JP2022547435A - Pmas方法を利用したパーソナライズ腸内環境改善物質スクリーニング方法 - Google Patents
Pmas方法を利用したパーソナライズ腸内環境改善物質スクリーニング方法 Download PDFInfo
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- JP2022547435A JP2022547435A JP2022513541A JP2022513541A JP2022547435A JP 2022547435 A JP2022547435 A JP 2022547435A JP 2022513541 A JP2022513541 A JP 2022513541A JP 2022513541 A JP2022513541 A JP 2022513541A JP 2022547435 A JP2022547435 A JP 2022547435A
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Abstract
Description
実施例1.PMAS(Personalized Pharmaceutical Meta-Analysis Screening)技法を利用したパーソナライズ候補物質スクリーニングシステムの全般的な過程
本願は、個人の糞便などの試料を利用してパーソナライズプロバイオティクス、食品、健康機能性食品及び医薬品などを体外(in vitro)条件でスクリーニングするための組成物及び方法に関し、本願においては、上記スクリーニングシステムをPMAS(Personalized Pharmaceutical Meta-Analysis Screening)と称しながら説明する。
ヒト又は動物の糞便とPMAS培地とを1:12の割合で混合し、ストマッカー(stomacher)を利用して均質化した後、フィルタネットを利用して便の残余物は取り除く。プロバイオティクス、食品、健康機能性食品及び医薬品候補物質の処置に先立ち、便と培地が混ざった混合物を嫌気チャンバ内において4時間還元する。
嫌気チャンバ内において糞便と培地の均質化された混合物を96-ウェルプレートなどの培養プレートにそれぞれ同一量ずつ分注する。
処理するプロバイオティクス、食品、健康機能性食品及び医薬品候補物質は滅菌済みの1xPBSに懸濁させ、濃度と量を均一化して糞便-培地混合物が入っている培養プレートにそれぞれ分注する。
温度、湿度及びモーションを腸内環境と類似に形成したまま嫌気条件においてプレートを培養することで、各実験群を醗酵培養させる。
培養されたそれぞれの実験群を遠心分離することで上澄液と沈殿物(pallet)とを分離した後、上澄液から代謝体、短鎖脂肪酸、毒性物質などを分析し、沈殿物から腸内細菌叢の分析を行う。
上記実施例1のPMAS技法において、サンプル分析ステップの過程の一例示図を図2及び図3に示す。
上記実施例2のサンプル分析結果を基にパーソナライズプロバイオティクス、食品、健康機能性食品及び医薬品などをスクリーニングする過程の一例示図を図4に示す。
上記実施例1のPMAS技法で使用するためのPMAS培地の最適の組成を確認するために、下記のような実験を行った。
上記実施例1のPMAS技法において、嫌気培養ステップにおける最適の培養時間を確認するために、下記のような実験を行った。
本願のPMAS技法を利用し、個々人の腸内環境を体外(in vitro)条件で形成してパーソナライズ候補物質を正確にスクリーニングできるか否かの有効性を確認するために、下記の実験を行うことで確認した。
本願のPMAS技法を利用した分析結果が同一個体で繰り返して再現されるか否かを確認するために、下記のような実験を行った。
本願のPMAS技法を利用した分析結果が実際の臨床結果と同一性を示しているか否かを確認するために、下記のような実験を行った。
上記実施例1乃至実施例3及び実験例1乃至3で説明したPMAS技法を利用すれば、下記のように個々人の腸内環境を体外条件で迅速で且つ正確に分析し、それを基に腸内環境を改善できる候補物質をスクリーニングすることができる。下記の説明は、PMAS技法を利用したスクリーニングシステムの一例示であり、当該技術分野において通常の知識を有する者であれば、上記記載から様々な修正及び変形が可能である。例えば、説明された技術が説明された方法とは異なる順番で行われるか、及び/又は説明されたシステム、構造、装置、過程などの構成要素が説明された方法とは異なる形態で結合又は組み合わせられるか、他の構成要素又は均等物によって対置あるいは置換されても適切な結果が達成されることができる。
96-ウェルプレートに8つの互いに異なるヒトの糞便サンプルをそれぞれPMAS培地と混合及び均質化して同一量ずつ分配し(横軸)、上記糞便サンプルが分注された96-ウェルプレートに縦に互いに異なる候補物質を処理した(図9)。
上記(1)と同じ方法で100名の互いに異なる成人の糞便サンプルを利用してPMAS検査を行い、それによる酪酸量の変化を分析して、それを図10に示した。
上記(1)と同じ方法で100名の互いに異なる成人の糞便サンプルを利用してPMAS検査を行い、このうち一部のサンプルに対する腸内微生物多様性の変化を分析して、それを図11に示した。
上記(1)と同じ方法で100名の互いに異なる成人の糞便サンプルを利用してPMAS検査を行い、それによる初期糞便サンプルの微生物構成とPMAS検査後の酪酸量変化の相関関係を分析して、それを図12に示した。
Claims (11)
- L-システイン(L-cystein)を含む、腸内環境改善物質スクリーニング用組成物。
- 前記組成物は、ムチン(Mucin)をさらに含む、請求項1に記載の腸内環境改善物質スクリーニング用組成物。
- 前記組成物は、タンパク質及び炭水化物を含まないことを特徴とする、請求項1に記載の腸内環境改善物質スクリーニング用組成物。
- 前記腸内環境改善物質は、プロバイオティクス、プレバイオティクス、食品、健康機能性食品及び医薬品からなる群より選択される1つ以上である、請求項1に記載の腸内環境改善物質スクリーニング用組成物。
- (a)個体から得られた試料を第1項の組成物と混合するステップと、
(b)前記ステップ(a)の混合物に1つ以上の腸内環境改善候補物質を処理し、培養するステップと、
(c)前記ステップ(b)の培養物を分析するステップと
を含む、腸内環境改善物質スクリーニング方法。 - 前記方法は、体外(in vitro)条件で行うことを特徴とする、請求項5に記載の腸内環境改善物質スクリーニング方法。
- 前記ステップ(b)の培養は、嫌気条件で18時間~24時間行う、請求項5に記載の腸内環境改善物質スクリーニング方法。
- 前記ステップ(c)において培養物を分析することは、培養物に含まれた内毒素(endotoxin)、硫化水素(hydrogen sulfide)、短鎖脂肪酸(Short-chain fatty acids、SCFAs)及び腸内細菌叢来由代謝体のうち1つ以上の含量、濃度又は種類を分析する、請求項5に記載の腸内環境改善物質スクリーニング方法。
- 前記短鎖脂肪酸は、酢酸(Acetate)、プロピオン酸(Propionate)、酪酸(Butyrate)、イソ酪酸(Isobutyrate)、吉草酸(Valerate)、及びイソ吉草酸(Iso-valerate)からなる群より選択される1つ以上を含む、請求項8に記載の腸内環境改善物質スクリーニング方法。
- 前記ステップ(c)において培養物を分析することは、培養物に含まれた腸内微生物の種類、濃度、含量又は多様性変化を分析する、請求項5に記載の腸内環境改善候補物質スクリーニング方法。
- 前記方法は、
(d)前記ステップ(c)の分析結果を対照群の分析結果と比較して短鎖脂肪酸の含量を増加させたり、腸内細菌叢内の有益菌の種類及び含量を増加させたり、内毒素及び硫化水素の含量を減少させたり、あるいは腸内細菌叢内の有害菌の種類及び含量を減少させる候補物質を選別するステップ
をさらに含む、請求項5に記載の腸内環境改善候補物質スクリーニング方法。
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