JP2014185172A - Catechin metabolite-containing composition - Google Patents
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本発明は、抗炎症作用やがん抑制作用が期待されるカテキン類の代謝産物であるフェニル−４−ハイドロキシ吉草酸を含有することを特徴とする組成物、該組成物を含有する口腔適用対象物に関する。 The present invention relates to a composition comprising phenyl-4-hydroxyvaleric acid which is a metabolite of catechins expected to have an anti-inflammatory action and a cancer suppressing action, and an oral application target containing the composition Related to things.
式(II)及び式(III)で表される化合物は、カテキン類を経口摂取したときに尿から検出されるカテキン類の代謝産物として知られている。最近、茶カテキン類の代謝産物である式(III)に示される化合物の一つである５−（３’，４’，５’−トリハイドロキシフェニル）γ−バレロラクトンに抗炎症作用やがん抑制作用が期待できるという報告がなされている（非特許文献１）。このようなことから、主要なカテキン類の代謝物である式(II)及び式(III)に示される化合物の生理活性機能が注目されている。 Compounds represented by formula (II) and formula (III) are known as metabolites of catechins detected from urine when catechins are taken orally. Recently, 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) γ-valerolactone, which is one of the compounds represented by formula (III), which is a metabolite of tea catechins, has anti-inflammatory activity and cancer. It has been reported that an inhibitory action can be expected (Non-Patent Document 1). For these reasons, bioactive functions of the compounds represented by formula (II) and formula (III), which are metabolites of main catechins, have attracted attention.
従来、式(II)及び式(III)に示される化合物は、カテキン類の代謝産物として同定されているだけであったため、その製造方法に関する知見はほとんどなく、先に示した非特許文献１に、式(III)に示される化合物である５−（３’，４’，５’−トリハイドロキシフェニル）γ−バレロラクトンおよび５−（３’，４’−ジハイドロキシフェニル）γ−バレロラクトンの化学合成法が開示されているに過ぎない。しかし、この化学合成法では合成のステップが多く煩雑であるばかりでなく、合成した化合物がラセミ体であるという欠点を有していた。 Conventionally, since the compounds represented by formula (II) and formula (III) have only been identified as metabolites of catechins, there is almost no knowledge about the production method, and Non-Patent Document 1 shown above. Of 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) γ-valerolactone and 5- (3 ′, 4′-dihydroxyphenyl) γ-valerolactone, which are compounds represented by the formula (III) Only chemical synthesis methods are disclosed. However, this chemical synthesis method has not only many complicated steps, but also has a drawback that the synthesized compound is a racemate.
そこで、本発明は、下記式(I)に示すカテキン誘導体を選択的に下記式(II)および／または式（III）の化合物に変換できる微生物を見出すとともに、該微生物を用いて極めて簡便に式(II)及び式(III)の化合物を製造する方法を見出し、本発明を完成した。 Therefore, the present invention finds a microorganism that can selectively convert a catechin derivative represented by the following formula (I) into a compound of the following formula (II) and / or formula (III), and uses the microorganism to formulate the formula very simply. A method for producing the compounds of (II) and formula (III) has been found and the present invention has been completed.
すなわち、請求項１記載の本発明は、
式（II）
式中、Ｒ１は水酸基（ＯＨ）、Ｒ２は水素（Ｈ）で表される５−（３’、５’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および
式中、Ｒ１、Ｒ２は水酸基（ＯＨ）で表される５−（３’、４’、５’−トリハイドロキシフェニル）−４−ハイドロキシ吉草酸の少なくとも一つを必須成分として含有することを特徴とする組成物を提供するものである。なお本発明においては式（II）で示される５−フェニル−４−ハイドロキシ吉草酸は、その塩をも包含し、例えば式（II）化合物のナトリウム塩、カリウム塩、アンモニウム塩、カルシウム塩、マグネシウム塩などを挙げることが出来る。
That is, the present invention according to claim 1
Formula (II)
In the formula, R1 is a hydroxyl group (OH), R2 is 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid represented by hydrogen (H), and in the formula, R1 and R2 are hydroxyl groups (OH And a composition containing at least one of 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -4-hydroxyvaleric acid as an essential component. . In the present invention, 5-phenyl-4-hydroxyvaleric acid represented by the formula (II) includes a salt thereof, for example, a sodium salt, potassium salt, ammonium salt, calcium salt, magnesium of the compound of the formula (II). Examples include salt.
また、請求項２記載の本発明は、請求項１記載の組成物を含有してなる口腔適用対象物である。 Moreover, this invention of Claim 2 is an oral application target object containing the composition of Claim 1.
本発明によれば、式（II）
式中、Ｒ１は水酸基（ＯＨ）、Ｒ２は水素（Ｈ）で表される５−（３’、５’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および
式中、Ｒ１、Ｒ２は水酸基（ＯＨ）で表される５−（３’、４’、５’−トリハイドロキシフェニル）−４−ハイドロキシ吉草酸の少なくとも一つを必須成分として含有することを特徴とする組成物および該組成物を含有してなる口腔適用対象物を得ることができる。
これらの化合物は、抗炎症作用やがん抑制作用のほか、他の生理活性作用も期待できるものである。
According to the invention, the formula (II)
In the formula, R1 is a hydroxyl group (OH), R2 is 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid represented by hydrogen (H), and in the formula, R1 and R2 are hydroxyl groups (OH And a composition containing at least one of 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -4-hydroxyvaleric acid represented by Thus, an oral application object can be obtained.
These compounds can be expected to have other physiological activity in addition to anti-inflammatory action and cancer suppression action.
本発明によれば式(I)
で示されるカテキン誘導体を、微生物の作用により簡単に式(II)
で表される５−フェニル−４−ハイドロキシ吉草酸および／または式（III）
で表される５−フェニルγ−バレロラクトンに変換できる。
また、式(IV)
で示されるカテキン類を式(I)のカテキン誘導体に変換する微生物を併用することで、該カテキン類から容易に上記に示した５−フェニル−４−ハイドロキシ吉草酸および／または５−フェニルγ−バレロラクトンを得ることができる。
According to the invention, the formula (I)
A catechin derivative represented by the formula (II)
5-phenyl-4-hydroxyvaleric acid and / or formula (III)
It can convert into 5-phenyl gamma-valerolactone represented by these.
And the formula (IV)
In combination with a microorganism that converts the catechins represented by formula (I) into a catechin derivative of the formula (I), 5-phenyl-4-hydroxyvaleric acid and / or 5-phenylγ- Valerolactone can be obtained.
本発明の生物学的変換方法では、前記式(I)で示されるカテキン誘導体を前記式(II)で示される５−フェニル−４−ハイドロキシ吉草酸および／または前記式（III）で表される５−フェニルγ−バレロラクトンへ変換する能力を有する微生物であれば、微生物の種類を問うことなく使用することができる。そのような微生物の好ましい例としては、ユウバクテリウム属細菌およびクロスリジウム属細菌を上げることができ、さらに好ましくは、ユウバクテリウム・プラウティＡＴＣＣ２９８６３（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＡＴＣＣ２９８６３）、ユウバクテリウム・プラウティＭＴ４２（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＭＴ４２，ＦＥＲＭ Ｐ−２１７６５）およびクロストリジウム・オルビシンデンスＡＴＣＣ４９５３１（Ｃｌｏｓｔｒｉｄｉｕｍ ｏｒｂｉｓｃｉｎｄｅｎｓ ＡＴＣＣ４９５３１）を挙げることができる。このうち、ユウバクテリウム・プラウティＭＴ４２（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＭＴ４２）株は、特許生物寄託センターに、受託番号ＦＥＲＭ Ｐ−２１７６５として寄託されているラットの糞中から分離された微生物である。 In the biological conversion method of the present invention, the catechin derivative represented by the formula (I) is represented by 5-phenyl-4-hydroxyvaleric acid represented by the formula (II) and / or the formula (III). Any microorganism having the ability to convert to 5-phenyl γ-valerolactone can be used regardless of the type of microorganism. Preferable examples of such microorganisms include Eubacterium spp. And Clostridium spp., More preferably Eubacterium pluti ATCC 29863 (Eubacterium plautii ATCC 29863), Eubacterium pluti MT42 (Eubacterium) Platii MT42, FERM P-21765) and Clostridium orubicindens ATCC49531 (Clostridium orbiscindens ATCC49531). Among these, the strain of Eubacterium pluti MT42 (Eubacterium plautii MT42) is a microorganism isolated from the feces of rats deposited at the Patent Biodeposition Center under the deposit number FERM P-21765.
上記ＭＴ４２株の１６ＳｒＲＮＡ遺伝子の全塩基配列（１４８４ｂｐ）は、配列番号１の通りである。得られたＭＴ４２株の１６ＳｒＲＮＡ遺伝子の塩基配列を国際塩基配列データベース（ＤＤＢＪ／ＥＭＢＬ／ＧｅｎＢａｎｋ）の配列データと比較し相同性を調べた。その結果、ＭＴ４２菌株の１６ＳｒＲＮＡ遺伝子の塩基配列はＥｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉＣＣＵＧ２８０９３Ｔ（ＡＴＣＣ２９８６３）と９９．７％、Ｃｌｏｓｔｒｉｄｉｕｍ ｏｒｂｉｓｃｉｎｄｅｎｓ ＤＳＭ６７４０Ｔ（ＡＴＣＣ４９５３１）と９９．５％の相同性を示した。以上の結果から本発明者らはＭＴ４２菌株がユウバクテリウム・プラウティ(Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ)であると同定した。 The entire base sequence (1484 bp) of the 16S rRNA gene of the MT42 strain is as shown in SEQ ID NO: 1. The base sequence of the 16S rRNA gene of the obtained MT42 strain was compared with the sequence data of the international base sequence database (DDBJ / EMBL / GenBank) to examine homology. As a result, the nucleotide sequence of 16SrRNA gene MT42 strain Eubacterium plautiiCCUG28093 T (ATCC29863) and 99.7% showed homology Clostridium orbiscindens DSM6740 T (ATCC49531) and 99.5%. From the above results, the present inventors have identified that the MT42 strain is Eubacterium plutii.
なお、１６ＳｒＲＮＡ遺伝子の全塩基配列（１４８４ｂｐ）の解析は以下の通りに行った。ＧＡＭ寒天培地（日水製薬（株）社製）により３７℃の嫌気条件下で２日間培養したＭＴ４２株ＰｒｅｐＭａｎ Ｕｌｔｒａ Ｒｅａｇｅｎｔ (Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製)を用いてＤＮＡ抽出を行った。得られたＤＮＡ溶液をＰＣＲ反応用ＤＮＡｔｅｍｐｌａｔｅとして用い、ＭｉｃｒｏＳｅｑ Ｆｕｌｌ Ｇｅｎｅ １６ＳｒＤＮＡ Ｂａｃｔｅｒｉａｌ ＩｄｅｎｔｉｆｉｃａｔｉｏｎＰＣＲ Ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）により、１６ＳｒＲＮＡ遺伝子領域１４８４ｐをＰＣＲ反応で増幅させた。得られたＰＣＲ反応産物はＱｕｉｃｋＳｔｅｐTM２ＰＣＲＰｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ(ＥｄｇｅＢｉｏＳｙｓｔｅｍｓ社製)を用いて精製した。精製したＰＣＲ反応産物をＢｉｇ−Ｄｙｅ Ｔｅｒｍｉｎａｔｉｏｎ反応用ｔｅｍｐｌａｔｅとして用い、ＭｉｃｒｏＳｅｑ Ｆｕｌｌ Ｇｅｎｅ １６ＳｒＤＮＡ Ｂａｃｔｅｒｉａｌ Ｉｄｅｎｔｉｆｉｃａｔｉｏｎ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）によりシークエンス反応（サイクルシークエンス）を行った。サーマサイクラーには、ＧｅｎｅＡｍｐ ＰＣＲ Ｓｙｓｔｅｍ ９７００(Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製)を使用した。得られた反応液はＡｕｔｏＳｅｑTMＧ-５０（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社製)を用いて精製し、ＡＢＩ ＰＲＩＳＭ（登録商標）３１００Ｇｅｎｅｔｉｃ Ａｎａｌｙｚｅｒ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）を用いて塩基配列の解読を行った。得られた本菌株の１６ＳｒＲＮＡ遺伝子(１４８４ｂｐ)の塩基配列は配列番号１のとおりである。 The analysis of the entire base sequence (1484 bp) of the 16S rRNA gene was performed as follows. DNA extraction was performed using MT42 strain PrepMan Ultra Reagent (manufactured by Applied Biosystems) cultured for 2 days under anaerobic conditions at 37 ° C. using a GAM agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.). The obtained DNA solution was used as a DNA template for PCR reaction, and 16SrRNA gene region 1484p was amplified by PCR reaction using MicroSeq Full Gene 16SrDNA Bacterial Identification PCR Kit (manufactured by Applied Biosystems). The obtained PCR reaction product was purified using QuickStep ™ 2PCR Purification Kit (manufactured by EdgeBioSystems). The purified PCR reaction product was used as a template for the Big-Dye Termination reaction, and a sequence reaction (cycle sequence) was performed by MicroSeq Full Gene 16SrDNA Bacterial Identification Sequencing Kit (manufactured by Applied Biosystems). A GeneAmp PCR System 9700 (Applied Biosystems) was used as the thermacycler. The obtained reaction solution was purified using AutoSeq ™ G-50 (manufactured by Amersham Pharmacia Biotech), and the base sequence was decoded using ABI PRISM (registered trademark) 3100 Genetic Analyzer (manufactured by Applied Biosystems). The base sequence of the obtained 16S rRNA gene (1484 bp) of this strain is as shown in SEQ ID NO: 1.
また、本発明の生物学的変換方法では、式(IV)で示されるカテキン類を式(I)のカテキン誘導体に変換する微生物を併用することで、該カテキン類含有物から容易に上記に示した５−フェニル−４−ハイドロキシ吉草酸および／または５−フェニルγ−バレロラクトン含有物を得ることができる。本発明の方法では、エガーテラ（Ｅｇｇｅｒｔｈｅｌｌａ）属およびアドラークルーツィア（Ａｄｌｅｒｃｒｅｕｔｚｉａ）属に属し、式(IV)で示されるカテキン類を式(I)のカテキン誘導体に変換する能力を有する微生物であれば、種および株の種類を問うことなく使用することができる。 Further, in the biological conversion method of the present invention, by using together with a microorganism that converts the catechin represented by the formula (IV) into a catechin derivative represented by the formula (I), the catechin-containing product can be easily shown above. In addition, a 5-phenyl-4-hydroxyvaleric acid and / or 5-phenyl γ-valerolactone-containing product can be obtained. In the method of the present invention, any microorganism belonging to the genus Eggerella and the genus Adlercreutzia and having the ability to convert a catechin represented by the formula (IV) into a catechin derivative of the formula (I), It can be used without regard to species and strain types.
そのような微生物の好ましい例として、エガーテラ・レンタＪＣＭ９９７９（Ｅｇｇｅｒｔｈｅｌｌａ ｌｅｎｔａ ＪＣＭ９９７９）およびアドラークルーツィア・エクオーリファシエンスＭＴ４ｓ−５（Ａｄｌｅｒｃｒｅｕｔｚｉａ ｅｑｕｏｌｉｆａｃｉｅｎｓ ＭＴ４ｓ−５）を挙げることができる。このうち、アドラークルーツィア・エクオーリファシエンスＭＴ４ｓ−５（Ａｄｌｅｒｃｒｅｕｔｚｉａ ｅｑｕｏｌｉｆａｃｉｅｎｓ ＭＴ４ｓ−５）株は、特許生物寄託センターに、受託番号ＦＥＲＭ Ｐ−２１７３８として寄託されているラットの糞中から分離された微生物である。 Preferable examples of such microorganisms include Egerterra Renta JCM9979 (Eggerella lenta JCM9979) and Adlercruzia equolifaciens MT4s-5 (Adlercreutzia equifaciens MT4s-5). Among these, the Adlercreutzia equalifaciens MT4s-5 strain is a microorganism isolated from the feces of rats deposited under the accession number FERM P-21738 at the Patent Organism Depositary. It is.
上記ＭＴ４ｓ−５株の１６ＳｒＲＮＡ遺伝子の全塩基配列（１４６０ｂｐ）は、配列番号２の通りである。得られたＭＴ４ｓ−５株の１６ＳｒＲＮＡ遺伝子の塩基配列を国際塩基配列データベース（ＤＤＢＪ／ＥＭＢＬ／ＧｅｎＢａｎｋ）の配列データと比較し相同性を調べた。その結果、ＭＴ４ｓ―５菌株の１６ＳｒＲＮＡ遺伝子の塩基配列は、Ａｄｌｅｒｃｒｅｕｔｚｉａ ｅｑｕｏｌｉｆａｃｉｅｎｓＦＪＣ−Ｂ９Ｔと９９．９％、Ａｄｌｅｒｃｒｅｕｔｚｉａ ｅｑｕｏｌｉｆａｃｉｅｎｓ ＦＪＣ−Ｄ５３およびＡｄｌｅｒｃｒｅｕｔｚｉａ ｅｑｕｏｌｉｆａｃｉｅｎｓ ＦＪＣ−Ａ１０と９９．８％、Ａｄｌｅｒｃｒｅｕｔｚｉａ ｅｑｕｏｌｉｆａｃｉｅｎｓ ＦＪＣ−Ｂ２０と９９．５％の相同性を示した。以上の結果から、ＭＴ４ｓ−５菌株はアドラークルーツィア・エクオーリファシエンス(Ａｄｌｅｒｃｒｅｕｔｚｉａ ｅｑｕｏｌｉｆａｃｉｅｎｓ)またはアサッカロバクター・セラタス（Ａｓａｃｃｈａｒｏｂａｃｔｅｒ ｃｅｌａｔｕｓ）であると同定した。 The entire base sequence (1460 bp) of the 16S rRNA gene of the MT4s-5 strain is as shown in SEQ ID NO: 2. The base sequence of the 16S rRNA gene of the obtained MT4s-5 strain was compared with the sequence data of the international base sequence database (DDBJ / EMBL / GenBank) to examine homology. As a result, the base sequence of the 16S rRNA gene of MT4s-5 strain was determined by Adlercreutzia equolifaciens FJC-B9T and 99.9%, Adlercreutzia equiquacienciFacienciFlAciocl. It showed 5% homology. Based on the above results, the MT4s-5 strain was identified as Adlercreutzia equalifaciens or Asaccharobacter ceratas.
なお、ＭＴ４ｓ−５菌株の１６ＳｒＲＮＡ遺伝子の全塩基配列（１４６０ｂｐ）の解析は以下の通りに行った。ＧＡＭ寒天培地（日水製薬（株）製）により３７℃の嫌気条件下で２日間培養したＭＴ４ｓ−５株ＰｒｅｐＭａｎ Ｕｌｔｒａ Ｒｅａｇｅｎｔ (Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製)を用いてＤＮＡ抽出を行った。得られたＤＮＡ溶液をＰＣＲ反応用ＤＮＡ ｔｅｍｐｌａｔｅとして用い、ＭｉｃｒｏＳｅｑ Ｆｕｌｌ Ｇｅｎｅ １６ＳｒＤＮＡ Ｂａｃｔｅｒｉａｌ ＩｄｅｎｔｉｆｉｃａｔｉｏｎＰＣＲ Ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）により、１６ＳｒＲＮＡ遺伝子領域１４６０ｐをＰＣＲ反応で増幅させた。得られたＰＣＲ反応産物はＱｕｉｃｋＳｔｅｐTM２ＰＣＲＰｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ(ＥｄｇｅＢｉｏＳｙｓｔｅｍｓ社製)を用いて精製した。精製したＰＣＲ反応産物をＢｉｇ−Ｄｙｅ Ｔｅｒｍｉｎａｔｉｏｎ反応用ｔｅｍｐｌａｔｅとして用い、ＭｉｃｒｏＳｅｑ Ｆｕｌｌ Ｇｅｎｅ １６ＳｒＤＮＡ Ｂａｃｔｅｒｉａｌ Ｉｄｅｎｔｉｆｉｃａｔｉｏｎ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）によりシークエンス反応（サイクルシークエンス）を行った。サーマサイクラーには、ＧｅｎｅＡｍｐＰＣＲ Ｓｙｓｔｅｍ ９７００(Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製)を使用した。得られた反応液はＡｕｔｏＳｅｑTMＧ-５０（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ社製)を用いて精製し、ＡＢＩ ＰＲＩＳＭ（登録商標）３１００Ｇｅｎｅｔｉｃ Ａｎａｌｙｚｅｒ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ社製）を用いて塩基配列の解読を行った。得られたＭＴ４ｓ−５菌株の１６ＳｒＲＮＡ遺伝子(１４６０ｂｐ)の塩基配列は配列番号２のとおりである。 The analysis of the entire base sequence (1460 bp) of the 16S rRNA gene of MT4s-5 strain was performed as follows. DNA extraction was performed using MT4s-5 strain PrepMan Ultra Reagent (manufactured by Applied Biosystems) cultured for 2 days under anaerobic conditions at 37 ° C. on a GAM agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.). Using the obtained DNA solution as a DNA template for PCR reaction, 16SrRNA gene region 1460p was amplified by PCR reaction using MicroSeq Full Gene 16SrDNA Bacterial Identification PCR Kit (manufactured by Applied Biosystems). The obtained PCR reaction product was purified using QuickStep ™ 2PCR Purification Kit (manufactured by EdgeBioSystems). The purified PCR reaction product was used as a template for the Big-Dye Termination reaction, and a sequence reaction (cycle sequence) was performed by MicroSeq Full Gene 16SrDNA Bacterial Identification Sequencing Kit (manufactured by Applied Biosystems). A GeneAmpPCR System 9700 (Applied Biosystems) was used as the thermacycler. The obtained reaction solution was purified using AutoSeq ™ G-50 (manufactured by Amersham Pharmacia Biotech), and the base sequence was decoded using ABI PRISM (registered trademark) 3100 Genetic Analyzer (manufactured by Applied Biosystems). The base sequence of the 16S rRNA gene (1460 bp) of the obtained MT4s-5 strain is as shown in SEQ ID NO: 2.
本発明によれば、前記式(I)で示されるカテキン誘導体を前記式(II)で示される５−フェニル−４−ハイドロキシ吉草酸および／または前記式（III）で表される５−フェニルγ−バレロラクトンへ変換する能力を有する微生物の培養菌体の存在下または当該微生物が生育する培養液中に、出発原料（基質）である式（I）で示されるカテキン誘導体含有物を添加してインキュベーション処理することにより式（II）で示される５―フェニル―４−ハイドロキシ吉草酸および／または式（III）で示される５−フェニルγ−バレロラクトン含有物を得ることができる。このインキュベーション処理は、前記微生物を培養後、
培養菌体を集菌し、この菌体を緩衝液、生理食塩水、水などに懸濁させた後に基質を添加するか、前記微生物を培養する際あるいは培養開始後一定期間経過した培養液に基質を添加して行うことができる。培養開始後、基質を添加する時期は特に限定されないが、好ましくは培養２〜１２０時間の間に、より好ましくは４〜７２時間、さらに好ましくは６〜４８時間の間に添加すると効果的である。
According to the present invention, the catechin derivative represented by the formula (I) is converted to 5-phenyl-4-hydroxyvaleric acid represented by the formula (II) and / or 5-phenyl γ represented by the formula (III). -Addition of a catechin derivative-containing substance represented by formula (I), which is a starting material (substrate), in the presence of cultured microorganisms having the ability to convert to valerolactone or in the culture medium in which the microorganisms grow By carrying out the incubation treatment, a 5-phenyl-4-hydroxyvaleric acid represented by the formula (II) and / or a 5-phenyl γ-valerolactone-containing product represented by the formula (III) can be obtained. This incubation treatment is performed after culturing the microorganism,
Collect the cultured cells and suspend the cells in a buffer solution, physiological saline, water, etc., and then add a substrate, or when culturing the microorganisms, It can be performed by adding a substrate. The timing of adding the substrate after the start of the culture is not particularly limited, but it is effective if it is added preferably during 2 to 120 hours, more preferably from 4 to 72 hours, and even more preferably from 6 to 48 hours. .
さらに、本発明では、式(IV)で示されるカテキン類を式(I)のカテキン誘導体に変換する微生物を、式（I）で示されるカテキン誘導体含有物を式（II）で示される５―フェニル―４−ハイドロキシ吉草酸および／または式（III）で示される５−フェニルγ−バレロラクトンに変換する能力を有する微生物の培養菌体懸濁液または培養液に共存させ、式（IV）で示されるカテキン類含有物を基質として添加し、インキュベーション処理することにより、容易に上記に示した５−フェニル−４−ハイドロキシ吉草酸および／または５−フェニルγ−バレロラクトン含有物を得ることができる。このインキュベーション処理は、前記微生物を培養後、培養菌体を集菌し、この菌体を緩衝液、生理食塩水、水などに懸濁させた後に基質を添加するか、前記微生物を培養する際あるいは培養開始後一定期間経過した培養液に基質を添加して行うことができる。培養開始後、基質を添加する時期は特に限定されないが、好ましくは培養２〜１２０時間の間に、より好ましくは４〜７２時間、さらに好ましくは６〜４８時間の間に添加すると効果的である。 Furthermore, in the present invention, a microorganism that converts a catechin represented by the formula (IV) into a catechin derivative represented by the formula (I) is represented by a microorganism containing a catechin derivative represented by the formula (I) represented by the formula (II) 5- Coexist with a cultured cell suspension or culture solution of a microorganism having the ability to convert to phenyl-4-hydroxyvaleric acid and / or 5-phenyl γ-valerolactone represented by the formula (III). By adding the indicated catechins-containing substance as a substrate and incubating it, the above-mentioned 5-phenyl-4-hydroxyvaleric acid and / or 5-phenylγ-valerolactone-containing substance can be easily obtained. . In this incubation treatment, after culturing the microorganism, the cultured cells are collected and suspended in a buffer solution, physiological saline, water, etc., and then a substrate is added or the microorganism is cultured. Or it can carry out by adding a substrate to the culture solution which passed for a fixed period after the culture | cultivation start. The timing of adding the substrate after the start of the culture is not particularly limited, but it is effective if it is added preferably during 2 to 120 hours, more preferably from 4 to 72 hours, and even more preferably from 6 to 48 hours. .
また、式(IV)で示されるカテキン類を式(I)のカテキン誘導体に変換する微生物の培養菌体の存在下または当該微生物が生育する培養液中に、出発原料（基質）である式（IV）で示されるカテキン類含有物を添加してインキュベーション処理することにより、式（I）で示されるカテキン誘導体を生成させた後、式（I）で示されるカテキン誘導体含有物を式（II）で示される５−フェニル−４−ハイドロキシ吉草酸および／または式（III）で示される５−フェニルγーバレロラクトンに変換する能力を有する微生物の培養菌体懸濁液または培養液を添加してインキュベーション処理することで目的とする式（II）および／または式（III）の化合物を得ることも可能である。さらに、式（IV）で示されるカテキン類含有物から微生物変換で得られる式（I）のカテキン誘導体を単離して、これを式（II）で示される５−フェニル−４−ハイドロキシ吉草酸および／または式（III）で示される５−フェニルγ−バレロラクトンに変換する能力を有する微生物の出発材料（基質）として用いることも可能である。 In addition, in the presence of a cultured cell of a microorganism that converts a catechin represented by the formula (IV) into a catechin derivative of the formula (I) or in a culture solution in which the microorganism grows, a formula (substrate) The catechin derivative-containing compound represented by the formula (I) is produced by adding the catechin-containing compound represented by the formula IV) to the incubation treatment, and then the catechin derivative-containing compound represented by the formula (I) is represented by the formula (II) Incubation treatment by adding a suspension or culture medium of a microorganism having the ability to convert to 5-phenyl-4-hydroxyvaleric acid represented by formula (5) and / or 5-phenyl γ-valerolactone represented by formula (III) By doing so, it is also possible to obtain the target compound of formula (II) and / or formula (III). Further, the catechin derivative of the formula (I) obtained by microbial conversion is isolated from the catechins-containing product represented by the formula (IV), and this is converted into 5-phenyl-4-hydroxyvaleric acid represented by the formula (II) and It is also possible to use it as a starting material (substrate) of a microorganism having the ability to convert to 5-phenyl γ-valerolactone represented by the formula (III).
上記の方法で生成した式（I）で示されるカテキン誘導体を単離するには、種々の既知精製手段を選択し、組み合わせて行うことができる。例えば、酢酸エチル、ブタノール、エーテルなどを用いた溶媒抽出、合成樹脂吸着剤の脱吸着を利用する方法、シリカゲルなどのカラムクロマトグラフィーや高速液体クロマトグラフィーを単独あるいは適宜組み合わせて分離・精製することができる。 In order to isolate the catechin derivative represented by the formula (I) produced by the above method, various known purification means can be selected and combined. For example, solvent extraction using ethyl acetate, butanol, ether, etc., a method using desorption of a synthetic resin adsorbent, column chromatography such as silica gel or high performance liquid chromatography can be separated or purified alone or in appropriate combination. it can.
前記に示した微生物を培養する場合には、該微生物が生育できる栄養源含有培地に接種し、嫌気的条件下で培養する。培養菌体を得るための微生物の培養および基質存在下での微生物の培養は、一般的な嫌気性微生物の培養方法を採用することができる。また、培養菌体を集菌した後、前記基質の存在下でインキュベーション処理する場合にも、嫌気条件下で行うことが望ましい。 When culturing the microorganisms described above, they are inoculated into a nutrient source-containing medium in which the microorganisms can grow and cultured under anaerobic conditions. A general anaerobic microorganism culture method can be employed for culturing microorganisms for obtaining cultured cells and culturing microorganisms in the presence of a substrate. In addition, it is desirable to carry out the incubation under anaerobic conditions also when incubation is performed in the presence of the substrate after collecting the cultured cells.
培養に用いられる培地としては、前記微生物が生育できる培地であれば特に限定されないが、例を挙げればＧＡＭブイヨン（日水製薬（株）製）などが利用可能である。なお、式（IV）のうちＲが水素であるカテキン類を式（I）のＲ１およびＲ２が水素であるカテキン誘導体に微生物変換するためには、培地中に水素及び蟻酸を添加することが望ましい。また、式（IV）のうちＲが水酸基であるカテキン類を式（I）のＲ１が水酸基、Ｒ２が水素であるカテキン誘導体に微生物変換するためには、培地中に水素を添加する必要がある。さらに、水素や蟻酸を培地中に添加しない場合には、式（IV）および式（I）の化合物には作用しない水素および／または蟻酸生成微生物を共存させることも可能である。このような微生物の例として、大腸菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）を挙げることができる。 The medium used for the culture is not particularly limited as long as the microorganism can grow therein. For example, GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) can be used. In order to microbially convert catechins in which R is hydrogen in formula (IV) to catechin derivatives in which R1 and R2 in formula (I) are hydrogen, it is desirable to add hydrogen and formic acid to the medium. . In addition, in order to microbially convert catechins of formula (IV) in which R is a hydroxyl group into catechin derivatives in which R1 of formula (I) is a hydroxyl group and R2 is hydrogen, it is necessary to add hydrogen to the medium. . Furthermore, when hydrogen and formic acid are not added to the medium, it is possible to coexist hydrogen and / or formic acid-producing microorganisms that do not act on the compounds of formula (IV) and formula (I). Examples of such microorganisms include Escherichia coli.
培養条件は、前記微生物が生育しうる範囲内で適宜選択することができる。通常、ｐＨ６．０〜７．５、３５〜４０℃であり、好ましくはｐＨ６．５〜７．３、３７〜３９℃である。培養時間は通常２４〜１２０時間、好ましくは４８〜７２時間である。上述した各種の培養条件は、使用する微生物の種類や特性、外部条件などに応じて適宜変更でき、最適条件を選択することができる。 The culture conditions can be appropriately selected within the range in which the microorganism can grow. Usually, it is pH 6.0-7.5, 35-40 degreeC, Preferably it is pH 6.5-7.3, 37-39 degreeC. The culture time is usually 24 to 120 hours, preferably 48 to 72 hours. The various culture conditions described above can be appropriately changed according to the type and characteristics of microorganisms used, external conditions, and the like, and optimal conditions can be selected.
基質となる式（I）で示されるカテキン誘導体あるいは式（IV）で示されるカテキン類は、水、食塩水、緩衝液などのほか、水溶性有機溶媒、例えばメタノール、エタノール、アセトン、アセトニトリル、ジメチルホルムアミド、ジメチルスルホキシドなどに溶解後、微生物の生育を阻害しない濃度範囲で培養液あるいは培養菌体の懸濁液に添加することができる。該基質の添加量は、微生物による変換が可能な濃度あるいは量の範囲内で適宜選択することができる。通常、培養液の場合には、培養液１リットル当たり０．３〜５ｇであり、好ましくは０．５〜２ｇ、より好ましくは０．６〜１ｇである。また、培養後の菌
体の懸濁液を用いる場合には、懸濁液１リットル当たり５０〜５００ｍｇ、好ましくは１００〜２５０ｍｇである。
The catechin derivative represented by the formula (I) or the catechin represented by the formula (IV) serving as a substrate includes water, saline, buffer, and other water-soluble organic solvents such as methanol, ethanol, acetone, acetonitrile, dimethyl After being dissolved in formamide, dimethyl sulfoxide, etc., it can be added to a culture solution or a suspension of cultured cells in a concentration range that does not inhibit the growth of microorganisms. The amount of the substrate added can be appropriately selected within the range of concentration or amount that can be converted by microorganisms. Usually, in the case of a culture solution, it is 0.3-5g per liter of culture solution, Preferably it is 0.5-2g, More preferably, it is 0.6-1g. Moreover, when using the suspension of the microbial cell after culture | cultivation, it is 50-500 mg per liter of suspension, Preferably it is 100-250 mg.
本発明の生物学的変換方法で生成した目的の式（II）で示される５−フェニル−４−ハイドロキシ吉草酸および／または式（III）で示される５−フェニルγーバレロラクトン含有物から該目的化合物を単離するには、種々の既知精製手段を選択し、組合わせて行うことができる。例えば、酢酸エチル、ブタノールなどを用いた溶媒抽出、合成樹脂吸着剤の
脱吸着を利用する方法、シリカゲルなどのカラムクロマトグラフィーや高速液体クロマトグラフィーを単独あるいは適宜組み合わせて分離・精製することができる。
The target compound produced from the desired 5-phenyl-4-hydroxyvaleric acid represented by the formula (II) and / or 5-phenyl γ-valerolactone represented by the formula (III) produced by the biological conversion method of the present invention In order to isolate, various known purification means can be selected and combined. For example, it can be separated and purified by solvent extraction using ethyl acetate, butanol or the like, a method utilizing desorption of a synthetic resin adsorbent, column chromatography such as silica gel, or high performance liquid chromatography alone or in combination.
本発明によれば、生物学的変換方法によって得られる式（II）の５−フェニル−４−ハイドロキシ吉草酸を式（III）で示される５−フェニルγ−バレロラクトンに変換することも可能である。この変換は、式（II）の化合物や式（II）および式（III）の化合物の混合物のｐＨを酸性とすることにより達成できる。例を挙げれば、生物学的変換方法によって得られる式（II）の５−フェニル−４−ハイドロキシ吉草酸および／または式（III）で示される５−フェニルγ−バレロラクトン含有物に酸（塩酸、蟻酸、酢酸、トリフルオロ酢酸など）を添加して、ｐＨ３以下、好ましくはｐＨ２以下において、０〜５０℃、好ましくは４〜４０℃で１〜７２時間、好ましくは２〜４８時間インキュベーション処理することにより、式（II）に示される５−フェニル−４−ハイドロキシ吉草酸を式（III）の５−フェニルγ−バレロラクトンに変換することが可能である。このような方法を用いることにより、式（II）の５−フェニル−４−ハイドロキシ吉草酸および式（III）で示される５−フェニルγ−バレロラクトン含有物から、式（III）の化合物を式（II）および式（III）の化合物総量の少なくとも９０％以上含有する組成物を得ることができる。 According to the present invention, it is also possible to convert 5-phenyl-4-hydroxyvaleric acid of the formula (II) obtained by the biological conversion method into 5-phenyl γ-valerolactone represented by the formula (III). is there. This conversion can be achieved by acidifying the pH of the compound of formula (II) or a mixture of compounds of formula (II) and formula (III). For example, 5-phenyl-4-hydroxyvaleric acid of the formula (II) obtained by a biotransformation method and / or 5-phenyl γ-valerolactone-containing product of the formula (III) , Formic acid, acetic acid, trifluoroacetic acid, etc.) at pH 3 or lower, preferably pH 2 or lower, and incubated at 0 to 50 ° C., preferably 4 to 40 ° C. for 1 to 72 hours, preferably 2 to 48 hours. This makes it possible to convert 5-phenyl-4-hydroxyvaleric acid represented by the formula (II) into 5-phenyl γ-valerolactone represented by the formula (III). By using such a method, the compound of the formula (III) is represented by the formula from the 5-phenyl-4-hydroxyvaleric acid of the formula (II) and the 5-phenyl γ-valerolactone-containing product represented by the formula (III). A composition containing at least 90% of the total amount of the compounds of (II) and formula (III) can be obtained.
また、本発明によれば、生物学的変換方法によって得られる（III）で示される５−フェニルγ−バレロラクトンを式（II）の５−フェニル−４−ハイドロキシ吉草酸に変換することも可能である。この場合には、式（III）の化合物や式（II）および式（III）の化合物の混合物のｐＨをアルカリ性とすることにより達成できる。例を挙げれば、生物学的変換方法によって得られる式（II）の５−フェニル−４−ハイドロキシ吉草酸および／または式（III）で示される５−フェニルγ−バレロラクトン含有物にアルカリ（水酸化ナトリウム、水酸化カリウム、アンモニア、炭酸ナトリウム、炭酸水素ナトリウム、炭酸カリウム、炭酸水素カリウムなど）を添加して、ｐＨ８以上、好ましくはｐＨ９以上、さらに好ましくはｐＨ１０以上とし、０〜５０℃、好ましくは４〜４０℃で１〜７２時間、好ましくは２〜４８時間インキュベーション処理することにより、式（III）の５−フェニルγ−バレロラクトンを式（II）に示される５−フェニル−４−ハイドロキシ吉草酸に変換することが可能である。このような方法を用いることにより、式（II）の５−フェニル−４−ハイドロキシ吉草酸および式（III）で示される５−フェニルγ−バレロラクトン含有物から、式（II）の化合物を少なくとも式（II）および式（III）の化合物総量の９０％以上含有する組成物を得ることができる。 According to the present invention, it is also possible to convert 5-phenyl γ-valerolactone represented by (III) obtained by the biological conversion method into 5-phenyl-4-hydroxyvaleric acid of formula (II). It is. In this case, it can be achieved by making the pH of the compound of the formula (III) or the mixture of the compounds of the formula (II) and the formula (III) alkaline. For example, 5-phenyl-4-hydroxyvaleric acid of formula (II) and / or 5-phenyl γ-valerolactone-containing product of formula (III) obtained by a biotransformation method may be added to an alkali (water Sodium oxide, potassium hydroxide, ammonia, sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate, etc.) to add pH 8 or higher, preferably pH 9 or higher, more preferably pH 10 or higher, 0 to 50 ° C., preferably Is incubated at 4 to 40 ° C. for 1 to 72 hours, preferably 2 to 48 hours, whereby 5-phenyl γ-valerolactone of formula (III) is converted to 5-phenyl-4-hydroxy represented by formula (II). It can be converted to valeric acid. By using such a method, at least the compound of the formula (II) is obtained from the 5-phenyl-4-hydroxyvaleric acid of the formula (II) and the 5-phenyl γ-valerolactone-containing product represented by the formula (III). A composition containing 90% or more of the total amount of the compounds of formula (II) and formula (III) can be obtained.
上記の、式（III）で示される化合物を式（II）の化合物に変換する方法としては、微生物学的変換方法も利用できる。例えば、ラットの糞あるいは盲腸内容物から嫌気培養によって得られる腸内細菌の培養菌体を式（III）で示される５−フェニルγ−バレロラクトン含有物に作用させるか、あるいは上記の腸内細菌を式（III）で示される化合物の存在下で培養する。培養菌体との反応や培養条件は、前記の生物学的変換方法による（III）で示される５−フェニルγ−バレロラクトンを式（II）の５−フェニル−４−ハイドロキシ吉草酸の生成条件や式(IV)で示されるカテキン類を式(I)のカテキン誘導体に変換するための条件等を適用することができる。 As a method for converting the compound represented by the formula (III) into the compound of the formula (II), a microbiological conversion method can also be used. For example, a culture of enteric bacteria obtained by anaerobic culture from rat feces or caecal contents is allowed to act on a 5-phenyl γ-valerolactone-containing product represented by formula (III), or the above enteric bacteria Is cultured in the presence of a compound represented by the formula (III). The reaction with the cultured cells and the culture conditions are the conditions for producing 5-phenyl-4-hydroxyvaleric acid of formula (II) from 5-phenyl γ-valerolactone represented by (III) by the biological conversion method described above. In addition, conditions for converting catechins represented by formula (IV) into catechin derivatives of formula (I) can be applied.
本発明の式（II）に示される５−フェニル−４−ハイドロキシ吉草酸あるいは（III）で示される５−フェニルγ−バレロラクトンの少なくとも一つを必須成分として含有する組成物は、上記成分の粗製品でも精製品でもよく、また液状であっても乾燥品等であってもよい。また、組成物の必須成分の含量も特に限定されるものではないが、通常０．１以上、好ましくは１〜１０％、より好ましくは１５〜３０％、さらに好ましくは４０％以上含有する。 The composition containing at least one of 5-phenyl-4-hydroxyvaleric acid represented by formula (II) of the present invention or 5-phenyl γ-valerolactone represented by (III) as an essential component is The product may be a crude product or a purified product, and may be liquid or dry. Moreover, the content of the essential components of the composition is not particularly limited, but is usually 0.1 or more, preferably 1 to 10%, more preferably 15 to 30%, and further preferably 40% or more.
本発明の前記組成物は、どのような形態であってもよく、例えば、粉末状であってもよいし液状であってもよい。また、本発明の組成物は、必要に応じて、抗酸化剤、着色剤、香料、矯味剤、界面活性剤、溶解補助剤、保存剤、甘味料などと併用して用いてもよい。抗酸化剤としては、例えば、クエン酸トコフェロール（三栄源・エイ・エフ・アイ社製）などが挙げられる。界面活性剤としては、例えば、ショ糖脂肪酸エステル、ポリグリセリン脂肪酸エステル、ソルビタン脂肪酸エステル、ポリグリセリン縮合リシノレイン酸エステル、レシチン、ポリオキシエチレン硬化ヒマシ油、ポリエチレングリコール脂肪酸エステル、グリセリン脂肪酸エステル、ポリオキシエチレン系非界面活性剤などが挙げられる。甘味料としては、砂糖、ブドウ糖、果糖、異性化液糖、グリチルリチン、ステビア、アスパラテーム、フラクトオリゴ糖、ガラクトオリゴ糖、その他のオリゴ糖としてシクロデキストリンが挙げられる。シクロデキストリンとしては、α−、β−、γ−シクロデキストリンおよび分岐α−、β−、γ−シクロデキストリンが使用できる。また、人工甘味料も使用できる。酸味料としては、天然成分から抽出した果汁類のほか、クエン酸、酒石酸、リンゴ酸、乳酸、フマル酸、リン酸が挙げられる。 The composition of the present invention may be in any form, and may be, for example, a powder or a liquid. Moreover, you may use the composition of this invention together with an antioxidant, a coloring agent, a fragrance | flavor, a corrigent, a surfactant, a solubilizing agent, a preservative, a sweetener, etc. as needed. As the antioxidant, for example, tocopherol citrate (manufactured by San-Eigen AFI Co., Ltd.) and the like can be mentioned. Examples of the surfactant include sucrose fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, polyglycerin condensed ricinoleic acid ester, lecithin, polyoxyethylene hydrogenated castor oil, polyethylene glycol fatty acid ester, glycerin fatty acid ester, polyoxyethylene. Non-surfactant and the like. Examples of the sweetener include sugar, glucose, fructose, isomerized liquid sugar, glycyrrhizin, stevia, aspartame, fructooligosaccharide, galactooligosaccharide, and other oligosaccharides such as cyclodextrin. As the cyclodextrin, α-, β-, γ-cyclodextrin and branched α-, β-, γ-cyclodextrin can be used. Artificial sweeteners can also be used. Examples of acidulants include fruit juices extracted from natural ingredients, citric acid, tartaric acid, malic acid, lactic acid, fumaric acid, and phosphoric acid.
本発明の飲食品は、式（II）に示される５−フェニル−４−ハイドロキシ吉草酸あるいは（III）で示される５−フェニルγ−バレロラクトンの少なくとも一つを必須成分として含有する組成物を配合することができるものであればどのような形態であってもよく、例えば、水溶液や混濁物や乳化物などの液状形態であっても、ゲル状やペースト状の半固形状形態であっても、粉末や顆粒やカプセルやタブレットなどの固形状形態であってもよい。 The food or drink of the present invention comprises a composition containing at least one of 5-phenyl-4-hydroxyvaleric acid represented by formula (II) or 5-phenyl γ-valerolactone represented by (III) as an essential component. Any form may be used as long as it can be blended, for example, it may be a liquid form such as an aqueous solution, a turbid product, an emulsion, or a semi-solid form such as a gel or paste. Alternatively, it may be in a solid form such as powder, granule, capsule or tablet.
本発明における飲食品としては、例えば、即席食品類（即席めん、カップめん、レトルト・調理食品、調理缶詰め、電子レンジ食品、即席味噌汁・吸い物、スープ缶詰め、フリーズドライ食品など）、炭酸飲料、柑橘類（グレープフルーツ、オレンジ、レモンなど）の果汁や果汁飲料や果汁入り清涼飲料、柑橘類の果肉飲料や果粒入り果実飲料、トマト、ピーマン、セロリ、ウリ、ニンジン、ジャガイモ、アスパラガスなどの野菜を含む野菜系飲料、豆乳・豆乳飲料、コーヒー飲料、お茶飲料、粉末飲料、濃縮飲料、スポーツ飲料、栄養飲料、アルコール飲料やタバコなどの嗜好飲料・嗜好品類、マカロニ・スパゲッティ、麺類、ケーキミックス、唐揚げ粉、パン粉、ギョーザの皮などの小麦粉製品、キャラメル・キャンディー、チューイングガム、チョコレート、クッキー・ビスケット、ケーキ・パイ、スナック・クラッカー、和菓子・米菓子・豆菓子、デザート菓子などの菓子類、しょうゆ、みそ、ソース類、トマト加工調味料、みりん類、食酢類、甘味料などの基礎調味料、風味調味料、調理ミックス、カレーの素類、たれ類、ドレッシング類、めんつゆ類、スパイス類などの複合調味料・食品類、バター、マーガリン類、マヨネーズ類、植物油などの油脂類、牛乳・加工乳、乳飲料、ヨーグルト類、乳酸菌飲料、チーズ、アイスクリーム類、調製粉乳類、クリームなどの乳・乳製品、素材冷凍食品、半調理冷凍食品、調理済み冷凍食品などの冷凍食品、水産缶詰め、果実缶詰め・ペースト類、魚肉ハム・ソーセージ、水産練り製品、水産珍味類、水産乾物類、佃煮類などの水産加工品、畜産缶詰め・ペースト類、畜肉缶詰め、果実缶詰め、ジャム・マーマレード類、漬物・煮豆類、農産乾物類、シリアル（穀物加工品）などの農産加工品、ベビーフード、ふりかけ・お茶漬けのりなどの市販食品などが挙げられる。 Examples of the food and drink in the present invention include instant foods (immediate noodles, cup noodles, retort / cooked foods, canned foods, microwave foods, instant miso soup / soup, canned soup, freeze-dried foods), carbonated drinks, citrus ( Grapefruit, orange, lemon, etc.) Fruit juices, fruit drinks, soft drinks with fruit juices, citrus fruit drinks, fruit drinks with fruits, vegetables such as tomatoes, peppers, celery, cucumbers, carrots, potatoes, asparagus Beverages, soy milk and soy milk drinks, coffee drinks, tea drinks, powdered drinks, concentrated drinks, sports drinks, nutrition drinks, liquor drinks and liquor products such as alcoholic drinks and tobacco, macaroni spaghetti, noodles, cake mix, fried powder, Bread crumbs, flour products such as gyoza peel, caramel candy, chewing Gum, chocolate, cookies / biscuits, cakes / pies, snacks / crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, soy sauce, miso, sauces, tomato processing seasonings, mirin, vinegar, sweetness Basic seasonings such as seasonings, flavor seasonings, cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices and other complex seasonings and foods, butter, margarines, mayonnaise, vegetable oils, etc. Fats and oils, milk / processed milk, milk drinks, yogurts, lactic acid bacteria drinks, cheese, ice creams, prepared milk powder, milk and dairy products such as cream, frozen foods, semi-cooked frozen foods, cooked frozen foods, etc. Frozen food, canned fish, canned fruits and pastes, fish ham and sausages, fish paste products, fish delicacy, fish dried products, boiled fish, etc. Processed products, canned livestock products / pastes, canned livestock meat, canned fruit, jams / marmalades, pickles / boiled beans, dried agricultural products, cereals (processed cereals), baby food, sprinkles, tea pickles, etc. And other commercially available foods.
また、本発明の飲食品は口に含むことのできる医薬品、医薬部外品、化粧品なども含むことができる。医薬品としては日本薬局方に収められている医薬品で口に含むことができれば特に限定されるものではなく、その製剤形態としては、例えば、エアゾール剤、液剤、エキス剤、エリキシル剤、カプセル剤、顆粒剤、丸剤、散剤、酒精剤、錠剤、シロップ剤、浸剤・煎剤、トローチ剤、芳香水剤、リモナーゼ剤などが挙げられる。医薬部外品としては厚生労働大臣が指定した医薬部外品で口に含むことができれば特に限定されるものではなく、例えば、内服液剤、健康飲料、消毒剤、消毒保護剤、ビタミン含有保健剤などが挙げられる。 Moreover, the food / beverage products of this invention can also contain the pharmaceutical which can be included in a mouth, a quasi-drug, cosmetics, etc. The pharmaceutical is not particularly limited as long as it is contained in the Japanese Pharmacopoeia and can be included in the mouth. Examples of the dosage form include aerosols, solutions, extracts, elixirs, capsules, granules Pills, pills, powders, spirits, tablets, syrups, dipping / decoction, troches, fragrances, limonase and the like. The quasi-drug is not particularly limited as long as it is a quasi-drug specified by the Minister of Health, Labor and Welfare and can be included in the mouth. For example, oral liquids, health drinks, disinfectants, antiseptics, vitamin-containing health agents Etc.
飲食品への本発明の組成物の配合方法は特に制限されるものではない。例えば、飲食品の形態が液状形態や半固形状形態である場合には、その調製段階において本発明の組成物をそのまま、あるいは水に溶解させた水溶液などとして添加し、均一化することにより行えばよい。また、本発明の組成物をアルコール水などの含水有機溶媒やエタノールなどの有機溶媒などに分散させた分散液として添加し、十分に攪拌してこれを分散させることも可能である。なお、このようにして得られた調製物を、噴霧乾燥機や凍結乾燥機などを用いて乾燥することで、粉末などの固形状形態としてもよい。また、飲食品の形態が固形状形態である場合には、その調製段階において本発明の組成物をそのまま、あるいは水に溶解させた水溶液などとして添加し、均一化することにより行えばよい。また、本発明の組成物をアルコール水などの含水有機溶媒やエタノールなどの有機溶媒などに分散あるいは溶解させた液として添加し、十分に混合させることも可能である。水難溶性の飲食品に本発明の組成物を配合する場合、必要に応じて飲食品にアルコール水などの含水有機溶媒やエタノールなどの有機溶媒などを添加してこれを溶解あるいは希釈し、ここに本発明の組成物を添加し、十分に攪拌して組成物を混合させるようにしてもよい。 The method for blending the composition of the present invention into a food or drink is not particularly limited. For example, when the form of the food or drink is a liquid form or a semi-solid form, the composition of the present invention is added as it is or in the form of an aqueous solution dissolved in water in the preparation stage and homogenized. Just do it. It is also possible to add the composition of the present invention as a dispersion in which the composition is dispersed in a water-containing organic solvent such as alcohol water or an organic solvent such as ethanol, and disperse this by sufficiently stirring. In addition, it is good also as solid forms, such as a powder, by drying the preparation obtained in this way using a spray dryer, a freeze dryer, etc. Moreover, when the form of food / beverage products is a solid form, it may be performed by adding the composition of the present invention as it is or in the form of an aqueous solution dissolved in water and homogenizing it. Moreover, it is also possible to add the composition of the present invention as a liquid dispersed or dissolved in a water-containing organic solvent such as alcohol water or an organic solvent such as ethanol, and sufficiently mix them. When the composition of the present invention is blended with a poorly water-soluble food or drink, if necessary, a water-containing organic solvent such as alcohol water or an organic solvent such as ethanol is added to the food or drink to dissolve or dilute it. The composition of the present invention may be added and mixed sufficiently with stirring.
飲食品に対する本発明の組成物の配合量は、特に制限されないが、対象となる飲食品により配合量を適宜設定する。一般的には、最終製品中で０.０１〜２０重量％であることが好ましく、０．０５〜１０重量％であることがより好ましく、０．１〜５重量％であることがさらに好ましい。 Although the compounding quantity of the composition of this invention with respect to food / beverage products is not restrict | limited in particular, A compounding quantity is suitably set with the food / beverage products used as object. Generally, it is preferably 0.01 to 20% by weight in the final product, more preferably 0.05 to 10% by weight, and still more preferably 0.1 to 5% by weight.
以下、本発明について具体的な例を挙げてより詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail with specific examples, but the present invention is not limited to these examples.
式（I）で示されるカテキン誘導体を式（II）の５−フェニル−４−ハイドロキシ吉草酸および／または式（III）で示される５−フェニルγ−バレロラクトンに変換する微生物のスクリーニング
細菌の分離源としてＷｉｓｔａｒ系のラット（日本チャールスリバー（株））を使用した。ラットから新鮮な糞便約２ｇを採取し、ＧＡＭブイヨン（組成（１Ｌ中）：ペプトン１０ｇ、ダイズペプトン３ｇ、プロテオーゼペプトン１０ｇ、消化血清末１３．５ｇ、酵母エキス５ｇ、肉エキス２．２ｇ、肝臓エキス１．２ｇ、ブドウ糖３ｇ、リン酸二水素カリウム２．５ｇ、塩化ナトリウム３ｇ、溶性デンプン５ｇ、Ｌ−システイン塩酸塩０．３ｇ、チオグリコール酸ナトリウム０．３ｇ、ｐＨ７．１、日水製薬（株）社製）３ｍｌに懸濁して糞溶液を調製した。糞溶液０．１ｍｌをマイクロチューブに入れＧＡＭブイヨン０．９ｍｌを加えて混合し１０倍に希釈した。この溶液を段階的に１０7倍まで希釈し、各希釈倍率の糞溶液１０μｌをそれぞれ式（I）で示されるカテキン誘導体（Ｒ１は水素（Ｈ）、Ｒ２は水酸基（ＯＨ）を示す）０．２ｍＭを含むＧＡＭ寒天培地（（組成（１Ｌ中）：ペプトン１０ｇ、ダイズペプトン３ｇ、プロテオーゼペプトン１０ｇ、消化血清末１３．５ｇ、酵母エキス５ｇ、肉エキス２．２ｇ、肝臓エキス１．２ｇ、ブドウ糖３ｇ、リン酸二水素カリウム２．５ｇ、塩化ナトリウム３ｇ、溶性デンプン５ｇ、Ｌ−システイン塩酸塩０．３ｇ、チオグリコール酸ナトリウム０．３ｇ、カンテン１５ｇ、ｐＨ７．１、日水製薬（株）社製）２０ｍｌ中に混濁培養した。３７℃で２日間嫌気培養（アネロパックケンキ（三菱ガス化学（株）社製）使用）を行った後、寒天培地中に出現したシングルコロニーをそれぞれ個別に５ｍｌのＧＡＭブイヨンに植菌した。この培地にフィルター滅菌（ＤＩＳＭＩＣ−２５ｃｓ ０．２μｍ アドバンテック東洋（株）社製）した（I）で示されるカテキン誘導体（Ｒ１は水酸基（ＯＨ）、Ｒ２は水素（Ｈ）を示す）水溶液（５０ｍＭ）を５０μｌ添加し、３７℃で２日間嫌気培養を行った。培養液を遠心分離（１５０００×ｇ、１５分）し、上清を高速液体クロマトグラフィー（ＨＰＬＣ）で分析し、式（II）の５−フェニル−４−ハイドロキシ吉草酸（Ｒ１は水素（Ｈ）、Ｒ２は水酸基（ＯＨ）を示す）を生成する菌株のスクリーニングを行った。高速液体クロマトグラフィー（ＨＰＬＣ）による分析は以下の条件で行った。
Screening of microorganisms that convert catechin derivatives of formula (I) into 5-phenyl-4-hydroxyvaleric acid of formula (II) and / or 5-phenyl γ-valerolactone of formula (III) Isolation of bacteria Wistar rats (Nippon Charles River Co., Ltd.) were used as a source. Approximately 2 g of fresh stool was collected from the rat, GAM bouillon (composition (in 1 L): 10 g peptone, 3 g soybean peptone, 10 g proteose peptone, digested serum powder 13.5 g, yeast extract 5 g, meat extract 2.2 g, liver Extract 1.2 g, glucose 3 g, potassium dihydrogen phosphate 2.5 g, sodium chloride 3 g, soluble starch 5 g, L-cysteine hydrochloride 0.3 g, sodium thioglycolate 0.3 g, pH 7.1, Nissui Pharmaceutical ( A feces solution was prepared by suspending in 3 ml). 0.1 ml of fecal solution was placed in a microtube and 0.9 ml of GAM broth was added and mixed to dilute 10 times. This solution was diluted stepwise to 10 7 times, and 10 μl of each fecal solution at each dilution ratio was added to each catechin derivative represented by the formula (I) (R1 represents hydrogen (H), R2 represents hydroxyl group (OH)). GAM agar medium containing 2 mM ((composition (in 1 L): peptone 10 g, soybean peptone 3 g, proteose peptone 10 g, digested serum powder 13.5 g, yeast extract 5 g, meat extract 2.2 g, liver extract 1.2 g, glucose) 3 g, potassium dihydrogen phosphate 2.5 g, sodium chloride 3 g, soluble starch 5 g, L-cysteine hydrochloride 0.3 g, sodium thioglycolate 0.3 g, agar 15 g, pH 7.1, Nissui Pharmaceutical Co., Ltd. The product was turbidly cultured in 20 ml and after anaerobic culture at 37 ° C. for 2 days (using Aneropack Kenki (Mitsubishi Gas Chemical Co., Ltd.)) appeared in the agar medium. Each single colony was inoculated individually in 5 ml of GAM broth.The catechin derivative represented by (I) (R1 is a hydroxyl group (R1) obtained by filter sterilization (DISMIC-25cs 0.2 μm, manufactured by Advantech Toyo Co., Ltd.) in this medium. OH) and R2 represent hydrogen (H) (50 μl) and anaerobic culture was performed for 2 days at 37 ° C. The culture was centrifuged (15000 × g, 15 minutes), and the supernatant was removed. Analysis by high performance liquid chromatography (HPLC) and screening for strains producing 5-phenyl-4-hydroxyvaleric acid of formula (II) (R1 is hydrogen (H), R2 is hydroxyl group (OH)) Analysis by high performance liquid chromatography (HPLC) was performed under the following conditions.
使用カラム：ＣＡＰＣＥＬＬＰＡＫ ＵＧ１２０（４．６ｍｍ×２５０ｍｍ、５μｍ、資生堂（株）社製）、カラム温度：４０℃、流速：１ｍｌ／分、移動相：水／メタノール／アセトニトリル／リン酸＝８５ /１０ /５ / ０．１（容量比（v/v/v/v））、検出器：ＵＶ２７０ｎｍ。
分析の結果、式（I）で示されるカテキン誘導体を式（II）の５―フェニル―４−ハイドロキシ吉草酸へ変換する能力を持つ微生物ユウバクテリウム・プラウティＭＴ４２（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＭＴ４２ ＦＥＲＭ Ｐ−２１７６５）株を単離した。
Column used: CAPCELLPAK UG120 (4.6 mm × 250 mm, 5 μm, manufactured by Shiseido Co., Ltd.), column temperature: 40 ° C., flow rate: 1 ml / min, mobile phase: water / methanol / acetonitrile / phosphoric acid = 85/10 / 5 / 0.1 (volume ratio (v / v / v / v)), detector: UV 270 nm.
As a result of the analysis, the microorganism Eutrobacterium plutii MT42 FERM P-21765 having the ability to convert the catechin derivative represented by the formula (I) into 5-phenyl-4-hydroxyvalerate of the formula (II) The strain was isolated.
ユウバクテリウム・プラウティＭＴ４２（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＭＴ４２ ＦＥＲＭ Ｐ−２１７６５）株による式（I）で示されるカテキン誘導体（Ｒ１は水酸基（ＯＨ）、Ｒ２は水素（Ｈ）を示す）からの５−（３’、５’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、５’−ジハイドロキシフェニル）γ−バレロラクトン含有物の生産
（緩衝液中での生産）
ＧＡＭ寒天培地上に生育したＭＴ４２株を１０ｍｌのＧＡＭブイヨンに植菌し、３７℃で２４時間嫌気培養した。この前培養液を新たに調整した同培地３００ｍｌに植菌し、３７℃で２４時間嫌気培養を行った。培養液を高速遠心分離（１５０００×ｇ、２０分）により集菌し、得られた菌体を滅菌水１００ｍｌで洗浄後、２０ｍｌのリン酸緩衝液（０．１Ｍ、ｐＨ７．１）に懸濁した。この懸濁液に基質となる式（I）のカテキン誘導体（Ｒ１は水酸基（ＯＨ）、Ｒ２は水素（Ｈ）を示す）を１０ｍｇになるように添加し、３７℃の嫌気条件下で４８時間インキュベーションした。反応液を高速遠心分離（１５０００×ｇ、２０分）し、菌体を除去した。上清に塩酸を適量添加しｐＨを３〜４に調整した後、２０ｍｌの酢酸エチルで３回抽出を行った。酢酸エチル相を合わせて減圧下で濃縮乾固し、乾固物を少量の純水に溶解後、凍結乾燥を行った。その結果、５−（３’、５’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、５’−ジハイドロキシフェニル）γ−バレロラクトンを含む含有物８．３ｍｇが得られた。
5- (3 ′) from a catechin derivative represented by the formula (I) (R1 is a hydroxyl group (OH) and R2 is hydrogen (H)) by a strain of Eubacterium plutii MT42 FERM P-21765. Production of 5′-dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 5′-dihydroxyphenyl) γ-valerolactone-containing product (production in buffer)
The MT42 strain grown on the GAM agar medium was inoculated into 10 ml of GAM broth and anaerobically cultured at 37 ° C. for 24 hours. This preculture was inoculated into 300 ml of the newly prepared medium and anaerobically cultured at 37 ° C. for 24 hours. The culture solution is collected by high-speed centrifugation (15000 × g, 20 minutes), and the resulting cells are washed with 100 ml of sterilized water and then suspended in 20 ml of a phosphate buffer (0.1 M, pH 7.1). did. A catechin derivative of the formula (I) as a substrate (R1 represents a hydroxyl group (OH) and R2 represents hydrogen (H)) was added to this suspension so as to be 10 mg, and the mixture was subjected to anaerobic conditions at 37 ° C. for 48 hours. Incubated. The reaction solution was centrifuged at high speed (15000 × g, 20 minutes) to remove the cells. An appropriate amount of hydrochloric acid was added to the supernatant to adjust the pH to 3-4, and then extracted three times with 20 ml of ethyl acetate. The ethyl acetate phases were combined and concentrated to dryness under reduced pressure. The dried product was dissolved in a small amount of pure water and then freeze-dried. As a result, 8.3 mg of a content containing 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 5′-dihydroxyphenyl) γ-valerolactone was obtained. It was.
凍結乾燥後の乾燥粉末の分析はＬＣ／ＭＳ分析によって行った。ＬＣ／ＭＳの分析条件は以下の通りである。
使用カラム：ＣＡＰＣＥＬＬＰＡＫ Ｃ１８ ＭＧ（２．０×１００．０ｍｍ，５μｍ 資生堂（株）社製）、カラム温度：４０℃、流速：０．２ｍｌ／分、移動相Ａ（水／アセトニトリル／酢酸＝１００／２．５／０．１ 容量比(v/v/v）)，Ｂ（水／アセトニトリル／メタノール／酢酸＝５０／２．５／５０／０．１ 容量比（v/v/v/v））、グラジエント：０−２分 アイソクラティック Ａ１００％、２−２５分 リニアグラジエント Ａ １００−０％、Ｂ０−１００％、２５．１−３３分 アイソクラティック Ａ１００％、検出器：ＵＶ２７０ｎｍ、インターフェース：ＥＳＩ、ポラリティ：ネガティブ。
Analysis of the dry powder after lyophilization was performed by LC / MS analysis. The analysis conditions of LC / MS are as follows.
Column used: CAPCELLPAK C18 MG (2.0 × 100.0 mm, 5 μm, manufactured by Shiseido Co., Ltd.), column temperature: 40 ° C., flow rate: 0.2 ml / min, mobile phase A (water / acetonitrile / acetic acid = 100 / 2.5 / 0.1 volume ratio (v / v / v)), B (water / acetonitrile / methanol / acetic acid = 50 / 2.5 / 50 / 0.1 volume ratio (v / v / v / v) ), Gradient: 0-2 minutes Isocratic A 100%, 2-25 minutes Linear gradient A 100-0%, B0-100%, 25.1-33 minutes Isocratic A 100%, detector: UV 270 nm, interface: ESI, polarity: negative.
ユウバクテリウム・プラウティＭＴ４２（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＭＴ４２ ＦＥＲＭ Ｐ−２１７６５）株による式（I）で示されるカテキン誘導体（Ｒ１は水素（Ｈ）、Ｒ２は水酸基（ＯＨ）を示す）からの５−（３’、４’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、４’−ジハイドロキシフェニル）γ―バレロラクトン含有物の生産
（培養液での生産）
ＧＡＭ寒天培地上に生育したＭＴ４２株をＧＡＭブイヨン１０ｍｌに植菌し３７℃で２４時間嫌気培養した。この前培養液を新たに調整した同培地１００ｍｌに植菌し、式（I）で示されるカテキン誘導体（Ｒ１は水素（Ｈ）、Ｒ２は水酸基（ＯＨ）を表す）を３０ｍｇ添加した。この培地を３７℃で４８時間嫌気培養して変換反応を行った。反応液を高速遠心分離（１５０００×ｇ、２０分）し、菌体を除去した後、上清に酢酸を添加しｐＨを３．５に調整した。１００ｍｌの酢酸エチルで３回抽出を行った後、酢酸エチル溶液を合わせて減圧下で濃縮乾固した。乾固物に少量の純水を加えて溶解し、凍結乾燥を行った結果、５−（３’、４’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、４’−ジハイドロキシフェニル）γ−バレロラクトン含有物５６ｍｇを得た。
5- (3 ′) from a catechin derivative represented by the formula (I) by Eubacterium plutii MT42 (Eubacterium plautii MT42 FERM P-21765) strain (R1 represents hydrogen (H), R2 represents hydroxyl group (OH)) Production of 4′-dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 4′-dihydroxyphenyl) γ-valerolactone-containing product (production in culture)
The MT42 strain grown on the GAM agar medium was inoculated into 10 ml of GAM broth and anaerobically cultured at 37 ° C. for 24 hours. This preculture was inoculated into 100 ml of the freshly prepared medium, and 30 mg of a catechin derivative represented by the formula (I) (R1 represents hydrogen (H) and R2 represents a hydroxyl group (OH)) was added. This medium was anaerobically cultured at 37 ° C. for 48 hours to carry out a conversion reaction. The reaction solution was centrifuged at a high speed (15000 × g, 20 minutes) to remove the cells, and then acetic acid was added to the supernatant to adjust the pH to 3.5. After extraction three times with 100 ml of ethyl acetate, the ethyl acetate solutions were combined and concentrated to dryness under reduced pressure. As a result of adding a small amount of pure water to the dried solid and dissolving and freeze-drying, 5- (3 ′, 4′-dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 4′-) were obtained. 56 mg of dihydroxyphenyl) γ-valerolactone-containing product was obtained.
ユウバクテリウム・プラウティＡＴＣＣ２９８６３（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＡＴＣＣ２９８６３）株による式（I）で示されるカテキン誘導体（Ｒ１，Ｒ２は水素を表す）からの５−（３’−ハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’−ハイドロキシフェニル）γ−バレロラクトン含有物の生産
（培養液での生産）
ＧＡＭ寒天培地上に生育したＡＴＣＣ２９８６３株をＧＡＭブイヨン１０ｍｌに植菌し、３７℃で２４時間嫌気培養した。この前培養液を新たに調整した同培地１００ｍｌに植菌し、式（I）で示されるカテキン誘導体（Ｒ１、Ｒ２は水素（Ｈ）を表す）を３０ｍｇ添加した。３７℃で４８時間嫌気培養して変換反応を行った後、高速遠心分離１５０００×ｇ、２０分）で菌体を除去した。上清に酢酸を加えｐＨ３．５に調整した。１００ｍｌの酢酸エチルで３回抽出を行った後、酢酸エチルを合わせて無水硫酸マグネシウムを適量添加して脱水を行った。ろ過により硫酸マグネシウムを除去し後、ろ液を減圧下で濃縮乾固した。乾固物に少量の純水を加えて溶解し、凍結乾燥を行った結果、５−（３’−ハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’−ハイドロキシフェニル）γ−バレロラクトン含有物４２ｍｇを得た。
5- (3′-Hydroxyphenyl) -4-hydroxyvaleric acid and 5 from the catechin derivative represented by the formula (I) (R1, R2 represents hydrogen) by the strain of Eubacterium pluti ATCC 29863 (Eubacterium plautii ATCC 29863) -Production of (3'-hydroxyphenyl) γ-valerolactone-containing product (production in culture)
The ATCC 29863 strain grown on the GAM agar medium was inoculated into 10 ml of GAM broth and anaerobically cultured at 37 ° C. for 24 hours. This preculture was inoculated into 100 ml of the freshly prepared medium, and 30 mg of a catechin derivative represented by the formula (I) (R1, R2 represents hydrogen (H)) was added. After anaerobic culture at 37 ° C. for 48 hours to carry out a conversion reaction, the cells were removed by high-speed centrifugation (15000 × g, 20 minutes). Acetic acid was added to the supernatant to adjust the pH to 3.5. After extraction with 100 ml of ethyl acetate three times, ethyl acetate was combined and an appropriate amount of anhydrous magnesium sulfate was added for dehydration. After removing magnesium sulfate by filtration, the filtrate was concentrated to dryness under reduced pressure. As a result of adding a small amount of pure water to the dried product and dissolving and freeze-drying, 5- (3′-hydroxyphenyl) -4-hydroxyvaleric acid and 5- (3′-hydroxyphenyl) γ-valerolactone were obtained. The content was 42 mg.
クロストリジウム・オルビシンデンスＡＴＣＣ４９５３１（Ｃｌｏｓｔｒｉｄｉｕｍ ｏｒｂｉｓｃｉｎｄｅｎｓ ＡＴＣＣ４９５３１）株による式（I）で示されるカテキン誘導体（Ｒ１，Ｒ２は水酸基（ＯＨ）を表す）からの５−（３’、４’、５’−トリハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、４’、５’−トリハイドロキシフェニル）γ−バレロラクトン含有物の生産
（培養液での生産）
ＧＡＭ寒天培地上に生育したＡＴＣＣ４９５３１株をＧＡＭブイヨン１０ｍｌに植菌し、３７℃で２４時間嫌気培養した。この前培養液を新たに調製した同培地１００ｍｌに植菌し、式（I）で示されるカテキン誘導体（Ｒ１、Ｒ２は水酸基（ＯＨ）を表す）を３０ｍｇ添加した。３７℃で４８時間嫌気培養して変換反応を行った後、高速遠心分離機（１５０００×ｇ、２０分）に供し菌体を除去した。上清にリン酸を加えｐＨ１．５に調整した後、８０ｍｌの酢酸エチル：ブタノール（１：１、溶量比（v/v））を加えてよく混合した。遠心分離（５０００×ｇ、５分）により２相に分けた後、有機溶媒相を回収した。この抽出を３回繰り返し行った。有機溶媒相を合わせて減圧下で濃縮乾固した。乾固物に５ｍｌの純水を加えて溶解し、再度減圧下で濃縮乾固した。この操作を３回繰り返し行い、有機溶媒相に含まれていた酸を完全に除去した。乾固物に少量の純水５ｍｌを加えて溶解し、凍結乾燥した。その結果、５−（３’、４’、５’−トリハイドロキシフェニル）−４−ハイドロキシ吉草酸、５−（３’、４’、５’−トリハイドロキシフェニル）γ−バレロラクトン含有物４１ｍｇを得た。
Catechin derivatives represented by the formula (I) by Clostridium orubicindens ATCC49531 strain (R1, R2 represents hydroxyl group (OH)) from 5- (3 ′, 4 ′, 5′-trihydroxy) Production of phenyl) -4-hydroxyvaleric acid and 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) γ-valerolactone-containing product (production in culture)
The ATCC49531 strain grown on the GAM agar medium was inoculated into 10 ml of GAM broth, and anaerobically cultured at 37 ° C. for 24 hours. This preculture was inoculated into 100 ml of the newly prepared medium, and 30 mg of a catechin derivative represented by the formula (I) (R1, R2 represents a hydroxyl group (OH)) was added. After anaerobic culture at 37 ° C. for 48 hours to carry out a conversion reaction, the cells were removed by using a high-speed centrifuge (15000 × g, 20 minutes). After adjusting the pH to 1.5 by adding phosphoric acid to the supernatant, 80 ml of ethyl acetate: butanol (1: 1, solubility ratio (v / v)) was added and mixed well. After separation into two phases by centrifugation (5000 × g, 5 minutes), the organic solvent phase was recovered. This extraction was repeated three times. The organic solvent phases were combined and concentrated to dryness under reduced pressure. 5 ml of pure water was added to the dried product to dissolve it, and the solution was again concentrated to dryness under reduced pressure. This operation was repeated three times to completely remove the acid contained in the organic solvent phase. A small amount of 5 ml of pure water was added to the dried product to dissolve it and freeze-dried. As a result, 41 mg of 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -4-hydroxyvaleric acid, 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) γ-valerolactone-containing product was obtained. Obtained.
式（IV）のカテキン類を式（I）に示されるカテキン誘導体に変換する微生物のスクリーニング
細菌の分離源としてＷｉｓｔａｒ系ラット（♂、日本チャールスリバー（株））を使用した。ラットから新鮮な糞便を採取し、採取直後に白金耳で糞を適量取り、ＧＡＭ寒天培地上に画線した。３７℃で４８時間嫌気培養して菌を充分生育させた後、培地上に生育したコロニー群を数十箇所から掻き取り、式（IV）で示されるエピガロカテキン（ＲはＯＨ（水酸基）を示す）を０．５ｍＭ含むＧＡＭブイヨン２ｍｌにそれぞれ植菌した。この培養液を３７℃で４８時間嫌気培養した後、高速遠心分離（１５０００×ｇ、１５分）し、得られた上清を高速液体クロマトグラフィー（ＨＰＬＣ）で分析し、式（I）のカテキン誘導体（Ｒ１、Ｒ２は水酸基（ＯＨ）を示す）を生成している培養液を選択した。ＨＰＬＣ分析は実施例１の段落００３８に示した条件と同じ条件で行った。目的のカテキン誘導体が生成されていた培養液から菌を取り、再度新たに調製したＧＡＭ寒天培地上に画線した。単菌になるまで上記と同様の操作を繰り返し行った。その結果、（I）のカテキン誘導体を生成する能力をもつアドラークルーツィア・エクオーリファシエンスＭＴ４ｓ−５（Ａｄｌｅｒｃｒｅｕｔｚｉａ ｅｑｕｏｌｉｆａｃｉｅｎｓ ＭＴ４ｓ−５，ＦＥＲＭ Ｐ−２１７３８）株を単離した。
Screening for microorganisms that convert catechins of formula (IV) into catechin derivatives represented by formula (I) Wistar rats (Nada, Charles River Japan, Inc.) were used as the bacterial separation source. Fresh feces were collected from rats, and immediately after collection, an appropriate amount of feces was collected with a platinum ear and streaked on a GAM agar medium. After anaerobic culture at 37 ° C. for 48 hours to sufficiently grow the fungus, colonies grown on the medium were scraped from several tens of places, and epigallocatechin represented by the formula (IV) (R is OH (hydroxyl group) 2 ml of GAM broth containing 0.5 mM. This culture solution was subjected to anaerobic culture at 37 ° C. for 48 hours, followed by high-speed centrifugation (15000 × g, 15 minutes), and the resulting supernatant was analyzed by high-performance liquid chromatography (HPLC). A culture solution in which a derivative (R1, R2 represents a hydroxyl group (OH)) was selected. The HPLC analysis was performed under the same conditions as shown in paragraph 0038 of Example 1. Bacteria were taken from the culture solution in which the target catechin derivative had been produced, and streaked on a freshly prepared GAM agar medium. The same operation as described above was repeated until single bacteria were obtained. As a result, an Adlercruzia equolifaciens MT4s-5 (Adlercreutzia equolifaciens MT4s-5, FERM P-21738) strain having the ability to produce the catechin derivative of (I) was isolated.
エガーテラ・レンタＪＣＭ９９７９（Ｅｇｇｅｒｔｈｅｌｌａ ｌｅｎｔａ ＪＣＭ９９７９）株とユウバクテリウム・プラウティＡＴＣＣ２９８６３（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＡＴＣＣ２９８６３）株および大腸菌Ｋ１２（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ Ｋ１２）株共存下での式（IV）で示される（Ｒは水素（Ｈ）を示す）エピカテキンからの５−（３’−ハイドロキシフェニル）−４−ハイドロキシ吉草酸ナトリウム塩の生産
（培養液）
ＪＣＭ９９７９株を１０ｍｌのＧＡＭブイヨンに植菌し、３７℃で４８時間嫌気培養し、前培養液とした。大腸菌Ｋ１２株およびＡＴＣＣ２９８６３株は５ｍｌのＧＡＭブイヨンで２４時間嫌気培養し、前培養液とした。式（IV）で示されるエピカテキン（Ｒは水素（Ｈ）を示す）２００ｍｇを含む１００ｍｌのＧＡＭブイヨンにＪＣＭ９９７９株、大腸菌Ｋ１２株の前培養液を加え、３７℃で４８時間嫌気培養した。１ｍｌをサンプリングし、高速遠心分離（１５０００×ｇ、１０分）して、菌体を除去し、上清をＬＣ／ＭＳ分析に供した。式（I）で示されるカテキン誘導体（Ｒ１、Ｒ２は水素（Ｈ）を示す）が生成されているのを確認した後、さらにＡＴＣＣ２９８６３株の前培養液を加え、３７℃で４８時間嫌気培養を行い、５−（３’−ハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’−ハイドロキシフェニル）γ−バレロラクトンへの変換を行った。ＬＣ／ＭＳ分析は実施例２の段落００４０記載の条件で行った。
Egerterra lenta JCM9979 (Eggerella lenta JCM9979) strain, Eubacterium pluti ATCC29863 (Eubacterium plautii ATCC29863) strain and E. coli K12 (Escherichia coli K12) strain (H) represented by the formula (IV) Production of 5- (3′-hydroxyphenyl) -4-hydroxyvaleric acid sodium salt from epicatechin (culture medium)
The JCM9979 strain was inoculated into 10 ml of GAM broth and anaerobically cultured at 37 ° C. for 48 hours to obtain a preculture solution. The Escherichia coli K12 strain and ATCC 29863 strain were anaerobically cultured in 5 ml of GAM broth for 24 hours to obtain a preculture solution. Precultures of JCM9979 strain and Escherichia coli K12 strain were added to 100 ml of GAM broth containing 200 mg of epicatechin represented by formula (IV) (R represents hydrogen (H)), followed by anaerobic culture at 37 ° C. for 48 hours. 1 ml was sampled and centrifuged at high speed (15000 × g, 10 minutes) to remove the cells and the supernatant was subjected to LC / MS analysis. After confirming that the catechin derivative represented by the formula (I) (R1 and R2 indicate hydrogen (H)) was produced, the ATCC29863 strain was further added, followed by anaerobic culture at 37 ° C. for 48 hours. And converted to 5- (3′-hydroxyphenyl) -4-hydroxyvaleric acid and 5- (3′-hydroxyphenyl) γ-valerolactone. LC / MS analysis was performed under the conditions described in paragraph 0040 of Example 2.
培養液を高速遠心分離（１５０００×ｇ、１０分、１０℃）し、菌体を除去した。上清にリン酸を加えｐＨ３．５に調整し、１００ｍｌの酢酸エチルを加え、目的化合物を抽出した。有機溶媒相を回収し、水相に再度酢酸エチル１００ｍｌを加えて抽出を行った。この抽出を３回繰り返し行った。回収した酢酸エチル相を合わせ、減圧下で濃縮乾固した。乾固物に３０ｍｌの純水を加えて溶解し、１Ｍの炭酸ナトリウム水溶液を適量加えてｐＨ９．５に調整した。室温で２０時間放置し、５−（３’−ハイドロキシフェニル）γ−バレロラクトンを５−（３’−ハイドロキシフェニル）−４−ハイドロキシ吉草酸に変換した。２Ｍ塩酸を適量加えてｐＨ７．０付近に調整した後、減圧下で濃縮を行った。５ｍｌ程度まで濃縮した後、塩酸で再度ｐＨを２〜５に調整した。この濃縮液を高速遠心分離（１５０００×ｇ、１０分、４℃）後、上清を分取ＨＰＬＣに供した。分取用ＨＰＬＣの条件は以下の通りである。 The culture solution was centrifuged at high speed (15000 × g, 10 minutes, 10 ° C.) to remove the cells. Phosphoric acid was added to the supernatant to adjust the pH to 3.5, and 100 ml of ethyl acetate was added to extract the target compound. The organic solvent phase was recovered, and extraction was performed again by adding 100 ml of ethyl acetate to the aqueous phase. This extraction was repeated three times. The collected ethyl acetate phases were combined and concentrated to dryness under reduced pressure. 30 ml of pure water was added to the dried product and dissolved, and an appropriate amount of 1M aqueous sodium carbonate solution was added to adjust the pH to 9.5. After standing at room temperature for 20 hours, 5- (3'-hydroxyphenyl) γ-valerolactone was converted to 5- (3'-hydroxyphenyl) -4-hydroxyvaleric acid. An appropriate amount of 2M hydrochloric acid was added to adjust the pH to around 7.0, followed by concentration under reduced pressure. After concentrating to about 5 ml, the pH was adjusted again to 2-5 with hydrochloric acid. The concentrated solution was subjected to high-speed centrifugation (15000 × g, 10 minutes, 4 ° C.), and the supernatant was subjected to preparative HPLC. Preparative HPLC conditions are as follows.
カラム：Ｃａｐｃｅｌｌｐａｋ ＭＧ（２０×１５０ｍｍ、５μｍ、（資生堂（株）社製）、流速９．５ｍｌ/分、温度４０℃、溶媒Ａ：アセトニトリル：メタノール：水（５：５：９０ 容量比（v/v/v））、溶媒Ｂ：アセトニトリル：メタノール：水（５：６０：３５ 容量比（v/v/v））、グラジエント；０分：Ａ７０％ Ｂ３０％、５分：Ａ７０％ Ｂ３０％、１５分：Ａ２０％ Ｂ８０％、１８分：Ａ２０％ Ｂ８０％、１９分：Ａ７０％ Ｂ３０％、２４分：Ａ７０％ Ｂ３０％、検出器：ＵＶ２３０ｎｍとした。 Column: Capcellpak MG (20 × 150 mm, 5 μm, manufactured by Shiseido Co., Ltd.), flow rate 9.5 ml / min, temperature 40 ° C., solvent A: acetonitrile: methanol: water (5: 5: 90 volume ratio (v / v / v)), solvent B: acetonitrile: methanol: water (5:60:35 volume ratio (v / v / v)), gradient; 0 min: A70% B30%, 5 min: A70% B30%, 15 Minute: A20% B80%, 18 minutes: A20% B80%, 19 minutes: A70% B30%, 24 minutes: A70% B30%, detector: UV 230 nm.
得られた５−（３’−ハイドロキシフェニル）−４−ハイドロキシ吉草酸画分（約５０ｍｌ）に１／２量の純水（２５ｍｌ）を添加後、減圧下で濃縮し、溶液中の溶媒を除去して水溶液にした。この水溶液を純水で平衡化した陽イオン交換樹脂（ダイアイオンＳＫ１Ｂ ナトリウム型、１０×６５ｍｍ）に通液し、さらに純水で溶出した。得られた溶液を減圧濃縮し凍結乾燥した結果、５−（３’−ハイドロキシフェニル）−４−ハイドロキシ吉草酸のナトリウム塩９５ｍｇが得られた。 After adding 1/2 volume of pure water (25 ml) to the obtained 5- (3′-hydroxyphenyl) -4-hydroxyvaleric acid fraction (about 50 ml), the mixture was concentrated under reduced pressure to remove the solvent in the solution. Removed to make an aqueous solution. This aqueous solution was passed through a cation exchange resin (diaion SK1B sodium type, 10 × 65 mm) equilibrated with pure water, and further eluted with pure water. The resulting solution was concentrated under reduced pressure and lyophilized to obtain 95 mg of sodium salt of 5- (3'-hydroxyphenyl) -4-hydroxyvaleric acid.
アドラークルーツィア・エクオーリファシエンスＭＴ４ｓ−５（Ａｄｌｅｒｃｒｅｕｔｚｉａ ｅｑｕｏｌｉｆａｃｉｅｎｓ ＭＴ４ｓ−５，ＦＥＲＭ Ｐ−２１７３８）株、ユウバクテリウム・プラウティ ＭＴ４２（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＭＴ４２ ＦＥＲＭ Ｐ−２１７６５）株および大腸菌Ｋ１２（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ Ｋ１２）株の共存下での式（IV）で示された（Ｒは水酸基（ＯＨ）を示す）エピガロカテキンからの５−（３’、５’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、５’−ジハイドロキシフェニル）γ―バレロラクトン含有物の生産
（培地中）
ＭＴ４ｓ−５株を３０ｍｌのＧＡＭブイヨンに植菌し、３７℃で４８時間嫌気培養し、前培養液とした。大腸菌Ｋ１２株およびＭＴ４２株は１０ｍｌのＧＡＭブイヨンで２４時間嫌気培養し、前培養液とした。式（IV）で示されたエピガロカテキン（Ｒは水酸基（ＯＨ）を示す）２９０ｍｇを含む１００ｍｌのＧＡＭブイヨンに上記３菌株の前培養液を加え、３７℃で４８時間嫌気培養して変換反応を行い、５−（３’、５’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、５’−ジハイドロキシフェニル）γ−バレロラクトン含有物を得た。培養液を高速遠心分離（１５０００×ｇ、１０分、１０℃）し、菌体を除去した。上清に塩酸を加えｐＨ３．５に調整した後、１２０ｍｌの酢酸エチルで３回抽出した。酢酸エチル相（約３５０ｍｌ）に４０ｍＭ炭酸ナトリウム水溶液１００ｍｌを加えよく混合した。遠心分離（５０００×ｇ、５分）により２相に分離させ、水相を回収した。再度酢酸エチル相に４０ｍＭ炭酸ナトリウム水溶液１００ｍｌを加え、同様に２相に分離した。回収した炭酸ナトリウム水溶液のｐＨを７．０付近に調整した後、約５ｍｌになるまで減圧濃縮した。さらに塩酸を加えてｐＨ２．０〜５．０に調製し、高速遠心分離（１５０００×ｇ、２０分、４℃）後、上清を分取用ＨＰＬＣに供した。分取ＨＰＬＣの条件は実施例７の段落００４７に示した方法と同様である。ただし、グラジエントは０分：Ａ８０％ Ｂ２０％、５分：Ａ８０％ Ｂ２０％、１５分：Ａ２０％ Ｂ８０％、１８分：Ａ２０％ Ｂ８０％、１９分：Ａ８０％ Ｂ２０％、２４分：Ａ８０％ Ｂ２０％に設定した。得られた５−（３’、５’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸の画分は実施例７の段落００４８に示した方法で処理した。その結果、５−（３’、５’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸のナトリウム塩が１０３ｍｇ得られた。
Adlercruzia equolifaciens MT4s-5 (Adlercreutzia equifaciens MT4s-5, FERM P-21738) strain, Eubacterium pluti MT42 (Eubacterium plautii MT42 FERM P-21765) strain E. coli 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 5′-dihydroxyphenyl) from epigallocatechin represented by the formula (IV) (R represents a hydroxyl group (OH)) in the presence of Production of (3 ′, 5′-dihydroxyphenyl) γ-valerolactone-containing product (in medium)
The MT4s-5 strain was inoculated into 30 ml of GAM broth and anaerobically cultured at 37 ° C. for 48 hours to obtain a preculture solution. The Escherichia coli K12 strain and MT42 strain were anaerobically cultured in 10 ml of GAM broth for 24 hours to prepare a preculture solution. The preculture solution of the above three strains was added to 100 ml of GAM broth containing 290 mg of epigallocatechin represented by the formula (IV) (R represents hydroxyl group (OH)), and anaerobically cultured at 37 ° C. for 48 hours for conversion reaction. To give 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 5′-dihydroxyphenyl) γ-valerolactone-containing product. The culture solution was centrifuged at high speed (15000 × g, 10 minutes, 10 ° C.) to remove the cells. Hydrochloric acid was added to the supernatant to adjust the pH to 3.5, followed by extraction with 120 ml of ethyl acetate three times. To the ethyl acetate phase (about 350 ml), 100 ml of 40 mM sodium carbonate aqueous solution was added and mixed well. Centrifugation (5000 × g, 5 minutes) separated into two phases, and the aqueous phase was recovered. Again, 100 ml of 40 mM aqueous sodium carbonate solution was added to the ethyl acetate phase, and the two phases were similarly separated. After adjusting the pH of the collected aqueous sodium carbonate solution to around 7.0, the solution was concentrated under reduced pressure to about 5 ml. Further, hydrochloric acid was added to adjust the pH to 2.0 to 5.0, and after high speed centrifugation (15000 × g, 20 minutes, 4 ° C.), the supernatant was subjected to preparative HPLC. Preparative HPLC conditions are the same as those described in Example 7, paragraph 0047. However, the gradient is 0 min: A80% B20%, 5 min: A80% B20%, 15 min: A20% B80%, 18 min: A20% B80%, 19 min: A80% B20%, 24 min: A80% B20 %. The fraction of 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid obtained was treated by the method shown in paragraph 0048 of Example 7. As a result, 103 mg of 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid sodium salt was obtained.
エガーテラ・レンタＪＣＭ９９７９（Ｅｇｇｅｒｔｈｅｌｌａ ｌｅｎｔａ ＪＣＭ９９７９）株およびユウバクテリウム・プラウティＭＴ４２（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＭＴ４２ ＦＥＲＭ Ｐ−２１７６５）株の共存下での式（IV）のエピカテキンからの５−（３’、４’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、４’−ジハイドロキシフェニル）γ―バレロラクトン含有物の生産
（緩衝液中）
ＧＡＭブイヨン３０ｍｌにＪＣＭ９９７９株を植菌し、３７℃で４８時間嫌気培養を行った。この前培養液を新たに調製した同培地５００ｍｌに植菌し、３７℃で４８時間嫌気培養を行った。同様にＭＴ４２株を１０ｍｌのＧＡＭブイヨンで２４時間嫌気培養し、この前培養液を同培地３００ｍｌに植菌し、３７℃で２４時間嫌気培養した。この２菌株の培養液を合わせて高速遠心分離（１５０００×ｇ、１５分、４℃）し、得られた菌体を滅菌水２００ｍｌで一度洗浄した。得られた菌体を予め高圧蒸気滅菌（１１５℃、１５分）した５０ｍｌのリン酸緩衝液（０．１Ｍ、ｐＨ７．１）に懸濁し、式（IV）で示される（Ｒは水素（Ｈ）を示す）エピカテキン水溶液（３０ｍｇ/３ｍｌ純水）をフィルター滅菌（ＤＩＳＭＩＣ−２５ｃｓ ０．２μｍ アドバンテック東洋（株）社製）処理をして加え、３７℃で３日間嫌気培養を行った。ＬＣ／ＭＳ分析の結果、５−（３’、４’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸（面積値５５％、ＵＶ２７０ｎｍ）、５−（３’、４’−ジハイドロキシフェニル）γ―バレロラクトン（面積値１４％、ＵＶ２７０ｎｍ）の生成が確認された。ＬＣ／ＭＳ分析は実施例２の段落００４０に示した条件と同様の方法で行った。この溶液を高速遠心分離（１５０００×ｇ、１０分）し、上清に酢酸を添加してｐＨ３．５に調整した。５０ｍｌの酢酸エチルで３回抽出した後、酢酸エチル相を減圧下で濃縮乾固した。乾固物に少量の純水を加えて凍結乾燥に供した結果、５−（３’、４’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、４’−ジハイドロキシフェニル）γ―バレロラクトン含有物２７ｍｇが得られた。
5- (3 ′, 4′−) from epicatechin of formula (IV) in the coexistence of strains Eggerella lenta JCM9979 and Eubacterium plutii MT42 FERM P-21765 Production of dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 4′-dihydroxyphenyl) γ-valerolactone-containing product (in buffer)
The JCM9979 strain was inoculated into 30 ml of GAM bouillon and anaerobic culture was performed at 37 ° C. for 48 hours. This preculture was inoculated into 500 ml of the same freshly prepared medium and anaerobically cultured at 37 ° C. for 48 hours. Similarly, the MT42 strain was anaerobically cultured in 10 ml of GAM broth for 24 hours. This preculture was inoculated into 300 ml of the same medium and anaerobically cultured at 37 ° C. for 24 hours. The culture solutions of these two strains were combined and subjected to high-speed centrifugation (15000 × g, 15 minutes, 4 ° C.), and the resulting cells were washed once with 200 ml of sterilized water. The obtained bacterial cells were suspended in 50 ml of a phosphate buffer (0.1 M, pH 7.1) that had been previously autoclaved (115 ° C., 15 minutes), and represented by the formula (IV) (R is hydrogen (H )) Epicatechin aqueous solution (30 mg / 3 ml pure water) was subjected to filter sterilization (DISMIC-25cs 0.2 μm, manufactured by Advantech Toyo Co., Ltd.) and anaerobic culture was performed at 37 ° C. for 3 days. As a result of LC / MS analysis, 5- (3 ′, 4′-dihydroxyphenyl) -4-hydroxyvaleric acid (area value 55%, UV270 nm), 5- (3 ′, 4′-dihydroxyphenyl) γ- Formation of valerolactone (area value 14%, UV 270 nm) was confirmed. LC / MS analysis was performed in the same manner as in the conditions shown in paragraph 0040 of Example 2. This solution was subjected to high-speed centrifugation (15000 × g, 10 minutes), and acetic acid was added to the supernatant to adjust the pH to 3.5. After extraction three times with 50 ml of ethyl acetate, the ethyl acetate phase was concentrated to dryness under reduced pressure. As a result of adding a small amount of pure water to the dried product and subjecting to freeze-drying, 5- (3 ′, 4′-dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 4′-dihydroxyphenyl) were obtained. ) 27 mg of γ-valerolactone-containing product was obtained.
エガーテラ・レンタＪＣＭ９９７９（Ｅｇｇｅｒｔｈｅｌｌａ ｌｅｎｔａ ＪＣＭ９９７９）株およびユウバクテリウム・プラウティＭＴ４２（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＭＴ４２ ＦＥＲＭ Ｐ−２１７６５）株の共存下での式（IV）で示される（Ｒは水素（Ｈ）を示す）エピカテキンからの５−（３’、４’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、４’−ジハイドロキシフェニル）γ−バレロラクトン含有物の生産
（培養液中）
ＪＣＭ９９７９株を３０ｍｌのＧＡＭブイヨンに植菌し、３７℃で４８時間嫌気培養した。またＭＴ４２株は１０ｍｌのＧＡＭブイヨンで２４時間嫌気培養した。式（IV）で示される（Ｒは水素（Ｈ）を示す）エピカテキン２１５ｍｇを含む１００ｍｌのＧＡＭブイヨンに上記２株の前培養液を加え、３７℃で４８時間嫌気培養して変換反応を行い、５−（３’、４’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、４’−ジハイドロキシフェニル）γ―バレロラクトンを生成させた。培養液を高速遠心分離（１５０００×ｇ、１０分、１０℃）して、菌体を除去後、上清に塩酸を加えｐＨ３．５に調整した。この上清液を１２０ｍｌの酢酸エチルで３回抽出し、酢酸エチル相を減圧下で濃縮乾固した。沈殿を少量の純水に溶解し、凍結乾燥した結果、５−（３’、４’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸、５−（３’、４’−ジハイドロキシフェニル）γ―バレロラクトン含有物５０６ｍｇを得た。
Epi (R represents hydrogen (H)) represented by the formula (IV) in the coexistence of the strain Eggerella lenta JCM9979 (Eggerella lenta JCM9979) and Eubacterium plutii MT42 (Eubacterium plauti MT42 FERM P-21765) Production of 5- (3 ′, 4′-dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 4′-dihydroxyphenyl) γ-valerolactone-containing product from catechin (in culture medium)
JCM9979 strain was inoculated into 30 ml of GAM bouillon and anaerobically cultured at 37 ° C. for 48 hours. The MT42 strain was anaerobically cultured in 10 ml of GAM broth for 24 hours. The above two strains of pre-cultured solution are added to 100 ml of GAM broth containing 215 mg of epicatechin represented by the formula (IV) (R represents hydrogen (H)), and an anaerobic culture is carried out at 37 ° C. for 48 hours to carry out a conversion reaction. 5- (3 ′, 4′-dihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 4′-dihydroxyphenyl) γ-valerolactone were produced. The culture solution was centrifuged at high speed (15000 × g, 10 minutes, 10 ° C.) to remove the cells, and then hydrochloric acid was added to the supernatant to adjust the pH to 3.5. This supernatant was extracted three times with 120 ml of ethyl acetate, and the ethyl acetate phase was concentrated to dryness under reduced pressure. The precipitate was dissolved in a small amount of pure water and freeze-dried. As a result, 5- (3 ′, 4′-dihydroxyphenyl) -4-hydroxyvaleric acid, 5- (3 ′, 4′-dihydroxyphenyl) γ- 506 mg of valerolactone-containing product was obtained.
実施例１０で得られた含有物を純水３０ｍｌに溶解後、２Ｍ塩酸を添加してｐＨ１．５に調整した。この水溶液を室温で２４時間放置し、５−（３’、４’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸を５−（３’、４’−ジハイドロキシフェニル）γ―バレロラクトンへ変換した。水溶液のｐＨを１Ｍ炭酸ナトリウムで４．０に調整後、５倍量の純水で平衡化した合成吸着剤ダイアイオンＨＰ−２０（１１０ｍｌ、三菱化学（株）社製）カラムに展開し、カラム容量の５倍量の純水で洗浄した後、吸着画分を５倍量の５０％メタノール水溶液で溶出した。この５０％メタノール溶出画分を減圧下で濃縮乾固し、凍結乾燥した結果、５−（３’、４’−ジハイドロキシフェニル）γ−バレロラクトン２７３ｍｇを得た。 The contents obtained in Example 10 were dissolved in 30 ml of pure water, and then adjusted to pH 1.5 by adding 2M hydrochloric acid. This aqueous solution was allowed to stand at room temperature for 24 hours to convert 5- (3 ′, 4′-dihydroxyphenyl) -4-hydroxyvaleric acid into 5- (3 ′, 4′-dihydroxyphenyl) γ-valerolactone. . The pH of the aqueous solution was adjusted to 4.0 with 1M sodium carbonate, and then developed on a synthetic adsorbent Diaion HP-20 (110 ml, manufactured by Mitsubishi Chemical Corporation) column equilibrated with 5 times the amount of pure water. After washing with 5 volumes of pure water, the adsorbed fraction was eluted with 5 volumes of 50% aqueous methanol. This 50% methanol-eluted fraction was concentrated to dryness under reduced pressure and freeze-dried to obtain 273 mg of 5- (3 ′, 4′-dihydroxyphenyl) γ-valerolactone.
エガーテラ・レンタＪＣＭ９９７９（Ｅｇｇｅｒｔｈｅｌｌａ ｌｅｎｔａ ＪＣＭ９９７９）株とユウバクテリウム・プラウティＭＴ４２（Ｅｕｂａｃｔｅｒｉｕｍ ｐｌａｕｔｉｉ ＭＴ４２ ＦＥＲＭ Ｐ−２１７６５）株の共存下での式（IV）で示される（Ｒは水酸基（ＯＨ）を示す）エピガロカテキンからの５−（３’、４’、５’−トリハイドロキシフェニル）−４−ハイドロキシ吉草酸および５−（３’、４’、５’−トリハイドロキシフェニル）γ―バレロラクトン含有物の生産
（培地中）
ＪＣＭ９９７９株を３０ｍｌのＧＡＭブイヨンに植菌し、３７℃で４８時間嫌気培養し、前培養液とした。大腸菌Ｋ１２株およびＭＴ４２株は１０ｍｌのＧＡＭブイヨンで２４時間嫌気培養し、前培養液とした。式（IV）で示される（Ｒは水酸基（ＯＨ）を示す）エピガロカテキン２２１．４ｍｇを含む１０％メタノール水溶液１０ｍｌをフィルター滅菌（ＤＩＳＭＩＣ−２５ｃｓ ０．２μｍ アドバンテック東洋（株）社製）し、９．５ｍｌをＧＡＭブイヨン１００ｍｌに加えた。上記３菌株の前培養液を加え、３７℃で３日間嫌気培養を行った。高速遠心分離（１５０００×ｇ、１０分、１０℃）により菌体を除去した後、得られた上清にリン酸を添加してｐＨ１．５に調整した。この溶液に１００ｍｌの酢酸エチル：ブタノール（１：１、v/v）を加えてよく混合した。遠心分離機（５０００×ｇ、５分）で２相に分けた後、有機溶媒相を回収した。この抽出操作を３回繰り返し行った。有機溶媒相（３００ｍｌ）に１／２量（１５０ｍｌ）の０．１Ｍ炭酸ナトリウム水溶液（０．１％アスコルビン酸ナトリウムを含む）を加えよく混合した後、遠心分離機（５０００×ｇ、５分）で２相に分け、水相を回収した。再度有機溶媒相に０．１Ｍ炭酸ナトリウム水溶液を加え同様の操作を繰り返した。得られた水相（３００ｍｌ）のｐＨを７．０に調整後、約３０ｍｌになるまで減圧下で濃縮した。この濃縮液に５倍量のエタノールを添加し、高速遠心分離（１５０００×ｇ、１５分、４℃）で不溶物を除去した。得られた上清をさらに減圧下で約１ｍｌになるまで濃縮し、２ＭＨＣｌでｐＨ２.０−３．０に調整した。この溶液を分取ＨＰＬＣに供した。分取ＨＰＬＣの条件は実施例７の段落００４７に記した方法と同じ条件で行った。但し、グラジエントはＡ８０％ Ｂ２０％のアイソクラティックとした。また得られた５−（３’、４’、５’−トリハイドロキシフェニル）γ−バレロラクトンの画分は実施例７の段落００４８に示した同様の方法で処理をした。その結果、５−（３’、４’、５’−トリハイドロキシフェニル）−４−ハイドロキシ吉草酸ナトリウム塩６５ｍｇを得た。
Egerterra Renta JCM9979 (Eggerella lenta JCM9979) and Eubacterium plutii MT42 (Eubacterium plautii MT42 FERM P-21765) strains represented by the formula (IV) (R represents hydroxyl (OH)) epi 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -4-hydroxyvaleric acid and 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) γ-valerolactone-containing products from gallocatechin Production (in medium)
The JCM9979 strain was inoculated into 30 ml of GAM broth and anaerobically cultured at 37 ° C. for 48 hours to obtain a preculture solution. The Escherichia coli K12 strain and MT42 strain were anaerobically cultured in 10 ml of GAM broth for 24 hours to prepare a preculture solution. 10 ml of 10% aqueous methanol solution containing 221.4 mg of epigallocatechin represented by the formula (IV) (R represents hydroxyl group (OH)) was sterilized by filter (DISMIC-25cs 0.2 μm, manufactured by Advantech Toyo Co., Ltd.) 9.5 ml was added to 100 ml of GAM bouillon. A preculture solution of the above three strains was added, and anaerobic culture was performed at 37 ° C. for 3 days. After removing the cells by high-speed centrifugation (15000 × g, 10 minutes, 10 ° C.), the resulting supernatant was adjusted to pH 1.5 by adding phosphoric acid. To this solution, 100 ml of ethyl acetate: butanol (1: 1, v / v) was added and mixed well. After separating into two phases with a centrifuge (5000 × g, 5 minutes), the organic solvent phase was recovered. This extraction operation was repeated three times. After adding 1/2 volume (150 ml) of 0.1M aqueous sodium carbonate solution (containing 0.1% sodium ascorbate) to the organic solvent phase (300 ml) and mixing well, centrifuge (5000 × g, 5 minutes) The aqueous phase was recovered. Again, a 0.1 M sodium carbonate aqueous solution was added to the organic solvent phase and the same operation was repeated. The pH of the obtained aqueous phase (300 ml) was adjusted to 7.0, and then concentrated under reduced pressure until it became about 30 ml. Five times the amount of ethanol was added to this concentrated solution, and insoluble matters were removed by high-speed centrifugation (15000 × g, 15 minutes, 4 ° C.). The obtained supernatant was further concentrated to about 1 ml under reduced pressure, and adjusted to pH 2.0-3.0 with 2M HCl. This solution was subjected to preparative HPLC. Preparative HPLC conditions were the same as those described in Example 7, paragraph 0047. However, the gradient was A80% B20% isocratic. The obtained 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) γ-valerolactone fraction was treated in the same manner as shown in paragraph 0048 of Example 7. As a result, 65 mg of 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -4-hydroxyvaleric acid sodium salt was obtained.
以上の通り、本発明の生物学的変換方法によって、抗炎症作用やガン抑制作用が期待できるカテキン類の代謝産物を簡便に製造することが出来る。 As described above, by the biological conversion method of the present invention, a catechin metabolite that can be expected to have an anti-inflammatory action or a cancer suppressing action can be easily produced.
受託番号ＦＥＲＭ Ｐ−２１７６５
受託番号ＦＥＲＭ Ｐ−２１７３８
Accession Number FERM P-21765
Accession Number FERM P-21738
Claims (2)
式中、Ｒ１は水酸基（ＯＨ）、Ｒ２は水素（Ｈ）で表される５−（３’、５’−ジハイドロキシフェニル）−４−ハイドロキシ吉草酸および
式中、Ｒ１、Ｒ２は水酸基（ＯＨ）で表される５−（３’、４’、５’−トリハイドロキシフェニル）−４−ハイドロキシ吉草酸の少なくとも一つを必須成分として含有することを特徴とする組成物。 Formula (II)
In the formula, R1 is a hydroxyl group (OH), R2 is 5- (3 ′, 5′-dihydroxyphenyl) -4-hydroxyvaleric acid represented by hydrogen (H), and in the formula, R1 and R2 are hydroxyl groups (OH ) -Containing 5- (3 ′, 4 ′, 5′-trihydroxyphenyl) -4-hydroxyvaleric acid as an essential component.
An oral application object comprising the composition according to claim 1.
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| CN111989318A (en) * | 2018-02-08 | 2020-11-24 | 医疗法人圣光医疗财团 | Novel synthesis method of DHPV |
| CN111989318B (en) * | 2018-02-08 | 2023-04-04 | 医疗法人圣光医疗财团 | Novel synthesis method of DHPV |
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