JP6290080B2 - 新規乳酸菌 - Google Patents
新規乳酸菌 Download PDFInfo
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- JP6290080B2 JP6290080B2 JP2014528112A JP2014528112A JP6290080B2 JP 6290080 B2 JP6290080 B2 JP 6290080B2 JP 2014528112 A JP2014528112 A JP 2014528112A JP 2014528112 A JP2014528112 A JP 2014528112A JP 6290080 B2 JP6290080 B2 JP 6290080B2
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- lactic acid
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- bacteria
- cells
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Description
形態学的性質
1)グラム染色:陽性
2)胞子形成能:なし
3)運動性:なし
生理学的性質
1)カタラーゼ:陰性
2)ピルピン酸ナトリウムの分解:陽性
3)エスクリンの分解:陽性
4)ピロリドニル−2−ナフチルアミドの分解:陽性
5)2−ナフチル−β−D−ガラクトピラノシドの分解:陽性
6)L−ロイシン−2−ナフチルアミドの分解:陽性
7)L−アルギン酸の分解:陽性
8)各種炭水化物の分解性
D−リボース:+
D−マンニトール:+
乳糖:+
D−ソルビトール:−
D−トレハロース:−
D−ラフィノース:−
化学分類学的性質
配列番号1に示す塩基配列、又は配列番号1と90%以上の配列同一性を示す塩基配列を有する。
本発明の乳酸菌は、分散媒非存在下で凍結乾燥を行った際の生菌の生残率が40%以上であり、且つ40℃で4ヶ月間保存した際における生菌製剤中の生菌の生残率が80%以上、好ましくは分散媒非存在下で凍結乾燥を行った際の生菌の生残率が40%以上であり、且つ40℃で4ヶ月間保存した際における生菌製剤中の生菌の生残率が90%以上、より好ましくは分散媒非存在下で凍結乾燥を行った際の生菌の生残率が40%以上であり、且つ40℃で4ヶ月間保存した際における生菌製剤中の生菌の生残率が95%以上であるエンテロカッコス・フェシウム(Enterococcus faecium)に属する乳酸菌である。
乳酸菌が生育可能な培地を用いてスクリーニング株を37℃で24時間培養し、遠心分離により培養液と菌体を分離させ、培養液を除去し菌体を回収する。前記回収した菌体に、分散媒を添加しないで、適量の水を加え、分散させた菌体濃縮液を得る。当該菌体濃縮液中の総生菌数をコロニーカウント法などの常法により求める。次いで菌体濃縮液を凍結乾燥し、得られた凍結乾燥菌体中の総生菌数をコロニーカウント法などの常法により求める。そして、前記菌体濃縮液中の総生菌数と前記凍結乾燥菌体中の総生菌数により、分散媒非存在下で凍結乾燥を行った際の生菌の生残率を算出し、前記生残率が40%以上である乳酸菌を選抜する。
1次スクリーニングにおいて分散媒非存在下で凍結乾燥を行った際の生菌の生残率が40%以上であった乳酸菌を37℃で24時間培養し、遠心分離により培養液と菌体を分離させ、培養液を除去し湿菌体を得る。前記湿菌体に分散媒を添加した後、凍結乾燥し凍結乾燥菌体を得る。得られた凍結乾燥菌体を生菌数が1.0×1010cfu/gになるよう賦形剤を添加し生菌製剤を調製する。調製した生菌製剤を40℃のインキュベータ内で4ヶ月間保存した後、生菌製剤1g当たりの生菌数をコロニーカウントなどの常法により求める。そして、保存前の生菌製剤中の生菌数(1.0×1010cfu/g)と40℃で4ヶ月保存した生菌製剤1g当たりの生菌数により、40℃で4ヶ月保存した際における生菌製剤中の生菌の生残率を算出し、前記生残率が80%以上である乳酸菌を選抜する。
形態学的性質
1)グラム染色:陽性
2)胞子形成能:なし
3)運動性:なし
生理学的性質
1)カタラーゼ:陰性
2)ピルピン酸ナトリウムの分解:陽性
3)エスクリンの分解:陽性
4)ピロリドニル−2−ナフチルアミドの分解:陽性
5)2−ナフチル−β−D−ガラクトピラノシドの分解:陽性
6)L−ロイシン−2−ナフチルアミドの分解:陽性
7)L−アルギン酸の分解:陽性
8)各種炭水化物の分解性
D−リボース:+
D−マンニトール:+
乳糖:+
D−ソルビトール:−
D−トレハロース:−
D−ラフィノース:−
配列番号1に示す塩基配列、又は配列番号1と90%以上の配列同一性を示す塩基配列を有する。
本発明の組成物は、前記の本発明の乳酸菌若しくはその変異体を含有する。また、本発明の組成物に含まれる乳酸菌若しくはその変異体は、菌体そのものであってもよく、当該乳酸菌若しくはその変異体の処理物若しくは抽出残渣であってもよい。
本発明の組成物は、食品、機能性食品またはサプリメント、飼料(ペットフードを含む)、動物用医薬品、または医薬品として使用できる。
乳酸菌を10mLのMRS液体培地(MRSブイヨン、関東化学株式会社製0.52gを10mLの水に溶解し121℃、15分オートクレーブ滅菌したもの)で、37℃、24時間培養し前培養液を得た。前培養液1mLを100mLのMRS液体培地に加え、37℃、24時間培養した。培養終了後、8000rpm、10分間遠心分離し、培養液と菌体を分離させた。菌体を100mLの滅菌水で洗浄後、再度遠心分離により、滅菌水と菌体を分離し、菌体に滅菌水2mLを加え、菌体濃縮液を得た。前記菌体濃縮液0.1mLを0.9mLの生理食塩水に懸濁し、さらに生理食塩水で106倍希釈した希釈液0.1mLをMRS寒天培地(MRS寒天培地、関東化学株式会社製1.24gを20mLの水に溶解し121℃、15分オートクレーブ滅菌したものを、90φ×20の滅菌シャーレに加え、固まらせたもの)に塗抹し、37℃、2日間培養し出現するコロニー数をカウントし、菌体濃縮液中の総生菌数を求めた。また、前記菌体濃縮液を、凍結乾燥(凍結乾燥機 VirTis社製 advantage Plus、凍結条件; −30℃、12時間、乾燥条件; 10×10−1torr、棚温20℃、48時間)し、凍結乾燥菌体を得た。前記凍結乾燥菌体20mgを、1mLの生理食塩水に分散した菌体懸濁液を生理食塩水で106倍希釈した希釈液0.1mLをMRS寒天培地に塗抹し、37℃、2日間培養し出現するコロニー数をカウントし、凍結乾燥菌体中の総生菌数を求め、分散媒非存在下で凍結乾燥を行った際の生菌の生残率を次式より算出し、生残率が40%以上の乳酸菌を選抜した。
分散媒非存在下で凍結乾燥した際の生菌の生残率(%)=(乾燥菌体中の総生菌数/菌体濃縮液の総生菌数)×100
1次スクリーニング法で選抜した乳酸菌を10mLのMRS液体培地で37℃、24時間培養し前培養液を得た。前培養液10mLを1LのMRS液体培地に加え、37℃、24時間培養した。培養終了後、8000rpm、10分間遠心分離し、培養液と菌体を分離させた。菌体を1Lの滅菌水で洗浄後、再度遠心分離により、滅菌水と菌体を分離し、菌体を滅菌水で20mLにメスアップし、菌体濃縮液を得た。該菌体濃縮液にショ糖が0.4重量%、トレハロースが0.2重量%、グルタミン酸ナトリウムが0.2重量%、ヒスチジンが0.2重量%、リンゴ酸が0.2重量%からなる保護剤を溶解した溶液2mL加えて、上記と同条件で凍結乾燥し、凍結乾燥菌体を得た。該凍結乾燥菌体を生菌数が1.0×1010cfu/gになるようにデキストリンに混合し、生菌製剤を得た。生菌製剤を10gずつチャック付きポリエチレン袋(ユニパック B−8、株式会社セイニチ社製)に分注したものを、シリカゲル1g(富士シリシア化学株式会社製)を入れたアルミパウチ(ラミジップ、株式会社セイニチ社製)に梱包し、ヒートシールにより密閉したものを保存サンプルとした。保存サンプルを40℃のインキュベーターで4ヶ月間保存した後、生菌製剤100mgを1mLの生理食塩水に分散した菌体懸濁液を生理食塩水で107倍希釈した希釈液0.1mLをMRS寒天培地に塗抹し、37℃、2日間培養し出現するコロニー数をカウントし、40℃で4ヶ月間保存した際における生菌製剤中の生菌の生残率を次式より算出した。そして前記生残率が80%以上である乳酸菌を選抜した。
40℃で4ヶ月間保存した際における生菌製剤中の生菌の生残率(%)=(40℃で4ヶ月間保存後の生菌製剤1gあたりの生菌数/1.0×1010)×100
疲労改善効果は、トレッドミル装置(5連式形式 MK−680S−02A、室町機会社製)を用いて、7〜10週齢のSD系雄性ラット(1群;n=10)を10m/minの速度で走行させ、3分ごとに5m/minずつ段階的に速度を上げていき、ラットが走行不能となるまでの時間(最大走行時間)を測定することで評価した。被検物質投与前の最大走行時間と被検物質投与後2時間の最大走行時間を測定し、被検物質投与後2時間の最大走行時間から被検物質投与前の最大走行時間を引いた値(被検物質投与前後における最大走行時間の差)を算出し、被検物質投与により、被検物質投与前後における最大走行時間の差が増加しているか否かで評価した。
血流改善効果は、20〜50歳の健常男性(1群;n=5)を対象に、被検物質を1週間摂取後に冷水負荷試験を実施することで評価した。冷水負荷試験は、被検者の左手を15℃の冷水に10秒間浸し、冷水負荷直後及び冷水負荷後10分の皮膚温度をサーモグラフィー(TVS 600、日本アビオニクス社製)を用い測定した。被検物質摂取により、冷水負荷後10分の平均皮膚温度から、冷水負荷直後の平均皮膚温度を引いた値(平均皮膚温度の差)が有意に増加しているか否かで評価した。平均皮膚温度は手背部の中指の指先、中指基節骨の中間点及び第3中指手骨の中間点の3ポイントの皮膚温度の平均値とした。
便臭改善効果は、8日齢の雄の鶏ひな(1群;n=10)に試験飼料を6日間与え、試験開始前及び試験終了時に回収した糞便の便臭を測定し、試験前後の便臭の低減率を算出することで評価した。被検物質を混合していない試験飼料(標準飼料)を与えた群の便臭の低減率と比べ、被検物質を混合した試験飼料(被検物質混合飼料)を与えた群の便臭の低減率が大きいか否かで評価した。便臭は、糞便の臭気指数、硫化水素ガス濃度、酢酸ガス濃度で評価し、試験前後の臭気指数、硫化水素ガス濃度、酢酸ガス濃度の低減率を算出した。臭気指数は、糞便1gを100mLの無臭蒸留水に懸濁し25℃30分間静置後、さらに10倍希釈したものを試験水とし、3点比較式フラスコ法にて求めた。また、硫化水素ガス濃度及び酢酸ガス濃度は、糞便100gをガラスシャーレに乗せ10Lのポリスチレン製袋に入れ、袋内の空気を全て排出した後、活性炭を通し精製した無臭空気を袋内に充満させ密閉し、約22℃の室温で1時間静置した後、袋内の硫化水素ガス及び酢酸ガス濃度をガス検知管で測定することで求めた。尚、回収した糞便は測定時まで密閉し、−80℃で保管した。
成長促進効果は、5週齢のICR雄性マウス(1群;n=10)に試験飼料を1ヶ月間与え、試験終了時の体重を測定することで評価した。被検物質を混合していない試験飼料(標準飼料)を与えた群と比べ、被検物質を混合した試験飼料(被検物質混合飼料)を与えた群の体重が大きいか否かで評価した。試験期間中の各マウスの摂餌量を測定するため、マウスは個別飼育とし、試験飼料はローデンカフェ(オリエンタル酵母社製)付き粉末給餌器を用いて自由摂取させた。
食品素材等から分離した乳酸菌125株(分離株1−分離株125)を対象に、上記1次スクリーニング法により、各分離株の分散媒非存在下で凍結乾燥を行った際の生菌の生残率を求めた。
2次スクリーニング法に記載の方法に準じてR30株及び基準株の凍結乾燥菌体を調製し、各々の凍結乾燥菌体を生菌数が5.0×1010cfu/gになるようにデキストリンに混合した生菌製剤を得た。R30株の生菌製剤を1g/kg投与するR30株投与群、基準株の生菌製剤を1g/kg投与する基準株投与群、及びデキストリンを1g/kg投与するデキストリン投与群について上記疲労改善効果の評価法を実施した。その結果を表2に示す。
実施例2で調製したR30株の生菌製剤を1g/日摂取するR30株摂取群、実施例2で調製した基準株の生菌製剤を1g/日摂取する基準株摂取群及びデキストリンを1g/日摂取するデキストリン摂取群について、上記血流改善効果の評価法を実施した。その結果を表3に示す。
2次スクリーニング法に記載の方法に準じてR30株の凍結乾燥菌体を調製し、該凍結乾燥菌体を生菌数が1.0×1010cfu/gになるようにデキストリンに混合した生菌製剤を得た。当該R30株の生菌製剤が0.3重量%になるように標準飼料(ブロイラー肥育前期用標準飼料;日本配合飼料株式会社製)に混合した試験飼料(R30株混合飼料)を調製し、上記便臭改善効果の評価法を実施した。その結果を表4に示す。
被検物質として実施例4で調製したR30株の生菌製剤が0.1重量%となるように標準飼料(CE−2粉末飼料;日本クレア社製)に混合した試験飼料(R30株混合飼料)を調製し、上記成長促進効果の評価法を実施した。試験終了時の標準飼料を与えた群の体重を100%とした時のR30株混合飼料を与えた群の体重の割合を算出した。その結果を表5に示す。
食品素材等から分離した乳酸菌を、API20ストレップ(シスメックス・ビオメリュー株式会社製)を用いてエンテロコッカス・フェシウム属と同一の菌学的性質を示した10株(エンテロコッカス・フェシウム株1〜10)を選抜した。各株に対して上記1次スクリーニング法を実施し、分散媒非存在下で凍結乾燥を行った際の生菌の生残率が40%以上であった3株(エンテロコッカス・フェシウム株1、エンテロコッカス・フェシウム株2、エンテロコッカス・フェシウム株3)について2次スクリーニングを実施し、40℃で4カ月間保存した際における生菌製剤中の生菌の生残率を求めた。その結果を表6に示す。
実施例6により得られたR28株の疲労改善効果及び血流改善効果について、実施例2及び実施例3と同様に調べた。尚、エンテロコッカス・フェシウム株2及びエンテロコッカス・フェシウム株4はR28株の比較対象とした。その結果を表7に示す。
R28株の便臭改善効果及び成長促進効果について、実施例4及び実施例5と同様に調べた。その結果を表8に示す。
R30株を1LのMRS液体培地で37℃、24時間培養した。培養終了後、遠心分離した菌体を1Lの滅菌水で洗浄後、再度遠心分離することにより、菌体と滅菌水を分離させた。菌体を滅菌水で20mLにメスアップした菌体濃縮液に対して、ショ糖、トレハロース、グルタミン酸ナトリウム、ヒスチジン、リンゴ酸からなる保護剤が菌体濃縮液中の乾燥菌体重量に対して、0、1、3、10、100、1000、10000重量%になるよう保護剤を溶解した溶液を添加し、凍結乾燥した。該凍結乾燥菌体を生菌数が1.0×1010cfu/gになるようにデキストリンに混合し、生菌製剤を調整し、40℃で4ヶ月間保存した際における生菌製剤中の生菌の生残率を求めた。また、R28株についても同様の実験を行った。その結果を表9に示す。
Claims (10)
- エンテロコッカス・フェシウムR30株(NITE BP−01362)、エンテロコッカス・フェシウムR28株(NITE BP−01361)、又は、該菌株がDNA変異処理された変異体若しくは該菌株の子孫株である乳酸菌であって、該変異体若しくは該子孫株は、以下の要件:
(i) 分散媒非存在下で凍結乾燥を行った際の生菌の生残率が40%以上であり、且つ、ショ糖、トレハロース、グルタミン酸ナトリウム、ヒスチジン、リンゴ酸から成る保護剤を乾燥菌体重量に対して1重量%以上含む菌体濃縮液を凍結乾燥した菌体を含む生菌製剤を40℃で4ヶ月間保存した際における該生菌製剤中の生菌の生残率が80%以上であること;
(ii) トレッドミル装置を用いてラットを10m/minの速度で走行させ、3分毎に5m/minずつ段階的に速度を上げていき、ラットが走行不能となるまでの最大走行時間の差を指標とする疲労改善効果の評価試験において、被検菌投与前のラットの最大走行時間と、被検菌投与後2時間の同ラットの最大走行時間の差が、基準株であるエンテロコッカス・フェシウムDSM20477投与ラットの同最大走行時間の差よりも増加すること;及び
(iii)健常人(n=5)に被検菌を1週間摂取させた後、15℃の冷水に手を10秒間浸水させ、冷水負荷直後の平均皮膚温度と冷水負荷後10分の平均皮膚温度の差を指標とする血流改善効果の評価試験において、被検菌を摂取した健常人の平均皮膚温度の差が、基準株であるエンテロコッカス・フェシウムDSM20477を摂取した健常人の同平均皮膚温度の差よりも増加すること;
を満たすものである、上記乳酸菌。 - 請求項1に記載の乳酸菌、又は該乳酸菌の処理物若しくは抽出残渣を含有する組成物。
- さらに保護剤を含むことを特徴とする、請求項2に記載の組成物。
- 保護剤の含量が乾燥菌体重量に対して1重量%以上であることを特徴とする請求項3に記載の組成物。
- 保護剤が、ショ糖、トレハロース、グルタミン酸ナトリウム、ヒスチジン、リンゴ酸の群から選択される1種以上であることを特徴とする請求項3に記載の組成物。
- 請求項2〜5のいずれかに記載の組成物を有効成分とする疲労改善剤。
- 請求項2〜5のいずれかに記載の組成物を有効成分とする血流改善剤。
- 請求項2〜5のいずれかに記載の組成物を含有する食品。
- 請求項2〜5のいずれかに記載の組成物を含有する飼料または動物用医薬品。
- 請項2〜5のいずれかに記載の組成物を含有する医薬品。
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