JP2004222516A

JP2004222516A - Fermented soybean fungus and method for producing fermented soybean

Info

Publication number: JP2004222516A
Application number: JP2003010640A
Authority: JP
Inventors: Arinori Taya; 有紀田谷; Masaaki Kawane; 政昭川根; Yoshitaka Takeshita; 義隆竹下
Original assignee: Takano Foods Co Ltd
Current assignee: Takano Foods Co Ltd
Priority date: 2003-01-20
Filing date: 2003-01-20
Publication date: 2004-08-12
Anticipated expiration: 2023-01-20
Also published as: JP3564121B2

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fermented soybean fungus having an ability for highly producing a thrombolytic enzyme, to provide a method for producing fermented soybeans, and to provide a fermented soybean product. <P>SOLUTION: This fermented soybean strain is TTCC 865 strain (accession number: FERM P-19176) belonging to Bacillus subtilis having an ability for highly producing the thrombolytic enzyme or its similar strain. This method for producing the soybeans highly containing the thrombolytic enzyme is characterized by using the above-described fermented soybean strain to produce the fermented soybeans. The fermented soybeans highly containing the thrombolytic enzyme is characterized by being produced using the above-described fermented soybean fungus and containing the thrombolytic enzyme in an amount of at least 90 FU/wet weight g. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【０００１】
【発明の属する技術分野】
本発明は、特定の納豆菌株と、この納豆菌株を用いて納豆を製造する方法、及び得られた納豆製品に関するものであり、更に詳しくは、血栓溶解酵素を高産生する能力を有する新規な納豆菌株、及びこの菌株を使用した血栓溶解酵素を多く含有する納豆の製造方法、及びその納豆製品に関するものである。
【０００２】
【従来の技術】
一般に、納豆は、高い栄養価を有する伝統食品として広く摂取されているが、近年、納豆中に含まれる酵素の一種（ナットウキナーゼ）が血栓溶解作用を有することが知られるに至り、ナットウキナーゼを含む納豆製品の機能性とその健康食品としての有用性が特に注目を集めている。このナットウキナーゼは、納豆の製造過程で用いられる納豆菌（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）の産生する酵素であり、フィブリン溶解能（血栓溶解作用）を有し、例えば、血栓症、脳卒中、心筋梗塞等の循環器系疾患の予防に有効であるとされている。このナットウキナーゼは、例えば、宮城野菌、高橋菌、成瀬菌等の従来の市販納豆菌株を用いて製造した納豆に含有されている。また、従来、ナットウキナーゼを比較的多く含有する納豆製品も製造、販売されている。更に、従来、納豆及び納豆菌等から他の機能性を有する酵素が見出されている。
【０００３】
このように、従来、納豆及び納豆菌の培養液由来のナットウキナーゼ等の機能性を有する酵素及びその応用例が種々報告されている。ここで、それらの代表的なものを幾つか例示してみると、例えば、フィブリンに対し非常に強い分解活性を持つ酵素として、納豆及び納豆菌の培養液から抽出された新規な線溶酵素及びそれを培養液から取得する方法（特許文献１参照）、納豆酵素（ナットウキナーゼ）活性の高い新しい無臭・酵素賦活納豆、及びこのような納豆を容易かつ廉価に製造するための方法（特許文献２参照）、ナットウキナーゼを多量に含有し、かつ品質の優れた納豆を特殊な方法を用いることなく製造する方法（特許文献３参照）、納豆、あるいは納豆菌培養液より抽出される新規なプロウロキナーゼ活性化酵素及びその取得方法（特許文献４参照）等が提案されている。更に、納豆、あるいは納豆菌培養液から抽出される血小板凝集阻害物質及びそれを生産する方法（特許文献５参照）、笹及びその他の広葉植物の葉等より採取した納豆菌を用いて製造されるナットウキナーゼ等の酵素力の高い納豆（特許文献６参照）、納豆の発酵をよくし、食感を改善し、更に栄養的にも付加価値をつけた、ナットウキナーゼ、プロテアーゼ等の酵素力と機能性を示す、無臭で食感のよい栄養価の高い納豆の製造（特許文献７参照）、等が提案されている。
【０００４】
【特許文献１】
特開昭６１−１６２１８４号公報
【特許文献２】
特開平５−３３６９１７号公報
【特許文献３】
特開平６−２６１７４４号公報
【特許文献４】
特開平９−１８２５８３号公報
【特許文献５】
特開平９−３０９８３９号公報
【特許文献６】
特開２０００−１５２７７９号公報
【特許文献７】
特開２００１−１２０２１２号公報
【０００５】
上述のように、納豆及び納豆菌は、納豆に含まれる血栓溶解酵素の発見により、特に、それらの心筋梗塞や脳梗塞等の血栓性疾患の予防効果に期待が高まってきている。しかし、納豆中の血栓溶解酵素は、納豆菌が発酵中に出す酵素の１つであるが、菌の種類、大豆の種類、製法によって納豆に含まれる酵素量は大きく相違する。また、発酵における微妙な温度、湿度、そして時間によっても納豆菌の酵素活性は大きく異なることから、従来、安定的に血栓溶解酵素を含んだ納豆を大量生産するのはかなり困難であった。また、納豆中の血栓溶解酵素含量を増加させる方法の１つとして、例えば、納豆に酵素活性を高めると報告がある麦やとうもろこし、おからなどを混ぜる方法があるが、この種の方法では、従来の納豆品質と同じ食感や風味を味わうことができる納豆を供給することは困難であり、その製品は、嗜好の相違により敬遠されることがあった。また、何らかの原料を添加、混合することは、製造工程を煩雑にし、商品コストが上がることになる。更に、均一な添加ができないことにより血栓溶解酵素量のバラツキを生じる原因ともなるため、良策ではない。
【０００６】
血栓症、脳梗塞の予防効果を期待するために、安定的、効率的に多くの血栓溶解酵素を納豆と共に摂取したいと考えられる。従って、当技術分野においては、納豆中の血栓溶解酵素含量を通常のものより増加させ、効率的に血栓溶解酵素が摂取できる納豆を供給することを可能とする血栓溶解酵素高含有納豆、及びその生産方法の開発が強く要請されていた。また、従来、ナットウキナーゼ産生能の高い納豆菌株を用いて、ナットウキナーゼ高含有納豆が製造、販売されているが、従来の高酵素活性菌株を利用して納豆を製造するには、例えば、厳密な温度管理や発酵管理等が必要とされ、また、そのために、製造工程が特殊なものとなり、高コストの原因になるため、それらの改善が強く求められていた。
【０００７】
【発明が解決しようとする課題】
このような状況の中で、本発明者らは、上記従来技術に鑑みて、血栓溶解酵素を高含有すると共に、品質的にも高品質の納豆製品を、従来のような特殊な製造工程を用いることなく、また、従来の方法と比べて製造ライン及び製造条件等をほとんど変えることなく、簡便なプロセスで大量生産することができる新しい血栓溶解酵素高含有納豆の製造方法、及びその製品を開発することを目標として鋭意研究を積み重ねた結果、従来知られていなかった、血栓溶解酵素高産生能を有する新規な納豆菌株を単離することに成功すると共に、該納豆菌株を用いて納豆を生産することにより所期の目的を達成し得ることを見出し、更に研究を重ねて、本発明を完成するに至った。
すなわち、本発明は、従来の納豆菌と比べて、血栓溶解酵素産生量がはるかに多く、しかも、優れた品質の納豆を生産することを可能とする新規な血栓溶解酵素高産生能を有する納豆菌、該納豆菌を用いた血栓溶解酵素高含有納豆の製造方法、及びその納豆製品を提供することを目的とするものである。
【０００８】
【課題を解決するための手段】
上記課題を解決するための本発明は、以下の技術的手段から構成される。
（１）血栓溶解酵素を高産生する能力を有するバチルス・ズブチリス（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）に属するＴＴＣＣ８６５株（受託番号ＦＥＲＭ
Ｐ−１９１７６）及びその類似株である納豆菌株。
（２）前記（１）記載の納豆菌株を用いて納豆を製造することを特徴とする、血栓溶解酵素を高含有する納豆の製造方法。
（３）前記（１）記載の納豆菌を用いて製造した、血栓溶解酵素を少なくとも９０ＦＵ／湿重量ｇ含有することを特徴とする、血栓溶解酵素高含有納豆。
【０００９】
【発明の実施の形態】
次に、本発明について更に具体的に説明する。
本発明は、上述のように、血栓溶解酵素を高産生する能力を有するバチルス・ズブチリス（Ｂａｃｉｌｌｕｓｓｕｂｔｉｌｉｓ）に属するＴＴＣＣ８６５株及びその類似株である納豆菌株、該納豆菌株を用いて血栓溶解酵素を高含有する納豆を製造すること、及び該方法により製造した血栓溶解酵素を９０ＦＵ／ｇ以上含有する血栓溶解酵素高含有納豆、により特徴づけられるものである。本発明の納豆菌株は、広く自然界より得た種々の稲わら等の材料より納豆菌を採取してそれらの酵素活性、菌学的性質及び納豆生産力等を広く調査して、従来知られていない新規な納豆菌株を選抜して単離されるに至ったものである。
【００１０】
本発明において、納豆の原料は、例えば、大豆と納豆菌で構成され、納豆は、基本的には、吸水した大豆を高圧で蒸し、納豆菌を摂取した煮豆を発酵させるという製造工程で作られる。本発明において、納豆の原料及び製造工程は、特に制限されるものではなく、必要に応じて、これらに適宜の構成を付加することができる。本発明の方法では、通常、発酵の１０時間目ごろより明らかな酵素活性が確認され、発酵の経過に伴ない酵素量も増加してくる。発酵が終了する頃には、酵素量の増加は少なくなり、納豆の含有酵素量が平衡になってくる。
【００１１】
納豆に含まれるナットウキナーゼは、納豆菌が産生したものである。よって、ナットウキナーゼを含む納豆中の血栓溶解酵素活性を増加させるには、通常の納豆菌に比べて、血栓溶解活性の高い納豆菌を使用し、納豆を製造する方法が最適であると考えられるが、本発明では、血栓溶解酵素活性の高い納豆菌株を使用することで、より高濃度に血栓溶解酵素力を高めた納豆を製造し、提供することを可能とするものである。
【００１２】
本発明によって、良好な品質を維持したまま、血栓溶解酵素を高含有する納豆を製造することができる。すなわち、本発明は、納豆菌としてバチルス・ズブチリスに属するＴＴＣＣ８６５株（ＦＥＲＭＰ−１９１７６）を用いることで、血栓溶解酵素を高含有する納豆を製造し、提供すること、及び血栓溶解酵素を高含有する納豆を製造するためのバチルス・ズブチリスＴＴＣＣ８６５株（ＦＥＲＭＰ−１９１７６）及びその類似の株である納豆菌株を提供することを特徴とするものである。
【００１３】
次に、本発明において、新しい納豆菌株を単離した手順について説明する。本発明では、広く自然界より野生株を採集し、それらの酵素活性、菌学的性質及び納豆生産性能を調べて目的の納豆菌株を取得した。まず、上記野生株をプロテアーゼ力価測定培地に接種してプロテアーゼ産生が最も速く出現した株をスクリーニングし、これらの菌株を用いて納豆を製造し、納豆の品質が良好でＮＫ（ナットウキナーゼ）量が高い株を選抜し、更に、これらの菌株を用いて納豆を製造し、ＮＫ量が高い株を選抜する工程を繰り返して、血栓溶解酵素高産生能を有する納豆菌株を取得した。
【００１４】
一方、上記野生株に紫外線照射処理した後、一例として、例えば、後記する培地組成の粘質物生産能判定培地に接種し、粘質物生産力のある株を選抜し、ＬＢ培地を用いた試験管培養を行い、遠心処理して得た培養液を用いて血栓溶解活性の測定を行い、血栓溶解活性の値が高い株を選抜し、これを粘質物生産能判定培地に移し、粘質物生産力がある株を選抜し、これらの株を用いて納豆を製造して納豆品質が良好でＮＫ量が高い株を選抜する工程を繰り返して、血栓溶解酵素高産生能を有する納豆菌を取得した。
【００１５】
以上の通りの各種スクリーニング工程を経て単離した納豆菌株のうちから、該納豆菌を用いて製造した納豆が血栓溶解酵素を９０ＦＵ／湿重量ｇ以上含有し、かつ納豆の品質が良好な条件を満たす血栓溶解酵素高含有納豆を製造可能にする納豆菌株を取得することに成功し、それらの代表的な菌株として、ＴＴＣＣ８６５株を取得した。本発明の納豆菌株には、このＴＴＣＣ８６５株とその類似株である納豆菌株が含まれるが、ここで言う類似株とは、血栓溶解酵素を９０ＦＵ／ｇ以上含有する納豆生産能を有し、かつ得られる納豆が従来品以上の良好な品質（外観、糸引き、香り、味、硬さ）を有し、上記ＴＴＣＣ８６５株と同等の菌学的性質を有する納豆菌株を意味するものとして定義される。
【００１６】
次に、ＮＫ量の測定法について説明すると、ＮＫ活性の測定法としては、現在、例えば、ＦＵ法、フィブリン平板法、合成基質（プラスミン）法等に代表される種々の方法が提案され、利用されている。しかし、現時点での公定法がなく、納豆の血栓溶解活性を正確に測定する方法がない状況にある。そこで、本発明では、ＮＫ量の測定を、測定値が安定で、定量的な測定が可能となるようにＦＵ法を一部改良した、以下の測定方法により行った。
【００１７】
（ＮＫ活性の測定）
納豆２０ｇに生理食塩水１８０ｇを加え、１８０００ｒｐｍ、１．５分間ホモジナイズした後、３０００ｒｐｍ、１０分間、遠心分離した上清を検体とした。
次に、１）氷中、１．７×１０ｃｍ試験管にトロンビン（５０Ｕ／ｍｌ、ｐＨ８．５ホウ酸緩衝液に溶解）を０．１ｍｌ分注し、２）これに検体溶液０．１ｍｌを加え、３）０．５％フィブリノーゲン（ｐＨ８．５ホウ酸緩衝液に溶解）１ｍｌを加え、チューブミキサーで攪拌後、すぐに３７℃湯浴に入れ、正確に６０分間インキュベートした。
【００１８】
次いで、４）インキュベート終了後、８％トリクロロ酢酸（ＴＣＡ）を１．２ｍｌ添加し、６０分間、３７℃に放置し、５）ＴＣＡにより変性した蛋白質を遠心分離により除去し、６）上清を採取し、２７５ｎｍの吸光度を測定し、７）得られた吸光度から下式を用いてナットウキナーゼ活性（ＮＫ量）とした。
ＮＫ量（ＦＵ／ｇ）＝（Ａ_ｔ −Ａ_ｂ）／０．０１×１／６０×１／０．１×Ｄ
Ａ_ｔ：反応液の吸光度
Ａ_ｂ：ブランクの吸光度
Ｄ：希釈倍率
尚、ブランクの吸光度の測定は、上記１）、２）の後、８％ＴＣＡを加え、３）以降の操作は同様に行った。また、測定時の吸光度差（Ａ_ｔ −Ａ_ｂ）が０．１５〜０．３となるように試料の調製を行った。
【００１９】
次に、本発明の納豆菌株の選抜、単離の過程及び菌学的性質について具体的に説明すると、本発明における新規な納豆菌は、上述のように、多くの地域から採集された、土、枯草、稲藁等から分離選別されたものである。まず、土や枯草等の試料を生理食塩水に浸潰し、煮沸して得た浸漬水を一般寒天培地に塗沫して培養し、耐熱性胞子を形成する菌を選抜した。次いで、ショ糖及びグルタミン酸ナトリウムを含む、以下のような組成の培地（前記した粘質物生産能判定培地に相当する）で培養し、糸引き性を示した菌株を分離した。
【００２０】
（培地組成）
成分濃度（１００ｍｌ）
サッカロース０．２ｇ
グルタミン酸ナトリウム１．５ｇ
フィトン（ソイペプトン）１．５ｇ
リン酸２水素カリウム０．２７ｇ
リン酸水素２カリウム１２水塩０．４２ｇ
塩化ナトリウム０．０５ｇ
硫酸マグネシウム７水塩０．０５ｇ
ビオチン１０μｇ
寒天２．０ｇ
水
ｐＨ６．８
【００２１】
こうして分離した菌を、更に、煮豆上で培養し、粘質物を生産する菌を選抜した。このような手順で得た分離株に対して、カタラーゼ反応、レシチナーゼ反応、硝酸塩の還元Ｖ−Ｐ反応、デンプンの分解、ゼラチンの分解、クエン酸・プロピオン酸の利用、チロシンの分解による簡易同定試験を経て、枯草菌であることの判定を行った。
【００２２】
以上の選抜試験によって、納豆菌と判定された菌株を用いて、納豆を製造し、官能評価及び血栓溶解酵素力価の測定を行い、従来の納豆に比べて、高い血栓溶解酵素活性を示し、また、官能的にも従来の納豆と同等以上の品質となる、バチルス・ズブチリスＴＴＣＣ８６５株を得て、平成１４年１２月２６日付で独立行政法人産業技術総合研究所特許生物寄託センターに、ＢａｃｉｌｌｕｓｓｕｂｔｉｌｉｓＴＴＣＣ８６５株、受託番号ＦＥＲＭＰ−１９１７６として寄託した。
【００２３】
この納豆菌は、以下の様な菌学的性質を備えた菌である。

【００２４】
上記菌学的性質からも明らかなように、この寄託された菌は、納豆菌に属しているものである。この納豆菌を用いて、納豆を製造して、発酵工程及び得られた製品における特徴を調べたところ、従来の方法では、発酵の中期及び後期の段階で血栓溶解酵素活性が高くなる傾向を示すのに対し、本発明の方法では、発酵の初期及び中期の段階で血栓溶解酵素活性が急激に高くなる傾向を示し、発酵過程における血栓溶解酵素産生のパターンが従来のものと大きく異なることが示された。また、後記する実施例に具体的に示したように、得られた納豆は、血栓溶解酵素含量が格別に高く、しかも、外観、糸引き、硬さ、その他の官能評価についても優れた品質を有していることが分かった。従来の方法では、発酵時間を長くすることにより、血栓溶解酵素活性をなるべく高い値にすることが行われており、そのために、発酵時間の短縮化が難しくなり、過度の発酵により製品への影響が出てくる等の問題があったが、本発明の方法では、それらの問題を全て解消することができる利点がある。
【００２５】
次に、本発明の納豆菌株を用いた納豆の製造方法は、主に、（１）従来の製造装置及びラインをそのまま使用することができる、（２）血栓溶解酵素の活性を高めるために、格別の原料を配合する必要がない、（３）発酵の初期及び中期の段階から高い血栓溶解活性が得られるので、発酵時間の短縮化とそれによる省エネルギー生産が可能となる、（４）血栓溶解酵素を高含有すると共に、優れた品質を有する高付加価値の納豆製品を安定的に、効率良く製造できる、（５）血栓溶解酵素高産生の本発明の納豆菌を使用するだけで、製造原料、製造工程及び製造条件等を格別に変更することなく、血栓溶解酵素高含有（従来品の約１．５−２倍）の納豆を製造し、提供することができる、等の特徴点を有する。
【００２６】
次に、本発明の納豆菌を用いて製造した納豆製品は、主に、（１）血栓溶解酵素量は、９０ＦＵ／ｇ以上であり、従来の納豆の約１．５〜２倍である、（２）産生された粘質物に基づく、優れた糸引き性を有する、（３）アンモニア臭などの不快臭がなく、適度な納豆臭を有する、（４）苦味、異味がなく、優れた旨味を有する、（５）良好な硬さを有する、（６）発酵の初期段階から高い血栓溶解酵素量を含有する、（７）短い発酵時間で、高い血栓溶解酵素量を有する納豆製品を生産できる、（８）従来の納豆製品と比べて、血栓溶解酵素量が多く、しかも、従来品以上の品質を持つ納豆製品を提供できる、（９）従来の製造ライン及び製造条件をほとんど変更することなく、本発明の納豆製品を生産できる、（１０）従来製品の約１．５〜２倍の血栓溶解酵素を含有する血栓溶解酵素高含有納豆を提供できる、（１１）製品間の血栓溶解酵素量のバラツキがなく、安定して、効率良く多くの血栓溶解酵素を摂取可能な納豆を提供できる、（１２）格別の原料を配合することなしに、通常の原料を用いて、血栓溶解酵素含量を増加させた納豆を製造できる、等の特徴点を有する。
【００２７】
【実施例】
次に、実施例に基づいて本発明を具体的に説明するが、本発明は、以下の実施例によって何ら限定されるものではない。
実施例１
本発明の納豆菌株のＴＴＣＣ８６５株、及び市販納豆菌の胞子懸濁液を用いて、常法通り納豆を製造した。市販納豆菌として、宮城野納豆菌を使用した。納豆用極小粒大豆に３倍量の水を加え、１５〜２０℃で１８〜２４時間浸潰した浸漬大豆を加圧釜に入れ、ゲージ圧２ｋｇ／ｃｍ^２２０分前後で蒸煮した。蒸煮大豆がまだ熱いうちに、蒸煮大豆ｌｋｇあたり１０^６個の胞子を接種し、これを納豆用ＰＳＰ容器に充填後、表面を有孔ポリエチレンフィルムで覆い、蓋をかぶせて、３７〜４０℃で１４〜１８時間発酵させた。発酵終了後に熟成させ目的とする納豆を得た。
【００２８】
上記方法で得られた納豆について、それぞれの官能評価、及び納豆中の血栓溶解酵素量を測定した。尚、官能評価は、５段階評価法にて行い、平均値を示した。５点を良い、４点をやや良い、３点を普通、２点をやや悪い、１点を悪いとし、下表の項目について評価した。

上記方法で得られた納豆の官能評価結果、及び血栓溶解酵素含量を、以下の表２及び表３に示す。
【００２９】

【００３０】

【００３１】
上記表２、及び表３の結果から分かるように、本発明の納豆菌バチルス・ズブチリスＴＴＣＣ８６５株を用いて製造した納豆の品質は、市販納豆菌を用いて製造した納豆の品質と同様に良好であり、かつ血栓溶解酵素量は、９０ＦＵ／ｇ以上であり、通常の納豆の約１．５〜２倍となっていることが確認された。
【００３２】
次に、本発明の納豆菌（ＴＴＣＣ８６５株）、及び市販納豆菌（Ｍ）による納豆製造工程における発酵時間と血栓溶解酵素量の関係を調べた。ＴＴＣＣ８６５株は、市販納豆菌の１２時間目の血栓溶解酵素量に相当する３４ＦＵ／ｇを発酵１０時間目で産生し、発酵１４時間目で市販納豆菌の製品の血栓溶解酵素量（５０ＦＵ／ｇ）に達していた。すなわち、市販納豆菌を用いた場合に比べて、本発明の納豆菌を用いた場合、発酵開始後、約１０時間から１４時間の発酵の初期及び中期の段階で高い血栓溶解酵素量が得られること、及び本発明の納豆菌は、従来の納豆菌とは異なる血栓溶解酵素産生パターンを示すことが分かった。
【００３３】
また、本発明の納豆菌（ＴＴＣＣ８６５株）、及び市販納豆菌（Ｍ）による納豆製造工程におけるプロテアーゼ活性との関係を調べたところ、血栓溶解酵素と同じ産生パターンを示した。すなわち、本発明の納豆菌の製品のプロテアーゼ活性は、市販納豆菌の製品のプロテアーゼ活性（１３３．３Ｕｎｉｔ／ｇ）に発酵１２時間目〜１４時間目達しており、本発明の納豆菌は、発酵の早い段階で高いプロテアーゼ産生能を有することが分かった。プロテアーゼを早い段階より産生することで、従来より短い発酵時間で、血栓溶解酵素量が多く、従来と同等な旨味や糸引きを有する品質の納豆製品の生産を可能とすることが分かった。
【００３４】
【発明の効果】
以上詳述したように、本発明は、血栓溶解酵素を高生産する新規な納豆菌株、この菌を用いて血栓溶解酵素を高含有する納豆を製造する方法、及び納豆製品に係るものであり、本発明により、以下のような効果が奏される。
（１）本発明の納豆菌株は、市販納豆菌に比べて、血栓溶解酵素を多く生産する血栓溶解酵素高産生能を有する菌株である。
（２）この納豆菌株を用いて製造された納豆では、血栓溶解酵素量が市販納豆に比べて、約１．５〜２倍となっている。
（３）この菌株を用いて納豆を製造すれば、従来の納豆と同様で、品質は変わることなく良好で、血栓溶解酵素を多く含む納豆を得ることができる。
（４）本発明の納豆の製法は、従来の製法において、納豆菌としてバチルス・ズプチリスＴＴＣＣ８６５株を用いることを特徴とするので、新たな装置を必要とすることなく、製造コストも高くならず、従来どおりの生産体制で対応できる。
（５）納豆菌株の性質も、市販納豆菌とほぼ同等であるため、製造条件の変更もほとんど必要ない。
【図面の簡単な説明】
【図１】本発明の納豆菌（ＴＴＣＣ８６５株）、及び市販納豆菌（Ｍ）による納豆製造工程（発酵を３７℃、湿度９０％の一定条件で行った場合）における発酵時間と血栓溶解酵素活性（ＮＫ活性）の関係を示す。
【図２】本発明の納豆菌（ＴＴＣＣ８６５株）、及び市販納豆菌（Ｍ）による納豆製造工程（発酵を３７℃、湿度９０％の一定条件で行った場合）における発酵時間とプロテアーゼ活性の関係を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a specific natto strain, a method for producing natto using this natto strain, and an obtained natto product. More specifically, the present invention relates to a novel natto having an ability to highly produce thrombolytic enzymes. The present invention relates to a strain, a method for producing natto containing a large amount of thrombolytic enzyme using the strain, and a natto product thereof.
[0002]
[Prior art]
In general, natto is widely ingested as a traditional food with high nutritional value. In recent years, it has been known that one of the enzymes (nattokinase) contained in natto has a thrombolytic effect, and natto containing nattokinase has been known. Special attention has been paid to the functionality of the product and its usefulness as a health food. This nattokinase is an enzyme produced by Bacillus subtilis used in the process of producing natto, has fibrinolytic ability (thrombolytic action), and includes, for example, circulatory system such as thrombosis, stroke, and myocardial infarction. It is said to be effective in preventing disease. This nattokinase is contained, for example, in natto produced using a conventional commercial natto strain such as Miyagi field bacterium, Takahashi bacterium, and Naruse bacterium. Conventionally, natto products containing a relatively large amount of nattokinase have also been manufactured and sold. Further, conventionally, enzymes having other functions have been found from natto and natto fungi.
[0003]
As described above, conventionally, various functional enzymes such as nattokinase derived from a culture solution of natto and Bacillus natto and applications thereof have been reported. Here, when some typical examples thereof are illustrated, for example, a novel fibrinolytic enzyme extracted from a culture solution of natto and Bacillus natto, as an enzyme having a very strong degrading activity for fibrin, A method for obtaining it from a culture solution (see Patent Document 1), a new odorless and enzyme-activated natto having high natto enzyme (nattokinase) activity, and a method for easily and inexpensively producing such natto (see Patent Document 2) ), A method for producing natto containing a large amount of nattokinase and having excellent quality without using a special method (see Patent Document 3), a novel pro-urokinase activation extracted from natto or a culture solution of Bacillus natto An enzyme and a method for obtaining the enzyme (see Patent Document 4) have been proposed. Further, a platelet aggregation inhibitor extracted from natto or a natto fungus culture solution and a method for producing the same (see Patent Document 5), and manufactured using natto bacteria collected from leaves of bamboo grass and other broadleaf plants, etc. Nattokinase and other natto with high enzymatic power (see Patent Document 6), improved fermentation of natto, improved texture, and added nutritional value. The production of odorless odor-free, nutritious natto with good texture (see Patent Literature 7) has been proposed.
[0004]
[Patent Document 1]
JP-A-61-162184 [Patent Document 2]
JP-A-5-336917 [Patent Document 3]
JP-A-6-261744 [Patent Document 4]
Japanese Patent Application Laid-Open No. 9-182585 [Patent Document 5]
Japanese Patent Application Laid-Open No. 9-309839 [Patent Document 6]
JP 2000-152779 A [Patent Document 7]
JP 2001-120212 A [0005]
As described above, natto and Bacillus natto are expected to be particularly effective in preventing thrombotic diseases such as myocardial infarction and cerebral infarction due to the discovery of thrombolytic enzymes contained in natto. However, the thrombolytic enzyme in natto is one of the enzymes released by natto during fermentation, but the amount of enzyme contained in natto varies greatly depending on the type of bacteria, the type of soybean, and the production method. In addition, since the enzymatic activity of Bacillus natto greatly varies depending on the delicate temperature, humidity, and time during fermentation, it has conventionally been quite difficult to stably mass-produce natto containing thrombolytic enzymes. In addition, as one of the methods for increasing the thrombolytic enzyme content in natto, for example, there is a method of mixing natto with wheat, corn, okara, etc., which has been reported to increase the enzyme activity. It is difficult to supply natto that can have the same texture and flavor as conventional natto quality, and the product may be avoided due to differences in taste. In addition, adding and mixing some raw materials complicates the manufacturing process and increases the product cost. In addition, it is not a good measure because the inability to uniformly add the same causes a variation in the amount of thrombolytic enzyme.
[0006]
In order to expect a preventive effect on thrombosis and cerebral infarction, it is considered that many thrombolytic enzymes should be stably and efficiently taken together with natto. Therefore, in the art, the thrombolytic enzyme-rich natto that increases the thrombolytic enzyme content in natto more than usual so that it is possible to supply natto that can be efficiently ingested by thrombolytic enzyme, and the like. Development of production methods was strongly demanded. Conventionally, nattokinase-rich natto has been produced and sold using a nattokinase-producing natto strain, but natto is produced using a conventional high-enzyme-active bacterium, for example, at a strict temperature. Management, fermentation management, and the like are required, and because of this, the manufacturing process becomes special and causes high cost. Therefore, improvement thereof has been strongly demanded.
[0007]
[Problems to be solved by the invention]
Under these circumstances, the present inventors have taken into account the above-mentioned prior art, and at the same time, have a high content of thrombolytic enzyme and have a high quality natto product in terms of quality. Developed a new method for producing natto with high content of thrombolytic enzyme and its product, which can be mass-produced by a simple process without using it and with little change in production line and production conditions compared to conventional methods As a result of intensive studies with the aim of doing so, as a result, we succeeded in isolating a novel natto strain having a high thrombolytic enzyme high producing ability and producing natto using the natto strain, which was previously unknown. It has been found that the intended purpose can be achieved by doing so, and further studies have been made to complete the present invention.
In other words, the present invention provides a natto having a novel thrombolytic enzyme high-producing ability, which has a much larger amount of thrombolytic enzyme production than conventional natto bacteria, and is capable of producing natto of excellent quality. It is an object of the present invention to provide a bacterium, a method for producing natto containing a high amount of thrombolytic enzyme using the natto, and a natto product thereof.
[0008]
[Means for Solving the Problems]
The present invention for solving the above-mentioned problems includes the following technical means.
(1) TTCC865 strain belonging to Bacillus subtilis (accession number: FERM) having the ability to highly produce thrombolytic enzymes
P-19176) and a natto strain which is a similar strain thereof.
(2) A method for producing natto containing a high amount of thrombolytic enzyme, wherein natto is produced using the natto strain according to (1).
(3) A natto containing a high amount of thrombolytic enzyme, characterized by containing at least 90 FU / g of wet weight of thrombolytic enzyme produced using the Bacillus natto according to (1).
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
Next, the present invention will be described more specifically.
As described above, the present invention provides a method for increasing the amount of thrombolytic enzyme by using the TTCC 865 strain belonging to Bacillus subtilis having an ability to highly produce thrombolytic enzyme, a natto strain that is a similar strain thereof, and the natto strain. The present invention is characterized by producing natto containing natto, and natto having a high thrombolytic enzyme content of 90 FU / g or more containing the thrombolytic enzyme produced by the method. The natto strain of the present invention has been conventionally known by widely collecting natto bacteria from various rice straw and other materials obtained from the natural world and extensively investigating their enzymatic activities, mycological properties, natto productivity, and the like. No new natto strain was selected and isolated.
[0010]
In the present invention, the raw material of natto is composed of, for example, soybeans and natto bacteria, and natto is basically produced by a manufacturing process of steaming soybeans having absorbed water under high pressure and fermenting boiled soybeans ingesting natto bacteria. . In the present invention, the raw material and the production process of natto are not particularly limited, and an appropriate configuration can be added to these as needed. In the method of the present invention, usually, a clear enzyme activity is confirmed from about 10 hours after fermentation, and the amount of enzyme increases with the progress of fermentation. By the end of the fermentation, the increase in the amount of the enzyme becomes small, and the amount of the enzyme contained in natto becomes equilibrium.
[0011]
Nattokinase contained in natto is produced by Bacillus natto. Therefore, in order to increase the thrombolytic enzyme activity in natto containing nattokinase, it is considered that a method of producing natto using a natto having a high thrombolytic activity, compared to normal natto, is most suitable. In the present invention, it is possible to manufacture and provide natto having a higher concentration of thrombolytic enzyme by using a natto strain having a high thrombolytic enzyme activity.
[0012]
According to the present invention, natto containing a high amount of thrombolytic enzyme can be produced while maintaining good quality. That is, the present invention uses TTCC865 strain (FERM P-19176) belonging to Bacillus subtilis as a Bacillus subtilis to produce and provide natto containing a high amount of thrombolytic enzyme, and to provide a high content of thrombolytic enzyme. Bacillus subtilis TTCC 865 strain (FERM P-19176) and a natto strain similar thereto are provided.
[0013]
Next, a procedure for isolating a new natto strain in the present invention will be described. In the present invention, wild strains are widely collected from nature and their enzyme activities, mycological properties and natto production performance are examined to obtain the desired natto strain. First, the wild-type strain was inoculated into a protease titer measurement medium, and the strain in which the production of protease was the fastest was screened, natto was produced using these strains, and the quality of natto was good and the amount of NK (nattokinase) was low. High strains were selected, natto was produced using these strains, and the process of selecting strains having a high NK content was repeated to obtain a natto strain having a high thrombolytic enzyme-producing ability.
[0014]
On the other hand, after irradiating the wild strain with ultraviolet light, as an example, for example, a strain having a mucilage-producing ability having a medium composition described below is inoculated, a strain having a mucilage-producing ability is selected, and a test tube using an LB medium is selected. After culturing, the thrombolytic activity is measured using a culture solution obtained by centrifugation, a strain having a high value of thrombolytic activity is selected, and the strain is transferred to a mucous substance-producing ability determination medium, and the mucous substance producing ability is determined. A certain strain was selected, natto was produced using these strains, and the process of selecting a strain having good natto quality and a high NK amount was repeated to obtain a natto bacterium having a high thrombolytic enzyme-producing ability.
[0015]
Among the natto strains isolated through the various screening steps as described above, natto produced using the natto strain contains thrombolytic enzyme in an amount of 90 FU / g wet weight or more and natto with good quality. Succeeded in obtaining natto strains capable of producing thrombolytic enzyme-rich natto, and obtained TTCC 865 strain as a representative strain thereof. The natto strain of the present invention includes the TTCC 865 strain and a natto strain which is a similar strain thereof. The term "similar strain" as used herein refers to a natto-producing ability containing thrombolytic enzyme of 90 FU / g or more, and The resulting natto has a better quality (appearance, stringiness, aroma, taste, hardness) than conventional products and is defined as a natto strain having the same microbiological properties as the above TTCC865 strain. .
[0016]
Next, the method of measuring the amount of NK will be described. As the method of measuring the NK activity, various methods represented by the FU method, the fibrin plate method, the synthetic substrate (plasmin) method and the like have been proposed and used. Have been. However, there is no official method at present and there is no method for accurately measuring the thrombolytic activity of natto. Therefore, in the present invention, the measurement of the amount of NK was carried out by the following measurement method in which the FU method was partially improved so that the measured value was stable and quantitative measurement was possible.
[0017]
(Measurement of NK activity)
180 g of physiological saline was added to 20 g of natto, homogenized at 18,000 rpm for 1.5 minutes, and then centrifuged at 3000 rpm for 10 minutes to obtain a supernatant.
Next, 1) 0.1 ml of thrombin (50 U / ml, dissolved in a pH 8.5 borate buffer) was dispensed into a 1.7 × 10 cm test tube in ice, and 2) 0.1 ml of the sample solution was added thereto. In addition, 3) 1 ml of 0.5% fibrinogen (dissolved in a borate buffer of pH 8.5) was added, and the mixture was stirred with a tube mixer, immediately placed in a 37 ° C water bath, and incubated for exactly 60 minutes.
[0018]
Next, 4) After completion of the incubation, 1.2 ml of 8% trichloroacetic acid (TCA) is added, and the mixture is left at 37 ° C. for 60 minutes. 5) The protein denatured by TCA is removed by centrifugation, and 6) the supernatant is removed. The sample was collected, the absorbance at 275 nm was measured, and 7) the nattokinase activity (NK amount) was determined from the obtained absorbance using the following formula.
NK amount (FU / g) = (A _t −A _b ) /0.01×1/60×1/0.1×D
A _t: reaction absorbance _{A b:} blank absorbance D: dilution ratio The measurement of the absorbance of the blank, after the 1), 2), added 8% TCA, 3) subsequent operations are carried out in the same manner Was. Further, absorbance difference at the time of measurement _(A t -A _b) is carried out sample preparation so that 0.15 to 0.3.
[0019]
Next, the process of selection, isolation and mycological properties of the Bacillus natto strain of the present invention will be specifically described. As described above, the novel Bacillus natto in the present invention is obtained by collecting soil from many regions as described above. , Hay, rice straw and the like. First, samples such as soil and hay were immersed in physiological saline, and the immersion water obtained by boiling was applied to a general agar medium and cultured, and bacteria that formed heat-resistant spores were selected. Subsequently, the cells were cultured in a medium containing sucrose and sodium glutamate and having the following composition (corresponding to the above-mentioned medium for judging mucilage-producing ability), and a strain showing a stringiness was isolated.
[0020]
(Medium composition)
Ingredient concentration (100ml)
Saccharose 0.2g
1.5 g of sodium glutamate
Phytone (Soypeptone) 1.5g
Potassium dihydrogen phosphate 0.27g
Dipotassium hydrogen phosphate dodecahydrate 0.42g
Sodium chloride 0.05g
Magnesium sulfate heptahydrate 0.05g
Biotin 10μg
Agar 2.0g
Water pH 6.8
[0021]
The bacteria thus isolated were further cultured on boiled beans, and bacteria producing mucilage were selected. Catalase reaction, lecithinase reaction, reduction VP reaction of nitrate, decomposition of starch, decomposition of gelatin, utilization of citric acid / propionic acid, simple identification test by decomposition of tyrosine on the isolate obtained by such procedure After that, it was determined to be Bacillus subtilis.
[0022]
By the above selection test, using the strain determined to be Bacillus natto, natto was manufactured, and the sensory evaluation and measurement of the thrombolytic enzyme titer were performed. In addition, Bacillus subtilis TTCC 865 strain having a quality equivalent to or better than conventional natto was obtained, and Bacillus subtilis was registered with the National Institute of Advanced Industrial Science and Technology (AIST) on December 26, 2002. TTCC 865 strain, accession number FERM P-19176.
[0023]
This Bacillus natto is a bacterium having the following mycological properties.

[0024]
As is clear from the above mycological properties, the deposited microorganism belongs to Bacillus natto. Using this natto fungus, natto was produced, and the characteristics of the fermentation process and the resulting product were examined. In the conventional method, the thrombolytic enzyme activity tended to increase in the middle and late stages of fermentation. On the other hand, in the method of the present invention, the thrombolytic enzyme activity tends to increase rapidly in the initial and middle stages of fermentation, indicating that the pattern of thrombolytic enzyme production in the fermentation process is significantly different from the conventional one. Was done. In addition, as specifically shown in Examples described later, the obtained natto has a particularly high thrombolytic enzyme content, and also has excellent quality in appearance, stringing, hardness, and other sensory evaluations. It was found to have. In the conventional method, the thrombolytic enzyme activity is made as high as possible by increasing the fermentation time, which makes it difficult to shorten the fermentation time, and excessive fermentation affects the product. However, the method of the present invention has an advantage that all of these problems can be solved.
[0025]
Next, the method for producing natto using the natto strain of the present invention mainly comprises (1) the conventional production apparatus and line can be used as they are, and (2) in order to increase the activity of the thrombolytic enzyme, There is no need to mix special raw materials. (3) Since high thrombolytic activity can be obtained from the initial and middle stages of fermentation, it is possible to shorten fermentation time and thereby achieve energy-saving production. (4) Thrombolysis It is possible to stably and efficiently produce high-value-added natto products with high content of enzymes and excellent quality. It is possible to manufacture and provide natto with a high content of thrombolytic enzyme (about 1.5 to 2 times that of conventional products) without specially changing the manufacturing process and manufacturing conditions. .
[0026]
Next, the natto product produced using the natto fungus of the present invention mainly comprises (1) a thrombolytic enzyme amount of 90 FU / g or more, which is about 1.5 to 2 times that of conventional natto. (2) It has excellent stringiness based on the produced mucilage, (3) has no unpleasant odor such as ammonia odor, has an appropriate natto odor, (4) has no bitterness, off-flavor, and excellent umami (5) having good hardness, (6) containing a high amount of thrombolytic enzyme from the initial stage of fermentation, and (7) producing a natto product having a high amount of thrombolytic enzyme in a short fermentation time. (8) Compared with conventional natto products, the amount of thrombolytic enzyme is larger, and natto products having higher quality than conventional products can be provided. (9) Conventional production line and production conditions are hardly changed. Can produce the natto product of the present invention. It is possible to provide natto containing a high amount of thrombolytic enzyme containing up to twice as much thrombolytic enzyme. (11) There is no variation in the amount of thrombolytic enzyme among products, and stable and efficient ingestion of many thrombolytic enzymes is possible. Natto can be provided, and (12) Natto with an increased thrombolytic enzyme content can be produced using ordinary raw materials without blending special raw materials.
[0027]
【Example】
Next, the present invention will be specifically described based on examples, but the present invention is not limited to the following examples.
Example 1
Using the natto strain TTCC 865 of the present invention and a commercially available spore suspension of Bacillus natto, natto was produced in the usual manner. Miyagino natto was used as a commercially available natto. Three times the amount of water was added to ultra-small soybeans for natto, and the immersed soybeans immersed at 15 to 20 ° C. for 18 to 24 hours were placed in a pressure cooker and steamed at a gauge pressure of about 2 kg / cm ^{2 for about} 20 minutes. While still hot steamed soybean was inoculated with 10 ⁶ spores per steamed soybeans LKG, which after filling the natto for PSP container, cover the surface with a perforated polyethylene film, covered with a lid, at 37 to 40 ° C. Fermented for 14-18 hours. After fermentation was completed, it was aged to obtain the desired natto.
[0028]
With respect to the natto obtained by the above method, each sensory evaluation and the amount of thrombolytic enzyme in the natto were measured. In addition, the sensory evaluation was performed by a five-step evaluation method and the average value was shown. Five points were good, four points were slightly good, three points were normal, two points were slightly bad, and one point was bad, and the items in the following table were evaluated.

The sensory evaluation results and the thrombolytic enzyme content of natto obtained by the above method are shown in Tables 2 and 3 below.
[0029]

[0030]

[0031]
As can be seen from the results of Tables 2 and 3, the quality of natto produced using the Bacillus subtilis TTCC865 strain of the present invention is as good as the quality of natto produced using commercially available Bacillus natto. In addition, it was confirmed that the amount of thrombolytic enzyme was 90 FU / g or more, which was about 1.5 to 2 times that of normal natto.
[0032]
Next, the relationship between the fermentation time and the amount of thrombolytic enzyme in the natto production process using the Bacillus natto of the present invention (TTCC865 strain) and a commercially available Bacillus natto (M) was examined. The TTCC 865 strain produced 34 FU / g corresponding to the amount of thrombolytic enzyme at 12 hours of the commercially available Bacillus natto at 10 hours of fermentation, and the thrombolytic enzyme amount (50 FU / g) of the product of the commercially available Bacillus natto at 14 hours of fermentation ) Had been reached. That is, compared to the case of using a commercially available natto, when the natto of the present invention is used, a high amount of thrombolytic enzyme is obtained at the initial and middle stages of fermentation for about 10 to 14 hours after the start of fermentation. In addition, it was found that the Bacillus natto of the present invention exhibited a different thrombolytic enzyme production pattern than conventional Bacillus natto.
[0033]
Further, when the relationship with the protease activity in the natto production process using the Bacillus natto of the present invention (TTCC865 strain) and the commercially available Bacillus natto (M) was examined, the same production pattern as that of the thrombolytic enzyme was shown. That is, the protease activity of the product of Bacillus natto of the present invention reaches the protease activity (133.3 Unit / g) of the product of Bacillus natto on the 12th to 14th hour of fermentation. It was found that it had a high protease-producing ability at an early stage of fermentation. It was found that by producing the protease from an earlier stage, it was possible to produce a natto product having a large amount of thrombolytic enzyme and a tasting quality equivalent to that of a conventional product in a shorter fermentation time than before.
[0034]
【The invention's effect】
As described in detail above, the present invention relates to a novel natto strain that highly produces thrombolytic enzyme, a method for producing natto containing thrombolytic enzyme at a high content using this bacterium, and a natto product, According to the present invention, the following effects can be obtained.
(1) The natto strain of the present invention is a strain having a high thrombolytic enzyme-producing ability that produces a large amount of thrombolytic enzyme as compared with commercially available natto bacteria.
(2) In the natto produced using this natto strain, the amount of thrombolytic enzyme is about 1.5 to 2 times that of the commercially available natto.
(3) If natto is produced using this strain, it is possible to obtain natto having the same quality as conventional natto without changing the quality and containing a large amount of thrombolytic enzyme.
(4) The method for producing natto of the present invention is characterized in that Bacillus subtilis TTCC865 strain is used as a natto bacterium in the conventional production method, so that a new apparatus is not required and the production cost is not increased. Can be handled with the same production system as before.
(5) Since the properties of the natto strain are almost the same as those of the commercially available natto strain, there is almost no need to change the production conditions.
[Brief description of the drawings]
FIG. 1: Fermentation time and thrombolytic enzyme activity in the natto production process (when fermentation is carried out at a constant temperature of 37 ° C. and 90% humidity) using the Bacillus natto (TTCC865 strain) of the present invention and a commercially available Bacillus natto (M) (NK activity).
FIG. 2 Relationship between fermentation time and protease activity in a natto production process (when fermentation is carried out under constant conditions of 37 ° C. and 90% humidity) using the Bacillus natto (TTCC865 strain) of the present invention and a commercially available Bacillus natto (M). Is shown.

Claims

A natto strain which is a strain TTCC865 belonging to Bacillus subtilis (accession number: FERM P-19176) and a similar strain belonging to Bacillus subtilis having an ability to produce thrombolytic enzymes at a high level.

A method for producing natto containing a high amount of thrombolytic enzyme, wherein natto is produced using the natto strain according to claim 1.

A natto containing a high amount of thrombolytic enzyme, wherein the natto contains thrombolytic enzyme at least 90 FU / g wet weight, produced using the Bacillus natto according to claim 1.