JP2018082701A - 酵素生成能、胆汁抵抗性、及びヘリコバクターピロリに対する抗菌能及び血栓溶解能を有するバチルスサブチリス(Bacillus subtilis)LRCC1002菌株 - Google Patents
酵素生成能、胆汁抵抗性、及びヘリコバクターピロリに対する抗菌能及び血栓溶解能を有するバチルスサブチリス(Bacillus subtilis)LRCC1002菌株 Download PDFInfo
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- 150000003722 vitamin derivatives Chemical class 0.000 description 1
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Abstract
【解決手段】本発明によるバチルスサブチリス(Bacillus subtilis)LRCC1002菌株は、優れた酵素生成能を有し、優れた胆汁抵抗性、腸粘膜付着性及びヘリコバクターピロリに対する優れた抗菌能を有するので、腸機能改善用健康食品などの高機能性食品に適用可能である。また、本発明によるバチルスサブチリス(Bacillus subtilis)LRCC1002菌株は優れた血栓溶解能を有するので、血栓症予防又は改善用健康食品に適用可能であり、ひいてはこのような健康食品は血栓によって発生し得る生活習慣病に対する予防効果を期待することができる。
【選択図】 図4
Description
そして、前記酵素はアミラーゼ又はプロテアーゼである。
本発明の他の側面による血栓症の予防又は改善用健康食品は、バチルスサブチリス(Bacillus subtilis)LRCC1002菌株(受託番号:KCCM11880P)又は前記菌株の培養液のいずれか一方又は両方を有効成分として含むことができる。
本発明のさらに他の側面による腸機能改善用健康食品は、バチルスサブチリス(Bacillus subtilis)LRCC1002菌株(受託番号:KCCM11880P)又は前記菌株の培養液のいずれか一方又は両方を有効成分として含むことができる。
伝統発酵食品から菌株を分離した後、生育がよくコロニー形状が鮮やかな9種の菌株を選抜した。選抜した菌株は食品原料として使用可能な微生物であるかを確認するために(株)バイオファクト(BIOFACT Co.Ltd.,大韓民国)に依頼して16sリボソームDNA塩基配列に基づいて菌株を同定した分析結果を得た。同定を実施した結果、いずれもBacillus属の微生物であり、このうち食用可能な微生物は4種で、いずれもBacillus subtilisであり、その細胞外酵素分泌能を確認した結果、Bacillus subtilis LRCC1002菌株においてアミラーゼ(Amylase)及びプロテアーゼ(Protease)酵素活性が一番優秀であった(図1)。実験の対照群は韓国細胞株銀行から分譲されたBacillus subtilis KCCM12512を用いた。
分離菌株の人工胃液に対する抵抗性評価のために菌株はトリプシンソイ培地(Tryptic Soy Broth)(Difco、Detroit、USA)にて前培養した後、遠心分離して菌体のみ回収した。回収した菌体は滅菌した0.5%NaClで洗浄し、洗浄の最終段階で0.5%NaClに分散させて菌株懸濁液を準備した。人工胃液の抵抗性測定は、0.5%NaCl(pH2.5)に0.04%(w/v)ペプシンを添加して製造した人工胃液に菌株懸濁液を接種し、3時間反応させたのち寒天(Agar)培地に平板塗抹して生菌数を測定した。対照群の生菌数は、人工胃液に接種した後、直ちに寒天(Agar)培地に平板塗抹して測定した。分離菌株の人工胃液に対する抵抗性は初期生菌数と3時間反応後の生菌数の割合で計算してLog%で示した。
本実験に使用されたCaco2 Cellは人の大腸癌に由来する腸管上皮細胞であって、ATCC(American Type Culture Collection)から分譲されたものを用いた。Caco2 Cell(HTB−37、ATCC)は10%ウシ胎児血清(FBS;Fetal bovine serum)(Welgene co.,デグ,大韓民国)、1%ペニシリン/ストレプトマイシン(Gibco、NY、USA)を添加したEagle’s Minimum Essential Medium(EMEM、Welgene co.,デグ,大韓民国)培地を用いて37℃で5%CO2が供給される培養器で培養した。細胞が単一層(Monolayer)を形成したものについて、継代培養し、隔日で培地を交換しながら実験に使った。腸内付着性実験のために、Caco2 Cellは24Well PlateにWell当たり1×105cells/mLの濃度で分取し、37℃で5%CO2培養器で24時間培養した。分離菌株はトリプシンソイ培地(Tryptic Soy Broth)に接種し、24時間の間37℃で前培養した後、菌株を回収し、0.1Mリン酸緩衝生理食塩水(PBS;Phosphate Buffered Saline)で3回洗浄し、FBSとペニシリン/ストレプトマイシンが無添加のEMEM培地に分散した。Caco2Cellを培養した24Well Plateに分離された菌株培養液を500μlずつ接種し、37℃で5%CO2条件の下で2時間培養した後、付着しなかった菌株を除去するために0.1Mリン酸緩衝生理食塩水で洗浄した。これを回収して希釈した後、トリプシンソイ寒天(Tryptic Soy Agar)培地に平板塗抹し、37℃で24時間培養して生菌数を確認した。腸内付着性(Log%)は接種した初期菌数に対するCaco2 Cellに処理されて付着した菌数の割合で計算した。
ヘリコバクターピロリ(Helicobacter pylori ATCC 40449)は韓国微生物保存センター(KCCM;Korean Culture Center of Microorganisms)から分譲された。ヘリコバクターピロリは10%羊血液(Sheep Blood)を含む血液寒天培地(BAP;Blood Agar Plate)(ハンイルコメド、大韓民国)に接種し、10%CO2培養器で増菌した。3日乃至4日間培養されたヘリコバクターピロリを0.85%生理食塩水に懸濁して準備した。実験用培地はBottom Agar(Agar 1.2%含有)及びTop Agar(Agar 0.3%含有)に区分して作成した。BBLTM Brucella Broth(Becton、Dickinson and Company、USA)を滅菌し、40℃まで冷やし、全体の10%の体積でウシ胎児血清(FBS;Fetal Bovine Serum)を追加した。Bottom Agar10mLをペトリ皿(Petri Dish)に先に分取し、常温で2時間程度固化させた。滅菌したTop Agarは40℃まで冷やし、全体の10%の体積でウシ胎児血清(FBS)と用意したヘリコバクターピロリ菌を接種し、固化させたBottom Agar上に3mL分株し、30分間固化させた後、10mmペーパーディスク(Toyo Roshi Kaisha、Ltd、Japan)を前記プレート上に載せた。LRCC 1002菌株の培養液を遠心分離して(8,000rpm、10分)上澄液と菌体液を分離した後、それぞれの試料を30μLずつペーパーディスクに分取し、37℃10%CO2培養器で3日乃至4日間培養して、阻止円形成の有無を観察した(図3)。伝統発酵食品から分離したBacillus subtilis LRCC 1002菌株はプロバイオティックスとして適用可能な有害菌の抑制力に対する特性を持っていると判断される。
Astrup,T.and Mullertz方法[The Fibrin Plate Method for Estimating Fibrinolytic Activity、 Archivesbiochem. Biophysics,1952]を変形してフィブリノゲン(Fibrinogen)溶液(0.17M ほう酸緩衝溶液、0.3%フィブリノゲン10mL)をペトリ皿に分取し、これにトロンビン(Thrombin)溶液(0.17Mほう酸緩衝溶液、500U/mLトロンビン)を0.5mL添加した後、1分間撹拌し、1時間以上固化させた。固化したフィブリンプレートにペーパーディスクを載せ、測定試料20μLを分株した後、室温で3時間放置し、ペーパーディスクの周りに生成された透明な部位の直径を測定した。対照群は、精製された血栓溶解酵素であるプラスミン(Plasmin、1.0units/mL)を用い、次の式で血栓溶解能を計算した。分離した菌株LRCC1002と比較群としてのKCCM12512、KCCM12027、KCCM12511の血栓溶解酵素活性度の測定結果は図4に示す通りである。
前記のように、本発明によるバチルスサブチリス(Bacillus subtilis)LRCC1002菌株は、優れた酵素生成能を有し、優れた胆汁抵抗性、腸粘膜付着性及びヘリコバクターピロリに対する優れた抗菌能を有するので、腸機能改善用健康食品などの高機能性食品に適用可能であることが分かった。さらに、本発明によるバチルスサブチリス(Bacillus subtilis)LRCC1002菌株は優れた血栓溶解能を有するので、血栓症の予防又は改善用健康食品に適用可能であり、ひいてはこのような健康食品は血栓によって発生し得る生活習慣病に対する予防効果を期待することができる。
受託番号:KCCM11880P
寄託日:2016.08.12
Claims (4)
- 酵素生成能、胆汁抵抗性、及びヘリコバクターピロリに対する抗菌能及び血栓溶解能を有する、バチルスサブチリス(Bacillus subtilis)LRCC1002菌株(受託番号KCCM11880P)。
- 前記酵素生成能はアミラーゼ又はプロテアーゼを生成する能力である、請求項1に記載のバチルスサブチリス(Bacillus subtilis)LRCC1002菌株(受託番号KCCM11880P)。
- 酵素生成能、胆汁抵抗性、及びヘリコバクターピロリに対する抗菌能及び血栓溶解能を有するバチルスサブチリス(Bacillus subtilis)LRCC1002菌株(受託番号KCCM11880P)又は前記菌株の培養液のいずれか一方又は両方を有効成分として含む、血栓症予防又は改善用健康食品。
- 酵素生成能、胆汁抵抗性、及びヘリコバクターピロリに対する抗菌能及び血栓溶解能を有するバチルスサブチリス(Bacillus subtilis)LRCC1002菌株(受託番号KCCM11880P)又は前記菌株の培養液のいずれか一方又は両方を有効成分として含む、腸機能改善用健康食品。
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KR101865177B1 (ko) | 2018-06-08 |
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