JP7440407B2 - エコールの製造方法 - Google Patents
エコールの製造方法 Download PDFInfo
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- JP7440407B2 JP7440407B2 JP2020509208A JP2020509208A JP7440407B2 JP 7440407 B2 JP7440407 B2 JP 7440407B2 JP 2020509208 A JP2020509208 A JP 2020509208A JP 2020509208 A JP2020509208 A JP 2020509208A JP 7440407 B2 JP7440407 B2 JP 7440407B2
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- Prior art keywords
- echol
- strain
- yit
- medium
- culture
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Description
しかしながら、当該YIT 9029株をエコール産生菌と組み合わせて使用することについては、全く知られていない。
1)ダイゼイン類を含有する培地中で、エコール産生菌及びラクトバチルス・カゼイ YIT 9029株(FERM BP-1366)を培養するエコールの製造方法。
2)ダイゼイン類が、ダイゼイン、ダイゼイン配糖体及びジヒドロダイゼインから選択される1種以上である、1)の方法。
3)ダイゼイン類を含有する培地が、豆乳を主成分とする培地である、1)又は2)の方法。
4)エコール産生菌がコリオバクテリア科に属する細菌である、1)~3)のいずれかの方法。
5)エコール産生菌がスラッキア属細菌である、1)~4)のいずれかの方法。
6)エコール産生菌がSlackia sp.YIT 11861株(FERM BP-1366)である、1)~5)のいずれかの方法。
7)ダイゼイン類を含有する培地中で、エコール産生菌及びラクトバチルス・カゼイ YIT 9029株(FERM BP―1366)を培養するエコール含有発酵組成物の製造方法。
8)ダイゼイン類を含有する培地が、豆乳を主成分とする培地である、7)の方法。
9)ダイゼイン類を含有する培地中で、エコール産生菌及びラクトバチルス・カゼイYIT 9029株を培養してなるエコール含有発酵組成物。
10)ダイゼイン類を含有する培地が、豆乳を主成分とする培地である、9)のエコール含有発酵組成物。
このうち、コリオバクテリア科に属するスラッキア属細菌、アドレクロイチア属細菌、エガセラ属細菌が好ましく、より好ましくはスラッキア属細菌である。具体的には、例えばスラッキア・イソフラボニコンバーテンス、スラッキア・エクオリファシエンス、Slackia spp.TM-30株が挙げられ、より好ましくは、Slackia sp.YIT 11861(FERM BP-11231)株である。
ダイゼイン類を含有する培地は、ダイゼイン類又はダイゼイン類を分散又は溶解させた液を培地に添加することにより調製できる。この場合に、ダイゼイン類の水への溶解性を高めるべく、培地に可溶化剤を適宜含有させることができる。また、ダイゼイン類を含有する培地は、ダイゼイン類を含有する原料、例えば豆乳を主成分とする培地(以下、「豆乳培地」と称する)であってもよい。
また、上記培地のpH(培養開始時)は、通常5.0~8.0、好ましくは5.5~7.5、更に好ましくは6.0~7.0である。
また、上記培地には、例えば、水溶性の有機物を炭素源として加えることができる。水溶性の有機物としては、グルコース、ガラクトース、フルクトース、アラビノース、キシロース、マンノース、ラムノース、リボース、ソルボース、トレハロース、セロビオース、ラクトース、マルトース、シュクロース、ラフィノース、メリビオース、メレジトース、ラクチュロース、グリコーゲン、エリスリトール、ソルビトール、アドニトール、マンニトール、イノシトール、ラクチトール、ガラクトオリゴ糖、フラクトオリゴ糖、イヌリン、可溶性でんぷん等の糖類の他、ピルビン酸、リンゴ酸、コハク酸、乳酸、吉草酸、イソ吉草酸、酪酸、イソ酪酸、プロピオン酸及び酢酸など有機酸類等の化合物を挙げることができる。
上記の炭素源に加えて、培地には、窒素源を加えることができる。好ましい無機窒素源としては、アンモニウム塩や硝酸塩が挙げられ、例えば、硫安、塩化アンモニウム、リン酸アンモニウム、リン酸水素アンモニウム、クエン酸アンモニウム、硝酸カリウム、硝酸ソーダ等が挙げられる。有機窒素源としては、アミノ酸類、酵母エキス、ペプトン類、肉エキス、肝臓エキス、消化血清末等が挙げられ、好ましくは、アルギニン、システイン、シトルリン、オルニチン、リジン、酵母エキス、ペプトン類等が挙げられる。特に、エコール産生菌の増殖を向上させる点から、アルギニンを添加するのが好ましい。アルギニンとしては、L体、D体、それらの混合物でもよいが、好ましくはL-アルギニンである。アルギニンは塩の形態をとっても良く、塩としては、例えば塩酸塩、グルタミン酸塩、クエン酸塩等を挙げることができ、好ましくは塩酸塩である。培地中のアルギニン含有量は、0.4~2.5w/v%、好ましくは0.8~2.1w/v%である。尚、アルギニンの塩を使用した場合、アルギニンの含有量とは、遊離体のアルギニンに換算した量である。
また、ビタミン類としては、例えば、ビオチン、葉酸、ピリドキシン、チアミン、リボフラビン、ニコチン酸、パントテン酸、ビタミンB12、チオオクト酸、p-アミノ安息香酸が挙げられる。また、ポルフィリン化合物であるヘミンを添加することも可能である。
また、培養は通常、窒素ガス、炭酸ガス、水素ガスもしくはこれらの混合ガスを用いた嫌気条件下で静置培養することにより行われるが、攪拌培養を行ってもよい。
また、例えば、豆乳培地やダイゼイン類を含有する乳培地を用いて培養を行った場合、当該培養物は、そのまま、あるいは通常発酵豆乳食品に添加される他の食品素材と混合することによりエコールを含有する発酵組成物とすることができる。
(1)使用菌株
エコール産生菌:Slackia sp. YIT 11861(以下、NATTS株)
乳酸菌:Lactobacillus acidophilus YIT 0198
Lactobacillus casei YIT 0180T
Lactobacillus casei YIT 9029(以下、LcS)
Lactococcus lactis ss lactis YIT 2008T
Lactococcus lactis ss lactis YIT 2027
Streptococcus thermophilus YIT 2037T
Streptococcus thermophilus YIT 2001
Streptococcus thermophilus YIT 2021
NATTS株の培養には変法GAM平板培地(日水製薬)を、各種乳酸菌の培養にはMRS培地(ベクトン・ディッキンソン:BD)を使用した。
豆乳培地は、表1に示す組成とし、システイン塩酸塩及びアルギニン塩酸塩以外の成分を精製水に溶解し、100℃、30分加熱して脱気した。システイン塩酸塩を加えた後にN2ガスを吹き込み冷却し、試験管に12.6mLずつ分注した後、115℃、20分オートクレーブした。培養前にフィルター滅菌済みの15w/v%アルギニン塩酸塩を1.4mL加えたものを培地とした。
NATTS株は、嫌気グローブボックス内で変法GAM平板培地(日水製薬)にNATTS株の凍結保存液(分散媒:20%グリセロール溶液)を200μL播種し、37℃で24時間培養した。生育したコロニーを平板1枚あたり2mLの変法GAM液体培地に懸濁し、接種菌液とした。
各種乳酸菌は、凍結保存菌株(マイクロバンク:イワキ)を4mLのMRS培地に接種して、37℃で24時間好気培養したものを前培養液とした。
NATTS株と乳酸菌の共培養は、NATTS株は1.0%、乳酸菌株は0.1%となるように豆乳培地に接種し、37℃で72時間嫌気培養した(各組み合わせは、n=3で実施)。嫌気ガスは、窒素ガスを使用した。
1)培養液の前処理
培養液200μLに800μLの1M Tris-HCl(pH8.0)を加え、そこに1M Tris-HCl(pH8.0)に38.3mg/mLとなるように溶解したプロテアーゼP「アマノ」3SD(天野エンザイム)を150μL添加し、45℃で3分間インキュベートした。4℃のもと20,400×gで5分間遠心し、上清をピペッティングで除去した後、ペレットを200μLのTEバッファー(pH8.0)に再懸濁した。そこに400μLのRNAprotect bacterial reagent(QIAGEN)を添加し5分間室温で放置した後、20,400×gで5分間遠心し、上清を除去した。得られた沈殿はRNA抽出まで-80℃で保存した。
上記の沈殿から既報[1)Matsuda K et al. Establishment of an analytical system for the human fecal microbiota, based on reverse transcription-quantitative PCR targeting of multicopy rRNA molecules. Appl Environ Microbiol. 75:1961-1969 (2009)]に従ってRNAを抽出し、RT-qPCR法[2)Tsuji, H. et al. Isolation and characterization of the equol-producing bacterium Slackia sp. strain NATTS. Arch Microbiol. 192, 279-87 (2010)]によりNATTS株の菌数を測定した。使用したプライマーは表2に示した。なお、標準曲線の作成には、純粋培養菌液のDAPIカウントの結果をもとに培養液の菌数調整を行い、上記1)と同様に酵素処理を加えた後に抽出したRNAを用いた。
培養72時間後の嫌気豆乳培地0.5mLをねじ口試験管に移し、2.5mLのジエチルエーテルを加えて激しく混合した。試験管を1,000×gで10分間遠心し、エーテル相を新しい試験管に回収した後、残りの水相に再度2.5mLのジエチルエーテルを加えて激しく混合した。試験管を1,000×gで10分間遠心し、エーテル相を上記の試験管に回収した後、40℃に加温したブロックヒーターにより窒素気流下で乾固した。乾固させた抽出物を250μLの80v/v%メタノールに溶解した後、0.45μmフィルターでろ過した。ろ液中のダイゼイン及びその誘導体の定量には液体クロマトグラフ質量分析計(LC/MS)を用いた。システムとして、質量分析計ZQ 4000を備えたアライアンスHPLCシステム(日本ウォーターズ)を用いた。分離カラムにはCadenzaCD-C18(内径:3.0mm、長さ:75mm、粒径3μm、インタクト)を、移動相には0.1%ギ酸/アセトニトリル混液(70:30)を使用し、サンプルアプライ量は10μLとした。解析にはWaters Empower 2ソフトウエア(日本ウォーターズ)を用いた。標準曲線は、ダイゼイン、エコール、ジヒドロダイゼイン(DHD)及びo-デスメチルアンゴレンシン(o-DMA)の標品のメタノール溶液を用いて作成した。各物質の定量範囲は10~1,000ng/mLとした。
なお、豆乳培地中に配糖体として含まれているイソフラボンアグリコン量は、菌株未接種の豆乳培地0.5mLにアーモンド由来β-グルコシダーゼ(100U/mL,SIGMA)を含む0.2M酢酸バッファー(pH5.0)を0.5mL加えて、37℃で16時間反応させ、遊離したイソフラボンアグリコンの濃度を、反応液0.5mLを用いて上記と同様の方法で測定した。
1)供試した8株の乳酸菌のすべての菌株との共培養により、培養後のNATTS株の菌数が、NATTS株単独培養と比べて有意に高かった(図1-A,B)。共培養に用いた菌株のなかでもL.casei YIT 9029株(LcS)と組み合わせた場合に、1mLあたり109cells以上の菌数まで到達した。
(1)培養方法
NATTS株を1.0%、LcSを0.1%となるように豆乳培地に接種し、実施例1(3)と同様の方法で培養を行った(n=3)。培養開始0、24、48、72時間後に培養液の一部をサンプリングした。
1)NATTS株単独培養では培養24時間で菌数の僅かな増加が認められたが、それ以降では経時的に減少した。一方、LcSとの共培養では、NATTS株の菌数は培養24時間で最大となり、以降72時間までほぼ一定数を維持した(図3)。
Claims (3)
- 豆乳を主成分とする培地中で、エコール産生菌及びラクトバチルス・カゼイ YIT 9029株(FERM BP―1366)を培養するエコールの製造方法であって、エコール産生菌がSlackia sp.YIT 11861株(FERM BP-11231)である、方法。
- 豆乳を主成分とする培地中で、エコール産生菌及びラクトバチルス・カゼイ YIT 9029株(FERM BP―1366)を培養するエコール含有発酵組成物の製造方法であって、エコール産生菌がSlackia sp.YIT 11861株(FERM BP-11231)である、方法。
- 豆乳を主成分とする培地中で、エコール産生菌及びラクトバチルス・カゼイ YIT 9029株(FERM BP―1366)を培養してなるエコール含有発酵組成物であって、エコール産生菌がSlackia sp.YIT 11861株(FERM BP-11231)である、組成物。
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