JP2016147881A - 肥満治療効果及び抗酸化活性を有する酵母加水分解物を含む組成物及びその製造方法 - Google Patents
肥満治療効果及び抗酸化活性を有する酵母加水分解物を含む組成物及びその製造方法 Download PDFInfo
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Abstract
Description
タンパク質分解酵素
タンパク質分解酵素KH−15は、高麗大学食品栄養学科で味噌から分離して保存中のバチルス属菌(Bacillus sp.)KH−15をタンパク質分解酵素の生産菌株に用いて得た。即ち、タンパク質分解酵素の生産のためにLB培地(tryptone10 g/L、酵母エキス5 g/L、NaCl 10 g/L)にスキムミルク 1%を添加した液体培地をタンパク質分解酵素生産用培地として用いた。タンパク質分解酵素生産用培地はpH 6.5に調整後、121℃、15分間加圧殺菌し、30℃で24時間培養し、上澄液を遠心分離して回収し、粗酵素液として用いた。このように得たタンパク質分解酵素KH−15のアミノ酸配列は配列番号1の通りである。
MNQKAMIVIA AGSMLFGGAG VYAGINLLEM DKPQTAAVPA TAQADSERDK AMDKIEKAYE
LISNEYVEKV DREKLLEGAI QGMLSTLNDP YSVYMDKQTA KGGSDSLDSS FEGIGAEVGM
EDGKIIIVSP FKKSPAEKAG LKPLSTIISI NGESMAGKDL NHAVLKIRGK KGSSVSMKIQ
RPGTKKQLSF RIKRAEIPLE TVFASEKKVQ GHSVGYIAIS TFSEHTAEDF AKALRELEKK
EIEGLVIDVR GNPGGYLQSV EEILKHFVTK DQPYIQIAER NGDKKRYFST LTHKKAYPVN
VITDKGSASA SEILAGALKE AGHYDVVGDT SFGKGTVQQA VPMGDGSNIK LTLYKWLTPN
GNWIHKKGIE PTIAIKQPDY FSAGPLQLKE PLKVDMNNED VKHAQVLLKG LSFDPGREDG
YFSKDMKKAV MAFQDQNKLN KTGVIDTRTA ETLNQQIEKK KSDEKNDLQL QTALKASFVN
実験結果はSPSS programを用いて平均と誤差を提示し、各処理別有意性はDuncans multiple range testでp<0.05水準で検定した。
Saccharomyces cerevisiae (ATCC4126)を2%モラセス(廃糖蜜)、0.6%(NH4)2SO4、0.1%MgSO4・7H2O、0.2%KH2PO4、0.03%K2HPO4と0.1%NaClを含有した液体培地を用いて30℃で3日間培養した。培養後、3,000 rpmで20分間遠心分離して菌体を回収し、20 mMリン酸塩緩衝溶液(pH 7.0)を用いて10%懸濁液を作った。懸濁液にプロタメックス(Protamex)、フレーバーザイム(Flavourzyme)、プロテアーゼA(Protease A)、アロアーゼAP−10(Aroase AP-10)、ペスカラーゼ(Pescalase)、パパイン(Papain)及びKH−15をそれぞれ0.5%ずつ添加し、35℃で6時間加水分解後、95℃で10分間加熱処理し、遠心分離して上澄液を回収した。酵母加水分解物は10kDa分離膜(Satocon cassette, Sartorius, Germany)を用いて処理し凍結乾燥して製造した。
商業用酵素とバチルス属菌KH−15が生産したタンパク質分解酵素KH−15を用いて酵母加水分解物を製造してタンパク質回収率を測定した(図1、式1)。
タンパク質回収率(%)=(加水分解後のタンパク質/加水分解前のタンパク質)×100
脂肪細胞での脂肪分解効果
3T3−L1前駆脂肪細胞(preadipocytes)(ATCC#F8979, Manassas, VA)を10%ウシ胎児血清、100 unit/mL ペニシリン、100mg/mL ストレプトマイシンが含まれたDMEM培養液を用いて5%CO2下にインキュベーターで培養した。3, 4日後に細胞が融合するようになると、0.05%トリプシン/EDTAで処理して細胞を分離した後、遠心分離(1,000rpm, 5分)して細胞を集め、細胞密度が3.3×103cell/cm2になるように懸濁溶液を作って12ウェルプレートに1 mlずつ分注して2次培養した。3, 4日後に細胞を増殖させた後、分化培地(DMEM培養液に5μg/mlインスリン、0.25 μMデキサメタゾン及び0.5 mM IBMX(3−イソブチル−1−メチルキサンチン)が添加された培地)を添加して分化を誘導した。
レプティンは、脂肪細胞の肥満遺伝子により生成されるホルモンで、視床下部に作用して飲食物の摂取を抑制させてエネルギー消費を増加させ、肥満を調節するタンパク質で(Caro et al., 1996)、レプティンは体脂肪量と密接な関連関係があり(Considine et al., 1996)、血中レプティン濃度は体脂肪量を示す指標として知られ、最近の肥満研究で多く適用されている。レプティンは脂肪細胞内の脂肪の蓄積量が増加するほどレプティンの分泌量が増加すると知られている(Considine et al., 1996)。従って、レプティン量の減少は脂肪蓄積が少ないということを意味する。
試料に対する細胞生存率は、MTT〔3-(4,5-メチルチアゾ-2-イル)-2,5-ジフェニルテトラゾリウムブロマイド〕比色法で実験した(Camichael et al., 1978)。細胞は1×105 cell/200μLの濃度で96ウェル プレートに分注して24時間培養後に培地を除去した。ここで新たなDMEM培地200μLに濃度別に希釈した試料をそれぞれ添加して24時間培養した後、2.5mg/mLで製造したMTT溶液20μLを各ウェルに加えて4時間培養した。培養終了後、上澄液を除去して各ウェルに100μLのDMSOを添加して生成されたformazan結晶を溶解させてマイクロプレートリーダーを用いて550nmで吸光度を測定した。この時、細胞生存率は次の通り計算して示した。
細胞生存率(%)={100−(対照群の吸光度−試料処理群の吸光度)/対照群の吸光度}×100
酵母KH−15加水分解物
実施例2で抗肥満効果が最も優れていたBacillus sp. KH−15が生産したタンパク質分解酵素であるKH−15を用いて製造した酵母加水分解物(以下、Eatlessということがある)をもって生理活性を測定した。
実験動物は体重180−185g程度のS/D雄ラットを大韓実験動物センター(忠清北道陰城)から購入して用いた。食餌(表5)と水は自由に摂取するようにし、飼育場温度は22℃、そして湿度は40−60%に維持し、照明は12時間明暗周期とした。
血清中の総コレステロール、HDL−コレステロール、中性脂質濃度は酵素法による測定キット試薬で分析した。
(※)HTR = HDLコレステロール/総コレステロール
酵母KH−15加水分解物のDPPHラジカルとABTSラジカル消去能
DPPHラジカル消去能の測定は、Cheung等(2003)の方法を用いた。1,1-ジフェニル-2-ピクリルヒドラジルをエタノールに溶解させた0.2mM DPPH溶液0.4mLと試料0.1mLを暗所で10分間反応させた後、520nmで吸光度を測定した。
ラジカル消去活性(%)=(1-Asample/A対照群)×100
Asample:試料添加時の吸光度、 A対照群:試料未添加時の吸光度
酸素遊離基(酸素フリーラジカル、活性酸素)は酸素を用いて生存する細胞内で生成される毒性が強い物質で、細胞のDNAや細胞膜脂質及びタンパク質等を損傷させるので、細胞内ではスーパーオキシドディスムターゼ(SOD)、カタラーゼ、グルタチオンペルオキシダーゼ等の抗酸化酵素とグルタチオン、尿酸等の様々な非酵素的抗酸化物質があり、酸素遊離基による酸化的損傷から各種細胞構造物を保護する。SODは遊離酸素をH2O2とO2に転換し、生成されたH2O2はカタラーゼとグルタチオンペルオキシダーゼにより消去されるが、グルタチオンペルオキシダーゼは還元型グルタチオン(GSH)を酸化型グルタチオン(GSSG)に酸化させながらH2O2を除去し、GSSGはグルタチオンリダクターゼにより再びGSHに還元され、人体内でグルタチオンはGSHとGSSGの2つの形態で均衡をなしながら存在するようになる。もし、細胞内に毒性又は酸化的損傷が生じた場合には、GSSGが徐々に増加するようになって、GSH/GSSGの不均衡をもたらすようになり防御機作としての役割が消失する。GSHは酸素遊離基による細胞の損傷を防止し、細胞内解毒作用を担当する重要な物質とみなされている。従って、健康な個体の細胞は酸化的細胞損傷を引き起こし得る酸素遊離基を消去する防御機作により十分に保護を受けることができる(De Haan et al., 1995)。
体重増加量及び食餌摂取率
高脂肪食餌をしたラットと酵母KH−15加水分解物を経口投与(100mg/kg, 24日投与)したラット間の体重増加量を測定した結果、経口投与12日以後から高脂肪食餌群と酵母KH−15加水分解物投与群との間に体重増加量が有意的差を示し、酵母KH−15加水分解物投与群の体重増加量が少ないことが確認された(図5)。
各実験群の臓器の重さの変化を測定した結果、肝、脾臓と腎臓の臓器間の重量変化は有意的差がなかったが、副睾丸周辺の脂肪(epididymal fat pad)と腎臓周辺の脂肪(perirenal fat pad)は対照群の場合、5.38 gと3.78 gである一方、酵母KH−15加水分解物経口投与群の場合、4.38 gと3.05 gの脂肪含量を示した。
酵母KH−15加水分解物
以上の結果により、抗肥満効果が最も優れたバチルス属菌KH−15が生産したタンパク質分解酵素KH−15を用いて製造した酵母KH−15加水分解物(Eatless)を用いて生理活性を測定した。
犬(ビーグル(beagle))(2〜5年)を標準飼料(ピュリナ(登録商標))と水は自由に摂取するようにし、飼育場温度は24℃、そして湿度は40−60%に維持し、照明は12時間明暗周期とした。酵母加水分解物が入っているカプセルを100mg/kgに該当する量を経口投与した。ビーグルを3群(対照群、実験群1、実験群2)に分類し、群当り5匹を用いた。対照群は標準飼料を給餌、実験群1は30日間標準試料及び酵母KH−15加水分解物を経口投与し(100 mg/kg)、実験群2は初期10日間は標準飼料、11−20日の間には、標準飼料及び酵母KH−15加水分解物を(100 mg/kg)、21−30日の間には、標準飼料をそれぞれ給餌した。各群間の飼料摂取は次の通りであり(表9)、酵母KH−15加水分解物に対して犬の最初の食いつき(嗜好性)が相当良かったことから、飼料添加物に適するものと見られる。
体重増加
30日間標準飼料、酵母KH−15加水分解物をそれぞれ経口投与し、実験前、実験開始10,20,30日後に体重増加量を測定した結果(図7)、対照群は実験期間が経過するほど体重が増加する傾向を示し、10,20,30日での体重増加量はそれぞれ0.88, 1.46, 3.06 kgの増加量を示した。一方、実験群1(Eatless-A)では30日経過後に0.2 kgの体重増加量を示し、実験群2(Eatless-B)ではむしろ1.64 kgの体重減少量を示した。
腹囲を測定した結果(図8)、標準飼料のみ摂取した対照群では体重の増加量と共に腹囲も10日目は2.2 cm、20日と30日目は2.86と3.43 cmの増加を示した一方、実験群1では30日目1.8 cm減少し、実験群2では30日目に腹囲の変化がなかった。
前述した通り、酵母KH−15加水分解物の経口投与は体重増加を抑制又はむしろ減少させる効果があることを確認したが、これは酵母KH−15加水分解物が有する毒性による可能性があるので、血液を採取してペットの健康点検時に測定するいくつかの血液中の成分を測定した(表10, 11)。
血液内で抗酸化活性の指標であるMDAとGSH含量をそれぞれ測定した結果、酸化生成物であるMDA(マロンジアルデヒド)の含量は、対照群が278.4μmol/Lと高い数値を示した一方、酵母KH−15加水分解物投与群である実験群1, 2は、それぞれ213.6と224.4 μmol/Lと低い数値を示した。また、抗酸化活性物質であるGSH含量は、対照群が2.0 mmol/Lである一方、実験群1, 2はそれぞれ2.7と2.6 mmol/Lとして多少高い含量を示した(図9)。なお、図9において、各群間で記号bの値は記号aの値に対して有意差(p<0.05)があることを示す。
本発明の酵母KH−15 加水分解物(Eatless)170部、ミツロウ30部及びコーン油50部を約50℃に加熱混合して均質にした後、カプセル充填機に供して、常法により1粒当り内容量が250mgのゼラチン被覆ソフトカプセル製剤を試作した。このカプセル製剤は、経口摂取が可能な栄養補助食品、飼料又は医薬品として利用できる。
本発明の酵母KH−15 加水分解物(Eatless)100部、化工デンプン(松谷化学工業社製、商品名:「パインフロー」(登録商標))80部、リン酸三カルシウム85部及びセルロース35部を混合機に仕込み、10分間攪拌混合した。この混合物を直打式打錠機に供して直径7mm、高さ4mm、質量150mg/個の素錠を作成し、次いでコーティング機でシェラック被膜を形成させて錠剤形状の経口摂取用組成物を試作した。
市販の緑茶飲料水1000部に本発明の酵母KH−15 加水分解物(Eatless)10部を加えて十分に混合し均質な飲料水を試作した。これは冷蔵庫で1週間保存しても外観及び風味に異状や違和感は認められなかった。
Claims (11)
- 酵母(Saccharomyces cerevisiae)をタンパク質分解酵素で分解した酵母加水分解物を有効成分として含み、体重減少効果及び抗酸化活性を有することを特徴とする組成物。
- 上記タンパク質分解酵素は、プロタメックス(Protamex)、フレーバーザイム(Flavourzyme)、プロテアーゼA(Protease A)、アロアーゼAP−10(Aroase AP-10)、ペスカラーゼ(Pescalase)、パパイン(Papain)及び配列番号1のアミノ酸配列を含むKH−15で構成される群から選択される1つ又はそれ以上であることを特徴とする、請求項1に記載の組成物。
- 上記タンパク質分解酵素は、配列番号1のアミノ酸配列を含むKH−15であることを特徴とする、請求項2に記載の組成物。
- 上記組成物は、食品組成物、飼料組成物、薬学的組成物及び化粧料組成物から選択されるいずれか1つであることを特徴とする、請求項1に記載の組成物。
- 上記薬学的組成物は、動脈硬化、内臓肥満、腹部肥満、高脂血症、脂肪肝及び肥満で構成された群から選択されるいずれか1つを予防又は治療することを特徴とする、請求項4に記載の組成物。
- 請求項1〜請求項5のいずれか1項の組成物を動物に投与する段階を含む、動物の動脈硬化、内臓肥満、腹部肥満、高脂血症、脂肪肝及び肥満で構成された群から選択されるいずれか1つを予防又は治療する方法。
- 酵母(Saccharomyces cerevisiae)にタンパク質分解酵素を加える段階を含む、体重減少効果及び抗酸化活性を有する組成物の製造方法。
- 上記タンパク質分解酵素は、プロタメックス(Protamex)、フレーバーザイム(Flavourzyme)、プロテアーゼA(Protease A)、アロアーゼAP−10(Aroase AP-10)、ペスカラーゼ(Pescalase)、パパイン(Papain)及び配列番号1のアミノ酸配列を含むKH−15で構成される群から選択される1つ又はそれ以上であることを特徴とする、請求項7に記載の製造方法。
- 上記タンパク質分解酵素は、配列番号1のアミノ酸配列を含むKH−15であることを特徴とする、請求項8に記載の製造方法。
- 上記組成物は、食品組成物、飼料組成物、薬学的組成物及び化粧料組成物から選択されるいずれか1つであることを特徴とする、請求項7に記載の製造方法。
- 上記薬学的組成物は、動脈硬化、内臓肥満、腹部肥満、高脂血症、脂肪肝及び肥満で構成された群から選択されるいずれか1つを予防又は治療することを特徴とする、請求項10に記載の製造方法。
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- 2011-04-27 WO PCT/KR2011/003116 patent/WO2011136573A2/ko active Application Filing
- 2011-04-27 EP EP11775278.2A patent/EP2564860B1/en active Active
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2016
- 2016-03-10 JP JP2016046918A patent/JP2016147881A/ja active Pending
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Also Published As
Publication number | Publication date |
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WO2011136573A2 (ko) | 2011-11-03 |
EP2564860B1 (en) | 2015-03-04 |
EP2564860A4 (en) | 2014-01-08 |
KR100999429B1 (ko) | 2010-12-09 |
US20130052185A1 (en) | 2013-02-28 |
JP2011231097A (ja) | 2011-11-17 |
JP5902382B2 (ja) | 2016-04-13 |
EP2564860A2 (en) | 2013-03-06 |
WO2011136573A3 (ko) | 2012-04-19 |
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