JP4574103B2 - Glutamic acid high productivity natto strain and natto with high glutamic acid content produced using the same - Google Patents
Glutamic acid high productivity natto strain and natto with high glutamic acid content produced using the same Download PDFInfo
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Description
【０００１】
【発明の属する技術分野】
本発明は、納豆の旨味の主成分であるグルタミン酸の生産能が高い新規納豆菌の創製、並びに、その納豆菌を利用した、グルタミン酸含有量が高く、チロシン含有量が低い、美味しくて、かつ品質の安定した納豆を製造する方法に関するものである。
【０００２】
【従来の技術】
納豆は、大豆の煮豆に納豆菌を接種して発酵させて得られる日本独自の伝統食品である。取扱いが簡便で、安価な栄養食品であることに加えて、近年血栓症や骨粗鬆症の予防効果を有することが発見されて以来、需要の伸びが期待できる食品である。
納豆の旨味の基は、納豆菌が産生する蛋白分解酵素によって大豆蛋白が分解されて生じるアミノ酸であるが、このアミノ酸の中でもグルタミン酸が旨味の中心であり、通常の納豆には１００ｇ当り５０乃至１５０ｍｇ程度のグルタミン酸が含有されている。この観点から、納豆の旨味を増強することを目的とした研究が従来より種々報告されている。例えば、発酵時間と熟成時間を長くして大豆蛋白の酵素による分解を促進する方法や、特開平０３−２５４６５９、特開平０４−４０８８９等に開示されているような、自然界からのスクリ−ニングや育種により蛋白分解酵素活性の強い納豆菌を開発して納豆を製造する方法等が報告されている。
しかし、これらの方法で納豆の旨味の増強化を図った場合、いずれも、グルタミン酸ばかりでなく、ほとんどすべてのアミノ酸量が増加し、その結果、例えば、シャリの原因となるチロシンのような水に溶けにくいアミノ酸が、製品の保存中に結晶化して食感の劣化を引き起こしたり、或いは、苦みのあるペプチドが生成して味を悪くする等、マイナスの面が多々認められていた。このように、蛋白質の分解を促進して旨味を増強するという従来の方法には限度があり、よって、グルタミン酸を納豆１００ｇ当り１５０ｍｇ以上含有し、且つチロシン含量を、結晶化による食感の劣化が認め難くなる、納豆１００ｇ当り１１０ｍｇ以下とした、旨味が強くて品質の良い納豆の製造は、実際のところ非常に困難であるのが現状であった。
【０００３】
【発明が解決しようとする課題】
本発明は、従来の技術上の問題点を解決し、納豆需要の更なる拡大を図るために、品質の劣化を伴うことなく旨味が一段と増強された納豆を製造しうる技術を開発することを主な目的とする。
【０００４】
【課題を解決するための手段】
本発明者らは、上記の目的に即して鋭意研究を重ねたところ、品質の劣化を生じさせることなく納豆中のグルタミン酸を増強する方法として、納豆の発酵中に、煮豆中に存在する糖から納豆菌によりグルタミン酸を生産させることを思考するに至った。従来の納豆菌では、糖からグルタミン酸を生産する能力は非常に低いので、まず、突然変異やスクリ−ニングの手段により、グルタミン酸の生産能が高い納豆菌の創製／育種を行い、次いで、こうして得られた納豆菌を実際に用いて、グルタミン酸を納豆１００ｇ当り１５０ｍｇ以上含有し、かつチロシン含量の低い、即ち、従来の納豆におけるチロシン含量或いはそれ以下である、品質の安定した納豆を製造することを種々試みたところ、下記の方法によるならば、所期の目的が達成されることがわかった。
【０００５】
本発明者らは、まず、通常用いられている納豆菌を、蛋白源を全く含まない、糖、無機窒素源およびミネラルを含む最小液体培地で培養したところ、極少量ではあるがグルタミン酸が生産されることを確認した。このことから、納豆菌においてもグルタミン酸の合成経路が存在すると思考するに至り、更に、突然変異によりグルタミン酸アナログ耐性株を創製し、グルタミン酸生産能の増強化を種々試みた結果、親株として市販の納豆から分離した菌株を用いて、後述において詳説するような方法により、グルタミン酸高生産能を有する菌株を得ることに成功した。
ここにおいて、グルタミン酸アナログとして、Ｌ−グルタミン酸−γ−メチルエステル、α−メチルグルタミン酸、β−ヒドロキシグルタミン酸、メチオニンスルホキシミン、グルタミン酸−γ−モノヒドロキサメ−ト、２−アミノ−４−ホスホノ酪酸、Ｌ−グルタミン酸−γ−モノエチルエステル、Ｌ−グルタミン酸ジメチルエステル、Ｌ−グルタミン酸−ジ−ｔ−ブチルエステル、モノフロログルタミン酸、Ｌ−グルタミン酸ジエチルエステル、Ｄ−グルタミン酸等、納豆菌の生育を抑制するが、Ｌ‐グルタミン酸が併存すれば生育抑制が解除される様な物質を試みた。
よって、本発明は、第一義的に、グルタミン酸アナログに耐性を有し、かつグルタミン酸高生産能を有する納豆菌株を提供するものである。
上記の納豆菌を、通常の納豆製造条件下で用いて実際に納豆を製造させたところ、旨味の成分であるグルタミン酸を大量に含有していても、品質を劣化させるチロシンなどの難水溶性のアミノ酸の含量が低く、即ち、従来の納豆における程度或いはそれ以下であり、また、苦味ペプチドによる苦味も少ない、美味しくて、品質の安定した納豆が得られることを確認した。
よって、本発明は、第二義的に、納豆を製造するに際して、グルタミン酸アナログに耐性を有し、かつグルタミン酸高生産能を有する納豆菌株を用いることを特徴とするグルタミン酸高含有納豆の製造方法を提供するものである。
また、本発明は、このような製造方法により得られるグルタミン酸高含有納豆を提供するものである。
【０００６】
【発明の実施の形態】
以下、本発明を詳しく説明する。
Ｉ．突然変異作出手段
市販の納豆菌を親株としてＬＢ培地（トリプトン１０ｇ、酵母エキス５ｇ、食塩５ｇおよび純水１Ｌ：ｐＨ６．８）で前培養し、５ｍｌのＬＢ培地を入れた試験管にこの前培溶液５０μｌを接種し、３７℃、１２０ｒｐｍで２時間振盪培養する。次いで、遠心機で集菌した菌体をトリス・マレイン酸緩衝液〔トリス（ヒドロキシメチル）アミノメタン ６．１ｇ、 マレイン酸 ５．８ｇ、 ＭｇＳＯ４・７Ｈ２Ｏ ０．１ｇ、 クエン酸ナトリウム０．５ｇおよび純水１Ｌ：ｐＨ６．０〕で３回洗浄後、Ｎ−メチル−Ｎ′−ニトロ−Ｎ−ニトロソグアニジン（ＮＴＧ）溶液（最終濃度３００μｇ/ml）に再懸濁し、３０℃で３０分間静置して変異処理を行う。
なお、変異株の作出は、エチルエタンスルホン酸（ＥＭＳ）等の化学的な方法、紫外線やＸ線照射等の物理的な処理、遺伝子工学的な手法等を用いて実施することも可能であり、特に限定されるものではない。
【０００７】
変異処理後の菌体はＬＢ培地で２回洗浄後、５mlのＬＢ培地に再懸濁し、３７℃で一晩振盪培養を行う。翌朝、Ｓｐｉｚｚｅｎ最少培地〔（ＮＨ４）２ＳＯ４２g、K２HPO４ １４g、ＫＨ２ＰＯ４ ６g、クエン酸ナトリウム１g、ＭｇＳＯ４ ０.２g、グルコ−ス ５g、ビオチン ０．１ｍｇおよび純粋１L：ｐＨ６．８〕で３回洗浄後、適当量を、メチオニンスルホキシミンまたはＬ−グルタミン酸−γ−メチルエステルを１００μｇ／ｍｌ含むＳｐｉｚｚｅｎ最少寒天培地にプレ−ティングし、３７℃で３日間の培養により生育してきたコロニ−をグルタミン酸アナログ耐性株として取得する。なお、ここにおいて、グルタミン酸アナログとしては、Ｌ−グルタミン酸−γ−メチルエステル、α−メチルグルタミン酸、β−ヒドロキシグルタミン酸、メチオニンスルホキシミン、グルタミン酸−γ−モノヒドロキサメ−ト、２−アミノ−４−ホスホノ酪酸、Ｌ−グルタミン酸−γ−モノエチルエステル、Ｌ−グルタミン酸ジメチルエステル、Ｌ−グルタミン酸−ジ−ｔ−ブチルエステル、モノフロログルタミン酸、Ｌ−グルタミン酸ジエチルエステル、Ｄ−グルタミン酸等、納豆菌の生育を抑制するが、Ｌ−グルタミン酸が併存すれば生育抑制が解除される様な物質から選ばれる少なくとも１つであればよく、特に限定されるものではない。
【０００８】
これら耐性株を、いずれも２倍濃度のＳｐｉｚｚｅｎ最小液体培地で、３７℃の条件下１日間振盪培養した後、培養液中のグルタミン酸濃度を、F−キット L−グルタミン酸（Ｊ．Ｋ．インターナショナル製）を用いて測定し、コントロ−ルの親株よりも多くL−グルタミン酸を生産した株を、グルタミン酸高生産性菌株とする。
これらグルタミン酸高生産性菌株を使用して、常法に従い納豆を製造し、白粉のかぶり、糸引き、味の官能評価を行い、また、同時に１０℃で保存試験を行い、経時的にシャリの発生（結晶の発生）を官能評価し、変異株の選抜を行う。これらグルタミン酸高生産性菌株のうち最もグルタミン酸を多く生産し、納豆の品質のよかったものを、目的を達成した変異株とする。
【０００９】
II ．変異体の同定
後述の実施例で示すように、上記の方法に従って、グルタミン酸アナログに耐性を示し、グルタミン酸生産能が従来の３倍以上も増強された変異株ＤＧ１１９９ 株が得られた。この菌株は、グルタミン酸アナログに耐性であること以外の菌学的性質は、市販の納豆菌のもの〔食総研報（Ｒｅｐ．Ｎａｔｌ．Ｆｏｏｄ Ｒｅｓ．Ｉｎｓｔ．）Ｎｏ．５０，１８〜２１（１９８７）および大豆月報、１２月号、２１〜２９（１９８５）参照〕と大差はなかった。即ち、この変異株ＤＧ１１９９は、好気性、グラム染色陽性の桿菌であり、菌（栄養細胞）の大きさ（１×２〜３μｍ）、生育適温（３５〜４５℃）、各種の糖の発酵性、ＤＮＡのＧＣ含量等の性質がＢｅｒｇｅｙ´ｓ Ｍａｎｕａｌ ８版の枯草菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）の性質と一致しており、かつ粘質物を生成し、ビオチン要求性であること、及び、納豆菌のファ−ジに対して感受性であることから、いわゆる納豆菌（Ｂａｃｉｌｌｕｓ ｎａｔｔｏ）に属しているものである。このＤＧ１１９９株 （Ｂａｃｉｌｌｕｓ ｓｐ．ＤＧ１１９９）は、平成１３年１１月２９日、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、 ＦＥＲＭ Ｐ−１８６４４号として寄託されている。
【００１０】
【実施例】
以下、本発明を実施例をもってさらに詳しく説明する。
実施例１
市販の納豆種菌（宮城野納豆製造所製）から良質の納豆を作る菌株を分離し、親株とした。この親株をＬＢ培地で前培養し、５mlのＬＢ培地を入れた試験管にこの前培溶液５０μｌを接種し、３７℃、１２０rpmで２時間振盪培養した。次いで、遠心機で集菌した菌体をトリス・マレイン酸緩衝液で３回洗浄後、ＮＴＧ溶液（最終濃度３００μｇ／ｍｌ）に再懸濁し、３０℃で３０分間静置して変異処理を行った。
変異処理後の菌体はＬＢ培地で２回洗浄後、５mlのＬＢ培地に再懸濁し、３７℃で一晩振盪培養を行った。翌朝、Ｓｐｉｚｚｅｎ最少培地で３回洗浄後、適当量をメチオニンスルホキシミン１００μｇ／ｍｌを含むＳｐｉｚｚｅｎ最少寒天培地にプレ−ティングし、３７℃で３日間培養した。このプレ−トからグルタミン酸アナログ耐性株ＤＬＳ３４２、ＤＬＳ４０４、ＤＧ１１９９を得た。
また、同様に、Ｌ−グルタミン酸−γ−メチルエステル１００μｇ／ｍｌを含むＳｐｉｚｚｅｎ最少寒天培地から、M５４、Ｍ６５株を得た。
これら耐性株を、いずれも２倍濃度のＳｐｉｚｚｅｎ最小液体培地で、３７℃の条件下１日間振盪培養した後、培養液中のグルタミン酸濃度を測定した。その結果を、下記の表１に示す。
【００１１】
【表１】
実施例２
実施例１で取得したグルタミン酸高生産性菌株のうち最も多く（培地１００ｍｌ当り２９．２ｍｇ）グルタミン酸を生産したＤＧ１１９９株を、２０%大豆煮汁寒天培地の試験管スラントで培養し、保存した。この試験管スラントから１白金耳の胞子をかき取り、２００mlの滅菌水に懸濁し、８０℃程度の熱い蒸煮大豆に噴霧した。この蒸煮大豆を小型の発泡スチロ−ル容器に入れ、大豆の上を薄いポリエチレンフィルムで覆い、蓋をして４０℃で１８時間発酵させた後、４℃で１日間熟成させた。
こうして出来上がった納豆は、白粉のかぶりがきれいで、糸引きも良く、強い旨味を有していた。また、この納豆のアミノ酸組成を分析したところ、下記の表２に示すように、グルタミン酸は納豆１００ｇ当り２１０ｍｇで、親株の納豆に比べ非常に高かったが、チロシンは納豆１００ｇ当り１０２．５ｍｇで、親株の１１２．０ｍｇと大差はなかった。次いで、この納豆を１０℃に保存して品質の変化を調べたところ、チロシンの結晶化による食感の劣化は１７日目に起こり、親株の納豆と変わらなかった。
【００１３】
【表２】
実施例３
親株、ＤＧ１１９９、および、蛋白分解酵素活性の強い納豆菌株ＮＮ−１（特開平０３−２５４６５９）を使用して、実施例２と同様にして納豆を製造した。ただし、発酵時間は１７時間とした。また、４℃での熟成は２日間とし、その後１０℃に保存して保存試験を行った。
熟成終了後の納豆中の遊離アミノ酸の量をアミノ酸分析装置で分析してみたところ、下記の表３に示すような結果が得られた。すなわち、ＤＧ１１９９は親株と比べると、グルタミン酸のみが特に増加しているのに対して、ＮＮ−１は遊離アミノ酸が全体的に増加している。
また、１０℃での保存中にシャリの発生の有無を観察した結果を、下記の表４に示す。この表から明らかなように、ＮＮ−１では、７日目当たりからシャリの発生が確認され始め、１０日目では明らかに確認されているのに対して、親株、ＤＧ１１９９では、２５日目になって初めて食感の変化が認められている。シャリの発生はチロシンの結晶化に起因すると一般的に考えられている。ＮＮ−１では遊離アミノ酸全体が増えてしまうため、シャリの発生の原因となるチロシンも増加してしまい、その結果、シャリの発生が他の株の場合よりも早く起きてしまうと考えられる。
【００１５】
【表３】
【表４】
【発明の効果】
納豆菌変異株ＤＧ１１９９株で作った納豆は、旨味の成分であるグルタミン酸を納豆１００ｇ当り１５０ｍｇ以上、好ましくは２００ｍｇ以上含有するため、美味しく、また、シャリの原因となるチロシン含量は従来の納豆と同程度、或いはそれ以下であることから、品質も安定である。よって、このような納豆は消費者に好まれると考えられることから、納豆の消費及び市場の拡大、即ち、納豆需要の更なる拡大が期待できる。[0001]
BACKGROUND OF THE INVENTION
The present invention is the creation of a novel natto bacterium having a high ability to produce glutamic acid, which is the main ingredient of natto umami, and a high glutamic acid content, low tyrosine content, tasty and quality using the natto bacterium. It is related with the method of manufacturing the stable natto.
[0002]
[Prior art]
Natto is a Japanese traditional food obtained by inoculating fermented natto bacteria on boiled soybeans. In addition to being an easy-to-handle and inexpensive nutritional food, it is a food that can be expected to grow in demand since it was recently discovered to have a preventive effect on thrombosis and osteoporosis.
The umami group of natto is an amino acid produced by degrading soy protein by a proteolytic enzyme produced by Bacillus natto. Among these amino acids, glutamic acid is the center of umami, and in normal natto, 50 to 150 mg per 100 g. A degree of glutamic acid is contained. From this point of view, various studies have been reported for the purpose of enhancing the umami taste of natto. For example, a method in which fermentation time and ripening time are lengthened to promote degradation of soybean protein by an enzyme, screening from the natural world as disclosed in JP-A-03-254659, JP-A-04-40889, etc. A method for producing natto by developing natto bacteria having strong protease activity by breeding has been reported.
However, when these methods are used to enhance the umami taste of natto, not only glutamic acid but also almost all amino acids are increased, resulting in, for example, water such as tyrosine that causes shari. Many negative aspects were observed, such as amino acids that are difficult to dissolve crystallize during storage of the product to cause a deterioration in food texture, or that a bitter peptide is produced to make the taste worse. As described above, there is a limit to the conventional method of enhancing the umami by promoting the degradation of protein. Therefore, glutamic acid is contained in an amount of 150 mg or more per 100 g of natto, and the tyrosine content is reduced due to crystallization. Production of natto with high umami and high quality, which is less than 110 mg per 100 g of natto, which is difficult to recognize, was actually very difficult.
[0003]
[Problems to be solved by the invention]
In order to solve the conventional technical problems and to further increase the demand for natto, the present invention develops a technology capable of producing natto with further enhanced umami without deteriorating quality. Main purpose.
[0004]
[Means for Solving the Problems]
As a method for enhancing glutamic acid in natto without causing deterioration in quality, the present inventors have conducted extensive research in line with the above-mentioned purpose. Came to think about producing glutamic acid by natto bacteria. In conventional natto bacteria, the ability to produce glutamic acid from sugar is very low. First, by means of mutation or screening, natto bacteria with high glutamic acid producing ability are created / breeded, and then obtained. To produce stable natto containing glutamic acid in an amount of 150 mg or more per 100 g of natto and having a low tyrosine content, that is, a tyrosine content in conventional natto or less. As a result of various attempts, it was found that the intended purpose was achieved by the following method.
[0005]
First, the inventors cultured a commonly used natto bacterium in a minimal liquid medium containing no protein source and containing sugar, an inorganic nitrogen source and minerals, but produced a very small amount of glutamic acid. I was sure that. This led to the thought that the synthesis pathway of glutamic acid also exists in Bacillus natto.Furthermore, as a result of various attempts to create glutamic acid analog-resistant strains by mutation and enhance glutamic acid production ability, commercially available natto as a parent strain Using the strain isolated from the above, the inventors succeeded in obtaining a strain having a high glutamate producing ability by a method described in detail later.
Here, as the glutamic acid analog, L-glutamic acid-γ-methyl ester, α-methyl glutamic acid, β-hydroxyglutamic acid, methionine sulfoximine, glutamic acid-γ-monohydroxamate, 2-amino-4-phosphonobutyric acid, L-glutamic acid-γ-monoethyl ester, L-glutamic acid dimethyl ester, L-glutamic acid-di-t-butyl ester, monofluoroglutamic acid, L-glutamic acid diethyl ester, D-glutamic acid, etc. In addition, an attempt was made to develop a substance capable of releasing the growth inhibition if L-glutamic acid coexists.
Therefore, the present invention primarily provides a natto strain that is resistant to glutamic acid analogs and has a high ability to produce glutamic acid.
When natto was actually produced using the above natto bacteria under normal natto production conditions, even though it contains a large amount of umami, glutamic acid, it has poor water solubility such as tyrosine, which degrades the quality. It was confirmed that natto having a low amino acid content, that is, a level lower than or equal to that of conventional natto and having a bitter taste due to a bitter peptide, having a good quality and stable quality.
Therefore, the present invention, secondarily, in producing natto, a method for producing natto with a high glutamate content, characterized by using a natto strain that is resistant to glutamic acid analogs and has a high ability to produce glutamate. It is to provide.
Moreover, this invention provides the glutamic acid high content natto obtained by such a manufacturing method.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described in detail below.
I. Mutation production means A test tube containing 5 ml of LB medium pre-cultured in LB medium (tryptone 10 g, yeast extract 5 g, salt 5 g and pure water 1 L: pH 6.8) using commercially available natto bacteria as a parent strain Is inoculated with 50 μl of this pre-culture solution, and cultured with shaking at 37 ° C. and 120 rpm for 2 hours. Subsequently, the cells collected by the centrifuge were tris-maleic acid buffer solution [tris (hydroxymethyl) aminomethane 6.1 g, maleic acid 5.8 g, MgSO 4 .7H 2 O 0.1 g, sodium citrate 0. 5 g and 1 L of pure water: pH 6.0], and then resuspended in an N-methyl-N′-nitro-N-nitrosoguanidine (NTG) solution (final concentration 300 μg / ml) for 30 minutes at 30 ° C. Let stand and perform mutation treatment.
It is also possible to produce mutant strains using chemical methods such as ethyl ethane sulfonic acid (EMS), physical treatment such as ultraviolet rays and X-ray irradiation, genetic engineering techniques, and the like. There is no particular limitation.
[0007]
The cells after the mutation treatment are washed twice with LB medium, resuspended in 5 ml of LB medium, and cultured with shaking at 37 ° C. overnight. The next morning, Spizzen minimal medium [(NH 4 ) 2 SO 4 2 g, K 2 HPO 4 14 g, KH 2 PO 4 6 g, sodium citrate 1 g, MgSO 4 0.2 g, glucose 5 g, biotin 0.1 mg and pure 1 L : PH 6.8] after washing three times, an appropriate amount was plated on a Spizzen minimal agar medium containing 100 μg / ml of methionine sulfoximine or L-glutamic acid-γ-methyl ester, and cultured at 37 ° C. for 3 days The colony that has grown by the above is obtained as a glutamate analog resistant strain. Here, as the glutamic acid analog, L-glutamic acid-γ-methyl ester, α-methyl glutamic acid, β-hydroxyglutamic acid, methionine sulfoximine, glutamic acid-γ-monohydroxamate, 2-amino-4- Phosphonobutyric acid, L-glutamic acid-γ-monoethyl ester, L-glutamic acid dimethyl ester, L-glutamic acid-di-t-butyl ester, monophloglutamic acid, L-glutamic acid diethyl ester, D-glutamic acid, etc. Although it suppresses, if L-glutamic acid coexists, it should just be at least 1 chosen from the substance from which growth suppression is cancelled | released, It does not specifically limit.
[0008]
All of these resistant strains were shake-cultured in a Spizzen minimal liquid medium with a double concentration at 37 ° C. for 1 day, and then the glutamic acid concentration in the culture solution was changed to F-kit L-glutamic acid (manufactured by JK International). ), And a strain that produced more L-glutamic acid than the control parent strain is defined as a glutamic acid high-producing strain.
Using these glutamic acid high-producing strains, natto is produced according to a conventional method, and white powder fogging, stringing, and taste sensory evaluation are performed. At the same time, a storage test is performed at 10 ° C. Sensory evaluation of (crystal generation) and selection of mutant strains. Among these glutamic acid high-producing strains, the one that produces the most glutamic acid and has the best quality of natto is defined as a mutant that achieves the purpose.
[0009]
II . Identification of mutants As shown in the examples described later, according to the above-described method, a mutant DG1199 strain which was resistant to glutamic acid analogs and whose glutamic acid production ability was enhanced by 3 times or more than the conventional strain was obtained. . This strain has the bacteriological properties other than being resistant to glutamic acid analogs from those of commercially available natto bacteria [Rep. Natl. Food Res. Inst. 50, 18-21 (1987) and soybean monthly report, December issue, 21-29 (1985)]. That is, this mutant strain DG1199 is an aerobic, Gram-staining-positive gonococcus, the size of bacteria (vegetative cells) (1 × 2 to 3 μm), the optimum growth temperature (35 to 45 ° C.), and the fermentability of various sugars The properties of the DNA, such as the GC content, are consistent with those of the Bergey's Manual 8th edition, Bacillus subtilis, and it produces a sticky substance and is biotin-requiring, and -Since it is sensitive to bismuth, it belongs to the so-called Bacillus natto. This DG1199 strain (Bacillus sp. DG1199) was deposited as FERM P-18644 on November 29, 2001 at the National Institute of Advanced Industrial Science and Technology.
[0010]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
A bacterial strain that produces high quality natto was isolated from a commercially available natto inoculum (manufactured by Miyagino Natto Factory) and used as a parent strain. This parent strain was precultured in LB medium, 50 μl of this preculture solution was inoculated into a test tube containing 5 ml of LB medium, and cultured with shaking at 37 ° C. and 120 rpm for 2 hours. Next, the cells collected by the centrifuge are washed three times with Tris / maleic acid buffer, then resuspended in NTG solution (final concentration 300 μg / ml), and left at 30 ° C. for 30 minutes for mutation treatment. It was.
The cells after the mutation treatment were washed twice with LB medium, resuspended in 5 ml of LB medium, and cultured with shaking at 37 ° C. overnight. The next morning, after washing three times with Spizzen minimal medium, an appropriate amount was plated on Spizzen minimal agar medium containing 100 μg / ml methionine sulfoximine and cultured at 37 ° C. for 3 days. From this plate, glutamic acid analog resistant strains DLS342, DLS404, and DG1199 were obtained.
Similarly, M54 and M65 strains were obtained from Spizzen minimal agar medium containing 100 μg / ml of L-glutamic acid-γ-methyl ester.
All of these resistant strains were cultured with shaking at a double concentration of Spizzen minimal liquid medium at 37 ° C. for 1 day, and then the glutamic acid concentration in the culture solution was measured. The results are shown in Table 1 below.
[0011]
[Table 1]
Example 2
Among the glutamic acid high-producing strains obtained in Example 1, the most DG1199 strain that produced glutamic acid (29.2 mg per 100 ml medium) was cultured in a test tube slant of a 20% soybean broth agar medium and stored. One platinum ear spore was scraped from this test tube slant, suspended in 200 ml of sterilized water, and sprayed on hot steamed soybean at about 80 ° C. This steamed soybean was placed in a small foamed polystyrene container, the soybean was covered with a thin polyethylene film, covered, fermented at 40 ° C. for 18 hours, and then aged at 4 ° C. for 1 day.
The finished natto had a white powder cover, good stringiness, and a strong taste. In addition, when the amino acid composition of this natto was analyzed, as shown in Table 2 below, glutamic acid was 210 mg per 100 g of natto, which was very high compared to natto of the parent strain, but tyrosine was 102.5 mg per 100 g of natto, There was no big difference with 112.0mg of the parent strain. Subsequently, when this natto was stored at 10 ° C. and the change in quality was examined, the deterioration of the texture due to crystallization of tyrosine occurred on the 17th day and was not different from that of the parent strain.
[0013]
[Table 2]
Example 3
Natto was produced in the same manner as in Example 2 using the parent strain, DG1199, and natto strain NN-1 (JP-A-03-254659) having strong protease activity. However, the fermentation time was 17 hours. The aging at 4 ° C. was performed for 2 days, and then stored at 10 ° C. for a storage test.
When the amount of free amino acids in natto after ripening was analyzed with an amino acid analyzer, the results shown in Table 3 below were obtained. That is, DG1199 has a particularly increased amount of glutamic acid as compared to the parent strain, whereas NN-1 has an overall increased free amino acid.
In addition, Table 4 below shows the results of observing the presence or absence of shaving during storage at 10 ° C. As is clear from this table, in NN-1, the occurrence of shari began to be confirmed around day 7, whereas it was clearly confirmed on day 10, whereas the parent strain, DG1199, on day 25. It is not until the change of texture is recognized. The occurrence of shari is generally considered to result from crystallization of tyrosine. In NN-1, since the total free amino acids increase, tyrosine causing the occurrence of shari also increases, and as a result, the occurrence of shari is likely to occur earlier than in other strains.
[0015]
[Table 3]
[Table 4]
【The invention's effect】
Natto made with natto fungus mutant DG1199 contains 150mg or more, preferably 200mg or more of glutamic acid, which is an umami ingredient, per 100g of natto, so it is delicious and has the same tyrosine content as the cause of shari. The quality is stable because it is about or less. Therefore, since it is considered that such natto is preferred by consumers, it is expected that natto consumption and market expansion, that is, further expansion of natto demand will be expected.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03254659A (en) * | 1990-03-05 | 1991-11-13 | Asahi Kogyo Kk | Production of high viscosity natto (fermented soybeans) having good taste |
| JPH08275772A (en) * | 1995-04-06 | 1996-10-22 | Fujitsuko Kk | Bacillus natto, fermented soybean and its production |
| JPH10262655A (en) * | 1997-03-25 | 1998-10-06 | Hoshino Kagaku Kk | New bacillus natto and its acquisition and poly-gamma-glutamic acid produced with new bacillus natto and seasoning utilizing the same |
| JPH11253123A (en) * | 1998-03-12 | 1999-09-21 | Fujicco Co Ltd | Fungus for fermented soybeans and production of fermented soybeans therewith and fermented soybeans |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03254659A (en) * | 1990-03-05 | 1991-11-13 | Asahi Kogyo Kk | Production of high viscosity natto (fermented soybeans) having good taste |
| JPH08275772A (en) * | 1995-04-06 | 1996-10-22 | Fujitsuko Kk | Bacillus natto, fermented soybean and its production |
| JPH10262655A (en) * | 1997-03-25 | 1998-10-06 | Hoshino Kagaku Kk | New bacillus natto and its acquisition and poly-gamma-glutamic acid produced with new bacillus natto and seasoning utilizing the same |
| JPH11253123A (en) * | 1998-03-12 | 1999-09-21 | Fujicco Co Ltd | Fungus for fermented soybeans and production of fermented soybeans therewith and fermented soybeans |
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