JP6165185B2 - 乳酸菌又はその処理物を含む脂質代謝及び/又は糖代謝改善剤 - Google Patents
乳酸菌又はその処理物を含む脂質代謝及び/又は糖代謝改善剤 Download PDFInfo
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Description
本発明は、第1の態様により、ペルオキシソーム増殖剤応答性受容体(PPAR)α及びペルオキシソーム増殖剤応答性受容体(PPAR)γに対するデュアルアゴニスト活性をもつ菌体、好ましくは乳酸産生菌、より好ましくはラクトバチルス属及びビフィドバクテリウム属から選択される菌体、その菌体処理物又はそれらの混合物を有効成分として含むことを特徴とする、脂質代謝及び/又は糖代謝改善剤を提供する。
本発明はさらに、本発明の脂質代謝及び/又は糖代謝改善剤を食品添加物として含んでなる飲食品を提供する。その実施形態によれば、飲食品は、脂質代謝及び糖代謝改善用の機能性食品又は健康食品である。
<菌体粉末の作製>
各乳酸菌の菌株の凍結ストックを平板培地に起こした後、液体培地を用いて、前々培養、前培養、本培養を実施した(5ml→40ml→2L)。表1に使用菌種、菌株、培地、培養温度を示す。なお、植菌量は各液体培地重量の1%とし、18時間培養した(使用培地及び培養温度は表2参照)。培養後、10℃にて12000 g 、 7分間遠心分離し、培養上清を除去した。イオン交換水を加え、同様に遠心分離後、凍結乾燥した菌体をミル(TESCOM)で分散し、菌体粉末を得た。
2gの菌体粉末を500mlの0.5mol/l水酸化カリウム・エタノール溶液(関東化学)に懸濁後、2分間超音波破砕処理(出力40%、最大750W中プローブ使用、VC-750(東京理化器械))した。処理後の液を500mlの赤キャップ付広口メディウム瓶(耐熱性、三商)に移し、密栓した。100℃の沸騰水中に1時間静置加温後、流水にて冷却した。冷却した菌液に濃塩酸(和光純薬工業)を加え、pHを2以下に調製した。
<PPARαレポーターアッセイ>
アフリカミドリザル腎臓由来培養細胞CV-1を5x104個/mlの濃度に調製し、10%(v/v)FBSを含むDMEM培地(SIGMA)に懸濁後、平底24wellプレート(Corning)を用いて500μL/ウェル濃度にて、5%(v/v)CO2雰囲気下、37℃、24時間培養した。24時間後、顕微鏡下に80〜90%コンフルエントであることを確認後、以下の手順でトランスフェクションを実施した。
評価サンプルの調製は以下のとおり行った。各種乳酸菌のジエチルエーテル抽出物をDMSOの終濃度が0.1%となるようOpti-MEMで希釈した。PPARαリガンドのポジティブコントロールとしてGW7647(SIGMA)を、ネガティブコントロールとしてDMSOを使用した。アッセイ時の濃度は、乳酸菌抽出物サンプルは2.5μM(脂肪酸換算値)、GW7647は10nMとした。
結果を表2に示す。
<PPARγレポーターアッセイ>
アフリカミドリザル腎臓由来培養細胞CV-1を5x104個/mlの濃度に調製し、10%(v/v)FBSを含むDMEM培地(SIGMA)に懸濁後、平底24wellプレート(Corning)を用いて500μL/ウェル濃度にて、5%(v/v)CO2雰囲気下、37℃、24時間培養した。24時間後、顕微鏡下に80〜90%コンフルエントであることを確認後、以下の手順でトランスフェクションを実施した。
PPARγリガンドのポジティブコントロールとしてTroglitazone(和光純薬工業)を、ネガティブコントロールとしてDMSOを使用した。アッセイ時の濃度は、乳酸菌抽出物サンプルは2.5μM(脂肪酸換算値)、Troglitazoneは1μMとした。
結果を表3に示す。
<CP1563株の脂質代謝改善(マウス実験)>
乳酸菌Lactobacillus amylovorus CP1563株(FERM BP-11255)を以下のとおり調製した。
周速:14.0m/s
処理流速:1L/10min
処理回数:5回
破砕槽温度:15℃
使用ガラスビーズ:直径0.5mm 0.4L
上記破砕(破壊)処理により、乳酸菌懸濁液中の菌体の平均長径が処理前の68%に縮小した(2.77μ→1.89μm)。破砕後、懸濁液を凍結乾燥し、破砕乳酸菌凍結乾燥粉末を得た。
動脈硬化指数=(総コレステロール−HDLコレステロール)÷HDLコレステロール
結果を図1(HDL-コレステロール)及び図2(動脈硬化指数)に示す。CP1563株破砕菌体の投与により、HDL-コレステロール及動脈硬化指数を改善し、その効果は用量依存的であることを確認した。
<CP1563株の脂質代謝改善(ヒトでの有効性の確認)>
Lactobacillus amylovorus CP1563株破砕菌体のHDLコレステロール等の脂質関連マーカー及び体内脂肪への影響を確認するため、12週間摂取試験を実施した。試験は二重盲検並行群間比較試験とし、ヘルシンキ宣言に基づく倫理的原則を遵守して実施した。
周速:14.0m/s
原料投入量:200g仕込み/ポット
使用メディア:φ2(3kg/ポット)
回転数:110rpm(ポット、テーブルとも)
粉砕時間:10時間
上記破砕(破壊)処理により、菌体の平均長径が処理前の47%に縮小した(2.77μ→1.30μm)CP1563株破砕菌体を得た。
株式会社タニタ社の体内脂肪計TBF-310を用いて測定した。
CTスキャンは株式会社日立メディコのCTスキャナシステム(CT−W450)を用いた。
管電圧; 120kVp
mAs値; 90mAs
ウインドウレベル; 0
ウインドウ幅; 1000
撮影方法:以下の(a)から(i)の順に行った。
(b)天板を撮影位置付近まで移動させた。
(c)臍部を露出させ、スリットランプ(ライトローカライザー)で撮影位置あわせをした。
(d)CT撮影時の呼吸の練習を2〜3回させた。
(e)天板の上昇、下降させてランプを確認しながら最終的な撮影位置合わせをした。
(f)1枚を臍の中心部位においてマニュアル設定で撮影した。
(g)次いで同位置から3枚連続のオート設定として臍中心部とその上下部3mmを撮影した。
(h)撮影した3枚の画像を確認して、臍の中心部に近いものを1枚保存。前回測定画像がある場合は、前回画像と測定位置の近いものを採用した。
(i)内臓脂肪計測PCソフト(Fat ScanTM Ver.3.0,N2システム(株))を用いて内臓脂肪面積及び皮下脂肪面積を算出した。
(1)検査日前日はオリゴ糖や食物繊維の多い食事や炭酸飲料の摂取は極力控えるよう指導した。
(2)検査日は被験者にガードル・ボディスーツなどの補正下着は着てこないよう指導した。
(3)検査着に着替える時、男女問わず検査着の下は下着のみとし、パンツは臍から離れるように下げておいてもらう。下着で腹部を絞めつけていた跡がある場合は腰周辺をもみほぐした。
(4)撮影時、天板に被験者が仰向けになったら、真っ直ぐになるよう姿勢を直した。
(5)位置あわせをする前に、被験者の腰を数回浮かして、天板に皮膚の張りが無いようにした。
(6)呼気時に臍部を撮影できるよう、撮影場所を示すライトを当てながら呼吸の練習をさせて位置あわせをした。
(7)前回測定時の撮影画像がある場合は、前回画像を参照しながらモニターで確認し、撮影位置を確かめた。
Claims (5)
- 乳酸産生菌の菌体又は菌体の処理物のペルオキシソーム増殖剤応答性受容体(PPAR)α活性化能及びペルオキシソーム増殖剤応答性受容体(PPAR)γ活性化能を測定し、PPARαアゴニスト活性が、菌体のジエチルエーテル抽出物についてのPPARαレポーターアッセイでのDMSOネガティブコントロール0(ゼロ)及びGW7647(10nM、SIGMA社)ポジティブコントロール100に対して70以上であり、PPARγアゴニスト活性が、菌体のジエチルエーテル抽出物についてのPPARγレポーターアッセイでのDMSOネガティブコントロール0(ゼロ)及びトログリタゾン(1μM、Troglitazone、和光純薬工業社)ポジティブコントロール100に対して30以上の陽性を示す菌体又は菌体処理物を選択することを特徴とする、脂質代謝及び/又は糖代謝改善用組成物の製造に使用するための乳酸産生菌の菌体又は菌体処理物の選択方法。
- 乳酸産生菌の菌体又は菌体の処理物のペルオキシソーム増殖剤応答性受容体(PPAR)α活性化能及びペルオキシソーム増殖剤応答性受容体(PPAR)γ活性化能を測定し、PPARαアゴニスト活性が、菌体のジエチルエーテル抽出物についてのPPARαレポーターアッセイでのDMSOネガティブコントロール0(ゼロ)及びGW7647(10nM、SIGMA社)ポジティブコントロール100に対して70以上であり、PPARγアゴニスト活性が、菌体のジエチルエーテル抽出物についてのPPARγレポーターアッセイでのDMSOネガティブコントロール0(ゼロ)及びトログリタゾン(1μM、Troglitazone、和光純薬工業社)ポジティブコントロール100に対して30以上の陽性を示す菌体又は菌体処理物を選択することを特徴とする、脂質代謝予防及び/又は糖代謝予防用組成物の製造に使用するための乳酸産生菌の菌体又は菌体処理物の選択方法。
- 乳酸産生菌の菌体又は菌体の処理物のペルオキシソーム増殖剤応答性受容体(PPAR)α活性化能及びペルオキシソーム増殖剤応答性受容体(PPAR)γ活性化能を測定し、PPARαアゴニスト活性が、菌体のジエチルエーテル抽出物についてのPPARαレポーターアッセイでのDMSOネガティブコントロール0(ゼロ)及びGW7647(10nM、SIGMA社)ポジティブコントロール100に対して70以上であり、PPARγアゴニスト活性が、菌体のジエチルエーテル抽出物についてのPPARγレポーターアッセイでのDMSOネガティブコントロール0(ゼロ)及びトログリタゾン(1μM、Troglitazone、和光純薬工業社)ポジティブコントロール100に対して30以上の陽性を示す菌体又は菌体処理物を選択することを特徴とする、皮下脂肪低減及び/又は内臓脂肪低減用組成物の製造に使用するための乳酸産生菌の菌体又は菌体処理物の選択方法。
- 乳酸産生菌の菌体又は菌体の処理物のペルオキシソーム増殖剤応答性受容体(PPAR)α活性化能及びペルオキシソーム増殖剤応答性受容体(PPAR)γ活性化能を測定し、PPARαアゴニスト活性が、菌体のジエチルエーテル抽出物についてのPPARαレポーターアッセイでのDMSOネガティブコントロール0(ゼロ)及びGW7647(10nM、SIGMA社)ポジティブコントロール100に対して70以上であり、PPARγアゴニスト活性が、菌体のジエチルエーテル抽出物についてのPPARγレポーターアッセイでのDMSOネガティブコントロール0(ゼロ)及びトログリタゾン(1μM、Troglitazone、和光純薬工業社)ポジティブコントロール100に対して30以上の陽性を示す菌体又は菌体処理物を選択することを特徴とする、皮下脂肪蓄積予防及び/又は内臓脂肪蓄積予防用組成物の製造に使用するための乳酸産生菌の菌体又は菌体処理物の選択方法。
- 乳酸産生菌の菌体が、ラクトバチルス(Lactobacillus)属に属する菌体及びビフィドバクテリウム(Bifidobacterium)属に属する菌体からなる群より選択される少なくとも1種の菌体である、請求項1〜4のいずれか1項に記載の選択方法。
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KR102128098B1 (ko) * | 2020-03-23 | 2020-06-30 | 주식회사 종근당바이오 | 락토바실러스 델브루키 subsp. 락티스 CKDB001 균주, 및 이를 포함하는 비알코올성 지방간의 예방, 개선, 또는 치료용 조성물 |
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SG11201402993XA (en) | 2014-09-26 |
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CN104010648A (zh) | 2014-08-27 |
JP2015108010A (ja) | 2015-06-11 |
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PH12014501254A1 (en) | 2014-09-08 |
JP5690435B2 (ja) | 2015-03-25 |
KR101809695B1 (ko) | 2017-12-15 |
AU2012349340A1 (en) | 2014-07-24 |
EP2789340A4 (en) | 2015-07-22 |
CA2861533A1 (en) | 2013-06-13 |
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