JP6618792B2 - Natto that can be heat sterilized - Google Patents
Natto that can be heat sterilized Download PDFInfo
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- JP6618792B2 JP6618792B2 JP2015244194A JP2015244194A JP6618792B2 JP 6618792 B2 JP6618792 B2 JP 6618792B2 JP 2015244194 A JP2015244194 A JP 2015244194A JP 2015244194 A JP2015244194 A JP 2015244194A JP 6618792 B2 JP6618792 B2 JP 6618792B2
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Agronomy & Crop Science (AREA)
- Botany (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Beans For Foods Or Fodder (AREA)
Description
本発明は、納豆上での胞子形成能を低下させた納豆菌変異株、及び、当該変異株を用いて製造された加熱殺菌が可能な納豆に関する。より詳しくは、ジピコリン酸合成酵素遺伝子が欠損されているために、納豆発酵時には胞子形成能が低下しているが、ジピコリン酸を添加した培地での培養時には正常な胞子形成能を有する納豆菌変異株、当該変異株を用いて製造された納豆、その製造方法、並びに、当該変異株を用いる納豆の胞子及び栄養細胞の低減方法に関する。 The present invention relates to a Bacillus natto mutant strain having reduced spore-forming ability on natto, and to natto that can be heat-sterilized and produced using the mutant strain. More specifically, because of the deficiency of the dipicolinate synthase gene, the spore-forming ability is reduced during natto fermentation, but the natto-mutant that has normal spore-forming ability when cultured in a medium supplemented with dipicolinic acid The present invention relates to a strain, natto produced using the mutant, a method for producing the same, and a method for reducing spore and vegetative cells of natto using the mutant.
食品の消費又は賞味期限内において、一般的な加工食品１ｇあたりの生菌数は１×１０５から１×１０６以下に抑える必要がある。 It is necessary to keep the number of viable bacteria per gram of general processed foods from 1 × 10 5 to 1 × 10 6 or less within the consumption of food or the expiration date.
納豆菌の胞子は、物理的、化学的に安定である。従って、納豆を加工食品に利用する場合は、当該加工食品はもとより、当該食品の製造ラインにおいても納豆菌胞子の付着が問題となる。例えば、納豆菌胞子の除菌・殺菌が不十分であると、上記加工食品の製造ラインにおいて納豆を利用しない加工食品を製造する場合、納豆菌胞子の混入の危険性が生じ、その結果納豆の加工食品への利用は限られることになる。 Bacillus natto spores are physically and chemically stable. Therefore, when using natto as a processed food, adhesion of natto fungal spores becomes a problem not only in the processed food but also in the food production line. For example, if the sterilization and sterilization of natto fungus spores are insufficient, when manufacturing processed foods that do not use natto in the processed food production line, there is a risk of contamination with natto fungal spores. Use in processed foods will be limited.
納豆中の胞子を減らすためには１２０℃程度の加熱が必要であるが、当該加熱条件では納豆の品質を保持することは困難である。胞子を形成し難い納豆菌の使用により、あるいは、胞子を形成させない発酵条件によって、納豆中の胞子数を減らすことは可能であっても、納豆の品質は十分には確保し難い。 In order to reduce the spores in natto, heating at about 120 ° C. is necessary, but it is difficult to maintain the quality of natto under the heating conditions. Although it is possible to reduce the number of spores in natto by using natto bacteria that do not easily form spores, or by fermentation conditions that do not allow the formation of spores, it is difficult to ensure sufficient quality of natto.
従来、納豆中の胞子数が少ない納豆を製造する方法としては、胞子形成率の低い納豆菌を利用した乾燥納豆に殺菌処理を施すという方法（特許文献１参照）や、胞子形成率の低いタイミングで殺菌を施すという方法（特許文献２参照）が報告されている。乾燥納豆を利用した方法では、生の納豆ではないため、食感の違いが出てしまい、また、胞子形成率の低いタイミングでの殺菌は、栄養細胞の殺菌を主とするものであることから、胞子数を減少させるのには不十分であった。 Conventionally, as a method for producing natto with a small number of spores in natto, a method of sterilizing dried natto using natto bacteria with a low spore formation rate (see Patent Document 1), or a timing with a low spore formation rate A method of performing sterilization (see Patent Document 2) has been reported. Because the method using dry natto is not raw natto, the texture is different, and sterilization at a low spore formation rate is mainly due to sterilization of vegetative cells. It was not enough to reduce the number of spores.
さらに、ｓｉｇＫ遺伝子などの胞子形成遺伝子が欠損されており、該欠損が、該遺伝子の薬剤耐性遺伝子での置換によるものである納豆菌変異株を用いて製造される納豆が報告されている（特許文献３参照）。しかしながら、これらの遺伝子はコート形成など多くの胞子形成期特異的遺伝子の発現制御に関与するため、当該遺伝子破壊株では正常な胞子形成能を回復することができない、というのが当業者の一般常識であり、純粋培養において胞子を調製することが困難である。 Furthermore, natto produced using a Bacillus natto mutant strain in which a spore-forming gene such as the sigK gene is deficient and the deficiency is due to substitution of the gene with a drug resistance gene has been reported (patent) Reference 3). However, since these genes are involved in the control of the expression of many sporulation phase-specific genes such as coat formation, it is common knowledge of those skilled in the art that the gene-disrupted strain cannot restore normal sporulation ability. And it is difficult to prepare spores in pure culture.
一般的な納豆製造法では、蒸煮工程後の高温の大豆に、純粋培養した納豆菌胞子を植菌する盛り込み工程を行い、納豆菌による発酵工程を行う。ここで、純粋培養した上記納豆菌変異株を高温条件で植菌すると、耐熱性の低い栄養細胞が死滅してしまい納豆発酵ができない。そのため、蒸煮した大豆の温度を下げて低温条件にする工程を追加しなければ盛り込みができず、さらに低温条件であるために雑菌汚染が生じやすいという問題があった。 In a general natto manufacturing method, a high-temperature soybean after the cooking process is inoculated with purely cultured natto spores and a fermentation process using natto is performed. Here, when the natto bacillus mutant strain that has been purely cultured is inoculated under high temperature conditions, vegetative cells with low heat resistance are killed and natto fermentation cannot be performed. For this reason, there is a problem in that it cannot be incorporated unless a step for lowering the temperature of the steamed soybean to lower the temperature is added, and contamination with germs is likely to occur due to the lower temperature.
通常の納豆菌を用いて製造した納豆中には耐熱性のある胞子が多く含まれているため、既存の設備においては納豆の加熱殺菌（胞子数の低減）は不可能な状況にある。また、通常の納豆製造法では盛り込みに胞子を用いる。蒸煮した熱い大豆に栄養細胞の納豆菌を噴霧すると、納豆菌が死んでしまい良好な発酵が得られない。そのため、従来の胞子化しない納豆菌株を用いると、盛り込みを低い温度で行う必要が生じるため、雑菌汚染の問題が生じ、衛生的な納豆を製造できない、などの課題があった。 Since natto produced using ordinary natto bacteria contains many heat-resistant spores, it is impossible to heat sterilize natto (reduce the number of spores) with existing facilities. In addition, the usual natto manufacturing method uses spores for incorporation. When natto bacteria of vegetative cells are sprayed on hot steamed soybeans, natto bacteria die and good fermentation cannot be obtained. For this reason, when a conventional spore-forming natto strain is used, it is necessary to add the natto at a low temperature, which causes problems such as contamination of germs, and hygienic natto cannot be produced.
そこで、本発明は、培地では胞子化して胞子液を調製することができ、かつ納豆上では胞子化しにくい納豆菌を取得することにより、上記の従来技術の問題を解決することを目的とした。 Therefore, the present invention aims to solve the above-mentioned problems of the prior art by obtaining natto bacteria that can be spore-prepared in a medium to prepare a spore solution and that are difficult to spore on natto.
本発明者は、上記課題を解決するには、通常の培地では胞子化するが、納豆中では胞子化しない変異株を作製し、納豆を殺菌すればよいと考えた。 In order to solve the above-mentioned problem, the present inventor considered that a mutant strain that is spore-forming in a normal medium but not spore-forming in natto should be prepared and natto should be sterilized.
納豆菌の胞子に含まれるジピコリン酸は耐熱性を生じる成分であり、これを欠くと耐熱性が低下する（胞子ができにくい）。それゆえ、納豆菌のジピコリン酸合成酵素遺伝子(spoVFAまたはspoVFB)を破壊すれば、自分でジピコリン酸を合成できなくなり、胞子化がジピコリン酸の存在下でのみ起こるようになると考えられた。 Dipicolinic acid contained in the spore of Bacillus natto is a component that produces heat resistance, and lacking this reduces heat resistance (it is difficult to produce spores). Therefore, it was thought that if the dipicolinate synthase gene (spoVFA or spoVFB) of Bacillus natto was destroyed, dipicolinic acid could not be synthesized by itself, and sporulation would occur only in the presence of dipicolinic acid.
そこで、本発明者は納豆菌のspoVFA遺伝子破壊株を作製し、それを用いて納豆を製造したところ、納豆にはジピコリン酸が含まれていないため、納豆発酵中に新たに胞子形成されず、胞子の少ない納豆が得られた。 Therefore, the present inventors produced a spoVFA gene disruption strain of Bacillus natto, and produced natto using it, because natto does not contain dipicolinic acid, so no new spores are formed during natto fermentation, Natto with few spores was obtained.
一方、上述の株をジピコリン酸を含有する培地で培養すると、通常の納豆菌と同様に胞子形成能を有しており、従来の納豆製造法に用いうる植菌用納豆菌を調製することができた。この植菌用納豆菌は胞子化しているため、高温で盛り込みを行っても十分に発酵の進んだ納豆ができ、豆を低温化する工程を追加する必要がなく、雑菌汚染の問題もない。 On the other hand, when the above-mentioned strain is cultured in a medium containing dipicolinic acid, it has spore-forming ability in the same manner as ordinary natto bacteria, and it is possible to prepare natto bacteria for inoculation that can be used in conventional natto production methods. did it. Since the Bacillus natto for inoculation is sporulated, it can be fermented natto even if it is added at a high temperature, and it is not necessary to add a process for lowering the temperature of the bean and there is no problem of contamination with bacteria.
本発明は、当該知見に基づいてなされたものである。
すなわち、請求項１に係る本発明は、ジピコリン酸を含まない培地での胞子形成能が、ジピコリン酸を２００μｇ／ｍＬ以上含む培地での胞子形成能に対して５％以下であり、且つ、糸引きが良好であると共に、菌膜が均一に広がっている納豆を製造できる性質を有する、納豆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）変異株を、ジピコリン酸を含有する液体培地にて培養して胞子懸濁液を調製し、得られた胞子懸濁液を、蒸煮大豆又は煮大豆に植菌し発酵させて製造されたことを特徴とする納豆に関する。
請求項２に係る本発明は、請求項１に記載の納豆であって、発酵後、５５〜１００℃で３０〜２００分加熱殺菌されたことを特徴とする納豆に関する。
請求項３に係る本発明は、請求項２に記載の納豆であって、胞子数が１０５/納豆ｇ以下であり、総菌数が１０６/納豆ｇ以下である納豆に関する。
請求項４に係る本発明は、納豆を製造するにあたり、ジピコリン酸を含まない培地での胞子形成能が、ジピコリン酸を２００μｇ／ｍＬ以上含む培地での胞子形成能に対して５％以下であり、且つ、糸引きが良好であると共に、菌膜が均一に広がっている納豆を製造できる性質を有する、納豆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）変異株を、ジピコリン酸を含有する液体培地にて培養して胞子懸濁液を調製し、得られた胞子懸濁液を、蒸煮大豆又は煮大豆に植菌し発酵させることを特徴とする、納豆の製造方法に関する。
請求項５に係る本発明は、発酵後、５５〜１００℃で３０〜２００分加熱殺菌する、請求項４に記載の納豆の製造方法に関する。
請求項６に係る本発明は、納豆における納豆菌の胞子及び栄養細胞を低減するにあたり、ジピコリン酸を含まない培地での胞子形成能が、ジピコリン酸を２００μｇ／ｍＬ以上含む培地での胞子形成能に対して５％以下であり、且つ、糸引きが良好であると共に、菌膜が均一に広がっている納豆を製造できる性質を有する、納豆菌（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）変異株を、ジピコリン酸を含有する液体培地にて培養して胞子懸濁液を調製し、得られた胞子懸濁液を、蒸煮大豆又は煮大豆に植菌し発酵させ、納豆を製造することを特徴とする、納豆における納豆菌の胞子及び栄養細胞の低減方法に関する。
請求項７に係る本発明は、発酵後、５５〜１００℃で３０〜２００分加熱殺菌する、請求項６に記載の納豆における納豆菌の胞子及び栄養細胞の低減方法に関する。
This invention is made | formed based on the said knowledge.
That is, the present invention according to claim 1 is characterized in that the spore-forming ability in a medium not containing dipicolinic acid is 5% or less with respect to the spore-forming ability in a medium containing 200 μg / mL or more of dipicolinic acid. A spore suspension obtained by culturing a Bacillus subtilis mutant strain in a liquid medium containing dipicolinic acid, which has good pulling properties and has the property of producing natto with an evenly spread bacterial membrane. The present invention relates to natto, which is produced by inoculating and fermenting the prepared and obtained spore suspension into steamed soybeans or cooked soybeans .
The present invention according to claim 2 relates to natto according to claim 1 , which is sterilized by heating at 55 to 100 ° C. for 30 to 200 minutes after fermentation.
The present invention according to claim 3 relates to natto according to claim 2 , wherein the spore count is 10 5 / natto g or less and the total number of bacteria is 10 6 / natto g or less.
In the present invention according to claim 4 , when producing natto, the spore forming ability in a medium not containing dipicolinic acid is 5% or less relative to the spore forming ability in a medium containing 200 μg / mL or more of dipicolinic acid. And cultivating a Bacillus subtilis mutant strain in a liquid medium containing dipicolinic acid, which has the property of producing natto with good stringiness and a uniform spread of mycelium. The present invention relates to a method for producing natto characterized by preparing a suspension and inoculating and fermenting the obtained spore suspension into steamed soybeans or cooked soybeans .
This invention which concerns on Claim 5 is related with the manufacturing method of the natto of Claim 4 which heat-sterilizes for 30 to 200 minutes at 55-100 degreeC after fermentation.
The present invention according to claim 6 is characterized in that the spore-forming ability in a medium not containing dipicolinic acid is the spore-forming ability in a medium containing 200 μg / mL or more of dipicolinic acid. A Bacillus subtilis mutant strain containing dipicolinic acid, which has a property of producing natto with 5% or less and good stringing and having a fungus membrane spread uniformly. A spore suspension is prepared by culturing in a liquid medium, and the resulting spore suspension is inoculated into fermented soybeans or boiled soybeans and fermented to produce natto. The present invention relates to a method for reducing spores and vegetative cells.
The present invention according to claim 7 relates to a method for reducing spore and vegetative cells in natto according to claim 6, which is sterilized by heating at 55 to 100 ° C for 30 to 200 minutes after fermentation.
本発明によって、発酵不良や雑菌汚染といった問題がなく、既存の設備、工程によって容易に胞子の含有量が少ない納豆を提供することができる。
また、本発明の納豆は胞子が少ないため、栄養細胞が死滅する温度で加熱殺菌（胞子数の低減）が可能である。それゆえ、本発明の納豆は、納豆以外の製造ラインなどにおいて納豆菌胞子の混入の危険性が少なく、各種加工食品の製造に供することができる。
According to the present invention, it is possible to provide natto with low content of spores by existing facilities and processes without problems such as poor fermentation and contamination with bacteria.
Further, since the natto of the present invention has few spores, it can be sterilized by heating (reducing the number of spores) at a temperature at which the vegetative cells die. Therefore, the natto of the present invention is less likely to be mixed with natto fungal spores in a production line other than natto and can be used for manufacturing various processed foods.
以下、具体的な実施形態を挙げて本発明を詳細に説明する。
本発明は、納豆発酵時には胞子形成能が低下しているが、ジピコリン酸を添加した培地での培養時には胞子形成が正常な納豆菌変異株に関する。
Hereinafter, the present invention will be described in detail with specific embodiments.
The present invention relates to a Bacillus natto mutant strain having reduced spore-forming ability during natto fermentation but having normal spore formation when cultured in a medium supplemented with dipicolinic acid.
［新規納豆菌変異株の菌学的性質］
本発明の新規納豆菌変異株は、ジピコリン酸を含まない納豆上では胞子形成能が著しく低下しているが、ジピコリン酸を添加した培地では正常な胞子形成能を有する納豆菌変異株である。
[Mycological properties of new natto mutants]
The novel Bacillus natto mutant of the present invention is a Bacillus natto mutant that has a spore-forming ability that is remarkably reduced on natto that does not contain dipicolinic acid, but that has a normal spore-forming ability in a medium supplemented with dipicolinic acid.
本発明の新規納豆菌変異株は、胞子形成能以外の点においては、以下に示す通り、親株である納豆菌と同等の菌学的性質を有している。したがって、通常の納豆菌と同様に、糸引き及び外観が良好な納豆を製造できる性質を有している。 The novel Bacillus natto mutant of the present invention has bacteriological properties equivalent to those of the parent strain Bacillus natto, as shown below, except for the ability to form spores. Therefore, like ordinary natto bacteria, it has the property of producing natto with good stringiness and appearance.
ここで納豆菌とは、枯草菌バチルス・サチリス（Bacillus subtilis）の変種（B. subtilis var.natto、B. subtilis (natto)）に分類される細菌である。分類体系によっては、枯草菌の近縁種バチルス・ナットウ（B. natto）として分類される場合もある。 Here, Bacillus natto is a bacterium classified as a variant of Bacillus subtilis (B. subtilis var. Natto, B. subtilis (natto)). Depending on the classification system, it may be classified as B. natto, a closely related species of Bacillus subtilis.
・形態的特徴
当該新規納豆菌変異株の栄養細胞は、大きさ2〜3μm程度の桿菌であり運動性を有する。また、グラム染色性を有する。胞子の形状は楕円形であり、大きさは1.12〜1.28μm程度である。
-Morphological features The vegetative cells of the new Bacillus natto mutant are gonococci having a size of 2 to 3 μm and have motility. Moreover, it has gram dyeability. The shape of the spore is elliptical, and the size is about 1.12 to 1.28 μm.
・培養的性質
当該新規納豆菌変異株の寒天平板培地上でのコロニーの形状は環状である。当該コロニーは、表面に皺があり、光沢が無く、色調が不透明〜乳白色であり、中央部の隆起がない特徴を有する。なお、グルタミン酸に富む培地上でのコロニーは粘調性を有する。
当該新規納豆菌変異株を液体培地で培養すると、培養後の培地表面に菌膜が形成される。また、培養液は混濁する。
-Culture characteristics The shape of the colonies on the agar plate of the new Bacillus natto mutant is circular. The colony has characteristics of having wrinkles on the surface, no gloss, a color tone of opaque to milky white, and no bulge in the central part. Note that colonies on a medium rich in glutamic acid are viscous.
When the novel Bacillus natto mutant is cultured in a liquid medium, a fungal membrane is formed on the surface of the cultured medium. Moreover, the culture solution becomes turbid.
・炭素源資化性
当該新規納豆菌変異株は、グルコース, シュクロースに対する資化性を有する。一方、ラクトース, アラビノースに対する資化性を有さない。
-Carbon source assimilation The new Bacillus natto mutant has an ability to assimilate glucose and sucrose. On the other hand, it has no assimilability for lactose and arabinose.
・生理学的性質
当該新規納豆菌変異株は、好気性細菌であり、ビオチン要求性を示し、最少培地での生育が可能である。また、プロテアーゼ活性を有する。また、クエン酸塩を利用して生育が可能である。胞子形成にジピコリン酸を要求する。
-Physiological properties The novel Bacillus natto mutant is an aerobic bacterium, exhibits biotin requirement, and can grow on a minimal medium. It also has protease activity. It can also grow using citrate. Dipicolinic acid is required for sporulation.
・温度特性
当該新規納豆菌変異株は、通常の納豆菌の生育温度帯である37〜40℃付近で好適な生育能を有し、通常の納豆菌と同等の増殖速度により増殖する。
-Temperature characteristics The new Bacillus natto mutant has a suitable growth ability at around 37-40 ° C, which is the normal temperature range of Bacillus natto, and grows at a growth rate equivalent to that of ordinary Bacillus natto.
［胞子形成能］
当該新規納豆菌変異株の胞子形成能は、培養液中の胞子数の測定結果を指標として判定することができる。胞子数の測定は、培養液を加熱処理した後、寒天培地を用いて耐熱性菌数を求めることにより行うことができる。
具体的には、胞子を形成しうる液体培地（例えば後述の実施例１（表４）に記載の胞子形成培地）に植菌し、液相温度37℃にて24時間振盪培養して得られた培養液を、７５℃で１５分間加熱処理した後、LB寒天培地に塗末培養して出現したコロニー数から算出することができる。
[Spore formation ability]
The spore forming ability of the novel Bacillus natto mutant can be determined using the measurement result of the number of spores in the culture solution as an index. The number of spores can be measured by heat-treating the culture solution and then determining the number of heat-resistant bacteria using an agar medium.
Specifically, it is obtained by inoculating a liquid medium capable of forming spores (for example, a spore-forming medium described in Example 1 (Table 4) described later) and shaking culture at a liquidus temperature of 37 ° C. for 24 hours. The obtained culture broth can be calculated from the number of colonies that appeared after heat treatment at 75 ° C. for 15 minutes and then smear culture on LB agar medium.
当該新規納豆菌変異株は、胞子形成にジピコリン酸を必要とし、ジピコリン酸不存在下では胞子形成能が著しく低下する。具体的には、ジピコリン酸を含まない培地での胞子形成能が、ジピコリン酸を２００μｇ／ｍＬ以上含む培地での胞子形成能に対して、好ましくは５％以下、より好ましくは４％以下、さらに好ましくは３％以下、特に好ましくは２％以下、最も好ましくは１％以下である。 The novel Bacillus natto mutant strain requires dipicolinic acid for sporulation, and its spore-forming ability is significantly reduced in the absence of dipicolinic acid. Specifically, the spore-forming ability in a medium not containing dipicolinic acid is preferably 5% or less, more preferably 4% or less, more preferably spore-forming ability in a medium containing 200 μg / mL or more of dipicolinic acid. It is preferably 3% or less, particularly preferably 2% or less, and most preferably 1% or less.
また、当該新規納豆菌変異株は、ジピコリン酸を添加した培地では正常な胞子形成能を有する。ここで「正常な胞子形成能」とは、通常の納豆菌と同等の胞子形成能を指し、具体的には培養液中の胞子数を総菌数で割ることにより算出される胞子形成率（％）が好ましくは７０％以上、より好ましくは７３％以上、特に好ましくは７５％以上であることを指す。 The novel Bacillus natto mutant has normal spore-forming ability in a medium supplemented with dipicolinic acid. Here, “normal spore-forming ability” refers to a spore-forming ability equivalent to that of normal natto bacteria, and specifically, a spore formation rate calculated by dividing the number of spores in a culture solution by the total number of bacteria ( %) Is preferably 70% or more, more preferably 73% or more, and particularly preferably 75% or more.
ここで、総菌数（胞子と栄養細胞の総和）の測定は、培養液を適切に希釈した後、寒天培地に塗抹培養することにより行うことができる。具体的には、液体培地（例えば胞子形成培地）に植菌し、液相温度37℃にて24時間振盪培養して得られた培養液を、リン酸緩衝液にて希釈し、LB寒天培地に塗抹培養して出現したコロニー数から算出することができる。 Here, the total number of bacteria (total of spores and vegetative cells) can be measured by appropriately diluting the culture solution and then smearing it on an agar medium. Specifically, a culture medium obtained by inoculating a liquid medium (for example, a sporulation medium) and shaking culture at a liquidus temperature of 37 ° C. for 24 hours is diluted with a phosphate buffer, and an LB agar medium is obtained. It can be calculated from the number of colonies that appeared after smear culture.
［選抜菌株］
本発明の納豆上での胞子形成能が低下した新規納豆菌変異株は、上記のような胞子数の測定法により自然界からスクリーニングすることができる。また、人為的に変異を導入することによっても得ることができる。人為的に変異を導入する方法としては、例えばニトロソグアニジン（NTG）処理、エチルメタンスルホン酸（EMS）処理などの化学的手法、紫外線処理などの物理的手法、後述する実施例1に記載の方法のような遺伝子工学的手法を採用することができ、特に限定されるものではない。
[Selected strains]
A novel Bacillus natto mutant strain having reduced spore-forming ability on natto of the present invention can be screened from the natural world by the method for measuring the number of spores as described above. It can also be obtained by artificially introducing mutations. Examples of methods for artificially introducing mutations include chemical methods such as nitrosoguanidine (NTG) treatment, ethylmethanesulfonic acid (EMS) treatment, physical methods such as ultraviolet treatment, and the method described in Example 1 described later. A genetic engineering technique such as can be employed, and is not particularly limited.
遺伝子工学的手法において、ターゲットとなる遺伝子としては、ジピコリン酸合成酵素（spoVFA及び／またはspoVFB）遺伝子を用いることが好ましい。ジピコリン酸は納豆菌の胞子に含まれ、耐熱性を生じる成分であり、この遺伝子が欠損されると耐熱性が低下する（胞子ができにくい）。spoVFA及び／またはspoVFB遺伝子を破壊すると、自身でジピコリン酸を合成できないため、胞子化がジピコリン酸の存在下でのみ起こるようになる。 In the genetic engineering technique, it is preferable to use a dipicolinate synthase (spoVFA and / or spoVFB) gene as a target gene. Dipicolinic acid is a component that is contained in the spore of Bacillus natto and produces heat resistance. When this gene is deleted, heat resistance is reduced (it is difficult to produce spores). When spoVFA and / or spoVFB genes are disrupted, sporelation occurs only in the presence of dipicolinic acid because dipicolinic acid cannot be synthesized by itself.
遺伝子工学的手法においてターゲット遺伝子に変異を導入する方法としては、ポイントミューテーションや、欠失、置換、挿入失活など遺伝子産物の活性を低下させ得る方法であれば何でもよく、特に限定されるものではない。 As a method for introducing a mutation into a target gene in a genetic engineering technique, any method can be used as long as it can reduce the activity of a gene product such as point mutation, deletion, substitution, insertion inactivation, and the like. is not.
遺伝子置換又は挿入による変異導入法において、ターゲット遺伝子を置換する又はターゲット遺伝子に挿入する変異用遺伝子としては、クロラムフェニコール耐性遺伝子、テトラサイクリン耐性遺伝子、エリスロマイシン耐性遺伝子などの薬剤耐性遺伝子を用いることが好ましい。 In the mutation introduction method by gene replacement or insertion, a drug resistance gene such as a chloramphenicol resistance gene, a tetracycline resistance gene, an erythromycin resistance gene, etc. may be used as a mutation gene to replace the target gene or to be inserted into the target gene. preferable.
また、遺伝子工学的手法を納豆菌に利用するためには、納豆菌への変異用遺伝子の導入が必要であるが、そのような方法としては、自然形質転換能を利用したコンピテンス法や、プロトプラスト法、エレクトロポレーション法、ファージを利用した形質導入法などがあるが、変異用遺伝子を導入することが可能な方法であれば何であっても良く、特に限定されるものではない。 In addition, in order to use genetic engineering techniques for Bacillus natto, it is necessary to introduce a gene for mutation into Bacillus natto. Such methods include competency methods using natural transformation ability, protoplasts, and the like. Methods, electroporation methods, transduction methods using phages, etc., but any method can be used as long as it can introduce a gene for mutation, and is not particularly limited.
当該新規納豆菌変異株の親株としては、如何なる納豆菌も採用することができる。例えば、一般的な市販菌である宮城野菌、高橋菌、成瀬菌等を用いることができるが、特定の性質を有する突然変異株、遺伝子組み換え株などの各種菌株を利用することもできる。なお、後述する実施例で例示したr22/pDG-comK株は、正常な胞子形成能を有する菌株であり、出願人が自社開発した菌株である。 Any Bacillus natto can be adopted as a parent strain of the new Bacillus natto mutant. For example, common commercially available bacteria such as Miyagino, Takahashi, Naruse and the like can be used, but various strains such as mutant strains and genetically modified strains having specific properties can also be used. The r22 / pDG-comK strain exemplified in the examples described later is a strain having normal spore-forming ability, and is a strain developed by the applicant in-house.
本発明の新規納豆菌変異株として具体的な菌株としては、DPA2株,DPA3株,DPA6株を挙げることができる。これらの菌株は、コンピテンス法を利用して、後述する実施例1に記載の方法により、納豆菌r22/pDG-comK株のspoVFA遺伝子をクロラムフェニコール耐性遺伝子で置換した納豆菌変異株の中から、胞子形成能及び発酵品質を指標として何段階もの選抜を経て選抜された菌株である。当該選抜株は、納豆上での胞子形成能が極めて低く、且つ納豆の品質特性が優れた新規納豆菌変異株である。 Specific strains of the novel Bacillus natto mutant of the present invention include DPA2, DPA3 and DPA6 strains. These strains are among the mutants of Bacillus natto strains in which the spoVFA gene of the Bacillus natto r22 / pDG-comK strain is replaced with a chloramphenicol resistance gene by the method described in Example 1 described later using the competence method. Therefore, it is a strain selected through several stages of selection using spore-forming ability and fermentation quality as indicators. The selected strain is a novel Bacillus natto mutant strain that has extremely low spore-forming ability on natto and has excellent natto quality characteristics.
当該選抜株のうち、特にDPA6株は総合的に最も優れた性質を有する菌株であったため、本願出願人は、当該菌株を独立行政法人製品評価技術基盤機構（千葉県木更津市かずさ鎌足2-5-8）に寄託申請した。当該菌株は、受託番号 NITE BP-02166としてDPA6の名称で2015年11月27日付で国際寄託が認められている。 Among the selected strains, the DPA6 strain in particular was the most comprehensive strain, so the applicant of the present application identified the strain as an independent administrative agency, Product Evaluation Technology Foundation (Kazusa Kamashizu, Kisarazu City, Chiba Prefecture). 5-8) applied for deposit. This strain is accepted as an international deposit on November 27, 2015 under the name DPA6 under the deposit number NITE BP-02166.
また、本発明の納豆上での胞子形成能低下納豆菌変異株としては、前記選抜菌に由来する納豆菌変異株、又は、前記選抜菌を突然変異させて作出した納豆菌変異株、又は、前記選抜菌に遺伝子導入し形質転換して作出した納豆菌変異株であって、；胞子形成能及び発酵品質に関する上記性質を有する納豆菌株であれば、；本発明の新規納豆菌変異株に該当する。 The spore-forming ability reduced natto mutant strain on natto of the present invention is a natto mutant strain derived from the selected bacteria, or a natto mutant mutant produced by mutating the selected bacteria, or A Bacillus natto mutant produced by gene transfer into the selected bacterium and transformed; if it is a Bacillus natto strain having the above-mentioned properties relating to spore-forming ability and fermentation quality, it corresponds to the novel Bacillus natto mutant of the present invention To do.
［納豆の製造］
本発明では、上記納豆菌変異株を用いて納豆を製造することによって、加熱処理による殺菌又は滅菌が可能な納豆を製造することが可能となる。ここで、殺菌、滅菌とは、納豆菌胞子数及び栄養細胞数の低減を意味する。
本発明において、納豆の製造方法としては、納豆菌として上記納豆菌変異株を用いることを除いては、常法に従って納豆を製造することができる。
[Manufacture of natto]
In the present invention, it is possible to produce natto that can be sterilized or sterilized by heat treatment, by producing natto using the mutant strain of Bacillus natto. Here, sterilization and sterilization mean a reduction in the number of Bacillus natto spores and the number of vegetative cells.
In the present invention, as a method for producing natto, natto can be produced according to a conventional method, except that the natto mutant is used as natto.
・原料大豆
本発明の納豆の製造方法では、通常の納豆の製造に用いることができる如何なる原料をも用いることができる。例えば、丸大豆、半割大豆、割砕大豆（引き割り納豆の原料）、脱脂大豆などを使用できる。特に高品質の納豆製造時に使用される中粒や大粒のものが好適である。これらの大豆は、生のまま用いることもできるが、乾燥処理を行ったもの（乾燥品）を用いることが一般的である。
-Raw material soybean In the manufacturing method of natto of this invention, any raw material which can be used for manufacture of normal natto can be used. For example, whole soybean, half soybean, cracked soybean (raw natto raw material), defatted soybean, etc. can be used. Particularly preferred are medium and large grains used in the production of high quality natto. These soybeans can be used as they are, but it is common to use those that have been dried (dried products).
本発明では、原料の大豆を常法により蒸煮大豆又は煮大豆にして用いる。成分の流亡を防ぐ意味では、蒸煮大豆が好適である。なお、蒸煮や煮る操作を行う前には、原料大豆を水に浸漬し、膨潤させて用いることが望ましい。
ここで、蒸煮大豆の具体的な調製手順としては、大豆を水中に6〜24時間程度浸漬した後、水切りして、100〜135℃の蒸気で10〜30分の蒸煮処理する方法を採用することができる。また、0.12〜0.22Mpaの高圧条件にて、加圧蒸煮する方法を採用することもできる。
また、煮大豆の具体的な調製手順としては、大豆を水中に6〜24時間程度浸漬した後、90〜100℃の湯で20〜50分間煮込む方法を採用することができる。
In the present invention, the raw soybean is used as a steamed soybean or boiled soybean by a conventional method. Steamed soybeans are preferable in terms of preventing the components from flowing away. In addition, it is desirable to immerse the raw material soybeans in water and swell them before performing the cooking or cooking operation.
Here, as a specific preparation procedure of the steamed soybean, a method is adopted in which the soybean is immersed in water for about 6 to 24 hours, drained, and steamed at 100 to 135 ° C. for 10 to 30 minutes. be able to. Moreover, the method of pressure-steaming on the high pressure conditions of 0.12-0.22Mpa is also employable.
Moreover, as a specific preparation procedure of boiled soybeans, a method of immersing soybeans in water for about 6 to 24 hours and then simmering in 90 to 100 ° C. hot water for 20 to 50 minutes can be employed.
・植菌（盛り込み）
本発明の納豆の製造方法において、上記納豆菌変異株の接種に用いる納豆菌の状態としては、胞子状態のものを用いることが好適である。胞子状態の納豆菌は、安定保存が可能で取扱いが容易であり、熱い煮豆等への接種の際にも死滅しないため、豆の雑菌汚染を防ぐことができる。また、胞子状態の納豆菌は、熱によるヒートショックにより、大豆への接種後速やかに発芽させることが可能となる。
・ Inoculation (including)
In the natto production method of the present invention, it is preferable to use a spore state as the state of Bacillus natto used for inoculation with the mutant of Bacillus natto. The spore-shaped Bacillus natto can be stably stored and is easy to handle, and since it does not die even when inoculated with hot boiled beans or the like, it can prevent contamination of beans with various bacteria. In addition, spore-shaped Bacillus natto can be germinated promptly after inoculation with soybeans by heat shock caused by heat.
大豆等への上記納豆菌変異株の植菌は、発酵を均一に行うため、大豆等と納豆菌が均一になるように添加（又は、接種, 散布など）した後、混合等を行うことが望ましい。好ましくは、上記納豆菌変異株の胞子懸濁液を調製し、液体状態にて添加して用いることが好適である。 Inoculation of the above-mentioned Bacillus natto mutants into soybeans etc. may be performed after addition (or inoculation, spraying, etc.) so that soybeans etc. and natto bacteria are evenly mixed in order to perform fermentation uniformly. desirable. It is preferable to prepare a spore suspension of the above-mentioned Bacillus natto mutant and add it in a liquid state.
ここで胞子懸濁液としては、ジピコリン酸を含有する液体培地（例えばグルタミン酸やグルコースを主原料とした合成培地、大豆煮汁、豆乳、酵母エキスなどを含む液体培地等）にて上記納豆菌変異株を培養した培養液を用いることができる。 Here, as the spore suspension, the Bacillus natto mutant strain described above is used in a liquid medium containing dipicolinic acid (for example, a synthetic medium containing glutamic acid or glucose as a main raw material, a liquid medium containing soybean broth, soy milk, yeast extract, or the like). Can be used.
植菌する納豆菌の数としては、常法に準じた菌濃度で特に限定はないが、蒸煮大豆1gあたり10３〜10６個、好ましくは10３〜10５個、最も好ましくは10３〜10４個であることが望ましい。 The number of Bacillus natto to be inoculated is not particularly limited by the bacterial concentration according to the conventional method, but is 10 3 to 10 6 , preferably 10 3 to 10 5 , most preferably 10 3 to 1 g of steamed soybean. 10 4 is desirable.
植菌する際の大豆の品温としては、55〜95℃、好ましくは60〜95℃、より好ましくは65〜95℃、さらに好ましくは70〜90℃、特に好ましくは75〜90℃とすることができる。上記範囲より大豆の品温が低い場合は雑菌汚染が生じる恐れがあり、また上記範囲より大豆の品温が高い場合は納豆菌胞子が死滅してしまい発酵不良となる恐れがある。 The temperature of soybean when inoculated is 55 to 95 ° C, preferably 60 to 95 ° C, more preferably 65 to 95 ° C, still more preferably 70 to 90 ° C, and particularly preferably 75 to 90 ° C. Can do. If the product temperature of soybean is lower than the above range, there is a risk of contamination with germs, and if the product temperature of soybean is higher than the above range, natto fungal spores may be killed, resulting in poor fermentation.
上記納豆菌変異株を植菌した大豆は、1〜数食分用の個容器に充填した後、個容器内にて後述する発酵を行うことが好適である。また、伝統的な方法として、煮沸した藁苞に充填して行うことも可能である。
また、数リットル体積容の容器等にて発酵を行うことも可能であるが、表面積に対する体積の値が大きくなると、中央部の豆に温度変化が伝わりにくくなることを考慮すると、大きめの容器を用いることは望ましくない。
It is preferable that the soybean inoculated with the Bacillus natto mutant is filled in an individual container for one to several meals and then subjected to fermentation described later in the individual container. In addition, as a traditional method, it is possible to fill a boiled rice cake.
It is also possible to perform fermentation in a container of several liters volume, etc., but considering that the temperature change is difficult to be transmitted to the beans in the center when the volume value relative to the surface area increases, a larger container is used. It is not desirable to use it.
ここで個容器としては、豆の充填が可能であれば、どんな容器を用いることもできる。また、後述する加熱殺菌を行う場合は、その加熱処理に耐えうる容器を用いる。具体的には納豆で一般に用いられるようなオパレイ(非塩ビ系ポリオレフィン樹脂)、PP、PSP、PS等を用いた合成樹脂性の容器や、カップ状の紙製の容器を用いることができる。
また、容器の形状として、当該容器を用いて直接、喫食のための掻き混ぜ（攪拌）ができるような形状のものが好適である。
また、発酵後は、蓋やシーリングによる封を行うことができる態様のものが好適である。
Here, any container can be used as long as it can be filled with beans. Moreover, when performing the heat sterilization mentioned later, the container which can endure the heat processing is used. Specifically, synthetic resin containers using opalay (non-vinyl chloride polyolefin resin), PP, PSP, PS, etc., which are generally used in natto, and cup-shaped paper containers can be used.
Moreover, the thing of the shape which can be stirred (stirring) for eating directly using the said container as a shape of a container is suitable.
Moreover, the thing of the aspect which can be sealed by a lid | cover or sealing after fermentation is suitable.
・発酵
本発明の納豆の製造方法において、納豆の発酵は、豆の品温を実質的に37〜53℃（好ましくは40〜50℃）である通常の納豆発酵温度帯に維持することで行うことが可能である。
また、本発明においては、室温を30〜50℃（好ましくは35〜45℃）の温度帯に維持することによって、発酵中の豆の品温を上記温度帯に維持することが可能となる。発酵熱によって豆の品温が上昇し、上記温度帯に維持されるためである。
豆の品温を、当該発酵温度帯に維持する所定時間（発酵時間）としては、特に制限はないが、12〜24時間、好ましくは16〜20時間を挙げることができる。
当該温度帯で発酵が進行することによって、納豆らしい風味の付与、柔らかい納豆らしい食感の付与、菌膜形成により白みがかった色調の付与、糸引き性の付与（ネバの付与）などが促進される。
-Fermentation In the natto production method of the present invention, natto fermentation is performed by maintaining the product temperature of the beans at a normal natto fermentation temperature range of substantially 37 to 53 ° C (preferably 40 to 50 ° C). It is possible.
Moreover, in this invention, it becomes possible to maintain the product temperature of the beans during fermentation in the said temperature range by maintaining room temperature in the temperature range of 30-50 degreeC (preferably 35-45 degreeC). This is because the product temperature of the beans rises due to the heat of fermentation and is maintained in the above temperature range.
Although there is no restriction | limiting in particular as predetermined time (fermentation time) which maintains the product temperature of a bean in the said fermentation temperature range, 12 to 24 hours, Preferably 16 to 20 hours can be mentioned.
Fermentation in the temperature range promotes the natto-like flavor, the soft natto-like texture, the whitish color due to the formation of the fungus, and the stringiness (neva). Is done.
なお、当該発酵において「実質的に温度帯に維持する」とは、完全に当該温度帯を外れないことを意味するものではなく、例えば、若干の温度範囲（例えば、2℃以内、好ましくは1℃以内）で、若干の時間（例えば、10分以内、好ましくは5分以内）であれば、当該温度帯を外れた品温となった場合も、当該発酵条件を満たすことを意味する。 In the fermentation, “maintaining substantially in the temperature range” does not mean that the temperature range is not completely removed. For example, a slight temperature range (for example, within 2 ° C., preferably 1 (Within ° C.) for a short time (for example, within 10 minutes, preferably within 5 minutes), it means that the fermentation condition is satisfied even when the product temperature is out of the temperature range.
・加熱殺菌
上述の納豆菌変異株はジピコリン酸を含まない納豆上では新たに胞子形成できないことから、上記発酵後に納豆菌栄養細胞を死滅させうる条件で加熱処理することにより、効果的に納豆を殺菌又は滅菌することが可能である。従って、本発明の納豆の製造方法においては、上記発酵後に加熱殺菌処理を行うことが好ましい。
-Heat sterilization As the above-mentioned mutants of Bacillus natto cannot newly form spores on natto that does not contain dipicolinic acid, natto can be effectively treated by heat treatment under conditions that can kill natto-vegetative vegetative cells after the fermentation. It can be sterilized or sterilized. Therefore, in the method for producing natto of the present invention, it is preferable to perform heat sterilization after the fermentation.
本発明の加熱殺菌における処理条件は、通常の納豆菌（親株）の栄養細胞を死滅させ得る条件とすることができる。具体的な処理条件としては55〜100℃、好ましくは60〜90℃、より好ましくは60〜75℃で、30〜200分間、好ましくは60〜180分間、より好ましくは90〜150分間、特に好ましくは110〜130分間である。このとき、湿度は相対湿度で0〜60％とすることができる。 The treatment condition in the heat sterilization of the present invention can be a condition capable of killing vegetative cells of normal natto bacteria (parent strain). Specific treatment conditions are 55 to 100 ° C., preferably 60 to 90 ° C., more preferably 60 to 75 ° C., 30 to 200 minutes, preferably 60 to 180 minutes, more preferably 90 to 150 minutes, particularly preferably. Is 110-130 minutes. At this time, the relative humidity can be 0 to 60%.
当該加熱殺菌は、処理条件を変えて2段階で行ってもよい。例えば、1段階目の加熱殺菌処理を55〜75℃、好ましくは60〜70℃、より好ましくは65±3℃で、相対湿度30〜90％、好ましくは40〜80％、より好ましくは50〜70％、さらに好ましくは55〜65％、特に好ましくは60±3％で、60〜120分間、好ましくは70〜110分間、より好ましくは80〜100分間、さらに好ましくは85〜95分間の条件で行った後に、2段階目の加熱殺菌処理を60〜100℃、65〜90℃、好ましくは70〜80℃、より好ましくは75±3℃で、相対湿度0〜20％、好ましくは0〜10％、より好ましくは0％で、10〜80分間、好ましくは20〜70分間、より好ましくは30〜60分間、さらに好ましくは40〜50分間の条件で行うことができる。 The heat sterilization may be performed in two stages by changing processing conditions. For example, the first stage heat sterilization treatment is 55 to 75 ° C., preferably 60 to 70 ° C., more preferably 65 ± 3 ° C., relative humidity 30 to 90%, preferably 40 to 80%, more preferably 50 to 70%, more preferably 55 to 65%, particularly preferably 60 ± 3%, for 60 to 120 minutes, preferably 70 to 110 minutes, more preferably 80 to 100 minutes, more preferably 85 to 95 minutes. After performing the second stage heat sterilization treatment at 60-100 ° C., 65-90 ° C., preferably 70-80 ° C., more preferably 75 ± 3 ° C., relative humidity 0-20%, preferably 0-10 %, More preferably 0%, for 10 to 80 minutes, preferably 20 to 70 minutes, more preferably 30 to 60 minutes, still more preferably 40 to 50 minutes.
・熟成
上記の加熱殺菌処理終了後、二次発酵を抑制するために、通常、3℃以上10℃未満、好ましくは3℃以上8℃未満、より好ましくは3℃以上6℃未満の低温になるようにして6時間〜3日間、好ましくは8時間〜2日間、より好ましくは24時間程度、熟成を行い、製造は完了する。
・ Aging After completion of the above heat sterilization treatment, in order to suppress secondary fermentation, the temperature is usually 3 ° C. or higher and lower than 10 ° C., preferably 3 ° C. or higher and lower than 8 ° C., more preferably 3 ° C. or higher and lower than 6 ° C. Thus, aging is performed for 6 hours to 3 days, preferably 8 hours to 2 days, more preferably about 24 hours, and the production is completed.
［製造された納豆の性質］
・胞子含有量
上記の方法により製造された本発明の納豆は、通常の納豆と比べて納豆菌胞子含有量が少ないものである。なぜなら、納豆にはジピコリン酸が含まれていないため、発酵工程中に当該納豆菌変異株が新たに胞子形成できない（胞子形成能が極めて低い）ためである。通常の納豆中の納豆菌胞子数は納豆１ｇ当たり１０７個から１０９個程度であるが、本発明の納豆中の納豆菌胞子数は納豆１ｇ当たり１０５個以下、好ましくは５×１０４個以下、より好ましくは３×１０４個以下とすることができる。また、上記納豆菌変異株は新たに胞子形成できない（胞子形成能が極めて低い）ため、本発明の納豆中の胞子数は納豆の賞味又は消費期限内を通じて上記範囲内に維持される。
従って、本発明の納豆は、加工食品の原料として利用した場合でも、工場の製造ラインを胞子で汚染するおそれが少ないため、幅広い加工食品に適用可能である。
[Properties of manufactured natto]
-Spore content The natto of the present invention produced by the above method has a low natto fungal spore content as compared with normal natto. This is because natto does not contain dipicolinic acid, so that the mutant of Bacillus natto cannot newly form spores during the fermentation process (the ability to form spores is extremely low). Although the number of Bacillus natto spores during normal natto is 10 9 about 10 7 per natto 1g, natto number of bacteria spores in natto of the present invention is 10 5 or less per natto 1g, preferably 5 × 10 4 Or less, more preferably 3 × 10 4 or less. Moreover, since the said Bacillus natto mutant cannot newly form a spore (the spore-forming ability is extremely low), the number of spores in the natto of the present invention is maintained within the above range throughout the natto taste or the expiration date.
Therefore, even when the natto of the present invention is used as a raw material for processed foods, it can be applied to a wide range of processed foods because there is little risk of contaminating a factory production line with spores.
なお、納豆中の胞子数の測定は、例えば納豆懸濁液を加熱処理した後、寒天培地を用いて耐熱性菌数を求めることにより行うことができる。具体的には、納豆をストマッカー袋と呼ばれるフィルターの付いた袋に入れてリン酸緩衝液を注入し、ストマッカーと呼ばれる機器にて懸濁した納豆懸濁液をさらにリン酸緩衝液にて希釈し、それを75℃で15分間加熱処理した後、LB寒天培地に塗抹培養して、そこに出現したコロニー数から算出することができる。 In addition, the measurement of the spore number in natto can be performed by calculating | requiring the number of heat-resistant bacteria using an agar medium, for example after heat-processing a natto suspension. Specifically, natto is put into a bag with a filter called a stomacher bag, a phosphate buffer solution is injected, and the natto suspension suspended in a device called a stomacher is further diluted with a phosphate buffer solution. It can be calculated from the number of colonies that appeared after being heat-treated at 75 ° C. for 15 minutes, smeared on LB agar medium.
・栄養細胞含有量
上記の製造方法において発酵後に加熱殺菌工程を行った場合に製造される本発明の納豆は、発酵後に加熱殺菌を行わない通常の納豆と比べて、納豆菌栄養細胞含有量が少ないものである。具体的には、加熱殺菌処理条件によって異なるが、納豆製造直後の総菌数として、納豆１ｇ当たり１０６個以下、好ましくは５×１０５個以下、より好ましくは３×１０５個以下、さらに好ましくは１０５個以下、特に好ましくは８×１０４個以下とすることができる。
・ Nutrition cell content Natto of the present invention produced when the heat sterilization process is performed after fermentation in the above production method has a natto fungus vegetative cell content compared to normal natto that is not heat sterilized after fermentation. There are few things. Specifically, although depending on the heat sterilization treatment conditions, the total number of bacteria immediately after natto production is 10 6 or less per 1 g of natto, preferably 5 × 10 5 or less, more preferably 3 × 10 5 or less, The number is preferably 10 5 or less, and particularly preferably 8 × 10 4 or less.
ここで、総菌数（胞子と栄養細胞の総和）の測定は、納豆懸濁液の菌数を寒天培地を用いて求めることにより行うことができる。具体的には、納豆をストマッカー袋に入れてリン酸緩衝液を注入し、ストマッカーにて懸濁した納豆懸濁液をさらにリン酸緩衝液にて希釈した後、LB寒天培地に塗抹培養して、そこに出現したコロニー数から算出することができる。 Here, the total number of bacteria (total of spores and vegetative cells) can be measured by determining the number of bacteria in the natto suspension using an agar medium. Specifically, natto is put into a stomacher bag, a phosphate buffer solution is injected, and the natto suspension suspended in the stomacher is further diluted with a phosphate buffer solution, and then smeared on an LB agar medium. It can be calculated from the number of colonies appearing there.
・納豆の品質
上記の方法により製造された本発明の納豆は、十分な糸引き性（ネバ）を有し、菌膜が均一に広がって納豆らしい良好な外観を有する納豆となる。
-Quality of natto The natto of the present invention produced by the above method has a sufficient stringiness (neva), and the fungus membrane spreads uniformly and has a good appearance like natto.
［胞子及び栄養細胞の低減方法］
本発明においては、上記の方法に従い納豆を製造することによって、納豆菌の胞子及び栄養細胞の含有量が低減された納豆を製造することができる。従って、本発明は、上記納豆菌変異株を用いて納豆を製造することを特徴とする、納豆における納豆菌の胞子及び栄養細胞の低減方法を提供するものである。
[Method for reducing spores and vegetative cells]
In the present invention, by producing natto according to the above method, it is possible to produce natto with a reduced content of spore and vegetative cells of Bacillus natto. Accordingly, the present invention provides a method for reducing the spore and vegetative cells of natto bacteria in natto, characterized in that natto is produced using the natto bacteria mutant.
本発明においては、納豆発酵後に上記の加熱殺菌工程を行うことで、納豆における納豆菌の胞子及び栄養細胞（特に栄養細胞）の含有量を顕著に低減することができる。当該加熱殺菌における処理条件は上述の通りであるが、例えば55〜100℃、好ましくは60〜90℃、より好ましくは60〜75℃で、30〜200分間、好ましくは60〜180分間、より好ましくは90〜150分間、特に好ましくは110〜130分間とすることができる。このとき、湿度は相対湿度で0〜60％とすることができる。また、上述したように、当該加熱殺菌は処理条件を変えて2段階で行うこともできる。 In the present invention, by performing the heat sterilization step after natto fermentation, the contents of natto bacteria spores and vegetative cells (particularly vegetative cells) in natto can be significantly reduced. The treatment conditions in the heat sterilization are as described above. For example, 55 to 100 ° C, preferably 60 to 90 ° C, more preferably 60 to 75 ° C, 30 to 200 minutes, preferably 60 to 180 minutes, more preferably. Can be 90 to 150 minutes, particularly preferably 110 to 130 minutes. At this time, the relative humidity can be 0 to 60%. Further, as described above, the heat sterilization can be performed in two stages by changing the processing conditions.
以下、実施例を挙げて本発明を説明するが、本発明の範囲はこれらにより限定されるものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, the scope of the present invention is not limited by these.
実施例１（ジピコリン酸合成能欠損納豆菌の作成）
出願人が自社開発した納豆菌r22/pDG-comK株を宿主に用いて、相同組換えによりspoVFA遺伝子をクロラムフェニコール耐性遺伝子で置換したspoVFA遺伝子破壊株を作製した。
Example 1 (Preparation of dipicolinic acid synthesis ability deficient natto)
Using the natto r22 / pDG-comK strain developed in-house by the applicant as a host, a spoVFA gene-disrupted strain was prepared by replacing the spoVFA gene with a chloramphenicol resistance gene by homologous recombination.
１．spoVFA遺伝子破壊株の作製
（1）核酸供与体の調製
まず、定法に従いr22/pDG-comK株ゲノムDNAを調製した。次に、spoVFA遺伝子上流部（約2.5kb）を配列表の配列番号1、2に記載のプライマーを用いて、spoVFA遺伝子下流部（約3.0kb）を配列番号3、4に記載のプライマーを用いて、r22/pDG-comK株ゲノムDNAを鋳型としたPCRにより増幅した。また、クロラムフェニコール耐性遺伝子（約0.7kbp）は、pC194（ATCC 37128株保有のプラスミド）を鋳型として、配列番号5、6に記載のプライマーを用いてPCRにより増幅した。尚、PCRはTakara ExTaq（タカラバイオ）を用いて添付のマニュアルの推奨条件にて行った（以下同様）。
1. Preparation of spoVFA gene disrupted strain (1) Preparation of nucleic acid donor First, r22 / pDG-comK genomic DNA was prepared according to a conventional method. Next, the spoVFA gene upstream part (about 2.5 kb) is used with the primers shown in SEQ ID Nos. 1 and 2 in the sequence listing, and the spoVFA gene downstream part (about 3.0 kb) is used with the primers shown in SEQ ID Nos. 3 and 4. The r22 / pDG-comK strain genomic DNA was amplified by PCR. The chloramphenicol resistance gene (approximately 0.7 kbp) was amplified by PCR using pC194 (a plasmid possessed by the ATCC 37128 strain) as a template and the primers described in SEQ ID NOs: 5 and 6. PCR was performed using Takara ExTaq (Takara Bio) under the recommended conditions in the attached manual (the same applies hereinafter).
得られたPCR産物はGenElute PCR Clean-Up Kit（シグマ）を用いて精製を行い、FusionPCRに供した。尚、非特異増幅が見られたPCR産物は、QIAquick Gel Extraction Kit（Qiagen社製）を用いて精製を行い、FusionPCRに供した。当該FusionPCRにより、spoVFA遺伝子の代わりとしてクロラムフェニコール耐性遺伝子を挟むように、それぞれ増幅したspoVFA遺伝子の上流、クロラムフェニコール耐性遺伝子、およびspoVFA遺伝子の下流を連結した。 The obtained PCR product was purified using GenElute PCR Clean-Up Kit (Sigma) and subjected to Fusion PCR. The PCR product in which non-specific amplification was observed was purified using QIAquick Gel Extraction Kit (manufactured by Qiagen) and subjected to Fusion PCR. By the Fusion PCR, the upstream of the amplified spoVFA gene, the downstream of the chloramphenicol resistance gene, and the downstream of the spoVFA gene were linked so that the chloramphenicol resistance gene was inserted instead of the spoVFA gene.
（FusionPCR条件）
・組成：×10ExTaqBuffer 10μL、dNTP 8μL、各100μM Primer 1μL、鋳型2.5μL、ExTaq 0.5μL／100μL
・PCRサイクル：95℃、5min.→（95℃、0.5min.→55℃、0.5min.→72℃、7min.）×30→72℃、7min.
(FusionPCR conditions)
Composition: x10 ExTaqBuffer 10 μL, dNTP 8 μL, each 100 μM Primer 1 μL, template 2.5 μL, ExTaq 0.5 μL / 100 μL
PCR cycle: 95 ° C, 5 min. → (95 ° C, 0.5 min. → 55 ° C, 0.5 min. → 72 ° C, 7 min.) × 30 → 72 ° C, 7 min.
次いで、FusionPCRにより得られたPCR産物を用いてアガロースゲル電気泳動を行い、ゲル中の目的のバンド（約6.2kbp）を切り出してQIAquick Gel Extraction Kit（キアゲン）で精製を行った。さらに、得られた精製物を鋳型として、配列番号7、8に記載のプライマーを用いて以下の条件でnested PCRを行うことにより、遺伝子破壊用の核酸供与体を調製した。 Subsequently, agarose gel electrophoresis was performed using the PCR product obtained by Fusion PCR, and the target band (about 6.2 kbp) in the gel was cut out and purified by QIAquick Gel Extraction Kit (Qiagen). Furthermore, a nucleic acid donor for gene disruption was prepared by performing nested PCR under the following conditions using the obtained purified product as a template and the primers described in SEQ ID NOs: 7 and 8.
（nested PCR条件）
・組成：×10ExTaqBuffer 10μL、dNTP 8μL、各100μM Primer 1μL、鋳型1μL、ExTaq 0.5μL／100μL
・PCRサイクル：95℃、5min.→（95℃、0.5min.→55℃、0.5min.→72℃、7min.)×30→72℃、7min.
(Nested PCR conditions)
Composition: x10 ExTaqBuffer 10 μL, dNTP 8 μL, each 100 μM Primer 1 μL, template 1 μL, ExTaq 0.5 μL / 100 μL
PCR cycle: 95 ° C, 5 min. → (95 ° C, 0.5 min. → 55 ° C, 0.5 min. → 72 ° C, 7 min.) × 30 → 72 ° C, 7 min.
（2）spoVFA遺伝子破壊株の取得
r22/pDG-comK株からCI培地を用いてコンピテントセルを調製し、上記(1)で調製した核酸供与体を使用して形質転換を行った。
(2) Acquisition of spoVFA gene disruption strain
Competent cells were prepared from r22 / pDG-comK strain using CI medium and transformed using the nucleic acid donor prepared in (1) above.
すなわち、r22/pDG-comK株をカナマイシン（7.5μg/ml）入りLB寒天培地に植菌し、28℃、16hr培養し、出現したコロニーを再度カナマイシン入りLB寒天培地6枚に植菌し28℃、16hr培養した。LB寒天培地に出現したコロニーのうち若いコロニーを選び、白金耳でカナマイシン（7.5μg/ml）入りCI培地 5.5ml（MM培地 10ml、50％(w/v)グルコース水溶液 0.1ml、1M硫酸マグネシウム7水和物水溶液 0.1ml、5％(w/v)酵母エキス水溶液 0.1ml、0.1g/lビオチン水溶液 0.01ml）に懸濁し、ボルテックスミキサーにて激しく攪拌した。2〜3分間自然沈降させ不溶物を取り除いて得られた懸濁液を、カナマイシン入りCI培地44.5mlを含む坂口フラスコに添加し37℃、120rpmで振盪培養した。
尚、懸濁液の添加量は濁度がOD660＝0.04〜0.06となるように調整し、濁度は分光光度計（HITACHI U-2000）を用い660nmにて測定した。
Specifically, the r22 / pDG-comK strain was inoculated into an LB agar medium containing kanamycin (7.5 μg / ml), cultured at 28 ° C. for 16 hours, and the emerged colony was again inoculated into 6 LB agar media containing kanamycin at 28 ° C. For 16 hours. Select a young colony from the colonies that appeared on the LB agar medium, and 5.5 ml of CI medium containing kanamycin (7.5 μg / ml) with a platinum loop (10 ml of MM medium, 0.1 ml of 50% (w / v) glucose solution, 1M magnesium sulfate 7 The suspension was suspended in 0.1 ml of a hydrate aqueous solution, 0.1 ml of a 5% (w / v) yeast extract aqueous solution and 0.01 ml of a 0.1 g / l biotin aqueous solution, and vigorously stirred with a vortex mixer. The suspension obtained by natural sedimentation for 2 to 3 minutes to remove insolubles was added to a Sakaguchi flask containing 44.5 ml of CI medium containing kanamycin, and cultured with shaking at 37 ° C. and 120 rpm.
The added amount of the suspension was adjusted so that the turbidity was OD660 = 0.04 to 0.06, and the turbidity was measured at 660 nm using a spectrophotometer (HITACHI U-2000).
培養液は経時的にその濁度を測定し、対数増殖期から定常期になった時点から１時間後に、培養液から菌体を集菌した。尚、集菌は培養液0.5mlをエッペンドルフチューブに分取し、遠心分離（10,000rpm、4℃、1min.）し、上清の一部を除いた後、再度同条件で遠心分離し上清を完全に除去し、コンピテントセルを沈殿として得た。 The turbidity of the culture broth was measured over time, and the cells were collected from the culture broth 1 hour after the time from the logarithmic growth phase to the stationary phase. Collect 0.5 ml of the culture solution in an Eppendorf tube, centrifuge (10,000 rpm, 4 ° C, 1 min.), Remove a portion of the supernatant, and centrifuge again under the same conditions. Was completely removed and a competent cell was obtained as a precipitate.
次に、得られたコンピテントセルにカナマイシン（7.5μg/ml）入りCII培地（MM培地 10ml、50％グルコース水溶液 0.1ml、1M硫酸マグネシウム7水和物水溶液 0.1ml、5％酵母エキス水溶液 0.05ml、0.1g/lビオチン水溶液 0.01ml）1.0mlを加え穏やかに懸濁した。この懸濁液0.1mlに上記(1)で調製した供与DNA1μgを加え、37℃、150rpm、1.5hr振盪培養しコンピテントセルにDNAを取り込ませた。さらに、この培養液にLB培地0．3mlを加え、37℃、150rpm、2hr振盪培養し、カナマイシン及びクロラムフェニコール耐性遺伝子を発現させた後、カナマイシン及びクロラムフェニコールを含むLB寒天培地に塗抹培養（37℃、一夜）した。 Next, CII medium containing kanamycin (7.5 μg / ml) was added to the obtained competent cell (MM medium 10 ml, 50% glucose aqueous solution 0.1 ml, 1M magnesium sulfate heptahydrate aqueous solution 0.1 ml, 5% yeast extract aqueous solution 0.05 ml. Then, 1.0 ml of 0.1 g / l biotin aqueous solution 0.01 ml) was added and gently suspended. 1 μg of the donor DNA prepared in (1) above was added to 0.1 ml of this suspension, and the suspension was cultured with shaking at 37 ° C., 150 rpm for 1.5 hr, and the DNA was incorporated into competent cells. Further, 0.3 ml of LB medium was added to this culture solution, and cultured with shaking at 37 ° C., 150 rpm, 2 hours to express kanamycin and chloramphenicol resistance genes. Then, the LB agar medium containing kanamycin and chloramphenicol was added. Smear culture (37 ° C., overnight) was performed.
（3）形質転換体の確認
カナマイシン及びクロラムフェニコールを含むLB寒天培地にて単一コロニー分離後、クロラムフェニコールを含むLB培地5mlに植菌し、37℃、150rpm、一夜振盪培養した。得られた培養液約0.3mlから遠心分離（12000rpm、4℃、1min.）により菌体を取得し、DNeasy Tissue Kit(キアゲン製)を用いてゲノムを抽出した。このゲノムを鋳型として、配列番号9、10に記載のプライマーを用いてPCR増幅を行い、目的とする0.75kbpのPCR産物の増幅の有無で組換えを確認した。
得られた形質転換体20株の中から、ジピコリン酸非添加培地での胞子形成能が低く、且つ、納豆の製造が可能であった６株（DAP1〜6）を選抜し、以下の試験に供試した。
(3) Confirmation of transformant Single colonies were isolated on LB agar medium containing kanamycin and chloramphenicol, then inoculated into 5 ml of LB medium containing chloramphenicol, and cultured with shaking at 37 ° C, 150 rpm overnight. . Bacteria were obtained from about 0.3 ml of the obtained culture solution by centrifugation (12000 rpm, 4 ° C., 1 min.), And the genome was extracted using DNeasy Tissue Kit (manufactured by Qiagen). Using this genome as a template, PCR amplification was performed using the primers described in SEQ ID NOs: 9 and 10, and recombination was confirmed by the presence or absence of amplification of the desired 0.75 kbp PCR product.
From the 20 transformants obtained, 6 strains (DAP1-6) that had low sporulation ability in dipicolinic acid-free medium and were able to produce natto were selected for the following test. I tried it.
（3）他の遺伝子破壊株
なお、比較例として、上記と同様の方法により、r22/pDG-comK株を宿主とし、sigK（胞子形成後期に作用するシグマ因子）遺伝子をエリスロマイシン耐性遺伝子（pE194由来）により置換した遺伝子破壊株（sigK2〜4）、dacB（胞子の耐熱性に関与するペニシリン結合蛋白質）遺伝子をクロラムフェニコール耐性遺伝子（pC194由来）により置換した遺伝子破壊株（dacB1〜4）、およびasnO（胞子形成期に作用するアスパラギン合成酵素）遺伝子をクロラムフェニコール耐性遺伝子（pC194由来）により置換した遺伝子破壊株（asnO3,6）を作製し、以下の試験に供試した。
(3) Other gene-disrupted strains As a comparative example, the sigK (sigma factor acting in the late sporulation stage) gene was derived from the erythromycin resistance gene (derived from pE194) using the r22 / pDG-comK strain as a host by the same method as above ) Gene-disrupted strains (sigK2-4), gene-disrupted strains (dacB1-4) in which dacB (penicillin-binding protein involved in spore heat resistance) gene is replaced by a chloramphenicol resistance gene (derived from pC194), And a gene-disrupted strain (asnO3,6) in which the asnO (asparagine synthase acting in the spore-forming stage) gene was replaced with a chloramphenicol resistance gene (derived from pC194) was prepared for the following test.
２．胞子液の調製と耐熱性評価
作製した遺伝子破壊株を5ml酵母エキス培地に植菌し37℃、150rpm、一夜振盪培養した。次に酵母エキス培養液0.1mlを10ml胞子形成培地（Y入り）／試験管に植菌し37℃、150rpm、24hr振盪培養することで胞子液を調製した。また、r22/pDG-comK株についても同様の手順で胞子液を調製した。
2. Preparation of Spore Solution and Evaluation of Heat Resistance The prepared gene disruption strain was inoculated into a 5 ml yeast extract medium and cultured overnight at 37 ° C. and 150 rpm with shaking. Next, 0.1 ml of the yeast extract culture solution was inoculated into a 10 ml spore formation medium (with Y) / test tube, and cultured at 37 ° C., 150 rpm, for 24 hours with shaking to prepare a spore solution. A spore solution was also prepared for the r22 / pDG-comK strain in the same manner.
調製した胞子液について、75℃での耐熱性を評価した。
胞子液中に含まれる胞子数は、適切に50mM燐酸Na緩衝液（pH7）で希釈した胞子液を75℃、15min.加熱した後、LB寒天培地に塗抹培養し出現したコロニー数から算出した。それぞれの胞子数測定結果を表５、６に示す。
About the prepared spore solution, the heat resistance at 75 ° C. was evaluated.
The number of spores contained in the spore solution was calculated from the number of colonies that appeared after the spore solution appropriately diluted with 50 mM Na phosphate buffer (pH 7) was heated at 75 ° C. for 15 min and smeared on the LB agar medium. The results of measuring the number of spores are shown in Tables 5 and 6.
表５、６から、sigK破壊株、dacB破壊株、asnO破壊株およびspoVFA破壊株において、リン酸緩衝液での胞子の耐熱性低下が確認できた。 From Tables 5 and 6, in the sigK disrupted strain, dacB disrupted strain, asnO disrupted strain, and spoVFA disrupted strain, it was confirmed that the heat resistance of the spore was reduced in the phosphate buffer.
３．耐熱性評価（ジピコリン酸の有無の影響）
上記のspoVFA破壊株のうち、最も胞子形成率が低かったDPA2株、最も胞子形成率が高かったDPA3株、及び胞子形成率が最も平均値に近かったDPA6株について、ジピコリン酸（DPA）存在下で胞子形成が復帰するかどうか調べた。
spoVFA破壊株を5ml酵母エキス培地に植菌し37℃、150rpm、一夜振盪培養した。次に終夜培養液0.1mlを200μg/mLのDPAを含む10ml胞子形成培地に植菌し37℃、150rpm、24hr振盪培養することで胞子液を調製した。
調製した胞子液を4℃にて4日間保存した後、胞子液中に含まれる胞子数は、適当に50mMリン酸Na緩衝液（pH7）で希釈した胞子液を75℃、15min.加熱した後、LB寒天培地に塗抹培養し出現したコロニー数から算出した。胞子数測定結果を表７に示す。
3. Evaluation of heat resistance (effect of presence or absence of dipicolinic acid)
Among the above spoVFA-disrupted strains, DPA2 strain with the lowest sporulation rate, DPA3 strain with the highest sporulation rate, and DPA6 strain with the highest sporulation rate in the presence of dipicolinic acid (DPA) It was investigated whether sporulation returned.
The spoVFA-disrupted strain was inoculated into 5 ml yeast extract medium and cultured with shaking at 37 ° C., 150 rpm overnight. Next, 0.1 ml of the overnight culture solution was inoculated into a 10 ml spore formation medium containing 200 μg / mL of DPA, and the spore solution was prepared by shaking culture at 37 ° C., 150 rpm, 24 hours.
After storing the prepared spore solution at 4 ° C for 4 days, the number of spores contained in the spore solution was determined by heating the spore solution diluted with 50 mM Na phosphate buffer (pH 7) at 75 ° C for 15 min. It was calculated from the number of colonies that appeared after smear culture on LB agar medium. The spore count measurement results are shown in Table 7.
表７から、spoVFA破壊株のDPA非添加の胞子形成培地での胞子数はいずれも10の6乗オーダーであったが、DPAを添加した胞子形成培地では10の8乗オーダーに回復し、親株と同等の胞子数であった。中でも、DPA6株は、DPA添加時と比較したDPA無添加での胞子形成能の低下率が最も高かったことから、以降の実施例ではこの菌株を使用した。なお、この菌株は、受託番号 NITE BP-02166として「DPA6」の名称で国際寄託されている。
また、今回は4℃で4日間保存した胞子液を用いており、DPA添加培地で大量生産した胞子を長期保存しておけば、納豆の製造において植菌用胞子として利用可能であると考えられた。
From Table 7, the spoVFA-disrupted strains in sporulation medium without DPA were all on the order of 10 6, but in the sporulation medium with DPA added, it recovered to the order of 10 to the 8th order. The number of spores was the same. Among these strains, DPA6 strain had the highest rate of decrease in sporulation capacity without DPA compared to when DPA was added, so this strain was used in the following examples. This strain has been deposited internationally under the name “DPA6” under the accession number NITE BP-02166.
In addition, this time, spore solution stored at 4 ° C for 4 days is used, and if the spores mass-produced in DPA-added medium are stored for a long time, it can be used as spore for inoculation in the production of natto. It was.
なお、胞子形成に関与するsigK遺伝子、asnO遺伝子を破壊した枯草菌株については、不可逆的に胞子形成が阻害されるということが、当業者の一般常識となっている。従って、上記２．でspoVFA破壊株と同様に胞子の耐熱性低下が認められたsigK破壊株及びasnO破壊株についても、これらはいずれも胞子形成に関与する遺伝子破壊株であることから、胞子形成の復帰は不可能であると考えられる。 In addition, it is common knowledge of those skilled in the art that spore formation is irreversibly inhibited in Bacillus subtilis strains in which the sigK gene and asnO gene involved in sporulation are disrupted. Therefore, the above 2. In both the sigK disrupted strain and the asnO disrupted strain, in which a decrease in heat resistance of the spore was observed in the same manner as the spoVFA disrupted strain, these are gene disrupted strains that are involved in sporulation, so spore formation cannot be restored. It is thought that.
実施例２（ジピコリン酸合成能欠損納豆菌を用いた納豆製造試験）
(1)納豆の製造
実施例１の３．に記載の方法（DPA添加）で調製したspoVFA破壊株の胞子液を添加して納豆を製造した。
乾燥大豆を水に終夜浸漬し、水切りした後、1.65kg/cm２で30分間加圧蒸煮した。蒸煮した大豆（75℃）50gあたり、胞子液1ml（蒸煮大豆1gあたり納豆菌胞子数５０００個）を添加し、軽く均一化した。その後、50gずつ個容器に入れて蓋をし、46℃ 16時間の温度条件にて発酵させた。発酵後、65℃ 1.5時間 湿度60％の条件で加熱処理し、さらに75℃ 30分〜1時間 湿度0％の条件で加熱処理を行うことで、納豆を製造した。
また、比較例として、r22/pDG-comK株（親株）の胞子液を用いて上記と同様に納豆を製造した。
Example 2 (Natto production test using dipicolinic acid synthesis ability deficient natto)
(1) Manufacture of natto The spore solution of spoVFA-disrupted strain prepared by the method described above (DPA addition) was added to produce natto.
Dried soybeans were soaked in water overnight, drained, and then steamed under pressure at 1.65 kg / cm 2 for 30 minutes. 1 ml of spore solution (5,000 natto spores per 1 g of steamed soybeans) was added per 50 g of steamed soybean (75 ° C.), and lightly homogenized. Thereafter, 50 g each was put into an individual container, capped, and fermented at 46 ° C. for 16 hours. After fermentation, natto was produced by heat treatment at 65 ° C. for 1.5 hours and humidity of 60%, and further by heat treatment at 75 ° C. for 30 minutes to 1 hour and humidity of 0%.
As a comparative example, natto was produced in the same manner as described above using a spore solution of r22 / pDG-comK strain (parent strain).
(2)胞子数、総菌数の測定
各加熱条件における納豆中の胞子数、総菌数を測定した。
胞子数の測定は、納豆50gをストマッカー袋に入れて、50mMリン酸緩衝液(pH7.0)を150gを投入し、ストマッカーで1分懸濁することにより納豆懸濁液を調製した。この納豆懸濁液中の胞子数を実施例１の３．と同様にして測定した。
総菌数の測定は、上記納豆懸濁液を75℃で15分処理する前に、適切に希釈しLB寒天培地に塗抹培養することにより行った。
胞子数、総菌数の測定結果を表８に示す。
(2) Measurement of spore count and total bacterial count The spore count and total bacterial count in natto under each heating condition were measured.
For the measurement of the number of spores, natto suspension was prepared by putting 50 g of natto into a stomacher bag, adding 150 g of 50 mM phosphate buffer (pH 7.0), and suspending in the stomacher for 1 minute. The number of spores in this natto suspension was determined according to 3 in Example 1. Measured in the same manner as above.
The total number of bacteria was measured by appropriately diluting the natto suspension and treating it on a LB agar medium before being treated at 75 ° C. for 15 minutes.
Table 8 shows the measurement results of the number of spores and the total number of bacteria.
表８から、DPA6株を用いた納豆は、65℃1.5時間加熱後でも、親株を用いた納豆と比べて胞子数が約1/1000であり、胞子数が顕著に少ないことがわかった。総菌数においても、親株と比べてDPA6株の納豆は1/10未満と非常に少なかった。また、さらに75℃30分〜1時間加熱することにより、親株と比べて総菌数を1/100〜1/1000低減することができた。 From Table 8, it was found that natto using the DPA6 strain had a spore count of about 1/1000 compared with natto using the parent strain even after heating at 65 ° C. for 1.5 hours, and the number of spores was significantly smaller. In terms of the total number of bacteria, the natto of the DPA6 strain was much less than 1/10 compared to the parent strain. Furthermore, by further heating at 75 ° C. for 30 minutes to 1 hour, the total number of bacteria could be reduced by 1/100 to 1/1000 compared to the parent strain.
(3)納豆の品質評価
以下の評価基準に基づいて、各加熱条件における納豆の品質（糸引き、外観）を評価した。評価結果を表９、１０に示す。
(3) Quality evaluation of natto Based on the following evaluation criteria, the quality (string drawing, appearance) of natto under each heating condition was evaluated. The evaluation results are shown in Tables 9 and 10.
［糸引きの評価基準］
◎：非常に強い
○：強い
△：普通（混ぜた塊を薬さじで持ち上げても空中で保持できずに粒が落ちる）
×：弱い（混ぜても塊が形成されない）
[Evaluation criteria for stringing]
◎: Extremely strong ○: Strong △: Normal (Even if the mixed lump is lifted with a spoon, it cannot be held in the air and the grains fall)
X: weak (no lump is formed even when mixed)
［外観の評価基準］
○：菌膜が均一に広がっている
△：菌膜は認められるがごく一部であり不均一
[Evaluation criteria for appearance]
○: Bacterial membrane spread evenly △: Bacterial membrane is observed but only a small part is uneven
表９、１０から、DPA6株を用いた納豆は、親株を用いた納豆と同様に、納豆らしい良好な糸引き、外観を有していることが示された。 From Tables 9 and 10, it was shown that natto using DPA6 strain had good stringiness and appearance similar to natto, similar to natto using parent strain.
本発明によれば、納豆中では胞子を形成しないが、ジピコリン酸を含有する培地では通常の納豆菌と同様に胞子形成可能である、新規な納豆菌株が提供される。本発明の納豆菌株によれば、従来の納豆製造工程によって、胞子数は少ないが、その他の点においては従来の一般的な納豆と同等の品質を備えた納豆を製造することができる。
また、本発明の納豆は、納豆の品質を損なわない程度の加熱条件による殺菌が可能であるため、納豆を利用した各種加工食品の開発を促進することが期待される。
従って、本発明は、納豆およびそれを利用した加工食品の製造において利用可能である。
ADVANTAGE OF THE INVENTION According to this invention, the novel natto strain which does not form a spore in natto but can form a spore similarly to normal natto bacteria in the culture medium containing dipicolinic acid is provided. According to the natto strain of the present invention, natto having the same quality as conventional general natto can be produced in other respects, although the number of spores is small by the conventional natto production process.
In addition, since the natto of the present invention can be sterilized under heating conditions that do not impair the quality of natto, it is expected to promote the development of various processed foods using natto.
Therefore, the present invention can be used in the manufacture of natto and processed foods using the same.
ＮＩＴＥ ＢＰ−０２１６６ NITE BP-02166
配列番号１：spoVFA遺伝子の上流のDNA断片を増幅するためのフォワードプライマー
配列番号２：spoVFA遺伝子の上流のDNA断片を増幅するためのリバースプライマー
配列番号３：spoVFA遺伝子の下流のDNA断片を増幅するためのフォワードプライマー
配列番号４：spoVFA遺伝子の下流のDNA断片を増幅するためのリバースプライマー
配列番号５：クロラムフェニコール耐性遺伝子を含むDNA断片を増幅するためのフォワードプライマー
配列番号６：クロラムフェニコール耐性遺伝子を含むDNA断片を増幅するためのリバースプライマー
配列番号７：spoVFA遺伝子破壊用のDNA断片を増幅するためのフォワードプライマー
配列番号８：spoVFA遺伝子破壊用のDNA断片を増幅するためのリバースプライマー
配列番号９：spoVFA遺伝子破壊を確認するためのフォワードプライマー
配列番号１０：spoVFA遺伝子破壊を確認するためのリバースプライマー
SEQ ID NO: 1: Forward primer for amplifying a DNA fragment upstream of the spoVFA gene SEQ ID NO: 2: Reverse primer for amplifying a DNA fragment upstream of the spoVFA gene SEQ ID NO: 3: Amplifying a DNA fragment downstream of the spoVFA gene Forward primer SEQ ID NO: 4: reverse primer for amplifying a DNA fragment downstream of the spoVFA gene SEQ ID NO: 5: forward primer for amplifying a DNA fragment containing a chloramphenicol resistance gene SEQ ID NO: 6: chloramphenic Reverse primer for amplifying a DNA fragment containing a call resistance gene SEQ ID NO: 7: Forward primer for amplifying a DNA fragment for spoVFA gene disruption SEQ ID NO: 8: Reverse primer for amplifying a DNA fragment for spoVFA gene disruption Sequence number 9: Forward primer for confirming spoVFA gene disruption Reverse primer to confirm the spoVFA gene disruption: the column number 10
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