularly limited as long as lactic acid bacteria grow in plant materials and can produce reduced cinnamic acid analogs from plant materials. Not. For example, it can be cultured by adding about 0.5 to 10% of a lactic acid bacteria culture to a plant material and fermenting it for 1 to 7 days in a temperature range where the lactic acid bacteria can grow, but is not limited thereto.
The reduced cinnamic acid analog-containing fraction obtained by the method for producing the reduced cinnamic acid analog-containing fraction of the present invention may be a culture itself obtained by culturing lactic acid bacteria in a plant material. Alternatively, it may be obtained by adding an additive or the like to the culture or removing impurities. In addition, the cinnamic acid analog compound reduced from the culture can be fractionated and purified to obtain a reduced cinnamic acid analog-containing fraction having a higher reduced cinnamic acid analog concentration.
(2) Lactic acid bacteria fermented food / beverage product of the present invention The lactic acid bacteria fermented food / beverage product of the present invention contains at least a lactic acid bacterium having the ability to produce a reduced cinnamic acid-related compound and a reduced cinnamic acid-related compound, and is derived from a plant material. It is.
As lactic acid bacteria having the ability to produce reduced cinnamic acid analogs, reduced cinnamic acid analogs can be released when cultured under conditions suitable for the production of free reduced cinnamic acid analogs in plant materials. It is not particularly limited as long as it is a lactic acid bacterium, and specifically, those described in the above (1) method for producing a fraction containing a reduced cinnamic acid analog compound of the present invention can be used.
Examples of reduced cinnamic acid analogs include dihydroferulic acid, dihydrocaffeic acid, and dihydroparacoumaric acid.
As the plant material, those described in the above (1) method for producing a fraction containing a cinnamic acid-related compound according to the present invention can be used. For example, when vegetable juice, fruit juice, or the like is used, a lactic acid bacteria fermented beverage excellent in flavor and nutritional value can be provided.
The concentration of cinnamic acid analogs in the fermented foods and drinks of the present invention depends on how much is contained in the plant material, but the amount of reduced cinnamic acid analogs in the material can be increased by the lactic acid bacteria of the present invention. it can.
Although the manufacturing method of this invention lactic-acid-bacteria fermented food-drinks is not specifically limited, It can manufacture with the manufacturing method of this invention reduced cinnamic-acid related compound containing fraction.
The plant material-derived lactic acid bacteria fermented food and drink of the present invention can be provided in the form of beverages, fermented milk, desserts, etc. by mixing with foods such as milk and fermented milk.
(3) Screening method of the present invention The screening method of the present invention is a screening method for lactic acid bacteria having the ability to produce reduced cinnamic acid analogs, comprising at least the following steps;
Step 1: culturing a specimen lactic acid bacterium in a medium containing ethyl ferulate and quantifying the produced dihydroferulic acid compound;
Step 2: A step of determining a lactic acid bacterium having a property of producing a certain proportion or more of dihydroferulic acid with respect to ethyl ferulate in
According to the screening method of the present invention, it is possible to screen for lactic acid bacteria that can act on esters such as caffeic acid and paracoumaric acid, and that are excellent in the ability to produce reduced cinnamic acid analogs.
In
Thus, lactic acid bacteria having the ability to produce a desired reduced cinnamic acid analog can be screened. For example, a lactic acid bacterium having the following properties can be obtained as a lactic acid bacterium having reduced cinnamic acid analog production ability. .
Properties: A property in which 30% or more of the ethyl ferulate is liberated as dihydroferulic acid when cultured for 48 hours at an optimum temperature of the lactic acid bacteria in a medium supplemented with 0.05% by weight of ethyl ferulate.
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
（ジヒドロフェルラ酸遊離活性を持つ菌株の選抜試験）
試験には表１に示した菌株を使用し、ＭＲＳ−Ｂｒｏｔｈ培地（ＤＩＦＣＯ）にて３７℃（ラクトコッカス・ラクティス及びラクトバチルス・ケフィラノファシエンスは３０℃）で１６時間培養した。０．０５％フェルラ酸エチル（東京化成工業株式会社）を添加したＭＲＳ−Ｂｒｏｔｈ培地に菌液を３％接種し、３７℃（ラクトコッカス・ラクティス及びラクトバチルス・ケフィラノファシエンスは３０℃）にて４８時間培養した。培養上清を分画分子量３ｋＤａのＵＦフィルターに通し、通過液を高速液体クロマトグラフィーにて分析した。Ａｇｉｌｅｎｔ ｔｅｃｈｎｏｌｏｇｙ Ｓｅｒｉｅｓ １２００ ＨＰＬＣ ｓｙｓｔｅｍを用い、カラムはＨｙｄｒｏｓｐｈｅｒｅ Ｃ１８，Ｓ−５μｍ，１２ｎｍ （４．６ ｍｍ ＩＤ×２５０ｍｍ： ＹＭＣ） を用いた。溶媒はＡ液：０．０５％ ＴＦＡ ｉｎ ５％アセトニトリル、Ｂ液：０．０５％ ＴＦＡ ｉｎ ９５％アセトニトリルを用い、Ｂ液の量が、０→３分は２５％、８分までに３０％、９分までに１００％、９→１４分は１００％、１５分までに２５％、１５→２０分は２５％のグラジエントで行った。溶媒流量は１ｍｌ／分、インジェクション量は１０μｌとし、フェルラ酸の検出は３２０ｎｍで、フェルラ酸の代謝産物であるジヒドロフェルラ酸の検出は２１０ｎｍで行った。同定は保持時間で行い、そのピーク面積値からモル濃度を算出した。
図１に示す通り、供試した３９株のうち２５株が、添加したフェルラ酸エチルの３０％以上をジヒドロフェルラ酸に変換した。特に、ラクトバチルス・ガセリＦＥＲＭ−ＢＰ１０９５３、ＪＣＭ１０２５、ＪＣＭ１１３１、ＪＣＭ８７８７、ＪＣＭ８７８８、ラクトバチルス・アシドフィルスＪＣＭ１１３２、ラクトバチルス・クリスパタスＪＣＭ１０３７、ＪＣＭ１１８５、ＪＣＭ５８０６、ラクトバチルス・アミロボラスＪＣＭ１０３３、ＪＣＭ１１２６、ＪＣＭ１１２７、ラクトバチルス・ジョンソニーＪＣＭ２０１０、ＪＣＭ２１２２、ラクトバチルス・ガリナラムＪＣＭ１０３６、ラクトバチルス・ファーメンタムＳＢＴ１８４６、ＳＢＴ２５３７、ラクトバチルス・デルブルイッキーＳＢＴ１３７１、ラクトバチルス・ブクネリＳＢＴ１１４１１及びエンテロコッカス・フェカリスＳＢＴ１１２０、ラクトバチルス・ケフィラノファシエンスＳＢＴ１１２２３はフェルラ酸エチルの８０％以上をジヒドロフェルラ酸に変換した。一方、フェルラ酸エチルの３０％以上をフェルラ酸に代謝・蓄積した株が７株みられ、この内４株はジヒドロフェルラ酸を産生しなかった（図２）。また、供試した１０株はフェルラ酸エチルをほとんど代謝しなかった。以上の結果から、ある一定の乳酸菌はフェルラ酸エステルからフェルラ酸を遊離する活性を持っているが、その中の一部の乳酸菌がフェルラ酸をジヒドロフェルラ酸に還元する活性を持っていることが見出された。
For the test, the strains shown in Table 1 were used and cultured in MRS-Broth medium (DIFCO) at 37 ° C. (Lactococcus lactis and Lactobacillus kefiranofaciens were 30 ° C.) for 16 hours. MRS-Broth medium supplemented with 0.05% ethyl ferulate (Tokyo Kasei Kogyo Co., Ltd.) is inoculated with 3% of the bacterial solution and incubated at 37 ° C. (Lactococcus lactis and Lactobacillus kefiranofaciens are 30 ° C.) For 48 hours. The culture supernatant was passed through a UF filter having a fractional molecular weight of 3 kDa, and the passing solution was analyzed by high performance liquid chromatography. Agilent technology Series 1200 HPLC system was used, and the column was Hydrosphere C18, S-5 μm, 12 nm (4.6 mm ID × 250 mm: YMC). As the solvent, A liquid: 0.05% TFA in 5% acetonitrile, B liquid: 0.05% TFA in 95% acetonitrile, the amount of B liquid is 25% from 0 to 3 minutes, 30% by 8 minutes The gradient was 100% by 9 minutes, 100% from 9 to 14 minutes, 25% by 15 minutes, and 25% from 15 to 20 minutes. The solvent flow rate was 1 ml / min, the injection amount was 10 μl, ferulic acid was detected at 320 nm, and dihydroferulic acid, which is a metabolite of ferulic acid, was detected at 210 nm. Identification was performed based on the retention time, and the molar concentration was calculated from the peak area value.
As shown in FIG. 1, 25 of the 39 strains tested converted 30% or more of the added ethyl ferulate to dihydroferulic acid. In particular, Lactobacillus gasseri FERM-BP10953, JCM1025, JCM1131, JCM8787, JCM8788, Lactobacillus acidophilus JCM1132, Lactobacillus crispatus JCM1037, JCM1185, JCM5806, Lactobacillus amyloboras JCM1013, JCM1126 JCM1126 , JCM2122, Lactobacillus gallinarum JCM1036, Lactobacillus fermentum SBT1846, SBT2537, Lactobacillus delbruicki SBT1371, Lactobacillus bukuneri SBT11411 and Enterococcus faecalis SBT1120, Lactobacillus kefiranofii T112 3 was converted more than 80% of ethyl ferulic acid dihydro ferulic acid. On the other hand, there were 7 strains that metabolized and accumulated 30% or more of ethyl ferulate into ferulic acid, and 4 of these strains did not produce dihydroferulic acid (FIG. 2). Further, the 10 strains tested hardly metabolized ethyl ferulate. From the above results, certain lactic acid bacteria have the activity of releasing ferulic acid from ferulic acid esters, but some of the lactic acid bacteria have the activity of reducing ferulic acid to dihydroferulic acid. It was found.
（フェルラ酸リダクターゼ活性の評価）
実施例１で評価した菌株のうち、ジヒドロフェルラ酸を産生した５株と、コントロールとしてフェルラ酸を生成した３株を試験に用いた。ＭＲＳ−Ｂｒｏｔｈ培地にて３７℃で１６時間培養した菌液を、０．０５％フェルラ酸を添加したＭＲＳ−Ｂｒｏｔｈ培地に３％接種し、３７℃にて４８時間培養した。培養上清を実施例１と同様の方法で処理し、ＨＰＬＣにてフェルラ酸、ジヒドロフェルラ酸を定量した。
図３に示す通り、実施例１でジヒロドフェルラ酸のみを産生していたＪＣＭ１１３１、ＪＣＭ１１３２、ＪＣＭ１１８５、ＪＣＭ１１２６、ＦＥＲＭ−ＢＰ１０９５３は、添加したフェルラ酸をほぼ全てジヒドロフェルラ酸に変換した。一方、実施例１でフェルラ酸のみを産生していた３株ではフェルラ酸をほとんど代謝・分解せず、ジヒドロフェルラ酸も産生しなかった。以上の方法により、実施例１でフェルラ酸エチルからジヒドロフェルラ酸を産生した株は、フェルラ酸リダクターゼ活性を持つことが確認された。
(Evaluation of ferulic acid reductase activity)
Among the strains evaluated in Example 1, 5 strains that produced dihydroferulic acid and 3 strains that produced ferulic acid as a control were used in the test. The bacterial solution cultured for 16 hours at 37 ° C. in MRS-Broth medium was inoculated into 3% of MRS-Broth medium supplemented with 0.05% ferulic acid and cultured at 37 ° C. for 48 hours. The culture supernatant was treated in the same manner as in Example 1, and ferulic acid and dihydroferulic acid were quantified by HPLC.
As shown in FIG. 3, JCM1131, JCM1132, JCM1185, JCM1126, and FERM-BP10953 that produced only dihydroferulic acid in Example 1 converted almost all of the added ferulic acid into dihydroferulic acid. On the other hand, the three strains that produced only ferulic acid in Example 1 hardly metabolized or decomposed ferulic acid and did not produce dihydroferulic acid. By the above method, it was confirmed that the strain that produced dihydroferulic acid from ethyl ferulate in Example 1 had ferulic acid reductase activity.
（選抜した株による他の桂皮酸類縁化合物の代謝）
植物素材には、フェルラ酸以外にもコーヒー酸やパラクマル酸のような桂皮酸類縁化合物も存在し、フェルラ酸同様にその抗酸化能が知られている。そこで今回選抜した株について、コーヒー酸についてもエステルからの遊離活性・還元活性を示すか評価した。実施例２で使用した菌株を用い、ＭＲＳ−Ｂｒｏｔｈ培地にて３７℃で１６時間培養した菌液を、０．０５％コーヒー酸エチルを添加したＭＲＳ−Ｂｒｏｔｈ培地に３％接種し、３７℃にて４８時間培養した。培養上清を実施例１と同様の方法で処理し、ＨＰＬＣにて分析した。コーヒー酸の検出は３２０ｎｍで、代謝産物であるジヒドロコーヒー酸の検出は２１０ｎｍで行った。同定は保持時間で行い、そのピーク面積値から濃度を算出した。
その結果、図４に示すように、実施例１にてジヒドロフェルラ酸を産生した株はジヒドロコーヒー酸を産生し、実施例１でフェルラ酸を産生した株はコーヒー酸を産生した。以上のことから、本発明により選抜された乳酸菌は、フェルラ酸以外にも多様な桂皮酸類縁化合物をエステル体から切り出し、ジヒドロ体へ還元する能力を有することが判明した。よって本発明の乳酸菌を用いることにより、植物素材から多様な還元された桂皮酸類縁化合物を産生することが期待できる。
(Metabolism of other cinnamic acid-related compounds by selected strains)
In addition to ferulic acid, there are also cinnamic acid-related compounds such as caffeic acid and paracoumaric acid, and their antioxidant ability is known in the same way as ferulic acid. Therefore, the strains selected this time were evaluated whether caffeic acid also showed free activity / reduction activity from esters. Using the bacterial strain used in Example 2, 3% of the bacterial solution cultured at 37 ° C. for 16 hours in MRS-Broth medium was inoculated into MRS-Broth medium supplemented with 0.05% ethyl caffeate and brought to 37 ° C. For 48 hours. The culture supernatant was treated in the same manner as in Example 1 and analyzed by HPLC. Caffeic acid was detected at 320 nm, and metabolite dihydrocaffeic acid was detected at 210 nm. Identification was performed based on the retention time, and the concentration was calculated from the peak area value.
As a result, as shown in FIG. 4, the strain that produced dihydroferulic acid in Example 1 produced dihydrocaffeic acid, and the strain that produced ferulic acid in Example 1 produced caffeic acid. From the above, it was found that the lactic acid bacteria selected according to the present invention have the ability to cut out various cinnamic acid analogs other than ferulic acid from the ester form and reduce them to the dihydro form. Therefore, by using the lactic acid bacteria of the present invention, it can be expected to produce various reduced cinnamic acid analogues from plant materials.
（選抜した株による野菜汁の発酵）
実施例１にてジヒドロフェルラ酸の産生能が高かったラクトバチルス・ガセリＦＥＲＭ−ＢＰ１０９５３を用いて、アスパラガス汁の発酵試験を行った。対照として、実施例１にてフェルラ酸の産生能が高かったラクトバチルス・ヘルベティカスＦＥＲＭ−ＢＰ０５４４５を用いた。ＭＲＳ−Ｂｒｏｔｈ培地にて３７℃で１６時間培養した菌液を、０．１％ホエーパウダーを添加した還元アスパラガス汁（２００%還元、９５℃、３０分殺菌）に１％接種し、３７℃にて１６時間培養した。発酵物の上清を実施例１と同様の方法で処理し、ＨＰＬＣにて分析した。フェルラ酸、パラクマル酸の検出は３２０ｎｍで、代謝産物であるジヒドロフェルラ酸、ジヒドロパラクマル酸の検出は２１０ｎｍで行った。各化合物の同定は保持時間で行い、そのピーク面積値から濃度を算出した。
その結果、図５に示すように、ラクトバチルス・ガセリＦＥＲＭ−ＢＰ１０９５３にて発酵した場合にはジヒドロフェルラ酸とジヒドロパラクマル酸が１２９μＭ生成・蓄積した。一方、ラクトバチルス・ヘルベティカスＦＥＲＭ−ＢＰ０５４４５にて発酵した場合には、フェルラ酸とパラクマル酸が生成・蓄積した。以上のことから、本発明により選抜された乳酸菌株を用いることにより、植物素材から多様な還元された桂皮酸類縁化合物を産生できることが証明された。
(Fermentation of vegetable juice by selected strains)
The fermentation test of asparagus juice was performed using Lactobacillus gasseri FERM-BP10953, which had a high ability to produce dihydroferulic acid in Example 1. As a control, Lactobacillus helveticus FERM-BP05445, which had high ferulic acid production ability in Example 1, was used. Bacteria obtained by culturing at 37 ° C. for 16 hours in MRS-Broth medium is inoculated with 1% of reduced asparagus juice (200% reduced, 95 ° C., sterilized for 30 minutes) supplemented with 0.1% whey powder. For 16 hours. The supernatant of the fermented product was treated in the same manner as in Example 1 and analyzed by HPLC. Ferulic acid and paracoumaric acid were detected at 320 nm, and metabolites dihydroferulic acid and dihydroparacoumaric acid were detected at 210 nm. Each compound was identified by the retention time, and the concentration was calculated from the peak area value.
As a result, as shown in FIG. 5, when fermented with Lactobacillus gasseri FERM-BP10953, 129 μM of dihydroferulic acid and dihydroparacoumaric acid were generated and accumulated. On the other hand, when fermented with Lactobacillus helveticus FERM-BP05445, ferulic acid and paracoumaric acid were produced and accumulated. From the above, it was proved that various reduced cinnamic acid analogues can be produced from plant materials by using the lactic acid strain selected according to the present invention.
（選抜した株による植物素材の発酵）
実施例１にてジヒドロフェルラ酸の産生能が高かったラクトバチルス・ガセリＦＥＲＭ−ＢＰ１０９５３を用いて、米糠抽出液の発酵試験を行った。対照として、実施例１にてフェルラ酸の産生能が高かったラクトバチルス・ヘルベティカスＦＥＲＭ−ＢＰ０５４４５を用いた。新鮮な米ぬかを１０倍量の水に懸濁し、９０℃で１時間煮出した後、ろ紙でろ過した。ろ液 １ＬにＭＲＳ−Ｂｒｏｔｈ ５５ｇを溶解し、滅菌後、米ぬか抽出物として発酵試験に用いた。供試菌株はＭＲＳ−Ｂｒｏｔｈ培地にて３７℃で１６時間培養し、米ぬか抽出物に３％接種して、３７℃にて４８時間発酵した。培養上清を実施例１と同様の方法で処理し、ＨＰＬＣにてジヒドロフェルラ酸を定量した。
その結果、図６に示すように、ラクトバチルス・ガセリＦＥＲＭ−ＢＰ１０９５３にて発酵した場合にのみジヒドロフェルラ酸が生成・蓄積した。以上のことから、本発明により選抜された乳酸菌株を用いることにより、米糠などの植物素材からも還元された桂皮酸類縁化合物を産生できることが証明された。
(Fermentation of plant material by selected strains)
A fermentation test of rice bran extract was performed using Lactobacillus gasseri FERM-BP10953, which had high dihydroferulic acid production ability in Example 1. As a control, Lactobacillus helveticus FERM-BP05445, which had high ferulic acid production ability in Example 1, was used. Fresh rice bran was suspended in 10 times the amount of water, boiled at 90 ° C. for 1 hour, and then filtered through filter paper. 55 g of MRS-Broth was dissolved in 1 L of the filtrate, and after sterilization, it was used as a rice bran extract for the fermentation test. The test strain was cultured in MRS-Broth medium at 37 ° C. for 16 hours, inoculated with 3% of rice bran extract, and fermented at 37 ° C. for 48 hours. The culture supernatant was treated in the same manner as in Example 1, and dihydroferulic acid was quantified by HPLC.
As a result, as shown in FIG. 6, dihydroferulic acid was produced and accumulated only when fermented with Lactobacillus gasseri FERM-BP10953. From the above, it was proved that by using a lactic acid strain selected according to the present invention, a cinnamic acid-related compound reduced from plant materials such as rice bran can be produced.
（フェルラ酸、ジヒドロフェルラ酸のORAC法による抗酸化活性の評価）
フェルラ酸、ジヒドロフェルラ酸の抗酸化性を比較するため、ORAC法による測定を行なった。測定はOxiSelect ORAC Activity Assay Kit (CELL BIOLABS)にて、添付のプロトコルに準じて行った。フェルラ酸、ジヒドロフェルラ酸は50μMとなるようにキット付属のアッセイ用希釈液を用いて調製した。検量線の作成には、キット付属のTrolox標品水溶液を段階希釈したものを使用した。Free radical initiator試薬添加後、速やかに蛍光マイクロプレートリーダーにより蛍光（励起波長480 nm、発光波長520 nm）の測定を開始し、120分まで経時的に測定した。測定後、蛍光強度曲線下の面積を求めてAUC (Antioxidant)とし、それからブランクのAUC (Blank) を差し引いたNet AUCを各サンプルについて算出した。Trolox標品の濃度とNet AUCの検量線を作成し、サンプルのNet AUCに対応するTrolox濃度：TE値(Trolox Equivalent値)を求め、ORAC値とした。測定の結果、フェルラ酸が7.7、ジヒドロフェルラ酸が6.2 mmol TE/gであり、両者で大きな差異は認められなかった。
(Evaluation of antioxidant activity of ferulic acid and dihydroferulic acid by ORAC method)
In order to compare the antioxidative properties of ferulic acid and dihydroferulic acid, measurement was performed by the ORAC method. The measurement was performed using OxiSelect ORAC Activity Assay Kit (CELL BIOLABS) according to the attached protocol. Ferulic acid and dihydroferulic acid were prepared using an assay diluent attached to the kit so as to be 50 μM. For the preparation of the calibration curve, a serial dilution of the Trolox standard aqueous solution included in the kit was used. After the addition of the free radical initiator reagent, measurement of fluorescence (excitation wavelength: 480 nm, emission wavelength: 520 nm) was immediately started with a fluorescence microplate reader, and the measurement was performed over time until 120 minutes. After the measurement, the area under the fluorescence intensity curve was determined to be AUC (Antioxidant), and then Net AUC was calculated by subtracting blank AUC (Blank) for each sample. A calibration curve of Trolox preparation of concentration and Net AUC, Trolox corresponding to Net AUC of sample concentration: TE value (T rolox E quivalent value) was determined and the ORAC value. As a result of the measurement, ferulic acid was 7.7 and dihydroferulic acid was 6.2 mmol TE / g, and no significant difference was observed between the two.
（フェルラ酸とジヒドロフェルラ酸の血中への移行性・安定性の評価）
フェルラ酸とジヒドロフェルラ酸の血中への移行性・安定性を評価するため、これらの化合物のラットへの投与試験を行い、末梢血からの回収を行なった。試験には７週齢のラット（Wister、オス：Charles river）を用い、飼育は温度と湿度をコントロール（温度23 ℃ 、相対湿度50 ± 5 % ）した12時間毎の明暗サイクル下で行い、粗飼料Certified Rodent Diet 5002（Lab diet:SLC）及び脱イオン水を自由摂取させた。試験６時間前より絶食させ、体重を測定して平均が同じとなるよう2群に分けた（n=4）。7ｍＭのフェルラ酸またはジヒドロフェルラ酸溶液を１ｍｌ／１００ｇラット体重、ゾンデにて胃内投与し、投与５，１０，１５，３０，６０分後に尾部静脈より採血した。
生体内に吸収されたフェルラ酸、ジヒドロフェルラ酸は代謝を受け、糖や硫酸と結合した抱合型を形成することが知られているため、血漿中の遊離型と抱合型のフェルラ酸／ジヒドロフェルラ酸の総量を測定した。まず血漿25μlをβ-Glcuronidase Type H-2（SIGMA）500Uを含む酵素液（pH5.0の0.1M酢酸ナトリウム溶液にて希釈）25μlと混和し、37℃で2.5時間反応した。反応液を希釈後、分画分子量3,000ｋＤaのUltrafree-MC 3,000 NMWL Filter Unit（MILLIPORE）に供し、通過液を実施例１に記載の方法でＨＰＬＣにて分析した。最終的に投与前の血中濃度を0とした場合の変化量を算出した。
フェルラ酸及びジヒドロフェルラ酸投与後のラット血漿中フェルラ酸及びジヒドロフェルラ酸の総量の経時変化を図７に示す。フェルラ酸又はジヒドロフェルラ酸のどちらを投与した場合も、投与後１５分〜３０分をピークに増加し、その後減少した。しかし、血漿中の最高濃度はフェルラ酸で５．７μＭであったのに対し、ジヒドロフェルラ酸は８．７μＭと高値であった。また、０分から６０分までの血漿中フェルラ酸及びジヒドロフェルラ酸の総量の積分値を求めると（Σ（Δフェルラ酸＋Δジヒドロフェルラ酸）、μＭ×６
０分）フェルラ酸投与が２６２．６μＭ×６０分なのに対し、ジヒドロフェルラ酸投与が３９７．４μＭ×６０分と高値を示した。以上の結果から、ジヒドロフェルラ酸は、フェルラ酸と比較して血中へより多く移行し、血中での保持時間が長いことから、全体として血中でより高い抗酸化性を発揮できると考えられた。よって、本発明の乳酸菌を用いてフェルラ酸を含む素材を発酵させることにより、血中における素材の抗酸化性を高めることができると考えられる。
(Evaluation of migration and stability of ferulic acid and dihydroferulic acid into blood)
In order to evaluate the transfer and stability of ferulic acid and dihydroferulic acid into the blood, administration tests of these compounds to rats were conducted and recovery from peripheral blood was performed. For the test, 7-week-old rats (Wister, male: Charles river) were used, and breeding was carried out under a light / dark cycle every 12 hours under controlled temperature and humidity (temperature 23 ° C,
It is known that ferulic acid and dihydroferulic acid absorbed in the body are metabolized and form a conjugated form combined with sugar and sulfuric acid. Therefore, free and conjugated ferulic acid / dihydroferula in plasma are known. The total amount of acid was measured. First, 25 μl of plasma was mixed with 25 μl of an enzyme solution containing 500 U of β-Glcuronidase Type H-2 (SIGMA) (diluted with 0.1 M sodium acetate solution at pH 5.0) and reacted at 37 ° C. for 2.5 hours. After the reaction solution was diluted, it was applied to an Ultrafree-MC 3,000 NMWL Filter Unit (MILLIPORE) having a molecular weight cut-off of 3,000 kDa, and the passing solution was analyzed by HPLC by the method described in Example 1. Finally, the amount of change when the blood concentration before administration was 0 was calculated.
FIG. 7 shows the time course of the total amount of ferulic acid and dihydroferulic acid in rat plasma after administration of ferulic acid and dihydroferulic acid. When either ferulic acid or dihydroferulic acid was administered, the peak increased from 15 to 30 minutes after administration, and then decreased. However, the highest concentration in plasma was 5.7 μM for ferulic acid, whereas dihydroferulic acid was as high as 8.7 μM. Further, when the integral value of the total amount of ferulic acid and dihydroferulic acid in plasma from 0 to 60 minutes is determined (Σ (Δ ferulic acid + Δ dihydroferulic acid), μM × 6
0 minutes) Ferulic acid administration was 262.6 μM × 60 minutes, while dihydroferulic acid administration was high, 397.4 μM × 60 minutes. Based on the above results, dihydroferulic acid migrates more into the blood than ferulic acid and has a longer retention time in the blood. It was. Therefore, it is thought that the antioxidant property of the raw material in blood can be improved by fermenting the raw material containing ferulic acid using the lactic acid bacteria of the present invention.
（野菜発酵飲料の試作）
選択された乳酸菌を用いて、実際に野菜を発酵した際の発酵性と風味への影響を調べた。市販のキャロット混濁濃縮汁、ブロッコリー混濁濃縮汁、ケール混濁濃縮汁を水で還元し、９５℃で１５分加熱殺菌した。乳酸菌として実施例１でジヒドロフェルラ酸遊離能が高かったラクトバチルス・ガセリＦＥＲＭ−ＢＰ１０９５３を用い、０．５％の酵母エキスを含む１２％還元脱脂乳で３７℃、１６時間培養した後、野菜汁に３％接種して３７℃で１６時間発酵した。
表２に示すように、選択された乳酸菌は様々な野菜汁中でも成育し、適度な酸度の上昇、ｐＨの低下が見られた。風味についても酸味が強すぎず、非常に良好であった。以上の結果から、このようにして選択された乳酸菌によって、野菜の良好な発酵が可能であることが確認された。
Using the selected lactic acid bacteria, the effect on fermentability and flavor when actually fermenting vegetables was investigated. Commercially available carrot turbid concentrate, broccoli turbid concentrate, and kale turbid concentrate were reduced with water and sterilized by heating at 95 ° C. for 15 minutes. Lactobacillus gasseri FERM-BP10953, which had a high ability to release dihydroferulic acid in Example 1 as lactic acid bacteria, was cultured in 12% reduced skim milk containing 0.5% yeast extract at 37 ° C. for 16 hours, and then vegetable juice 3% inoculated and fermented at 37 ° C. for 16 hours.
As shown in Table 2, the selected lactic acid bacteria grew in various vegetable juices, and an appropriate increase in acidity and a decrease in pH were observed. The flavor was not so strong and very good. From the above results, it was confirmed that the lactic acid bacteria selected in this way can satisfactorily ferment vegetables.
本発明は、フェルラ酸などの桂皮酸類縁化合物を産生する能力を有し、かつフェルラ酸などの桂皮酸類縁化合物をより生体への移行性・安定性が高い化合物に変換する能力を有する乳酸菌を植物素材中で培養する工程を含むことを特徴とする還元された桂皮酸類縁化合物含有画分の製造方法、還元された桂皮酸類縁化合物産生能を有する乳酸菌及び還元された桂皮酸類縁化合物を含有する植物素材由来の乳酸菌発酵飲食品、還元された桂皮酸類縁化合物産生能を有する乳酸菌のスクリーニング方法等に関する。本発明は食品分野、医薬分野等に利用可能である。
The present invention relates to a lactic acid bacterium having the ability to produce cinnamic acid analogues such as ferulic acid, and the ability to convert cinnamic acid analogues such as ferulic acid into compounds with higher migration and stability to living bodies. A method for producing a fraction containing a reduced cinnamic acid-related compound, comprising a step of culturing in a plant material, containing a lactic acid bacterium having the ability to produce reduced cinnamic acid-related compounds, and a reduced cinnamic acid-related compound The present invention relates to lactic acid bacteria fermented foods and beverages derived from plant materials, screening methods for lactic acid bacteria having the ability to produce reduced cinnamic acid analogs, and the like. The present invention can be used in the food field, pharmaceutical field, and the like.
ＦＥＲＭ ＢＰ−１０９５３
[寄託生物材料への言及]
（１）還元された桂皮酸類縁化合物産生能を有するラクトバチルス・ガセリ菌株
イ 当該生物材料を寄託した寄託機関の名称及び住所
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
日本国茨城県つくば市東１丁目１番地１中央第６（郵便番号305-8566）
ロ イの寄託機関に生物材料を寄託した日付
平成８年３月２７日（１９９６年３月２７日）（原寄託日）
平成２０年２月２６日（２００８年２月２６日）（原寄託によりブタペスト条約に基づく寄託への移管日）
ハ イの寄託機関が寄託について付した受託番号
ＦＥＲＭ ＢＰ−１０９５３
FERM BP-10953
[Reference to deposited biological materials]
(1) Lactobacillus gasseri strain having the ability to produce reduced cinnamic acid analogs Name and address of the depositary institution depositing the biological material National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center East of Tsukuba City, Ibaraki Prefecture,
Date of deposit of biological materials at Loi depository March 27, 1996 (March 27, 1996) (original deposit date)
February 26, 2008 (February 26, 2008) (Date of transfer to deposit under the Budapest Treaty by original deposit)
Deposit number FERM BP-10953 attached to the depositary by the high depository
Claims (1)
前記還元された桂皮酸類縁化合物は、ジヒドロフェルラ酸、ジヒドロパラクマル酸又はジヒドロコーヒー酸であり、
前記還元された桂皮酸類縁化合物産生能を有する乳酸菌菌株が下記性質を有し、かつ、下記乳酸菌群から選択される乳酸菌菌株である、前記方法；
性質：フェルラ酸エチルを０．０５重量％添加した培地において、前記乳酸菌の至適温度で４８時間培養したとき、前記フェルラ酸エチルの３０％以上をジヒドロフェルラ酸として遊離する性質
乳酸菌群：
ラクトバチルス・アシドフィルス（ただし、ＪＣＭ１１３２を除く）、
ラクトバチルス・ガセリ（ただし、ＪＣＭ１１３１を除く）、
ラクトバチルス・クリスパタス（ただし、ＪＣＭ１１８５を除く）、
ラクトバチルス・アミロボラス（ただし、ＪＣＭ１１２６及びＪＣＭ１１２７を除く）、
ラクトバチルス・ジョンソニー（ただし、ＪＣＭ２０１２を除く）、
ラクトバチルス・ケフィラノファシエンス、
ラクトバチルス・ガリナラム（ただし、ＪＣＭ２０１１を除く）、
ラクトバチルス・ファーメンタム（ただし、ＪＣＭ１５６０及びＪＣＭ１１３７
を除く）、
ラクトバチルス・デルブルイッキー、
ラクトバチルス・ブクネリ、
エンテロコッカス・フェカリス。 A method for increasing the concentration of reduced cinnamic acid analog in a plant material, comprising a step of culturing a lactic acid bacterium strain having reduced cinnamic acid analog compound-producing ability in the plant material,
The reduced cinnamic acid analog is dihydroferulic acid, dihydroparacoumaric acid or dihydrocaffeic acid;
The method, wherein the reduced cinnamate-related compound producing ability is a lactic acid bacterium strain having the following properties and selected from the following lactic acid bacteria group:
Properties: Properties of lactic acid bacteria that liberate 30% or more of the ethyl ferulates as dihydroferulic acid when cultured for 48 hours at the optimum temperature of the lactic acid bacteria in a medium supplemented with 0.05% by weight of ethyl ferulate.
Lactobacillus acidophilus (excluding JCM1132),
Lactobacillus gasseri (excluding JCM1131),
Lactobacillus crispatas (except JCM1185),
Lactobacillus amylovorus (excluding JCM1126 and JCM1127),
Lactobacillus johnsonii (excluding JCM2012),
Lactobacillus kefiranofaciens,
Lactobacillus gallinarum (except JCM2011),
Lactobacillus fermentum (however, JCM1560 and JCM1137
except for),
Lactobacillus delbruickey,
Lactobacillus bukuneri,
Enterococcus faecalis.
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| JP7303509B2 (en) * | 2017-07-19 | 2023-07-05 | 丸善製薬株式会社 | Method for producing 3-(4-hydroxyphenyl)propanoic acid analogue |
| WO2022065330A1 (en) * | 2020-09-23 | 2022-03-31 | 雪印メグミルク株式会社 | Muscle atrophy prevention agent |
| JP2022130294A (en) * | 2021-02-25 | 2022-09-06 | 株式会社ニコリオ | Intestinal environment improver and composition |
| TWI802009B (en) * | 2021-09-16 | 2023-05-11 | 創百股份有限公司 | Treatment and/or prevention of particulate matter-induced lung injury using heat-killed strains of lactic acid bacteria |
| JP7126731B1 (en) * | 2021-10-19 | 2022-08-29 | 丸善製薬株式会社 | AMPK activator, motor function improver, muscle endurance improver and muscle atrophy inhibitor |
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