JP6029242B2 - クロストリジウム・ディフィシル増殖阻害剤 - Google Patents
クロストリジウム・ディフィシル増殖阻害剤 Download PDFInfo
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- JP6029242B2 JP6029242B2 JP2013507369A JP2013507369A JP6029242B2 JP 6029242 B2 JP6029242 B2 JP 6029242B2 JP 2013507369 A JP2013507369 A JP 2013507369A JP 2013507369 A JP2013507369 A JP 2013507369A JP 6029242 B2 JP6029242 B2 JP 6029242B2
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- clostridium difficile
- growth
- propionibacterium
- present
- prebiotic composition
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- A61K31/19—Carboxylic acids, e.g. valproic acid
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Description
また、本発明の一態様は、プロピオン酸菌及び/又はその発酵物を含んでなるクロストリジウム・ディフィシル(Clostridium difficile)増殖阻害作用を有するプレバイオティクス組成物である。
また本発明の別の態様は、1,4−ジヒドロキシ−2−ナフトエ酸(DHNA)および/または有機酸を含んでなるクロストリジウム・ディフィシル(Clostridium difficile)増殖阻害作用を有するプレバイオティクス組成物である。
さらに本発明は、1,4−ジヒドロキシ−2−ナフトエ酸(DHNA)及び/又は有機酸の、クロストリジウム・ディフィシル(Clostridium difficile)増殖阻害剤の製造のための使用に関する。
さらに本発明は、クロストリジウム・ディフィシル(Clostridium difficile)増殖阻害に用いるための1,4−ジヒドロキシ−2−ナフトエ酸(DHNA)に関する。
これらの菌体については単独または複数のものを組み合わせて使用しても良い。
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。
・本発明のプレバイオティクス組成物であるプロピオン酸菌培養上清の調製
プロピオニバクテリウム・フロイデンライヒ ET−3株(平成13年8月9日付で、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))にFERM BP−8115として寄託されている)、プロピオニバクテリウム・フロイデンライヒ ATCC9614T株(プロピオニバクテリウム・フロイデンライヒ基準株、American Type Culture Collection(ATCC)より購入)、市販のエメンタールチーズより分離したプロピオニバクテリウム・フロイデンライヒ株(No.32−1)を30℃嫌気条件下で48時間賦活及び前培養した後、さらに30℃嫌気条件下で72時間培養した。培養物を遠心分離し(遠心分離条件:8000rpm、4℃、10分)、培養上清を回収し、フィルター滅菌し、得られた培養上清を本発明のプレバイオティクス組成物として−80℃で保存した。
クロストリジウム・ディフィシルJCM1296T株(クロストリジウム・ディフィシル基準株)は理研バイオリソースセンターより購入し、添付のマニュアルに従って復元した。
上記において調整したクロストリジウム・ディフィシルを予め嫌気状態に保ったBHI培地に1%接種した。これに本発明のプレバイオティクス組成物又はDHNAを添加し、37℃嫌気条件下で24時間培養した。培養液をよく攪拌した後、OD650を測定した(島津分光光度計BioSpec1600、(株)島津製作所製)。結果をグラフ図として図1に示す。なお得られたデータについては、Tukey−Kramer法による多重比較検定を行い、各群n=3、*p<0.05、**p<0.01とした。なお、プロピオニバクテリウム・フロイデンライヒ ATCC9614T及びプロピオニバクテリウム・フロイデンライヒ ET−3についてはcontrol群に対してp<0.01とした。
本発明のプレバイオティクス組成物を5%添加した系において、24時間培養後のOD650が有意に低下したことから、本発明のプレバイオティクス組成物が効果的にクロストリジウム・ディフィシルの増殖阻害をすることがわかった。
・本発明のプレバイオティクス組成物に含むDHNAの調製
DHNA(Sigma−Aldrich製)100mgを0.5mlのDMSOに溶解し、1MのDMSO溶液を作成した。BHI培地で10倍希釈し100mMとした後、フィルター滅菌した。同時にDMSOをBHI培地で10倍希釈した溶液も作成し、フィルター滅菌した。その後の希釈は全て10倍希釈DMSO溶液で行い、全濃度のDHNA溶液にDMSOが10%含まれるようにした。これらのDHNA溶液を本発明のプレバイオティクス組成物として菌培養液に0.1%添加するため、DMSOの最終濃度はいずれも0.01%とした。
実施例1において用いたクロストリジウム・ディフィシルに対する本発明のプレバイオティクス組成物の増殖阻害効果について実施例1と同様の阻害試験を行った。具体的には各試料に対してDHNA含有の本発明のプレバイオティクス組成物をそれぞれの濃度で添加した(1μM、10μM、20μM、50μM)。得られた結果を図2のグラフに示す。なお、Tukey−Kramer法による多重比較検定を行い、各群n=3、*p<0.05とした。なお、10μM〜50μMについてはDMSO群に対して**p<0.01とした。
・ビフィズス菌及び乳酸菌培養上清の調整
B.longum OLB6001株(FERM P−13610)及び「明治ブルガリアヨーグルト」(商品名:明治乳業社製)より分離したL.bulgaricusは37℃嫌気条件下で18時間培養した。S.thermophilus OLS3059株(FERM P−15487)は30℃嫌気条件下で30時間培養した。培養物を遠心分離し(遠心分離条件:8000rpm、4℃、10分)、培養上清を回収し、フィルター滅菌して、得られた培養上清を比較組成物として−80℃で保存した(乳酸菌は実施例4において使用)。
Bifidobacterium longum OLB6001株を実施例2において用いたDHNAもしくは0.01%DMSOを(対照)添加し、37℃嫌気条件下で12時間培養した。培養液をよく攪拌した後、OD650を測定した(島津分光光度計BioSpec1600、(株)島津製作所製)。結果をグラフ図として図3に示す。なお、Tukey−Kramer法による多重比較検定を行い、各群n=3、*p<0.05とした。なお、10μM〜50μMについてはDMSO群に対して**p<0.01とした。
本発明のDHNAを含有するプレバイオティクス組成物の添加によりOLB6001株の増殖が有意に促進されることがわかった。即ち、本発明のプレバイオティクス組成物は有用細菌に対して影響を及ぼさずむしろ増殖促進効果があることが示された。したがって、クロストリジウム・ディフィシルの増殖阻害を行う際には有用細菌についてその増殖に影響を及ぼさない(むしろ増殖促進効果がある)ことがわかった。
・本発明のプレバイオティクス組成物が乳酸菌の増殖に及ぼす影響
本発明のDHNAを含有するプレバイオティクス組成物(DHNA濃度:1μM〜50μM)の、実施例3で用いたL.bulgaricus及びS.thermophilus OLS3059株に対する影響を調べるため、実施例3と同様の試験を行い、結果を図4及び図5に示す。実施例3と同様に、本発明のDHNAを含有するプレバイオティクス組成物の添加によりL.bulgaricus及びS.thermophilus OLS3059株の増殖に対して影響を及ぼさないことがわかった。したがって、クロストリジウム・ディフィシルの増殖阻害する際には有用細菌についてその増殖に影響を及ぼさないことがわかった。
[実施例5]
・本発明のプロピオン酸がクロストリジウム・ディフィシルの増殖に及ぼす影響
24時間培養してOD650が1.2まで増殖したクロストリジウム・ディフィシル(C.difficile JCM1296株)培養液を培地に1%添加すると同時に、各種有機酸を終濃度100μMとなるように添加し、24時間後のOD650値を測定した。結果をグラフ図として図6に示す。
図6に示すようにプロピオン酸の添加により、クロストリジウム・ディフィシルの増殖が阻害されることが示された。
Claims (3)
- プロピオン酸菌及び/又はその発酵物を有効成分として含む、クロストリジウム・ディフィシル(Clostridium difficile)増殖阻害剤であって、
前記プロピオン酸菌が、プロピオニバクテリウム・フロイデンライヒ ET−3(Propionibacterium freudenreichii ET−3)(FERM BP−8115)またはプロピオニバクテリウム・フロイデンライヒ ATCC9614T(Propionibacterium freudenreichii ATCC9614T)である、剤。 - 請求項1に記載の剤を含んでなる、抗生物質もしくは抗菌薬との併用剤。
- クロストリジウム・ディフィシル(Clostridium difficile)増殖阻害剤の製造における、有効量のプロピオン酸菌及び/又はその発酵物の使用であって、
前記プロピオン酸菌が、プロピオニバクテリウム・フロイデンライヒ ET−3(Propionibacterium freudenreichii ET−3)(FERM BP−8115)またはプロピオニバクテリウム・フロイデンライヒ ATCC9614T(Propionibacterium freudenreichii ATCC9614T)であることを特徴とする使用。
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WO2006098447A1 (ja) * | 2005-03-18 | 2006-09-21 | Meiji Feed. Co., Ltd. | ビフィズス菌増殖促進成分を含有する飼料組成物及びその使用方法 |
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