JP2020515296A - 終末糖化産物の低減活性を有する新規な菌株およびその用途 - Google Patents
終末糖化産物の低減活性を有する新規な菌株およびその用途 Download PDFInfo
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Abstract
Description
本発明のまた他の目的は、本発明の前記新菌株、その破砕物またはその培養物を有効成分として含む、終末糖化産物関連疾患の予防または治療用の薬学的組成物を提供することにある。
終末糖化産物の低減活性を有する新菌株の分離および同定
<1−1>新菌株ラクトコッカス・ラクチスKF140の分離および同定
本発明者らは、終末糖化産物の低減活性を有する新しい菌株をキムチから分離するために次のような実験を行った。キムチを0.85%生理食塩水に懸濁し、懸濁液の一部をMRS固体培地に塗抹した後に37℃で培養した。培養後、形成されたコロニーを取ってMRS培地に画線培養して、汚染されていない純粋な単一コロニーを確保した。確保された菌株から16S rDNAを分離してシークエンシング分析を行った。
終末糖化産物の低減活性を有する新しい菌株を味噌から分離するために次のような実験を行った。味噌を0.85%生理食塩水に懸濁し、懸濁液の一部をTSB固体培地に塗抹した後に37℃で培養した。培養後、形成されたコロニーを取ってTSB培地に画線培養して、汚染されていない純粋な単一コロニーを確保した。確保された菌株から16S rDNAを分離してシークエンシング分析を行った。
キムチからラクトバチルス・パラカゼイKF00816菌株を分離および同定し、伝統式で製造されたキムチを0.85%生理食塩水に懸濁し、懸濁液の一部をMRS固体培地に塗抹した後に37℃で培養した。培養後、形成されたコロニーを取ってMRS培地に画線培養して、汚染されていない純粋な単一コロニーを確保した。確保された菌株から16S rDNAを分離してシークエンシング分析を行った。
終末糖化産物の低減活性を有するラクトバチルス・ペントーサスKF8菌株は伝統式の醤油から分離し、伝統式の醤油を0.85%生理食塩水に懸濁し、懸濁液の一部をMRS固体培地に塗抹した後に37℃で培養した。培養後、形成されたコロニーを取ってMRS培地に画線培養して、汚染されていない純粋な単一コロニーを確保した。確保された菌株から16S rDNAを分離してシークエンシング分析を行った。
本発明の新菌株の終末糖化産物の低減効能の分析
本発明者らは、前記実施例1において分離および同定した4個の新菌株が各々終末糖化産物の低減効能があるか否かを分析するために、牛乳に含まれた終末糖化産物に対する低減活性の程度を次のような方法によって確認した。
本発明の新菌株が非蛍光性の終末糖化産物の一種類であるCML(Nε−(Carboxymethyl)−L−lysine)を低減させる活性があるか否かを分析するためにCML含有食品を製造した。すなわち、牛乳の熱処理工程中に生成されるCMLを低減するために乳タンパク質であるカゼインと乳糖であるラクトースを用い、sodium caseinateとD−lactoseは(株)ES食品原料とSigma−Aldrichから各々購入して用いた。牛乳内のカゼインとラクトースの比率を考慮して牛乳タンパク質(カゼイン)と糖(ラクトース)を1:7の比率で混ぜて140℃の温度で80分間反応し、それを本発明の新菌株に対するCML低減効能の評価に用いた。この時、カゼインとラクトースのメイラード反応によるCML含量は8.24ug/mLとして確認された。
本発明に係る新菌株のCML低減効能を下記表1に記載された他の菌株および商用化されたプロバイオティクス製品を対照群にして分析を行った。下記表1の対照群として用いられた他の菌株としては韓国食品研究院の伝統食品微生物菌株銀行から購入したものを用い、プロバイオティクス製品としては市販中のものを購入して用い、プロバイオティクス製品は図1に示す。また、下記実験に用いられた菌株を培養するための培地はDifco社から購入し、乳酸菌株の培養のためにLactobacilli MRS agarとLactobacilli MRS brothを用い、バチルス属菌株の培養培地としてTSA(tryptic soy agar)とTSB(tryptic soy broth)を用いた。乳酸菌およびバチルス菌のCML低減のための本培養にはM9 minimal medium(MB cell)を用い、M9培地を製造する時、glucose 2g/L、CaCl2 0.015g/L、MgSO4 0.5g/Lを添加して用いた。
表1の乳酸菌およびプロバイオティクス製品をCMLが含まれた培養液において24時間本培養させた後、Nε−(carboxymethyl)lysine(CML)の変化量をCML ELISA kit(CircuLex、Ina、Nagano、Japan)を用いて測定した。CML−BSAで事前コーティングされた96wellに上清または標準試料とanti−CML−adduct monoclonal antibodyを添加して室温で1時間反応させた。洗浄バッファで4回洗浄した後、HRP conjugated detection antibody 100μlを添加して1時間反応させた後、洗浄バッファで4回洗浄し、基質含有溶液100μlを添加して20分間光を遮断した条件下で反応させた後、停止液100μlを添加し、マイクロプレートリーダーを用いて450nmで吸光度を測定し、その結果を下記表に示す。この時、下記表2において、陰性対照群(CON;control)は乳酸菌を全く添加していない前記<2−1>のメイラード反応で測定されたCMLが8.24ug/mL含量で含まれた群を用いたものであり、陰性対照群をCML含量100%に基準にして測定された結果である。
CML標準形式を用いた本発明に係る新菌株の終末糖化産物の低減効能の検証
前記実施例の結果から、本発明者らは、本発明の新菌株が従来のCML低減活性を有する菌株および市販品に比べて遥かに優れたCML低減効能があることを確認した。そこで、CML低減効能の再検証のために、終末糖化産物のうち最も代表的な非蛍光性の終末糖化産物であるNε−(1−carboxymethyl)−L−lysine(CAS:5746−04−3)をSanta Cruz Biotechnology社から購入し、その低減活性を再度分析した。純度99%のCML標準形式を用い、CMLの低減分析は前記実施例2と同様の方法により分析を行った。
動物実験を通じた本発明の新菌株の血中CML低減効能の分析
さらに、本発明者らは、動物実験を通じて、本発明の新菌株を用いる場合、直接的に血中内CMLの含量を低減できるか否かを確認する実験を行った。このために、牛乳に含まれたタンパク質成分であるカゼインと糖成分であるラクトースのメイラード反応によるCML高含有食品を前記実施例で記述された方法により製造した後、それをSDマウス(6週齢、雄)に摂取させた後、血中CML含量の変化を確認し、この時、1週間は本発明の新菌株を各々50mg/kg摂取させ、12時間の空腹状態を維持したラットにCML高含有食品を摂取させた後、8時間の経過後に血中CML濃度を測定した。また、陰性対照群としては、本発明の新菌株を摂取させず、CML高含有食品だけを摂取させた群を用いた。
寄託機関名:韓国微生物保存センター(国外)
受託番号:KCCM11997P
受託日時:20170324
寄託機関名:韓国微生物保存センター(国外)
受託番号:KCCM11998P
受託日時:20170324
寄託機関名:韓国微生物保存センター(国外)
受託番号:KCCM11673P
受託日時:20150306
寄託機関名:韓国微生物保存センター(国外)
受託番号:KCCM11981P
受託日時:20170224
Claims (11)
- ラクトコッカス・ラクチスKF140(受託番号KCCM11673P)菌株、バチルス・サブティリスKF11(受託番号KCCM11981P)菌株、ラクトバチルス・ペントーサスKF8(受託番号KCCM11997P)菌株およびラクトバチルス・パラカゼイKF00816(受託番号KCCM11998P)菌株からなる群の中から選択されることを特徴とする、終末糖化産物(AGEs;Advanced glycation end products)の低減活性を有する新菌株。
- 前記終末糖化産物(AGEs;Advanced glycation end products)は、非蛍光性の終末糖化産物であることを特徴とする、請求項1に記載の終末糖化産物の低減活性を有する新菌株。
- 請求項1に記載の新菌株、その破砕物またはその培養物を有効成分として含むことを特徴とする、終末糖化産物の低減活性を有する食品組成物。
- 前記組成物は、終末糖化産物により引き起こされる糖尿病、慢性腎臓疾患、心臓疾患、血管疾患、糖尿病性網膜症(retinopathy)、糖尿病性神経障害(diabetic neuropathy)、糖尿病性白内障(cataract)または糖尿病性腎症(nephropathy)を予防または改善することを特徴とする、請求項3に記載の食品組成物。
- 請求項1に記載の新菌株、その破砕物またはその培養物を有効成分として含むことを特徴とする、終末糖化産物関連疾患の予防または治療用の薬学的組成物。
- 前記疾患は、糖尿病、慢性腎臓疾患、心臓疾患、血管疾患、糖尿病性網膜症(retinopathy)、糖尿病性神経障害(diabetic neuropathy)、糖尿病性白内障(cataract)および糖尿病性腎症(nephropathy)からなる群の中から選択されることを特徴とする、請求項5に記載の終末糖化産物関連疾患の予防または治療用の薬学的組成物。
- 請求項1に記載の新菌株、その破砕物またはその培養物を有効成分として含むことを特徴とする、整腸用組成物。
- 請求項1に記載の新菌株、その破砕物またはその培養物を有効成分として含むことを特徴とする、プロバイオティクス組成物。
- 請求項1に記載の新菌株、その破砕物またはその培養物を有効成分として含むことを特徴とする、飼料用組成物。
- 請求項1に記載の新菌株、その破砕物またはその培養物を有効成分として含むことを特徴とする、発酵製品。
- 請求項1に記載の新菌株、その破砕物またはその培養物を用いた終末糖化産物の抑制方法。
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KR1020170044938A KR101841021B1 (ko) | 2017-04-06 | 2017-04-06 | 최종당화산물 저감 활성을 갖는 신규한 락토코쿠스 락티스 균주 및 이의 용도 |
KR10-2017-0044949 | 2017-04-06 | ||
KR10-2017-0044938 | 2017-04-06 | ||
KR1020170044949A KR101841023B1 (ko) | 2017-04-06 | 2017-04-06 | 최종당화산물 저감 활성을 갖는 신규한 바실러스 서브틸리스 균주 및 이의 용도 |
KR10-2017-0079726 | 2017-06-23 | ||
KR1020170079735A KR101869740B1 (ko) | 2017-06-23 | 2017-06-23 | 최종당화산물 저감 활성을 갖는 신규한 락토바실러스 파라카제이 균주 및 이의 용도 |
KR1020170079726A KR101869739B1 (ko) | 2017-06-23 | 2017-06-23 | 최종당화산물 저감 활성을 갖는 신규한 락토바실러스 펜토서스 균주 및 이의 용도 |
KR10-2017-0079735 | 2017-06-23 | ||
PCT/KR2018/004076 WO2018186710A1 (ko) | 2017-04-06 | 2018-04-06 | 최종당화산물 저감 활성을 갖는 신규한 균주 및 이의 용도 |
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Free format text: JAPANESE INTERMEDIATE CODE: R250 |