JP2022063337A - 微生物共同体の投与により乳生産を改善する方法 - Google Patents
微生物共同体の投与により乳生産を改善する方法 Download PDFInfo
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Abstract
Description
本願は、2016年1月7日に出願された米国特許仮出願第62/276,142号、2016年1月8日に出願された米国特許仮出願第62/276,531号、2016年5月11日に出願された米国特許仮出願第62/334,816号、及び2016年11月1日に出願された米国特許仮出願第62/415,908号の優先権を主張し、それらの全体を参照により本明細書に援用する。
本開示は、とりわけ乳製品生産における用途をもつ、単離された生物学的に純粋な微生物に関する。本開示の微生物は、単離された生物学的に純粋な状態で利用することができ、また製剤して組成物とすることもできる。さらに、本開示は、本開示の微生物のメンバーを少なくとも2つは含む微生物共同体、ならびに該共同体を利用する方法を提供する。さらに、本開示は、第一胃ミクロビオームを調節する方法を提供する。
本願に関する配列一覧表を書類の代わりにテキスト形式で提供し、参照により本明細書に援用する。配列一覧表が収められたテキストファイル名は、ASBI_002_02US_SeqList_ST25.txtである。このテキストファイルは893kb、作成日は2016年10月31日であり、EFS-Webにより電子提出される。
本出願の一部の微生物を2016年4月25日1、United States Department of Agriculture(USDA)Agricultural Research Service(ARS)Culture Collection(NRRL(登録商標))(所在地 1815 N.University St.,Peoria,IL 61604,USA)に寄託した。本出願の一部の微生物を、Bigelow National Center for Marine Algae and Microbiota(所在地 60 Bigelow Drive,East Boothbay,Maine 04544,USA)に寄託した。
以下の各用語は当業者であれば十分理解していようが、以下の各定義は、本開示の主題の説明を容易にするために示すものである。
F.2d 1394,(CCPA 1970)(精製プロスタグランジンについて記述)、また、In re Bergy,596 F.2d 952(CCPA 1979)(精製微生物についての記述)、また、Parke-Davis & Co.v.H.K.Mulford & Co.,189 F.95(S.D.N.Y.1911)(Learned Handの精製アドレナリンについての記述)、aff’d in part,rev’d in part,196 F.496(2d Cir.1912)を参照されたい(これらをすべて参照により本明細書に援用する)。さらに、いくつかの態様では、本開示は、単離された生物学的に純粋な微生物培養物において見られるべき濃度つまり純度限界のある特定の量的尺度を提供する。これらの純度値の存在は、ある特定の実施形態では、本開示の微生物を自然な状態で存在する微生物と区別するさらなる属性である。たとえば、Merck & Co.v.Olin Mathieson Chemical Corp.,253 F.2d 156(4th Cir.1958)(微生物が生産したビタミンB12の純度限界について記述)を参照されたい(参照により本明細書に援用する)。
ECM=(0.327x乳ポンド数)+(12.95x脂肪ポンド数)+(7.2xタンパク質ポンド数)
et al.2014.Nature Rev.Micro.12:635-45)。
Laboratory Manual(2nd ed.,Cold Spring Harbor Laboratory Press,Plainview,New York)に開示されている。また、Innis et al.,eds.(1990)PCR
Protocols:A Guide to Methods and Applications(Academic Press,New York)、 Innis and Gelfand,eds.(1995)PCR Strategies(Academic Press,New York)、及びInnis and Gelfand,eds.(1999)PCR Methods Manual(Academic Press,New York)も参照されたい。知られているPCR方法としては、限定ではないが、ペアのプライマー、入れ子式プライマー、単一特異的プライマー、変性プライマー、遺伝子特異的プライマー、ベクター特異的プライマー、部分ミスマッチプライマー等を用いる方法が挙げられる。
関係の総リストのファイルをリンクとして読み込む
いくつかの態様では、本開示は、表1及び表3に示す、微生物の新規株を含む単離微生物を提供する。
いくつかの態様では、本開示は、表1及び/または表3で特定される微生物から選択される少なくとも2微生物の組合せを含む微生物共同体を提供する。
本開示の微生物は、たとえば、米国内の様々な場所で、ウシの胃腸管から入手した。
表1及び表3の微生物を、16s rRNA配列については分類ツールRibosomal Database Project(RDP)を用いて、ITS rRNA配列についてはUser-friendly Nordic ITS Ectomycorrhiza(UNITE)データベースを用いて、最も近い分類学的グループとマッチさせた。微生物を最も近い分類群にマッチングする例は、Lan et al.(2012.PLOS one.7(3):e32491),Schloss and Westcott(2011.Appl.Environ.Microbiol.77(10):3219-3226)、及びKoljalg et al.(2005.New Phytologist.166(3):1063-1068)に見出すことができる。
for General and Molecular Microbiology.American Society for Microbiology,Washington,D.C.(1994)及びLennette,E.H.(ed.)Manual of Clinical Microbiology,Third Edition.American Society for Microbiology,Washington,D.C.(1980)で見出すことができ、これらを参照により本明細書に援用する。
微生物は、あらゆる有効な表現型データ及び遺伝子型データをコンセンサス分類に組み込む多相分類学に基づいて、属に区分することができる(Vandamme et al.1996.Polyphasic taxonomy,a consensus approach to bacterial systematics.Microbiol Rev 1996,60:407-438)。種を定義するための、一般に認められている1つの遺伝子型法は、全体的ゲノム関連性に基づくものであり、DNA-DNAハイブリダイゼーションにより、標準条件下、ΔTm(相同及び異種ハイブリッド間の溶解温度の差)は5℃以下で、およそ70%以上の関連性がある株を、同種のメンバーとみなす。したがって、この70%閾値超を共有する集団を、同種のバリアントとみなすことができる。種を定義するための、一般に認められている別の遺伝子型法は、本開示のマーカー遺伝子を単離し、これらの遺伝子を配列決定し、これらの配列決定した複数の単離物またはバリアントからの遺伝子をアラインメントすることである。それらの微生物は、配列決定した遺伝子の1つ以上が少なくとも97%の配列同一性を有する場合、同種に属すると解釈される。
いくつかの実施形態では、本開示の微生物は微生物組成物中に混合される。
1~26 1~24 1~22 1~20 1~18 1~16 1~14 1~12 1~10 1~8 1~6 1 1 4 1~2 4~36 4~34 4~32 4~30 4~28 4~26 4~24 4~22 4~20 4~18 4~16 4~14 4~12 4~10 4~8 4~6 6~36 6~34 6~32 6~30 6~28 6~26 6~24 6~22 6~20 6~18 6~16 6~14 6~12 6~10 6~8 8~36 8~34 8~32 8~30 8~28
8~26 8~24 8~22 8~20 8~18 8~16 8~14 8~12
8~10 10~36 10~34 10~32 10~30 10~28 10~26 10~24 10~22 10~20 10~18 10~16 10~14 10~12 12~36 12~34 12~32 12~30 12~28 12~26 12~24 12~22 12~20 12~18 12~16 12~14 14~36 14~34 14~32 14~30 14~28 14~26 14~24 14~22 14~20 14~18 14~16 16~36 16~34 16~32 16~30 16~28 16~26 16~24 16~22 16~20 16~18 18~36 18~34 18~32 18~30 18~28 18~26 18~24 18~22 18~20 20~36 20~34 20~32 20~30 20~28 20~26 20~24 20~22 22~36 22~34 22~32 22~30 22~28 22~26 22~24 24~36 24~34 24~32 24~30 24~28 24~26 26~36 26~34 26~32 26~30 26~28 28~36 28~34 28~32 28~30 30~36 30~34 30~32 32~36 32~34、または約34~36の期間は保存可能である。
いくつかの実施形態では、本開示の微生物または微生物組成物は、封入組成物に封入されている。封入組成物は、有蹄動物の胃腸管に入るまで微生物を外部ストレッサーから保護する。封入組成物はさらに、有酸素環境が嫌気性微生物に及ぼす酸化ストレスを最小限化するなど、微生物にとって有益であり得る環境を作る。微生物の封入組成物、及び微生物を封入する方法については、Kalsta et al.(米国特許第5,104,662A号)、Ford(同第5,733,568A号)、及びMosbach and Nilsson(同第4,647,536A号)を参照されたい。
いくつかの実施形態では、本開示の組成物を動物飼料と混合する。いくつかの実施形態では、動物飼料は、ペレット、カプセル、顆粒、粉末、液体、または半液体など、様々な形態で存在し得る。
いくつかの実施形態では、本開示の微生物組成物は、経口経路で反芻動物に投与される。いくつかの実施形態では、微生物組成物は、胃腸管への直接注入経路で投与される。さらなる実施形態では、直接注入による投与で、微生物組成物は第一胃に直接送達される。いくつかの実施形態では、本開示の微生物組成物は、動物に肛門経由で投与される。さらなる実施形態では、肛門投与は坐剤挿入という形をとる。
1967.Weeds.Vol.15,pp.20-22を参照されたい(その内容を参照により本明細書に援用する)。したがって、「相乗的」とは、相加量よりも増加した結果/パラメーター/効果を反映することを意図する。
本明細書では、「微生物」という用語は、広義に解釈すべきである。この用語には、限定ではないが、原核生物の2ドメインの細菌及び古細菌、ならびに真核生物の真菌、原生生物、及びウイルスが含まれる。
いくつかの実施形態では、第一胃ミクロビオームなどの反芻動物のミクロビオームは、様々な代謝能力をもつ多様な微生物の集積を含む。ミクロビオームは、食餌、動物の代謝の変化、品種など様々な要因に影響される。ほとんどのウシの食餌は植物系であり、食餌に含まれる特定の高分子成分を分解できる微生物の胃腸微生物群集を増強する複合ポリサッカライドが豊富である。一次分解の最終産物には微生物連鎖が保持されており、これら微生物は最終的に様々な有機酸を水素及び二酸化炭素と一緒に生産する。ミクロビオームは複合的で相互連鎖的な性質なので、食餌の変化、したがって一次分解の基質の変化は、第一胃微生物の代謝にカスケード的効果を与え、有機酸プロファイルと生産メタンレベルの両方が変化し得、したがって動物の生産及び/または動物が生産する産物の質及び量に影響を及ぼし得る。Menezes et al.(2011.FEMS Microbiol.Ecol.78(2):256-265.)を参照されたい。
50Sリボソームタンパク質L20(rplT)、ruvAホリデイジャンクションDNAヘリカーゼ、ruvBホリデイジャンクションDNAヘリカーゼB、serSセリルtRNAシンセターゼ、rplU 50Sリボソームタンパク質L21、rpsR 30Sリボソームタンパク質S18、DNAミスマッチ修復タンパク質MutS、rpsT 30Sリボソームタンパク質S20、DNA修復タンパク質RecN、frrリボソームリサイクリング因子(frr)、組換タンパク質RecR、未知機能タンパク質UPF0054、miaA tRNAイソペンテニルトランスフェラーゼ、GTP結合性タンパク質YchF、染色体複製イニシエータータンパク質DnaA、デホスホ-CoAキナーゼ、16S rRNA加工タンパク質RimM、ATP-coneドメインタンパク質、1-デオキシ-D-キシルロース5-リン酸レダクトイソメラーゼ、2C-メチル-D-エリスリトール2,4-シクロジホスファートシンターゼ、脂肪酸/リン脂質合成タンパク質PlsX、tRNA(Ile)ライシジンシンセターゼ、dnaG DNAプライマーゼ(dnaG)、ruvCホリデイジャンクションリゾルバーゼ、rpsP 30Sリボソームタンパク質S16、リコンビナーゼA recA、リボフラビン生合成タンパク質RibF、グリシルtRNAシンセターゼベータサブユニット、trmU tRNA(5-メチルアミノメチル-2-チオウリジラート)-メチルトランスフェラーゼ、rpmI
50Sリボソームタンパク質L35、hemEウロポルフィリノーゲンデカルボキシラーゼ、Rod形状決定タンパク質、rpmA 50Sリボソームタンパク質L27(rpmA)、ペプチジルtRNAヒドロラーゼ、翻訳開始因子IF-3(infC)、UDP-N-アセチルムラミル-トリペプチドシンセターゼ、rpmF 50Sリボソームタンパク質L32、rpIL 50Sリボソームタンパク質L7/L12(rpIL)、leuSロイシルtRNAシンセターゼ、ligA NAD依存性DNAリガーゼ、細胞分裂タンパク質FtsA、GTP結合性タンパク質TypA、ATP依存性Clpプロテアーゼ、ATP結合性サブユニットClpX、DNA複製及び修復タンパク質RecF、ならびにUDP-N-アセチレノールピルボイルグルコサミンレダクターゼが挙げられる。
the intestinal microbiota using SOLiD 16S rRNA gene sequencing and SOLiD shotgun sequencing.BMC Genomics,14(Suppl 5):S16;Mardis(2008)Next-generation DNA sequencing methods.Annu Rev Genomics Hum Genet,9:387-402;これらすべての内容をあらゆる目的で参照により本明細書に援用する)。配列を決定する試料からDNA断片のライブラリーを調製して、クローンビーズ集団の調製に用いるが、ここで各磁気ビーズ表面には1種の断片だけが存在することになる。磁気ビーズに結合する断片は、ユニバーサルP1アダプター配列を有するので、各断片の開始配列は既知かつ同一である。プライマーは、ライブラリー鋳型内のP1アダプター配列にハイブリダイズする。4つ一組の蛍光標識二塩基プローブが、シーケンシングプライマーへのライゲーションについて競合する。二塩基プローブの特異性は、各ライゲーション反応の第1と第2の塩基を全て調べることで得られる。ライゲーション、検出、及び切断の複数のサイクルを実行し、サイクル数が最終的なリード長を決める。SOLiDプラットフォームは、1回のランで、最大30億のリードを、75塩基のリード長で、生産できる。対合末端シーケンシングが可能であり、本明細書で用いることができるが、対の第2のリード長はわずか35塩基である。試料の多重増幅は、別途指標ランがある、Illuminaで使用するものと類似したシステムにより、可能である。
いくつかの態様では、本開示は、本明細書で説明する1つ以上の微生物組成物をウシに投与して、胃腸管の病原性微生物を排除することに関する。いくつかの実施形態では、本開示はさらに、本明細書で説明する微生物組成物を投与して、病原性微生物の胃腸管におけるコロニー形成を予防することに関する。いくつかの実施形態では、本明細書で説明する微生物組成物の投与により、さらに、ウシの外皮及び気道から病原体が排除され、及び/または外皮及び気道での病原体コロニー形成が予防される。いくつかの実施形態では、本明細書で説明する微生物組成物の投与により、リーキーガット/腸からの浸出、炎症、及び/または肝疾患発生が軽減する。
sp.、Bacillus cereus、Bacillus licheniformis、Streptococcus uberis、Staphylococcus aureus、ならびにEscherichia coli及びStaphylococcus aureusの病原性株を含み得る。いくつかの実施形態では、病原性微生物は、ウイルス病原体を含む。いくつかの実施形態では、病原性微生物は、ウシとヒト両方にとって病原性である。いくつかの実施形態では、病原性微生物は、ウシかヒトのいずれかにとって病原性である。
いくつかの態様では、本開示は、本明細書で説明する微生物組成物を反芻動物に投与して、乳生産、乳量、乳質、反芻動物の消化化学、ならびに飼料利用及び消化の効率の側面の調節により1つ以上の形質を改善することに関する。
第一胃は、反芻動物において飼料成分を消化するための特別な胃である。多様な微生物集団が第一胃内に生息しており、それらの主な機能は繊維質及び非繊維質の炭水化物成分を利用可能なエネルギー源及びタンパク質源に変換することである(図16)。なかでもセルロースは、植物バイオマスの最大40%を占め、哺乳類には消化できないと考えられている。ヘミセルロース、ペクチン、及びリグニンなど他の構造性炭水化物とも密接な関係がある。第一胃内のセルロース分解性微生物は、強力な酵素活性を利用して、これらの分子を単糖及び揮発性脂肪酸に分解する。この酵素活性は飼料からのエネルギー抽出に重要であり、より高効率な分解は最終的により多くのエネルギーを動物に与える。飼料の非繊維質部分に見られる可溶性糖はまた、発酵して、ガスや、酪酸エステル(butyrate)、プロピオン酸エステル(propionate)、及び酢酸エステル(acetate)などの揮発性脂肪酸になる。飼料の繊維質及び非繊維質両方の成分の消化で生じる揮発性脂肪酸は、最終的に、反芻動物の主なエネルギー源となる。
ネットワーク及び/またはクラスター分析法は、一実施形態では、ネットワーク(共通または類似の環境パラメーターを共有する2つ以上の試料の総体)内の1つ以上の株の関連性を測定するのに用いられる。一実施形態では、ネットワーク分析には、リンケージ分析、モジュラリティー分析、頑強性測定、介在性測定、関連性測定、推移性測定、中心性測定またはそれらの組合せが含まれる。別の実施形態では、ネットワーク分析には、リンク・マイニング及び予測、ソーシャル・ネットワーク・セオリー、集団分類、リンクベースのクラスタリング、関係類似性、またはそれらの組合せによるネットワークの予測モデリングが含まれる。別の実施形態では、ネットワーク分析には、関連性を構築するため、相互情報量、最大情報係数(MIC)の計算、または他の変数間ノンパラメトリック方法が含まれる。別の実施形態では、ネットワーク分析には、微分方程式ベースの集団のモデリングが含まれる。さらに別の実施形態では、ネットワーク分析にはLotka-Volterraモデリングが含まれる。
実施例Iの方法は、泌乳反芻動物が生産する乳脂肪及び乳タンパク質の総量、ならびにECM計算値の増加を目的とする。
本開示のある特定の実施形態では、本方法は、泌乳反芻動物が生産する乳脂肪及び乳タンパク質の総量の増加を目的とする。
ウシを無作為に8頭ずつ15群に割り当て、1つの群を対照群として微生物共同体を含まない緩衝液を与える。残りの7群は実験群であり、それぞれ13の微生物バイオ共同体のうちの1つを1日1回、6週間にわたり投与される。ウシは、相互汚染を避けるために1頭ずつ囲いに収容し、飼料と水を自由に摂らせる。餌は、高乳収量用の餌である。ウシに1日2回給餌し、給餌のたびに飼料の重量を測定し、前日残した飼料の重量を測定して廃棄する。重量測定には、Salter Brecknell(Fairmont,MN)の秤PS-2000を用いる。
本開示のある特定の実施形態では、本方法は、1つ以上の微生物を反芻動物の胃腸管に投与することにより、反芻動物のミクロビオームを調節することを目的とする。
乳牛の乳脂肪生産に影響する第一胃微生物群集の構成員を決定する。
本出願で用いられるAscus Biosciencesの技術の一構成要素は、相互情報量を利用して、動物の胃腸管に生息する天然の微生物株の、動物の特定の形質に対する重要性を順位付けする。最大情報係数(MIC)スコアを全微生物と動物の所望の形質について計算する。1が微生物株と動物の形質との強い関係を表し、0が無関係を表す、0から1のスケールで、関係をスコア付けした。このスコアに基づくカットオフにより、特定の形質の改善について有用な微生物と非有用な微生物とを定める。図9及び図10は、乳牛の乳脂肪効率との関係を共有する第一胃微生物株のMICスコア分布を示す。曲線が指数的から線形に移行する点(細菌では約0.45~0.5、真菌では約0.3)は、乳脂肪効率に関する有用な微生物と非有用な微生物間のカットオフ点を表す。図11及び図12は、酪農効率との関係を共有する第一胃微生物株のMICスコア分布を示す。曲線が指数的から線形に移行するポイント(細菌では約0.45~0.5、真菌では約0.25)は、有用な微生物と非有用な微生物間のカットオフ点を表す。
Ascus Biosciencesの技術を用いて、現在入手が可能な微生物飼料添加物の性能を予測した。
MIC計算値は、Lactobacillus plantarumは乳脂肪効率との関連性が低いと予測しており、当業界はL.plantarumを接種しても乳脂肪生産は増加しないことを開示しており、少なくとも1つの研究が、L.plantarumのいくつかの株は乳脂肪低下の原因となる分子を生じることを開示している。Lee et
al.2007.J.Appl.Microbiol.103(4):1140-1146及びMohammed et al.2012.J.Dairy Sci.95(1):328-339を参照されたい。
MIC計算値は、Lactobacillus acidophilusは乳脂肪効率との関連性が低いと予測しており、当業界はL.acidophilusを乳牛/仔牛に投与しても乳収量/乳成分収量の様々な側面に対し効果がなかったことを開示している。Higginbotham and Bath.1993.J.Dairy Sci.76(2):615-620、Abu-Tarboush et al.1996.Animal Feed Sci.Tech.57(1-2):39-49;McGilliard and Stallings.1998.J.Dairy Sci.81(5):1353-1357、及びRaeth-Knight et al.2007.J.Dairy Sci.90(4):1802-1809を参照されたいが、Boyd et al.2011.94(9):4616-4622も参照されたい(乳収量及び乳タンパク質収量の増加を開示している)。Boyd et al.は、乳及び乳タンパク質の収量の増加を開示してはいるが、この一研究の対照は、増加の原因としてL.acidophilusの存在を適切に切り離しているようには見えない。先行技術の主論はBoyd
et al.の知見と一致しない。
MIC計算値は、Megasphaera elsdeniiは乳脂肪効率との関連性が低いと予測しており、当業界はMegasphaera elsdeniiは乳脂肪効率に関し好ましい効果がないことを示す確固たる証拠を提供しているが、複数の参考文献が、乳脂肪効率について負の効果があることを示している。Kim et al.2002.J.Appl.Micro.92(5):976-982;Hagg.2008.Dissertation,University of Pretoria.1-72;Hagg et al.2010.S.African.J.Animal Sci.40(2):101-112;Zebeli et al.2011.J.Dairy Res.79(1):16-25、Aikman et al.2011.J.Dairy Sci.94(6):2840-2849;Mohammed et al.2012.J.Dairy Sci.95(1):328-339、及びCacite and Weimer.2016.J.Animal Sci.Poster Abstract.94(sup.5):784を参照されたい。
MIC計算値は、Prevotella bryantiiは乳脂肪効率と高い関連性はないと予測しており、当業界はP.bryantiiを亜急性酸血症の攻撃中、泌乳中期の乳牛に投与しても、乳収量に特に効果がないことの証拠を提供しているが、この微生物を泌乳初期の乳牛に投与すると、乳脂肪濃度の改善が得られる。それぞれ、Chiquette et al.2012.J.Dairy Sci.95(10):5985-5995と、Chiquette et al.2008.91(9):3536-3543を参照されたい。
この実施例の方法は、泌乳反芻動物が生産する乳脂肪及び乳タンパク質の総量、ならびにエネルギー補正乳(ECM)計算値の増加を目的とする。
A.揮発性脂肪酸(VFA)の生産
株が揮発性脂肪酸を生産する能力を評価するため、高速液体クロマトグラフィー(HPLC)を用いて消費培地での酢酸エステル、酪酸エステル、及びプロピオン酸エステルの濃度を測定した。アッセイでは第一胃の条件を可能なかぎり模倣してM2GSC培地を用いた。
株が様々な炭素源を分解する能力を評価するために、光学密度(OD600)を用いて、複数の炭素源での経時的な株の増殖を測定した。
株が様々な不溶性炭素源を分解する能力を評価するために、株の分解能力を目視により質的に判定した。
実験計画及び材料及び方法
目的:乳牛の乳脂肪生産に影響する第一胃微生物群集の構成員の決定
相対数度と絶対細胞数
上位15ターゲット種を、細胞数データを含むデータセットについて(絶対細胞数、表21)、また、MICスコアに基づいて、細胞数データを含まないデータセット(相対数度、表20)について特定した。細胞数データが最終的なターゲット選択に及ぼす影響を切り離すために、活性データはこの分析では用いなかった。結局、2つのデータセット間で上位8ターゲットが同じであった。残りの7株のうち、5株が異なる順位で両リストに登場した。これら5株は、順位は異なっていたが、各株のMICスコア計算値は、2つのリストで同一であった。絶対細胞数リストには登場するが、相対数度リストには登場しない2株、すなわちascus_111とascus_288は、それぞれ相対数度リストでは91位と16位であった。相対数度リストには登場するが、絶対細胞数リストには登場しない2株、すなわちascus_102とascus_252は、それぞれ絶対細胞数リストでは50位と19位であった。これらの4株は、各リストで異なるMICスコアを有しており、順位の変動と、それに伴うリスト内の他の株への影響を説明するものである。
活性データに基づき株をフィルタリングする影響を評価するために、ターゲット種を、活性データとともに(表22)、及び活性データ無しで(表20)相対数度を用いたデータセット、ならびに活性データとともに(表23)、及び活性データ無して(表21)絶対細胞数を用いたデータセットから、特定した。
最終的に、本明細書で定める方法は、細胞数データと活性データの両方を使って、関係のあるメタデータ特徴と強い関連のある微生物を特定する。両方法により選択した上位15ターゲット(表23、表20)のうち、両リストに見られるのは7株だけである。8株(53%)は、絶対細胞数リストと活性リストでそれぞれ特有であった。両リストの上位3ターゲットは、株も順位も一致していた。ところが、3株のうち2株は両リストで同じMICスコアをもたず、順位が変わるほどではないが、活性データセットの統合が影響したことを示唆している。
微生物群集を分析する従来の方法は、活性情報は組み込まず、相対数度データの使用に依存するものであり、結局は微生物種とメタデータの単純な相関をとるだけである(たとえば、米国特許第9,206,680号を参照されたい。この内容をあらゆる目的で参照により本明細書に援用する)。本明細書では、各データセットの組込みが、最終的なターゲットリストにいかに増分的に影響するかを示した。本明細書で説明する方法を全て用いると、従来の方法と比べて全く異なるターゲットセットが選択された(表24a及び表24c)。従来の方法で選択された第1位ターゲット株Ascus_3038を乳脂肪に対してプロットして相関の強さを視覚化した(図21)。前述の例と同じく、Ascus_3038も乳脂肪との相関性が弱いことが示された。
表24:相互情報量または相関による上位15ターゲット株
本開示が意図する主題は、以下の番号付けされた実施形態に示される。
a)配列番号32と少なくとも約97%同一であるITS核酸配列を有する真菌を含む精製されたPichia真菌集団、及び
b)反芻動物への投与に適した保存可能な担体を含み、
前記a)の精製されたPichia真菌集団は、前記サプリメントを投与されていない反芻動物と比べて、前記サプリメントを投与された反芻動物の乳生産を増加させるかまたは乳成分特徴を改善するのに有効な量で前記サプリメント中に存在する、前記保存可能な反芻動物用サプリメント。
i.配列番号1~30及び2045~2103からなる群より選択される核酸配列と少なくとも約97%同一である16S核酸配列を有する細菌を含む精製された細菌集団、及び/または
ii.配列番号31、33~60及び2104~2107からなる群より選択される核酸配列と少なくとも約97%同一であるITS核酸配列を有する真菌を含む真菌の精製集団を含む、請求項1に記載の保存可能な反芻動物用サプリメント。
a)NRRL Y-67249で寄託された精製された真菌集団、及び
b)反芻動物への投与に適した担体を含み、
前記a)の精製された真菌集団は、前記組成物を投与されていない反芻動物と比べて、前記組成物を投与された反芻動物の乳生産を増加させるかまたは乳成分特徴を改善するのに有効な量で前記組成物中に存在している、前記組成物。
a)NRRL Y-67249で寄託された精製された真菌集団、
b)NRRL B-67248で寄託された精製された細菌集団、及び
c)反芻動物への投与に適した担体を含み、
前記a)の精製された真菌集団及び前記b)の精製された細菌集団は、前記組成物を投与されていない反芻動物と比べて、前記組成物を投与された反芻動物の乳生産を増加させるかまたは乳成分特徴を改善するのに有効な量で前記組成物中に存在している、前記組成物。
本開示が意図する主題は、以下の番号付けされた実施形態に示される。
a)配列番号32と少なくとも約97%同一であるITS核酸配列を有する真菌を含む精製されたPichia真菌集団、及び
b)反芻動物への投与に適した担体を含み、
前記a)の精製されたPichia真菌集団は、前記組成物を投与された反芻動物の第一胃のミクロビオームを変化させるのに有効な量で、前記組成物中に存在している、前記組成物。
a)
i.配列番号1~30及び2045~2103からなる群より選択される核酸配列と少なくとも約97%同一である16S核酸配列を有する細菌を含む精製された細菌集団、及び/または
ii.配列番号31~60及び2104~2107からなる群より選択される核酸配列と少なくとも約97%同一であるITS核酸配列を有する真菌を含む真菌の精製集団を含む、
精製された微生物集団、及び
b)反芻動物への投与に適した担体を含み、
前記有効量の前記組成物を投与された前記反芻動物は、第一胃のミクロビオーム内の変化を示す、前記方法。
本開示が意図する主題は、以下の番号付けされた実施形態に示される。
a)有効量の保存可能な反芻動物用サプリメントを反芻動物に投与することを含み、前記サプリメントは、
i.配列番号1~60及び2045~2107からなる群より選択される核酸配列と少なくとも約97%同一である16S核酸配列を有する細菌及び/またはITS核酸配列を有する真菌を含む精製された微生物集団であって、前記細菌は少なくとも約0.4のMICスコアを有し、前記真菌は少なくとも約0.2のMICスコアを有する、前記微生物集団、及び
ii.反芻動物への投与に適した保存可能な担体を含み、
前記細菌または真菌の少なくとも一方は、炭素源を酢酸エステル、酪酸エステル、プロピオン酸エステル、またはそれらの組合せからなる群より選択される揮発性脂肪酸に変換することができ、前記細菌または真菌の少なくとも一方は、可溶性または不溶性炭素源を分解することができ、
前記有効量の前記保存可能な反芻動物用サプリメントを投与された前記反芻動物は、前記反芻動物用サプリメントを投与されていない反芻動物と比べて、乳生産の増加または乳成分特徴の改善を示す、前記方法。
前記反芻動物用サプリメントの投与前に第一胃に存在していた微生物集団は、前記反芻動物用サプリメントの投与後に数度が増加する、請求項1に記載の方法。
前記反芻動物用サプリメントの投与前に第一胃に存在していた微生物集団は、前記反芻動物用サプリメントの投与後に数度が減少する、請求項1に記載の方法。
前記反芻動物用サプリメントの投与前に第一胃に存在していた微生物の第1の集団は、前記反芻動物用サプリメントの投与後に数度が増加し、
前記反芻動物用サプリメントの投与前に第一胃に存在していた微生物の第2の集団は、前記反芻動物用サプリメントの投与後に数度が減少する、請求項1に記載の方法。
本明細書で引用した参照文献、論文、発行物、特許、特許公報、及び特許出願は全て、あらゆる目的でそれらの全体を参照により本明細書に援用する。
特定の実施形態では、例えば以下の項目が提供される。
(項目1)
反芻動物に有効量の組成物を投与することを含む、反芻動物の乳生産を増加させるかまたは乳成分特徴を改善する方法であって、前記組成物は、
a)
i.配列番号1~30及び2045~2103からなる群より選択される核酸配列と少なくとも約97%同一である16S核酸配列を有する細菌を含む精製された細菌集団、及び/または
ii.配列番号31~60及び2104~2107からなる群より選択される核酸配列と少なくとも約97%同一であるITS核酸配列を有する真菌を含む真菌の精製集団を含む、
精製された微生物集団、及び
b)反芻動物への投与に適した担体を含み、
前記有効量の前記組成物を投与された前記反芻動物は、前記組成物を投与されていない反芻動物と比べて、乳生産の増加または乳成分特徴の改善を示す、
前記方法。
(項目2)
前記反芻動物はウシである、項目1に記載の方法。
(項目3)
前記組成物は周囲条件下で少なくとも1日は安定している、項目1に記載の方法。
(項目4)
前記組成物は、封入物、錠剤、カプセル、丸薬、飼料添加物、食品成分、食品添加物、食品調製物、食品サプリメント、消耗溶液、消耗スプレー添加物、消耗固体、消耗ゲル、注射、坐剤、ボーラス、水薬、またはそれらの組合せとして製剤されている、項目1に記載の方法。
(項目5)
前記組成物はポリマーまたは炭水化物に封入されている、項目1に記載の方法。
(項目6)
投与することには、前記組成物を反芻動物に給餌することが含まれる、項目1に記載の方法。
(項目7)
投与することには、前記組成物を反芻動物に注入することが含まれる、項目1に記載の方法。
(項目8)
前記精製された微生物集団は、前記組成物中に少なくとも102細胞の濃度で存在する、項目1に記載の方法。
(項目9)
前記精製された微生物集団は、配列番号1~30及び2045~2103からなる群より選択される核酸配列と少なくとも約97%同一である16S核酸配列を有する細菌を含む、項目1に記載の方法。
(項目10)
前記精製された微生物集団は、配列番号31~60及び2104~2107からなる群より選択される核酸配列と少なくとも約97%同一であるITS核酸配列を有する真菌を含む、項目1に記載の方法。
(項目11)
前記精製された微生物集団は、配列番号1~30及び2045~2103からなる群より選択される核酸配列と少なくとも約99%同一である16S核酸配列を有する細菌を含む、項目1に記載の方法。
(項目12)
前記精製された微生物集団は、配列番号31~60及び2104~2107からなる群より選択される核酸配列と少なくとも約99%同一であるITS核酸配列を有する真菌を含む、項目1に記載の方法。
(項目13)
前記精製された微生物集団は、配列番号1~30及び2045~2103からなる群より選択される16S核酸配列を有する細菌を含む、項目1に記載の方法。
(項目14)
前記精製された微生物集団は、配列番号31~60及び2104~2107からなる群より選択されるITS核酸配列を有する真菌を含む、項目1に記載の方法。
(項目15)
前記精製された微生物集団は、配列番号1~60及び2045~2107からなる群より選択される核酸配列と少なくとも約97%同一である16S核酸配列を有する細菌及びITS核酸配列を有する真菌を含む、項目1に記載の方法。
(項目16)
前記精製された微生物集団は、配列番号28と少なくとも約97%同一である16S核酸配列を有する細菌を含む、項目1に記載の方法。項目1に記載の方法。
(項目17)
前記精製された微生物集団は、配列番号32と少なくとも約97%同一であるITS核酸配列を有する真菌を含む、項目1に記載の方法。
(項目18)
前記精製された微生物集団は、NRRL Y-67249で寄託されたPichia真菌を含む、項目1に記載の方法。
(項目19)
前記精製された微生物集団は、
Intestinimonas、Anaerolinea、Pseudobutyrivibrio、Olsenella、Eubacterium、Catenisphaera、Faecalibacterium、Solobacterium、Blautia、Ralsonia、Coprococcus、Casaltella、Anaeroplasma、Acholeplasma、Aminiphilus、Mitsuokella、Alistipes、Sharpea、Oscillibacter、Neocallimastix、Odoribacter、Pichia、Tannerella、Candida、Hydrogenoanaerobacterium、Orpinomyces、Succinivibrio、Sugiyamaella、Ruminobacter、Lachnospira、Caecomyces、Sinimarinibacterium、Tremella、Hydrogenoanaerobacterium、Turicibacter、Clostridium_XlVa、Anaerolinea、Saccharofermentans、Butyricicoccus、Olsenella、Papillibacter、Clostridium_XIa、Pelotomaculum、Erysipelotrichaceae_incertae_sedis、Lachnospiracea_incertae_sedis、Solobacterium、Anaeroplasma、Ralstonia、Clostridium_sensu_stricto、Eubacterium、Rikenella、Lachnobacterium、Tannerella、Acholeplasma、Howardella、Selenomonas、Butyricimonas、Sharpea、Succinivibrio、Ruminobacter、Candida、Syntrophococcus、Pseudobutyrivibrio、Orpinomyces、Cyllamyces、Saccharomycetales、Phyllosticta、Ascomycota、及びPiromycesから選択される群のメンバーである生物のみを含む、項目1に記載の方法。
(項目20)
前記有効量の前記組成物を投与された前記反芻動物は、実測乳収量の増加につながる乳生産の増加を示す、項目1に記載の方法。
(項目21)
前記有効量の前記組成物を投与された前記反芻動物は、実測エネルギー補正乳の増加につながる乳生産の増加及び乳成分特徴の改善を示す、項目1に記載の方法。
(項目22)
前記有効量の前記組成物を投与された前記反芻動物は、乳脂肪の増加、乳タンパク質の増加、乳中炭水化物の増加、乳中ビタミンの増加、乳中ミネラルの増加、またはそれらの組合せからなる群より選択される乳成分特徴の改善を示す、項目1に記載の方法。
(項目23)
前記有効量の前記組成物を投与された前記反芻動物は、乳脂肪、乳タンパク質、エネルギー補正乳、またはそれらの組合せの平均生産の少なくとも1%の増加を示す、項目1に記載の方法。
(項目24)
前記有効量の前記組成物を投与された前記反芻動物は、乳脂肪、乳タンパク質、エネルギー補正乳、またはそれらの組合せの平均生産の少なくとも10%の増加を示す、項目1に記載の方法。
(項目25)
前記有効量の前記組成物を投与された前記反芻動物は、乳脂肪、乳タンパク質、エネルギー補正乳、またはそれらの組合せの平均生産の少なくとも20%の増加を示す、項目1に記載の方法。
(項目26)
前記有効量の前記組成物を投与された前記反芻動物はさらに、改善された飼料利用効率、改善された消化率、ポリサッカライド及びリグニン分解率の増加、第一胃内の脂肪酸濃度の上昇、第一胃内のpHバランス、メタン放出量の減少、糞生産の減少、改善された乾物摂取量、改善された窒素利用効率、またはそれらの組合せからなる群より選択される少なくとも1つの改善された表現型形質を示す、項目1に記載の方法。
(項目27)
前記有効量の前記組成物を投与された前記反芻動物は、さらに、第一胃のミクロビオームの変化を示す、項目1に記載の方法。
(項目28)
前記有効量の前記組成物を投与された前記反芻動物は、さらに、第一胃のミクロビオームの変化を示し、
前記組成物の投与前に第一胃に存在していた微生物集団は、前記組成物の投与後に数度が増加する、項目1に記載の方法。
(項目29)
前記有効量の前記組成物を投与された前記反芻動物は、さらに、第一胃のミクロビオームの変化を示し、
前記組成物の投与前に第一胃に存在していた微生物集団は、前記組成物の投与後に数度が減少する、項目1に記載の方法。
(項目30)
前記有効量の前記組成物を投与された前記反芻動物は、さらに、第一胃のミクロビオームの変化を示し、
前記組成物の投与前に第一胃に存在していた微生物の第1の集団は、前記組成物の投与後に数度が増加し、
前記組成物の投与前に第一胃に存在していた微生物の第2の集団は、前記組成物の投与後に数度が減少する、項目1に記載の方法。
(項目31)
表1及び/または表3から選択される少なくとも1つの微生物株を含む、微生物共同体。
(項目32)
少なくとも1つの微生物株を含む微生物共同体であって、前記少なくとも1つの微生物株は、配列番号1~30及び2045~2103から選択される配列によりコードされる16S rRNA配列、または配列番号31~60及び2104~2107から選択されるITS配列を含む、前記微生物共同体。
(項目33)
前記少なくとも1つの微生物株は、Ascusb_7、Ascusb_32、Ascusf_45、及びAscusf_24を含む、項目32に記載の微生物共同体。
(項目34)
前記少なくとも1つの微生物株は、Ascusb_7、Ascusb_1801、Ascusf_45、及びAscusf_24を含む、項目32に記載の微生物共同体。
(項目35)
前記少なくとも1つの微生物株は、Ascusb_7、Ascusb_268、Ascusf_45、及びAscusf_24を含む、項目32に記載の微生物共同体。
(項目36)
前記少なくとも1つの微生物株は、Ascusb_7、Ascusb_232、Ascusf_45、及びAscusf_24を含む、項目32に記載の微生物共同体。
(項目37)
前記少なくとも1つの微生物株は、Ascusb_7、Ascusb_32、Ascusf_45、及びAscusf_249を含む、項目32に記載の微生物共同体。
(項目38)
前記少なくとも1つの微生物株は、Ascusb_7、Ascusb_32、Ascusf_45、及びAscusf_353を含む、項目32に記載の微生物共同体。
(項目39)
前記少なくとも1つの微生物株は、Ascusb_7、Ascusb_32、Ascusf_45、及びAscusf_23を含む、項目32に記載の微生物共同体。
(項目40)
前記少なくとも1つの微生物株は、Ascusb_3138を含む、項目32に記載の微生物共同体。
(項目41)
前記少なくとも1つの微生物株は、Ascusf_15を含む、項目32に記載の微生物共同体。
(項目42)
前記少なくとも1つの微生物株は、Ascusb_3138及びAscusf_15を含む、項目32に記載の微生物共同体。
(項目43)
前記共同体は封入されている、項目31~42のいずれか1項に記載の微生物共同体。(項目44)
(a)項目31~43のいずれか1項に記載の微生物共同体、及び
(b)許容される担体
を含む、組成物。
(項目45)
前記微生物共同体は封入されている、項目44に記載の組成物。
(項目46)
前記封入微生物共同体は、サッカライドポリマー、寒天ポリマー、アガロースポリマー、タンパク質ポリマー、及び脂質ポリマーから選択されるポリマーを含む、項目44に記載の組成物。
(項目47)
前記許容される担体は、食用飼料等級物質、ミネラル混合物、水、グリコール、モラセス、及びトウモロコシ油からなる群より選択される、項目44に記載の組成物。
(項目48)
前記微生物共同体を形成する前記少なくとも1つの微生物株は、前記組成物中、前記組成物1グラムあたり102~1015細胞で存在する、項目44に記載の組成物。
(項目49)
前記組成物は家畜飼料と混合される、項目44に記載の組成物。
(項目50)
動物に少なくとも1つの改善された形質を与える方法であって、項目44の組成物を前記動物に投与することを含む、前記方法。
(項目51)
前記動物は反芻動物である、項目50に記載の方法。
(項目52)
前記反芻動物はウシである、項目51に記載の方法。
(項目53)
前記投与には、前記組成物を前記動物の第一胃に注入することが含まれる、項目51に記載の方法。
(項目54)
前記組成物は少なくとも月1回投与される、項目50に記載の方法。
(項目55)
前記組成物は少なくとも週1回投与される、項目54に記載の方法。
(項目56)
前記組成物は少なくとも1日1回投与される、項目55に記載の方法。
(項目57)
前記投与は動物に給餌するたびに行われる、項目50に記載の方法。
(項目58)
前記投与は肛門投与である、項目50に記載の方法。
(項目59)
前記肛門投与には、前記組成物を含む坐剤を前記動物の直腸に挿入することが含まれる、項目58に記載の方法。
(項目60)
前記投与は経口投与である、項目50に記載の方法。
(項目61)
前記経口投与には、前記組成物を、前記動物の飼料、水、薬、またはワクチンと合わせて投与することが含まれる、項目60に記載の方法。
(項目62)
前記経口投与には、前記動物の体の一部に前記組成物のゲルまたは粘性液を塗ることが含まれ、前記動物は前記組成物を舐めて摂取する、項目60に記載の方法。
(項目63)
前記投与には、前記組成物を前記動物に噴霧することが含まれ、前記動物は前記組成物を摂取する、項目50に記載の方法。
(項目64)
前記少なくとも1つの改善された形質は、乳中の脂肪の増加、乳中の炭水化物の増加、乳中のタンパク質の増加、乳中のビタミンの増加、乳中のミネラルの増加、乳体積の増加、改善された飼料利用効率及び消化率、ポリサッカライド及びリグニン分解率の増加、第一胃内の脂肪酸濃度の上昇、第一胃内のpHバランス、メタン放出量の減少、糞生産の減少、改善された乾物摂取量、エネルギー補正乳(ECM)の重量及び/または体積の増加、ならびに改善された窒素利用効率からなる群より選択され、前記増加または減少は、前記組成物を投与されていない動物との比較により決定される、項目50に記載の方法。
(項目65)
前記乳中の脂肪の増加は、トリグリセリド、トリアシルグリセリド、ジアシルグリセリド、モノアシルグリセリド、リン脂質、コレステロール、糖脂質、及び/または脂肪酸の増加である、項目64に記載の方法。
(項目66)
前記炭水化物の増加は、オリゴサッカライド、ラクトース、グルコース、及び/またはガラクトースの増加である、項目64に記載の方法。
(項目67)
前記ポリサッカライド分解率の増加は、リグニン、セルロース及び/またはヘミセルロースの分解率の増加である、項目64に記載の方法。
(項目68)
前記脂肪酸濃度の上昇は、酢酸、プロピオン酸、及び/または酪酸の増加である、項目64に記載の方法。
(項目69)
前記少なくとも1つの微生物株は、前記株が単独で出現するとき、または前記株が自然発生する濃度で存在するときには示さない、高い有用性を示す、項目44に記載の組成物。
(項目70)
前記少なくとも1つの微生物株は、動物において少なくとも1つの改善された形質を与えることにおいて相乗効果を示す、項目44に記載の組成物。
(項目71)
反芻動物において望ましい表現型形質を増加させることができる反芻動物飼料サプリメントであって、
(a)自然発生しない濃度で存在する、項目31~42のいずれか1項に記載の微生物共同体、及び
(b)許容される担体
を含む、前記反芻動物飼料サプリメント。
(項目72)
前記微生物共同体は封入されている、項目71に記載の反芻動物飼料サプリメント。
(項目73)
表1及び/または表3の微生物のいずれかから選択される、単離微生物株。
(項目74)
(a)Bigelow登録寄託番号Patent201612011として寄託されたAscusb_7、
(b)Bigelow登録寄託番号Patent201612007として寄託されたAscusb_32、
(c)Bigelow登録寄託番号Patent201612012として寄託されたAscusb_82、
(d)Bigelow登録寄託番号Patent201612009として寄託されたAscusb_119、
(e)Bigelow登録寄託番号Patent201612009として寄託されたAscusb_1801、
(f)Bigelow登録寄託番号Patent201612003として寄託されたAscusf_206、
(g)Bigelow登録寄託番号Patent201612014として寄託されたAscusf_23、
(h)Bigelow登録寄託番号Patent201612004として寄託されたAscusf_24、
(i)Bigelow登録寄託番号Patent201612002として寄託されたAscusf_45、
(j)Bigelow登録寄託番号Patent201612003として寄託されたAscusf_208、
(k)NRRL登録寄託番号B-67248として寄託されたAscusb_3138、及び
(l)NRRL登録寄託番号Y-67249として寄託されたAscusf_15
からなる群より選択される単離微生物株。
(項目75)
配列番号1~60及び2045~2107のいずれかと少なくとも90%の配列同一性をもつポリヌクレオチド配列を含む、単離微生物株。
(項目76)
項目73~75のいずれか1項に記載の単離微生物株の実質的に純粋な培養物。
(項目77)
項目45の組成物を投与することが含まれる、反芻動物のミクロビオームを調節する方法。
(項目78)
前記組成物の前記投与により、前記反芻動物に少なくとも1つの改善された形質が与えられる、項目77に記載の方法。
(項目79)
前記少なくとも1つの改善された形質は、乳中の脂肪の増加、乳中の炭水化物の増加、乳中のタンパク質の増加、乳中のビタミンの増加、乳中のミネラルの増加、乳体積の増加、改善された飼料利用効率及び消化率、ポリサッカライド及びリグニン分解率の増加、第一胃内の脂肪酸濃度の上昇、第一胃内のpHバランス、メタン放出量の減少、糞生産の減少、改善された乾物摂取量、エネルギー補正乳(ECM)の重量及び/または体積の増加、ならびに改善された窒素利用効率からなる群より選択され、前記増加または減少は、前記組成物を投与されていない動物との比較により決定される、項目78に記載の方法。
(項目80)
前記乳中の脂肪の増加は、トリグリセリド、トリアシルグリセリド、ジアシルグリセリド、モノアシルグリセリド、リン脂質、コレステロール、糖脂質、及び/または脂肪酸の増加である、項目79に記載の方法。
(項目81)
前記炭水化物の増加は、オリゴサッカライド、ラクトース、グルコース、及び/またはガラクトースの増加である、項目79に記載の方法。
(項目82)
前記ポリサッカライド分解率の増加は、リグニン、セルロース及び/またはヘミセルロースの分解率の増加である、項目79に記載の方法。
(項目83)
前記脂肪酸濃度の上昇は、酢酸、プロピオン酸、及び/または酪酸の増加である、項目79に記載の方法。
(項目84)
前記ミクロビオームの調節は、前記ミクロビオームの少なくとも1つの微生物株の割合の増加であり、この増加は、前記少なくとも1つの微生物株を投与されなかった反芻動物に対して測定される、項目78に記載の方法。
(項目85)
前記ミクロビオームの調節は、前記組成物の投与前に前記ミクロビオームに存在していた微生物株の割合の減少であり、この減少は、前記組成物の投与前の前記反芻動物のミクロビオームに対して測定される、項目78に記載の方法。
(項目86)
病原性微生物のコロニー形成に対するウシの耐性を増加させる方法であって、項目44に記載の組成物を投与することを含み、前記病原体はウシの胃腸管内でコロニー形成ができない、前記方法。
(項目87)
項目44に記載の組成物を投与することが含まれる、少なくとも1つの病原性微生物の存在に関してウシを治療する方法。
(項目88)
前記組成物の投与後、胃腸管内で前記少なくとも1つの病原性微生物の相対数度が5%未満の相対数度に減少する、項目87に記載の方法。
(項目89)
胃腸管内で前記少なくとも1つの病原性微生物の相対数度が1%未満の相対数度に減少する、項目88に記載の方法。
(項目90)
前記少なくとも1つの病原性微生物は胃腸管内で検出不能である、項目88に記載の方法。
(項目91)
前記微生物共同体は、細菌及び/または真菌を、胞子の形態で、栄養細胞の形態で、及び/または溶解物の形態で含む、項目44に記載の組成物。
Claims (1)
- 本願明細書に記載の発明。
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