JP5160889B2 - 表皮細胞分化及び角化促進剤 - Google Patents
表皮細胞分化及び角化促進剤 Download PDFInfo
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- JP5160889B2 JP5160889B2 JP2007522377A JP2007522377A JP5160889B2 JP 5160889 B2 JP5160889 B2 JP 5160889B2 JP 2007522377 A JP2007522377 A JP 2007522377A JP 2007522377 A JP2007522377 A JP 2007522377A JP 5160889 B2 JP5160889 B2 JP 5160889B2
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- fermented milk
- keratinization
- cell differentiation
- milk
- epidermal cell
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
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Description
ラクトバチルス・ヘルベティカスを含む菌体により乳を発酵させて得た発酵乳ホエーを有効成分として含む、経口摂取用の表皮細胞分化及び角化促進剤が提供される。
本発明の表皮細胞分化及び角化促進剤は、乳酸菌としてラクトバチルス・ヘルベティカスを含む菌体により乳を発酵させて得た発酵乳ホエーを有効成分として含む。
前記乳酸菌としては、ストレプトコッカス属、ラクトコッカス属、ラクトバチルス属、ビフィドバクテリウム属等に属する乳酸菌が挙げられるが、ラクトバチルス属が好ましい。具体的には、例えば、ラクトバチルス・ブルガリカス(Lactobacillus bulgaricus)、ラクトバチルス・ヘルベティカス(Lactobacillus helveticus)、ラクトバチルス・カゼイ(Lactobacillus casei)、ラクトバチルス・アシドフィラス(Lactobacillus acidophilus)、ラクトバチルス・ファーメンタム(Lactobacillus fermentum)等が挙げられ、特に、ラクトバチルス・ヘルベティカスを使用する。
菌学的性質
1.形態学的性質:1)細胞の形状;桿菌、2)運動性;なし、3)胞子の有無;なし、4)グラム染色性;陽性。
2.生理学的性質:1)カタラーゼ;陰性、2)インドール生成;陰性、3)硝酸塩の還元;陰性、4)酸素に対する態度;通性嫌気性菌、5)グルコースによりホモ乳酸発酵によりDL(−)乳酸を生成し、ガスの産生はない。
6)各種炭水化物の分解性:グルコース;+、ラクトース;+、マンノース;+、フラクトース;+、ガラクトース;+、シュークロース;−、マルトース;−、キシロース;−、ラムノース;−、セルビオース;−、トレハロース;−、メルビオース;−、ラフィノース;−、スタキオース;−、マンニトール;−、ソルビトール;−、エスクリン;−、サリシン;−。
有効成分である発酵乳ホエーは、得られる発酵乳から遠心分離、ろ過等の常法の分離操作によりホエーを分離することにより得られるが、使用に際しては、分離せずに発酵乳の状態で、また分離したホエーを適宜、分画、濃縮、精製等して用いることができる他、該ホエーやその濃縮物を凍結乾燥、噴霧乾燥等により粉末として用いることもできる。
有効成分である発酵乳ホエーを、風味を良好にし、嗜好性を良好とするために、前記乳酸菌に酵母を併用することもできる。酵母の菌種は特に限定されないが、例えば、サッカロマイセス・セレビシェ等のサッカロマイセス属酵母が好ましく挙げられる。酵母の含有割合は、その目的に応じて適宜選択することができる。
乳の固形分濃度は特に限定されないが、例えば、脱脂乳を用いる場合の無脂乳固形分濃度は、通常3〜15重量%程度であり、生産性的には6〜15重量%が好ましい。
本発明の表皮細胞分化及び角化促進剤は、必須の有効成分が前記発酵乳ホエーであるので、その経口摂取量は、投与期間やその継続性等に応じて所望の効果が得られるよう適宜選択できるが、通常、前記発酵乳ホエー量で、1日あたり1〜1000ml/ヒト程度であり、好ましくは10〜200ml/ヒト程度である。
本発明の表皮細胞分化及び角化促進剤の投与は、皮膚の不全角化による症状が生じた後であっても、また、そのような症状を予防する時期に継続して毎日、若しくは断続的に摂取することができる。
実施例1
固形率9重量%脱脂粉乳からなる乳培地にCM4株スターター(菌数5×108個/mL)を3%接種して、37℃で24時間、静置状態で発酵させ発酵乳を得た。この発酵乳を12000G、20分間遠心分離し、沈殿を除去し、発酵乳ホエーを調製した。得られた発酵乳ホエーを用いて以下の試験を行った。
1)方法
(a)培養表皮細胞および培地
ヒト正常表皮細胞及び培地(Humedia-KG2)は、市販品(クラボウ社製)を用いた。
(b)細胞培養
ヒト正常表皮細胞の細胞数を上記培地にて5.652×104cells/mLに調整し、60mmディッシュに5mLずつ播種し、95%空気(V/V)−5%(V/V)炭酸ガスの雰囲気下、37℃で24時間静置培養した。その後、終濃度0.03、0.1、0.3、1%の発酵乳ホエー及び対照として精製水を添加した上記培地に交換して、24時間静置培養した。また、タイムコース解析用としては、終濃度1%の試料含有培地と交換後、0、1、2、4、6、8日間静置培養した。
(c)抗体染色による分化マーカー発現解析
上記(b)において調製した1%の発酵乳ホエーの存在下、若しくは不存在下において24時間培養後、4%パラフォルムアルデヒド含有PBSにて細胞の固定を行った。10%ウサギ血清含有PBSでブロッキング後、マウス抗サイトケラチン10抗体(Dako社製)又はマウス抗インボルクリン抗体(YLEM社製)を室温で一時間反応させた。PBS洗浄後、ペルオキシダーゼ標識ウサギ抗マウス免疫グロブリン抗体(Dako社製)を室温で30分間反応させた。更にPBS洗浄を行った後、DAB Substrate-Chromogen System(Dako社製)を用いて発色反応を行った。結果を図1に示す。
図1において、aは発酵乳ホエーを添加していないKeratin10の発現観察結果、bは終濃度1%で発酵乳ホエーを添加した際のKeratin10の発現観察結果、cは発酵乳ホエーを添加していないInvolucrinの発現観察結果、dは終濃度1%で発酵乳ホエーを添加した際のInvolucrinの発現観察結果を示す。
上記(b)において諸濃度の発酵乳ホエーの存在下、若しくは不存在下で24時間培養後、及びタイムコース分析用の培養後、培養上清を吸引除去し、Hepes緩衝液で2回洗浄した後、RNeasy Mini Kit (QIAGEN社製)を用いて細胞から全RNAを抽出した。抽出した全RNA1ngについて、Smart Cycler II System(Cepheid社製)にて、One Step SYBR RT-PCR Kit(商品名、TaKaRa社製)及び表1に示すプライマーを用い、プロトコルに従いReal-time RT-PCRを実施し、ケラチン10(Keratin10)mRNA、インボルクリン(Involucrin)mRNA及びグリセルアルデヒド3リン酸脱水素酵素(GAPD)mRNAの発現量を定量化した。発現量は、どの細胞においても同量発現すると考えられるGAPDmRNA発現量で割ることにより規格化し、さらに、その値を、上記(b)において培地中の発酵乳ホエー濃度を0%としたもの(対照)を1とした相対値で表した。結果を図2〜5に示す。但し、図4及び図5において、黒丸が発酵乳ホエー添加による結果、白丸は発酵乳ホエー非添加による結果を示す。
(e)細胞傷害性評価試験
上記(b)と同様に、ヒト正常表皮細胞を試料存在下で48時間培養後、アラマーブルー試薬(ナカライテスク社製)を用いて、プロトコルに従い細胞傷害性評価を行った。結果を図6に示す。
図2及び3より、発酵乳ホエーの添加量依存的にKeratin10及びInvolucrinの発現が増大することが判った。
図4及び5より、発酵乳ホエーは、表皮細胞における分化マーカーであるKeratin10及びInvolucrinの発現を促進することが判った。
図6より、発酵乳ホエーの添加による細胞毒性は認められなかった。
実施例1で調製した発酵乳ホエーを飲用し易くするために、該発酵乳ホエー90質量部、香料0.25質量部、アスパルテーム0.05質量部及び水9.70質量部を混合し、発酵乳ホエー含有飲料を調製した。得られた飲料を用いて以下に示す評価を行った。
セロハンテープ(ニチバン社製)を用いたテープストリッピング法により、顔面左頬の最外層の角層細胞を剥離し、スライドグラスへ転写、定着させた。これを、各々ブリリアントグリーン・ゲンチアナバイオレット(BG)にて染色し、染色画像をコンピュータに取り込んだ。BG染色画像の画像解析により、角層細胞面積を測定した。
評価試験は、24〜43歳の平均年齢29.4歳である男性パネル16名で行った。まず、試料を飲用する前に実施し、各パネルの平均値を求めた。次いで、発酵乳ホエー含有飲料を、一日あたり150g、9週間毎日飲用してもらい、9週間目に再度同様に評価試験を行い、各パネルの平均値を求めた。結果を表2に示す。
Claims (2)
- 不全角化の改善を要する対象者に経口摂取する剤であって、
ラクトバチルス・ヘルベティカスを含む菌体により乳を発酵させて得た発酵乳ホエーを有効成分として含む、経口摂取用の表皮細胞分化及び角化促進剤。 - ラクトバチルス・ヘルベティカスが、ラクトバチルス・ヘルベティカス CM4株(独立行政法人産業技術総合研究所特許生物寄託センター寄託番号:FERM BP−6060)である請求項1記載の表皮細胞分化及び角化促進剤。
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EP2170088A2 (en) * | 2007-06-27 | 2010-04-07 | Chr. Hansen A/S | Lactobacillus helveticus bacterium composition for treatment of atopic dermatitis |
WO2010071132A1 (ja) | 2008-12-15 | 2010-06-24 | カルピス株式会社 | 皮膚老化抑制ペプチド |
EP2340803A1 (en) * | 2009-12-30 | 2011-07-06 | Brands Asia Pacific Limited | Dead skin exfoliating composition and pad for hands and feet |
EP2465507A1 (en) * | 2010-11-23 | 2012-06-20 | Nestec S.A. | Oligosaccharide composition for treating skin diseases |
KR101998210B1 (ko) * | 2012-10-31 | 2019-07-10 | (주)아모레퍼시픽 | 녹차 유산균을 포함하는 피부 각질 제거용 조성물 |
JP6326413B2 (ja) * | 2013-07-05 | 2018-05-16 | 株式会社ヤクルト本社 | ペントシジン生成阻害剤 |
CN106661546B (zh) | 2014-06-26 | 2020-03-03 | 日本瑞翁株式会社 | 培养细胞的分化促进方法以及培养细胞分化促进剂 |
WO2019158652A1 (fr) * | 2018-02-14 | 2019-08-22 | Danstar Ferment Ag | Composition cosmetique ou dermatologique et methode pour l'hydratation cutanee |
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JPH1198978A (ja) * | 1997-09-26 | 1999-04-13 | The Calpis Co Ltd | トリペプチド高生産性ラクトバチルス・ヘルベチカス乳酸菌 |
JP2000239175A (ja) * | 1999-02-18 | 2000-09-05 | Calpis Co Ltd | 抗アレルギー剤 |
JP2002241289A (ja) * | 2001-02-16 | 2002-08-28 | Ichimaru Pharcos Co Ltd | 肥厚化角質層柔軟化剤 |
JP2004501199A (ja) * | 2000-06-28 | 2004-01-15 | セリカウサ ベーヴェー | 異化乳清から成るまたは少なくとも異化乳清を含む組成物の使用 |
JP4680571B2 (ja) * | 2003-12-25 | 2011-05-11 | カルピス株式会社 | 経口摂取用保湿剤 |
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JP2826808B2 (ja) * | 1995-07-14 | 1998-11-18 | カルピス株式会社 | 脳機能改善、学習能力増強および記憶力増強作用を有する機能性食品 |
JP3227378B2 (ja) | 1996-03-29 | 2001-11-12 | カネボウ株式会社 | 表皮角質化促進剤 |
JP4065038B2 (ja) | 1996-08-07 | 2008-03-19 | カルピス株式会社 | 計算作業負荷ストレス緩和剤 |
AU2058702A (en) * | 2000-10-06 | 2002-04-15 | Nestle Sa | Use of probiotic lactic acid bacteria for balancing the skin's immune system |
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- 2006-06-23 AU AU2006260100A patent/AU2006260100A1/en not_active Abandoned
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- 2006-06-23 CA CA002614111A patent/CA2614111A1/en not_active Abandoned
- 2006-06-23 KR KR1020087000629A patent/KR20080019059A/ko not_active Application Discontinuation
- 2006-06-23 TW TW095122776A patent/TW200740378A/zh unknown
Patent Citations (5)
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JPH1198978A (ja) * | 1997-09-26 | 1999-04-13 | The Calpis Co Ltd | トリペプチド高生産性ラクトバチルス・ヘルベチカス乳酸菌 |
JP2000239175A (ja) * | 1999-02-18 | 2000-09-05 | Calpis Co Ltd | 抗アレルギー剤 |
JP2004501199A (ja) * | 2000-06-28 | 2004-01-15 | セリカウサ ベーヴェー | 異化乳清から成るまたは少なくとも異化乳清を含む組成物の使用 |
JP2002241289A (ja) * | 2001-02-16 | 2002-08-28 | Ichimaru Pharcos Co Ltd | 肥厚化角質層柔軟化剤 |
JP4680571B2 (ja) * | 2003-12-25 | 2011-05-11 | カルピス株式会社 | 経口摂取用保湿剤 |
Non-Patent Citations (1)
Title |
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JPN6012017482; Foods & Food Ingredients Journal of Japan Vol.209 , No.9, 2004, pp.780-792 * |
Also Published As
Publication number | Publication date |
---|---|
EP1902720A4 (en) | 2012-03-28 |
CN101242849A (zh) | 2008-08-13 |
AU2006260100A1 (en) | 2006-12-28 |
EA200800124A1 (ru) | 2008-06-30 |
TW200740378A (en) | 2007-11-01 |
CA2614111A1 (en) | 2006-12-28 |
KR20080019059A (ko) | 2008-02-29 |
US8097246B2 (en) | 2012-01-17 |
US20100166877A1 (en) | 2010-07-01 |
WO2006137513A1 (ja) | 2006-12-28 |
EP1902720A1 (en) | 2008-03-26 |
CN101242849B (zh) | 2011-07-27 |
EA013926B1 (ru) | 2010-08-30 |
EP1902720B1 (en) | 2014-09-17 |
JPWO2006137513A1 (ja) | 2009-01-22 |
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