JP5951604B2 - イソフラボン−アグリコン、エクオール及びルナシルを含む醗酵大豆ベース混合物、食品、医薬品及び化粧品分野におけるその調製方法及び使用 - Google Patents
イソフラボン−アグリコン、エクオール及びルナシルを含む醗酵大豆ベース混合物、食品、医薬品及び化粧品分野におけるその調製方法及び使用 Download PDFInfo
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- JP5951604B2 JP5951604B2 JP2013519219A JP2013519219A JP5951604B2 JP 5951604 B2 JP5951604 B2 JP 5951604B2 JP 2013519219 A JP2013519219 A JP 2013519219A JP 2013519219 A JP2013519219 A JP 2013519219A JP 5951604 B2 JP5951604 B2 JP 5951604B2
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- lactobacillus
- lactic acid
- dsm
- acid bacteria
- soy
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Description
;IFN−γと有機農業大豆粉から得られ醗酵していない豆乳の存在下
、又は選択された混合スターターで醗酵され10μMのエクオール最終濃度に希釈され滅菌ろ過された豆乳の存在下(−x−)で実施された。DMEM培養基を陰性対照として用いた
。データは3つの3回の実験の平均値である。星印は陰性対照と比較した有意の差(P<0.01)を示す。
b)該乳酸菌の水性懸濁液で大豆ベース基質をインキュベートする工程;好ましくは、基質は全基質体積の1〜4%の量の乳酸菌の水性懸濁液でインキュベートされ、該水性懸濁液は各バイオタイプについて約log 9.0 cfu/mlの細胞密度を有している;
c)30〜37℃、好ましくは30℃で、48〜96時間、好ましくは96時間インキュベートする工程。
Lactobacillus alimentarius(10N、2B、5A)、Lactobacillus brevis(5Z、DPPMA869)、Lactobacillus casei(LC10)、Lactobacillus casei subsp.casei(2047、2756、2763、2766)、Lactobacillus casei subsp.pseudoplantarum(2742、2745、2749、2750)、Lactobacillus curvatus(13H5、14H10、1Hd、2042、2081、2768、2770、2771、2775、SAL23、SAL35)、Lactobacillus delbrueckii subsp.bulgaricus(11842、B15Z)、Lactobacillus fermentum(DPPMA114、D13)、Lactobacillus gasseri(B30W)、Lactobacillus helveticus(15009、B26W、PR4)、Lactobacillus hilgardii(51B)、Lactobacillus paralimentarius(15α、15β、16R、8D、DPPMA238)、Lactobacillus paracasei(12H8、1Hb、B14F5、B18S、B25F3、PF6、B61F5)、Lactobacillus pacarbuckneri(B10F5、 B48F3、B48F5、B9F5t、BF1、BF2)、Lactobacillus paraplantarum(4DE、DPPMA667)、Lactobacillus pentosus(8CF、12H5、12H6)、Lactobacillus plantarum(14H4、17N、19A、1A7、2A、30、3DM、4H1、4H10、DB200、DPPMASL33、DPPMA24W)、Lactobacillus rhamnosus(11、19、DPPMAAZ1、DPPMAAZ21)、Lactobacillus sakei (9I、SAL1、SAL18)、Lactobacillus rossiae (10A、15R、3D、5C1、5α、CF51、CI35、CR20、E18)、Lactobacillus sanfranciscensis(16α、A17、BB12、DE9、E19、H10)、Lactococcus lactis subsp.lactis(10γ)、Pediococcus pentosaceus(C16F5、C25F3、C30F5t、C6F5、C7F3、C9F5t、C29F5)、及びWeissella cibaria(10XA16、3XA4、5S、5XF12)種に属する乳酸菌の103種のバイオタイプを本研究に用いた。すべてのバイオタイプはthe Collezione di Colture del Dipartimento di Protezione delle Piante e Microbiologia Applicata dell’Universita degli Studi di Bariに属し、あらかじめ食物マトリックス(パン製造及びチーズのための天然酵母)から単離された。乳酸菌のバイオタイプは30又は37℃で24時間MRS培地(Oxoid,Basingstoke,United Kingdom)で30℃で24時間増殖させた。
有機農業大豆(OFS)(ECorNaturaSi,Verona,Italy)、大豆タンパク質単離物(SPI)(Supro Soja 80 Aptonia,Villeneuve d’ Ascq,France)及び様々な市販大豆粉調製物(Cargill Texturizing Solutions Soy Protein,Gent,Belgium)を豆乳の製造に用いた。OFSは洗浄して蒸留水中に一晩静置した。湿潤して膨潤した大豆を手で皮を剥き、温水(約90℃)で1:10の比で希釈し、PBI国際ホモジナイザー(ミラノ、イタリア)で均一化した。均一化は10,000xgで2分間行い、次いで1分休止し、再び14,000xgで2分間行った。懸濁液を遠心分離し(7,000xg、10分、4℃)、豆乳を0.22μm孔径フィルター(Millipore Corporation,Bedford)で滅菌濾過した。pHは6.2であった。SPIを蒸留水(40℃)、0.4:10の比で蒸留し、約55℃で30分間攪拌下(120rpm)で熱処理した。室温で冷却後、pHをNaOH5M(Tsangalis et al.2002)を用いて6.7に調整した。滅菌をオートクレーブ中で121℃で15分間行った。市販の大豆粉調製物を、Chun et al.(Chun et al.,2007.乳酸菌での醗酵による豆乳中イソフラボングルコシドのアグリコンへの変換(Conversion of isoflavone glucoside to aglycones in soymilk by fermentation with lactic acid bacteria)J.Food Sci.72:39−44)に記載の方法により、蒸留水(40℃)で1:10の比で希釈した。pHは約6.3であった。滅菌はオートクレーブ中121℃で15分間行った。
様々な豆乳タイプの醗酵の間、混合スターターとして使用した乳酸菌(DPPMASL33に対応するLactobacillus plantarum DSM 23755、DPPMA24Wに対応するLactobacillus plantarum DSM 23756、DPPMA114に対応するLactobacillus fermentum DSM 23757、及びDPPMAAZ1に対応するLactobacillus rhamnosus DSM 23758)のモニタリングは、RAPD−PCR法を用いて行った。二つのプライマー(Invitrogen、ミラノ、イタリア)を、任意に選択された配列(P7 5’ AGCAGCGTGG 3’(配列番号:1)及びM13,5’ GAGGGTGGCGGTTCT 3’ (配列番号:2))とともに、プラスミドと染色体バクテリアDNAの様々な領域をランダムに増幅しながら(De Angelis et al.,2001.表現型、遺伝子型及び細胞壁タンパク質分析に基づく雌羊イタリアチーズ由来の非スターター乳酸菌(NSLAB)のキャラクタリゼーション(Characterization of non−starter lactic acid bacteria (NSLAB)from ewes’ Italian cheeses based on phenotypic,genotypic and cell−wall protein analyses.)Appl.Environ.Microbiol.67:2011−2020;Rossetti e Giraffa,2005.M13により生成したRAPD−PCR指紋データベースによる乳製品乳酸菌の迅速な同定(Rapid identification of dairy lactic acid bacteria by M13−generated,RAPD−PCR fingerprint databases.)J.Microbiol.Met.63:135−144)、タイピングに用いた。
ヒト再構成上皮SkinEthic(登録商標)(再構成ヒト上皮)は、多重層としてヒト上皮の正常ケラチノサイト(角化細胞)からなる。それは、化学的に規定された培地(MCDM153)中、ウシ胎児血清を添加せずに、不活性な多孔性ポリカーボネート支持体上の気液界面で17日間、ヒトケラチノサイトの培養後、完全に分化した上皮である。この成長段階で、形態的な分析により、生存する多層化した上皮と、10を超える数の緻密細胞層からなる角質層が示された。ヒト再構成上皮SkinEthic(登録商標)を先に記載された手順に従って用いた(Di Cagno et al.,2009.ラクトバチルス プランタルム DSMZ19463によるγ−アミノ酪酸(GABA)の合成:機能的ブドウの飲料及び皮膚科学的応用(Synthesis of γ−amino butyric acid (GABA)by Lactobacillus plantarum DSMZ19463:functional grape must beverage and dermatological application.)Appl Biotechnol Microbiol DOI:10.1007/s00253−009−23704)。
ヒト起源Caco−2細胞(クローンTC7)を、ウシ胎児血清(10%)を添加し、必須でないアミノ酸(1%)、ゲンタマイシン/ストレプトマイシン(50μg/ml)、グルタミン(2mM)及び4−2−ヒドロキシエチル−1−ピペラジニル−エタンスルホン酸(1%)を添加したダルベッコ(DMEM)培地で培養した(Di Cagno et al.,2010.サワードウのラクトバチルス プランタルムDC400における菌体密度感知機構:他の乳酸菌との共培養下でのプランタリシンA(PlnA)の誘導、並びにPlnAのバクテリア及びCaco−2細胞への効果(Quorum sensing in sourdough Lactobacillus plantarum DC400:induction of plantaricin A (PlnA)under co−cultivation with other lactic acid bacteria and effect of PlnA on bacterial and Caco−2 cells.)Proteomics in press)。細胞生存率はニュートラルレッド色素の取り込みアッセイにより決定した(Balls et al.,1987.毒性学における評価代替方法へのアプローチ(Approaches to validation alternative methods in toxicology.)In:Goldber A.M.(Ed).N.Y.Academic Press pp.45−58)。様々な調製物で24〜72時間の処理の後、細胞をPBS緩衝液で洗浄し、37℃で4時間ニュートラルレッド溶液(33mg/l)とともにインキュベートした。次いで、細胞を再度PBS緩衝液で洗浄し、溶菌溶液(1%酢酸を含む水中50%エタノール)で処理した。プレートの読み取りをNovapath reader (Biorad,Hercules,CA)(Di Cagno et al.,2010)を用いて行った。
(1)β−グルコシダーゼ活性に基づく乳酸菌の選択
予備的に、食物マトリクスから単離された乳酸菌の103種のバイオタイプのβ−グルコシダーゼ活性をpNPG合成基質で試験した。活性は0から202.3Uまで変化した(図1)。L.alimentarius、L.brevis、L.casei、L.delbrueckii subsp.bulgaricus、L.helveticus、L.hilgardiii、L.paralimentarius、L.paraplantarum、L.pentosus、L.sanfranciscensis、Lc.lactis subsp.lactis、L.parabuchneri e W.cibaria種にほぼ属する48種のバイオタイプは、β−グルコシダーゼ活性を示さなかった。活性平均値は3Uであり、25°と75°データパーセンタイルに対応する値は0及び45.5Uであった。乳酸菌の様々な種に対応する25種のバイオタイプは55Uより高いβ−グルコシダーゼ活性を示した。L.plantarum DPPMA24W、L.fermentum DPPMA114、L.rhamnosus DPPMAAZ1、及びL.plantarum DPPMASL33はより高い活性を示した(それぞれ、202.35±7.08、163.15±6.52、146.60±5.84、及び144.34±7.19U)。これらのバイオタイプのβ−グルコシダーゼ活性の値はボックスプロットのエラーバーの外に出ていた。これらの結果に基づいて4種の乳酸菌を選択し、様々な豆乳タイプの醗酵に用いるための混合スターターの配合のために用いた。
豆乳の調製に用いる様々な大豆粉タイプの化学組成、タンパク質分散性指数及び粒径を表1に報告する。表1は、DPPMASL33に対応するLactobacillus plantarum DSM 23755、DPPMA24Wに対応するLactobacillus plantarum DSM 23756、DPPMA114に対応するLactobacillus fermentum DSM 23757、及びDPPMAAZ1に対応するLactobacillus rhamnosus DSM 23758を含む選択された混合スターターによる機能性化合物製造に用いる14種の大豆粉の化学組成、タンパク質分散性及び粒径を示す。
有機農業大豆粉から得られ選択した混合スターターで醗酵したOFS豆乳をヒト再構成上皮をSkinEthic(登録商標)モデルに従い処理するためにエクオールの最終濃度10μMで使用した。このモデルは科学コミュニティで広く実験され受け入れられている(Di Cagno et al.,2009.Lactobacillus plantarum DSMZ19463によるγ−アミノ酪酸(GABA)の合成:機能的グレープは飲料及び皮膚科学的適用(Synthesis of γ−amino butyric acid (GABA)by Lactobacillus plantarum DSMZ19463):functional grape must beverage and dermatological application.Appl Biotechnol Microbiol DOI:10.1007/s00253−009−23704)。24時間の処理のあと、TEER測定を行った。このタイプの分析は国際的な科学コミュニティで広く受け入れられており、角質層の完全性やバリア機能を参照して組織の腐食能力を評価する。特にこの検出により、角質層レベルでのコンパクトなラメラ状構造の存在、完全緊密接合(integral tight junctions)及び上皮厚さについての情報を得ることができる。これらの要素が全体として効率的なバリア機能を定義する。図4は醗酵OFS豆乳の存在下でTEER値の顕著な増加(P<0.05)を示しており、これは皮膚レベルでの分子の保護作用を実証している。同様の結果が化学的に合成されたエクオールとルナシンの混合物で得られた。
有機農業大豆粉(OFS)から製造された豆乳に含まれるイソフラボン−アグリコンの免疫調整特性を試験する目的で、ニュートラルレッド(NR)取り込みアッセイを用いた、5〜100μMの濃度での標準化合物(エクオール、ダイゼイン、ゲニステイン、及びグリシテイン)によるCaco−2/TC7細胞に対する細胞毒性をまず評価した。ゲニステイン、グリシテイン、及びエクオールはメタノールとDMSO(陰性対照)に類似した振る舞いを示したが、細胞増殖には有意には影響しなかった。72時間の処理の後、ダイゼインは100μMより高い濃度で細胞増殖を顕著に阻害した(p<0.03)。
本文の他の箇所で先に概略を述べたように、イソフラボン−アグリコン(ダイゼイン、ゲニステイン、及びグリシテイン)、エクオール、及びルナシンの合成のためのバイオテクノロジー的プロトコル、並びに皮膚科学分野でのその使用を開発した。当該方法は以下を含む:
a)MRS培地上での純粋培養におけるL.plantarum DPPMA24W及びDPPMASL33,L.fermentum DPPMA114及びL.rhamnosus DPPMAAZ1の培養;
b)様々な豆乳タイプ、好ましくは農業生物学的方法に従い栽培され、実験室で皮をむいた大豆粉から調製された滅菌豆乳での細胞懸濁液の収集、洗浄及び接種;
c)選択された混合スターターによる48〜96時間、好ましくは96時間、30〜37℃、好ましくは30℃での豆乳の醗酵;
d)遠心分離による細胞の分離。方法変更に従って、その調製物は乳酸菌細胞を含んでいてもよい;
e)乾燥又は凍結乾燥プロセスによる調製物の脱水;
f)場合に応じて経口又は局所投与による使用にふさわしい形態を得るために適切な賦形剤の添加による組成物の調製。
毛髪成長のプロモータとしてルナシンを含むバイオマス(BL)をルナシンを含まないバイオマス(B)と比べたインビトロ研究
材料と方法
皮膚乳頭細胞(DPC)を2mMのL−グルタミン、1xの抗真菌性かつ抗生剤的溶液(1000u g/ml 硫酸ストレプトマイシン、1000 単位/ml ペニシリンG、及び2.5 μg/ml アンフォテリシンB)及び10%ウシ胎仔血清を含む培地(Dulbeccoの変性Eagle培地、DMEM)で培養した。合流して細胞を24時間血清なしでDMEM中で培養し、次いで様々な濃度のルナシンを含むか若しくは含まないバイオマスで処理した。
試験した濃度(0.01〜0.5μM)の範囲内で、ルナシンを含むバイオマスはインビトロでのDPC増殖の増大を用量依存的に誘発する(p<0.05)(図1)。
Claims (21)
- 以下の4種の乳酸菌:ラクトバチルス プランタルム(Lactobacillus plantarum)DSM 23755、ラクトバチルス プランタルム(Lactobacillus plantarum)DSM 23756、ラクトバチルス ファーメンツム(Lactobacillus fermentum)DSM 23757、及びラクトバチルス ラムノスス(Lactobacillus rhamnosus)DSM 23758の混合物を用いた大豆醗酵によるイソフラボン−アグリコン、エクオール、及びルナシンを含む醗酵大豆ベース混合物の調製方法。
- 以下の工程:
a)該4種のラクトバチルス プランタルム(Lactobacillus plantarum)DSM 23755、ラクトバチルス プランタルム(Lactobacillus plantarum)DSM 23756、ラクトバチルス ファーメンツム(Lactobacillus fermentum)DSM 23757、及びラクトバチルス ラムノスス(Lactobacillus rhamnosus)DSM 23758乳酸菌を培養増殖させること、
b)該乳酸菌の水性懸濁液で大豆ベース基質をインキュベートすること、
c)30〜37℃、48〜96時間インキュベートすること
を含む又はからなる、請求項1に記載の方法。 - 前記工程c)を30℃で96時間行う、請求項2に記載の方法。
- 基質が全基質体積の1〜4%の量の乳酸菌の水性懸濁液で接種され、該水性懸濁液がどの株についても約log 9.0 cfu/mlの細胞密度を有するものである、請求項2又は3に記載の方法。
- 大豆ベース基質が、大豆粉、豆乳からなる群から選ばれる、請求項2又は3に記載の方法。
- 前記大豆粉が有機農業大豆粉である、請求項5に記載の方法。
- 乳酸菌細胞を除去するためにブロス培養の遠心分離の工程d)をさらに含む、請求項1〜6のいずれか一項に記載の方法。
- ブロス培養の遠心分離が10,000xgで15分間4℃で行われる、請求項5又は6に記載の方法。
- 工程(d)で得られた上清、又は工程(c)で得られた培養物の乾燥又は凍結乾燥による脱水の工程(e)をさらに含む、請求項1〜8のいずれか一項に記載の方法。
- イソフラボン−アグリコン、エクオール、及びルナシン、並びに以下の4種の乳酸菌:ラクトバチルス プランタルム(Lactobacillus plantarum)DSM 23755、ラクトバチルス プランタルム(Lactobacillus plantarum)DSM 23756、ラクトバチルス ファーメンツム(Lactobacillus fermentum)DSM 23757、及びラクトバチルス ラムノスス(Lactobacillus rhamnosus)DSM 23758の混合物を含む、醗酵豆乳ベース混合物。
- 請求項10に定義された醗酵豆乳ベース混合物を、一つ以上の薬学的に若しくは化粧品的に許容可能な賦形剤及び/又はアジュバントとともに含む又はからなる医薬又は化粧品組成物。
- そのままで、又は一つ以上の賦形剤及び/又はアジュバントと組み合わせて、食物インテグレーターとして使用するための、請求項10に記載の醗酵豆乳ベース混合物。
- 皮膚又は腸壁の障害又は疾患の治療に使用するための請求項10に記載の醗酵豆乳ベース混合物又は請求項11に記載の組成物。
- 化粧品使用のための請求項10に記載の醗酵豆乳ベース混合物又は請求項11に記載の組成物。
- 脱毛の治療のための請求項14に記載の使用のための醗酵豆乳ベース混合物又は組成物。
- 脱毛症又は休止期脱毛の治療に使用するための請求項10に記載の醗酵豆乳ベース混合物又は請求項11に記載の組成物。
- 以下の4種の乳酸菌ラクトバチルス プランタルム(Lactobacillus plantarum)DSM 23755、ラクトバチルス プランタルム(Lactobacillus plantarum)DSM 23756、ラクトバチルス ファーメンツム(Lactobacillus fermentum)DSM 23757、及びラクトバチルス ラムノスス(Lactobacillus rhamnosus)DSM 23758の混合物。
- ラクトバチルス プランタルム(Lactobacillus plantarum)DSM 23755乳酸菌。
- ラクトバチルス プランタルム(Lactobacillus plantarum)DSM 23756乳酸菌。
- ラクトバチルス ファーメンツム(Lactobacillus fermentum)DSM 23757乳酸菌。
- ラクトバチルス ラムノスス(Lactobacillus rhamnosus)DSM 23758乳酸菌。
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Publication number | Priority date | Publication date | Assignee | Title |
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KR101840376B1 (ko) * | 2017-10-20 | 2018-03-20 | (주)코엔바이오 | 탈모 예방, 발모 촉진 또는 성기능 개선능이 있는 락토바실러스 퍼멘텀 균주 및 이를 포함하는 조성물 |
Also Published As
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JP2013538187A (ja) | 2013-10-10 |
MX2013000353A (es) | 2013-03-22 |
SI2593570T1 (sl) | 2017-08-31 |
EP2593570B1 (en) | 2017-03-01 |
HRP20170603T1 (hr) | 2017-06-30 |
CA2803322C (en) | 2020-02-25 |
HUE032690T2 (en) | 2017-10-30 |
LT2593570T (lt) | 2017-06-26 |
RU2013105731A (ru) | 2014-08-20 |
RU2564576C2 (ru) | 2015-10-10 |
WO2012007978A2 (en) | 2012-01-19 |
ITRM20100378A1 (it) | 2012-01-13 |
IT1405780B1 (it) | 2014-01-24 |
CN103140588A (zh) | 2013-06-05 |
EP2593570A2 (en) | 2013-05-22 |
RS55900B1 (sr) | 2017-09-29 |
WO2012007978A3 (en) | 2012-05-18 |
US20130231291A1 (en) | 2013-09-05 |
BR112013000778A2 (pt) | 2016-06-07 |
CA2803322A1 (en) | 2012-01-19 |
CN103140588B (zh) | 2015-09-30 |
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