JP2018111711A - リボフラビン、リン酸リボフラビン又は生理学上許容されるその塩 - Google Patents
リボフラビン、リン酸リボフラビン又は生理学上許容されるその塩 Download PDFInfo
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- JP2018111711A JP2018111711A JP2018043874A JP2018043874A JP2018111711A JP 2018111711 A JP2018111711 A JP 2018111711A JP 2018043874 A JP2018043874 A JP 2018043874A JP 2018043874 A JP2018043874 A JP 2018043874A JP 2018111711 A JP2018111711 A JP 2018111711A
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- Prior art keywords
- riboflavin
- phosphate
- physiologically acceptable
- acceptable salt
- bacteria
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Abstract
【解決手段】動物の胃腸管においてフィーカリバクテリウムプラウスニッツィイを選択的に刺激するための食品組成物、医薬組成物、食品又は健康補助食品の製造のためのリボフラビン、リン酸リボフラビン又は生理学上許容されるその塩の使用の提供。また、リボフラビン、リン酸リボフラビン又は生理学上許容されるその塩、及びシステインとともに調合された生きた有益なブチレート生成嫌気性細菌を含むシンバイオティクス組成物の提供。
【選択図】なし
Description
リボフラビンが腸のフィーカリバクテリウム プラウスニッツィイの個数に与える影響を調査するため、ヒト介入試験を実施した。志願者8名の群(体重60〜90kg)は、リボフラビンのサプリメント100mgを1日1回、14日間経口摂取するよう求められた。
好気条件下で少なくとも24時間安定である生きたフィーカリバクテリウムを備える製剤を生成するため、一連の実験を異なる成分の組み合わせで実施した。フィーカリバクテリウムは、ブロス培地中で一晩増殖させ、遠心分離し、ペレットを嫌気性リン酸緩衝食塩水(PBS)にて洗浄し再び遠心分離を行った。ペレットは、表1に示す成分を含有するPBSと混合した。混合物は−20℃で凍結し、次に凍結乾燥した。
・可逆性酸化還元反応を持つ、抗酸化剤のシステイン及びリボフラビンの存在。この保護効果は、クエルセチン又はアスコルビン酸のような他の抗酸化剤では観察されなかった。クエルセチンは不可逆的に酸化され、アスコルビン酸は水分が存在すると安定しないため、これらは保護効果を持たなかった。
・抗酸化性の酸化還元媒介剤により酸素透過から良好に保護するためには、細粒が小型であること又は例えばイヌリンといった凍結保存剤(cryo−preservant)でコーティングすることが好ましい。イヌリンでコーティングされた場合にのみ、小麦ふすま又はコーンスターチの追加が有効である。また、イヌリンは小麦ふすま又はコーンスターチより水分を引き付けない。
細菌の培養
F.プラウスニッツィイ株A2−165(DSM17677)を、酵母エキス、カシトン、脂肪酸及びグルコース(YCFAG)寒天培地上でサンプル10μlを培養することで、グリセロールストック(−80℃)から戻した。培養物は、37℃における、H210%、CO210%及びN280%(v/v)混合ガスを備える嫌気性のテント(tent)内の、YCFAG培地上で、培養及び通常通りの維持がなされた。種培養液は、F.プラウスニッツィイ株の単一コロニーをYCFAG培養液5mlに播種し、600nm(A600)の光学密度(OD)が0.8以下となるまで12時間〜16時間培養して得た。種培養液1mlを新鮮YCFAG培地50mlに播種した(2%v/vで播種)。培養物は、ODA600が0.8±0.2以下に到達するまで12時間〜16時間培養した。細胞は、17℃10分間、2700gで遠心分離し、採取した。ペレットは、殺菌した嫌気性食塩水(0.85%食塩と0.05%システイン)で1度洗浄した。
製剤手順
ステップ1に準じて得られた細菌ペレット(5×108〜2×109の細菌を含有)を、イヌリン10%(w/v)及びシステイン16.5mMを含有する溶液0.4mlに懸濁した。ペレットを再懸濁した後、リボフラビンの貯蔵液0.2ml(16.5mM)を追加した。完全に混合した後、小麦ふすま0.5gとコーンスターチ0.9gを追加し、その結果生じたスラリーを湿式造粒した。湿潤細粒は、少なくとも3時間、−20℃で凍結した。凍結後、細粒を少なくとも3時間凍結乾燥した。
ステップ1に準じて得られた細菌ペレットを、トレハロース10%及びシステイン16.5mMを含有する溶液0.4mlに懸濁した。ペレットを再懸濁した後、リボフラビンの貯蔵液0.2ml(16.5mM)を追加した。完全に混合した後、小麦ふすま0.5gとコーンスターチ0.9gを追加し、その結果生じたスラリーを湿式造粒した。湿潤細粒は、少なくとも3時間、−20℃で凍結した。凍結後、細粒を少なくとも3時間凍結乾燥した。
ステップ1に準じて得られた細菌ペレットを、イヌリン又はトレハロース10%及びシステイン16.5mMを含有する溶液1.6mlに懸濁した。溶液は、システインの追加前にN2でパージした。ペレットを再懸濁した後、リボフラビンの貯蔵液0.2ml(16.5mM)を追加した。完全に混合した後、その結果生じたスラリーを少なくとも3時間、−20℃で凍結した。凍結後、細粒を少なくとも3時間凍結乾燥した。
ステップ1に準じて得られた細菌ペレットを、システイン16.5mMの溶液0.4mlに懸濁した。ペレットを再懸濁した後、リボフラビンの貯蔵液0.2ml(16.5mM)を追加した。完全に混合した後、その結果生じたスラリーを少なくとも3時間、−20℃で凍結した。凍結後、細粒を少なくとも3時間凍結乾燥した。
ステップ1に準じて得られた細菌ペレット(5×108〜2×109の細菌を含有)をイヌリン10%(w/v)とシステイン16.5mMを含有する溶液0.4mlに懸濁した。ペレットを再懸濁した後、リボフラビンの貯蔵液0.2ml(100mM)を追加した。完全に混合した後、小麦ふすま0.5gとコーンスターチ0.9gを追加し、その結果生じたスラリーを湿式造粒した。湿潤細粒は、少なくとも3時間、−20℃で凍結した。凍結後、細粒を少なくとも3時間凍結乾燥した。
安定性及び生存率試験の手順
ステップ2に準じて得られた凍結乾燥した細粒は、周囲の好気的条件の下、気密容器に貯蔵した。安定性及び生存率試験のため、細粒を規定の期間、最大24時間、周囲空気に曝露した。好気への曝露後、細粒を嫌気性のテント(tent)下で嫌気的に処理した。細粒を希釈し、システイン0.05%が補充された無酸素リン酸緩衝食塩水(pH7.2)で水戻しし、10倍希釈系列をYCFAG寒天培地に置いた。コロニー形成単位は、37℃で24時間培養した後、寒天培地上に形成されたコロニーを計測することで定量した。
(付記1)
動物の胃腸管においてフィーカリバクテリウム プラウスニッツィイ(Faecalibacterium prausnitzii)を選択的に刺激するための食品組成物、医薬組成物、食品又は健康補助食品の製造のためのリボフラビン、リン酸リボフラビン又は生理学上許容されるその塩の使用であって、
リボフラビンは、体重1kg当たり1日0.01〜2mgの量で使用される、使用。
前記動物は哺乳類であり、好ましくはヒト対象である、付記1に記載の使用。
前記ヒト対象は、炎症性の胃腸疾患、特にクローン病に罹患している、付記2に記載の使用。
リボフラビンは、体重1kg当たり1日0.1〜2mgの量で使用される、付記1〜3のいずれか1つに記載の使用。
リボフラビンは、少なくとも3日間、好ましくは少なくとも7日間、より好ましくは少なくとも10日間投与される、付記1〜4のいずれか1つに記載の使用。
リボフラビンは、1日1回の用量として投与される、付記1〜5のいずれか1つに記載の使用。
フィーカリバクテリウム プラウスニッツィイのプレバイオティクスとしての、リボフラビン、リン酸リボフラビン又は生理学上許容されるその塩の使用。
フィーカリバクテリウム プラウスニッツィイを、それを必要とする動物の胃腸管において選択的に刺激する方法であって、
リボフラビン、リン酸リボフラビン又は生理学上許容されるその塩を、前記胃腸管においてフィーカリバクテリウム プラウスニッツィイの増殖を選択的に刺激するのに有効な量で、前記動物に投与することを含む、方法。
シンバイオティクス組成物であって、
(i)生きている有益なブチレート生成嫌気性細菌、
(ii)前記組成物の総乾燥重量に基づき少なくとも0.05%の量の、リボフラビン、リン酸リボフラビン又は生理学上許容されるその塩、及び、
(iii)システイン、
を含む、シンバイオティクス組成物。
リボフラビン、リン酸リボフラビン又はその塩は、前記組成物の総乾燥重量に基づき少なくとも1%、好ましくは少なくとも2%の量で存在する、付記9に記載の組成物。
システインは、前記組成物の総乾燥重量に基づき0.05〜2%の量で存在する、付記9又は10に記載の組成物。
イヌリン又はイヌリンタイプのフラクトオリゴ糖を、好ましくは前記組成物の総乾燥重量に基づき2〜10%の量で、さらに含む、付記9〜11のいずれか1つに記載の組成物。
充填剤を、好ましくは前記組成物の総乾燥重量に基づき40〜65%量で、さらに含む、付記9〜12のいずれか1つに記載の組成物。
前記ブチレート生成嫌気性細菌は、ファーミキューテス(Firmicutes)門、好ましくはクロストリジウムレプタム(Clostridium leptum)系統発生グループのメンバーである、付記9〜13のいずれか1つに記載の組成物。
前記ブチレート生成嫌気性細菌は、フィーカリバクテリウム プラウスニッツィイである、付記14に記載の組成物。
フィーカリバクテリウム プラウスニッツィイ細菌を周囲空気への曝露による有害な影響から保護するための方法であって、当該方法は、
フィーカリバクテリウム プラウスニッツィイ細菌を、
(i)リボフラビン、リン酸リボフラビン又は生理学上許容されるその塩、
(ii)システイン、
(iii)イヌリン、
(iv)コーンスターチ、及び、
(v)小麦ふすま又はソバふすま、
を含む組成物中に調剤することを含み、
前記調剤は、前記細菌、前記リボフラビン及び前記システインを含有する細粒を提供することと、前記コーンスターチ及びふすまを追加する前に前記細粒をイヌリンでコーティングすることとを含む、方法。
Claims (7)
- 動物の胃腸管におけるフィーカリバクテリウム プラウスニッツィイ(Faecalibacterium prausnitzii)の増殖を選択的に刺激するのに有効な量で、前記動物にリボフラビン、リン酸リボフラビン又は生理学上許容されるその塩を投与することを含む、動物の胃腸管におけるフィーカリバクテリウム プラウスニッツィイを選択的に刺激するための方法における使用のためのリボフラビン、リン酸リボフラビン又は生理学上許容されるその塩。
- 前記動物は、ヒト、ペット又は家畜である、
ことを特徴とする請求項1に記載のリボフラビン、リン酸リボフラビン又は生理学上許容されるその塩。 - 胃腸管におけるフィーカリバクテリウム プラウスニッツィイの増殖を維持、支援又は刺激するのに有効な量で、必要とするヒト対象にリボフラビンが投与することを含む、炎症性の胃腸障害に関連する症状を予防、治療又は低減するための方法における使用のためのリボフラビン。
- 前記炎症性の胃腸障害は、炎症性の胃腸疾患である、
ことを特徴とする請求項3に記載のリボフラビン。 - 前記炎症性の胃腸疾患は、クローン病及び潰瘍性結腸炎である、
ことを特徴とする請求項4に記載のリボフラビン。 - リボフラビンは、体重1kg当たり1日0.1〜1000mg、好ましくは体重1kg当たり1日1〜100mgの用量で投与される、
ことを特徴とする請求項1乃至5のいずれか1項に記載のリボフラビン、リン酸リボフラビン又は生理学上許容されるその塩。 - リボフラビンは、少なくとも3日間、好ましくは少なくとも7日間、より好ましくは少なくとも10日間投与される、
ことを特徴とする請求項1乃至6のいずれか1項に記載のリボフラビン、リン酸リボフラビン又は生理学上許容されるその塩。
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2013
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- 2013-11-01 WO PCT/NL2013/050781 patent/WO2014070014A1/en active Application Filing
- 2013-11-01 EP EP18157821.2A patent/EP3345606A1/en not_active Withdrawn
- 2013-11-01 BR BR112015009975A patent/BR112015009975A2/pt not_active Application Discontinuation
- 2013-11-01 AU AU2013338774A patent/AU2013338774B2/en not_active Ceased
- 2013-11-01 US US14/439,882 patent/US20150283144A1/en not_active Abandoned
- 2013-11-01 EP EP13795881.5A patent/EP2914135A1/en not_active Withdrawn
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2018
- 2018-03-09 US US15/916,704 patent/US20180207165A1/en not_active Abandoned
- 2018-03-12 JP JP2018043874A patent/JP2018111711A/ja active Pending
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EP2914135A1 (en) | 2015-09-09 |
BR112015009975A2 (pt) | 2017-07-11 |
AU2013338774B2 (en) | 2017-03-02 |
US20180207165A1 (en) | 2018-07-26 |
WO2014070014A1 (en) | 2014-05-08 |
US20150283144A1 (en) | 2015-10-08 |
AU2013338774A1 (en) | 2015-05-21 |
JP2015535280A (ja) | 2015-12-10 |
EP3345606A1 (en) | 2018-07-11 |
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