JP2021505211A - 腎臓疾患進行抑制及び予防用プロバイオティクス及びこれを含む腎臓疾患進行抑制及び予防用組成物 - Google Patents
腎臓疾患進行抑制及び予防用プロバイオティクス及びこれを含む腎臓疾患進行抑制及び予防用組成物 Download PDFInfo
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Classifications
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
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Abstract
Description
下記方法で嬰児の糞便から分離された菌株を含む腎臓疾患進行抑制及び予防用プロバイオティクスを提供する:
(a)生後12ヶ月以下の嬰児の糞便を滅菌水で希釈してMRS培地に接種して培養する段階;
(b)前記MRS培地で生成された乳白色の単一コロニーを継代培養する段階;及び
(c)前記(b)段階で継代培養されたコロニーから乳酸菌を選別して得る段階。
前記プロバイオティクス;及びその培養物、濃縮物、ペースト化物、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物、液状物、希釈物及び破砕物の中で選択される1種以上;の中で選択される1種以上を含む腎臓疾患進行抑制及び予防用組成物を提供する。
ラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)菌株(受託番号:KCCM12169P)を提供する。
前記ラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)菌株;及びその培養物、濃縮物、ペースト化物、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物、液状物、希釈物及び破砕物の中で選択される1種以上;の中で選択される1種以上を含む腎臓疾患進行抑制及び予防用組成物を提供する。
ラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株(受託番号:KCCM12170P)を提供する。
前記ラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株;及びその培養物、濃縮物、ペースト化物、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物、液状物、希釈物及び破砕物の中で選択される1種以上;の中で選択される1種以上を含む腎臓疾患進行抑制及び予防用組成物を提供する。
動物に前記プロバイオティクス、または前記プロバイオティクス及びその培養物、濃縮物、ペースト化物、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物、液状物、希釈物及び破砕物の中で選択される1種以上を含む薬剤学的組成物;
前記ラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)菌株(受託番号:KCCM12169P)または前記ラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)菌株及びその培養物、濃縮物、ペースト化物、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物、液状物、希釈物及び破砕物の中で選択される1種以上を含む薬剤学的組成物;または
前記ラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株(受託番号:KCCM12170P)、または前記ラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株及びその培養物、濃縮物、ペースト化物、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物、液状物、希釈物及び破砕物の中で選択される1種以上を含む薬剤学的組成物;を有効量で投与することを含む腎臓疾患がある動物を治療する方法を提供する。
(a)生後12ヶ月以下の嬰児の糞便を滅菌水で希釈してMRS培地に接種して培養する段階;
(b)前記MRS培地で生成された乳白色の単一コロニーを継代培養する段階;及び
(c)前記(b)段階で継代培養されたコロニーから乳酸菌を選別して得る段階。
1ヶ月になった嬰児の糞便を滅菌水で10段階ずつ希釈し、平板希釈法(Dilution plating method)で菌株を分離した。希釈した糞便サンプルをMRS培地(MRS broth agar;BD Difco)に塗抹接種した後、37℃で72時間嫌気培養した。MRS agar plateで表れた乳白色の単一コロニーを継代培養して本発明のプロバイオティクスを純粋分離した。
前記実施例1で純粋分離された菌株に対し、染色体DNA抽出及び錠剤を行った。2つのユニバーサルプライマー(Universal primers)である27F(5'‐AGAGTTTGATCMTGGCTCAG‐3')及び1492R(5'‐TACGGYTACCTTGTTACGACTT‐3')を使って、16s rRNA遺伝子を増幅した後、増幅された16s rRNA遺伝子のシークエンシングを分析した。分析した16s rRNAシーケンスデータとEzTaxon server(http://www.ezbiocloud.net)を利用してGRAS(Generally Recognized as Safe)に該当する15種の菌株のみを選別して下記表2に示す。
<16S rRNA of bacterial strain、L.acidophilus BP105>
CTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCTGAACCAACAGATTCACTTCGGTGATGACGTTGGGAACGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCTGCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAAGAAAGCAGATCGCATGATCAGCTTATAAAAGGCGGCGTAAGCTGTCGCTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAGGGTAACGGCCTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGGTTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTAGTGCAATCCGTAGAGATACGGAGTTCCCTTCGGGGACACTAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCCAGCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGTACAACGAGGAGCAAGCCTGCGAAGGCAAGCGAATCTCTTAAAGCTGTTCTCAGTTCGGACTGCAGTCTGCAACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGGCCCGTCACACCATGGGAAGTCTGCAATGCCCCAAACCCGG
<16S rRNA of bacterial strain、L.salivarius BP121>
CCTAGATATAGTTTTTTTAATGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAAACTTTCTTACACCGAATGCTTGCATTCATCGTAAGAAGTTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTAAAAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATATCTCTAAGGATCGCATGATCCTTAGATGAAAGATGGTTCTGCTATCGCTTTTAGATGGACCCGCGGCGTATTAACTAGTTGGTGGGGTAACGGCCTACCAAGGTGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACACGAGTGAGAGTAACTGTTCATTCGATGACGGTATCTAACCAGCAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGGGAACGCAGGCGGTCTTTTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGTAGTGCATTGGAAACTGGAAGACTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGTTCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCAATAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACCTAAGAGATTAGGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTGTCAGTTGCCAGCATTAAGTTGGGCACTCTGGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCGAGACCGCGAGGTTTAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGAATCAGCATGTCGCGGTGAATACGTT
腎臓で酸化ストレスを誘発するものとして知られているインドキシル硫酸を除去及び分解する微生物を分離するため、前記実施例2で分離された15種の菌株を対象にしてインドキシル硫酸除去能を評価した。MRS brothにそれぞれ24時間前培養した後、60μg/ml濃度のインドキシル硫酸が添加されたMRS brothに各1%で接種して24、48、72時間培養した。
心血管疾患を誘発するものとして知られているパラクレゾール(p‐cresol)を除去及び分解する微生物を選別するために、前記実施例2で分離された15種の微生物を各MRS brothに前培養した。前培養した微生物の1%を250μg/ml濃度のパラクレゾールが添加されたMRS brothに24、48、72時間培養した。
インドキシル硫酸阻害能及びパラクレゾール阻害能が確認された菌株であるラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)菌株(受託番号:KCCM12169P)及びラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株(受託番号:KCCM12170P)の腎臓機能が低下する時、高リン酸血症を引き起こすリン吸収能力を評価した。微生物のリン吸収能力は、5‐ブロモ‐4‐クロロ‐3‐インドリルリン酸塩ジナトリウム塩(5‐bromo‐4‐chloro‐3‐indolyl phosphate disodium salt、sigma)を使用し、比色法で行った。 前記比色法によれば、リン酸塩を生育に利用する微生物の場合、青緑を示す。前記実験結果を図1に図示した。
(1)菌株あたりリン吸収率測定
試験例3で確認されたリン吸収能力を定量的に評価するために、菌株別リン吸収率を評価した。それぞれラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)(受託番号:KCCM12169P)及びラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)(受託番号:KCCM12170P)菌株をMRS brothに前培養した後、20mM phosphateを含むMRS培地にOD値が1.0になるように接種した。
ラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)(受託番号:KCCM12169P)及びラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)(受託番号:KCCM12170P)菌株の時間によるリン吸収率を測定するために、前記と同様の方法で9時間菌株を培養した。その結果、BP105菌株は9時間まで1.63mMのリンを吸収し(図3参照)、BP121菌株は3.51mMのリンを吸収したことが確認された(図4参照)。
雄鼠(SD rat)に7mg/kgのシスプラチン(Cisplatin)を腹腔投与して急性腎臓疾患モデルを樹立した。ラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)菌株(受託番号:KCCM12169P)及びラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株(受託番号:KCCM12170P)を各1×109CFU、そして2つの菌株を1:1の割合で混合して1×109CFUの濃度で作った混合物をシスプラチン誘導前10日、誘導後4日間経口投与して総14日投与した。実験は2週間実施し、14日目に実験動物を犠牲にした。シスプラチンで急性腎臓毒性を誘導して生体内でBP105とBP121のインドキシル硫酸抑制効能を評価するために、14日目に最終血中インドキシル硫酸濃度を測定した。その結果、BP105、BP121及びこれらの混合物の投与群は、皆急性腎臓毒性誘発群に比べてインドキシル硫酸の有意義な減少を示した(図5参照)。
ラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株(受託番号:KCCM12170P)の腎臓保護効果を評価するために、雄鼠(SD rat)に7mg/kgのCisplatinを腹腔投与して急性腎臓疾患を誘導した後、腎臓機能を評価した。BP121を各1×108、1×109、1×1010CFUの3グループに投与し、陽性対照群で尿毒症治療剤であるクレメジン(Kremezin)を0.5g/匹経口投与した。各グループは、Cisplatin誘導前10日、誘導後4日間経口投与して総14日投与し、対照群はPBSを同量で使用した。
試験例6でラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株の投与時、糞便で排出された短鎖脂肪酸の量の変化がBP121の効果であるか否かを確認するために、ラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)菌株とラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株の単一培養を通じて時間による短鎖脂肪酸の量を測定した。MRS brothにそれぞれ24時間前に培養した後、MRS brothにBP105及びBP121を各1%で接種して48時間培養して0、6、12、24、48時間の培養上等液を測定した。
Claims (16)
- 下記方法で嬰児の糞便から分離された菌株を含む腎臓疾患進行抑制及び予防用プロバイオティクス:
(a)生後12ヶ月以下の嬰児の糞便を滅菌水で希釈してMRS培地に接種して培養する段階;
(b)前記MRS培地で生成された乳白色の単一コロニーを継代培養する段階;及び
(c)前記(b)段階で継代培養されたコロニーから乳酸菌を選別して得る段階。 - 前記プロバイオティクスは、ラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)菌株(受託番号:KCCM12169P)及びラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株(受託番号:KCCM12170P)の中で選択される1種以上を含むことを特徴とする請求項1に記載の腎臓疾患進行抑制及び予防用プロバイオティクス。
- 前記プロバイオティクスは、1×101〜1×1015CFU/gの生菌含量を有することを特徴とする請求項1に記載の腎臓疾患進行抑制及び予防用プロバイオティクス。
- 請求項1ないし請求項3のいずれか一項に記載のプロバイオティクス;及びその培養物、濃縮物、ペースト化物、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物、液状物、希釈物及び破砕物の中で選択される1種以上;の中で選択される1種以上を含む腎臓疾患進行抑制及び予防用組成物。
- 前記組成物は、薬剤学的組成物または食品組成物であることを特徴とする請求項4に記載の腎臓疾患進行抑制及び予防用組成物。
- 前記薬剤学的組成物は、顆粒剤(GRANULES)、リモナーデ剤(LEMONADES)、散剤(POWDERS)、シロップ剤(SYRUPS)、液剤(LIQUIDS AND SOLUTIONS)、エキス剤(EXTRACTS)、エリキシル剤(ELIXIRS)、流動エキス剤(FLUIDEXTRACTS)、懸濁剤(SUSPESIONS)、煎剤(DECOCTIONS)、浸剤(INFUSIONS)、錠剤(TABLETS)、酒精剤(SPIRITS)、カプセル剤(CAPSULES)、トローチ剤(TROCHES)、丸剤(PILLS)、軟質または硬質ゼラチンカプセルの中で選択されるいずれか一つであることを特徴とする請求項5に記載の腎臓疾患進行抑制及び予防用組成物。
- ラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)菌株(受託番号:KCCM12169P)。
- 前記菌株は、インドキシル硫酸除去能、パラクレゾール除去能及びリン吸収能を有することを特徴とする請求項7に記載のラクトバチルスアシドフィルスBP105(Lactobacillus acidophilus BP105)菌株(受託番号:KCCM12169P)。
- 請求項7に記載の菌株;及びその培養物、濃縮物、ペースト化物、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物、液状物、希釈物及び破砕物の中で選択される1種以上;の中で選択される1種以上を含む腎臓疾患進行抑制及び予防用組成物。
- 前記組成物は、薬剤学的組成物または食品組成物であることを特徴とする請求項9に記載の腎臓疾患進行抑制及び予防用組成物。
- 前記薬剤学的組成物は、顆粒剤(GRANULES)、リモナーデ剤(LEMONADES)、散剤(POWDERS)、シロップ剤(SYRUPS)、液剤(LIQUIDS AND SOLUTIONS)、エキス剤(EXTRACTS)、エリキシル剤(ELIXIRS)、流動エキス剤(FLUIDEXTRACTS)、懸濁剤(SUSPESIONS)、煎剤(DECOCTIONS)、浸剤(INFUSIONS)、錠剤(TABLETS)、酒精剤(SPIRITS)、カプセル剤(CAPSULES)、トローチ剤(TROCHES)、丸剤(PILLS)、軟質または硬質ゼラチンカプセルの中で選択されるいずれか一つであることを特徴とする請求項10に記載の腎臓疾患進行抑制及び予防用組成物。
- ラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株(受託番号:KCCM12170P)。
- 前記菌株は、インドキシル硫酸除去能、パラクレゾール除去能及びリン吸収能を有することを特徴とする請求項12に記載のラクトバシラスサリバリウスBP121(Lactobacillus salivarius BP121)菌株(受託番号:KCCM12170P)。
- 請求項12に記載の菌株;及びその培養物、濃縮物、ペースト化物、噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物、液状物、希釈物及び破砕物の中で選択される1種以上;の中で選択される1種以上を含む腎臓疾患進行抑制及び予防用組成物。
- 前記組成物は、薬剤学的組成物または食品組成物であることを特徴とする請求項14に記載の腎臓疾患進行抑制及び予防用組成物。
- 前記薬剤学的組成物は、顆粒剤(GRANULES)、リモナーデ剤(LEMONADES)、散剤(POWDERS)、シロップ剤(SYRUPS)、液剤(LIQUIDS AND SOLUTIONS)、エキス剤(EXTRACTS)、エリキシル剤(ELIXIRS)、流動エキス剤(FLUIDEXTRACTS)、懸濁剤(SUSPESIONS)、煎剤(DECOCTIONS)、浸剤(INFUSIONS)、錠剤(TABLETS)、酒精剤(SPIRITS)、カプセル剤(CAPSULES)、トローチ剤(TROCHES)、丸剤(PILLS)、軟質または硬質ゼラチンカプセルの中で選択されるいずれか一つであることを特徴とする請求項15に記載の腎臓疾患進行抑制及び予防用組成物。
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