JP2014516567A - チャノキ葉から分離した新規なラクトバチルス・プランタラム - Google Patents
チャノキ葉から分離した新規なラクトバチルス・プランタラム Download PDFInfo
- Publication number
- JP2014516567A JP2014516567A JP2014514809A JP2014514809A JP2014516567A JP 2014516567 A JP2014516567 A JP 2014516567A JP 2014514809 A JP2014514809 A JP 2014514809A JP 2014514809 A JP2014514809 A JP 2014514809A JP 2014516567 A JP2014516567 A JP 2014516567A
- Authority
- JP
- Japan
- Prior art keywords
- strain
- apsulloc
- lactobacillus plantarum
- apsuloc
- lactic acid
- 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.)
- Granted
Links
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Classifications
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- A—HUMAN NECESSITIES
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
Description
寄託機関名:韓国微生物保存センター(韓国国内)
受託番号:KCCM11179P
受託日:2011年03月28日
(2)ラクトバチルス・プランタラムAPsulloc 331263
寄託機関名:韓国微生物保存センター(韓国国内)
受託番号:KCCM11180P
受託日:2011年03月28日
(3)ラクトバチルス・プランタラムAPsulloc 331266
寄託機関名:韓国微生物保存センター(韓国国内)
受託番号:KCCM11181P
受託日:2011年03月28日
(4)ラクトバチルス・プランタラムAPsulloc 331269
寄託機関名:韓国微生物保存センター(韓国国内)
受託番号:KCCM11182P
受託日:2011年03月28日
チャノキ葉200gを、1次蒸留水に2回洗浄して異物を除去する。洗浄したチャノキ葉の水気を払い落とし、チャノキ葉重量の8%に該当する食塩と混合した後、3時間室温に放置する。食塩に漬け込まれたチャノキ葉を、1%フラクトオリゴ糖溶液1000mLに混合した後、3日間、32℃インキュベーターにおいて培養する。3日後、培養液のpHが5未満に下がったかを確認し、pH5未満である場合、これを採ってディフコラクトバシリMRSアガー(登録商標)(Difco Lactobacilli MRS Agar(登録商標))培地に培養する。このとき、培養は32℃、嫌気条件のチャンバーで2日間培養した後、白色集落を示すコロニーを採る。
(1)菌株の培養
実施例1において分離したAPsulloc 331261を、MRSアガープレートに塗抹(streaking)し、37℃で、2日間培養する。得られた単一コロニー(single colony)を、MRSブロス(broth)10mLに接種し、さらに37℃で一晩培養してラクトバチルス・プランタラム菌株培養液を製造する。APsulloc 331263,APsulloc 331266およびAPsulloc 331269についても、それぞれ実質的に同一の方法によりラクトバチルス・プランタラム菌株培養液を製造する。
前記(1)のように製造したAPsulloc 331261菌株の培養液を、MRSブロス10mLに0.5%接種して、37℃で一晩培養した。培養液を8,000rpmで5分間遠心分離し、上澄み液を取り除いて菌体のみを集めた後、0.85%緩衝生理食塩水(saline buffer)を2mL添加して懸濁した。その後、API 50CHLキット(Biomerieux)を使用して、製造社のプロトコルに従って進行した。その具体的な内容は、以下のとおりである。
前記(1)のように製造したAPsulloc 331261菌株培養液をMRSブロス10mLに0.5%接種し、37℃で一晩培養した。培養液を8,000rpmで5分間遠心分離し、上澄み液を取り除いて菌体のみを集めた後、0.85%緩衝生理食塩水(saline buffer)を2mL添加して懸濁した。その後、API ZYMキット(Biomerieux)を使用して、製造社のプロトコルに従って進行した。その具体的な内容は、以下のとおりである。
ペトリ皿(直径100mm)に、滅菌したMRSアガーを20mLずつ入れ、クリーンベンチで冷まして培地を製造した。MRSブロス10mLに、(1)で製造したAPsulloc 331261菌株培養液を0.5%接種し、37℃で6時間培養した。625nmにおける吸光度が0.08〜0.13程度となるように希釈した。希釈液を滅菌した綿棒に十分に浸した後、あらかじめ作られたMRSアガープレートに全体的に均等に塗抹した。抗生物質感受性評価ディスクをプレート上に適当な間隔を開けて落とした。37℃で24時間培養した後、クリーンゾーン(clear zone)の直径を測定した。
(1)pH別耐酸性能評価
MRSブロス10mLに、APsulloc 331261の菌株培養液を0.5%接種し、37℃で一晩培養した。HClでpHをそれぞれ2.0、2.5、3.0、3.5に調整して、滅菌したMRSブロス5mLに前記培養液50μlを接種した後、37℃で1時間培養した。1時間後、ペプトン食塩緩衝液で希釈して、1mLあたりの菌数を測定した。前記培養液の菌数を測定した後、0.01を乗じたものを対照群(Control)とし、対照群の菌数を100%として生存率を計算した。
MRSブロス10mLに、APsulloc 331261培養液を0.5%接種し、37℃で一晩培養した。滅菌したMRSブロス5mLに、HClでpHを2.5に調整した培養液50μlを接種した後、37℃で3時間培養した。1時間および3時間後、ペプトン食塩緩衝液で希釈してから、1mLあたりの菌数を測定した。前記培養液の菌数を測定した後、0.01を乗じたものを対照群(control)とし、対照群の菌数を100%として生存率を計算した。
MRSブロス10mLに、APsulloc 331261培養液を0.5%接種し、37℃で一晩培養した。ウシ胆汁(ox gall)をそれぞれ0.3%、0.5%ずつ添加して、MRSアガープレートを製造した。対照群(control)としては、ウシ胆汁を添加しなかったMRSアガーを使用した。前記菌株の培養液を希釈してMRSアガー培地に塗抹後、37℃で2日間培養した。各コロニーを計数した後、対照群の菌数を100%としてAPsulloc 331261の生存率(%)を計算した結果を、下表に示した。APsulloc 331263,APsulloc 331266およびAPsulloc 331269の菌株培養液についても、それぞれ実質的に同一の方法により耐胆汁酸能を評価し、その結果を下表に示した。(表17:APsulloc 331261とAPsulloc 331266、表18:APsulloc 331263とAPsulloc 331269)
MRSブロス10mLに、APsulloc 331261培養液を0.5%接種し、37℃で一晩培養した。培養液を8,000rpmで15分間遠心分離して、上澄み液のみを回収した。回収した上澄み液を80℃で15分間処理して、酵素反応を停止させた。蒸留水で熱処理した上澄み液を、100倍希釈した。D‐乳酸/L‐乳酸UV法キット(UV method kit)(R‐biopharm)を使用して、製造社のプロトコルに従って進行した。その具体的な内容は、以下のとおりである。
(1)APsulloc 331261
MRSブロス10mLに、APsulloc 331261培養液を0.5%接種し、37℃で一晩培養した。滅菌したMRSアガーを15mLずつペトリ皿に分注して培地を製造後、前記APsulloc 331261培養液を1μlずつ点滴して、37℃で24時間培養した。
MRSブロス10mLに、APsulloc 331266培養液を0.5%接種し、37℃で一晩培養した。
MRSブロス10mLに、APsulloc 331269培養液を0.5%接種し、37℃で一晩培養した。培養液を8,000rpmで5分間遠心分離した後、上澄み液のみを回収した。上澄み液10mLを、0.22μlシリンジフィルターで除菌して準備した。
Claims (12)
- ラクトバチルス・プランタラム(Lactobacillus plantarum)APsulloc 331261(寄託番号:KCCM11179P)菌株。
- ラクトバチルス・プランタラム(Lactobacillus plantarum)APsulloc 331263(寄託番号:KCCM11180P)菌株。
- ラクトバチルス・プランタラム(Lactobacillus plantarum)APsulloc 331266(寄託番号:KCCM11181P)菌株。
- ラクトバチルス・プランタラム(Lactobacillus plantarum)APsulloc 331269(寄託番号:KCCM11182P)菌株。
- 前記菌株は、チャノキ(Camellia sinensis)の葉から分離されたものであることを特徴とする、請求項1〜4のいずれか1項に記載の菌株。
- 前記菌株は、耐酸性能を有することを特徴とする、請求項1〜4のいずれか1項に記載の菌株。
- 前記菌株は、pH2〜pH4において0.5時間〜5時間の耐酸性能を有することを特徴とする、請求項6に記載の菌株。
- 前記菌株は、耐胆汁酸能を有することを特徴とする、請求項1〜4のいずれか1項に記載の菌株。
- 前記菌株は、乳酸(lactic acid)生成能を有することを特徴とする、請求項1に記載の菌株。
- 前記菌株は、生成した乳酸のうちD型が70%以下である乳酸生成能を有することを特徴とする、請求項9に記載の菌株。
- 請求項1〜4のいずれか1項による菌株またはその培養液を含む組成物。
- 組成物は、発酵食品組成物であることを特徴とする、請求項11に記載の組成物。
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KR10-2011-0056468 | 2011-06-10 | ||
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KR1020110056469A KR101769446B1 (ko) | 2011-06-10 | 2011-06-10 | 차나무 잎에서 분리한 신규 락토바실러스 플란타룸 |
KR1020110056465A KR101769444B1 (ko) | 2011-06-10 | 2011-06-10 | 차나무 잎에서 분리한 신규 락토바실러스 플란타룸 |
KR1020110056466A KR101769445B1 (ko) | 2011-06-10 | 2011-06-10 | 차나무 잎에서 분리한 신규 락토바실러스 플란타룸 |
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KR10-2011-0056469 | 2011-06-10 | ||
PCT/KR2012/004569 WO2012169842A2 (ko) | 2011-06-10 | 2012-06-08 | 차나무 잎에서 분리한 신규 락토바실러스 플란타룸 |
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US20150197722A1 (en) | 2015-07-16 |
JP6033290B2 (ja) | 2016-11-30 |
WO2012169842A2 (ko) | 2012-12-13 |
CN104245920A (zh) | 2014-12-24 |
US20160151434A1 (en) | 2016-06-02 |
US9889167B2 (en) | 2018-02-13 |
US20150197721A1 (en) | 2015-07-16 |
US20160228480A1 (en) | 2016-08-11 |
US20140106435A1 (en) | 2014-04-17 |
US9895402B2 (en) | 2018-02-20 |
US20160228479A1 (en) | 2016-08-11 |
US20160228478A1 (en) | 2016-08-11 |
US20150197723A1 (en) | 2015-07-16 |
US9895401B2 (en) | 2018-02-20 |
WO2012169842A3 (ko) | 2013-03-07 |
US9782446B2 (en) | 2017-10-10 |
CN113151045A (zh) | 2021-07-23 |
HK1199904A1 (en) | 2015-07-24 |
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