JP5916846B2 - 細菌性感冒に対する新規な乳酸菌及びそれを含有する組成物 - Google Patents
細菌性感冒に対する新規な乳酸菌及びそれを含有する組成物 Download PDFInfo
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- JP5916846B2 JP5916846B2 JP2014510815A JP2014510815A JP5916846B2 JP 5916846 B2 JP5916846 B2 JP 5916846B2 JP 2014510815 A JP2014510815 A JP 2014510815A JP 2014510815 A JP2014510815 A JP 2014510815A JP 5916846 B2 JP5916846 B2 JP 5916846B2
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Description
急性疾患の標準治療は抗生物質の投与である。とりわけ1980年代半ばからのペニシリン耐性の出現の増加により、ここ数十年になって再び、この疾患が先進国において増加してきている(非特許文献9、非特許文献10)。これまでに機能的なワクチンが開発されていないことから、予防法は存在していない。これは、使用される主な抗原の多様性、細菌細胞壁由来のMタンパク質、及び他の抗原候補物質とヒトタンパク質との免疫学的交差反応性によるものである。
a.生物膜を形成する病原微生物をインキュベーションする工程と、
b.試験対象の乳酸菌を添加するとともにインキュベーションして混合物を形成する工程であって、病原微生物と調査対象の乳酸菌との間で共凝集を起こす工程と、
c.上清を除去することによって結合していない乳酸菌を分離する工程と、
d.共凝集した乳酸菌に関して生物膜を測定する工程と、
を少なくとも含む、方法に関する。
病原微生物の生物膜の阻害を調査する工程であって、調査対象の乳酸菌を、生物膜を形成する病原微生物のインキュベーション中に添加する工程及び/又は好ましくは、
試験対象の乳酸菌を添加していない対照と比較して、光学密度を測定することによって、結合していない細胞の除去後の生物膜の形成を定量化する工程、
を更に含み得る。
本発明による微生物の特定及び選択に関して、ラクトバチルス株バンクからの様々な菌株を、4段階スクリーニングプロセスによって試験した。この4段階スクリーニングプロセスでは、初めに、標的病原微生物である化膿連鎖球菌(以後「標的微生物」とも称される)に結合する能力に関してスクリーニングし(結合アッセイ)、次に、第1の工程で特定された菌株を、マイクロタイタープレートスケールにおける共凝集アッセイにおいて試験し、各標的微生物との共凝集を、双眼実体顕微鏡を用いて定性的に測定した。本発明の意味において、「共凝集(co-aggregation)」という用語は「共凝集(coaggregation)」という用語と同義で用いられる。さらに、共凝集の強度及び標的微生物への結合の安定性、並びにフィブロネクチンとの結合防止能を調査して、最終的には、本発明により特定される例示的な微生物(ラクトバチルス)を導く。
選択されたラクトバチルス株の結合活性の定量化を可能にする結合アッセイを構築し、それにより96ウェルプレートでのラクトバチルス株の病原微生物への結合の定量的検出が可能になる。ラクトバチルス細胞の標的微生物への結合活性は共凝集活性及び/又は共凝集能に相当する。これは実験によって試験した。これに関して、図2はラクトバチルスDSM25972、DSM25987、DSM25988、DSM25989及びDSM25973と、化膿連鎖球菌ATCC19615との共凝集の例を示すものである。
共凝集アッセイ
1.0mLの体積及び/又は24ウェルプレートにおける以下の検証を用いて、選択したラクトバチルス株の共凝集活性を説明する。
ヒト唾液中の共凝集
ヒトの天然唾液中の本発明によるラクトバチルス株の共凝集能を調査するために、本発明によるラクトバチルス株を実施例2に記載のように16時間の培養後に回収し、PBSで2回洗浄して、ヒト唾液中に入れた。唾液は予め数人から回収しており、混合して、8000×gで20分間の遠心分離により粒子から分離した。標的微生物を実施例2に記載のように処理し、これもPBSで2回洗浄した後、ヒト唾液中に入れた。共凝集アッセイを行うために、24ウェルプレートにおいて500μLの一部の標的微生物の唾液懸濁液を1ウェル当たり500μLのラクトバチルス唾液懸濁液と合わせた。500μLの標的微生物+500μLの唾液、又は500μLの各ラクトバチルス懸濁液+500μLの唾液(対照1)を含む対照バッチを並行して試験した。デスクトップ型撹拌器での25℃での10分のインキュベーション後、バッチを写真記録システムを用いて肉眼で観察し、さらに顕微鏡で観察した。
フィブロネクチンへの結合の阻止
標的病原微生物である化膿連鎖球菌(ATCC19615)のフィブロネクチンへの結合に対する本発明によるラクトバチルス株の効果を調査するために、フィブロネクチンをコーティングした96ウェルプレートにおける蛍光標識した標的微生物の結合の定量的検出を可能にする結合アッセイを構築した。本発明によるラクトバチルス株をCFDAで標識した標的微生物に結合開始時に直接添加し、37℃で2時間インキュベートした。結合していない細胞を取り除き、PBSで2回洗浄した後、結合した標的微生物細胞の特異的定量化を、蛍光プレート型光度計でEm 485nm/Ex 535nmの蛍光を測定することによって行った。これらの試験を行うために、標的株と本発明によるラクトバチルス株とを標準的プロトコルに従って培養した。ラクトバチルス株の処理のために、培養後、ラクトバチルス株をPBSで2回洗浄した後、PBS中に入れた。PBSで洗浄後、ラクトバチルス株の一部を70℃で30分間の低温殺菌によって破壊した。標的株の処理のために、標的株を定常状態の増殖期に達するまで16時間培養した後、回収し、CFDAを添加することで蛍光標識して、OD600nmを3.0に調整した。結合アッセイを行うために、ラクトバチルス細胞を蛍光標識した標的微生物に結合開始時に直接添加し、37℃で2時間微好気性条件下でインキュベートした。ラクトバチルス懸濁液を添加しない対照バッチを並行して行った。浮遊細胞を取り除き、生物膜内に見られる細胞を洗浄した後、結合した標的微生物細胞の特異的定量化を、ラクトバチルス細胞の存在下及び非存在下での蛍光測定によって行った。ラクトバチルスの非存在下での標的微生物の対照と比較した上での蛍光の低減は、標的微生物である化膿連鎖球菌と、フィブロネクチンをコーティングしたマイクロタイタープレートとの結合の強度に相関するものであった。この低減は生物膜形成の阻害率で表される。図5に、フィブロネクチンへの結合、すなわち化膿連鎖球菌ATCC19615の阻害におけるラクトバチルス株DSM25972、DSM25987、DSM25988、DSM25989及びDSM25973(これらの株は本発明による微生物の例として試験された)で得られた結果を示す。実験から、これによって、生きている及び破壊された両方の形態の上記で挙げられた本発明によるラクトバチルス株に起因して標的微生物のフィブロネクチンへの結合の阻害がもたらされることが分かっている。そのためこのことによって、本発明によるラクトバチルス株が化膿連鎖球菌の宿主細胞への結合を防ぐことが可能であることが示される。さらに、同じ属及び種の更なる微生物による実験を行うことで、本発明によるラクトバチルス株の結合特性及び/又は凝集特性の特異性と、同じ属及び種の標的微生物に応じたフィブロネクチンへの結合を防ぐ能力とが示される。この目的のためにそれらの中で化膿連鎖球菌ATCC12344が選択された。標的微生物である化膿連鎖球菌ATCC12344のフィブロネクチンへの結合に対する効果を調査するために、本発明によるラクトバチルス株と標的微生物とを上記の標準的条件下で培養した後、処理し、アッセイに使用した。
ストレプトコッカス・サリバリウスの共凝集
共凝集アッセイにおいて本発明によるラクトバチルス株と、ストレプトコッカス・サリバリウスとを標準的な条件に従って培養し、PBSで3回洗浄し、ヒト唾液中で使用した。結果によって、ストレプトコッカス・サリバリウスは本発明によるラクトバチルス株によっては共凝集しないことが示唆される(図7)。このため、ここでは例としてストレプトコッカス・サリバリウスが挙げられる片利共生株は、ラクトバチルス株によっては共凝集せず、自然環境から取り除かれる可能性を排除することができる。これによって、とりわけ好ましい乳酸菌が細菌感染症に対して特異的な作用を有することが示唆される。
(S.=頁;und=及び;Hrsg.=編;Kapitel=章)
Claims (37)
- 主要有効成分として、乳酸菌目の微生物若しくはそのフラグメントを含み、それは、病原微生物としての化膿連鎖球菌と共凝集する、化膿連鎖球菌に関連する皮膚、粘膜、及び口腔の疾患の処置、予防及び/又は治療用剤若しくは組成物であって、ここで、
該微生物が、DSM25972、DSM25987、DSM25988、DSM25989及びDSM25973という番号でドイツ微生物細胞培養物コレクションに寄託された微生物を含む群から1以上選択されることを特徴とする、剤若しくは組成物。
- 前記微生物がラクトバチルス属に属する、請求項1に記載の微生物若しくはそのフラグメントを含む剤若しくは組成物。
- 生物学的処理、化学的処理又は物理的処理の後であっても前記病原微生物との共凝集能が現れる、請求項1又は2に記載の微生物若しくはそのフラグメントを含む剤若しくは組成物。
- 前記病原微生物との共凝集能がおよそ3〜およそ8のpHで現れる、請求項1〜3のいずれか一項に記載の微生物若しくはそのフラグメントを含む剤若しくは組成物。
- 前記病原微生物の生物膜の形成を防ぐ能力を有する、請求項1〜4のいずれか一項に記載の微生物若しくはそのフラグメントを含む剤若しくは組成物。
- 化膿連鎖球菌によるフィブロネクチンの結合を妨げる能力を有する、請求項1〜5のいずれか一項に記載の微生物若しくはそのフラグメントを含む剤若しくは組成物。
- 皮膚又は粘膜の任意の片利共生微生物と共凝集しない能力を有する、請求項1〜6のいずれか一項に記載の微生物若しくはそのフラグメントを含む剤若しくは組成物。
- 請求項1〜7のいずれか一項に記載の少なくとも1つの微生物若しくはそのフラグメントを含む組成物。
- 薬学上又は美容上許容可能な賦形剤又は補形薬を含む、請求項8に記載の組成物。
- 固体、液体若しくは粘性体であるか、又はエアロゾルである、請求項8又は9に記載の組成物。
- ペースト、軟質ゼラチンカプセル、硬質ゼラチンカプセル、粉末、顆粒、ビーズ、香錠、発泡錠、ロゼンジ、口腔錠、チュアブル錠、舌下錠、溶液、チンキ剤、エマルション、ジュース、濃縮液、シロップ、スプレー液、飲用アンプル、ゲル、洗口液、歯磨き粉、チューインガム、錠剤、コーティングピル又は糖菓の形態である、請求項8〜10のいずれか一項に記載の組成物。
- プロバイオティクス、防腐剤又は他の抗菌物質を含む、請求項8〜11のいずれか一項に記載の組成物。
- 医薬組成物、獣医組成物若しくは化粧品組成物又は栄養補助食品若しくは栄養補助食品組成物である、請求項8〜12のいずれか一項に記載の組成物。
- 酸化防止剤、ビタミン、補酵素、脂肪酸、アミノ酸及び補因子から選択される1つ又は複数の物質を更に含む、請求項8〜13のいずれか一項に記載の組成物。
- 1つ若しくは複数の増粘剤、1つ若しくは複数の甘味料、及び/又は1つ若しくは複数の人工甘味料を含む、請求項8〜14のいずれか一項に記載の組成物。
- 前記増粘剤が、セルロースエーテル、キサンタンガム、ゼラチン、高分散二酸化ケイ素、デンプン、アルギン酸塩、トラガカントゴム、寒天、アラビアゴム、ペクチンを含む群から選択される多糖類及びポリビニルエステルから選択され、前記甘味料が、グルコース、フルクトース、スクロース及びグルコースシロップ、ソルビトール、マンニトール、キシリトール及びマルチトール、サッカリン、シクラミン酸ナトリウム、アセスルファムK及び/又はアスパルテームを含む群から選択される、請求項15に記載の組成物。
- ビルダー物質、酵素、電解質、pH調整剤、増粘剤、防汚剤、蛍光増白剤、灰色化阻害剤、色移り阻害剤、整泡剤(foamregulators)及び/又は着色剤を更に含む、請求項8〜16のいずれか一項に記載の組成物。
- 前記微生物が前記組成物中で生きている又は破壊した微生物として存在する、請求項8〜17のいずれか一項に記載の組成物。
- 前記微生物が、封入形態、噴霧乾燥形態及び/又は凍結乾燥形態で存在する、請求項8〜18のいずれか一項に記載の組成物。
- 前記微生物が細胞可溶化物の形態で存在する、請求項8〜19のいずれか一項に記載の組成物。
- 前記微生物が、0.001wt%〜20wt%、又は0.005wt%〜10wt%、又は0.01wt%〜5wt%の量で存在する、請求項8〜20のいずれか一項に記載の組成物。
- 化膿連鎖球菌と共凝集する特性を有する乳酸菌を特定及び/又は選択する方法であって、
a.生物膜を形成する化膿連鎖球菌をインキュベーションする工程と、
b.試験対象の乳酸菌を添加するとともに混合物をインキュベーションする工程であって、前記化膿連鎖球菌と調査対象の該乳酸菌との間で共凝集を起こす工程と、
c.上清を除去することによって結合していない乳酸菌を分離するとともに、共凝集した乳酸菌に関して前記生物膜を測定する工程と、
を少なくとも含む、方法。
- 前記化膿連鎖球菌の生物膜の阻害を調査する工程であって、調査対象の前記乳酸菌を、生物膜を形成する前記化膿連鎖球菌のインキュベーション中に添加する工程、
を更に含む、請求項22に記載の方法。
- 試験対象の前記乳酸菌を添加していない対照と比較して、光学密度の測定を用いて結合していない細胞の除去後の生物膜の形成を定量化する工程、
を更に含む、請求項22又は23に記載の方法。
- 化膿連鎖球菌に関連する皮膚、粘膜、及び口腔の疾患の治療、予防及び/又は治療法用の調合薬、医療品又は化粧品の製造への請求項8〜21のいずれか一項に記載の組成物の使用。
- 化膿連鎖球菌に関連する皮膚、粘膜、及び口腔の疾患の局所予防及び/又は局所治療用の調合薬、医療品又は化粧品の製造での使用である請求項25に記載の使用。
- 前記組成物が1つ又は複数の香味物質を更に含む、請求項25又は26に記載の使用。
- 前記組成物が化膿連鎖球菌に関連する皮膚、粘膜、及び口腔の疾患、好ましくは口腔の化膿連鎖球菌に関連する疾患の局所予防及び/又は局所治療用途であり、かつ、前記組成物が、チュアブル化合物、チューインガム、糖菓、香錠、歯磨き粉、スプレー液又は洗口液の形態である、請求項25〜27のいずれか一項に記載の使用。
- 1つ又は複数の抗炎症物質及び/又は抗微生物物質が更に使用されて、調合薬、医療品又は化粧品の製造がされる、請求項25〜28のいずれか一項に記載の使用。
- 前記組成物が、溶媒、賦形剤、補形薬、充填剤、香味物質及び/又は芳香物質、及び/又は更なる成分と組み合わせて、調合薬、医療品又は化粧品の製造がされる、請求項25〜29のいずれか一項に記載の使用。
- 表面の処理用の清浄剤又は消毒剤への製造への請求項8〜21のいずれか一項に記載の組成物の使用。
- 医療品及び/又は予防の分野において使用される製品の製造への請求項8〜21のいずれか一項に記載の組成物の使用。
- 経口摂取、舌下摂取又は口腔摂取用の作用物質の製造への請求項8〜21のいずれか一項に記載の組成物の使用。
- 口腔咽頭域内の炎症の局所治療に(for)又は上気道及び皮膚の感染に特異的な作用を有する抗菌添加剤の製造への請求項8〜21のいずれか一項に記載の組成物の使用。
- 前記組成物が予防的に又は治癒的用組成物である、請求項25〜34のいずれか一項に記載の使用。
- 前記組成物が経口投与、舌下投与又は口腔投与用組成物である、請求項25〜35のいずれか一項に記載の使用。
- 請求項1〜7のいずれか一項に記載の剤若しくは組成物又は請求項8〜21のいずれか一項に組成物と、身体衛生用の機器若しくは装置、リンス及び/又はペーストとを含む衛生的な治療用キット。
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WO2008064893A1 (en) | 2006-12-01 | 2008-06-05 | Organobalance Gmbh | Compositions, kits and uses for protecting the skin against pathogenic microorganisms |
BRPI0719279B1 (pt) * | 2006-12-19 | 2018-12-04 | Basf Se | uso de um microorganismo pertencendo ao grupo de bactérias do ácido lático ou a um mutante ou derivado do mesmo |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US11382939B2 (en) | 2016-09-15 | 2022-07-12 | Kirin Holdings Kabushiki Kaisha | Composition which contains lactic acid bacterium as effective component and which is for preventing or ameliorating skin condition deterioration caused by abnormal proliferation of specific bacterium in skin |
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WO2012156491A1 (de) | 2012-11-22 |
ES2660775T3 (es) | 2018-03-26 |
KR20140032427A (ko) | 2014-03-14 |
EP2710115B1 (de) | 2017-12-27 |
US20140065218A1 (en) | 2014-03-06 |
RU2651466C2 (ru) | 2018-04-19 |
JP2014516957A (ja) | 2014-07-17 |
EP2710115A1 (de) | 2014-03-26 |
RU2013150691A (ru) | 2015-06-27 |
CN103547670A (zh) | 2014-01-29 |
CA2833937C (en) | 2022-04-05 |
US9308227B2 (en) | 2016-04-12 |
CA2833937A1 (en) | 2012-11-22 |
CN103547670B (zh) | 2017-06-16 |
KR101918792B1 (ko) | 2018-11-14 |
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