JP4649244B2 - Reduced stringiness natto bacteria, and reduced stringiness natto manufactured using the natto bacteria - Google Patents
Reduced stringiness natto bacteria, and reduced stringiness natto manufactured using the natto bacteria Download PDFInfo
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Description
本発明は、新規納豆菌及び該納豆菌を用いて製造された新規納豆に関し、さらに詳細には、納豆らしい外観を有し、かつ糸引性が低下している納豆を効率良く製造することができる糸引性低下納豆菌、及び該納豆菌を用いて製造され喫食性に優れた糸引性低下納豆に関する。 The present invention relates to a novel natto bacterium and a novel natto produced using the natto bacterium, and more specifically, it is possible to efficiently produce natto having a natto-like appearance and reduced stringiness. The present invention relates to a stringiness-reduced natto bacterium and a stringiness-reduced natto produced using the natto bacterium and having excellent eating properties.
納豆は日本古来の発酵食品であり、一般には糸引納豆と言われるように、納豆菌による発酵過程で生成されるグルタミン酸ポリペプチドを主体とする強い粘りを持つ糸引物質を豊富に有しており、糸を引けば引く程、良質の納豆であるとされている。
一方、納豆を食する際には、一般に、まず、納豆に添付された小袋入りの納豆調味用のタレや、或いは好みに応じて醤油や砂糖などの調味料を納豆にかける。さらに、添付のからしや、好みによっては、きざみネギなどの薬味を加えた後、箸などで納豆を良く攪拌して糸を引かせるのが常である。しかし、納豆の糸引物質に起因して喫食時の口中や唇のベタつきが生じたり、また糸引物質が衣服などに付着したりする等の理由から、糸引性が逆に嫌われる場合があった。
Natto is an ancient fermented food in Japan, and as it is generally called Itobiki Natto, it has an abundance of stringing substances with strong stickiness mainly composed of glutamic acid polypeptide produced in the fermentation process by Bacillus natto. The more you pull the thread, the better it is.
On the other hand, when eating natto, generally, a sachet of natto seasoning in a small bag attached to natto or a seasoning such as soy sauce or sugar is applied to natto according to taste. In addition, depending on the mustard and taste, it is usual to add a seasoning such as chopped leek and then stir the natto well with chopsticks to draw the thread. However, the stringiness of the natto is sometimes disliked due to stickiness of the mouth and lips at the time of eating, and the stringiness substance adheres to clothes.
このような観点から、納豆の糸引性を消失させる方法が種々提案されており、そのほとんどは、タレの組成を変更することによる納豆の糸引物質の飛散を抑制する方法であった。例えば、特許文献１には、納豆又は納豆菌の糸引物質をカルシウムイオン、マグネシウムイオンなどで処理してなる糸引き性のない納豆が開示されている。
しかしながら、特許文献１記載の納豆の場合、糸引性を充分低下させるために高濃度のイオンを添加する必要があり、このため、納豆を食する際苦味やエグミが発生し納豆の呈味性が変化するという欠点を有していた。また、タレの組成を変更することによる他の方法においても、同様の欠点があった。
From such a viewpoint, various methods for eliminating the stringiness of natto have been proposed, and most of them have been methods for suppressing scattering of the stringing material of natto by changing the composition of the sauce. For example, Patent Document 1 discloses natto having no stringiness, which is obtained by treating natto or natto fungus stringing substances with calcium ions, magnesium ions, or the like.
However, in the case of natto described in Patent Document 1, it is necessary to add a high concentration of ions in order to sufficiently reduce the stringiness. For this reason, when eating natto, bitterness and agility occur, and the taste of natto is reduced. It had the disadvantage of changing. Also, other methods by changing the composition of the sauce have the same drawbacks.
一方、納豆を生産する納豆菌の改良により納豆の糸引性を変化させる例は少なく、わずかに、γ−グルタミルトランスペプチダーゼ活性の低い納豆菌を用いた糸引性のない納豆を作製する方法（例えば、特許文献２及び特許文献３参照）が開示されている程度であった。
しかしながら、これらの改良例により得られる納豆菌は、例えば、納豆１００ｇを水２００ｍｌに溶解し、これに最終濃度が８５％となるようにエチルアルコールを添加して納豆の糸引物質を沈殿させた場合に、ガラス棒で巻き取ることができない程、全く糸引物質を生産しないものであった。
納豆らしさは、発酵過程で大豆の周囲に生産される糸引物質が付着した特有の量的外観に特徴を有するものであり、糸引物質の量が減ると、納豆としての外観が損なわれてしまう。
従って、糸引性は低下している反面、納豆としての外観上必要な糸引物質を、従来の納豆と大差ない程度の生産量で生産できる納豆菌が求められていた。
On the other hand, there are few examples of changing the stringiness of natto by improving natto bacteria that produce natto, and a method for producing natto having no stringiness using natto bacteria having a low γ-glutamyltranspeptidase activity (for example, Patent Document 2 and Patent Document 3) are disclosed.
However, the natto bacteria obtained by these improved examples are, for example, when 100 g of natto is dissolved in 200 ml of water, and ethyl alcohol is added to the final concentration of 85% to precipitate the natto stringing material. Furthermore, the yarn-drawn material was not produced at all so that it could not be wound with a glass rod.
The uniqueness of natto is characterized by a specific quantitative appearance in which the stringing material produced around soybeans in the fermentation process is attached. When the amount of stringing material is reduced, the appearance as natto is impaired.
Therefore, while the stringiness is reduced, there has been a demand for a natto bacterium that can produce a stringing material necessary for appearance as natto with a production amount not much different from that of conventional natto.
本発明は、納豆らしい特有の糸引物質の量的外観を損なわずに、かつ糸引性が低下した納豆を充分量生産できる納豆菌を開発し、該納豆菌を用いて製造される、周りに糸引物質が飛散しにくい糸引性低下納豆を提供することを目的とする。 The present invention has developed a natto bacterium that can produce a sufficient amount of natto with reduced stringiness without losing the quantitative appearance of a unique stringing material unique to natto. It aims at providing the stringiness fallen natto which a substance does not disperse easily.
上記課題に鑑み、本発明者らは、納豆の糸引性を変化させるべく検討を重ねる過程において、納豆菌の糸引物質の糸引性を、納豆らしい外観を損なわない程度に低下させるための指標について検討を行ない、その過程で、納豆のグルタミン酸ポリペプチド含有量、及び納豆から水等で抽出して得られる糸引物質抽出液の粘度に着目した。
そして、上記グルタミン酸ポリペプチド含有量と納豆中の糸引物質含有量との間の相関性、及び、糸引物質抽出液の粘度と納豆の糸引性との相関の有無を検討した結果、グルタミン酸ポリペプチドの含有量が従来の納豆と同程度以上であり、かつ、納豆の糸引物質抽出液の粘度を割った係数、すなわち粘度比が従来の納豆より低下している納豆は、糸引物質の含有量は従来のものと同等で納豆らしい外観を有し、かつ糸引性が低下していることを見出した。
そこで、これらの指標をもとに、上記のようなグルタミン酸ポリペプチドの含有量及び粘度比がそれぞれ所定の数値範囲である糸引性低下納豆を充分量生産する能力を有する納豆菌の育種改良に努めた結果、種々の糸引性低下納豆菌を得ることができた。
In view of the above problems, the present inventors examined an index for lowering the stringiness of the natto fungus stringing material to an extent that does not impair the natto-like appearance in the process of repeated examination to change the stringiness of natto. In the process, attention was paid to the glutamic acid polypeptide content of natto and the viscosity of the stringing substance extract obtained by extraction from natto with water or the like.
And, as a result of examining the correlation between the content of the glutamic acid polypeptide and the stringiness substance content in natto, and the presence or absence of the correlation between the viscosity of the stringing substance extract and the stringiness of natto, the glutamic acid polypeptide The natto content is equal to or higher than that of conventional natto and the viscosity of the natto string drawing material extract is divided, i.e., the viscosity ratio is lower than that of conventional natto. It was found to have a natto-like appearance that is similar to that of natto and has reduced stringiness.
Therefore, based on these indicators, efforts are made to improve the breeding of Bacillus natto having the ability to produce a sufficient amount of stringiness-reduced natto in which the content and viscosity ratio of the glutamic acid polypeptide are in the predetermined numerical ranges, respectively. As a result, various stringiness-reduced natto bacteria could be obtained.
さらに、上記したような納豆らしい外観を有し、かつ糸引性が低下している納豆を喫食するにあたり、納豆にかけるタレの組成を検討した結果、タレ中の２価カチオンの含量を特定割合に高めることにより、納豆としての呈味性に悪影響を与えない範囲で糸引性を更に低下させて、切れのよい糸引性を達成できることを見出した。
そしてこのような効果を有する２価カチオンとしては、食品として使用可能なカルシウムやマグネシウムがより好ましく、かつ使用可能なことを見出した。
Furthermore, as a result of examining the composition of the sauce to be applied to natto when eating natto having an appearance like the above-mentioned natto and having reduced stringiness, the content of divalent cations in the sauce was determined to a specific ratio. It has been found that, by increasing the thread drawability, the stringiness can be further reduced within a range that does not adversely affect the taste as natto, and a good stringiness can be achieved.
And as a bivalent cation which has such an effect, it discovered that calcium and magnesium which can be used as a foodstuff were more preferable, and can be used.
また、本発明者らは、更に進んで、納豆らしい外観を損なわない範囲で、納豆の糸引物質飛散抑制を十分に達成するための手段を鋭意検討した。
その過程で、本発明者らは、納豆の粘物質のレバンに注目した。そして、レバンの分解活性及び合成活性を、親株のそれとは変化させた納豆菌が生産する納豆は、親株の生産する納豆とはレバンの量及び性質の点で相違するのではないかと推察した。
本発明者らは上記観点から検討を重ねた結果、前述の糸引性低下納豆菌を親株として育種改良することによって得られたレバン分解酵素活性が低下した納豆菌は、納豆らしい外観を有することはもちろん、糸引性が十分に低下した納豆を生産することができ、２価カチオンを含むタレを添加しなくとも糸引性は十分に低下していることを見出した。さらに、レバン分解酵素の活性が低下している糸引低下納豆菌を用いて納豆を製造する際に、蒸煮大豆にショ糖を所定量添加することにより、納豆の糸引物質の飛散性がより一層抑制されることを見出した。
本発明は、係る知見に基くものである。
Further, the present inventors have further studied and intensively studied means for sufficiently achieving natto string-drawing material scattering suppression within a range that does not impair the natto-like appearance.
In the process, the present inventors paid attention to the natto mucilage levan. And, it was inferred that natto produced by Bacillus natto, in which the decomposition activity and synthesis activity of levan were changed from that of the parent strain, differed from the natto produced by the parent strain in terms of the amount and nature of levan.
As a result of repeated studies from the above viewpoints, the present inventors have demonstrated that the Bacillus natto having reduced levan-degrading enzyme activity obtained by improving the breeding of the above-described stringiness-reduced natto bacillus as a parent strain has a natto-like appearance. Of course, it was found that natto with sufficiently reduced stringiness could be produced, and the stringiness was sufficiently reduced without adding a sauce containing a divalent cation. In addition, when producing natto using natto-fermenting natto bacteria, the activity of levan-degrading enzyme is reduced, by adding a predetermined amount of sucrose to steamed soybeans, the scattering properties of natto stringing substances are further suppressed. I found out that
The present invention is based on such knowledge.
すなわち、請求項１に記載の本発明は、グルタミン酸ポリペプチドを６ｍｇ／ｇ納豆以上含有し、粘度比が１４ｃｐｓ／ｍｇ以下であり、かつ、親株であるバシルス・サチリス（Bacillus subtilis）ＯＵＶ２３４８１株（ＦＥＲＭ ＢＰ−６６５９）から製造される納豆と比較して、糸引物質の量が同程度以上であり、糸引物質の飛散性が低下した納豆を製造できることを特徴とするバシルス・サチリス（Bacillus subtilis）に分類される、糸引性低下納豆菌に関する。
さらに、請求項３に記載の本発明は、請求項１〜２のいずれかに記載の糸引性低下納豆菌を用いて製造されたことを特徴とする納豆に関する。
そして、請求項４に記載の本発明は、請求項３に記載の納豆と、２価カチオン濃度が納豆当り０．１〜０．３重量／重量％となるように調製されたタレとからなる容器入り納豆に関する。
さらにまた、請求項５に記載の本発明は、２価カチオンがカルシウム及び／又はマグネシウムであることを特徴とする上記請求項４に記載の容器入り納豆に関する。
That is, the present invention according to claim 1, the glutamic acid polypeptide containing more than 6 mg / g natto state, and are viscosity ratios 14 cps / mg or less, and a parent strain Bacillus subtilis (Bacillus subtilis) OUV23481 strain ( Compared with natto produced from FERM BP-6659), Bacillus subtilis is characterized in that it can produce natto with the same amount of string-drawing substance and reduced scattering of string-drawing substance. It relates to natto bacteria with reduced stringiness.
Furthermore, the present invention described in claim 3 relates to natto, characterized by being manufactured using the stringiness-reduced natto bacteria described in any one of claims 1 to 2.
And this invention of Claim 4 consists of natto of Claim 3, and sauce prepared so that a bivalent cation density | concentration may be 0.1-0.3 weight / weight% per natto. It relates to natto in containers.
Furthermore, the present invention according to claim 5 relates to natto in a container according to claim 4, wherein the divalent cation is calcium and / or magnesium.
また、請求項６に記載の本発明は、レバン分解酵素の活性が０．７Ｕ／ｍｇ以下であることを特徴とする請求項１に記載の糸引性低下納豆菌に関する。
そして、請求項７に記載の本発明は、バシルス・サチリス（Bacillus subtilis）ＤＣ６６株（ＦＥＲＭ ＢＰ−１０２３４）であることを特徴とする請求項６に記載の糸引性低下納豆菌に関する。
また、請求項８に記載の本発明は、請求項６または請求項７に記載の糸引性低下納豆菌を用いて製造されたことを特徴とする納豆に関する。
さらにまた、請求項９に記載の本発明は、蒸煮大豆あたり０．２〜２．０重量／重量％となるようにショ糖を添加して発酵させて製造されたことを特徴とする請求項８に記載の納豆に関する。
Moreover, this invention of Claim 6 is related with the stringiness fallen natto bacteria of Claim 1 whose activity of a levan degrading enzyme is 0.7 U / mg or less.
And this invention of Claim 7 is a Bacillus subtilis (Bacillus subtilis) DC66 strain | stump | stock (FERM BP-10234), It relates to the stringiness fallen natto bacteria of Claim 6.
The present invention according to claim 8 relates to natto characterized by being produced using the stringiness-reduced natto bacteria according to claim 6 or claim 7.
Furthermore, the present invention according to claim 9 is manufactured by adding sucrose and fermenting it so that it becomes 0.2 to 2.0 wt / wt% per steamed soybean. It relates to natto according to 8.
本発明により、納豆特有の糸引物質の量的外観は保持しつつ、糸引性が低下し、糸引物質の飛散性が抑制された納豆を充分量生産できる糸引性低下納豆菌が提供される。
また、本発明により、周りに糸引物質が飛散しにくく、喫食時に口中や唇のベタつきが生じたり、また糸引物質が衣服などに付着したりすることのない糸引性低下納豆が提供される。
さらに、本発明により、上記糸引性低下納豆と共に、該納豆を食する際に加えて攪拌すると、糸引性を更に低下させることのできるタレを添付させた容器入り納豆が提供される。
そして、本発明の糸引性低下納豆菌のうち、特にレバン分解酵素の活性が低下している糸引性低下納豆菌は、糸引物質の飛散性が顕著に抑制された納豆を生産することができる。従って、本発明によれば、該納豆菌を納豆の製造に用いることにより、２価カチオンを含むタレを添加しなくとも十分に糸引性の低い納豆が提供される。
さらにまた、本発明によれば、レバン分解酵素の活性が低下している糸引低下納豆菌を用いて納豆を製造する際に、蒸煮大豆にショ糖を所定量添加することにより、糸引物質の飛散性が一層抑制された納豆が提供される。
The present invention provides a stringiness-reduced natto bacterium that can produce a sufficient amount of natto with reduced stringiness and reduced stringiness of the stringing material while maintaining the quantitative appearance of the stringiness material unique to natto.
In addition, the present invention provides a stringiness-reduced natto in which the stringing substance is less likely to scatter around, the sticking of the mouth and lips during eating, and the stringing substance does not adhere to clothes or the like.
Furthermore, according to the present invention, there is provided a natto in a container to which a sauce that can further reduce the stringiness can be attached when the natto is eaten and stirred together with the above stringiness-reduced natto.
And among the stringiness-reduced natto bacteria of the present invention, the stringiness-reduced natto bacteria in which the activity of levan-degrading enzyme is reduced can produce natto in which the dispersibility of the stringiness substance is remarkably suppressed. Therefore, according to the present invention, by using the natto bacteria in the production of natto, natto having sufficiently low stringiness can be provided without adding a sauce containing a divalent cation.
Furthermore, according to the present invention, when producing natto using a string-reduced natto bacterium having a reduced activity of levan-degrading enzyme, a predetermined amount of sucrose is added to the steamed soybean, thereby dispersing the string-drawing substance. Natto with further suppressed properties is provided.
以下、本発明を詳細に説明する。
まず、本発明の糸引性低下納豆菌について説明する。
本発明の糸引性低下納豆菌は、請求項１に記載されているように、グルタミン酸ポリペプチドを６ｍｇ／ｇ納豆以上含有し、粘度比が１４ｃｐｓ／ｍｇ以下である納豆を製造できることを特徴とする。
一般に、粘度は、ある物質が溶解している場合に生じ、その物質の濃度がある特定の範囲内の場合、粘度は、その濃度に比例することが知られている。
後述の実施例に示すように、本発明者が納豆の糸引物質抽出液の粘度に関し、グルタミン酸ポリペプチド含有量の異なる納豆において糸引物質抽出液を得て、その粘度を測定したところ、得られる粘度は、グルタミン酸ポリペプチドの含有量に比例していた。このことから、グルタミン酸ポリペプチドの含有量が糸引物質抽出物の粘度に影響を与えていることが分かった。
Hereinafter, the present invention will be described in detail.
First, the stringiness-reduced natto bacteria of the present invention will be described.
As described in claim 1, the stringiness-reduced natto of the present invention is characterized in that natto containing 6 mg / g or more of glutamic acid polypeptide and having a viscosity ratio of 14 cps / mg or less can be produced. .
In general, viscosity is known to occur when a substance is dissolved, and when the concentration of the substance is within a certain range, the viscosity is known to be proportional to the concentration.
As shown in the examples below, the inventor obtained a stringing substance extract in natto with different glutamic acid polypeptide contents and measured the viscosity of the natto stringing substance extract with regard to the viscosity. Was proportional to the content of glutamic acid polypeptide. From this, it was found that the content of glutamic acid polypeptide affects the viscosity of the stringing material extract.
一方、納豆の糸引物質抽出液の粘度の低下のみで糸引物質の飛散の抑制された納豆菌を分離した場合、納豆中のグルタミン酸ポリペプチドの含有量が低下し、その結果、納豆らしい糸引物質の外観が失われる可能性がある。そこで、グルタミン酸ポリペプチドの納豆１ｇ当りの含有量（ｍｇ）で、納豆の糸引物質抽出液の粘度（ｃｐｓ）を割った係数、すなわち粘度比（ｃｐｓ／ｍｇ）も指標の一つとして適用できないかどうか、検討した。
その結果、粘度比が１４ｃｐｓ／ｍｇ以下、好ましくは１４〜４ｃｐｓ／ｍｇ、特に１２〜４ｃｐｓ／ｍｇと粘性物質抽出液の粘度が低下していると共に、納豆としての量的外観を呈するに充分なグルタミン酸ポリペプチドを含有している納豆、すなわち納豆１ｇ当りグルタミン酸ポリペプチドを６ｍｇ以上、好ましくは６〜１２ｍｇ、特に好ましくは８〜１２ｍｇ含有している納豆が糸引性低下納豆として充分量生産することができる糸引性低下納豆菌を取得することができた。
On the other hand, when natto bacteria whose scatter of the stringing material was suppressed only by reducing the viscosity of the natto stringing material extract, the content of glutamic acid polypeptide in natto decreased, and as a result, Appearance may be lost. Therefore, a coefficient obtained by dividing the viscosity (cps) of the natto string-drawing substance extract by the content (mg) per 1 g of natto of glutamic acid polypeptide, that is, the viscosity ratio (cps / mg) is not applicable as one of the indicators. Please consider.
As a result, the viscosity ratio is not more than 14 cps / mg, preferably 14 to 4 cps / mg, particularly 12 to 4 cps / mg, and the viscosity of the viscous substance extract is lowered, and sufficient to exhibit a quantitative appearance as natto. Natto containing glutamic acid polypeptide, that is, natto containing 6 mg or more, preferably 6 to 12 mg, particularly preferably 8 to 12 mg of glutamic acid polypeptide per gram of natto can be produced in a sufficient amount as natto with reduced stringiness. It was possible to obtain natto bacteria with reduced stringiness.
本発明の糸引性低下納豆菌は、親株としての納豆菌を、グルタミン酸ポリペプチドを６ｍｇ／ｇ納豆以上含有し、粘度比が１４ｃｐｓ／ｍｇ以下である納豆を製造できるように育種改良することにより得ることができる。
育種改良の対象である元の納豆菌（親株）としては、特に制限はないが、通常納豆工業で使用されている発酵能力に優れた納豆菌や、自然界から分離取得された納豆菌、およびさらに改良を重ねて得られた優れた納豆菌などを用いるのが望ましい。
納豆菌は、枯草菌バシルス・サチリス（Bacillus subtilis）に分類されているが、糸引物質（粘質物）などの納豆としての特徴を作り出すことができ、納豆発酵での主体をなす細菌であること、また、生育にビオチンを要求するとされるなどの特性を有していることなどから、バシルス・ナットウ（Bacillus natto）として分類されたり、枯草菌の変種としてBacillus subtilis var．nattoあるいはBacillus subtilis（natto）などと枯草菌と区別して分類する場合もある。このような納豆菌としては、Bacillus natto IFO3009株、Bacillus subtilis IFO3335株、同IFO3336株、同IFO3936株、同IFO13169株などがあるほか、各種の納豆菌が広く使用できる。
The stringiness-reduced natto of the present invention is obtained by improving the breeding of natto as a parent strain so that natto containing 6 mg / g or more of glutamic acid polypeptide and having a viscosity ratio of 14 cps / mg or less can be produced. be able to.
There are no particular restrictions on the original natto bacillus (parent strain) that is the subject of breeding improvement, but natto bacillus with excellent fermentative ability usually used in natto industry, natto bacillus isolated and acquired from nature, and It is desirable to use superior natto bacteria obtained through repeated improvements.
Bacillus natto is classified as Bacillus subtilis, but it can produce the characteristics of natto such as stringing substances (sticky material), and is a bacterium that forms the main body in natto fermentation, In addition, since it has characteristics such as requiring biotin for growth, it is classified as Bacillus natto, and Bacillus subtilis var. In some cases, natto or Bacillus subtilis (natto) and the like are distinguished from Bacillus subtilis. Examples of such Bacillus natto include Bacillus natto IFO3009, Bacillus subtilis IFO3335, IFO3336, IFO3936, and IFO13169, and various natto bacteria can be widely used.
具体的には、市販納豆から分離されたＯ２株や、該株の形質転換効率向上性変異株であるｒ２２株（例えば、特開２０００−２２４９８２号公報参照）などが挙げられ、また、市販の納豆種菌である高橋菌（Ｔ３株、東京農業大学菌株保存室）や宮城野菌（宮城野納豆製作所）、ＯＵＶ２３４８１株など各種の納豆菌が適宜使用可能である。
尚、ＯＵＶ２３４８１株は、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寄託されており、その受託番号は、ＦＥＲＭ ＢＰ−６６５９である。
Specific examples include O2 strains isolated from commercial natto and r22 strains (see, for example, JP-A-2000-224982), which are mutants that improve the transformation efficiency of the strains. natto inoculum in Takahashi bacteria (T 3 strains, TUA culture collection chamber) and Miyagino bacteria (Miyagino natto Seisakusho), a variety of Bacillus natto such OUV23481 strain can be suitably used.
The OUV23481 strain is deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and the deposit number is FERM BP-6659.
上記の親株としての納豆菌から、本発明の糸引性低下納豆菌を得るための育種改良法としては、自然界から選抜するいわゆるスクリーニング法や、ニトロソ化合物等を用いた薬剤変異法、紫外線などを用いた物理的変異処理など従来から実施されている方法の他、遺伝子組換え法などを含むあらゆる手段によることが可能である。
育種改良の結果、得られる菌の中から、グルタミン酸ポリペプチドを６ｍｇ／ｇ納豆以上含有し、粘度比が１４ｃｐｓ／ｍｇ以下である納豆を製造できる菌を分離して、本発明の糸引性低下納豆菌を得ることができる。
As a breeding improvement method for obtaining the stringiness-reduced Bacillus natto of the present invention from the above-mentioned parent strain Bacillus natto, a so-called screening method selected from the natural world, a drug mutation method using a nitroso compound, ultraviolet light, etc. are used. In addition to conventional methods such as physical mutation treatment, it is possible to use any means including genetic recombination.
As a result of breeding improvement, among the bacteria obtained, bacteria capable of producing natto having a glutamic acid polypeptide of 6 mg / g natto or more and a viscosity ratio of 14 cps / mg or less are isolated, and the stringiness reduced natto of the present invention is isolated. Bacteria can be obtained.
ここで、納豆のグルタミン酸ポリペプチドの含有量の測定は、例えば、トリクロロ酢酸などの抽出溶媒を用いて抽出して溶液を得て、該溶液からグルタミン酸ポリペプチドを含む抽出液を調製し、該抽出液に所定濃度のセチルメチルアンモニウムブロミド溶液を添加した反応液の吸光度を測定し、グルタミン酸ポリペプチドにて予め作成しておいた検量線で確認する測定法（日本食品科学工学会誌、４２巻、１１号、ｐ．８７８−８８６、１９９５年参照）によることができる。
該測定法の概要を説明すると、以下の通りである。まず、納豆にトリクロロ酢酸を添加して攪拌後、４０〜６０℃に加温して、上清を採取する操作を２回繰り返す。得られる上清に上記のと同様のトリクロロ酢酸を添加して、遠心等により莢雑物を除いてｐＨを中性に調整する。
この溶液に対し、水、エタノールの順に加えて混合し、氷上に放置した後、遠心等により溶液中に含まれるグルタミン酸ポリペプチドを沈殿させる。沈殿したグルタミン酸ポリペプチドに、リン酸ナトリウム緩衝液を溶解させて、抽出液を調製する。
該抽出液に、更にリン酸ナトリウム緩衝液を添加して攪拌、混合後、所定濃度に調製したセチルメチルアンモニウムブロミド溶液を添加し、攪拌混合後、吸光度を測定する。測定した吸光度を、予め精製したグルタミン酸ポリペプチドにて作成した検量線と照合して抽出液中のグルタミン酸ポリペプチド含有量を定量し、こうしてグルタミン酸ポリペプチドの納豆１ｇ当たりの含有量（ｍｇ）を求めることができる。
Here, the measurement of the content of glutamic acid polypeptide in natto is carried out, for example, by extracting using an extraction solvent such as trichloroacetic acid to obtain a solution, preparing an extract containing glutamic acid polypeptide from the solution, and extracting the solution. A method of measuring the absorbance of a reaction solution obtained by adding a cetylmethylammonium bromide solution of a predetermined concentration to the solution and confirming it with a calibration curve prepared in advance with a glutamic acid polypeptide (Japan Food Science and Technology Journal, Vol. 42, 11 No., p. 878-886, 1995).
The outline of the measurement method will be described as follows. First, trichloroacetic acid is added to natto and stirred, and then heated to 40 to 60 ° C. and the supernatant is collected twice. Trichloroacetic acid similar to the above is added to the resulting supernatant, and the pH is adjusted to neutral by removing impurities by centrifugation or the like.
To this solution, water and ethanol are added and mixed in this order, left on ice, and then the glutamic acid polypeptide contained in the solution is precipitated by centrifugation or the like. A sodium phosphate buffer is dissolved in the precipitated glutamic acid polypeptide to prepare an extract.
A sodium phosphate buffer solution is further added to the extract and stirred and mixed, and then a cetylmethylammonium bromide solution prepared to a predetermined concentration is added. After stirring and mixing, the absorbance is measured. The measured absorbance is collated with a calibration curve prepared with a prepurified glutamic acid polypeptide, and the glutamic acid polypeptide content in the extract is quantified. Thus, the glutamic acid polypeptide content per gram of natto (mg) is obtained. be able to.
一方、粘度比とは、納豆の糸引物質抽出液の粘度（ｃｐｓ）を、上記の測定方法で求められるグルタミン酸ポリペプチドの納豆１ｇ当りの含有量（ｍｇ）で割った係数を意味する。
ここで、糸引物質抽出液とは、納豆の糸引物質を水等の溶媒で抽出したものを意味し、例えば、納豆に脱イオン水を添加して攪拌して２０℃で９０分間水抽出を行い、ナイロンシャー等を用いて豆から分離することにより得ることができる。
糸引物質抽出液の粘度の測定は、例えば、上記のようにして得た糸引物質抽出液を、適当なサイズのガラス瓶に入れておいて、東京計器製のＢ型回転式粘度計などを用いて行なうことができる。
On the other hand, the viscosity ratio means a coefficient obtained by dividing the viscosity (cps) of the natto string-drawing substance extract by the content (mg) per 1 g of natto of glutamic acid polypeptide determined by the above-described measurement method.
Here, the stringing substance extract means a natto stringing substance extracted with a solvent such as water. For example, deionized water is added to natto and stirred for 90 minutes at 20 ° C. It can be obtained by separating from beans using a nylon shear or the like.
The viscosity of the stringing material extract can be measured, for example, by placing the stringing material extract obtained as described above in a glass bottle of an appropriate size and using a B-type rotary viscometer manufactured by Tokyo Keiki. Can be done.
本発明者らは、後述の実施例に示すように、バシルス・サチリス（Bacillus subtilis）ＯＵＶ２３４８１株（ＦＥＲＭ ＢＰ−６６５９）を親株として、これをＮ−メチル−Ｎ´−ニトロ−Ｎ−ニトロソグアニジンを用いて突然変異処理し、得られる変異株の中から、グルタミン酸ポリペプチドを６ｍｇ／ｇ納豆以上含有し、粘度比が１４ｃｐｓ／ｍｇ以下である納豆を製造できるものとして、バシルス・サチリス（Bacillus subtilis）Ｄ１８株、Ｄ３２株、Ｄ４０株、Ｄ４３株、Ｄ５０株、Ｄ７７株、Ｄ８３株、Ｄ８４株、Ｄ８６株、Ｄ１０３株、Ｄ１２３株、Ｄ１３８株、及びＤ１６６株の計１３株を得ることができた。これらの変異株から製造された納豆は、いずれも納豆らしい外観を有し、かつ糸引性が低下している納豆であった。
これらの変異株のうち、特に、請求項２に記載されるバシルス・サチリスＤ１０３株は、後述のグルタミン酸ポリペプチドを９ｍｇ／ｇ納豆以上含有し、粘度比が５ｃｐｓ／ｍｇであり、糸引物質の飛散性が充分に抑制された納豆を製造することができる。また、該納豆の糸引性以外の性質（例えば、呈味性）については親株から製造された納豆と同等である。
該納豆菌Ｄ１０３株は、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平成１６年５月１７日付で寄託されており、その受託番号は、ＦＥＲＭ ＢＰ−１００２６である。
As shown in the Examples below, the present inventors have used Bacillus subtilis OUV23481 strain (FERM BP-6659) as a parent strain, which is treated with N-methyl-N′-nitro-N-nitrosoguanidine. Bacillus subtilis (Bacillus subtilis) as a product capable of producing natto containing 6 mg / g natto or more of glutamic acid polypeptide and having a viscosity ratio of 14 cps / mg or less. A total of 13 strains of D18 strain, D32 strain, D40 strain, D43 strain, D50 strain, D77 strain, D83 strain, D84 strain, D86 strain, D103 strain, D123 strain, D138 strain, and D166 strain could be obtained. Natto produced from these mutant strains had a natto-like appearance and reduced stringiness.
Among these mutant strains, in particular, the Bacillus subtilis strain D103 described in claim 2 contains 9 mg / g natto or more of a glutamic acid polypeptide to be described later, has a viscosity ratio of 5 cps / mg, It is possible to produce natto with sufficiently suppressed properties. Moreover, properties (for example, taste) other than the stringiness of the natto are the same as those of natto manufactured from the parent strain.
The Bacillus natto strain D103 has been deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, on May 17, 2004, and its deposit number is FERM BP-10026.
このような本発明の納豆菌は、納豆生産において通常実施されている方法によって利用することができる。
特に、納豆中のグルタミン酸ポリペプチド含有量が親株と同程度以上で、かつ、糸引物質抽出液の粘度が親株より低下している納豆を生産できることから、納豆らしい外観を有し、かつ、糸引物質が飛散しにくい納豆の製造に好適に用いることができる。
このような製造方法により製造された納豆を提供するのが、請求項３に係る本発明である。
Such Bacillus natto of the present invention can be used by a method usually practiced in natto production.
In particular, it can produce natto with a glutamic acid polypeptide content in natto equal to or higher than that of the parent strain, and the viscosity of the stringing material extract is lower than that of the parent strain. Can be suitably used for the production of natto, which is difficult to scatter.
It is the present invention according to claim 3 that provides natto manufactured by such a manufacturing method.
すなわち、請求項３に係る本発明の納豆は、請求項１〜２のいずれかに記載の糸引性低下納豆菌を用いて製造されたことを特徴とする。
納豆は、丸大豆を原料として製造されたいわゆる丸大豆納豆が一般的であるが、一部には予め挽割った大豆を原料とする挽割り納豆もあり、本発明の納豆は、これらのいずれであっても良い。
丸大豆納豆の製造方法は、一般に原料である丸大豆を冷水に十数時間浸漬した後、蒸煮釜で加圧蒸気を用いて加圧蒸煮（０．１５〜０．２ＭＰａ・１２８〜１３３℃）して得られた蒸煮大豆に対して、高温状態（７０〜１００℃）で納豆菌を接種し混合した後、所定の容器に充填してから発酵室に搬入して比較的高温度（４０〜５５℃程度）で所定時間（１２〜４８時間程度）発酵させた後、５℃前後で冷蔵熟成（１２〜７２時間程度）して完成させるのが一般的である。また、挽割り納豆の場合は、予め挽割った大豆を水に浸漬する以外は、通常の丸大豆納豆の場合と同様の方法で製造される。
本発明の納豆は、納豆菌として、上述の請求項１〜２のいずれかに係る本発明の納豆菌を用いるのであれば、このような納豆生産において通常実施されている製造方法により得ることが可能である。
That is, the natto of the present invention according to claim 3 is manufactured using the stringiness-reduced natto bacterium according to any one of claims 1 and 2.
The natto is generally so-called whole soybean natto produced using whole soybeans as a raw material, but there is also some ground soybean natto using soybeans that have been previously ground, and the natto of the present invention is any of these. It may be.
The method for producing whole soybean natto is to immerse the whole soybean, which is the raw material, in cold water for more than 10 hours, and then use the steam in a steaming pot to cook under pressure (0.15-0.2 MPa, 128-133 ° C.) After inoculating and mixing natto bacteria in a high temperature state (70 to 100 ° C.) with respect to the steamed soybean obtained in this manner, it is filled into a predetermined container and then carried into the fermentation chamber (relatively high temperature (40 to 40 ° C.)). Generally, it is fermented at a temperature of about 55 ° C. for a predetermined time (about 12 to 48 hours) and then refrigerated at about 5 ° C. (about 12 to 72 hours) for completion. Further, in the case of ground natto, it is produced in the same manner as in the case of ordinary round soybean natto, except that the previously ground soybean is soaked in water.
The natto of the present invention can be obtained by a production method usually practiced in such natto production if the natto of the present invention according to any one of claims 1 to 2 described above is used as a natto bacterium. Is possible.
このように、請求項３に係る本発明によれば、本発明の納豆菌の特質が生かされた、グルタミン酸ポリペプチドを６ｍｇ／ｇ納豆以上含有し、粘度比が１４ｃｐｓ／ｍｇ以下である納豆が提供される。すなわち、通常の納豆菌が生産した納豆と比べて、納豆中のグルタミン酸ポリペプチド含有量が同程度以上で、かつ、糸引物質抽出液の粘度が低下していることから、納豆らしい外観を有しかつ糸引性が低下しており、喫食性に優れた糸引性低下納豆が得られる。
尚、請求項３に係る本発明の納豆は、糸引性以外の納豆としての品質、特に官能的な品質は、一般的な納豆と同等であり、この点については後述の実施例の結果によっても証明されている。
ここで、納豆の糸引物質の飛散性の評価は以下の方法によることができる。すなわち、納豆５０ｇ対し納豆の通常タレを６ｇ添加し、納豆を攪拌した後、約１０ｇ分の納豆を３０ｃｍ持ち上げて、５秒後に残存している糸の量を対照の納豆と比較して行なうことができる。
Thus, according to the third aspect of the present invention, there is provided a natto having a glutamic acid polypeptide of 6 mg / g or more and a viscosity ratio of 14 cps / mg or less, wherein the characteristics of the natto bacteria of the present invention are utilized. Provided. In other words, compared to natto produced by normal natto bacteria, the glutamic acid polypeptide content in natto is about the same or higher, and the viscosity of the stringing substance extract is reduced, so it has a natto-like appearance. In addition, the stringiness is lowered, and a stringiness-reduced natto excellent in eating properties can be obtained.
In addition, the natto of the present invention according to claim 3 has the same quality as natto other than the stringiness, particularly the sensual quality, which is equivalent to that of general natto. Proven.
Here, the evaluation of the scattering property of the stringing material of natto can be performed by the following method. That is, add 6g of normal natto sauce to 50g of natto, stir the natto, lift about 10g of natto 30cm, and compare the amount of yarn remaining after 5 seconds with the control natto Can do.
さらに、請求項３に係る本発明の納豆の糸引物質の飛散性は、２価カチオンの濃度が特定割合となるように調製されたタレと混合すると、一層抑制することができる。このような納豆とタレとからなる容器入り納豆を提供するのが、請求項４に係る本発明である。
すなわち、請求項４に係る発明は、請求項３記載の容器入り納豆と、２価カチオン濃度が納豆当り０．１〜０．３重量／重量％となるように調製されたタレとからなる容器入り納豆に関する。
Further, the scattering property of the natto stringing material of the present invention according to claim 3 can be further suppressed when mixed with sauce prepared so that the concentration of divalent cations becomes a specific ratio. It is the present invention according to claim 4 that provides such containered natto consisting of natto and sauce.
That is, the invention according to claim 4 is a container comprising natto in a container according to claim 3 and a sauce prepared such that the divalent cation concentration is 0.1 to 0.3 wt / wt% per natto. It is about entering natto.
本発明の容器入り納豆において、タレに使用する２価カチオンとしては、食品に使用可能な２価カチオンを適宜使用することができるが、中でも、請求項５に記載するようにカルシウムやマグネシウムなどを使用することが好ましい。これらの２価カチオンは、塩化物や水酸化物等の形の精製物やにがりなどの他の成分を含有する形で使用してもよい。また、複数の種類の２価カチオンを混合して使用することも可能である。
本発明の容器入り納豆において用いるタレは、２価カチオン濃度が、納豆あたり０．１〜０．３重量／重量％となるように調製されたものであれば、タレを納豆に注入した後の糸引物質の飛散を抑制することができる。
なお、タレの２価カチオン以外の成分として、納豆に注入した後の糸引物質の飛散を抑制するという機能が妨げられない限り、例えば鰹節のだしや、梅肉エキスなど従来から用いられてきた調味成分を好みに応じて添加して、調味機能を同時に果たさせることもできる。
In the natto in the container of the present invention, as the divalent cation used for the sauce, a divalent cation usable for foods can be used as appropriate, and among them, calcium, magnesium, etc. as described in claim 5 can be used. It is preferable to use it. These divalent cations may be used in a form containing other components such as purified products such as chlorides and hydroxides and bitterns. It is also possible to use a mixture of plural kinds of divalent cations.
The sauce used in the container-filled natto of the present invention is prepared so that the divalent cation concentration is 0.1 to 0.3 wt / wt% per natto, after the sauce is injected into the natto. Scattering of the stringing material can be suppressed.
In addition, as a component other than the divalent cation of sauce, as long as the function of suppressing the scattering of the stringing material after being injected into natto is not hindered, for example, the seasoning that has been used conventionally such as bonito soup stock and plum extract Ingredients may be added as desired to perform the seasoning function simultaneously.
通常、納豆は、発泡スチロール製などの納豆容器に所定量ずつ分納されると共に、所定量の調味用タレが納豆容器に添付される場合が殆どであるが、請求項４または請求項５に係る本発明の容器入り納豆も、納豆の重量と糸引物質の飛散を抑制するタレの添付量については、タレに含まれる２価カチオンの濃度が納豆あたり０．１〜０．３重量／重量％となるように調整されることを条件に、同様の形態で市販することができる。 Usually, natto is dispensed into a natto container made of foamed polystyrene or the like in a predetermined amount, and in most cases, a predetermined amount of seasoning sauce is attached to the natto container, but the book according to claim 4 or claim 5 As for the natto in the container of the invention, the amount of divalent cations contained in the sauce is 0.1 to 0.3% by weight / wt% per natto with respect to the weight of the natto and the attached amount of sauce that suppresses the scattering of the stringing material. It can be marketed in the same form on condition that it is adjusted.
また、一方で本発明者らは、本発明の糸引性低下納豆菌のうち、レバン分解酵素の活性が親株と比較して低い納豆菌は、糸引物質の飛散量が十分に抑制された納豆を製造することができることを見出した。このような糸引性低下納豆菌を提供するのが、請求項６に係る本発明である。
すなわち、請求項６に係る本発明の糸引性低下納豆菌は、上記の糸引性低下納豆菌において、更に、レバン分解酵素の活性が０．７Ｕ／ｍｇ以下であることを特徴とする。つまり、グルタミン酸ポリペプチドを６ｍｇ／ｇ納豆以上含有し、粘度比が１４ｃｐｓ／ｍｇ以下である納豆を製造でき、かつ、レバン分解酵素の活性が０．７Ｕ／ｍｇ以下である糸引性低下納豆菌である。
On the other hand, the inventors of the present invention, among the Bacillus natto bacteria with reduced stringiness of the present invention, Bacillus natto with a low levan-degrading enzyme activity compared to the parent strain, It has been found that it can be manufactured. The present invention according to claim 6 provides such a stringiness-reduced natto bacterium.
That is, the stringiness-reduced natto bacteria of the present invention according to claim 6 is characterized in that, in the above-described stringiness-reduced natto bacteria, the activity of levan-degrading enzyme is 0.7 U / mg or less. That is, it is possible to produce natto containing 6 mg / g natto or more of glutamic acid polypeptide, having a viscosity ratio of 14 cps / mg or less, and having a levan-degrading enzyme activity of 0.7 U / mg or less. is there.
請求項６に係る本発明の糸引性低下納豆菌は、レバン分解酵素の活性が親株と比較して低下していることから、レバンの生産量が親株と比べて変化しているため、ショ糖を炭素源に含むプレート上で生育させた時のコロニー形態が親株と異なる。従って、請求項６に係る本発明の糸引性低下納豆菌を分離するにあたっては、該コロニー形態を指標として変異株を選抜する方法などによって、該納豆菌を分離することが可能である。
レバン分解酵素の活性は、例えば次のような方法で測定することができる。すなわち、供試対象である納豆菌（親株及び変異株）をＬＣ培地により培養した後遠心して菌体を除き、培養上清を限外濾過して得られる粗酵素液を、レバンを含有する緩衝液に添加して反応させ、反応中に遊離するフルクトース量を測定し、１分間に１μｍｏｌフルクトースを遊離させるレバン分解酵素活性を１Ｕとする。一方、粗酵素液のタンパク質量を測定し、タンパク質１ｍｇあたりの活性（比活性：Ｕ／ｍｇ）として、レバン分解酵素活性を算出することができる。このような方法で測定された親株のレバン分解酵素活性は、通常、０．７〜１．０Ｕ／ｍｇ程度であることから、請求項６に係る本発明の糸引性低下納豆菌の該酵素の活性は、０．７Ｕ／ｍｇ以下、好ましくは０．５Ｕ／ｍｇ以下、より好ましくは０．２Ｕ／ｍｇ以下とすることができる。
In the stringiness-reduced natto of the present invention according to claim 6, since the activity of levan-degrading enzyme is lower than that of the parent strain, the production amount of levan is changed as compared with the parent strain. Is different from the parental strain when grown on a plate containing carbon in a carbon source. Therefore, when separating the stringiness-reduced natto bacteria of the present invention according to claim 6, it is possible to isolate the natto bacteria by a method of selecting mutants using the colony form as an index.
The activity of levan degrading enzyme can be measured, for example, by the following method. That is, natto bacteria (parent strain and mutant strain) to be tested were cultured in LC medium, centrifuged to remove the cells, and the crude enzyme solution obtained by ultrafiltration of the culture supernatant was used as a buffer containing levan. The amount of fructose released during the reaction is measured by adding to the solution, and the levan-degrading enzyme activity that liberates 1 μmol fructose per minute is defined as 1 U. On the other hand, the amount of protein in the crude enzyme solution is measured, and the activity of levan-degrading enzyme can be calculated as the activity per 1 mg of protein (specific activity: U / mg). Since the Levan-degrading enzyme activity of the parent strain measured by such a method is usually about 0.7 to 1.0 U / mg, the enzyme of the stringiness-reduced natto of the present invention according to claim 6 is used. The activity can be 0.7 U / mg or less, preferably 0.5 U / mg or less, more preferably 0.2 U / mg or less.
本発明者らは、後述の実施例で示すように、バシルス・サチリス（Bacillus subtilis）ＯＵＶ２３４８１株（ＦＥＲＭ ＢＰ−６６５９）から上述のようにして得られた糸引性低下納豆菌バシルス・サチリスＤ１０３（Bacillus subtilis D103）株（ＦＥＲＭ ＢＰ−１００２６）を親株として、これをＮ−メチル−Ｎ´−ニトロ−Ｎ−ニトロソグアニジンを用いて再び突然変異処理し、ショ糖を炭素源として含むプレート上で変異株を約２００株選抜した。
得られた約２００株の変異株の中から、グルタミン酸ポリペプチドを６ｍｇ／ｇ納豆以上含有し、かつ所望の糸引物質の飛散抑制を示した納豆を製造できるものとして、バシルス・サチリス（Bacillus subtilis）ＤＢ３１株、ＤＢ１０３株、ＤＢ１３７株、ＤＣ６６株、ＤＣ７１株及びＤＣ９８株の計６株を得ることができた。
As shown in Examples described later, the present inventors have used the above-mentioned Bacillus subtilis Bacillus subtilis D103 (Bacillus subtilis) OUV23481 strain (FERM BP-6659) obtained as described above. subtilis D103) strain (FERM BP-10026) as a parent strain, which was mutated again with N-methyl-N′-nitro-N-nitrosoguanidine and mutated on a plate containing sucrose as a carbon source About 200 strains were selected.
Among the approximately 200 mutant strains obtained, Bacillus subtilis (Bacillus subtilis) is one that can produce natto containing 6 mg / g natto or more of glutamic acid polypeptide and exhibiting the desired scattering suppression of stringing substances. A total of 6 strains of DB31 strain, DB103 strain, DB137 strain, DC66 strain, DC71 strain and DC98 strain could be obtained.
得られた６株のレバン分解酵素活性を測定したところ、すべての株において、親株であるバシルス・サチリス（Bacillus subtilis）Ｄ１０３株（ＦＥＲＭ ＢＰ−１００２６）や、前記Ｄ１０３株の親株であるバシルス・サチリス（Bacillus subtilis）ＯＵＶ２３４８１株と比較して低下していることが確認された。また、グルタミン酸ポリペプチドを６ｍｇ／ｇ納豆以上含有し、粘度比が１４ｃｐｓ／ｍｇ以下である納豆を作製することができることも確認された。
これらの変異株のうち、特に、請求項７に記載されるバシルス・サチリスＤＣ６６株は、後述するように、グルタミン酸ポリペプチドを１０ｍｇ／ｇ納豆以上含有し、粘度比が４．２ｃｐｓ／ｍｇであり、官能評価により糸引物質の飛散性が充分に抑制された納豆を製造することができる。また、該納豆の糸引性以外の性質（例えば、呈味性）については親株から製造された納豆と同等である。
該納豆菌ＤＣ６６株は、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バシルス・サチリスＤＣ６６（Bacillus subtilis DC66）として２００５（平成１７）年２月１４日付で寄託されており、その受託番号は、ＦＥＲＭ ＢＰ−１０２３４である。
When the levan-degrading enzyme activity of the obtained 6 strains was measured, in all strains, the parent strain Bacillus subtilis D103 strain (FERM BP-10026) and the D103 parent strain Bacillus subtilis It was confirmed that (Bacillus subtilis) OUV23481 strain was decreased. It was also confirmed that natto containing 6 mg / g natto or more of glutamic acid polypeptide and having a viscosity ratio of 14 cps / mg or less can be produced.
Among these mutant strains, in particular, the Bacillus subtilis strain DC66 described in claim 7 contains a glutamic acid polypeptide of 10 mg / g natto or more and a viscosity ratio of 4.2 cps / mg as described later. Thus, it is possible to produce natto in which the stringiness of the material is sufficiently suppressed by sensory evaluation. Moreover, properties (for example, taste) other than the stringiness of the natto are the same as those of natto manufactured from the parent strain.
The Bacillus natto strain DC66 was deposited on February 14, 2005 as Bacillus subtilis DC66 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center. , FERM BP-10234.
このような請求項６又は請求項７に係る本発明の納豆菌は、納豆生産において通常実施されている方法によって利用することができる。特に、レバン分解酵素の活性が親株と比較して低下していることから、２価カチオンを含まない通常の納豆のタレとの組み合わせでも、糸引物質の飛散量が十分に抑制された納豆の製造に好適に用いることができる。
このような製造方法により製造された納豆を提供するのが、請求項８に係る本発明である。
Such Bacillus natto of the present invention according to Claim 6 or Claim 7 can be used by a method usually practiced in natto production. In particular, since the activity of levan-degrading enzyme is lower than that of the parent strain, the production of natto in which the amount of string-drawing material is sufficiently suppressed even in combination with the usual natto sauce that does not contain divalent cations. Can be suitably used.
The present invention according to claim 8 provides natto manufactured by such a manufacturing method.
すなわち、請求項８に係る本発明の納豆は、請求項６または請求項７に記載の糸引性低下納豆菌を用いて製造されたことを特徴とする。
納豆については、請求項６または請求項７に記載の糸引性低下納豆菌を用いることを除き、請求項３に係る本発明の納豆において説明したのと同様であり、丸大豆納豆および挽割り納豆のいずれであっても良い。
また、その製造方法も、納豆菌として請求項６または請求項７に記載の糸引性低下納豆菌を用いるのであれば、納豆生産において通常実施されている製造方法により得ることが可能である。
That is, the natto of the present invention according to claim 8 is manufactured by using the stringiness-reduced natto bacterium according to claim 6 or claim 7.
The natto is the same as that described in the natto of the present invention according to claim 3, except that the stringiness-reduced natto bacteria according to claim 6 or 7 is used. Either may be sufficient.
Moreover, if the stringing property fallen natto bacteria of Claim 6 or Claim 7 are used also as the natto bacterium, the manufacturing method can be obtained by the manufacturing method currently normally implemented in natto production.
一方、請求項９に係る本発明のように、蒸煮大豆あたり０．２〜２．０重量／重量％となるようにショ糖を添加して発酵させて製造することにより、糸引物質の量がより少ない納豆を生産することができる。すなわち、請求項６または請求項７に記載の糸引性低下納豆菌を用いて納豆を製造する際に、蒸煮大豆にショ糖を０．２〜２．０重量／重量％となるように添加して上記納豆菌に発酵させることにより、納豆の糸引物質の飛散性が一層抑制された納豆を得ることができる。
ここで、ショ糖の添加量は、蒸煮大豆あたり０．２〜２．０重量／重量％とする必要がある。ショ糖の添加量が０．２重量／重量％よりも少ないと、糸引物質の飛散性を抑制する効果が不充分となり、逆に２．０重量／重量％よりも高濃度でショ糖を添加すると、糸引物質の量が少ない納豆が製造されるので、好ましくない。
このような方法で製造された納豆の糸引物質の飛散性は、以下の方法で評価した場合に、ショ糖を添加しない場合の糸引物質の飛散性に比較して、２〜３割程度低下する。
なお、ここで納豆の糸引物質の飛散性の評価は、納豆５０ｇ対し納豆の通常タレを６ｇ添加し、納豆を攪拌した後、約１０ｇ分の納豆を箸を用いて、約３０ｃｍ持ち上げ、そこで生じた糸が切れるまでの時間を測定することによって行なうことができる。
On the other hand, as in the present invention according to claim 9, by adding sucrose and fermenting it so that it becomes 0.2 to 2.0 wt / wt% per steamed soybean, the amount of the stringing substance is reduced. Less natto can be produced. That is, when producing natto using the stringiness-reduced natto bacteria according to claim 6 or 7, sucrose is added to the steamed soybean so as to be 0.2 to 2.0% by weight / weight. By fermenting to the natto bacteria, natto in which the scattering properties of the stringing material of natto is further suppressed can be obtained.
Here, the addition amount of sucrose needs to be 0.2-2.0 weight / weight% per steamed soybean. If the amount of sucrose added is less than 0.2 wt / wt%, the effect of suppressing the stringiness of the stringing material becomes insufficient, and conversely sucrose is added at a concentration higher than 2.0 wt / wt%. Then, since natto with a small amount of stringing material is produced, it is not preferable.
The scattering property of the stringing material of natto manufactured by such a method is reduced by about 20 to 30% compared to the scattering property of the stringing material when sucrose is not added, when evaluated by the following method. .
Here, the evaluation of the natto stringing material scattering property was made by adding 6g of normal natto sauce to 50g of natto, stirring the natto, and lifting about 10g of natto using chopsticks, about 30cm. This can be done by measuring the time until the chopped yarn breaks.
以下に、本発明を実施例により具体的に説明する。
実施例１（グルタミン酸ポリペプチド含有量と糸引物質抽出液の粘度との関係）
納豆中の糸引物質の主成分として知られているグルタミン酸ポリペプチドの含有量と糸引物質抽出液の粘度の関係を調べた。
すなわち、ＯＵＶ２３４８１株（ＦＥＲＭ ＢＰ−６６５９）を親株として用いて発酵室の温度を３６℃〜４２℃、及び発酵時間を１６時間〜２２時間に変化させることによりグルタミン酸ポリペプチドの含有量の異なる納豆を製造した。
Hereinafter, the present invention will be specifically described by way of examples.
Example 1 (Relationship between glutamic acid polypeptide content and viscosity of stringing substance extract)
The relationship between the content of glutamic acid polypeptide, known as the main component of stringing substances in natto, and the viscosity of the stringing substance extract was investigated.
That is, by using OUV23481 strain (FERM BP-6659) as a parent strain, changing the temperature of the fermentation chamber to 36 ° C. to 42 ° C., and changing the fermentation time from 16 hours to 22 hours, natto with different glutamic acid polypeptide contents was obtained. Manufactured.
納豆の製造は以下の方法により行なった。
極小大豆を水道水で４℃、１晩浸漬し、圧力蒸煮釜で蒸気圧０．２ＭＰａで８分間蒸煮した。このようにして調製した蒸煮大豆に対して、上記の親株の胞子液を滅菌水で１００倍に希釈し、蒸煮大豆１００ｇあたり０．８ｍｌ植菌し、所定の発酵温度及び所定の発酵時間で発酵した。
発酵後、４℃で２４時間保存して、納豆を製造した。製造した納豆について、グルタミン酸ポリペプチド含有量及び糸引物質抽出液の粘度を測定した。
Natto was manufactured by the following method.
Ultra-small soybeans were soaked in tap water at 4 ° C. overnight, and steamed in a pressure steamer at a steam pressure of 0.2 MPa for 8 minutes. For the cooked soybean thus prepared, the spore solution of the above parent strain is diluted 100-fold with sterilized water, inoculated 0.8 ml per 100 g of cooked soybean, and fermented at a predetermined fermentation temperature and a predetermined fermentation time. did.
After fermentation, it was stored at 4 ° C. for 24 hours to produce natto. About manufactured natto, glutamic acid polypeptide content and the viscosity of the stringing-substance extract were measured.
納豆中のグルタミン酸ポリペプチドの含有量の測定については、日本食品科学工学会誌、４２巻、１１号、ｐ．８７８−８８６、１９９５年記載の方法に従い、以下の手順で行なった。
まず、上記のようにして製造した納豆１０ｇに、２０ｍｌの２．５％トリクロロ酢酸を添加して、１分間攪拌後、５０℃で１０分間温めて、上清を５０ｍｌメスフラスコに移した。この納豆に、２０ｍｌの２．５％トリクロロ酢酸を再び添加して、５０℃で１０分間温めて、上清を上記メスフラスコに移した。２．５％トリクロロ酢酸にて５０ｍｌにメスアップした。
調製した溶液について、１２０００ｒｐｍで２０分間遠心して莢雑物を除き、２０ｍｌ上清を測り取りとった。これを、１Ｎの水酸化ナトリウムにてｐＨを７．０に調整して２５ｍｌのメスフラスコに移し、水で２５ｍｌにメスアップした。溶液を５ｍｌ測り取り、２０ｍｌの９９．５％エタノールを加えて混合させ、氷上に１０分間放置して、１２０００ｒｐｍ、１０分間遠心して、溶液中に含まれるグルタミン酸ポリペプチドを沈殿させた。
The measurement of the content of glutamic acid polypeptide in natto is described in Japanese Journal of Food Science and Technology, Vol. 42, No. 11, p. In accordance with the method described in 878-886, 1995, the following procedure was performed.
First, 20 ml of 2.5% trichloroacetic acid was added to 10 g of natto produced as described above, stirred for 1 minute, warmed at 50 ° C. for 10 minutes, and the supernatant was transferred to a 50 ml volumetric flask. To this natto, 20 ml of 2.5% trichloroacetic acid was added again, warmed at 50 ° C. for 10 minutes, and the supernatant was transferred to the volumetric flask. The volume was made up to 50 ml with 2.5% trichloroacetic acid.
The prepared solution was centrifuged at 12000 rpm for 20 minutes to remove contaminants, and 20 ml supernatant was measured. This was adjusted to 7.0 with 1N sodium hydroxide, transferred to a 25 ml volumetric flask, and made up to 25 ml with water. 5 ml of the solution was weighed, 20 ml of 99.5% ethanol was added and mixed, left on ice for 10 minutes, and centrifuged at 12,000 rpm for 10 minutes to precipitate the glutamic acid polypeptide contained in the solution.
沈殿したグルタミン酸ポリペプチドに、２０ｍｌの２０ｍＭ リン酸ナトリウム緩衝液（ｐＨ７．０）を溶解させて、抽出液を調製した。その後、０．５ｍｌの抽出液に、２ｍｌの２０ｍＭリン酸ナトリウム緩衝液（ｐＨ７．０）を添加して攪拌混合後、１Ｍ 塩化ナトリウム水溶液にて調製した０．１Ｍ セチルメチルアンモニウムブロミド溶液を０．５ｍｌ添加した。これを攪拌混合した後、４００ｎｍの吸光度を測定した。
予め、精製したグルタミン酸ポリペプチドにて作成した検量線にて、糸引物質中のグルタミン酸ポリペプチド含有量を定量し、グルタミン酸ポリペプチドの納豆１ｇ当たりの含有量（ｍｇ）を求めた。
20 ml of 20 mM sodium phosphate buffer (pH 7.0) was dissolved in the precipitated glutamic acid polypeptide to prepare an extract. Thereafter, 2 ml of 20 mM sodium phosphate buffer (pH 7.0) was added to 0.5 ml of the extract, and after stirring and mixing, 0.1 M cetylmethylammonium bromide solution prepared with 1 M sodium chloride aqueous solution was added to 0. 5 ml was added. After stirring and mixing this, the absorbance at 400 nm was measured.
The glutamic acid polypeptide content in the stringing material was quantified in advance using a calibration curve prepared with a purified glutamic acid polypeptide, and the glutamic acid polypeptide content per gram of natto (mg) was determined.
一方、糸引物質抽出液の粘度の測定については、以下の手順で行なった。
納豆５０ｇ当り脱イオン水２００ｍｌを添加してよく攪拌、３０分毎によく攪拌して２０℃で９０分間糸引物質の水抽出を行った後、ナイロンシャーを用いて豆と糸引物質抽出液を分離した。その後、調製した糸引物質抽出液について、直径５ｃｍ、高さ１４ｃｍのガラス瓶においてＢ型回転式粘度計（東京計器社製）にて粘度（ｃｐｓ）を測定した。
納豆中のグルタミン酸ポリペプチドの含有量と糸引物質抽出液の粘度との関係を、図１に示した。
On the other hand, the viscosity of the stringing material extract was measured according to the following procedure.
Add 200 ml of deionized water per 50 g of natto, stir well, stir well every 30 minutes, and after 90 minutes water extraction of stringing material at 20 ° C., separate the beans and stringing material extract using nylon shear did. Thereafter, the viscosity (cps) of the prepared string drawing material extract was measured with a B-type rotary viscometer (manufactured by Tokyo Keiki Co., Ltd.) in a glass bottle having a diameter of 5 cm and a height of 14 cm.
The relationship between the content of glutamic acid polypeptide in natto and the viscosity of the stringing substance extract is shown in FIG.
図１の結果から、グルタミン酸ポリペプチドの含有量が増加するにつれて、糸引物質抽出液の粘度が高くなることが明らかになると共に、グルタミン酸ポリペプチドの含有量と糸引物質抽出液の粘度との間には相関性があることが明らかになった。
しかし、糸引性低下納豆菌を分離するための指標を、糸引物質抽出液の粘度の低下のみとした場合、グルタミン酸ポリペプチドの含有量が低下する結果、納豆らしい糸引物質の外観が失われた納豆を生産するものしか選抜できない可能性がある。
そこで、下記の実施例２では、糸引物質抽出液の粘度（ｃｐｓ）を納豆１ｇ中のグルタミン酸ポリペプチドの含有量（ｍｇ）で割って得られる値（すなわち、粘度比）を、糸引性低下納豆菌を分離する際の指標の一つとして設定した場合に、糸引物質抽出液の粘度が低下した納豆を製造できる納豆菌が分離することができるかどうかについて検討した。
From the results of FIG. 1, it is clear that the viscosity of the stringing material extract increases as the glutamic acid polypeptide content increases, and between the glutamic acid polypeptide content and the viscosity of the stringing material extract. Were found to be correlated.
However, if the index for separating natto bacillus with reduced stringiness is only reduced in the viscosity of the extract of the stringing material, the content of glutamic acid polypeptide will decrease, resulting in a loss of the appearance of the stringing material that seems to be natto. There is a possibility that only those that produce can be selected.
Therefore, in Example 2 below, the value (that is, the viscosity ratio) obtained by dividing the viscosity (cps) of the yarn-drawing substance extract by the content (mg) of glutamic acid polypeptide in 1 g of natto is reduced to the yarn-drawing-reduced natto. We examined whether natto bacteria capable of producing natto with reduced viscosity of the stringing material extract could be isolated when set as one of the indicators for separating the fungus.
実施例２（糸引性低下納豆菌の分離）
（１）突然変異株の取得
納豆菌の培養にはＮＢ培地を用い、常法に従って実施した。ＯＵＶ２３４８１株（以下ＯＵＶ２３４８１株又は親株ということもある）を、一晩３７℃で振とう培養した後、遠心分離して滅菌生理食塩水で洗浄した。
ＮＢ培地の組成を以下の表１に示す。
Example 2 (Separation of reduced stringiness natto bacteria)
(1) Acquisition of Mutant Strains Natto bacillus was cultured according to a conventional method using NB medium. The OUV23481 strain (hereinafter sometimes referred to as OUV23481 strain or parent strain) was cultured with shaking at 37 ° C. overnight, and then centrifuged and washed with sterile physiological saline.
The composition of the NB medium is shown in Table 1 below.
この約１０７ｃｆｕ／ｍｌの濃度の洗浄菌体に、２０ｍｇ／ｌとなるようにＮ−メチル−Ｎ´−ニトロ−Ｎ−ニトロソグアニジンを添加し、３０分間振とうして、変異処理を行った。この時の生存率は２％前後であった。
変異処理した菌体を上記ＮＢ培地で培養して形質を発現させ、その後に滅菌生理食塩水で洗浄し、ＧＳ培地に塗抹した。
ＧＳ培地の組成を以下の表２に示す。
N-methyl-N′-nitro-N-nitrosoguanidine was added to the washed cells having a concentration of about 10 7 cfu / ml to a concentration of 20 mg / l, and the mixture was shaken for 30 minutes for mutation treatment. It was. The survival rate at this time was around 2%.
The mutated bacterial cells were cultured in the above NB medium to express their traits, and then washed with sterilized physiological saline and smeared on the GS medium.
The composition of the GS medium is shown in Table 2 below.
ＧＳ培地にて３７℃で１晩培養した後、コロニーの特性から糸引物質の生産が確認できた約２００株を取得した。ここで得られた約２００株について、常法に従い、胞子液を調製した。
すなわち、親株または得られた約２００株を表１に示したＮＢ培地に１白菌耳植菌し、３７℃、１晩培養後、表３に示す胞子形成培地に１％植菌し、３７℃、２４時間培養して胞子液を調製した。
胞子形成培地の組成を以下の表３に示す。
After culturing overnight at 37 ° C. in a GS medium, about 200 strains in which production of stringing substances could be confirmed from the characteristics of the colonies were obtained. About 200 strains obtained here were prepared according to a conventional method.
That is, the parent strain or about 200 strains obtained were inoculated with 1 white fungus ear in the NB medium shown in Table 1, cultured at 37 ° C. overnight, and then inoculated with 1% in the spore-forming medium shown in Table 3. A spore solution was prepared by culturing at 24 ° C. for 24 hours.
The composition of the sporulation medium is shown in Table 3 below.
（２）納豆の試作、評価
以下、それぞれの胞子液を種菌として使用し、実施例１記載の方法に従って納豆を試作した。試作した納豆について、糸引性が低下していると評価できる１９株を二次的に選択し、以下さらに詳細な検定を行った。
即ち、上記と同様にして、選択された１９株と親株ＯＵＶ２３４８１株の胞子液を作製し、納豆を試作して、納豆１ｇ中のグルタミン酸ポリペプチドの含有量、糸引物質の量、粘度比、及び糸引物質の飛散性の各項目について評価した。
なお、グルタミン酸ポリペプチドの含有量の測定については実施例１と同様にし、粘度比については、実施例１と同様にして測定した糸引物質抽出液の粘度（ｃｐｓ）を、上記グルタミン酸ポリペプチドの含有量（ｍｇ）で割って得られる値として算出した。
(2) Trial production and evaluation of natto Hereinafter, each spore solution was used as an inoculum, and natto was produced according to the method described in Example 1. Of the natto prototypes, 19 strains that could be evaluated as having reduced stringiness were secondarily selected, and the following detailed tests were conducted.
That is, in the same manner as described above, a spore solution of the selected 19 strains and the parent strain OUV23481 strain was prepared, and natto was produced as a prototype. The content of glutamic acid polypeptide in 1 g of natto, the amount of stringing substance, the viscosity ratio, and Each item of stringing material scattering was evaluated.
The content of glutamic acid polypeptide was measured in the same manner as in Example 1, and the viscosity ratio was measured in the same manner as in Example 1 by measuring the viscosity (cps) of the stringing material extract. It was calculated as a value obtained by dividing by the amount (mg).
糸引物質の量の評価は、各株を用いて試作された納豆を攪拌し、そこで生じる糸引物質の量を観察して、対照である親株を用いて試作された納豆と比較した。親株で製造した納豆よりも糸引物質の量が多い場合は○、同程度の場合は△、少ない場合は×、と評価した。 The amount of the stringing substance was evaluated by stirring the natto prototyped using each strain and observing the amount of stringing substance produced there, and comparing it with the natto prototyped using the parent strain as a control. When the amount of the stringing material was larger than that of the natto produced with the parent strain, it was evaluated as ◯, when the amount was the same as △, and when it was less, it was evaluated as ×.
糸引物質の飛散性の評価は、以下の方法によった。すなわち、納豆５０ｇに対して納豆の通常タレ（醤油３０重量％、味りん５重量％、かつおエキス１０重量％、食塩５重量％、水５０重量％）６ｇを添加し、攪拌した後、約１０ｇの納豆を３０ｃｍ持ち上げて、５秒後に残存している糸の量を対照の納豆と比較して行なった。糸引物質の飛散性は、対照としたＯＵＶ２３４８１株で作製した納豆よりも残存している糸量が明らかに少ない場合は○、やや少ない場合は△、同程度か若しくは多い場合は×、まったくない場合は◎というように４段階で評価した。
以上の評価結果を表４に示した。
The evaluation of the scattering property of the stringing material was based on the following method. That is, after adding 6 g of normal natto sauce (30% by weight of soy sauce, 5% by weight of taste phosphorus, 10% by weight of bonito extract, 5% by weight of salt and 50% by weight of water) to 50 g of natto and stirring, about 10 g The natto was lifted 30 cm and the amount of yarn remaining after 5 seconds was compared with the control natto. When the amount of yarn remaining is clearly smaller than that of natto prepared with the OUV23481 strain used as a control, the stringiness of the yarn-drawing substance is ○, when it is slightly less, △, when it is the same or more, ×, Was evaluated on a four-point scale.
The above evaluation results are shown in Table 4.
表４の結果から、グルタミン酸ポリペプチドの含有量が増加するにつれて、官能評価における糸引物質の量も増加し、グルタミン酸ポリペプチドの量が官能評価における糸引物質の量と相関関係にあることが確認された。
また、納豆の糸引物質の量が親株と同程度以上とされる納豆は、納豆１ｇ当り６ｍｇ以上であり、また、糸引物質の飛散性が有意に低下している納豆の粘度比は１４ｃｐｓ／ｍｇ以下となることが確認でき、このような条件にあてはまる糸引性低下納豆を生産する納豆菌変異株として、バシルス・サチリス（Bacillus subtilis）Ｄ１８株、Ｄ３２株、Ｄ４０株、Ｄ４３株、Ｄ５０株、Ｄ７７株、Ｄ８３株、Ｄ８４株、Ｄ８６株、Ｄ１０３株、Ｄ１２３株、Ｄ１３８株、及びＤ１６６株の合計１３株が分離できた。
From the results of Table 4, it was confirmed that as the content of glutamic acid polypeptide increased, the amount of stringing material in sensory evaluation also increased, and the amount of glutamic acid polypeptide correlated with the amount of stringing material in sensory evaluation. It was.
In addition, the amount of natto string-drawing substance is about 6 mg or more per gram of natto, and the viscosity ratio of natto, in which the stringiness of the natto is significantly reduced, is 14 cps / mg. As Bacillus subtilis D18 strain, D32 strain, D40 strain, D43 strain, D50 strain, D77 strain as Bacillus subtilis mutants producing Bacillus subtilis natto that meets the above conditions can be confirmed. A total of 13 strains of the strain, D83 strain, D84 strain, D86 strain, D103 strain, D123 strain, D138 strain, and D166 strain could be isolated.
これら１３株の納豆菌のうち、バシルス・サチリスＤ１０３（Bacillus subtilis D103）株（以下、Ｄ１０３株と称する場合もある）が生産した納豆は、最も低い粘度比を示し、親株の１／４以下であった。また、グルタミン酸ポリペプチドの含有量についても親株より多かった。更に、糸引物質の飛散性の評価については、親株が×であったのに対しＤ１０３株は○であったことから、親株よりも糸引物質の飛散性が抑制されることが確認できた。
なお、グルタミン酸ポリペプチド含量が多くかつ糸引物質の飛散性が抑制され、かつ粘度比の低下した納豆を生産することが確認されたＤ１０３株は、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バシルス・サチリスＤ１０３（Bacillus subtilis D103）として寄託されている（ＦＥＲＭ ＢＰ−１００２６）。
Of these 13 strains of natto, natto produced by the Bacillus subtilis D103 strain (hereinafter also referred to as the D103 strain) has the lowest viscosity ratio and is less than 1/4 of the parent strain. there were. Also, the content of glutamic acid polypeptide was higher than that of the parent strain. Furthermore, as for the evaluation of the dispersibility of the stringing material, the parent strain was x, whereas the D103 strain was ◯, so that it was confirmed that the dispersibility of the stringing material was suppressed more than that of the parent strain.
The D103 strain, which was confirmed to produce natto with a high glutamic acid polypeptide content, reduced stringiness of the stringing material, and a reduced viscosity ratio, was registered with the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. It has been deposited as Bacillus subtilis D103 (FERM BP-10026).
そこで、Ｄ１０３株で製造した納豆について、糸引物質の飛散性以外の品質、すなわち、菌膜の厚さ、豆色、香り及び熟度に関し、親株で製造した納豆との官能評価を行なったところ、各評価において違いが見られなかった。
このことから、Ｄ１０３株は、糸引物質の飛散が抑制されるとともに、納豆として必要な品質をも具備している糸引性低下納豆を生産することができ、糸引性低下納豆菌として有用であることが確認できた。
Therefore, for natto produced with the D103 strain, the sensory evaluation with the natto produced with the parent strain was performed on the quality other than the dispersibility of the stringing material, that is, the thickness, bean color, aroma and maturity of the fungal membrane, There was no difference in each evaluation.
From this fact, the strain D103 is capable of producing stringiness-reduced natto that has the necessary quality as natto, and is useful as a stringiness-reduced natto bacterium, while scattering of the stringing substance is suppressed. Was confirmed.
実施例３（糸引性低下納豆の糸切れを低下させるタレの組成の検討）
納豆を食する場合、ほとんどの場合において、タレをかけて食する。そこで、タレに含まれる塩類が、Ｄ１０３株で製造した糸引性低下納豆の糸引物質の飛散への影響を調べた。
納豆の糸引物質抽出液に２価カチオンを添加したところ、該抽出液の粘度が低下したことから、納豆に添加するタレに２価カチオンを含有させて納豆に添加した場合、糸引物質の飛散がより抑制されることが期待された。
そこで、納豆に２価カチオンを含有するタレを添加して、糸引物質の飛散性の官能評価を行なった。
Example 3 (Examination of the composition of the sauce that reduces thread breakage of natto with reduced stringiness)
When you eat natto, in most cases you eat it with sauce. Therefore, the influence of the salts contained in the sauce on the scattering of the stringiness substance of the stringiness-reduced natto produced by the strain D103 was examined.
When divalent cations were added to the natto string drawing material extract, the viscosity of the extract decreased, and when the sauce added to natto was mixed with divalent cations and added to natto, the stringing material was scattered. Expected to be more restrained.
Therefore, a sauce containing a divalent cation was added to natto, and sensory evaluation of the dispersibility of the stringing material was performed.
すなわち、納豆の通常タレ（醤油３０重量％、味りん５重量％、かつおエキス１０重量％、食塩５重量％、水５０重量％）６ｇに、２価カチオンであるカルシウムまたはマグネシウムを表６及び表７に示す種々の濃度で溶解して、各種のタレを調製した。
Ｄ１０３株を用い実施例１記載の方法に従って製造した納豆に、上記の各種のタレを納豆５０ｇに対して６ｇ添加して、糸引物質の飛散性の評価を実施例２記載の方法に従って行なった。一方、対照として、Ｄ１０３株に代えて親株（ＯＵＶ２３４８１株）を用いた他は同様に製造した納豆について、上記の各種のタレを添加した。
実施例２と同様な方法で、納豆の糸引物質の飛散性を評価すると同時に、各納豆の試食を行い、それぞれ○、△、×の３段階にて官能評価（呈味性）を行なった。官能評価の基準は、まったく苦味を感じない場合は○、やや苦味を感じる場合は△、はっきり苦味を感じる場合は×と評価した。
カルシウムを添加した場合の結果を表５に、マグネシウムを添加した場合の結果を表６に、それぞれ示す。
That is, 6 g of normal natto sauce (soy sauce 30% by weight, taste phosphorus 5% by weight, bonito extract 10% by weight, salt 5% by weight,
6 g of the above-mentioned various sauces were added to 50 g of natto to natto produced according to the method described in Example 1 using the D103 strain, and the scattering property of the stringing material was evaluated according to the method described in Example 2. On the other hand, as a control, various sauces described above were added to natto produced in the same manner except that the parent strain (OUV23481 strain) was used instead of the D103 strain.
In the same manner as in Example 2, the natto stringing substance scattering property was evaluated, and at the same time, each natto was sampled and subjected to sensory evaluation (taste) in three stages of ◯, Δ, and X, respectively. The criteria for sensory evaluation were evaluated as ○ when no bitterness was felt, Δ when slightly bitter, and × when slightly bitter.
Table 5 shows the results when calcium was added, and Table 6 shows the results when magnesium was added.
表５及び表６に示すように、カルシウムまたはマグネシウムを、納豆当り０．１重量／重量％以上になるようにタレに含有させて添加した場合、それ以下の濃度の場合と比較して納豆の糸引物質の飛散性がより強く抑制されることが分かった。
しかし、呈味性については、カルシウムやマグネシウムを納豆当り０．４重量／重量％以上になるように納豆に添加した場合は、納豆を食した場合において、苦味を感じることが分かり、好ましくないことが分かった。
このことから、タレに含まれる２価カチオンとして、カルシウムやマグネシウムが好ましいこと、及び２価カチオンの濃度は、納豆あたり０．１〜０．３重量／重量％となるように調製されることにより、呈味性への影響を与えずに納豆の糸引物質の飛散性を抑制することができることが明らかとなった。
As shown in Table 5 and Table 6, when calcium or magnesium is added to the sauce so as to be 0.1 wt / wt% or more per natto, the concentration of natto is lower than that of the lower concentration. It was found that the dispersibility of the stringing material was more strongly suppressed.
However, with regard to taste, when calcium or magnesium is added to natto so that it is 0.4 wt / wt% or more per natto, it can be seen that bitterness is felt when eating natto, which is not preferable. I understood.
From this, calcium and magnesium are preferable as the divalent cation contained in the sauce, and the concentration of the divalent cation is adjusted to be 0.1 to 0.3 wt / wt% per natto. It has been clarified that the scattering properties of the stringing material of natto can be suppressed without affecting the taste.
実施例４（レバンの分解活性及び合成活性が変化している納豆菌の分離）
（１）突然変異株の取得
レバンの合成及び分解が変化している菌株を取得するための親株として、実施例２で取得した納豆菌バシルス・サチリスＤ１０３（Bacillus subtilis D103）株（以下Ｄ１０３株又は親株ということもある：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 ＢＰ−１００２６として国際寄託されている）を用いた。
Ｄ１０３株を、実施例２記載の方法によりＮ−メチル−Ｎ´−ニトロ−Ｎ−ニトロソグアニジンを用いて化学変異処理した。この時の生存率は４％前後であった。
Example 4 (Separation of Bacillus natto in which the decomposition and synthesis activities of levan are changed)
(1) Acquisition of mutant strain As a parent strain for acquiring a strain in which the synthesis and degradation of levan is changed, the Bacillus subtilis D103 strain (hereinafter referred to as D103 strain or It is sometimes referred to as a parent strain: FERM BP-10026 is internationally deposited at the National Institute of Advanced Industrial Science and Technology (AIST).
The D103 strain was chemically mutated using N-methyl-N′-nitro-N-nitrosoguanidine by the method described in Example 2. The survival rate at this time was around 4%.
変異処理した菌体を、実施例２のＮＢ培地（表１）で培養して形質を発現させ、その後に滅菌生理食塩水で洗浄し、１０％ショ糖を含むＮＢ培地プレート（寒天１．５％含有）に塗抹した。
３７℃で１日間培養した後、親株のコロニーと比較し、コロニー表面に親株と異なる特徴を示すコロニーを約２００株取得した。ここで得られた約２００株について、実施例２に記載の方法に従い、胞子液を調製した。
The mutated bacterial cells are cultured in the NB medium (Table 1) of Example 2 to express the traits, and then washed with sterilized physiological saline, followed by NB medium plate containing 10% sucrose (agar 1.5 % Containing).
After culturing at 37 ° C. for 1 day, about 200 colonies having characteristics different from those of the parent strain were obtained on the surface of the colony as compared to the parent strain colonies. About 200 strains obtained here, a spore solution was prepared according to the method described in Example 2.
（２）納豆の試作
上記の（１）で調製した各胞子液を種菌として使用し、実施例１に記載の方法に従って納豆を作製し、糸引物質の飛散性について評価した。その結果、２００株の納豆菌変異株の中から、親株Ｄ１０３株の納豆よりも糸引物質の飛散性が親株より低下した納豆を生産できるものとして、バシルス・サチリス（Bacillus subtilis）ＤＢ３１、ＤＢ１０３、ＤＢ１３７、ＤＣ６６、ＤＣ７１、ＤＣ９８株の計６株を得ることができた。
(2) Trial production of natto Using each spore solution prepared in the above (1) as an inoculum, natto was produced according to the method described in Example 1, and the scattering property of the stringing substance was evaluated. As a result, among the 200 strains of Bacillus natto mutants, Bacillus subtilis DB31, DB103, DB137 can be produced that can produce natto in which the dispersibility of the stringing material is lower than that of the parent strain D103. , DC66, DC71, and DC98 were obtained in total.
（３）納豆の糸引き物質の飛散性評価及びグルタミン酸ポリペプチド含量測定
実施例１記載の方法に従ってＯＵＶ２３４８１株を用いて作製した納豆を対照とし、上記（２）で得られた６株及びそれらの親株（Ｄ１０３株）で作製した納豆について、納豆の糸引物質の飛散性評価、粘度比及びグルタミン酸ポリペプチド含量の測定を行った。
また、後述の方法に従い、レバン分解酵素活性についても測定した。
なお、糸引物質の飛散性については、納豆５０ｇ当り２価カチオンを添加していない納豆の通常タレ６ｇを添加、攪拌して、実施例２（２）記載の方法に従い糸引物質の飛散性を評価した。また、粘度比、及びグルタミン酸ポリペプチド含量については、実施例１に記載の方法に従って、納豆１ｇ当りのグルタミン酸ポリペプチド含量の測定を行なった。
それらの結果を表７に示した。
(3) Evaluation of scattering properties of natto stringing substance and measurement of glutamic acid polypeptide content Using the natto prepared using OUV23481 strain according to the method described in Example 1, as a control, 6 strains obtained in (2) above and those About natto produced in the parent strain (D103 strain), the scattering property evaluation, viscosity ratio, and glutamic acid polypeptide content of the natto stringing material were measured.
Further, levan-degrading enzyme activity was also measured according to the method described later.
In addition, as for the dispersibility of the stringing material, 6 g of normal natto sauce without adding a divalent cation per 50 g of natto was added and stirred, and the dispersibility of the stringing material was evaluated according to the method described in Example 2 (2). did. Moreover, about the viscosity ratio and glutamic acid polypeptide content, according to the method as described in Example 1, the glutamic acid polypeptide content per 1 g of natto was measured.
The results are shown in Table 7.
表７の結果から、（１）で得られる６つの菌株を用いて作製した納豆は、いずれもグルタミン酸ポリペプチド含量が６ｍｇ／ｇ納豆を超え、粘度比が１４ｃｐｓ／ｍｇ以下であった他、糸引物質の飛散性においても親株Ｄ１０３と比較して優れていた。中でも、ＤＣ６６株は、グルタミン酸ポリペプチド含量が顕著に多く、粘度比も非常に低く、かつ糸引物質の飛散性も明らかに低いことが分かる。
また、この結果からＤＣ６６株の納豆は２価カチオンを添加してない納豆の通常タレでも糸引き物質の飛散性が明らかに低くなることが分かった。
From the results in Table 7, natto produced using the six strains obtained in (1) all had a glutamic acid polypeptide content exceeding 6 mg / g natto and a viscosity ratio of 14 cps / mg or less. It was also superior to the parent strain D103 in terms of substance scattering. Among them, it can be seen that the DC66 strain has a significantly high glutamic acid polypeptide content, a very low viscosity ratio, and a clearly low scattering property of the stringing material.
Further, from this result, it was found that natto of the DC66 strain had a clearly reduced scattering property of the stringing substance even in the normal sauce of natto to which no divalent cation was added.
（４）レバン分解酵素活性測定
以下の方法によって、糸引き性の低下した納豆を生産できる納豆菌のレバン分解酵素活性が、ＯＵＶ２３４８１株に比べて低下していることを確認することができた。
すなわち、選抜した６株の糸引き性の低下した納豆菌及びそのＯＵＶ２３４８１株を、ＬＣ培地（１０ｇ／Ｌ ｔｒｙｐｔｏｎｅ、５ｇ／Ｌ ｙｅａｓｔ ｅｘｔｒａｃｔ、５ｇ／Ｌ ＮａＣｌ、１ｇ／Ｌ ショ糖）により培養した。ＯＤ６６０で調べた濁度が１．６になるまで培養し、遠心して菌体を除き、培養上清を採取した。
採取した培養上清を、ＭＩＣＲＯＣＯＮ ＹＭ−３（ミリポア社製）で限外濾過することにより、培養液中に含まれるタンパクを５倍程度濃縮し、これを粗酵素液とした。
(4) Levan-degrading enzyme activity measurement It was confirmed by the following method that the Levan-degrading enzyme activity of Bacillus natto capable of producing natto with reduced stringiness was lower than that of OUV23481 strain.
That is, the selected 6 strains of Bacillus natto with reduced stringiness and OUV23481 strain thereof were cultured in LC medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 1 g / L sucrose). Culturing was carried out until the turbidity determined by OD660 reached 1.6, centrifugation was performed to remove the cells, and the culture supernatant was collected.
The collected culture supernatant was subjected to ultrafiltration with MICROCON YM-3 (manufactured by Millipore) to concentrate the protein contained in the culture solution about 5 times, and this was used as a crude enzyme solution.
この粗酵素液０．１ｍｌを、３％レバン含有５０ｍＭリン酸カリウム緩衝液（ｐＨ６．０）０．５ｍｌに添加して、３７℃で６０分間反応させた。
反応中に遊離するフルクトース量をＦキット Ｄグルコース／果糖（ロッシュ製）で測定した。１分間に１μｍｏｌフルクトースを遊離させるレバン分解酵素活性を１Ｕ（酵素単位）として、レバン分解酵素活性を測定した。レバン分解酵素活性は、粗酵素液のタンパク量をｐｒｏｔｅｉｎ ａｓｓａｙ（Ｂｉｏ Ｒａｄ社製）で測定し、たんぱく質１ｍｇあたりの活性（比活性）として表した。結果を表７に示す。
0.1 ml of this crude enzyme solution was added to 0.5 ml of 50 mM potassium phosphate buffer (pH 6.0) containing 3% levan, and reacted at 37 ° C. for 60 minutes.
The amount of fructose released during the reaction was measured with F kit D glucose / fructose (Roche). The levan-degrading enzyme activity was measured with the levan-degrading enzyme activity that liberates 1 μmol fructose per minute as 1 U (enzyme unit). Levan degrading enzyme activity was expressed as activity (specific activity) per 1 mg of protein by measuring the amount of protein in the crude enzyme solution with protein assay (manufactured by Bio Rad). The results are shown in Table 7.
表７に示すように、前記（１）で得られる６つの菌株は、いずれもレバン分解酵素活性が親株と比較して低かった。特に、前記（３）において、特に優れた結果が得られたＤＣ６６株については、レバン分解酵素活性が他の菌株に比べて顕著に低下していることが明らかである。
以上の結果から、レバン分解活性が低下している納豆菌で作製した納豆において、糸引物質の飛散性が低下していることが分かった。
なお、グルタミン酸ポリペプチド含量が多くかつ糸引物質の飛散性が抑制され、最もレバン分解酵素活性が低下していたＤＣ６６株は、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バシルス・サチリスＤＣ６６（Bacillus subtilis DC66）として２００５（平成１７）年２月１４日付で寄託されており、その受託番号は、ＦＥＲＭ ＢＰ−１０２３４である。
As shown in Table 7, all of the six strains obtained in (1) had lower levan-degrading enzyme activity than the parent strain. In particular, in (3) above, regarding the DC66 strain that obtained particularly excellent results, it is clear that the levan-degrading enzyme activity is significantly reduced compared to other strains.
From the above results, it was found that in the natto produced with Bacillus natto having reduced levan degradation activity, the dispersibility of the stringing substance was lowered.
The DC66 strain, which has a high glutamic acid polypeptide content, has reduced stringiness, and has the lowest levan-degrading enzyme activity, has been transferred to the National Institute of Advanced Industrial Science and Technology Patent Biodeposition Center, Bacillus subtilis DC66 ( Bacillus subtilis DC66) was deposited on February 14, 2005, and the accession number is FERM BP-10234.
（５）納豆の品質評価
ＤＣ６６株を用いて発酵を行った場合、Ｄ１０３株を用いて発酵した場合と比べて、発酵時間の遅延、発酵中の納豆品温の低下等は見られなかった。このことから、レバン分解活性の低下は、納豆菌の生育に影響を及ぼさないことが分かった。
また、ＤＣ６６株及びＤ１０３株のそれぞれから作製された納豆における、糸引物質の飛散性以外の官能検査の結果を、表８に示した。
(5) Quality evaluation of natto When fermenting using DC66 strain, compared with the case of fermenting using D103 strain, delay of fermentation time, decrease in natto product temperature during fermentation, etc. were not seen. From this, it was found that the decrease in levan decomposition activity does not affect the growth of Bacillus natto.
In addition, Table 8 shows the results of sensory tests other than the stringiness of the natto produced from each of the DC66 strain and the D103 strain.
表８に示すように、親株（Ｄ１０３株）と変異株（ＤＣ６６株）との間で、納豆の官能評価に関しては全く違いが見られなかった。
このことから、ＤＣ６６株を種菌に用いて作製した納豆は、納豆として必要な品質を具備していることを確認することができた。
As shown in Table 8, there was no difference in the sensory evaluation of natto between the parent strain (D103 strain) and the mutant strain (DC66 strain).
From this, it was possible to confirm that natto produced using the DC66 strain as an inoculum had the quality required for natto.
実施例５（糸の飛散性へのショ糖添加発酵の影響）
実施例４において、レバン分解酵素活性が低下した株の生産する納豆は糸引物質の飛散性が低下していることが確認できたが、この結果から、糸引き物質の飛散性にレバンが大きな影響を与えていることが予想された。
そこで、レバンの合成酵素の基質であるショ糖を蒸煮大豆に添加してＤＣ６６株で発酵させた場合に、糸引物質の飛散性がどのように変化するかを調べた。
Example 5 (Effect of sucrose-added fermentation on yarn dispersibility)
In Example 4, it was confirmed that the natto produced by the strain having a reduced levan-degrading enzyme activity had a reduced scattering property of the stringing material. From this result, levan has a great influence on the scattering property of the stringing material. Was expected to give.
Therefore, it was investigated how the scattering property of the stringing material changes when sucrose, which is a substrate for Levan's synthase, is added to steamed soybeans and fermented with strain DC66.
すなわち、実施例１記載の方法に従って蒸煮大豆を調製し、調製した蒸煮大豆に５０％ショ糖水溶液を添加して、蒸煮大豆あたりのショ糖含量が表９に示す量になるように添加し、実施例１に記載の方法に従ってＤＣ６６株の胞子液を植菌し、発酵、及び熟成を行なった。
このようにして製造された納豆について、実施例１記載の方法に従いグルタミン酸ポリペプチド含量を測定し、実施例２記載の方法に従い糸引物質の量を評価するとともに糸引物質の飛散性の評価も行なった。
なお、糸引物質の飛散性の評価は、以下の方法によった。すなわち、納豆５０ｇ対し納豆の通常タレを６ｇ添加し、納豆を攪拌した後、約１０ｇ分の納豆を、箸を用いて約３０ｃｍ持ち上げ、そこで生じた糸が切れるまでの時間を測定し、これを３回繰り返した平均値を求めた。以上の評価結果を表９に示した。
That is, cooked soybeans were prepared according to the method described in Example 1, 50% aqueous sucrose solution was added to the prepared cooked soybeans, and the sucrose content per cooked soybean was added to the amount shown in Table 9, According to the method described in Example 1, the spore solution of DC66 strain was inoculated, fermented and matured.
For the natto produced in this way, the glutamic acid polypeptide content was measured according to the method described in Example 1, the amount of stringing material was evaluated according to the method described in Example 2, and the scattering property of the stringing material was also evaluated. .
In addition, the following method evaluated the scattering property of the stringing substance. That is, add 6g of normal natto sauce to 50g of natto, stir the natto, lift about 10g of natto using chopsticks, and measure the time until the resulting thread breaks. The average value repeated three times was determined. The above evaluation results are shown in Table 9.
表９の結果から、発酵前に添加するショ糖の量を増加させるにつれて、糸引物質が切れ易くなり、特にショ糖を０．２重量／重量％以上添加した場合には糸引物質の飛散性が大幅に低下することが分かる。一方、蒸煮大豆あたりショ糖を２．５重量／重量％添加した場合、グルタミン酸ポリペプチド含量が低下し、糸引物質の量も減少することが明らかになった。
このことから、蒸煮大豆にショ糖を添加して発酵させることにより糸引物質の飛散性を低下できることが確認された。
このことから、納豆を製造するにあたり、糸引性低下納豆菌を用いると共に、蒸煮大豆あたり０．２〜２．０重量／重量％になるようにショ糖を添加することにより、糸引物質の量を低下させず、かつ糸引物質の飛散性の抑制された納豆を得ることができることが明らかとなった。
From the results of Table 9, as the amount of sucrose added before fermentation increases, the stringing material becomes more easily cut, and in particular, when sucrose is added in an amount of 0.2% by weight or more, the dispersibility of the stringing material increases. It turns out that it falls significantly. On the other hand, when 2.5% w / w sucrose was added per steamed soybean, it was revealed that the glutamic acid polypeptide content was reduced and the amount of stringing substance was also reduced.
From this, it was confirmed that the dispersibility of the stringing substance can be reduced by adding sucrose to fermented steamed soybean and fermenting it.
From this, in producing natto, while using natto bacteria with reduced stringiness, and adding sucrose to 0.2-2.0 wt / wt% per steamed soybean, It has become clear that it is possible to obtain natto that does not decrease and in which the dispersibility of the stringing material is suppressed.
本発明により、納豆特有の糸引物質の量的外観は保持しつつ、糸引性が低下し、糸引物質の飛散性が抑制された納豆を充分量生産できる糸引性低下納豆菌が提供される。
また、本発明により、周りに糸引物質が飛散しにくく、喫食時に口中や唇のベタつきが生じたり、また糸引物質が衣服などに付着したりすることのない糸引性低下納豆が提供される。
さらに、本発明により、上記糸引性低下納豆と共に、該納豆を食する際に加えて攪拌すると、糸引性を更に低下させることのできるタレを添付させた容器入り納豆が提供される。
そして、本発明の糸引性低下納豆菌のうち、特にレバン分解酵素の活性が低下している糸引性低下納豆菌は、糸引物質の飛散性が顕著に抑制された納豆を生産することができる。従って、本発明によれば、該納豆菌を納豆の製造に用いることにより、２価カチオンを含むタレを添加しなくとも十分に糸引性の低い納豆が提供される。
さらにまた、本発明によれば、レバン分解酵素の活性が低下している糸引低下納豆菌を用いて納豆を製造する際に、蒸煮大豆にショ糖を所定量添加することにより、糸引物質の飛散性が一層抑制された納豆が提供される。
The present invention provides a stringiness-reduced natto bacterium that can produce a sufficient amount of natto with reduced stringiness and reduced stringiness of the stringing material while maintaining the quantitative appearance of the stringiness material unique to natto.
In addition, the present invention provides a stringiness-reduced natto in which the stringing substance is less likely to scatter around, the sticking of the mouth and lips during eating, and the stringing substance does not adhere to clothes or the like.
Furthermore, according to the present invention, there is provided a natto in a container to which a sauce that can further reduce the stringiness can be attached when the natto is eaten and stirred together with the above stringiness-reduced natto.
And among the stringiness-reduced natto bacteria of the present invention, the stringiness-reduced natto bacteria in which the activity of levan-degrading enzyme is reduced can produce natto in which the dispersibility of the stringiness substance is remarkably suppressed. Therefore, according to the present invention, by using the natto bacteria in the production of natto, natto having sufficiently low stringiness can be provided without adding a sauce containing a divalent cation.
Furthermore, according to the present invention, when producing natto using a string-reduced natto bacterium having a reduced activity of levan-degrading enzyme, a predetermined amount of sucrose is added to the steamed soybean, thereby dispersing the string-drawing substance. Natto with further suppressed properties is provided.
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