JP6245541B1 - Natto bacteria that produce nucleic acid-based umami ingredients in natto, a method for producing natto with high content of nucleic acid-based umami ingredients, and products thereof - Google Patents
Natto bacteria that produce nucleic acid-based umami ingredients in natto, a method for producing natto with high content of nucleic acid-based umami ingredients, and products thereof Download PDFInfo
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Abstract
【課題】核酸系うま味成分によるうま味が強く、同時に「被りの厚さ」、「糸の弾力」の劣化がない納豆発酵技術を提供する。【解決手段】納豆発酵の過程で納豆中に核酸系うまみ成分（アデニル酸）を産生させる特性と、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を劣化させることがない特性を備えた納豆菌、該納豆菌を使用して納豆発酵を行うことによる核酸系うま味成分高含有納豆の製造方法、及び納豆発酵の過程で納豆中に核酸系うま味成分を産生させた納豆であって、納豆に対するアデニル酸の含量が７ｍｇ／１００ｇ以上であり、該アデニル酸による強いうま味を呈する核酸系うま味成分高含有納豆。【効果】核酸系うま味成分（アデニル酸）によるうま味を呈する核酸系うま味成分高含有納豆を製造し、提供することができる。【選択図】図２The present invention provides a natto fermentation technology that has a strong umami taste due to a nucleic acid-based umami component, and at the same time, does not deteriorate “thickness of covering” and “elasticity of yarn”. [Solution] Degradation of natto fermented nucleic acid component (adenylic acid) in natto fermentation process, and basic quality characteristics of natto, "cover thickness" and "yarn elasticity" Natto bacteria with unique characteristics, a method for producing natto with a high content of nucleic acid umami ingredients by fermenting natto using the natto bacteria, and producing nucleic acid umami ingredients in natto during the natto fermentation process A natto with a high content of nucleic acid-based umami components, wherein the adenylic acid content relative to natto is 7 mg / 100 g or more and exhibits a strong umami taste due to the adenylic acid. [Effect] It is possible to produce and provide natto with a high content of nucleic acid-based umami component that exhibits umami taste due to a nucleic acid-based umami component (adenylic acid). [Selection] Figure 2
Description
本発明は、核酸系うま味成分を産生する特性を有する納豆菌、該納豆菌を使用して核酸系うま味成分（アデニル酸）を高い含有量で含有する核酸系うま味成分高含有納豆の製造方法、及びその製品に関するものであり、更に詳しくは、うま味を呈することが知られている核酸系うま味成分（アデニル酸）を従来の納豆よりも高い含有量で含有する納豆を製造することが可能な納豆菌、及び該納豆菌により、核酸系うま味成分（アデニル酸）が多く含まれ、従来の納豆とは異なる、うま味を強化した核酸系うま味成分高含有納豆を製造する方法並びにその製品に関するものである。 The present invention relates to a method for producing natto having a high content of nucleic acid-based umami components containing a high content of nucleic acid-based umami components (adenylic acid) using the natto bacteria, which has the property of producing nucleic acid-based umami components, And more specifically, it is possible to produce natto containing a nucleic acid-based umami component (adenylic acid), which is known to exhibit umami, at a higher content than conventional natto. The present invention relates to a method for producing natto with a high content of nucleic acid-based umami components, which contains a large amount of nucleic acid-based umami components (adenylic acid) and has enhanced umami, and a product thereof. .
すなわち、本発明は、グアニンアナログ耐性をもつ納豆菌であって、核酸系うま味成分のアデニル酸、グアニル酸を合計８ｍｇ／１００ｇ以上含み、強いうま味を呈する納豆を製造することを可能とする、核酸系うま味成分（アデニル酸）を納豆発酵の過程で納豆中に産生する納豆菌、該納豆菌を使用して核酸系のうま味成分（アデニル酸）を７ｍｇ／１００ｇ以上含み、強いうま味を呈する核酸系うま味成分高含有納豆を製造する方法及びその納豆製品に関する新技術・新製品を提供するものである。 That is, the present invention is a natto bacterium having resistance to guanine analogs, which comprises a total of 8 mg / 100 g or more of nucleic acid-based umami components adenylic acid and guanylic acid, and makes it possible to produce natto that exhibits a strong umami taste. Natto bacteria that produce umami components (adenylic acid) in natto during the natto fermentation process, nucleic acid systems that use nucleic acid-based umami components (adenylic acid) at least 7 mg / 100 g and exhibit a strong umami taste The present invention provides a method for manufacturing natto with a high content of umami ingredients, and new technologies and products related to the natto product.
近年、食の嗜好性の多様化に伴い、納豆においても、世代や地域性、季節性などを考慮した様々な商品が求められてきており、納豆業界においては、原料大豆や容器、納豆製品やその製造方法といった面で独自の技術を用いた研究／開発が種々なされてきている。納豆菌においても、各社独自の納豆菌株を保有し、各社独自の納豆の製造技術や商品の開発に利用されてきている。 In recent years, with the diversification of food preferences, various products that take generation, regionality, seasonality, etc. into consideration have been demanded for natto. In the natto industry, raw soybeans, containers, natto products, Various researches / developments using unique technologies have been made in terms of manufacturing methods. As for Bacillus natto, each company has its own natto strains, which have been used to develop their own natto manufacturing technology and products.
これまで、先行技術として、例えば、納豆特有のムレ臭を軽減して嗜好性を高めることを可能にした納豆菌や納豆製品に関する技術（特許文献１）、が報告されている。この技術は、ロイシンデヒドロゲナーゼの酵素活性を低下ないし失活せしめて短鎖分岐脂肪酸生産能が低下ないし消失した納豆菌を用いて、嗜好性を高めた、ムレ臭のないムレ臭非感納豆を製造することを特徴としている。しかし、この文献では、核酸系うま味成分や、その嗜好性、風味などについては何も言及されていない。 So far, for example, a technology relating to natto bacteria and natto products (patent document 1) that has made it possible to reduce the stuffy smell peculiar to natto and enhance its palatability has been reported. This technology produces natto odor-insensitive natto with no odor of odor, using natto bacteria that have reduced or inactivated the enzymatic activity of leucine dehydrogenase to reduce or eliminate the ability to produce short-chain branched fatty acids. It is characterized by doing. However, this document does not mention anything about the nucleic acid-based umami component, its palatability and flavor.
また、他の先行技術として、納豆の味について、グルタミン酸を高産生するグルタミン酸高生産性納豆菌及びそれを用いて作られるうま味成分（グルタミン酸）の含有量を増加させたグルタミン酸高含有納豆（特許文献２）、が報告されている。これは、グルタミン酸高産生能を有する納豆菌を使用することにより、グルタミン酸高含有納豆を製造し、アミノ酸系のうま味が一段と増強された納豆を提供することを特徴とするものである。しかし、グルタミン酸などのアミノ酸系うま味成分は、納豆に元から多く含まれている成分であるため、明確な嗜好性や風味などの差別化は難しいのが実情であった。 As another prior art, with regard to the taste of natto, glutamic acid-rich natto with increased glutamic acid-producing natto bacteria and glutamic acid-rich natto (glutamate) produced using the same (patent document) 2), has been reported. This is characterized by producing natto with a high glutamic acid content by using natto bacteria having high glutamic acid production ability and providing natto with further enhanced amino acid-based umami. However, since amino acid-based umami components such as glutamic acid are components that are originally contained in natto, it is difficult to differentiate between distinct palatability and flavor.
また、他の先行技術として、原料大豆に由来するラフィノース、スタキオースなどのオリゴ糖成分を高含有させた大豆オリゴ糖高含有納豆製品の製造技術（特許文献３）、が報告されている。この技術は、原料大豆又は添加したオリゴ糖に由来するショ糖、ラフィノース及びスタキオースのうち少なくともショ糖の分解を抑制して、それらのオリゴ糖を高含有させた納豆を製造することを特徴とするものである。しかし、この文献でも、核酸系うま味成分や、その嗜好性、風味などについては何も言及されていない。 In addition, as another prior art, there has been reported a technology for producing a soybean oligosaccharide-containing natto product with a high content of oligosaccharide components such as raffinose and stachyose derived from raw soybean (Patent Document 3). This technology is characterized by producing natto containing a high content of oligosaccharides by suppressing decomposition of at least sucrose among raw soybeans or sucrose, raffinose and stachyose derived from added oligosaccharides. Is. However, this document does not mention anything about the nucleic acid-based umami component, its palatability, and flavor.
また、他の先行技術として、選択培地としてＬ−プロリン、Ｌ−アルギニン、各種芳香族アミノ酸などのアミノ酸アナログ含有培地やプリン系アナログ含有培地などを使用して、これらのアミノ酸高分泌性の納豆菌や、イノシン及びイノシン酸高分泌性の納豆菌を使用した納豆の製造方法（特許文献４）、が報告されている。しかし、この文献には、イノシン、イノシン酸高含有納豆を製造する方法について記載されているが、本発明のようなイノシン酸を含まないアデニル酸高含有納豆の製造方法については何も言及されていない。 In addition, as other prior art, a medium containing amino acid analogs such as L-proline, L-arginine and various aromatic amino acids, a medium containing purine analogs, and the like are used as a selective medium. In addition, a method for producing natto using inosine and inocinic acid highly secreted natto bacteria (Patent Document 4) has been reported. However, this document describes a method for producing inosine and natto with a high content of inosinate, but does not mention anything about a method of producing a natto with a high content of adenylate that does not contain inosine as in the present invention. Absent.
また、他の先行技術として、本発明者は、甘味成分として知られる、グルコースをより高含有させることが可能なグルコース高含有納豆菌（特許文献５）、を開発し、特許出願している。この技術は、グルコースを資化しない又はその資化能力が市販の納豆菌を含む従来の納豆菌よりも低い納豆菌を使用して納豆発酵を行うことにより、納豆に特徴的な糸引き性を劣化させることなく、原料大豆由来のグルコースを残存させた納豆を提供するものである。該納豆は、納豆に対するグルコースの含量が１００ｍｇ／１００ｇ以上であり、該グルコースによる甘味性を呈することを特徴としている。 In addition, as another prior art, the present inventor has developed and has applied for a patent for a high glucose-containing Bacillus natto (Patent Document 5), which is known as a sweetening component and can contain a higher amount of glucose. This technology does not assimilate glucose or has a stringing property characteristic of natto by performing natto fermentation using natto bacteria that have lower assimilability than conventional natto bacteria, including commercially available natto bacteria. The present invention provides natto in which glucose derived from raw soybeans remains without deteriorating. The natto has a glucose content of 100 mg / 100 g or more with respect to natto and is characterized by sweetness due to the glucose.
また、従来から行われている納豆製造では、発酵や熟成の温度や長さによって、納豆に含まれるアミノ酸含有量を増やし、グルタミン酸などのアミノ酸系のうま味成分を多くすることが可能である。しかし、このような納豆製造技術では、その発酵や熟成の過程で、苦味や酸味などを感じる他のアミノ酸も同時に増えてしまうため、雑味が強くなってしまい、納豆製品の美味しさのバランスが損なわれてしまうという問題があった。 In addition, in conventional natto production, it is possible to increase the content of amino acids contained in natto and increase amino acid-based umami components such as glutamic acid depending on the temperature and length of fermentation and ripening. However, in such natto manufacturing technology, other amino acids that feel bitter and sour in the process of fermentation and ripening also increase at the same time, which increases the miscellaneous taste and balances the taste of natto products. There was a problem of being damaged.
以上詳述したように、これまでの先行技術を考慮しても、納豆の特徴である強い糸引き性と、バランスの良いうま味や風味、嗜好性などを満足させることができる納豆を開発することは難しい状況にあり、当技術分野においては、アミノ酸系のうま味に加え、核酸系のうま味を納豆中に産生する納豆菌や該納豆菌による核酸系うま味成分と他のうま味成分がバランス良く含まれた、核酸系うま味成分によるうま味や風味、嗜好性をバランス良く満たすことが可能な新しい納豆の製法を実現する新しい納豆発酵技術を開発することが強く要請されていた。 As described in detail above, to develop natto that can satisfy natto's strong stringiness and well-balanced umami, flavor, taste, etc. In this technical field, in addition to amino acid-based umami, natto bacteria that produce nucleic acid-based umami in natto, and nucleic acid-based umami components produced by the natto and other umami components are included in a balanced manner. In addition, there has been a strong demand for the development of a new natto fermentation technology that realizes a new natto manufacturing method that can satisfy a good balance of umami, flavor, and palatability with nucleic acid-based umami ingredients.
このような状況の中で、本発明者らは、上記従来技術に鑑みて、納豆において、核酸系うま味成分（アデニル酸）を強化した納豆を開発することを目標として鋭意研究／開発を積み重ねた結果、従来の納豆よりも、核酸系うま味成分（アデニル酸）によるうま味が強く、同時に、苦味や、雑味成分の増加による品質劣化や品質低下を抑えた新しい納豆発酵技術を確立することに成功し、本発明を完成するに至った。 Under such circumstances, the present inventors have conducted intensive research / development with the goal of developing natto with enhanced nucleic acid-based umami components (adenylic acid) in natto in view of the above-described conventional technology. As a result, the umami due to the nucleic acid-based umami component (adenylic acid) is stronger than conventional natto, and at the same time, we succeeded in establishing a new natto fermentation technology that suppresses quality deterioration and deterioration due to increased bitterness and miscellaneous components. Thus, the present invention has been completed.
本発明は、納豆において、うま味を呈することが知られている核酸系うま味成分（アデニル酸）を従来の納豆製品よりも高い含有量で含有させることを可能にした、グアニンアナログに耐性をもち、８−アザグアニンを含む最小寒天培地で生育することが可能な特性と、納豆発酵の過程で納豆中に核酸系うま味成分（アデニル酸）を産生させる特性と、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を通常の納豆と比べて劣化させることがない特性を備えたことを特徴とする納豆菌、該納豆菌を使用して核酸系うま味成分高含有納豆を製造する方法、及びその納豆製品を提供することを目的とするものである。
尚、本発明において、「核酸系うま味成分（アデニル酸）」とは、核酸系うま味成分のアデニル酸を意味する表現として用いたものである。
The present invention, in natto, has a nucleic acid-based umami component (adenylic acid) known to exhibit umami in a higher content than conventional natto products, and has resistance to guanine analogs, It is a characteristic that can grow on a minimum agar medium containing 8-azaguanine, a characteristic that a nucleic acid-based umami component (adenylic acid) is produced in natto during the natto fermentation, and a basic quality characteristic of natto. A natto bacterium characterized in that it does not deteriorate the thickness of the cover and the elasticity of the thread compared to ordinary natto, and a natto bacterium with a high content of nucleic acid-based umami components using the natto bacterium. It aims at providing the manufacturing method and its natto product.
In the present invention, “nucleic acid-based umami component (adenylic acid)” is used as an expression meaning nucleic acid-based umami component, adenylic acid.
上記課題を解決するための本発明は、以下の技術的手段から構成される。
（１）グアニンアナログに耐性をもち、８−アザグアニンを含む最小寒天培地で生育することが可能な特性と、納豆発酵の過程で納豆中に核酸系うま味成分（アデニル酸）を産生させる特性と、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を通常の納豆と比べて劣化させることがない特性とを備えた納豆菌であって、
前記納豆菌が、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２１株（受託番号 ＮＩＴＥ Ｐ−０２３８０）、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２２株（受託番号 ＮＩＴＥ Ｐ−０２３８１）から選択された菌株であることを特徴とする納豆菌。
（２）前記（１）に記載の納豆菌を使用して核酸系うま味成分高含有納豆を製造する方法であって、
原料の大豆から蒸煮大豆を調製する工程と、納豆発酵の過程で、この蒸煮大豆に、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高く、８−アザグアニンを含む最小寒天培地で生育することが可能な納豆菌を接種して蒸煮大豆を発酵させる工程と、発酵させた蒸煮大豆を熟成させる工程とを備え、
納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を通常の納豆と比べて劣化させることなく、核酸系うま味成分（アデニル酸）によるうま味を呈する納豆を製造することを特徴とする核酸系うま味成分高含有納豆の製造方法。
（３）核酸系うま味成分（アデニル酸）が納豆に含まれている核酸系うま味成分高含有納豆であって、
前記核酸系うま味成分（アデニル酸）が納豆発酵の過程で納豆中に産生されたものであり、通常の納豆と比べて、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」の劣化がなく、納豆発酵が、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２１株（受託番号 ＮＩＴＥ Ｐ−０２３８０）、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２２株（受託番号 ＮＩＴＥ Ｐ−０２３８１）から選択された菌株による発酵であり、該株菌を含み、納豆に対するアデニル酸の含量が７ｍｇ／１００ｇ以上であり、該アデニル酸によるうま味を呈することを特徴とする核酸系うま味成分高含有納豆。
（４）納豆に対するアデニル酸、グアニル酸の含量が合計８ｍｇ／１００ｇ以上である、前記（３）に記載の核酸系うま味成分高含有納豆。
（５）納豆発酵の過程で納豆中に産生された核酸系うま味成分（アデニル酸）と、納豆に含まれるアミノ酸系うま味成分（グルタミン酸）とのうま味の相乗効果により増強されたうま味を呈する納豆である、前記（３）又は（４）記載の核酸系うま味成分高含有納豆。
（６）納豆発酵の過程で納豆中に産生された核酸系うま味成分（アデニル酸）を含み、ナットウキナーゼによる血栓溶解活性を有する、前記（３）−（５）のいずれか一項に記載の核酸系うま味成分高含有納豆。
The present invention for solving the above-described problems comprises the following technical means.
(1) a characteristic that is resistant to guanine analogs and capable of growing on a minimum agar medium containing 8-azaguanine, and a characteristic that produces a nucleic acid-based umami component (adenylic acid) in natto during the natto fermentation process; It is a natto bacterium that has the basic quality characteristics of natto, such as "thickness of covering" and "elasticity of thread", which does not deteriorate compared to normal natto,
Bacillus subtilis TTCC2221 strain (Accession number NITE P-02380) and Bacillus subtilis TTCC2222 strain (Accession number NITE P-02381).
(2) A method for producing natto with a high content of nucleic acid-based umami components using the natto bacteria described in (1) above,
In the process of preparing cooked soybeans from raw soybeans and in the process of natto fermentation, this cooked soybean has a higher ability to resist guanine analogues than conventional natto bacteria, including commercially available natto bacteria, and the minimum containing 8-azaguanine Inoculating natto bacteria capable of growing on an agar medium and fermenting the steamed soybean, and aging the fermented steamed soybean,
Manufacture natto with umami taste due to nucleic acid-based umami ingredients (adenylic acid) without degrading the basic quality characteristics of natto, “thickness of covering” and “elasticity of yarn” compared to normal natto A method for producing natto containing a high content of nucleic acid-based umami components.
(3) A nucleic acid-based umami component-rich natto containing a nucleic acid-based umami component (adenylic acid) in natto,
The nucleic acid-based umami component (adenylic acid) is produced in natto during the process of natto fermentation. Compared to normal natto, the basic quality characteristics of natto are “thickness of covering”, “thread of elasticity "there is no deterioration of, paid bean fermentation, Bacillus subtilis TTCC2221 strain (accession number NITE P-02380), a fermentation with selected strains from Bacillus subtilis TTCC2222 strain (accession number NITE P-02381), strain Natto containing a high nucleic acid-based umami component , which contains a bacterium, has an adenylic acid content of 7 mg / 100 g or more with respect to natto, and exhibits an umami taste due to the adenylic acid.
(4) The high content of nucleic acid umami component natto according to (3), wherein the total content of adenylic acid and guanylic acid relative to natto is 8 mg / 100 g or more.
(5) Natto with enhanced umami due to the synergistic effect of the umami taste of the nucleic acid-based umami component (adenylic acid) produced in the natto fermentation process and the amino acid-based umami component (glutamic acid) contained in natto Natto with high content of nucleic acid-based umami component according to (3) or (4).
(6) a natto fermentation process in produced nucleic acid-based umami ingredients in natto (adenylate), to have a thrombolytic activity by nattokinase, wherein (3) - according to any one of (5) Natto with high content of umami components.
次に、本発明について更に詳細に説明する。
本発明は、核酸系うま味成分高含有納豆を製造するために使用する納豆菌であって、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高く、８−アザグアニンを含む最小寒天培地で生育可能な株を選抜して分離した選抜株であり、納豆発酵の過程で納豆中に核酸系うま味成分（アデニル酸）を産生させる特性と、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を劣化させることがない特性を備えたことを特徴とする納豆菌に係るものである。ここで、８−アザグアニン耐性をもつ納豆について説明すると、本発明では、２０μｇ／ｍｌ８−アザグアニンを含む最小寒天培地で生育できる株を耐性株（選抜株）とし、生育できない株を非耐性株とした。
Next, the present invention will be described in more detail.
The present invention is a natto bacterium used for producing natto containing a high content of nucleic acid-based umami components, and has a higher ability to have guanine analog resistance than a conventional natto bacterium containing a commercially available natto bacterium, and contains 8-azaguanine. This is a selected strain that has been selected and isolated from a strain that can grow on a minimum agar medium. It has the characteristics of producing a nucleic acid-based umami component (adenylic acid) in natto during the natto fermentation process and the basic quality characteristics of natto. The present invention relates to Bacillus natto, which has characteristics that do not deteriorate “thickness of covering” and “elasticity of yarn”. Here, natto having 8-azaguanine resistance will be described. In the present invention, a strain that can grow on a minimum agar medium containing 20 μg / ml 8-azaguanine is defined as a resistant strain (selected strain), and a strain that cannot grow is defined as a non-resistant strain. .
本発明では、該納豆菌として、好適には、例えば、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２１株（受託番号 ＮＩＴＥ Ｐ−０２３８０）、ＴＴＣＣ２２２２株（受託番号 ＮＩＴＥ Ｐ−０２３８１）が使用される。また、本発明では、納豆菌として、これらの耐性株（選抜株）を紫外線照射や化学変異剤などで常法により変異させた前記耐性株（選抜株）から派生した変異株についても、前記耐性株（選抜株）と同様の前記特性を備えたものであれば、同様に使用することができる。本発明では、「選抜株（菌株）又はその変異株」とは、前記耐性株（選抜株）ないし該耐性株（選抜株）を上記常法により変異させた該耐性株（選抜株）から派生した変異株であることを意味する。 In the present invention, for example, Bacillus subtilis TTCC2221 strain (Accession number NITE P-02380) and TTCC2222 strain (Accession number NITE P-02381) are preferably used as the Bacillus natto. Further, in the present invention, as a Bacillus natto, the resistant strains (selected strains) derived from the resistant strains (selected strains) obtained by mutating the resistant strains (selected strains) with ultraviolet irradiation, chemical mutagens, etc. in a conventional manner are also used. Any one having the same characteristics as the strain (selected strain) can be used in the same manner. In the present invention, the “selected strain (strain) or a mutant thereof” is derived from the resistant strain (selected strain) or the resistant strain (selected strain) obtained by mutating the resistant strain (selected strain) by the above-mentioned conventional method. It means that it is a mutant strain.
また、本発明は、上記納豆菌を使用して、核酸系うま味成分高含有納豆を製造する方法であって、原料の大豆から蒸煮大豆を調製する工程と、納豆発酵の過程で、この蒸煮大豆に、８−アザグアニンを含む最小寒天培地で生育可能な納豆菌を接種して該蒸煮大豆を発酵させる工程と、発酵させた蒸煮大豆を熟成させる工程とを備え、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を劣化させることなく、核酸系うま味成分（アデニル酸）による強いうま味を呈する納豆を製造する方法に係るものである。 Further, the present invention is a method for producing natto containing a high content of nucleic acid-based umami ingredients using the above-mentioned natto bacteria, wherein the steamed soybean is prepared in a process of preparing steamed soybeans from raw soybeans and a process of natto fermentation. And inoculating natto bacteria that can grow on a minimum agar medium containing 8-azaguanine and fermenting the steamed soybean, and aging the fermented steamed soybean, with the basic quality characteristics of natto The present invention relates to a method for producing natto that exhibits a strong umami taste due to a nucleic acid-based umami component (adenylic acid) without deteriorating certain “thickness of covering” and “elasticity of yarn”.
また、本発明は、上記納豆発酵により得られた核酸系うま味成分高含有納豆であって、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高い納豆菌を使用して納豆発酵を行うことにより、納豆発酵の過程で納豆中に核酸系うま味成分（アデニル酸）を産生させて、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を劣化させることなく、核酸系うま味成分を高い含有量で含有する核酸系うま味成分高含有納豆に係るものである。該納豆は、納豆に対するアデニル酸の含量が７ｍｇ／１００ｇ以上であり、該核酸系うま味成分による強いうま味を呈することを特徴とするものである。 In addition, the present invention is a natto containing a high nucleic acid-based umami component obtained by the above-mentioned natto fermentation, wherein the ability to have guanine analog resistance is higher than conventional natto bacteria including commercially available natto bacteria. By performing natto fermentation, nucleic acid-based umami ingredients (adenylic acid) are produced in natto during the natto fermentation process, and the basic quality characteristics of natto, “thickness of covering” and “yarn elasticity” The present invention relates to natto with a high content of nucleic acid-based umami components that contains a high content of nucleic acid-based umami components without deterioration. The natto has a content of adenylic acid relative to natto of 7 mg / 100 g or more, and exhibits a strong umami taste due to the nucleic acid-based umami component.
ここで、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を劣化させることなくとは、市販の納豆菌を含む従来の納豆菌と比べて、これらの品質特性が同等以上であり、納豆品質に遜色がないことを意味する。また、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高い納豆菌とは、グアニンアナログ耐性をもつ納豆菌であり、具体的には、２０μｇ／ｍｌの８−アザグアニンを含む最小寒天培地で生育可能な株を選抜して得られる耐性株（選抜株）として分離した納豆菌を意味する。 Here, without degrading the basic quality characteristics of natto, “thickness of covering” and “elasticity of yarn”, these quality characteristics are compared to conventional natto bacteria including commercially available natto bacteria. Means that the quality of natto is not inferior. In addition, natto bacteria having higher guanine analog resistance than conventional natto bacteria including commercially available natto bacteria are natto bacteria having guanine analog resistance, specifically, 20 μg / ml of 8-azaguanine. It means Bacillus natto isolated as a resistant strain (selected strain) obtained by selecting a strain that can grow on a minimal agar medium.
また、本発明は、核酸系うま味成分高含有納豆であって、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高い納豆菌を使用して、納豆発酵を行うことにより、納豆発酵の過程で納豆中に核酸系うま味成分（アデニル酸）を産生させて、アデニル酸を７ｍｇ／１００ｇ以上、あるいは、アデニル酸、グアニル酸を合計８ｍｇ／１００ｇ以上含み、該核酸系うま味成分による強いうま味を呈することを特徴とする核酸系うま味成分高含有納豆に係るものである。 In addition, the present invention is a natto containing a high content of nucleic acid-based umami components, wherein natto fermentation is performed using natto bacteria having higher guanine analog resistance than conventional natto bacteria including commercially available natto bacteria. The nucleic acid-based umami component (adenylic acid) is produced in natto during the natto fermentation process, and contains 7 mg / 100 g or more of adenylic acid or 8 mg / 100 g or more in total of adenylic acid and guanylic acid, and the nucleic acid-based umami component The present invention relates to natto containing a high content of nucleic acid-based umami components, which exhibits a strong umami taste.
核酸系うま味成分を含む納豆のうま味の強さについて試験をした結果、選抜株により納豆発酵の過程で納豆中に産生されるアデニル酸の量で、十分に納豆のうま味の増強効果があることが示された。また、核酸系うま味成分を含む納豆における当該うま味の増強効果は、核酸系うま味成分（アデニル酸）と、納豆に多く含まれるアミノ酸系のうま味成分（グルタミン酸）とのうま味の相乗効果によるものと考えられる。 As a result of testing the umami strength of natto containing nucleic acid-based umami ingredients, the amount of adenylic acid produced in the natto fermentation process during the natto fermentation process by the selected strain is sufficient to enhance the umami taste of natto. Indicated. In addition, the umami enhancement effect in natto containing nucleic acid-based umami components is considered to be due to the synergistic effect of umami between nucleic acid-based umami components (adenylic acid) and amino acid-based umami components (glutamic acid) that are abundant in natto. It is done.
次に、８−アザグアニン耐性株の選抜について説明する。
本発明では、当社納豆製品使用株、自然界や各種発酵食品から分離した分離株、又はそれらを紫外線照射や化学変異剤などで変異させた変異株などを用いて、本発明の納豆製造技術に適用できる納豆菌（優良株）の選抜を行った。該優良株（候補株）の選抜条件は、次の通りとした。
Next, selection of an 8-azaguanine resistant strain will be described.
In the present invention, it is applied to the natto production technology of the present invention using a strain using our natto product, an isolated strain isolated from nature or various fermented foods, or a mutant strain obtained by mutating them with ultraviolet irradiation or a chemical mutagen. A selection of possible natto bacteria (excellent strains) was made. The selection conditions for the excellent strain (candidate strain) were as follows.
（１）第１次の優良株（候補株）の選抜
当社納豆製品使用株、自然界や各種発酵食品から分離した分離株、又はそれらを紫外線照射や化学変異剤などで変異させた変異株を使用して、これらの分離株、変異株などを、それぞれ、８−アザグアニンを含む最小寒天培地に植菌し、当該培地で増殖が見られる株を優良株（候補株）として選抜した。
(1) Selection of primary excellent strains (candidate strains) Use natto product strains, isolates isolated from nature and various fermented foods, or mutant strains that have been mutated with UV irradiation or chemical mutagens Then, these isolates, mutants, and the like were each inoculated into a minimum agar medium containing 8-azaguanine, and a strain that showed growth in the medium was selected as an excellent strain (candidate strain).
具体的には、２０μｇ／ｍｌの８−アザグアニンを含む最小寒天培地を調製し、この培地に、白金耳などで、納豆菌を植菌し、生育可能な株を選抜した。植菌する納豆菌は、寒天培地で純化したコロニー、液体培地などで前培養した栄養細胞、休眠誘導した胞子など、どのような生育状態でも特に構わない。前記濃度の８−アザグアニンを含む最小寒天培地で生育することができる株を、第１次の優良株（候補株）とした。 Specifically, a minimum agar medium containing 20 μg / ml 8-azaguanine was prepared, and Bacillus natto was inoculated with a platinum loop or the like on this medium, and a viable strain was selected. The Bacillus natto to be inoculated can be in any growth state, such as colonies purified with an agar medium, vegetative cells pre-cultured with a liquid medium, or spore induced diapause. A strain capable of growing on a minimum agar medium containing 8-azaguanine at the above concentration was designated as a primary excellent strain (candidate strain).
（２）液体培地での培養
選抜した優良株（候補株）を、液体培地で培養した。
(2) Culture in liquid medium The selected excellent strain (candidate strain) was cultured in a liquid medium.
（３）第２次の優良株（候補株）の選抜
上記優良株（候補株）を用いて納豆を試作し、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を劣化させることなく、従来品よりも、核酸系うま味成分によるうま味の強い納豆を製造することが可能な納豆菌を、第２次の優良株（候補株）として選抜した。
(3) Selection of secondary superior strains (candidate strains) Using the above-mentioned superior strains (candidate strains), natto is prototyped, and the basic quality characteristics of natto are “cover thickness” and “yarn elasticity” As a secondary excellent strain (candidate strain), natto bacteria capable of producing natto having a stronger umami taste with a nucleic acid-based umami component than the conventional product without degrading the product was selected.
（４）第３次の優良株（候補株）の選抜
次に、これらの優良株（候補株）を用いて試作した納豆の核酸系うま味成分（アデニル酸、グアニル酸、イノシン酸）含量を、ＬＣ／ＭＳ／ＭＳ法によって分析し（日本ハム株式会社中央研究所に依頼）、核酸系うま味成分が顕著に含まれている納豆菌を、第３次の優良株（候補株）として選抜した。
(4) Selection of third excellent strains (candidate strains) Next, the content of nucleic acid-based umami components (adenylic acid, guanylic acid, inosinic acid) of natto prepared using these superior strains (candidate strains), Analysis was performed by LC / MS / MS method (requested from Central Research Laboratory, Nippon Ham Co., Ltd.), and Bacillus natto containing a significant nucleic acid-based umami component was selected as the third excellent strain (candidate strain).
次に、納豆の製造工程について説明する。
本発明において、納豆の製造工程は、原料の大豆から蒸煮大豆を調製する工程と、納豆発酵の過程で、この蒸煮大豆に、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高く、８−アザグアニンを含む最小寒天培地で生育することができる納豆菌を接種して蒸煮大豆を発酵させる工程と、発酵させた蒸煮大豆を熟成させる工程とを含むことを特徴としている。本発明では、上記納豆菌として、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高く、８−アザグアニンを含む最小寒天培地で生育することができる納豆菌を接種することを特徴としている。
Next, the manufacturing process of natto will be described.
In the present invention, the production process of natto includes a process of preparing cooked soybeans from raw soybeans, and a process of natto fermentation, wherein the cooked soybeans have conventional natto bacteria containing commercially available natto bacteria with the ability to have guanine analog resistance. It is characterized by including a step of inoculating natto bacteria that can be grown on a minimum agar medium containing 8-azaguanine and fermenting the cooked soybean, and a step of aging the fermented steamed soybean. In the present invention, as the above-mentioned natto bacteria, inoculating natto bacteria having a higher ability to have guanine analog resistance than conventional natto bacteria containing commercially available natto bacteria and capable of growing on a minimum agar medium containing 8-azaguanine. It is characterized by.
また、本発明では、納豆菌として、上述のグアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高く、８−アザグアニンを含む最小寒天培地で生育することができる納豆菌を接種することを除き、通常の納豆の製造技術を任意に適用することが可能である。 Moreover, in the present invention, as a natto bacterium, a natto bacterium that has the above-mentioned ability to have guanine analog resistance is higher than that of a conventional natto bacterium containing a commercially available natto bacterium and can grow on a minimum agar medium containing 8-azaguanine. Except for inoculation, it is possible to arbitrarily apply normal natto manufacturing techniques.
本発明の納豆製造工程では、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高い納豆菌として、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２１株（受託番号 ＮＩＴＥ Ｐ−０２３８０）、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２２株、（受託番号 ＮＩＴＥ Ｐ−０２３８１）を好適に使用することができる。また、これらの菌株は、ナットウキナーゼ活性について試験をした結果、９０ＦＵ／ｇ以上のナットウキナーゼ活性を有するナットウキナーゼ産生能力をもつ納豆菌であることが判明した。 In the natto production process of the present invention, Bacillus subtilis TTCC2221 strain (Accession number NITE P-02380), Bacillus subtilis TTCC2222 strain as natto bacteria having higher guanine analog resistance ability than conventional natto bacteria including commercially available natto bacteria. (Accession number NITE P-02381) can be preferably used. Further, as a result of testing for nattokinase activity, these strains were found to be natto bacteria having nattokinase production ability having nattokinase activity of 90 FU / g or more.
本発明では、納豆菌として、例えば、上述のように、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高く、８−アザグアニンを含む最小寒天培地で生育することができる納豆菌であれば適宜使用することができる。納豆発酵の過程で納豆中に産生された核酸系うま味成分の分析は、日本ハム株式会社中央研究所に依頼し、ＬＣ／ＭＳ／ＭＳ法によって行った。 In the present invention, as mentioned above, for example, as described above, the ability to have guanine analog resistance is higher than that of a conventional natto bacterium containing a commercially available natto bacterium, and can grow on a minimal agar medium containing 8-azaguanine. Any Bacillus natto can be used as appropriate. The analysis of the nucleic acid-based umami component produced in natto during the natto fermentation process was performed by the Central Research Laboratory of Nippon Ham Co., Ltd., and the LC / MS / MS method was used.
従来の納豆には、核酸系うま味成分は、ほとんど含まれないが、本発明の納豆には、該納豆に対するアデニル酸の含量は、７ｍｇ／１００ｇ以上であり、好ましくはアデニル酸、グアニル酸の合計が８ｍｇ／１００ｇ以上、あるいは核酸系うま味成分３種（アデニル酸、グアニル酸、イノシン酸）の合計が８ｍｇ／１００ｇ以上である。例えば、ＴＴＣＣ２２２１株は、納豆に対するアデニル酸含量は７.１ｍｇ／１００ｇであった。 The conventional natto contains almost no nucleic acid-based umami component, but the natto of the present invention has an adenylic acid content of 7 mg / 100 g or more, preferably a total of adenylic acid and guanylic acid. Is 8 mg / 100 g or more, or the total of three nucleic acid-based umami components (adenylic acid, guanylic acid, inosinic acid) is 8 mg / 100 g or more. For example, the TTCC 2221 strain had an adenylic acid content with respect to natto of 7.1 mg / 100 g.
本発明の核酸系うま味成分高含有納豆は、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高い納豆菌を使用して納豆発酵を行うことにより、納豆に対するアデニル酸の含量が７ｍｇ／１００ｇ以上であり、該アデニル酸による強いうま味を呈することを特徴としている。また、本発明は、それ以外の態様として、アデニル酸、グアニル酸の合計が８ｍｇ／１００ｇ以上、あるいは核酸系うま味成分３種（アデニル酸、グアニル酸、イノシン酸）を合計８ｍｇ／１００ｇ以上含み、核酸系うま味成分による強いうま味を呈する納豆であるとともに、９０ＦＵ／ｇ以上のナットウキナーゼによる血栓溶解活性をもつ納豆菌によるナットウキナーゼ高含有納豆であることを特徴とする納豆も本発明の対象としている。本発明では、核酸系うま味成分（アデニル酸）と、ナットウキナーゼによる血栓溶解活性を共に有するナットウキナーゼを内包する納豆製品も、本発明に係る「核酸系うま味成分高含有納豆」の範疇に含まれるものとする。 The natto with a high content of nucleic acid-based umami component of the present invention is obtained by performing natto fermentation using natto bacteria having higher guanine analog resistance than conventional natto bacteria including commercially available natto bacteria. The content is 7 mg / 100 g or more, and a strong umami taste due to the adenylic acid is exhibited. In addition, the present invention includes, as other embodiments, a total of 8 mg / 100 g or more of adenylic acid and guanylic acid, or a total of 8 mg / 100 g or more of nucleic acid-based umami components (adenylic acid, guanylic acid, inosinic acid), The present invention also includes natto, which is a natto that exhibits a strong umami taste due to a nucleic acid-based umami component and is a nattokinase-rich natto by natto bacteria having a thrombolytic activity by nattokinase of 90 FU / g or more. In the present invention, a natto product containing nattokinase having both a nucleic acid-based umami component (adenylic acid) and a thrombolytic activity by nattokinase is also included in the category of “nattokinase with a high content of nucleic acid-based umami component” according to the present invention. To do.
次に、本発明では、従来菌と、ＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株で発酵させた納豆との官能特性の比較を行った。得られた納豆菌で発酵させた納豆の官能特性の比較は、次の通りに行った。すなわち、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」と、「うま味の強さ」について、採点法によって官能評価を行い、官能特性の比較を行った。尺度は、従来菌１で発酵させた納豆をゼロとして、非常に強いから非常に弱いまでの両極７点の相対尺度（＋３非常に強い、＋２強い、＋１少し強い、０差がない、−１少し弱い、−２弱い、−３非常に弱い）を使用した。 Next, in the present invention, sensory characteristics were compared between conventional bacteria and natto fermented with TTCC 2221 strain and TTCC 2222 strain. Comparison of sensory characteristics of natto fermented with the obtained natto bacteria was performed as follows. In other words, the basic quality characteristics of natto, “thickness of covering”, “elasticity of thread”, and “strength of umami” were evaluated by a scoring method, and the sensory characteristics were compared. The scale is natto fermented with conventional fungus 1, with zero as a relative scale of 7 points from very strong to very weak (+3 very strong, +2 strong, +1 a little strong, no 0 difference, -1 Slightly weak, -2 weak, -3 very weak) were used.
評価は、官能評価士テキスト（日本官能評価学会編）に記載された方法に従って、基本五味（甘味、塩味、酸味、苦味、うま味）の識別が可能なパネラーを社内から選抜し、かつ、半年以上の納豆評価のトレーニングを繰り返し行った専門評価員によって行った。数値は、ｎ＝１２の評価結果の平均値として示した。得られた結果について、平均値の差の検討を行った。 For evaluation, according to the method described in the Sensory Evaluator Text (edited by the Japanese Society for Sensory Evaluation), panelists who can identify basic five tastes (sweet, salty, sour, bitter, umami) are selected from within the company, and more than half a year This was done by a professional evaluator who repeatedly conducted training on natto evaluation. Numerical values are shown as average values of the evaluation results of n = 12. About the obtained result, the difference of the average value was examined.
その結果、８−アザグアニン耐性選抜株として選抜されたＴＴＣＣ２２２１株、及びＴＴＣＣ２２２２株で発酵させた納豆は、従来菌と比べて、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」が同等以上であり、納豆品質に遜色がないことが示された。また、ＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株で発酵させた納豆は、従来菌と比べて、核酸系うま味成分による強いうま味を呈する納豆であることが示された。 As a result, TTCC2221 strain selected as an 8-azaguanine-resistant selection strain and natto fermented with TTCC2222 strain are the basic quality characteristics of natto “cover thickness”, “thread” It was shown that the elasticity of natto is equal to or better than natto quality. Moreover, it was shown that natto fermented with TTCC2221 stock | strain and TTCC2222 stock | strain is natto which exhibits the strong umami | taste by a nucleic acid type | system | group umami component compared with a conventional microbe.
このように、上記選抜株を用いて、納豆の試作を行い、納豆の官能特性について、納豆の官能評価に精通した専門評価員で評価（官能評価）した結果、従来菌と比べて、核酸系うま味成分によるうま味が有意に高い評価を得ると同時に、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」も同等以上であり、納豆品質に遜色がない納豆を製造することが可能であることが分かった。 In this way, using the selected strains, natto was prototyped, and the sensory characteristics of natto were evaluated (sensory evaluation) by a professional evaluator familiar with the sensory evaluation of natto. Manufactures natto that is not only inferior in terms of natto quality, but also has a significantly higher evaluation of umami due to its umami ingredients, as well as the basic quality characteristics of natto, such as “thickness of covering” and “elasticity of yarn”. It turns out that it is possible.
本発明では、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高い納豆菌として、８−アザグアニンを含む最小培地で生育することができる納豆菌が使用される。これらのうち、本発明では、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２１株（受託番号 ＮＩＴＥ Ｐ−０２３８０）、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２２株（受託番号 ＮＩＴＥ Ｐ−０２３８１）が好適に使用される。これらの納豆菌株（優良株）のＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株は、公的寄託機関である、独立行政法人製品評価技術基盤機構 特許微生物寄託センターに、２０１６年１１月３０日に寄託されている。選抜した優良株は、後記する表２〜４に示すような菌学的性質を有し、納豆菌であることが確認されている。 In the present invention, Bacillus natto that can grow on a minimal medium containing 8-azaguanine is used as Bacillus natto having higher ability to resist guanine analogs than conventional Bacillus natto containing commercially available Bacillus natto. Among these, Bacillus subtilis TTCC2221 strain (Accession number NITE P-02380) and Bacillus subtilis TTCC2222 strain (Accession number NITE P-02381) are preferably used in the present invention. These natto strains (excellent strains), TTCC 2221 and TTCC 2222 strains, were deposited on November 30, 2016 at the Patent Microorganism Depositary, National Institute for Product Evaluation and Technology, which is a public depositary organization. The selected excellent strain has bacteriological properties as shown in Tables 2 to 4 described later, and is confirmed to be Bacillus natto.
本発明により、以下に示すような格別の効果が奏される。
（１）従来の納豆菌に比べて、グアニンアナログ耐性をもつ能力が高く、８−アザグアニン耐性をもつ新規納豆菌を提供することができる。
（２）グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高い納豆菌を使用することで、納豆発酵の過程で納豆中に核酸系うま味成分（アデニル酸）を高い含有量で産生させた核酸系うま味成分高含有納豆を製造し、提供することができる。
（３）核酸系うま味成分（アデニル酸）の呈するうま味を特徴とした特有の嗜好性を有し、同時に納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」が通常の納豆と比べて劣化しない核酸系うま味成分高含有納豆の製造技術を確立することができる。
（４）納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」と、核酸系うま味成分（アデニル酸）の呈する強いうま味を有することを特徴とした新しいタイプの核酸系うま味成分高含有納豆を提供することができる。
（５）本発明の納豆は、核酸系うま味成分と、納豆に多く含まれるグルタミン酸などのアミノ酸系うま味成分とのうま味の相乗効果により、非常に強いうま味を呈する納豆製品として有用である。
（６）納豆菌として、９０ＦＵ／ｇ以上のナットウキナーゼによる血栓溶解活性を有する選抜株（ＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株）を使用することにより、核酸系うま味成分とナットウキナーゼを高い含有量で含有する核酸系うま味成分／ナットウキナーゼ高含有納豆を製造し、提供することができる。
According to the present invention, the following special effects are achieved.
(1) Compared with conventional Bacillus natto, the ability to have guanine analog resistance is high, and a novel Bacillus natto having 8-azaguanine resistance can be provided.
(2) By using natto bacteria having higher guanine analog resistance than conventional natto bacteria, including commercially available natto bacteria, the natto fermentation process contains a high content of nucleic acid-based umami components (adenylic acid). It is possible to produce and provide natto with a high content of nucleic acid-based umami components produced in an amount.
(3) have a specific preference was characterized umami exhibited by nucleoside umami (adenylate), simultaneously natto is the basic quality characteristics of "fog thick", "elasticity yarn" is normally It is possible to establish a production technology for natto with a high content of nucleic acid-based umami components that does not deteriorate compared to natto.
(4) A new type of nucleic acid system characterized by the basic quality characteristics of natto, “thickness of covering” and “elasticity of thread” and a strong umami taste exhibited by nucleic acid-based umami ingredients (adenylic acid) Natto with high content of umami ingredients can be provided.
(5) The natto of the present invention is useful as a natto product exhibiting a very strong umami taste due to a synergistic effect of umami with a nucleic acid-based umami component and an amino acid-based umami component such as glutamic acid contained in a large amount in natto.
(6) By using a selected strain having a thrombolytic activity by nattokinase of 90 FU / g or more (TTCC2221 strain, TTCC2222 strain) as a natto bacterium, a nucleic acid umami containing a high content of nucleic acid umami components and nattokinase Ingredients / nattokinase-rich natto can be manufactured and provided.
次に、実施例に基づいて本発明を具体的に説明するが、本発明は、以下の実施例によって何ら限定されるものではない。 EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited at all by the following Examples.
（１）８−アザグアニン耐性株の選抜（寒天培地）
２０μｇ／ｍｌの８−アザグアニン（ＭＰ Ｂｉｏｍｅｄｉｃａｌｓ、カタログＮｏ.
１００８３０）を含む最小寒天培地を使用して優良株（候補株）の選抜を実施した。最小培地の調整には、Ｄｉｆｃｏ Ｙｅａｓｔ Ｎｉｔｒｏｇｅｎ Ｂａｓｅ
Ｗ／Ｏ Ａｍｉｎｏ Ａｃｉｄｓ (ベクトン・ディッキンソン、カタログＮｏ．２９１９４０）を使用した。これを蒸留水に溶解後、ｐＨを６．８に調整し、２％の寒天を加えた後、オートクレーブで滅菌した。以下の実施例において、「％」は重量％を意味する。
(1) Selection of 8-azaguanine resistant strain (agar medium)
20 μg / ml 8-azaguanine (MP Biomedicals, catalog no.
10083) was used to select excellent strains (candidate strains). For adjustment of the minimum medium, Difco Yeast Nitrogen Base
W / O Amino Acids (Becton Dickinson, Catalog No. 291940) was used. This was dissolved in distilled water, adjusted to pH 6.8, added with 2% agar, and sterilized by autoclave. In the following examples, “%” means% by weight.
滅菌後、最終濃度が、０．５％になるように滅菌済みグルコース溶液、及び２０μｇ／ｍｌになるように滅菌済み８−アザグアニン溶液を添加して、最小寒天培地を調製した。８−アザグアニン溶液の滅菌は、適当な濃度の水酸化ナトリウム溶液やジメチルスルホキシドなどに溶解後、０．４５μｍの滅菌済みメンブランフィルターを通すことで実施した。 After sterilization, a minimal agar medium was prepared by adding a sterilized glucose solution to a final concentration of 0.5% and a sterilized 8-azaguanine solution to a concentration of 20 μg / ml. The 8-azaguanine solution was sterilized by dissolving in an appropriate concentration of sodium hydroxide solution or dimethyl sulfoxide, and then passing through a 0.45 μm sterilized membrane filter.
被験納豆菌として、当社納豆製品使用株、自然界や各種発酵食品から分離した分離株、又はそれらを紫外線照射や化学変異剤などで変異させた変異株を用いて、それぞれ、２０μｇ／ｍｌの８−アザグアニンを含む最小寒天培地に植菌し、増殖が見られる株を、耐性株として選抜した。 As test natto bacteria, using our natto product strains, isolates isolated from nature and various fermented foods, or mutants obtained by mutating them with ultraviolet irradiation or chemical mutagens, etc., 20 μg / ml 8- Strains that were inoculated on a minimal agar medium containing azaguanine and showed growth were selected as resistant strains.
なお、耐性株の選抜は、最小寒天培地に限らず、ＬＢ培地や肉汁培地など、一般的な微生物繁殖培地でも問題ないが、その場合は、培地によって非耐性株でも多少の生育が見られる場合があるため、耐性株は、コロニーの大きさ、高さなどで区別をする必要がある。選抜の結果、ＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株、ＴＴＣＣ２２２５株、ＴＴＣＣ２２２６株を選抜した。 The selection of resistant strains is not limited to the minimum agar medium, and there is no problem with general microbial growth media such as LB medium and gravy medium. Therefore, it is necessary to distinguish resistant strains by the size and height of colonies. As a result of selection, TTCC2221 strain, TTCC2222 strain, TTCC2225 strain, and TTCC2226 strain were selected.
本実施例において、従来菌１、従来菌２、及び選抜された耐性選抜株（４株）は、以下の納豆菌を示す。
従来菌１：
市販納豆菌（宮城野菌）から分離した納豆菌
従来菌２：
当社納豆製品使用株（受託番号 ＦＥＲＮ Ｐ−２１３２４）
ＴＴＣＣ２２２１株：
８−アザグアニン耐性選抜株；７．１ｍｇ／１００ｇのアデニル酸が含まれる。
ＴＴＣＣ２２２２株：
８−アザグアニン耐性選抜株；７．１ｍｇ／１００ｇのアデニル酸が含まれる。
ＴＴＣＣ２２２５株：
８−アザグアニン耐性選抜株；核酸系うま味成分は含まれず偽陽性。
ＴＴＣＣ２２２６株：
８−アザグアニン耐性選抜株；核酸系うま味成分は含まれず偽陽性。
In a present Example, the conventional microbe 1, the conventional microbe 2, and the selected resistant selection strain (4 strains) show the following Bacillus natto.
Conventional bacteria 1:
Bacillus natto conventional bacteria isolated from commercial Bacillus natto (Miyagino fungus) 2:
Our natto product use strain (Accession number FERN P-21324)
TTCC2221 strain:
8-Azaguanine-resistant selection strain; 7.1 mg / 100 g of adenylic acid is contained.
TTCC2222 strain:
8-Azaguanine-resistant selection strain; 7.1 mg / 100 g of adenylic acid is contained.
TTCC2225 strain:
8-Azaguanine-resistant selection strain: Nucleic acid-based umami component is not included and is false positive.
TTCC2226 strain:
8-Azaguanine-resistant selection strain: Nucleic acid-based umami component is not included and is false positive.
（２）納豆に含まれる核酸系うま味成分の分析
１）方法
選抜した納豆菌株を使用して、蒸煮大豆を発酵させ、納豆を製造し、納豆に含まれる核酸系うま味成分を調査した。
製造した納豆に含まれる核酸系うま味成分の分析は、日本ハム株式会社中央研究所に依頼し、ＬＣ／ＭＳ／ＭＳ法によって行った。
(2) Analysis of nucleic acid-based umami components contained in natto 1) Method Using the selected natto strains, fermented steamed soybeans were produced, and natto was produced, and nucleic acid-based umami components contained in natto were investigated.
Analysis of nucleic acid-based umami components contained in the manufactured natto was requested by the Central Research Laboratory of Nippon Ham Co., Ltd., and was performed by LC / MS / MS method.
２）結果
以上の結果を、表１、並びに、図１〜２にまとめて示す。
2) Results The above results are summarized in Table 1 and FIGS.
表１、並びに、図１〜２に示されるように、従来菌を使用した納豆には、１ｍｇ／１００ｇ程度のアデニル酸、グアニル酸しか含まれないが、選抜したＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株には、７ｍｇ／１００ｇ以上のアデニル酸、もしくは、合計８ｍｇ／１００ｇ以上のアデニル酸及びグアニル酸が含まれた。イノシン酸は、いずれの納豆にも含まれなかった。 As shown in Table 1 and FIGS. 1 and 2, natto using conventional bacteria contains only 1 mg / 100 g of adenylic acid and guanylic acid, but the selected TTCC2221 and TTCC2222 strains include: 7 mg / 100 g or more of adenylic acid or a total of 8 mg / 100 g or more of adenylic acid and guanylic acid were contained. Inosinic acid was not included in any natto.
一方、選抜株のうち、ＴＴＣＣ２２２５株、ＴＴＣＣ２２２６株には、核酸系うま味成分（アデニル酸、グアニル酸、イノシン酸）が検出されず、偽陽性株と考えられた。 On the other hand, among the selected strains, TTCC2225 strain and TTCC2226 strain were considered to be false positive strains because no nucleic acid-based umami components (adenylic acid, guanylic acid, inosinic acid) were detected.
（３）８−アザグアニン耐性選抜株の形態観察、生理・生化学試験
得られたＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株の菌株の同定は、株式会社テクノスルガ・ラボに依頼し、形態観察、生理・生化学試験によって実施した。
(3) Morphological observation, physiological and biochemical test of 8-azaguanine resistant selection strains The identification of the obtained strains of TTCC 2221 and TTCC 2222 strains was requested to Technosuruga Laboratories, Inc., Morphological observation, physiological and biochemical test Carried out by.
表２、表３〜４に、選抜株のＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株の形態観察、生理・生化学試験の結果を示す。以下の結果と、ビオチン要求性、及び納豆試作時に粘質物を産生する特性から、これらの菌が、納豆菌であることが確認された。 Tables 2 and 3 to 4 show the results of morphological observation and physiological / biochemical tests of the selected strains TTCC 2221 and TTCC 2222. From the following results, biotin requirement, and the property of producing mucilage during natto trial production, it was confirmed that these bacteria are Bacillus natto.
（４）従来菌と、選抜株（ＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株）で発酵させた納豆の官能特性の比較
１）方法
従来菌と、得られた選抜株で発酵させた納豆の官能特性の比較は、次の通りに行った。
納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」と、「うま味の強さ」について、採点法によって官能評価を行い、官能特性の比較を行った。
(4) Comparison of sensory characteristics of natto fermented with conventional bacteria and selected strains (TTCC 2221 strain, TTCC 2222 strain) 1) Method Comparison of sensory characteristics of natto fermented with conventional bacteria and the selected strain I went as follows.
The basic quality characteristics of “natto”, “thickness of covering”, “elasticity of thread” and “strength of umami” were subjected to sensory evaluation by scoring method, and sensory characteristics were compared.
尺度は、従来菌１で発酵させた納豆をゼロとして、非常に強いから非常に弱いまでの両極７点の相対尺度（＋３；非常に強い、＋２；強い、＋１；少し強い、０；差がない、−１；少し弱い、−２；弱い、−３；非常に弱い）を使用した。
評価は、基本五味（甘味、塩味、酸味、苦味、うま味）の識別が可能なパネラーを社内から選抜し、かつ、半年以上の納豆評価のトレーニングを繰り返し行った専門評価員によって行った。
The scale is a relative scale of 7 points from very strong to very weak natto fermented with conventional fungus 1 (+3; very strong, +2; strong, +1; slightly strong, 0; difference is No, -1; slightly weak, -2; weak, -3; very weak).
The evaluation was conducted by a professional evaluator who selected panelists capable of distinguishing basic five tastes (sweet, salty, sour, bitter, umami) from within the company and repeatedly conducted training on natto evaluation for more than half a year.
数値は、ｎ＝１２の評価結果の平均値を示した。
得られた結果について、平均値の差の検定を行った。
参考文献：官能評価士テキスト（日本官能評価学会編）
The numerical value showed the average value of the evaluation result of n = 12.
About the obtained result, the test of the difference of an average value was performed.
Reference: Text of Sensory Evaluation (Edited by Japanese Society for Sensory Evaluation)
２）結果
その結果を、表５に示す。ＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株で発酵させた納豆は、従来菌１、従来菌２で発酵させた納豆と比べて、被りの厚さ、糸の弾力が同等以上であり、納豆品質に遜色がないことが示された。
ＴＴＣＣ２２２１株、ＴＴＣＣ２２２２株で発酵させた納豆は、従来菌１、従来菌２で発酵させた納豆と比べて、強いうま味を感じる納豆を作ることが示された。
2) Results Table 5 shows the results. The natto fermented with TTCC 2221 and TTCC 2222 strains has the same thickness or more than that of fermented natto fermented with conventional fungus 1 and conventional fungus 2, and the natto quality is comparable to natto quality. Indicated.
It was shown that natto fermented with TTCC 2221 and TTCC 2222 strains produced natto with a strong umami taste compared to natto fermented with conventional bacteria 1 and 2.
以上詳述した通り、本発明は、納豆発酵の過程で納豆中に核酸系うま味成分（アデニル酸）を産生させる特性と、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を劣化させることがない特性を備えたことを特徴とする新規納豆菌、該納豆菌を使用して核酸系うま味成分高含有納豆を製造する方法及び該核酸系うま味成分高含有納豆に係るものであり、本発明により、従来の納豆菌に比べて、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高い納豆菌を使用することで、核酸系うま味成分（アデニル酸）の呈する強いうま味を特徴とした特有の嗜好性を有し、同時に納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」が劣化しない核酸系うま味成分高含有納豆の製造技術を確立することができる。また、本発明により、従来の納豆製品よりも納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」と、核酸系うま味成分（アデニル酸）による強いうま味を呈することを特徴とした特有の嗜好性を有する新しいタイプの核酸系うま味成分高含有納豆を提供することができる。本発明は、納豆に対する核酸系うま味成分（アデニル酸）の含量が７ｍｇ／１００ｇ以上で、該アデニル酸による強いうま味を呈する核酸系うま味成分高含有納豆及びその製造技術を提供するものとして産業上の利用可能性を有するものである。 As described above in detail, the present invention is characterized in that a nucleic acid-based umami component (adenylic acid) is produced in natto during the natto fermentation process, and the basic quality characteristics of natto are “thickness of covering”, “thread A novel natto bacterium characterized in that it does not deteriorate the elasticity of natto, a method for producing natto containing a high nucleic acid-based umami component using the natto bacterium, and a natto containing a high nucleic acid-based umami component Thus, according to the present invention, by using a natto bacterium having a higher ability to have guanine analog resistance than a conventional natto bacterium containing a commercially available natto bacterium, compared to a conventional natto bacterium, a nucleic acid-based umami component ( It has a unique taste characterized by the strong umami of adenylic acid), and at the same time contains a high content of nucleic acid-based umami ingredients that do not degrade the basic quality characteristics of natto, such as “thickness of covering” and “elasticity of yarn” Establishing natto manufacturing technology Kill. In addition, according to the present invention, the basic quality characteristics of natto, such as “thickness of covering”, “elasticity of yarn”, and a strong umami taste due to nucleic acid-based umami components (adenylic acid) are exhibited. It is possible to provide a new type of nucleic acid-based umami component-rich natto having a characteristic and unique taste. The present invention provides an natto with a high content of nucleic acid-based umami components, which has a nucleic acid-based umami component (adenylic acid) content of 7 mg / 100 g or more with respect to natto and exhibits a strong umami taste due to the adenylic acid, and its production technology. It has availability.
微生物の表示
＜識別の表示＞ Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ
ＴＴＣＣ２２２１
＜受託番号＞ ＮＩＴＥ Ｐ−０２３８０
＜識別の表示＞ Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ
ＴＴＣＣ２２２２
＜受託番号＞ ＮＩＴＥ Ｐ−０２３８１
Display of microorganisms <Display of identification> Bacillus subtilis
TTCC2221
<Trust number> NITE P-02380
<Indication display> Bacillus subtilis
TTCC2222
<Trust number> NITE P-02381
Claims (6)
前記納豆菌が、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２１株（受託番号 ＮＩＴＥ Ｐ−０２３８０）、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２２株（受託番号 ＮＩＴＥ Ｐ−０２３８１）から選択された菌株であることを特徴とする納豆菌。 Properties that are resistant to guanine analogs and that can grow on a minimal agar medium containing 8-azaguanine, and that produce nucleic acid-based umami components (adenylic acid) in natto during the natto fermentation process, and the basics of natto Natto fungus with characteristics that do not deteriorate the thickness of the cover, which is a typical quality characteristic, and the elasticity of the thread compared to normal natto,
Bacillus subtilis TTCC2221 strain (Accession number NITE P-02380) and Bacillus subtilis TTCC2222 strain (Accession number NITE P-02381).
原料の大豆から蒸煮大豆を調製する工程と、納豆発酵の過程で、この蒸煮大豆に、グアニンアナログ耐性をもつ能力が市販の納豆菌を含む従来の納豆菌よりも高く、８−アザグアニンを含む最小寒天培地で生育することが可能な納豆菌を接種して蒸煮大豆を発酵させる工程と、発酵させた蒸煮大豆を熟成させる工程とを備え、
納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」を通常の納豆と比べて劣化させることなく、核酸系うま味成分（アデニル酸）によるうま味を呈する納豆を製造することを特徴とする核酸系うま味成分高含有納豆の製造方法。 A method for producing natto containing a high content of nucleic acid-based umami components using the natto bacteria according to claim 1,
In the process of preparing cooked soybeans from raw soybeans and in the process of natto fermentation, this cooked soybean has a higher ability to resist guanine analogues than conventional natto bacteria, including commercially available natto bacteria, and the minimum containing 8-azaguanine Inoculating natto bacteria capable of growing on an agar medium and fermenting the steamed soybean, and aging the fermented steamed soybean,
Manufacture natto with umami taste due to nucleic acid-based umami ingredients (adenylic acid) without degrading the basic quality characteristics of natto, “thickness of covering” and “elasticity of yarn” compared to normal natto A method for producing natto containing a high content of nucleic acid-based umami components.
前記核酸系うま味成分（アデニル酸）が納豆発酵の過程で納豆中に産生されたものであり、通常の納豆と比べて、納豆の基本的な品質特性である「被りの厚さ」、「糸の弾力」の劣化がなく、納豆発酵が、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２１株（受託番号 ＮＩＴＥ Ｐ−０２３８０）、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ ＴＴＣＣ２２２２株（受託番号 ＮＩＴＥ Ｐ−０２３８１）から選択された菌株による発酵であり、該株菌を含み、納豆に対するアデニル酸の含量が７ｍｇ／１００ｇ以上であり、該アデニル酸によるうま味を呈することを特徴とする核酸系うま味成分高含有納豆。 Nucleic acid umami component high content natto containing nucleic acid umami component (adenylic acid) in natto,
The nucleic acid-based umami component (adenylic acid) is produced in natto during the process of natto fermentation. Compared to normal natto, the basic quality characteristics of natto are “thickness of covering”, “thread of elasticity "there is no deterioration of, paid bean fermentation, Bacillus subtilis TTCC2221 strain (accession number NITE P-02380), a fermentation with selected strains from Bacillus subtilis TTCC2222 strain (accession number NITE P-02381), strain Natto containing a high nucleic acid-based umami component , which contains a bacterium, has an adenylic acid content of 7 mg / 100 g or more with respect to natto, and exhibits an umami taste due to the adenylic acid.
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